Targeted Cell Therapies

Abstract

The invention relates to an artificial T cell receptor, wherein an antigen binding domain of the artificial T cell receptor specifically binds a complement pathway protein, nucleic acids encoding such artificial T cell receptors and cells engineered to express such nucleic acids. The invention also relates to targeting polypeptides comprising an extracellular ligand binding domain and an intracellular domain comprising a transcription factor, and wherein the transcription factor is configured to be released upon binding of the ligand binding domain by a ligand. The invention also relates to cells engineered to express the artificial T cell receptor and the targeting polypeptide, particularly where expression of the artificial T cell receptor is operatively linked to binding of the ligand binding domain. The cells of the invention are useful in medicine, particularly in the treatment of inflammatory conditions.

Claims

1. An artificial T cell receptor, wherein an antigen binding domain of the artificial T cell receptor specifically binds a complement pathway protein, preferably, wherein the artificial T cell receptor is a chimeric antigen receptor.

2. The artificial T cell receptor of claim 1, wherein the antigen binding domain comprises an antibody fragment or derivative thereof; preferably wherein the antibody fragment comprises a fragment selected from the group comprising Fab, Fab, F(ab).sub.2, Fv, scFv, disulfide-linked Fvs (sdFv), Fd, linear antibodies, and single domain antibodies; most preferably wherein the antigen binding domain comprises an scFv antibody fragment.

3. The artificial T cell receptor of claim 1 or 2, wherein the complement pathway protein is selected from the group comprising C1q, C1r, C1s, C2a, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, and C9; preferably wherein the complement pathway protein is C1q.

4. The artificial T cell receptor of any of claims 1 to 3, wherein the chimeric antigen receptor comprises an intracellular signalling domain comprising an intracellular signalling domain of CD3zeta, CD28, ICOS, OX-40, or a combination thereof.

5. A nucleic acid encoding the artificial T cell receptor of any of claims 1 to 4.

6. The nucleic acid of claim 5, wherein the nucleic acid is operatively linked to a transcriptional regulatory sequence, and wherein the transcriptional regulatory sequence is configured to bind a transcription factor; preferably wherein the transcriptional regulatory sequence configured to bind the transcription factor comprises a binding domain for Gal4-VP6, tetR-VP64 (tTA), ZFHD1-VP64, Gal4-KRAB, PIP-VP64, ZF21-16-VP64, ZF43-8-VP64 or FoxP3; most preferably wherein the transcriptional regulatory sequence comprises a binding domain for FoxP3.

7. A targeting polypeptide, wherein the targeting polypeptide comprises an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a ligand binding domain and wherein the intracellular domain comprises a transcription factor, and wherein the transcription factor is configured to be released upon binding of the ligand binding domain by a ligand; and preferably, wherein the extracellular domain and the intracellular domain are heterologous to the transmembrane domain.

8. The targeting polypeptide of claim 7, wherein the transcription factor is released by proteolytic cleavage.

9. The targeting polypeptide of claim 7 or 8, wherein the transmembrane domain comprises a notch minimal regulatory region or a notch extended regulatory region.

10. The targeting polypeptide of claim 7 or 8, wherein the intracellular domain comprises a cleavage domain configured to be cleaved by a protease; preferably a type II serine protease.

11. The targeting polypeptide of claim 10, wherein the intracellular domain comprises a cleavage domain configured to be cleaved by a type II serine protease, a type II serine protease domain comprising a catalytically active region of a serine protease, an inhibitory domain comprising an amino acid sequence that inhibits the catalytically active region of the type II serine protease when the ligand binding domain is not bound by a ligand, and the transcription factor; preferably wherein the catalytically active region of the serine protease comprises an active domain of thrombin, Hepatitis C virus Ns3 serine protease, or a TVMV protease.

12. The targeting polypeptide of claim 7 or 8, wherein the ligand binding domain comprises an amino acid sequence that specifically reacts with a benzyl guanine derivative or an O2-benzylcytosine (BC) derivative; preferably wherein the ligand binding domain comprises a SNAP-Tag or a CLIP-Tag.

13. A targeting polypeptide, wherein the targeting polypeptide comprises a ligand binding domain, a transmembrane domain, and a transcription factor, wherein the transmembrane domain is located between the ligand binding domain and the transcription factor, and wherein the transcription factor is cleavably linked to the transmembrane domain; preferably wherein the transmembrane domain and the transcription factor are linked by a cleavable peptide linker.

14. The targeting polypeptide of claim 13, wherein the cleavable linker comprises at least one self-cleaving peptide; preferably wherein the at least one self-cleaving peptide comprises a 2A self-cleaving peptide; more preferably wherein the 2A self-cleaving peptide comprises a P2A peptide, an E2A peptide, an F2A peptide, and/or a T2A peptide, or tandem or triple arrangements of such peptides.

15. The targeting polypeptide of any of claims 7 to 14, wherein the ligand binding domain specifically binds a tissue-associated antigen; preferably wherein the tissue-associated antigen is a tissue specific marker.

16. The targeting polypeptide of claim 15, wherein the tissue-associated antigen is a neuronal marker present at a neuronal synapse; preferably wherein the neuronal marker present at a neuronal synapse is a neuronal antigen; most preferably wherein the neuronal antigen is neurexin or neuroligin.

17. The targeting polypeptide of claim 16, wherein the ligand binding domain comprises a neurexin polypeptide or a neuroligin binding fragment of the neurexin polypeptide.

18. The targeting polypeptide of claim 17, wherein the ligand binding domain comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1.

19. The targeting polypeptide of claim 17 or 18, wherein the neurexin polypeptide or a neuroligin binding fragment thereof comprises an amino acid variant that reduces binding to neuroligin compared to a wild-type neurexin polypeptide or neuroligin binding fragment thereof.

20. The targeting polypeptide of claim 19, wherein the amino acid variant is selected from the group comprising S111A, D162A, I210A, N212A, I210A:D141A, and combinations thereof; preferably wherein the ligand binding domain comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 2 to 6.

21. The targeting polypeptide of any of claims 16 to 20, wherein the targeting polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in any one of SEQ ID NOs: 14 to 19.

22. The targeting polypeptide of claim 16, wherein the ligand binding domain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 7.

23. The targeting polypeptide of claim 15, wherein the tissue-associated antigen is an antigen associated with: i) inflammatory bowel disease, such as Carcinoembryonic antigens, GLUT2, or GLUT5; ii) Rheumatoid arthritis, such as Type II Collagen or Citrullinated vimentin; or iii) Type 1 diabetes, such as Insulin or pro-Insulin.

24. The targeting polypeptide of any of claims 7 to 23, wherein the transcription factor is heterologous to the extracellular domain, the transmembrane domain and/or the rest of the intracellular domain.

25. The targeting polypeptide of any of claims 7 to 24, wherein the transcription factor is selected from the group comprising Gal4-VP6, tetR-VP64 (tTA), ZFHD1-VP64, Gal4-KRAB, PIP-VP64, ZF21-16-VP64, ZF43-8-VP64, LAIR2, METTL7A, RTKN2, FoxP3, BACH2, Cish, ZEB2, EOMES, ZNF683 (Hobit), AML1, ReIA, RORt, TIP60/HDAC7, STAT3, IRF4, USP7, LEF1, GATA-1, GATA-3, and STAT5; preferably wherein the transcription factor is FoxP3.

26. A nucleic acid encoding the targeting polypeptide according to any of claims 7 to 25.

27. The nucleic acid according to claim 26, wherein the nucleic acid is operatively linked to a transcriptional regulatory sequence, and wherein the transcriptional regulatory sequence is configured to bind a transcription factor.

28. The nucleic acid of claim 27, wherein the transcriptional regulatory sequence configured to bind the transcription factor comprises a binding domain for Gal4-VP6, tetR-VP64 (tTA), ZFHD1-VP64, Gal4-KRAB, PIP-VP64, ZF21-16-VP64, ZF43-8-VP64 or FoxP3; preferably wherein the transcriptional regulatory sequence comprises a binding domain for FoxP3.

29. A cell engineered to express a nucleic acid encoding a targeting polypeptide, wherein the targeting polypeptide comprises a ligand binding domain, wherein the cell further comprises a nucleic acid encoding an artificial T cell receptor, and wherein an antigen binding domain of the artificial T cell receptor specifically binds a biomarker; optionally, wherein the nucleic acid encoding the artificial T cell receptor is operatively linked to a transcriptional regulatory sequence, and wherein the transcriptional regulatory sequence is configured to bind a transcription factor.

30. The cell according to claim 29, wherein the ligand binding domain specifically binds a tissue-associated antigen; preferably wherein the tissue-associated antigen is a tissue specific marker; and most preferably wherein the targeting polypeptide is a targeting polypeptide according to any of claims 7 to 25.

31. The cell according to claim 29 or 30, wherein the biomarker is a biomarker of inflammation, an inflammatory mediator, and/or a disease-associated biomarker.

32. The cell according to claim 31, wherein the biomarker of inflammation is a complement pathway protein; preferably wherein the complement pathway protein is selected from the group comprising C1q, C1r, C1s, C2a, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, and C9; more preferably wherein the complement pathway protein is C1q.

33. A cell comprising: i) the nucleic acid according to any of claims 5 or 6; and ii) the nucleic acid according to any of claims 26, 27 or 28.

34. The cell according to any of claims 29 to 33, wherein the nucleic acid encoding the targeting polypeptide comprises a constitutively active promoter or enhancer operatively coupled to the coding region for the targeting polypeptide.

35. The cell according to any of claims 29 to 34, wherein the transcriptional regulatory sequence that is operatively linked to the nucleic acid encoding the artificial T cell receptor is configured to bind the same transcription factor that is cleavably linked to the ligand binding domain of the targeting polypeptide; preferably wherein release of the transcription factor from the targeting polypeptide activates expression of the artificial T cell receptor.

36. The cell according to claim 35, wherein the transcription factor is selected from the group comprising Gal4-VP6, tetR-VP64 (tTA), ZFHD1-VP64, Gal4-KRAB, PIP-VP64, ZF21-16-VP64, ZF43-8-VP64 and FoxP3; preferably wherein the transcription factor is FoxP3.

37. The cell according to any of claims 29 to 36, wherein the cell is an immune cell; preferably wherein immune cell is a T lymphocyte; most preferably wherein the T lymphocyte is a regulatory T lymphocyte.

38. The cell according to any of claims 29 to 36, wherein the cell is a mesenchymal stem cell; optionally wherein the mesenchymal stem cell is a type II mesenchymal stem cell or an adipose-derived stem cell.

39. The cell according to any of claims 29 to 36, wherein the cell is a CD34+ hematopoietic stem cell, CD34+ progenitor cell or a CD34+ induced pluripotent stem cell.

40. A pharmaceutical composition comprising the cell according to any of claims 29 to 39 further comprising a pharmaceutically acceptable carrier, diluent, or excipient; preferably wherein the pharmaceutical composition is formulated for intravenous injection.

41. The cell according to any of claims 29 to 39, or the pharmaceutical composition according to claim 40, for use in medicine.

42. The cell or pharmaceutical composition for use according to claim 41, wherein the cell or pharmaceutical composition is for use in treating an inflammatory disorder in a subject.

43. The cell or pharmaceutical composition for use according to claim 42, wherein the inflammatory disorder is an inflammatory disorder of the nervous system; preferably wherein the inflammatory disorder of the nervous system is selected from the group comprising multiple sclerosis, chronic inflammatory demyelinating polyneuropathy, an encephalitis, a traumatic brain injury, myasthenia gravis, and amyotrophic lateral sclerosis; most preferably wherein the inflammatory disorder of the nervous system is amyotrophic lateral sclerosis.

44. The cell or pharmaceutical composition for use according to claim 42 or 43, wherein the cell is autologous to the subject, or wherein the cell is allogenic to the subject.

45. A nucleic acid vector comprising the nucleic acid according to claim 5 or 6, and/or the nucleic acid according to any of claims 26, 27 or 28.

46. The nucleic acid vector according to claim 45, wherein the nucleic acid vector is: i) a viral vector, preferably a retroviral vector, an adenoviral vector or an adeno-associated viral vector; ii) a non-polymeric vector, preferably a liposome or a gold nanoparticle; iii) a polymeric vector, preferably a dendrimer, a dendrigraft, a polymeric micelle or a poly(-amino ester) vector; iv) a transposon, such as a PiggyBack or Sleeping Beauty transposon; or v) a plasmid flanked by regions for homologous recombination for CRISPR/Cas knock-in.

47. A method of making a cell according to any of claims 29 to 39 comprising contacting a cell with: i) the nucleic acid according to claim 5 or 6; ii) the nucleic acid according to any of claims 26, 27 or 28; and/or iii) the nucleic acid vector of claim 45 or 46.

48. A biological targeting system comprising a) a targeting polypeptide comprising a domain that specifically binds to a tissue specific marker; b) an effector polypeptide wherein the effector polypeptide specifically binds a disease specific antigen or an immune effector molecule; and c) a cargo selected from an: extracellular vesicle, a protein-coated vesicle, a liposome, a dendrimer, a micelle, a biodegradable particle comprising P-selectin, endothelial selectin (E-selectin) and ICAM-1, an artificial nanostructure, an engineered viral particle, plasmid, a transposon, and a bacterial cell.

49. The biological targeting system according to claim 48 for use in medicine.

50. The biological targeting system for use according to claim 49, wherein the biological targeting system is for use in treating an inflammatory disorder in a subject; preferably wherein the inflammatory disorder is an inflammatory disorder of the nervous system; more preferably wherein the inflammatory disorder of the nervous system is selected from the group comprising multiple sclerosis, chronic inflammatory demyelinating polyneuropathy, an encephalitis, a traumatic brain injury, myasthenia gravis, and amyotrophic lateral sclerosis; most preferably wherein the inflammatory disorder of the nervous system is amyotrophic lateral sclerosis.

Description

BRIEF DESCRIPTION OF FIGURES

[0040] The novel features described herein are set forth with particularity in the appended claims. A better understanding of the features and advantages of the features described herein will be obtained by reference to the following detailed description that sets forth illustrative examples, in which the principles of the features described herein are utilized, and the accompanying drawings of which:

[0041] FIG. 1A illustrates a non-limiting embodiment of the mechanism of action of a targeting polypeptide.

[0042] FIG. 1B illustrates the mechanism of induction of a C1q CAR by a transcription factor liberated from a targeting polypeptide of the disclosure.

[0043] FIG. 2 shows a Western blot of expression of WT targeting polypeptide that comprise a neurexin targeting domain. WTA, WTB, WTC and WTD represent transfection optimization conditions. WTE and WTF are non-transfected negative controls

[0044] FIG. 3 shows induction of luciferase activity of targeting polypeptides expressed in HEK293 cells that have a luciferase gene under the control of the GAL4 response element.

[0045] FIG. 4 shows a comparison of the mechanisms of action of a SynNotch targeting polypeptide mediated system described herein with a SynNotch independent targeting polypeptide mediated system described herein. Thus, in the SynNotch dependent system Neuroligin binding could trigger transcription factor release by the targeting polypeptide, or in the SynNotch independent system transcription factor release could be constitutive.

[0046] FIG. 5 shows that C1q-coated tosyl-activated beads stimulate CD69 expression in Tregs by interacting with the constitutively expressed CAR of the invention.

[0047] FIG. 6 shows that binding of neuroligin coated beads to SynNotch receptor in T reg cells transfected with FLAG-tagged CAR gene, drives expression of CAR that is detectable by anti-FLAG antibody.

[0048] FIG. 7 shows that binding of neuroligin coated beads to SynNotch receptor in T reg cells transfected with C1q CAR gene, drives expression of CAR and the CAR is activated by C1q, detected by determination of CD69 expression.

[0049] FIG. 8 shows a comparison of the tissue location of SynNotch Tregs and SynNotch independent (neurexin tether combined with FoxP3 via P2A site) T regs in SOD1 mice (model animal for ALS).

[0050] FIG. 9 shows a comparison of the efficacy of SynNotch independent T regs in a murine experimental autoimmune encephalomyelitis (EAE) study.

[0051] FIG. 10 shows the open reading frame arrangement of the SynNotch construct according to an embodiment of the invention represented by SEQ ID NO: 14.

[0052] FIG. 11 shows a multiple sequence alignment of the neurexin fragments used in the examples represented by SEQ ID NOs: 1-6.

[0053] FIG. 12 shows a flow cytometry comparison of phenotypic Treg regulator, FoxP3 and CAR levels in human Tregs under normal conditions and under pro-inflammatory conditions. 1) Tregs have naturally high FoxP3 levels, but a significant fraction can lose FoxP3 expression. 2) Tregs with Reflection technology (RT) according to an embodiment of the present invention (here a tissue tether-P2A-FOXP3 transcript, under the control of a constitutive promoter, which triggers the expression of an -C1q CAR under the control of FOXP3 response elements) have consistently high FoxP3 levels across the population and express high levels of Chimeric Antigen Receptor (CAR). 3) Tregs lose FoxP3 expression when exposed to pro-inflammatory cytokines, which can cause them to lose their anti-inflammatory properties. 4) Tregs with according to the invention are resistant to pro-inflammatory cytokines and maintain high CAR expression.

DETAILED DESCRIPTION

[0054] The present targeting technology employs two receptors. The first receptor detects a tissue antigen, targeting cells to a tissue of interest. In a preferred embodiment this receptor targets Neuroligin, a receptor found on the post-synaptic membrane, to direct anti-inflammatory cells to neurons and neuromuscular junctions. The second receptor, an artificial T cell receptor such as a Chimeric Antigen Receptor (CAR) which is linked to the first via transcriptional regulation, targets an inflammatory antigen, in a preferred embodiment C1q. The combination of these two receptors targets anti-inflammatory cells to inflamed parts of an organ system, rather than to the whole organ, or to any inflamed area. A key advantage of the presently described system is that expression of the first receptor leads to expression of the second receptor via a transcription factor, allowing the anti-inflammatory activity to be restricted to target tissue. CAR T cells have previously been directed to single antigens (e.g. CD19 in treating cancer) or multiple cell surface antigens to improve specificity but the presently described combination of tissue-specific targeting with one receptor and diagnostic marker targeting with another receptor has not been previously described or contemplated. The targeting of a diagnostic marker of inflammation (e.g. C1q) does not need to be characteristic of a specific disease, but rather a sign that a particular tissue is suffering from inflammation. Targeting a membrane associated antigen (e.g. C1q) as opposed to a fixed receptor, represents a significant departure from previous approaches, as other CAR-T programs target receptors on the cell surface. In general it is known, a torque is required to trigger CARs- and that is normally met by membranous proteins, i.e. proteins that are anchored to the membrane. The C1 complex is only membrane associated when activated. The present inventors have surprisingly found that using a CAR to target a protein that is only associated with a membrane, provides sufficient torque to activate the CAR upon binding.

[0055] Further, it is also counter intuitive to target complement, such as C1q, as a diagnostic marker of disease with a CAR T cell. As these proteins are widely expressed throughout the body, they are not an obvious target for CAR, but when combined with the targeting aspects of the present invention in an AND gate configuration they become a powerful system for precisely directing anti-inflammatory activity to inflamed tissues. Neither Neuroligin/neurexin nor complement are indicative of ALS on their own, but in combination they highlight an inflamed neuron and neuroinflammation, which provides significant promise for treating ALS.

[0056] In one aspect, described herein is a mammalian cell comprising: (a) a targeting nucleic acid wherein the targeting nucleic acid comprises a coding region for a targeting polypeptide comprising: (i) an extracellular domain, wherein the extracellular domain specifically binds to a tissue specific marker; (ii) a transmembrane domain; and (iii) an intracellular domain comprising a transcription factor heterologous to the extracellular domain or the transmembrane domain; and (b) an effector nucleic acid, wherein the effector nucleic acid comprises: (i) a transcriptional regulatory sequence configured to be bound by the transcription factor heterologous to the extracellular domain; and (ii) an effector coding region operatively coupled to the transcriptional regulatory sequence configured to be bound by the transcription factor heterologous to the extracellular domain; wherein the effector coding region encodes a polypeptide that specifically binds an immune effector molecule.

[0057] In another aspect described herein is a targeting polypeptide, comprising an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a ligand binding domain and wherein the intracellular domain comprises a transcription factor, wherein the transcription factor is configured to be released upon binding of the ligand binding domain by a ligand.

[0058] In another aspect described herein is a targeting polypeptide, comprising a ligand binding domain, a transmembrane domain, and a transcription factor, wherein the transmembrane domain is located between the ligand binding domain and the transcription factor, and wherein the transcription factor is cleavably linked to the transmembrane domain, preferably with at least one self-cleaving peptide.

[0059] In another aspect described herein is an artificial T cell receptor, in particular a chimeric antigen receptor, wherein an antigen binding domain of the artificial T cell receptor specifically binds a complement pathway protein. In certain embodiments, the complement pathway protein comprises C1q, C1r, C1s, C2a, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, or C9. In certain embodiments, the complement pathway protein comprises C1q. In a preferred embodiment, the artificial T cell receptor is a CAR or other engineered T cell receptor.

[0060] In another aspect described herein is a biological targeting system comprising a) a targeting polypeptide comprising a domain that specifically binds to a tissue specific marker; b) an effector polypeptide wherein the effector polypeptide specifically binds an immune effector molecule; and c) and a cargo selected from an: extracellular vesicle, a protein-coated vesicle, a liposome, a dendrimer, a micelle, a biodegradable particle comprising P-selectin, endothelial selectin (E-selectin) and ICAM-1, an artificial nanostructure, an engineered viral particle, a plasmid, a transposon, and a bacterial cell.

Certain Definitions

[0061] In the present description, certain specific details are set forth in order to provide a thorough understanding of various embodiments. However, one skilled in the art will understand that the embodiments provided may be practiced without these details. Unless the context requires otherwise, throughout the specification and claims which follow, the word comprise and variations thereof, such as, comprises and comprising are to be construed in an open, inclusive sense, that is, as including, but not limited to. As used in this specification and the appended claims, the singular forms a, an, and the include plural referents unless the content clearly dictates otherwise. It should also be noted that the term or is generally employed in its sense including and/or unless the content clearly dictates otherwise. Further, headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0062] As used herein the term about refers to an amount that is near the stated amount by 10% or less.

[0063] As used herein the term individual, patient, or subject refers to individuals diagnosed with, suspected of being afflicted with, or at-risk of developing at least one disease for which the described compositions and method are useful for treating. In certain embodiments, the individual is a mammal. In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. In certain embodiments, the individual is a human.

[0064] As used herein a therapeutic amount is a dosage amount of a therapeutic intended to produce one or more beneficial effects useful for treating a condition for which the compounds and cells are provided. Some specific therapeutic amounts are discussed in detail herein.

[0065] As used herein treating or treatment refers to the intervention in a disease state intended to produce one or more beneficial effects. For neurodegenerative disease treatment includes methods that are intended to cause, or do cause, stable disease, partial response, complete response, extension of progression-free survival, extension of overall survival, improvement of numbness, improvement of paralysis, memory loss slow-down, delay in memory loss, delay in tremor progression or a prevention or reduction in neurodegeneration, delay in limb strength loss, delay of weakness, delay of atrophy. In certain cases, the therapeutic methods described herein may be used as maintenance after successful treatment or to prevent recurrence or relapse. It is understood that not all individuals will respond to the same degree, or at all, to a given administration of therapeutic cell therapy, however even if no response is detected these individuals are nonetheless considered to have been treated.

[0066] The terms polypeptide and protein are used interchangeably to refer to a polymer of amino acid residues, and are not limited to a minimum length. Polypeptides, including the provided antibodies and antibody chains, receptors and other peptides, e.g., linkers and binding peptides, may include amino acid residues including natural and/or non-natural amino acid residues. The terms also include post-expression modifications of the polypeptide, for example, glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, the polypeptides may contain modifications with respect to a native or natural sequence, as long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.

[0067] As used herein, the term receptor or receptors refer to protein molecules inside the target cell or on its surface that receive a chemical signal.

[0068] As used herein, the term tether, refers to a polypeptide chain expressed upon the cell surface with affinity for a target sufficient to link the cell to a structure containing the target. This structure could be another cell, extracellular matrix, bone, cartilage, tissue, or an artificial surface. In certain embodiments, the tether can be expressed on the cell's surface and link the cell for an extended amount of time to a tissue where the protein is present. In certain embodiments, the tether can be exogenously introduced to the surface of a cell, and link the cell to a target protein. In certain embodiments, the tether will be used as an anchor between one entity and another one.

[0069] As used herein, the term heterologous refers to a nucleotide or amino acid sequence that is from a different source (e.g., gene, polypeptide, or organism) compared to the amino acid or nucleotide sequence to which it refers to as being heterologous. Heterologous includes biological sequences derived from different organisms or to sequences derived from different sources (e.g., genes or proteins) of the same organism. Heterologous sequences include recombinant DNA molecules comprising nucleotide sequences from different sources, fusion proteins comprising amino acid sequences from different sources, and epitope or purification tags of natural or synthetic origin.

[0070] Percent (%) sequence identity with respect to a reference polypeptide sequence is the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are known for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences are able to be determined, including algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, Calif., or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.OD. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0071] In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows: 100 times the fraction X/Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0072] The term artificial T cell receptor refers to any T cell receptor that has either been modified from an naturally occurring T cell receptor, for example by mutation, or that has been engineered so as to have substantially the same properties of an artificial T cell receptor or CAR. The term Chimeric Antigen Receptor or CAR is used to refer generally to any engineered T cell receptor and refers in particular to a polypeptide or set of polypeptides which when in an immunomodulatory cell, provides the cell with specificity for a target location or molecule, for example, an inflamed synapse, and with intracellular signal generation. The term artificial T cell receptor herein is intended to include and encompass CAR as defined herein as the preferred embodiment, although other forms of artificial T cell receptor are also envisaged and included as embodiments of the invention, as would be understood by a person of skill in the art. In some embodiments, a CAR comprises at least an extracellular antigen binding domain, a transmembrane domain and a cytoplasmic signalling domain (also referred to herein as an intracellular signalling domain) comprising a functional signalling domain derived from a stimulatory molecule and/or costimulatory molecule as defined below. An antigen binding domain can suitably be derived from an antibody, an antibody fragment, the VH or VL chain of an antibody or any one or more CDRs associated with any one or more VH or VL chains, and in certain cases all six CDRs derived from an antibody molecule. In certain embodiments, the antigen binding domain of the CAR comprises an scFv derived from an antibody of a known and useful specificity. In some embodiments, the antigen binding domain of the artificial T cell receptor or CAR comprises a scFv derived from an isolated antibody that specifically binds to a C1q protein. For example, the antigen binding domain may be derived from a humanized version of antibody M1, described in WO/2016/073685, the teaching of which is incorporated herein by reference. An example humanized M1 has a light chain variable domain comprising the sequence of SEQ ID NO: 41 and a heavy chain variable domain comprising the sequence of SEQ ID NO: 42. Accordingly, the antibody fragment may comprise a sequence according to either SEQ ID NO: 41 or 42 or a fragment thereof. Preferably, the antigen binding fragment may comprise one or more, preferably all three, of the hyper variable sequences derived from SEQ ID NO: 41 or 42, which are underlined in the sequence table at the end of the examples of the present application and designated as SEQ ID Nos: 60 to 65. Thus, the antigen binding fragment of the artificial T cell receptor of any embodiment of the invention may comprise one, two or all three of SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62; and/or one two or all three of SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65. The hypervariable/complementarity determining regions may be determined according to the method of Chothia, Kabat, or IMGT. In some embodiments that may be combined with any of the preceding embodiments, the artificial T cell receptor comprises a light chain variable domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 41. In some embodiments that may be combined with any of the preceding embodiments, the artificial T cell receptor comprises a heavy chain variable domain comprising an amino acid sequence that is at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 42. In some embodiments, the polypeptide or set of polypeptides are in the same polypeptide chain (e.g., comprise a chimeric fusion protein). In some embodiments, the polypeptide or set of polypeptides are not contiguous with each other, e.g., are in different polypeptide chains. In some embodiments, the polypeptide or set of polypeptides include a dimerization switch that, upon the presence of a dimerization molecule, can couple the polypeptides to one another, e.g., can couple an antigen binding domain to an intracellular signalling domain. In one embodiment, the stimulatory molecule of the CAR is the zeta chain associated with the T cell receptor complex. In one aspect, the cytoplasmic signalling domain comprises a primary signalling domain (e.g., a primary signalling domain of CD3-zeta). In one embodiment, the cytoplasmic signalling domain further comprises one or more functional signalling domains of at least one costimulatory molecule as defined below. In one embodiment, the costimulatory molecule is a costimulatory molecule described herein, e.g., CD27, ICOS, and/or CD28. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signalling domain comprising a functional signalling domain of a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signalling domain comprising a functional signalling domain of a co-stimulatory molecule and a functional signalling domain of a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signalling domain comprising two functional signalling domains of one or more co-stimulatory molecule(s) and a functional signalling domain of a stimulatory molecule. In one embodiment, the CAR comprises a chimeric fusion protein comprising an extracellular antigen binding domain, a transmembrane domain and an intracellular signalling domain comprising at least two functional signalling domains of one or more co-stimulatory molecule(s) and a functional signalling domain of a stimulatory molecule. In one embodiment, the CAR comprises an optional leader sequence at the amino-terminus (N-terminus) of the CAR fusion protein. In one embodiment, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen binding domain, wherein the leader sequence is optionally cleaved from the antigen binding domain (e.g., a scFv) during cellular processing and localization of the CAR to the cellular membrane. The artificial T cell receptors/chimeric antigen receptors or targeting polypeptides described herein can be encoded by a nucleic acid for delivery to the cell of an individual to be treated. In an embodiment, the artificial T cell receptor or chimeric antigen receptor of the invention may be assembled in the cell, rather than expressed as a fusion protein in the cell. For example, the chimeric antigen domain expressed in the cell may comprise an extracellular domain, a transmembrane domain and an intracellular domain, where the extracellular domain comprises an amino acid sequence that specifically reacts with a benzyl guanine (BG) derivative or an O2-benzylcytosine (BC) derivative. In an embodiment the extracellular domain may comprise a SNAP-Tag or a CLIP-Tag. The complement binding functionality of the chimeric antigen receptor in this embodiment may be provided by a BG or BC conjugated antibody that specifically reacts with the complement target, and which is attached to the extracellular domain post-translationally. As the skilled person would appreciate, other variations on this system are possible and are intended to be encompassed by the terms artificial T cell receptor or chimeric antigen receptor. Thus, as indicted above in the context of this application, the term chimeric antigen receptor is intended to mean any polypeptide, or set of polypeptides, which when in an immunomodulatory cell, provides the cell with specificity for a target location or molecule.

[0073] The term biomarker of inflammation as used as used herein, refers to any molecule or cell that is associated with a pro-inflammatory state. This could include pro-inflammatory cytokines (such as interleukin-1 (IL-1), IL-6, IL-12, and IL-18, tumor necrosis factor alpha (TNF-), interferon gamma (IFN), and granulocyte-macrophage colony stimulating factor (GM-CSF)), Neurofilament Light chain (Nf-L), complement proteins as described herein, antibodies, T helper cells, cytotoxic T cells, Natural Killer T cells, gamma/delta T cells, macrophages, dendritic cells, bacterial or viral antigens, particles or cells, allergens and any other molecule or cell that can promote inflammation.

[0074] The term antibody, as used herein, refers to a protein, or polypeptide sequence which specifically binds a target molecule. The antibody may be derived from an immunoglobulin molecule or otherwise designed. The target molecule may comprise a protein, polypeptide, carbohydrate, or lipid.

[0075] The term antibody fragment refers to at least one portion of an antibody, that retains the ability to specifically interact with (e.g., by binding, steric hindrance, stabilizing/destabilizing, spatial distribution) an epitope of an antigen. Examples of antibody fragments include, but are not limited to, Fab, Fab, F(ab).sub.2, Fv fragments, scFv antibody fragments, disulphide-linked Fvs (sdFv), a Fd fragment consisting of the VH and CHI domains, linear antibodies, single domain antibodies such as sdAb (either VL or VH), camelid VHH domains, multi-specific antibodies formed from antibody fragments such as a bivalent fragment comprising two Fab fragments linked by a disulphide bridge at the hinge region, and an isolated CDR or other epitope binding fragments of an antibody. An antigen binding fragment can also be incorporated into single domain antibodies, maxibodies, minibodies, nanobodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis-scFv (see, e.g., Hollinger and Hudson, Nature Biotechnology 23: 1126-1136, 2005). Antigen binding fragments can also be grafted into scaffolds based on polypeptides such as a fibronectin type III (Fn3)(see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide minibodies). The term scFv or single chain variable fragment refers to a single-domain antibody-like construct. The ScFv may be a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chain variable regions are contiguously linked, e.g., via a synthetic linker, e.g., a short flexible polypeptide linker, and capable of being expressed as a single chain polypeptide, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein an scFv may have the VL and VH variable regions in either order, e.g., with respect to the N-terminal and C-terminal ends of the polypeptide, the scFv may comprise VL-linker-VH or may comprise VH-linker-VL. The portion of a CAR comprising an antibody or antibody fragment thereof may exist in a variety of forms where the antigen binding domain is expressed as part of a contiguous polypeptide chain including, for example, a single domain antibody fragment (sdAb), a single chain antibody (scFv) and a humanized antibody (Harlow et al., 1999, In: Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, In: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426). In one embodiment, the antigen binding domain of a CAR comprises an antibody fragment. In a further embodiment, the CAR comprises an antibody fragment that comprises an scFv.

[0076] A polynucleotide is a type of nucleic acid comprising two or more nucleotide bases. In certain embodiments, the nucleic acid is a component of a vector that can be used to transfer the polypeptide encoding polynucleotide into a cell. As used herein, the term vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as expression vectors. Suitable vectors comprise plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, transposons and the like. In the expression vectors regulatory elements such as promoters, enhancers, polyadenylation signals for use in controlling transcription can be derived from mammalian, microbial, viral or insect genes. The expression vectors described herein have one or more promoters or enhancers operatively coupled to a polypeptide to be expressed by the expression vector. In certain embodiments, the promoters are selectively inducible either by the administration of an agent to an individual that has been administered the vector, or in response to a biological stimulus, such as the liberation of transcription factor that can bind the promoter. One example of an inducible system is the Tet-On system, which utilizes the rtTA (reverse tetracycline-controlled transactivator) of Gossen et al. See Gossen M et al., Transcriptional activation by tetracyclines in mammalian cells. Science. 1995 Jun. 23; 268 (5218):1766-9. The expression vectors described herein can be replicated in a host to produce a sufficient amount of expression vector to administer to an individual or to transduce cells from an individual. The ability to replicate in a host, can be conferred by an origin of replication, and a selection gene to facilitate recognition of transformants may additionally be incorporated. Vectors derived from viruses, such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, and the like, may be employed as expression vectors. In certain embodiments, the viral vector is a gamma retrovirus. Viral expression vectors comprising nucleic acids encoding the molecules described herein can be generated in cell culture by the transfection of one or more plasmids that comprise the nucleic acid of interest, a packaging plasmid, and an envelope plasmid. Exemplary systems for lentivirus production are described in Dull T et al, A Third Generation Lentivirus Vector with a Conditional Packaging System. J Virol. 1998. 72(11):8463-8471; or Dull T. et al., Self-Inactivating Lentivirus for Safe and Efficient In Vivo Gene Delivery. J Virol. 1998. 72(12): 9873-9880. Plasmid vectors can be linearized for integration into a chromosomal location. Vectors can comprise sequences that direct site-specific integration into a defined location or restricted set of sites in the genome (e.g., AttP-AttB recombination). Additionally, vectors can comprise sequences derived from transposable elements.

[0077] One type of vector is a genomic integrated vector, or integrated vector, which can become integrated into the chromosomal DNA of the host cell. Another type of vector is an episomal vector, e.g., a nucleic acid capable of extra-chromosomal replication. Vectors that are for genomic integration can target several safe landing sites such as the AAVS1 gene in humans or another animal.

[0078] As used herein, the terms homologous, homology, or percent homology when used herein to describe an amino acid sequence or a nucleic acid sequence, relative to a reference sequence, can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such a formula is incorporated into the basic local alignment search tool (BLAST) programs of Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Percent homology of sequences can be determined using the most recent version of BLAST, as of the filing date of this application.

[0079] In certain embodiments, described herein, are cells in frozen form after transduction with vectors comprising a targeting and/or an immune effector polypeptide. The cells can be provided in a suitable vial such as a cryovial or other vessel capable of withstanding temperatures down to at least about 80 C. In certain embodiments, the cryoprotectant comprises glycerol, DMSO or a combination thereof. In certain embodiments, the frozen cells are contained in a suitable vial or container able to withstand freezing by liquid nitrogen.

[0080] As used herein, the term T reg phenotype refer to a general set of functions and behaviours of a cell exerting T reg capabilities. In some embodiments, it is FoxP3 expression. In some embodiments, it is anti-inflammatory cytokine production. In some embodiments, it is T cell suppression. In some embodiments, it is a regenerative function.

Therapeutic Targeting Systems

[0081] Described herein are systems of specific targeting polypeptides and immune effector molecules. Nucleic acids encoding the targeting polypeptides and immune effector molecules are introduced to a cell that is useful for treating a neuroinflammatory or an autoimmune disease, or an inflammatory condition in the body. Such cells are suitably any cell according to the claims, such as T cells, regulatory T cells, mesenchymal stem cells, and type II mesenchymal stem cells.

[0082] The cell systems described herein are suitably manufactured from an immune cell population, an immune cell population with regulatory characteristics, a T cell population, T cell precursors, or a stem cell population. In one embodiment, the cell systems described herein are manufactured from regulatory T cells (T regs). T regs are a specialized subpopulation cells that act to suppress an immune response, thereby maintaining homeostasis and self-tolerance. It has been shown that T regs are able to inhibit T cell proliferation and cytokine production and play a critical role in preventing autoimmunity and inflammation. Different subsets with various functions of T reg cells exist. T regs can be identified by flow cytometry among other methods. One marker of T reg cells is positivity for the FoxP3 transcription factor. Selected surface markers such as CD25high and CD127low can also serve as surrogate markers to detect T regs.

[0083] Referring to FIG. 1A, in a non-limiting embodiment a targeting polypeptide comprises an extracellular receptor, a transmembrane domain (TM), and an intracellular domain that comprises a transcription factor. Upon binding of the receptor to an appropriate target cell the transcription factor is released. The released transcription factor may then induce expression of a gene of interest. Referring to FIG. 1B, in degen-lock the gene of interest is a chimeric antigen receptor. This chimeric antigen receptor can target and bind an immune effector protein, such as those that participate in pro-inflammatory responses. Upon binding the CAR transduces a signal that activates the immunomodulatory function of the T cell. T regulatory cells for example, exert an immunomodulatory function by the release of the immunosuppressive cytokines IL-10 and TGF. The suppressive action of the Treg cell reduces inflammation in the tissue area surrounding the T cell. Such a system may have an improved safety profile. Two events are involved in the activation of the T cell effector function: 1) an appropriate tissue or disease specific marker bound by the targeting polypeptide; and 2) the presence of a disease associated antigen or inflammation (or inflammatory mediators) at the tissue.

[0084] Optionally, in order to increase the persistence of the transgenic Treg and/or endogenous Treg, or to increase the immune suppressive function of the transgenic Treg. The Treg may further comprise a nucleic acid encoding a regulatory T cell cytokine, chemokine or transcription factor that promotes regulatory T lymphocyte function. The nucleic acid can be inducible by the same transcription factor that induces the CAR, or may be constitutively active.

[0085] In certain embodiments, the cells used herein in the systems described are mammalian cells. In certain embodiments, the cells used herein in the cell systems described are human cells. In certain embodiments, the cells used herein in the systems described are immune cells. In certain embodiments, the immune cells used herein in the systems described are T lymphocytes. In certain embodiments, the immune cells used herein in the cell systems described are CD4+T lymphocytes. In certain embodiments, the T lymphocyte cells used herein in the cell systems described are regulatory T lymphocytes. In certain embodiments the regulatory T lymphocytes express FoxP3. Other such cells include non-T cell immune cells with regulatory function such as regulatory NK cell subsets, and regulatory B cell subsets.

[0086] In additional aspects, the cells used in the systems described herein are mesenchymal stem cells (MSC), adipose-derived stromal/stem cells (ADSCs), CD34+ hematopoietic stem or progenitor cells (such as those described in WO 2019/210042), or CD34+ induced pluripotent stem cells. Canonically, MSCs are CD73 CD90, CD105 and CD11b, CD14, CD19, CD79, CD45 and HLA-DR. ADSCs can be differentiated from MSCs based on lack of expression of CD105; ADSCs also show high expression of CD49d and low expression of Stro-1. These cells can be isolated from the bone marrow or adipose tissue, and further induced to develop an anti-inflammatory phenotype. Type II mesenchymal stem cells can also be used herein to target autoimmune and inflammatory responses. Type II MSCs express anti-inflammatory cytokines and molecules such as IL-10, IDO, and PGE2. Anti-inflammatory mesenchymal stem cells may be made by inducing primary MSC with TLR3 ligands (e.g., polyinosinic-polycytidylic acid (poly(I:C) or poly(rI):poly(rC)).

[0087] The targeting polypeptides and effector molecules herein can also comprise a component of an acellular system. The acellular system can comprise extracellular vesicles, protein-coated vesicles, liposomes, dendrimers, micelles, biodegradable particles based on P-selectin, endothelial selectin (E-selectin) and ICAM-1, artificial nanostructures, engineered viral particles, plasmids, transposons, or bacterial cells.

Targeting Polypeptides

[0088] There are broadly speaking two classes of targeting polypeptides described herein and forming part of the invention. The first comprises a chimeric receptor having an extracellular domain comprising a ligand binding domain for directing the cell to the target tissue, a transmembrane domain and an intracellular domain comprising a cleavable transcription factor. Binding of the ligand binding domain triggers release of the transcription factor and subsequent expression of downstream genes, such as the gene encoding the CAR. This type of receptor can comprise a SynNotch polypeptide, which facilitates the cleavage of the transcription factor in response to target binding. This first type of targeting polypeptide is not necessarily expressed as a complete fusion polypeptide but may be assembled post-translationally, such as via a SNAPtag receptor, as described herein. Thus, the key features are that it has an extracellular targeting domain and an intracellular cleavable transcription factor. In a preferred embodiment the transcription factor is FoxP3. The second type of targeting polypeptide may be independent of the SynNotch system and instead use a viral self-cleaving peptide to release the transcription factor. This type of targeting polypeptide may comprise a tether polypeptide alongside the transcription factor. When the peptide is cleaved, the tether inserts into the cell membrane to provide the targeting function while the cleaved transcription factor stimulates downstream gene expression, such as driving expression of the CAR. Thus, in this instance it is not binding of the external target ligand (e.g. polypeptide) that stimulates release of the transcription factor, but the presence of the external tether is associated with CAR expression by virtue of the co-expression of the targeting polypeptide with the transcription factor. Expression of the targeting polypeptide in this instance may be constitutive in the cell. FIG. 4 provides an illustration of these two systems in action.

[0089] Thus, some targeting polypeptides described herein comprise an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a ligand binding domain and wherein the intracellular domain comprises a transcription factor, wherein the transcription factor is configured to be released upon binding of the ligand binding domain by a ligand. The targeting polypeptide can comprise one or more variants that reduce binding to a specific target without completely abrogating such binding. In this way, a cell expressing the targeting polypeptide is allowed some movement around the site of disease or inflammation while being retained at the site. Such variants (mutants) may be used to tune the affinity of the targeting polypeptide to enhance therapeutic efficacy in any given therapeutic. Thus, the affinity of the targeting polypeptide may be tuned to specific therapeutic situations or targets to provide improved therapeutic outcomes. Such therapies could be personalised to the patient.

[0090] In certain embodiments, the targeting polypeptide comprises a portion of a neurexin polypeptide or a fragment of a neurexin polypeptide that binds neuroligin.

[0091] In certain embodiments, the targeting polypeptide comprises a portion of a neuroligin polypeptide or a fragment of a neuroligin polypeptide that binds neurexin.

[0092] The extracellular domain (or tether) of any targeting polypeptide of the invention can comprise one or more domains that target a neuronal marker. For the avoidance of doubt, any embodiment of the targeting portion (extracellular domain of the targeting polypeptide) is intended to relate to both classes of targeting polypeptide according to the invention. In certain embodiments, the neuronal marker is present at a neuronal synapse. In certain embodiments, the extracellular domain comprises a ligand binding domain that is neurexin or neuroligin. In certain embodiments, the extracellular domain comprises an amino acid sequence from neurexin. In order to reduce the affinity of the neurexin for the neuroligin receptor and contribute to degen-lock, the neurexin amino acid sequence may comprise one or more amino acid variants corresponding to S111A, D162A, I210A, N212A, D141A, I210A/D141A, compared to wild-type neurexin.

[0093] In certain embodiments, the extracellular domain of the targeting polypeptide comprises a neurexin amino acid sequence. In certain embodiments, the extracellular domain of the targeting polypeptide comprises a wild-type neurexin amino acid sequence. In certain embodiments, the extracellular domain comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 1. In certain embodiments, the neurexin amino acid sequence comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 1. The extracellular domain may comprise an amino acid sequence that is 100% identical to any 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 contiguous amino acids from SEQ ID NO: 1. The extracellular domain may comprise an amino acid sequence that comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100 or more amino acids from the N or C terminus of SEQ ID NO: 1. The extracellular domain may suitably comprise a signal sequence and/or a spacer sequence, such as a (G.sub.4S).sub.X spacer, where X is equal to 1, 2, 3, or 4.

[0094] In certain embodiments, the extracellular domain of the targeting polypeptide comprises a neurexin amino acid sequence. In certain embodiments, the extracellular domain of the targeting polypeptide comprises a mutant neurexin amino acid sequence. In certain embodiments, the neurexin amino acid sequence comprises a mutation from serine at amino acid 61 of SEQ ID NO: 1 (S111 in wild type neurexin). In certain embodiments, the neurexin amino acid sequence comprises an alanine at amino acid 61 of SEQ ID NO: 1. In certain embodiments, the neurexin amino acid sequence comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 2, wherein the mutation at position 61 of SEQ ID NO: 2 is preserved. In certain embodiments, the neurexin amino acid sequence comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 2. The extracellular domain may comprise an amino acid sequence that is 100% identical to any 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 contiguous amino acids from SEQ ID NO: 2. The extracellular domain may comprise an amino acid sequence that comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100 or more amino acids from the N or C terminus of SEQ ID NO: 2.

[0095] In certain embodiments, the extracellular domain of the targeting polypeptide comprises a neurexin amino acid sequence. In certain embodiments, the extracellular domain of the targeting polypeptide comprises a mutant neurexin amino acid sequence. In certain embodiments, the neurexin amino acid sequence comprises a mutation from aspartic acid at amino acid 111 of SEQ ID NO: 1 (D162 in wild type neurexin). In certain embodiments, the neurexin amino acid sequence comprises an alanine at amino acid 111 of SEQ ID NO: 1. In certain embodiments, the neurexin amino acid sequence comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 3, wherein the mutation at position 111 of SEQ ID NO: 3 is preserved. In certain embodiments, the neurexin amino acid sequence comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 3. The extracellular domain may comprise an amino acid sequence that is 100% identical to any 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 contiguous amino acids from SEQ ID NO: 3. The extracellular domain may comprise an amino acid sequence that comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100 or more amino acids from the N or C terminus of SEQ ID NO: 3.

[0096] In certain embodiments, the extracellular domain of the targeting polypeptide comprises a neurexin amino acid sequence. In certain embodiments, the extracellular domain of the targeting polypeptide comprises a mutant neurexin amino acid sequence. In certain embodiments, the neurexin amino acid sequence comprises a mutation from isoleucine at amino acid 160 of SEQ ID NO: 1 (I210 in wild type neurexin). In certain embodiments, the neurexin amino acid sequence comprises an alanine at amino acid 160 of SEQ ID NO: 1. In certain embodiments, the neurexin amino acid sequence comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 4, wherein the mutation at position 160 of SEQ ID NO: 4 is preserved. In certain embodiments, the neurexin amino acid sequence comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 4. The extracellular domain may comprise an amino acid sequence that is 100% identical to any 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 contiguous amino acids from SEQ ID NO: 4. The extracellular domain may comprise an amino acid sequence that comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100 or more amino acids from the N or C terminus of SEQ ID NO: 4.

[0097] In certain embodiments, the extracellular domain of the targeting polypeptide comprises a neurexin amino acid sequence. In certain embodiments, the extracellular domain of the targeting polypeptide comprises a mutant neurexin amino acid sequence. In certain embodiments, the neurexin amino acid sequence comprises a mutation from asparagine at amino acid 162 of SEQ ID NO: 1 (N212 in wild type neurexin). In certain embodiments, the neurexin amino acid sequence comprises an alanine at amino acid 162 of SEQ ID NO: 1. In certain embodiments, the neurexin amino acid sequence comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 5, wherein the mutation at position 162 of SEQ ID NO: 5 is preserved. In certain embodiments, the neurexin amino acid sequence comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 5. The extracellular domain may comprise an amino acid sequence that is 100% identical to any 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 contiguous amino acids from SEQ ID NO: 5. The extracellular domain may comprise an amino acid sequence that comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100 or more amino acids from the N or C terminus of SEQ ID NO: 5.

[0098] In certain embodiments, the extracellular domain of the targeting polypeptide comprises a neurexin amino acid sequence. In certain embodiments, the extracellular domain of the targeting polypeptide comprises a mutant neurexin amino acid sequence. In certain embodiments, the neurexin amino acid sequence comprises a mutation from aspartic acid at amino acid 91 and a mutation from isoleucine at amino acid 160 of SEQ ID NO: 1 (D141 and I210, respectively in wild type neurexin). In certain embodiments, the neurexin amino acid sequence comprises an alanine at amino acid 91 and 160 of SEQ ID NO: 1. In certain embodiments, the neurexin amino acid sequence comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 6. In certain embodiments, the neurexin amino acid sequence comprises an amino acid sequence identical to the amino acid sequence set forth in SEQ ID NO: 6. The extracellular domain may comprise an amino acid sequence that is 100% identical to any 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 100 contiguous amino acids from SEQ ID NO: 6. The extracellular domain may comprise an amino acid sequence that comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 100 or more amino acids from the N or C terminus of SEQ ID NO: 6.

[0099] In certain embodiments, the extracellular domain of the targeting polypeptide comprises an scFv that targets neuroligin. In certain embodiments, the scFv that targets neuroligin comprises an amino acid sequence at least about 90%, 95%, 97%, 98%, 99%, or 100% identical to the sequence set forth in SEQ ID NO: 7. In certain embodiments, the neuroligin scFv comprises the complementarity determining regions from SEQ ID NO: 7, wherein the complementarity determining regions are determined according to the method of Kabat. In certain embodiments, the neuroligin scFv comprises the complementarity determining regions from SEQ ID NO: 7, wherein the complementarity determining regions are determined according to the IMGT method. In certain embodiments, the neuroligin scFv comprises the complementarity determining regions from SEQ ID NO: 7, wherein the complementarity determining regions are determined according to the method of Chothia. In certain embodiments, the neuroligin scFv comprises the complementarity determining regions from SEQ ID NO: 6, wherein the complementarity determining regions are determined according to any combination of the method of Chothia, Kabat, or IMGT.

[0100] The extracellular domain of the targeting polypeptide may also comprise one or more fusions with a polypeptide labelling tag. Alternatively, the polypeptide labelling tag may comprise at least about 60%, 70%, 80% or 90% of the amino acid sequence of the extracellular domain. In certain embodiments, the polypeptide labelling tag comprises a SNAP-Tag or a CLIP-Tag. A SNAP-tag, is a 20 kDa mutant of the DNA repair protein 06-alkylguanine-DNA alkyltransferase that reacts specifically and rapidly with benzylguanine (BG) derivatives. See Keppler, A., Gendreizig, S., Gronemeyer, T. et al. (2003) Nat. Biotechnol. 21, 86. The CLIP-Tag is an engineered version of the SNAP-tag permitting it to react specifically with O2-benzylcytosine (BC) derivatives. See Gautier, A., Juillerat, A., Heinis, C. et al. (2008) Chem. Biol. 15, 128. Appropriately a benzylguanine or a derivative or benzylcytosine or a derivative can be used to modify a polypeptide such that introduction of the BC or OC labelled polypeptide into the body of an individual would label the cells of this disclosure with the desired BC or OC modified polypeptide. The polypeptide labelling tag may suitably comprise a leader sequence and/or a spacer domain allowing efficient cell surface expression of the targeting polypeptide comprising the labelling tag.

[0101] The targeting polypeptides described herein comprise a transmembrane domain to anchor the targeting polypeptide to the cell. Further, the transmembrane domain may contribute to the functional aspects of the targeting domain by providing catalytic activity that cleaves the intracellular domain releasing a polypeptide comprising an amino acid sequence configured to bind a regulatory sequence. Such regulated cleavable transmembrane systems may comprise a portion of a Notch protein.

[0102] Notch is transmembrane receptor protein that is present in vertebrates and invertebrates. Signal transduction through Notch from the extracellular space to the intracellular space requires the transmembrane portion of the Notch protein. The engagement of notch on cells leads to two-step proteolysis of the notch receptor that ultimately causes the release of the intracellular portion of the receptor from the membrane into the cytoplasm. The extracellular domain and the intracellular domains of notch can be replaced with heterologous polypeptides, while preserving this transmembrane based cleavage. Thus, creating a chimeric Notch protein by replacing the extracellular domain of Notch with a suitable antigen or ligand binding domain (e.g., a heterologous receptor or antibody-based polypeptide) allows for the construction of a molecular sensor that allows release of the intracellular portion by a non-delta-based ligand. Further, engineering of the intracellular domain, to replace the Notch intracellular domain with a heterologous transcription factor, allows the chimeric Notch to release the heterologous transcription factor. Such transcription factors and their targets are described herein in more detail, but in brief, can be used to induce activation of a variety of genes under the control of a regulatory sequence that binds the heterologous transcription factor.

[0103] In certain embodiments, the transmembrane domain comprises a portion of the mammalian Notch protein. The portion of the mammalian Notch protein may be derived from a human notch, such as that shown in SEQ ID NO: 8. In certain embodiments, the portion of the Notch protein is the Notch regulatory region. In certain embodiments, the notch regulatory region comprises a Lin 12-Notch repeat, an S2 proteolytic cleavage site, and an S3 proteolytic cleavage site The notch transmembrane domain may further comprise N and C terminal spacers to allow for efficient expression and activation of a polypeptide comprising heterologous extracellular and intracellular domains, these spacers may be at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids not derived from Notch or the corresponding intracellular or extracellular domains. In certain embodiments, the notch transmembrane domain comprises an amino acid sequence at least about 90%, 95%, 97%, 97%, 99%, or 100% identical to SEQ ID NO 9. In certain embodiments, the notch transmembrane domain comprises an amino acid sequence at least about 90%, 95%, 97%, 97%, 99%, or 100% identical to SEQ ID NO 10. In certain embodiments, the notch transmembrane domain comprises an amino acid sequence at least about 90%, 95%, 97%, 97%, 99%, or 100% identical to SEQ ID NO 11. Systems deploying a chimeric Notch protein are described in U.S. Pat. No. 9,834,608; Morsut L., et al. Engineering Customized Cell Sensing and Response Behaviors Using Synthetic Notch Receptors, Cell 2016 Feb. 11; 164(4):780-91; and Roybal K. T., et al. Engineering T Cells with Customized Therapeutic Response Programs Using Synthetic Notch Receptors Cell 2016 Oct. 6; 167(2):419-432.

[0104] The intracellular domain of the described targeting polypeptides comprises one or more polypeptides that are configured to bind a regulatory sequence. Such polypeptides can comprise all or part of a transcription factor. In certain embodiments, the transcription factor polypeptide comprises a DNA binding domain and an activating domain. The DNA binding domain and the activating domain may be derived from the same transcription factor or different transcription factors. In certain embodiments, the transcription factor may be configured to bind a regulatory sequence associated with an exogenous nucleic acid, a regulatory sequence associated with an endogenous gene, or both.

[0105] In certain embodiments, the transcription factor comprises Gal4-VP6, tetR-VP64 (tTA), ZFHD1-VP64, Gal4-KRAB, PIP-VP64, ZF21-16-VP64, ZF43-8-VP64 or FoxP3. In certain embodiments, the transcription factor comprises Gal4. In certain embodiments, the transcription factor comprises an amino acid sequence at least about 90%, 95%, 97%, 97%, 99%, or 100% identical to SEQ ID NO: 12. In certain embodiments, the transcription factor comprises Gal4-VP6. In certain embodiments, the transcription factor comprises FOXP3. In certain embodiments, the transcription factor comprises an amino acid sequence at least about 90%, 95%, 97%, 97%, 99%, or 100% identical to SEQ ID NO: 13.

[0106] Alternatively, if a notch-based system is not used for the targeting polypeptide a serine protease-based system can be used. In such a system the transmembrane domain lacks catalytic activity, and can be any suitable transmembrane domain that serves to anchor the extracellular domain to the cell. In a serine protease based system the intracellular domain comprises a cleavage domain configured to be cleaved by a type II serine protease, a type II serine protease domain comprising a catalytically active region of a serine protease, an inhibitory domain comprising an amino acid sequence that inhibits the catalytically active region of the type II serine protease when the ligand binding domain is bound by a ligand, and the transcription factor. Upon binding of ligand or antigen to the ligand binding domain, steric hinderance of the type II serine protease domain by the inhibitory domain is relieved, allowing the type II serine protease domain to cleave the cleavage domain releasing the transcription factor(s). This type of system is compatible with the same transcription factors already described herein including Gal4-VP6, tetR-VP64 (tTA), ZFHD1-VP64, Gal4-KRAB, or FoxP3. In certain embodiments, the catalytically active region of the serine protease comprises an active domain of thrombin, Hepatitis C virus Ns3 serine protease, or a TVMV protease.

[0107] In certain embodiments, the targeting polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 97%, 99%, or 100% identical to SEQ ID NO 14. In certain embodiments, the targeting polypeptide comprises an amino acid sequence identical to SEQ ID NO 14. In certain embodiments, the targeting polypeptide lack a signal sequence, and/or is expressed on the cell surface of a cell beginning with the amino acids ASSLGA.

[0108] In certain embodiments, the targeting polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 97%, 99%, or 100% identical to SEQ ID NO 15. In certain embodiments, the targeting polypeptide comprises an amino acid sequence identical to SEQ ID NO 15. In certain embodiments, the targeting polypeptide lacks a signal sequence, and/or is expressed on the cell surface of a cell beginning with the amino acids ASSLGA.

[0109] In certain embodiments, the targeting polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 97%, 99%, or 100% identical to SEQ ID NO 16. In certain embodiments, the targeting polypeptide comprises an amino acid sequence identical to SEQ ID NO 16. In certain embodiments, the targeting polypeptide lacks a signal sequence, and/or is expressed on the cell surface of a cell beginning with the amino acids ASSLGA.

[0110] In certain embodiments, the targeting polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 97%, 99%, or 100% identical to SEQ ID NO 17. In certain embodiments, the targeting polypeptide comprises an amino acid sequence identical to SEQ ID NO 17. In certain embodiments, the targeting polypeptide lacks a signal sequence, and/or is expressed on the cell surface of a cell beginning with the amino acids ASSLGA.

[0111] In certain embodiments, the targeting polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 97%, 99%, or 100% identical to SEQ ID NO 18. In certain embodiments, the targeting polypeptide comprises an amino acid sequence identical to SEQ ID NO 18. In certain embodiments, the targeting polypeptide lack a signal sequence, and/or is expressed on the cell surface of a cell beginning with the amino acids ASSLGA.

[0112] In certain embodiments, the targeting polypeptide comprises an amino acid sequence at least about 90%, 95%, 97%, 97%, 99%, or 100% identical to SEQ ID NO 19. In certain embodiments, the targeting polypeptide comprises an amino acid sequence identical to SEQ ID NO 19. In certain embodiments, the targeting polypeptide lack a signal sequence, and/or is expressed on the cell surface of a cell beginning with the amino acids ASSLGA.

[0113] The targeting polypeptides are encoded by nucleic acids and operatively coupled to a regulatory sequence that allows for constitutive or inducible activation of the targeting polypeptides. Such regulatory sequences comprise a CMV promoter, a -actin promoter, an RSV promoter, an EF1 promoter, an SV40 promoter, human ubiquitin C promoter, PGK promoter, doxycycline-inducible promoter, and any combination thereof.

[0114] The targeting polypeptides described herein can be encoded by nucleic acids and delivered to cells using the expression vectors described herein. In certain embodiments, the targeting polypeptides are encoded by nucleic acids and delivered to cells using a retroviral vector. In certain embodiments, the targeting polypeptides are encoded by nucleic acids and delivered to cells using an adenoviral vector or adeno associated viral (AAV) vector. In certain embodiments, the targeting polypeptides are encoded by nucleic acids and delivered to cells using a lentiviral vector. In certain embodiments, the targeting polypeptides are encoded by nucleic acids and delivered to cells using electroporation.

Immune Effector Molecules

[0115] In certain embodiments, described herein, are immune effector molecules and nucleic acids encoding the immune effector molecules. In certain embodiments, the immune effector molecule comprises an antigen binding domain, that is a component of an extracellular domain of the immune effector molecule. The immune effector molecules also comprise a transmembrane domain and one or more intracellular signalling domains. The one or more intracellular signalling domains may function in T cell signalling and comprise CD3-zeta, CD27, ICOS, and/or CD28. In certain embodiments, the immune effector molecule comprises a chimeric antigen receptor (CAR). In some embodiments, the immune effector molecule comprises a T cell receptor. In certain embodiments, the antigen binding domain of the chimeric antigen receptor comprises an scFv. In certain embodiments, the antigen binding domain of the chimeric antigen receptor comprises a binding domain that specifically binds to a disease associated antigen or a pro-inflammatory molecule. In certain embodiments, the pro inflammatory molecule is a cytokine, a chemokine, a bacterial derived molecule such as LPS or peptidoglycan, or a molecule derived from an apoptotic cells such as phosphatidyl serine. In certain embodiments, the antigen binding domain of the chimeric antigen receptor comprises a binding domain that specifically binds to a complement pathway protein or polypeptide. In certain embodiments, the complement pathway protein comprises C1q, C1 r, C1s, C2a, C3, C3a, C3b, C4, C4a, C4b, C5, C5a, C5b, C6, C7, C8, or C9. In certain embodiments, the complement pathway protein comprises C1q.

[0116] The antigen binding domain of the immune effector molecule may be derived from an antibody or an scFv. In certain embodiments, the antigen binding domain is derived from an anti-C1q monoclonal antibody or scFV. Certain C1q antibodies useable in the current immune effector molecules are described in WO 2008/035527 and U.S. Pat. No. 10,316,081. The complementarity determining regions from a C1q antibody can be used to construct the antigen binding domain of the described immune effector molecules, wherein the complementarity determining regions are determined according to any combination of the methods of Chothia, Kabat, or IMGT.

[0117] The immune effector molecules described herein are encoded by nucleic acids and can be operatively coupled to a regulatory sequence. This allows for inducible expression for the immune effector molecule. In certain embodiments, the regulatory sequence is configured to be bound by the transcription factor that is released upon engagement of the targeting polypeptide. In certain embodiments, the regulatory sequence is configured to be bound by Gal4-VP6, tetR-VP64 (tTA), ZFHD1-VP64, Gal4-KRAB, or FoxP3. In certain embodiments, the regulatory sequence is configured to be bound by FoxP3.

[0118] The immune effector molecules described herein can be encoded by nucleic acids and delivered to cells using the expression vectors described herein. In certain embodiments, the immune effector molecules are encoded by nucleic acids and delivered to cells using a retroviral vector. In certain embodiments, the immune effector molecules are encoded by nucleic acids and delivered to cells using a lentiviral vector. In certain embodiments, the immune effector molecules are encoded by nucleic acids and delivered to cells using an adenoviral vector or an AAV vector. In certain embodiments, the immune effector molecules are encoded by nucleic acids and delivered to cells using electroporation.

[0119] The cells described herein in addition to comprising a targeting polypeptide and/or an immune effector molecule may also comprise a nucleic acid encoding a regulatory T cell cytokine, chemokine or transcription factor that promotes regulatory T lymphocyte function. In certain embodiments, the transcription factor that promotes regulatory T lymphocyte function comprises STAT5b. In certain embodiments, the polypeptide that promotes regulatory T lymphocyte function comprises IL-2, IL-5, IL-4, IL-7, IL-15, or IL-37. In certain embodiments, the polypeptide that promotes regulatory T lymphocyte function comprises IL-2. Nucleic acids encoding a regulatory T cell cytokines, chemokines or transcription factors that promote regulatory T lymphocyte function are operatively coupled to a regulatory sequence. In certain embodiments, the regulatory sequence is inducible. In certain embodiments, the regulatory sequence is constitutive.

[0120] Cells administered comprising the targeting polypeptides and the immune effector molecules described herein can deliver highly-localized factors to promote regulatory T lymphocyte function, namely anti-inflammatory effects on surrounding tissue. In certain embodiments, the polypeptide that promotes regulatory T lymphocyte function is under control of a constitutive promoter. In certain embodiments, the polypeptide that promotes regulatory T lymphocyte function is under control of an inducible promoter. The inducible promoter may be under a promoter configured to be bound by a transcription factor associated with the targeting polypeptide and may be the same transcription factor that activates transcription of the immune effector molecule. Thus, activation of the targeting polypeptide by a suitable tissue specific marker or ligand results in activation of expression of both the immune effector molecule and expression of a polypeptide that promotes regulatory T lymphocyte function. In certain embodiments, the polypeptide that promotes regulatory T lymphocyte function comprises IL-2, IL-5, IL-4, IL-7, IL-15, or IL-37.

Methods of Making

[0121] Described herein are methods of making populations of cells comprising targeting polypeptides and immune effector molecules, the method comprising contacting the cell population with a nucleic acid encoding a chimeric antigen receptor and/or an immune effector molecule. In certain embodiments, the cell population is a population comprising CD4+ T cells that are at least about 75%, 80%, 85%, 90%, 95%, 98%, 99% or more CD4+. In certain embodiments, the nucleic acid or nucleic acids used to contact the cell is encoded in an expression vector such as a lentivirus vector, adenovirus, AAV or a retrovirus vector. In certain embodiments, the nucleic acids are introduced to the population of cells by electroporation.

[0122] The cell source of the population of cells can be from the individual ultimately treated, and therefore autologous. The cell source of the population of cells can be from an HLA-matched individual, and therefore heterologous. The cell source can be so called universal T cells, which have disruptions in the HLA locus or the TCR and chains.

[0123] Autologous or heterologous cells are isolated from the peripheral blood mononuclear cells (PBMC) of the individual. The leukocyte population can be isolated by leukapheresis, positive, or negative selection and then subjected to transduction or transfection with nucleic acid encoding targeting polypeptides and/or immune effector molecules of this disclosure.

[0124] Nucleic acids cells encoding targeting polypeptides and/or immune effector molecules can be introduced into cells in a variety of ways, including by the viral transduction, electroporation, or lipid assisted transfection of nucleic acids encoding each of the targeting polypeptide and the immune effector molecules.

[0125] In one such process, illustrated here by way of example, cells, for example, T cells, are collected from a donor (for example, a patient to be treated with an autologous chimeric antigen receptor T cell product) via apheresis (e.g., leukapheresis). Collected cells may then be optionally purified, for example, by an elutriation step. Paramagnetic particles, for example, anti-CD3/anti-CD28-coated paramagnetic particles, may then be added to the cell population, to expand the T cells. The process may also include a transduction step, wherein nucleic acid encoding one or more desired proteins, for example, a CAR, for example a CAR targeting C1q, is introduced into the cell. The nucleic acid may be introduced in a lentiviral vector. The cells, e.g., the lentiviral transduced cells, may then be expanded for a period of days, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more days. After expansion cells can be collected and washed by centrifugation, the overall transduction efficiency determined, by flow cytometry for example, suspended in an appropriate diluent and administered to a patient. Alternatively, cells may be refrigerated or frozen and shipped to a site where they are administered to the individual in need thereof.

[0126] Other cell types such as mesenchymal stem cells and adipose-derived stromal/stem cells can also be used in the method described herein. Isolation of mesenchymal stem cells can be from the bone-marrow or adipose tissue. Method of isolating mesenchymal stem cells are described for example in US 2002/0045260 A1 or US 2011/0076770 A1. Mesenchymal stem cells can further be induced to develop an anti-inflammatory phenotype by treatment with TLR3 ligand either before or after they have been transfected or transduced with nucleic acids encoding the targeting polypeptides. Method of inducing mesenchymal stem cells include those described in US 2014/0017787 A1.

Methods of Use and Medical Uses

[0127] Described herein are methods of delivering a cargo to a specific tissue and activating a desired effect. This effect may be a therapeutic effect, a diagnostic effect, or some other effect useful for research and discovery. The usefulness in such an approach is a result of the presence of a targeting polypeptide and a polypeptide that serves an effector function. The effector function can vary dependent on the exact application. The cargo may be a cell as described herein. Alternatively, the cargo may be extracellular vesicles, protein-coated vesicles, liposomes, dendrimers, micelles, biodegradable particles based on P-selectin, endothelial selectin (E-selectin) and ICAM-1, artificial nanostructures, engineered viral particles, plasmids, transposons, or bacterial cells. The cargo can further comprise therapeutically or diagnostically useful components such as fluorescent, radioactive, or luminescent cargo. Administration of the cargo comprising the targeting polypeptide and a polypeptide that serves an effector function targets the cargo to an appropriate location based upon specificity of the targeting polypeptide for a tissue specific marker.

[0128] In certain embodiments, described herein is a molecule with a targeting function and an effector function. In certain embodiments, the targeting function targets the antibody to neurexin or neuroligin and the effector function binds to a complement related protein.

[0129] The cells comprising targeting polypeptides and inducible immune effector molecules are for use in methods of treating a neuroinflammatory disease or an autoimmune neurological disease in a mammal. The mammal can be a human individual. In an embodiment, the method of treating the neuroinflammatory disease comprises administering a plurality or population of the cells that express a targeting polypeptide and comprise an inducible CAR construct activatable by the targeting polypeptide's engagement with an appropriate ligand to an individual in need thereof. In certain embodiments, the cells are autologous to the individual being administered the cells. In certain embodiments, the cells are heterologous to the individual being administered the cells, but are HLA matched. In certain embodiments, the cells are not HLA matched but are universal T cells. These are made by targeting genomic sequences in the constant regions of the endogenous a or R subunits of the TCR or disrupting the HLA-A locus of MHC gene complex, the expression of TCR or the HLA class I antigens is abrogated, and the resulting T cells are not capable of recognizing allogeneic antigens, thus leading to the elimination of GVHD. In certain embodiments, the plurality of cells or cell population is a population comprising CD4+ T cells that are at least about 75%, 80%, 85%, 90%, 95%, 98%, 99% or more CD4+. In certain embodiments, the plurality of cells or cell population is a population comprising CD4+ T cells that are at least about 75%, 80%, 85%, 90%, 95%, 98%, 99% or more positive for FoxP3 expression.

[0130] Also described are methods of treatment using other anti-inflammatory cell types such as regulatory NK cell subsets, regulatory B cell subsets, induced pluripotent stem cells, CD34+ stem cells or the mesenchymal stem cells and adipose-derived stromal/stem cells described herein.

[0131] Neuroinflammatory diseases generally display infiltration of cytotoxic T cells, Th1 cells, Th17 cells, or phagocytic cells into one or more neuronal tissues. Neuroinflammatory diseases can also be marked by the presence of autoantibodies, or elevated inflammatory mediators such as chemokines or cytokines at neuronal tissues. These neuronal tissues comprise, for example, the brain, spinal cord, neurons, or neuro-muscular junctions. The neuroinflammatory disease can suitably be a motor neuron disease, demyelinating disease, neurodegenerative disease, brain or spinal cord injury.

[0132] Neuroinflammatory diseases treatable by the cells, methods and medical uses described herein comprise acute disseminated encephalomyelitis (ADEM), acute Optic Neuritis (AON), transverse myelitis, neuromyelitis optica, multiple sclerosis, relapsing-remitting multiple sclerosis, secondary-progressive multiple sclerosis, primary-progressive multiple sclerosis (PPMS), progressive-relapsing multiple sclerosis (PRMS), Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease (amyotrophic lateral sclerosis), Creutzfeldt Jakob disease, multiple sclerosis, diffuse Lewy body disease, leukoencephalitis, autoimmune encephalitis, meningitis, temporal lobe epilepsy, traumatic brain injury, inflammatory spinal cord injury, myasthenia gravis. In certain embodiments, the neuroinflammatory disease treatable by the cells and methods described herein is a motor neuron disease selected from amyotrophic lateral sclerosis (ALS), progressive bulbar palsy (PBP), progressive muscular atrophy (PMA), primary lateral sclerosis (PLS), and combinations thereof. In a certain embodiment, the neuroinflammatory disease treatable by the cells and methods described herein is amyotrophic lateral sclerosis (ALS).

[0133] The cells described herein can also be used to treat autoimmune neurological diseases. Such diseases are defined by the development of adaptive immune responses to self-antigens expressed in neural tissues, such as myelin basic protein or myelin oligodendrocyte glycoprotein. Autoimmune neurological diseases treated by the cells and methods described herein comprise multiple sclerosis, myasthenia gravis, Guillain-Barre, and any combination thereof. The multiple sclerosis treated may be relapsing-remitting multiple sclerosis, secondary-progressive multiple sclerosis, primary-progressive multiple sclerosis, progressive-relapsing multiple sclerosis, or any combination thereof.

[0134] The cells comprising targeting polypeptides and inducible immune effector molecules are for use in treating, and can be used in the manufacture of a medicament for treating, a neuroinflammatory or autoimmune neurological disease. The neuroinflammatory diseases comprise acute disseminated encephalomyelitis (ADEM), acute Optic Neuritis (AON), transverse myelitis, neuromyelitis optica, multiple sclerosis, relapsing-remitting multiple sclerosis, secondary-progressive multiple sclerosis, primary-progressive multiple sclerosis (PPMS), progressive-relapsing multiple sclerosis (PRMS), Alzheimer's disease, Parkinson's disease, Huntington's disease, Lou Gehrig's disease (amyotrophic lateral sclerosis), Creutzfeldt Jakob disease, multiple sclerosis, diffuse Lewy body disease, leukoencephalitis, meningitis, temporal lobe epilepsy, traumatic brain injury, inflammatory spinal cord injury. The neuroinflammatory disease may be a motor neuron disease. The motor neuron disease may be selected from amyotrophic lateral sclerosis (ALS), progressive bulbar palsy (PBP), progressive muscular atrophy (PMA), primary lateral sclerosis (PLS), and combinations thereof. In a certain embodiment, the neuroinflammatory disease treatable by the cells and methods described herein is amyotrophic lateral sclerosis (ALS).

[0135] The cells comprising targeting polypeptides and inducible immune effector molecules described herein can be administered in a manner consistent with the disease being treated. Cells may be administered intravenously, subcutaneously, intradermally, or intrathecally as required by the neuroinflammatory/autoimmune neurological disease being treated. For example, to easily traffic to sites of inflammation in the brain or spinal cord the cells may be administered intrathecally. Intravenous, subcutaneous, or intradermal administration may be more suitable for administration to individuals with MS or motor neuron disease.

[0136] The cells comprising targeting polypeptides and inducible immune effector molecules described herein can be administered in a therapeutically effective amount. The total amount of cells administered to an individual can be administered in a single-dose or split over multiple doses to help minimize infusion reaction side-effects. In certain embodiments, a therapeutically effective dose of cells comprises about 110.sup.5 cells/kg to about 110.sup.7 cells/kg. These numbers refer to doses of positive cells, determined for example, by a parallel transduction of cells. The actual total number of cells given may be higher to account for the transduction efficiency. The number of cells above can refer to the number of CD4+ T cells or the number of FOXP3+CD4+ T cells.

[0137] Individuals may be administered a dosage of the cells comprising targeting polypeptides and inducible immune effector molecules based upon a diagnosis with or a suspected diagnosis with any of the neuroinflammatory disease or autoimmune neurological diseases described herein. Individuals can be administered one dose of cells or multiple doses based upon a reemergence of systems or no improvement in symptoms. A subsequent dose can comprise the same, 2, 3, 4, 5, 6, 7, 8, 9, or 10 more cells than the first dose. A subsequent dose may be administered after 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks.

Pharmaceutically Acceptable Excipients, Carriers, and Diluents

[0138] In certain embodiments, the cells of the current disclosure are included in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. In certain embodiments, the cells of the current disclosure are administered suspended in a sterile solution. In certain embodiments, the solution comprises about 0.9% NaCl. In certain embodiments, the solution comprises about 5.0% dextrose. In certain embodiments, the solution further comprises one or more of: buffers, for example, acetate, citrate, histidine, succinate, phosphate, bicarbonate and hydroxymethylaminomethane (Tris); surfactants, for example, polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; polyol/disaccharide/polysaccharides, for example, glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; amino acids, for example, glycine or arginine; antioxidants, for example, ascorbic acid, methionine; or chelating agents, for example, EDTA or EGTA.

[0139] Also described herein and forming part of the invention are kits comprising one or more of the vectors or nucleic acids described herein in a suitable container and one or more additional components selected from: instructions for use; a diluent, an excipient, a carrier, and a device for administration.

[0140] In certain embodiments, described herein is a method of preparing a neuroinflammatory treatment comprising admixing one or more pharmaceutically acceptable excipients, carriers, or diluents and a mammalian cell of the current disclosure. In certain embodiments, the cell expresses a suitable targeting polypeptide and comprises a nucleic acid(s) encoding an inducible CAR.

EXAMPLES

[0141] The following illustrative examples are representative of embodiments of compositions and methods/uses described herein and are not meant to be limiting in any way.

Example 1Design and Expression of Neurexin Targeting Polypeptides

[0142] An insert encoding a chimeric targeting polypeptide was designed as follows: a 20 amino-acid signal-peptide sequence (MALPVTALLLPLALLLHAARP (SEQ ID NO: 20)) was fused to a myc-tag sequence EQKLISEEDL (SEQ ID NO: 21) for ease of detection, the neurexin 1-b polypeptide, the linker and the transmembrane notch sequence and a transcription factor. The synthetic DNA was manufactured by Genescript Ltd, verified by sequencing, and cloned into the pcDNA3.1(+) backbone between the EcoRI and NotI restriction sites. pREF001 contains the natural sequence of the fragment encoding neurexin 1-b polypeptide (SEQ ID NO: 1). pREF002 encodes a neurexin 1-b variant, with a S111A mutation (SEQ ID NO: 2). pREF003 encodes a neurexin 1-b variant, with a D162A mutation (SEQ ID NO: 3). pREF004 encodes a neurexin 1-b variant, with a I210A mutation (SEQ ID NO: 4). pREF005 encodes a neurexin 1-b variant, with a N212A mutation (SEQ ID NO: 5). pREF006 encodes a neurexin 1-b double variant, with a D141A:I210A double mutation (SEQ ID NO: 6).

[0143] To assess functionality of the receptors, GAL4 Reporter (Luc)-HEK293 cells were transiently transfected with plasmid pREF001 to pREF006 as follows: the cell bank (BPS bioscience) was thawed and maintained in Growth Medium 1B: MEM medium supplemented with 10% FBS, 1% non-essential amino acids, 1 mM Na-pyruvate, 0.5% Penicillin/Streptomycin and 400 g/ml of geneticin. When cells reached around 90% confluency, they were split at 1:10 ratio. After the 3rd passage, cell stocks of 310.sup.6 cells were frozen in 10% DMSO. Cells were seeded onto a 6-well plate for 24 hours, at a density of 110.sup.6 cells per well in 2 ml of medium without antibiotics. 2 g of plasmid DNA (Genescript) was diluted into 200 l of medium without FBS. 6 l of X-tremeGENE HP DNA Transfection Reagent was added directly into medium containing the diluted DNA, mixed and incubated at 15 min at room temperature to allow complexes to form. Next, DNA complexes were added drop-wise into the medium. Cells were incubated at 37 C. in a 5% CO2 incubator for the next 24 h. Transfection efficiency was determined by detection with c-Myc (9E10) FITC antibody (Santa Cruz Biotechnology) after 24 h and anti-hc-Myc PE Conjugated antibody (R&D Systems) after 48 h. Expression by western blot was assessed and is shown in FIG. 2.

Example 2Functional Testing of Neurexin Targeting Polypeptides

[0144] For preliminary experiments, pREF020 was prepared from pREF001 as follows: primer pair AGCGAGGAGGATCTGATGTACCAGAGGATGCTGAGGTGC (SEQ ID NO: 25) and GCTGTAGTCCAGGATGGGCACCTCGCCCACCAGC (SEQ ID NO: 26) was used to amplify 828 bp fragment using template pREF001 and HiFi PCR Premix polymerase (Clontech-Takara) as follows: 12.5 L water was mixed with 12.5 L polymerase premix, 0.5 L plasmid DNA template, 1.25 L 10 uM primer GCCGCCGCGATCGCCatggcattgcccgt (SEQ ID NO: 27) and 1.25 L 10 uM GCGGCCGGCCGTTTAtcatgatccgagcatgtccag (SEQ ID NO: 28). Fragment was then run on 1% agarose 0.5TBE gel and was isolated from the gel using MN Cleanup kit. Then, the band was eluted in 15 L water. Then, 1 L insert was mixed with 1 uL vector backbone amplified as described above and the InFusion cloning reaction was performed in the presence of 2 L of InFusion 5 premix (Takara) and 6 L water at 50 C. for 15 minutes. 1 L was used to transform chemically competent Escherichia coli HST08 (Takara) by heat shock and cells were selected on LB with 100 g/mL ampicillin (Melford). Collect clones were identified and confirmed by sequencing.

[0145] Assay was performed as follows: Transfections were carried out with 7.5 L Fugene (Promega) and 2 g plasmid DNA from midi-prep per well. For pREF020 transfection, DNA concentration was 94.1 ng/uL and 53.1 microlitres of DNA solution were mixed with 18.8 uL Fugene and 3.1 L OptiMem (Gibco). Mixture was incubated for 20 minutes at room temperature and 30 L of mixture were added dropwise and plates were mixed gently to rocking from side to side. Plates were incubated for 48 hours at 37 C. 5% CO2.

[0146] To measure binding of the neurexin mutants to neuroligin, and activation of the GAL4 reporter, cells were seeded onto 96-well plates for a luciferase assay. White bottomed 96 well plate was coated with 100 ng neuroligin (Biotechne) suspended in PBS. After 24 hours PBS was removed. Cells were collected and counted, diluted in fresh DMEM medium and cells were plated at 18000 cells per well in 100 L volume. After 24 hours, luciferase assays using Promega kit E1500 were carried out on the wells. Media is removed from the plates and 20 L lysis buffer was added. After 5 minutes of lysis 100 L of luciferase assay reagent was mixed into each well and the light signal from each well was recorded with a plate reader. Exemplary induction using pREF001 is shown in FIG. 3.

Example 3Transduction of Lymphocytes with Nucleic Acids Encoding Neurexin Targeting Polypeptides

[0147] The ORFs of interest from vectors pREF001, pREF002, pREF003, pREF004, pREF005 or pREF006 are subcloned into a pBABE-puro vector using standard molecular biology methods.

[0148] 2.510.sup.6 Phoenix packaging cells are plated in 9 ml medium/10 cm dish in the afternoon. The cells are transfected with 20 micrograms of DNA 24 hrs after plating), using the CaPO.sub.4 precipitation method. Briefly, in a 2 ml eppendorf tube 50 microlitres of 2.5M CaCl.sub.2, 20 micrograms of DNA are mixed and distilled sterile water to 500 L is added. While vortexing the tube, slowly add 500 L of buffer consisting of 50 mM HEPES pH 7.05, 10 mM KCl, 12 mM D-glucose, 280 mM NaCl, 1.5 mM Na.sub.2HPO.sub.4, dropwise. Then, 1 mL is added to the Phoenix cells and cells are placed back into an incubator.

[0149] To produce a viral supernatant, selected virus producing Phoenix packaging cells are cultured overnight in 20-mm Petri dishes at 80% confluence with 5 mL complete Dulbecco's modified Eagle medium (DMEM) (DMEM supplemented with 10% FCS, 4 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, nonessential amino acids, and streptavidin/penicillin). CD4+CD25+ Tregs are isolated from murine splenocytes or human peripheral blood mononuclear cells using a CD4-CD25 Treg isolation kit (Miltenyi Biotech, Bergisch Gladbach, Germany) to achieve a purity of over 90%. The isolated cells are preactivated for 4 to 18 hours with 2 g/mL Concanavalin A or with plate bounded anti-CD3 and anti-CD28 antibodies (plated at 1 ng/mL and 5 ng/mL, respectively). Activation and following transduction steps are performed either in Biotarget-1serum-free medium (Biological Industries, Beit Haemek, Israel) or in complete medium (RPMI supplemented with 10% FCS); both media are supplemented with 750 U/mL IL-2, 4 mmol/L L-glutamine, 1 mmol/L sodium pyruvate, 0.01 mol/L HEPES, 50 mol/L 2-ME, non-essential amino acids, and streptavidin/penicillin. For retroviral transduction, the viral supernatant is filtered through a 0.45 filter and supplemented with 750 U/mL recombinant mouse IL-2. Next, 750 L cell-free viral supernatant is administered to each well of 24-well plates pre-coated with 5 g/mL retronectin (TakaraBio Inc, Shiga, Japan). Plates are centrifuged at 1,000G for 30 minutes at room temperature. The activated Tregs, resuspended in 750-L cell-free viral supernatant, are distributed to the same retronectin-coated, virus-containing wells (1.510.sup.6 activated Tregs/well). Plates are then centrifuged again at 1,000G for 1 hour at room temperature and incubated for 5 hours. After incubation, viral supernatant is replaced with either Biotarget-1 or RPMI 1640 complete medium. Plates are incubated in 5% CO.sub.2 for an additional 2 days before further experimentation.

Example 4Migration of Tregs Expressing Neurexin Targeting Polypeptides

[0150] To assess the ability of Treg cells to migrate to and persist at sites of neural inflammation using neurexin-neuroligin targeting, Tregs are transfected with the two-element degen-lock. (Applicant's term for the cells of the invention that are targeted to neuroligin and C1q). Treg cells are cultured with neuroligin-1 and culture supernatants are collected for assessment of IL-10 and TGF- by ELISA. Cells are collected after the initial culture and placed into 5 m pore 96 well polycarbonate membrane trans-well coated with or without neuroligin-1, the lower compartment of the trans-well plate is left untreated or treated with CCL4. Cells are cultured for 4 hours and cells in the top and bottom of the plates are counted. Tregs expressing neurexin targeting polypeptides should not migrate from the insert in the presence of neuroligin.

[0151] To assess the ability of T regs expressing neurexin targeting polypeptides to migrate to sites of inflammation in vivo, the transduced, expanded murine Tregs were genetically modified and delivered by intravenous infusion to the animals. Synthetic plasmid pREF061 was digested with EcoRI and NotI according to NEB double-digest protocol, and then following fragments were cloned in using InFusion cloning method (Clontech): REF138 TGATTTATGCGTAACGCCATTTTGCAAGGCATGG (SEQ ID NO: 29) and REF139 GATAAGCTTGATATCGAATTTTACTTGTACAGCTCGTCCATGCC (SEQ ID NO: 30) primers were used to amplify 1179 bp fragment from pREF061 containing GFP; REF140 ATGGCGTTACGCATAAATCAATATTGGCTATTGGCCATTGC (SEQ ID NO: 31) and REF141 ATGGCGTTACTTTATAGAGCTCATCCATCCCAAGTGTGA (SEQ ID NO: 32) were used to generate a 2648 bp fragment from pREF044. REF142 GCTCTATAAAGTAACGCCATTTTGCAAGGCA (SEQ ID NO: 33) and REF143 TCGACTCTAGAGTCGCGGCCGAGCATGTCCAG (SEQ ID NO: 34) were used to generate a 2090 bp fragment from pREF123.

[0152] pREF171 was prepared from pREF061 digested with EcoRI and NotI and contained fragment 1179 bp amplified with REF138 TGATTTATGCGTAACGCCATTTTGCAAGGCATGG (SEQ ID NO: 35) and REF139 GATAAGCTTGATATCGAATTTTACTTGTACAGCTCGTCCATGCC (SEQ ID NO: 36) from pREF061, a 2648 bp fragment amplified from pREF044 with REF140 ATGGCGTTACGCATAAATCAATATTGGCTATTGGCCATTGC (SEQ ID NO: 37) and REF144 AATCAATGTCTTTATAGAGCTCATCCATCCCAAGTGTGA (SEQ ID NO: 38) and a 3209 bp fragment amplified with REF145 GCTCTATAAAGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGG (SEQ ID NO: 39) and REF143 TCGACTCTAGAGTCGCGGCCGAGCATGTCCAG (SEQ ID NO: 40) from pREF031.

[0153] Briefly, PCRs were performed using 12.5 uL HiFi PCR premix (CLontech), 12.5 uL water, 1.25 uL 10 L of each primer, and the fragments were amplified using Thermofisher thermocycler according to the HiFi PCR premix manufacturers instructions. PCR products were then mixed 5:1 with loading dye (Promega) and run on 1% agarose (Melford) gel in 0.5TBE (Invitrogen) in the presence of 1SYBRSafe against Promega 1 kb plus molecular weight marker. Fragments of interest were identified using BioRad imager, and the fragments were cut out from the gel, and then isolated from the cut-outs using MN cleanup kit. Afterwards, 1 L of each fragment and vector were used alongside 2 uL InFusion 5 kit for 15 minutes at 50 C. Then, 2 L of each reaction was transformed into Escherichia coli HST08 (Stellar cells, Clonetech) and after overnight incubation at 37 C. on LB (Melford) with 50 g/mL ampicillin (Melford), the correct clones were selected and confirmed by restriction digest and sequencing. MidiPreps (QiaGene) were performed on LB cultures of correct clones.

[0154] Spleens from Male C57BL/6J MOUSEAge Range Cohort56 to 62 Days were isolated as per standard protocol at BIE Biological Support Unit. Splens were disrupted in PBS using a syringe plunger, and then were a subject of EasySep Mouse CD4+CD25+ Regulatory T Cell Isolation Kit II according to manufacturers' protocol without modifications. Briefly, PBS was removed from spleen and spleens were poured into a sterile, plastic petri dish. Spleens were disrupted in Treg sample preparation buffer (PBS with 2% fetal bovine serum (FBS), 1 mM EDTA) and filtered through a 70 m mesh nylon strainer (BD). Splenocytes centrifuged at 300g for 10 minutes and resuspend in 2 mL. 100 L of rat serum were added to the sample. 100 L/mL of CD4+ T Cell Isolation Cocktail were added to the sample. Sample was mixed by pipetting and incubated at RT for 10 minutes. Streptavidin RapidSpheres, which select for non CD4 lymphocytes, were thoroughly vortexed until no clumps were observed. 150 L of Streptavidin rapidspheres were added to the sample. Sample was then mixed and incubated at RT for 2.5 minutes. Tube was then placed on the magnet and incubated on the magnet at RT for 2.5 minutes. Then, the supernatant was decanted into a new tube while being kept in contact on the magnet, this removes non-CD4 splenocytes (these were kept on the beads). Supernatant was exposed to magnet and this step repeated to remove any remaining Streptavidin RapidSpheres. The subsequent pre-enriched cells were centrifuged at 200g for 10 minutes at RT and resuspended in 0.5 mL of Treg sample preparation buffer. 25 L of FcR blocker were added to the sample, and the tube was incubated at RT for 5 minutes. 25 L of the CD25 Regulatory T Cell Positive Selection Cocktail were added to the sample. The incubation time was at RT for 10 minutes. 10 L of the PE Selection Cocktail were added to the sample. Mix was incubated at RT for 5 minutes. Dextran rapidspheres were vortexed for 30 seconds. 30 L of Dextran rapid spheres were added to the sample. Incubation was performed at RT for 5 minutes. Tube was then placed on the magnet and incubated at RT for 5 minutesthe CD4+CD25+ sample (on beads) was topped up to 2.5 mL in Treg sample preparation buffer, and again incubated for RT for 5 minutes on the magnet. This enrichment step for CD25+ cells was repeated 4. Cells were suspended in complete RPMI-1640 containing 10% FCS, 2 mM L-glutamine, 1 mM pyruvate, 10 mM HEPES, 1MEM NEAAs, 0.05 mM 2-ME and activated with 4:1 Mouse T cell activator dynabeads (Invitrogen). 1000 u/mL human recombinant IL-2 (Peprotech) was added from 10 mM acetic acid solution.

[0155] Preparation of the ecotropic lentivirus was performed as follows: in each case 7 micrograms of the cargo vector (pREF170, to generate Alternate Tregs or pREF171, to generate SynNotch Tregs, or pREF061 (GFP only, to generate WT Tregs) from high-quality midiprep (Qiagen, prepared according to manufacturers' instruction) were mixed with 600 uL distilled water, and then added to the Lenti-X Packaging One Shot vial (Takara). The tube was vortexed, and incubated at room temperature for 10 minutes for the nanoparticle complexes to be formed, according to the manufacturer's instruction. Mixture was then applied to 80% confluent HEK293T culture, and after overnight incubation at 37 C. 5% CO2 the medium was replaced with fresh DMEM (Gibco) with 10% FCS (Sigma) and 1 mM pyruvate (Gibco). The lentivirus was collected after 48 hours and 72 hours and was pooled, and concentrated using Lenti-X Concentrator (Takara). Pooled stocks of the same cargo. Centrifuged at 500 g for 10 min to removed any debris. Transferred clarified supernatant to a sterile 50 mL falcons and combine 1 volume of Lenti-X Concentrator (10 mL) with 3 volumes of clarified supernatant (30 mL). Mixed by gentle inversion. Incubated mixture at 4 C. for 60 minutes. Centrifuged the sample at 1500 g for 45 minutes at 4 C. to concentrate the supernatant. After centrifugation, an off-white pellet was be visible. The pellet was resuspended in 500 L of complete RPMI to be added to the culture.

[0156] Protransduzin fibres were prepared as described in as follows: the 10 mg/mL DMSO solution of protransduzin (JPT) was mixed with PBS to make 1 mg/mL solution with 10 min incubation at RT. 50 L of 1 mg/mL protransduzin solution was added to 500 uL MLV-enveloped virus and incubated for 15 minutes at RT. Finally, 450 L of activated Tregs stimulated with CD3/CD28 were mixed with 550 uL complex of MLV-virus and protransduzin and were placed in 24 well plates at 37 C. and 5% CO2. Cells were expanded in the medium described above, 7 days after transduction the cells were sorted for eGFP expression, and re-stimulated and further expanded as described above for an additional 7 days prior to freezing in medium containing 90% FCS and 10% DMSO.

[0157] B6.Cg-Tg(SOD1*G93A)1Gur/J mice, stock number 004435, at 8 weeks old were purchased from The Jackson Laboratory, USA. Animals were acclimatised for 7 days prior to weekly weighing and health monitoring check. Food and water were provided Ad libitum and animals were kept in temperature and humidity-controlled environment, with 12 h/12 h light/dark cycle. All procedures were carried out under HO project licence PPL P15A1884A at Medicines Discovery Catapult facilities. .sup.89Zr was purchased from commercial radiotracer supplier (PETNET). Syringe activity was measured before and after the i.v. injection using the BriTec well counter and the times of measurement noted using a clock synchronised to the PET system. The injected dose was calculated as the difference of the pre and post injection syringe activities after decay, and corrected to the time of injection. Mice were injected with 210{circumflex over ()}6 Tregs labelled when mice reached 120 days of age. Cells were administered intravenously through the tail vein (in the total volume of 100 ul in PBS). Mice were euthanised and tissues were collected for biodistribution study. Blood, lungs, liver, brain, stomach, kidneys, small intestine, large intestine, right muscle, left muscle, spinal cord, and tail (injection site) were dissected from each animal for gamma counter ex-vivo analysis. Hydraulic extrusion of the spinal cord was performed for both lumbar and thoracic regions.

[0158] Biodistribution data revealed increased radiation dose in the liver in unmodified Tregs-treated group compared to SynNotch (pREF171-transduced Tregs)- and Alternate (pREF170-transduced Tregs)-treated groups. This result suggests that transduced Tregs might have lower risk to accumulate in the liver compared to a non-modified Tregs. The spinal cord appeared to be more susceptible to infiltration of to SynNotch (pREF171-transduced Tregs)- and Alternate (pREF170-transduced Tregs) Tregs (P=0.1 vs. WT Tregs), whereas the brain to infiltrations of WT Tregs. Results are shown in FIG. 8.

Example 5Targeting of Tregs In Vivo to Inflamed Synapse

[0159] CD4+ T cells are isolated, then are enriched for CD25+ cells prior to sorting live CD4+CD25hi Tregs using FACS. Sorted cells are stimulated. One day later, cells are transduced with lentivirus carrying the sequence encoding anti-neuroligin receptor and the anti-C1q CAR as well as a fluorescent protein co-expressed using a 2A self-cleaving peptide sequence, a multiplicity of infection of 10 virus particles:1 cell. At day 7, cells are purified with magnetic selection, re-stimulated and expanded for an additional 5 days prior to injection. 510.sup.6 murine CAR Tregs and untargeted T regs are injected to SOD1 G93A animals intravenously into the tail vein on day 50. Saline-injected (PBS) mice serve as a control. GFP fluorescence coming from the modified Tregs is used to document the localisation of the Tregs into inflamed synapses skeletal muscles and spinal cord.

Example 6Efficacy of Tregs Expressing Neurexin Targeting Polypeptides Gating a C1q CAR in a Mouse Models of ALS

[0160] In vivo efficacy of Tregs expressing neurexin targeting polypeptides gating a C1q CAR can be tested in the SOD1-G93A transgenic mouse. These mice express a G93A mutant form of human SOD1 and are useful in studying neuromuscular disorders such as Amyotrophic Lateral Sclerosis.

[0161] A cohort of 75 SOD1 G93A (TG) and 15 wild-type (WT) littermate female mice are divided into four experimental groups, each of 15 animals: WT mice are treated with vehicle, TG mice are treated with vehicle or pREF001 transduced T regs that also express the C1q CAR. Three other groups are treated with the best-performing groups of Tregs from example 5. The mice are analysed for 70 days between the 50.sup.th and 120.sup.th day. Body weight is monitored once per week between days 50 and 90, and three times per week between days 91 and 120. Wire hanging abilities are measured at three time-points: as a baseline, at week 14 and week 16. An open-field test is performed at two points, at baseline and week 16. Fine motor kinematic gait analysis at baseline and at week 15 is also tested. Terminal sampling is performed to confirm the efficacy by histology, including motor neuron count Iba-1 quantitation.

[0162] Tregs were prepared as described in Example 4. EAE kit EK-2110 (MOG35-55/CFA emulsion) and pertussis toxin were purchased from Hooke Laboratories, Inc. MA, USA. 21 L of stock solution containing 5 g pertussis toxin in 25 L glycerol buffer was diluted with 4.2 mL of sterile phosphate buffer saline (PBS calcium and magnesium free) to obtain 100 ng in 100 L dosing solution. Female C57BL/6J were purchased from Charles River, UK, and were housed at Pharmidex Pharmaceutical Ltd and used at five to eight weeks of age. Female C57BL/6J mice (n=35) were injected subcutaneously with total volume of 200 L of MOG35-55/CFA (Complete Freund Adjuvant) emulsion, 100 L each on upper back and on lower back on day 0. After 2 hrs of MOG35-55/CFA emulsion, 100 L containing 100 ng of pertussis toxin (PTX) was injected intraperitoneally on day 0 and repeated PTX injection again on day 2.

[0163] Unmodified Treg cells (1.5106) and pREF170-transduced Treg Cells (1.5106) were freeze in a mixture of FCS and DMSO. After thawed individually, they were transferred to 15 mL Falcon with 5 mL of 1PBS separately and centrifuged at 300 g for 5 minutes. Supernatant was completely removed and suspended the pellet with 1.5 mL of sterile PBS separately to obtain 1105 cells in 100 L.

[0164] A 100 L of unmodified Treg cells (100,000 cells) (n=11)/or pREF170-transduced Treg cells (100,000 cells) (n=11)/PBS (n=13) was injected into each mouse on day 11 intraperitoneally.

[0165] Daily clinical scores were recorded for each experimental group and the data was displayed as mean group clinical score standard error of the mean (SEM) for each day of the clinical score measurements.

[0166] During the experiment, total of 6 mice PBS group (n=3), WT Treg cells group (n=2) and AT Treg cells group (n=1) were euthanised for health reasons before the experimental endpoint. These mice clinical scores were included from the data set until euthanised and not scored 5 for those mice for the remaining days. Data was included for 7 mice which showed late onset of EAE symptoms PBS group (n=5) AT Treg cells group (n=2) to avoid between group and intra group variance.

[0167] Two way ordinary ANOVA with Dunnett's multiple comparisons test was used to test the hypothesis that treatment was ineffective in improving clinical scores of EAE and considered significant, if p<0.05.

[0168] In the untreated group (PBS), 12 mice had mild hind limb paresis in which 9 mice went into complete bilateral hind limb paralysis. Some mice showed mild hind limb paresis on day 18 (n=3) day 19 (n=1) and day 22 (n=1) only. 6 mice partially recovered from complete bilateral hind limb paralysis and 1 mouse fully recovered. 6 mice did not recover even partially from complete bilateral hind limb paralysis in which 3 were humanely killed on day 15 (n=1) and on day 18 (n=2) as the mice conditions did not show improvement.

[0169] In the pREF170-transduced-Treg treated group, 10 mice had mild hind limb paresis in which all mice went into complete bilateral hind limb paralysis. All mice showed mild hind limb paresis before 16 days. 8 mice partially recovered from complete bilateral hind limb paralysis and 1 mouse fully recovered. 2 mice did not recover even partially from complete bilateral hind limb paralysis and were humanely killed on day 20 (n=1) and on day 21 (n=1) as the mice conditions did not show improvement. 9 mice had mild hind limb paresis in which 2 mice showed mild hind limb paresis on day 18 and day 19 only and 6 mice went into complete bilateral hind limb paralysis. 2 mice showed only mild tail tone loss and never had any other symptoms. 6 mice partially recovered from complete bilateral hind limb paralysis and 4 mice fully recovered. 1 mouse recovered from complete bilateral hind limb paralysis to mild hind limb paresis however it was humanely killed on day 20 as the mouse showed very limited movement. The results are illustrated in the FIG. 9.

Example 7C1q Activates Tregs Through CAR Binding

[0170] Murine Tregs were transfected with a construct driving constitutive expression of a CAR of the invention with an antigen determining region comprising ScFv directed to C1q (construct pREF043). Beads were coated with C1q in the carbonate buffer. The carbonate buffer 0.05 M pH 9.5 was filter-sterilised through a Sartorius 0.2 um filter; The choice of the buffer was dictated by the isoelectric point of C1q chains (pI of chains A (8.87) and B (9.07). The pI of chain C is close to 7 (7.07)). Tosyl-activated Dynabeads (500 l) (610.sup.8 to 710.sup.8 beads per ml; M-280, Thermofisher, sold as 30 mg beads per mL) are pelleted by placing the tube in the powerful magnetic field of a magnetic particle concentrator (Stemcell).

[0171] Following removal of storage buffer, the beads are washed once with 1 ml of coating buffer (0.05 M carbonate, pH 9.5). After a final concentration, 250 l of coating buffer was added, the beads were suspended, and 250 l of human C1q (0.4 mg/ml of coating buffer) was added. Coupling of C1q was performed by gentle rotation for 24 h at 37 C. Wash beads three times with phosphate-buffered saline (PBS; pH 7.2) containing 2% bovine serum albumin (BSA). Following an overnight wash at 4 C. with the same buffer, the beads were suspended in sterile-filtered 0.5 ml of PBS-1% BSA and stored at 4 degC.

[0172] Cells were exposed to C1q-coated tosyl-activated beads and activation of T regs was determined by detection of CD69 expression. As a positive control, PHA0.25 ng/ml; in the 1 ml culture, 0.25 l added of the original stock (Cat No: 11082132001 from Sigma).

[0173] Briefly, after 24 hours, the Tregs were collected by centrifugation at 300 g for 5 minutes. The supernatant was removed and 500 uL PBS were added to the pellet. 1 L of Zombie violet in DMSO (Biolegend) were added, and the sample was incubated for 15 minutes at RT in the dark. Then, the sample was centrifugated at 300 g for 5 minutes. The sample was suspended in PBS with 10% FCS for staining with the anti-CD69 antibody. Biolegend antibody against CD69 conjugated with PE has been used for detection (the Armenian Hamster IgG H1.2F3 antibody conjugated with PE. Recommended concentration of this antibody is 50.25 g per sample. Original stock was 0.2 mg/ml, so 1.25 uL of antibody was used for staining condition. Staining was performed for 15 minutes at 4 C. Three washes by 300 g for 5 minutes. Then the samples were taken for flow analysis). Cytometer:LSRFortessa A (LSRFortessa) was used for the analysis. Zombie violet was detected using 405 nm excitation and 450/50 nm bandwidth emission filter. The fluorescence signal from the CD69 detected with the PE-conjugated antibody was measured using 561 nm excitation 586/15 emission bandwidth channel.

[0174] The results are displayed in FIG. 5, which shows that CD69 expression is significantly increased in cells stimulated with C1q beads over unstimulated cells and those stimulated with PHA/PMA

Example 8C1q CAR Induced by SynNotch Neurexin Receptor Binding Neuroligin

[0175] Mouse tregs transduced with ecotropic lentoivirus carrying pREF060 were placed on the magnet in 15 mL falcon for 2.5 minutes and decanted to a new tube, and spinned down for 5 minutes at 300 g and re-suspended in RPMI-1640 medium supplemented with 10% FCS (Hyclone), 2 mM L-glutamine, 1 mM Na-pyruvate, 10 mM HEPES, 1PenStrep, 1NEAAs (MEM) and 0.05 mM 2-mercaptoethanol, and cultivate ON without any added IL-2.

[0176] C1q beads were prepared and used as described in example 7.

[0177] Neuroligin ligand used here was derived from a mouse myeloma cell line, NS0-derived human Neuroligin 1/NLGN1 protein Gln46-Ser677, Deletion: aa 279-287, with a C-terminal 6-His tag (Biotechne, 6446-NL-050). Dynabeads His-Tag Isolation and Pulldown 10103D were coated with neuroligin as follows: beads were thoroughly resuspended in the vial (vortexed for 30 sec). Transferred 50 L (2 mg) Dynabeads magnetic beads to a microcentrifuge tube. Place the tube on a magnet for 2 min. Aspirate and discard the supernatant. Added neuroligin from 100 diluted in PBS, the total volume was 200 uL PBS and the final concentration was 1 g/mL. Incubated the beads with the ligand on the orbital shaker for 10 min at room temperature. Placed the tube on the magnet for 2 min, then discarded the supernatant. Washed the beads 4 times with 300 L PBS by placing the tube on a magnet for 2 min and discard the supernatant. Resuspended the beads thoroughly between each washing step. Kept until used in the fridge.

[0178] Stimulation of pREF060 Tregs was performed in duplicate. Neuroligin-coated beads were added to 6 well plate wells, the control stimulation was performed with uncoated beads. Wells contained 2 mL total volume of complete RPMI (with 2 mM glutamine, 1 mM Na-pyruvate, 1MEM NEAA, 1PenStrep, 0.05 mM 2-mercaptoethanol, 10% FCS (Hyclone), 10 mM HEPES buffer) at 110.sup.6 cells/mL (2 mln in total per well). Beads were added in 2:1 ratio of Tregs: beads calculated as follows: 2 mg of beads and these were suspended in 300 L of final volume; dynabeads are 6.510.sup.7 beads per mg, therefore the entire preparation contained 1.310.sup.8 beads. As a result, 5 L of beads suspension were added per well. For mock-stimulation, 50 L of bare beads were suspended in 250 L of PBS, washed once using 300 g 5 min spin-down and resuspended in 300 uL PBS. Also 5 L of beads were added when appropriate. Cells were returned to the 37 C. 5% CO2 incubator for 24 hours from stimulation.

[0179] Samples were centrifuged at 300 g for 5 minutes and resuspended in 500 uL PBS with 1 uL zombie violet viability dye in DMSO (Biolegend). Samples were incubated for 30 minutes at RT, and then washed in PBS+10% FCS. Then the samples were incubated with 500 uL PBS+10% FCS in the presence of 1 uL Anti-DDDDK tag (Binds to FLAG tag sequence) antibody [M2] (PerCP) (ab117514) for 15 minutes in the fridge. Samples were then washed three times, each time with resuspension in 1000 L of PBS+10% FCS, and were finally resuspended in 1000 L of PBS+10% FCS. Centrifugation steps were always 300 g for 5 minutes at RT. Samples were kept on ice until measured at Babraham Institute flow cytometry facility. Results of the flow cytometry are shown in FIG. 6. This demonstrates that the AND gate construct works, in that stimulation of the SynNotch receptor leads to release of FoxP3 and expression and display of CAR.

Example 9AND Gate Activates Murine T Regs Through C1q CAR

[0180] Murine T regs were transfected with the SynNotch neurexin targeting polypeptide of the invention and the anti-C1q CAR of the invention, operatively linked to a FoxP3 binding transcriptional activator. CD69 is an early activation marker of T lymphocytes. Detection of CD69 associated with full functionality of the AND-gate construct pREF060 was performed using PE-conjugated mouse-CD69 specific antibody. Induction was performed after 24 hours from stimulation with C1q-coated tosyl-activated beads upon previous NLGN-1 beads stimulation. The results are displayed in FIG. 7, which shows that the AND gate of the invention drives expression of CD69 and is therefore capable of activating T regs.

[0181] After the stimulation the neuroligin beads, as detailed in Example 8, beads were removed from the sample of 500000 Tregs by exposing through the strong magnetic field of the Stemcell EasySep magnet and incubation for 2.5 minutes at RT. The cells were then placed in 1 mLwells of 24 well plate in complete RPMI (with 2 mM glutamine (Gibco), 1 mM Na-pyruvate (Gibco), 1MEM NEAA (Gibco), 1PenStrep (Gibco), 0.05 mM 2-mercaptoethanol (Gibco), 10% FCS (Hyclone), 10 mM HEPES buffer). Note, that no IL-2 was added to the culture to avoid any non-specific stimulation. 50 L of C1q bead suspension prepared as described above were added when appropriate, when no stimulation was intended 50 L of beads not conjugated with C1q and washed with PBS were added as Mock beads for mock stimulation. For positive controls of activation and CD69 detection, PMA200 g/ml; in the 1 ml culture, 0.75 l added of the original stock (Ref: P1585-1 MG from Sigma). PHA0.25 ng/ml; in the 1 ml culture, 0.25 l added of the original stock (Cat No: 11082132001 from Sigma).

[0182] After 24 hours, the Tregs were collected by centrifugation at 300 g for 5 minutes. The supernatant was removed and 500 uL PBS were added to the pellet. 1 L of Zombie violet in DMSO (Biolegend) were added, and the sample was incubated for 15 minutes at RT in the dark. Then, the sample was centrifugated at 300 g for 5 minutes. The sample was suspended in PBS with 10% FCS for staining with the anti-CD69 antibody. Biolegend antibody against CD69 conjugated with PE has been used for detection (the Armenian Hamster IgG H1.2F3 antibody conjugated with PE. Recommended concentration of this antibody is <0.25 g per sample. Original stock was 0.2 mg/ml, so 1.25 uL of antibody was used for staining condition. Staining was performed for 15 minutes at 4 C. Three washes by 300 g for 5 minutes. Then the samples were taken for flow analysis). Cytometer:LSRFortessa A (LSRFortessa) was used for the analysis. Zombie violet was detected using 405 nm excitation and 450/50 nm bandwidth emission filter. The fluorescence signal from the CD69 detected with the PE-conjugated antibody was measured using 561 nm excitation 586/15 emission bandwidth channel. The results of the signal are showed in the FIG. 7.

[0183] The results here showed the full activation circuit of the pREF060 and-gate of our prototype cell therapeutic. We have shown here that the pREF060 Tregs after stimulation with neuroligin, but only in the presence of C1q, express CD69, an early activation marker promoting Treg immunosuppressive function. This means our cell therapy prototype can be used for targeting to inflamed synapse, as well as the similar system can be used for detection of any disease-specific set of two antigens.

Example 10Design of Neurexin Tether for SynNotch Independent Targeting

[0184] An insert comprising a chimeric targeting polypeptide to act as a membrane anchored tether was designed as follows: an 18 amino acid signal peptide sequence MSMLFYTLITAFLIGIQA (SEQ ID NO: 22) was fused to a myc-tag sequence EQKLISEEDL (SEQ ID NO: 21) for ease of detection, the neurexin 1-b polypeptide of SEQ ID NO: 1 and a membrane anchor polypeptide sequence GGGGSGGGGSGGGGS (SEQ ID NO: 23). The full tethered neurexin sequence comprising SEQ ID NO: 1 is provided as SEQ ID NO: 24.

Example 11SynNotch Independent Targeting System Demonstrates Significant Efficacy In Vivo

[0185] A study on the efficacy of synNotch independent (neurexin tether) targeting system (Alternate) of the invention was performed on an experimentally induced mouse model of multiple scleroses known as experimental autoimmune encephalomyelitis (EAE). This model is produced by administering a myelin basic protein peptide (MBP) fragment that induces an autoimmune response directed to the myelin sheath surrounding motor neurons. The EAE score gives a measurement of neurological impairment with 0 being no obvious changed over motor function in normal mice and 5 being the most severe, typically paralysis and recommended euthanasia. In this study, there were 3 treatment groups of mice PBS, Wildtype T regs (W) and Alternate Targeted Tregs (A) of the invention. EAE was induced at day 1 and mice were dosed with 100,000 cells by i.p. at day 15. The results are displayed in FIG. 9, which shows that cognitive impairment in all groups starts at around day 10 and continues up to day 15 when the cells were administered. In the two control groups (PBS and W) the cognitive impairment stabilized and drops slights up to the end of the study. However, in the test group who received the cells of the invention, the cognitive decline appears to reverse and finishes much lower than the control groups at the end of the study. This may suggest that the cells of the invention not only halted the cognitive decline but appear to have reversed it in part. This may be due to a reduction in inflammation at motor neurons.

Example 12FoxP3 Expression in Tregs Maintained in Inflammatory Conditions

[0186] In this experiment regulatory T cells were transfected with Reflection Therapeutics technology according an embodiment of the present invention (in this case a tissue tether-P2A-FOXP3 transcript, under the control of a constitutive promoter, which triggers the expression of an a-C1q CAR under the control of FOXP3 response elements). These cells were cultured under normal, or pro-inflammatory conditions, achieved by the addition of IL-1, IL-6 and TNF.

[0187] The results of this experiment are provided in FIG. 12. Human regulatory T cells, isolated from PBMCs, were either untransfected, or transfected with a plasmid bearing Reflection Therapeutics degen-lock targeting technology according to an embodiment of the invention which includes: a tissue targeting receptor, FOXP3, and a CAR which is under the control of a FOXP3 response element. 24, 48 and 72 hours after transfection cells were treated with IL-2 at 1000 /mL, and in addition cells in the pro-inflammatory condition were treated with IL-6 (100 ng/ml), IL-1b (100 ng/ml) and TNF-alpha (100 ng/ml). 96 hours after transfection cells were collected and analysed by flow cytometry. Data presented shows levels of FOXP3 and Chimeric Antigen Receptor (FLAG tag).

[0188] This experiment demonstrates that the present invention can increase and stabilise FOXP3 levels in regulatory T cells, even in highly inflammatory conditions (4), in contrast to untransfected cells which lose FOXP3 expression (3). Furthermore this stable and high level of FOXP3 expression is linked to Chimeric Antigen Receptor expression, ensuring cells without FOXP3, and thus the anti-inflammatory properties of Tregs, are unlikely to express CARs, preventing the development of a pro-inflammatory CAR-T subpopulation. This suggests that the cells of the present invention will maintain a stable regulatory T cell phenotype when targeted to neuronal tissue, thereby reducing inflammation in the target tissue.

[0189] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0190] All publications, patent applications, issued patents, and other documents referred to in this specification are herein incorporated by reference as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions that are contained in text incorporated by reference are excluded to the extent that they contradict definitions in this disclosure.

Sequences Forming Part of the Application as Filed:

TABLE-US-00001 SEQ ID NO: Sequence Origin Description 1 ASSLGAHHIHHFHGSSKHHSVPIAIYRSPASLRGG WT Neurexin-segment HAGTTYIFSKGGGQITYKWPPNDRPSTRADRLAI derivedfromtheWT GFSTVQKEAVLVRVDSSSGLGDYLELHIHQGKIGV sequence, KFNVGTDDIAIEESNAIINDGKYHVVRFTRSGGNA responsiblefor TLQVDSWPVIERYPAGRQLTIFNSQATIIIGGKEQG dockingtheCAR- QPFQGQLSGLYYNGLKVLNMAAENDANIAIVGNV Tregintoa RLVGEVP neuroligin- expressinglocus 2 ASSLGAHHIHHFHGSSKHHSVPIAIYRSPASLRGG S111A Neurexin-segment HAGTTYIFSKGGGQITYKWPPNDRPATRADRLAI derivedfromtheWT GFSTVQKEAVLVRVDSSSGLGDYLELHIHQGKIGV sequence, KFNVGTDDIAIEESNAIINDGKYHVVRFTRSGGNA responsiblefor TLQVDSWPVIERYPAGRQLTIFNSQATIIIGGKEQG dockingtheCAR- QPFQGQLSGLYYNGLKVLNMAAENDANIAIVGNV Tregintoa RLVGEVP neuroligin- expressinglocus- tunedaffinitymutant 3 ASSLGAHHIHHFHGSSKHHSVPIAIYRSPASLRGG D162A Neurexin-segment HAGTTYIFSKGGGQITYKWPPNDRPSTRADRLAI derivedfromtheWT GFSTVQKEAVLVRVDSSSGLGDYLELHIHQGKIGV sequence, KFNVGTDAIAIEESNAIINDGKYHVVRFTRSGGNA responsiblefor TLQVDSWPVIERYPAGRQLTIFNSQATIIIGGKEQG dockingtheCAR- QPFQGQLSGLYYNGLKVLNMAAENDANIAIVGNV Tregintoa RLVGEVP neuroligin- expressinglocus- tunedaffinitymutant 4 ASSLGAHHIHHFHGSSKHHSVPIAIYRSPASLRGG I210A Neurexin-segment HAGTTYIFSKGGGQITYKWPPNDRPSTRADRLAI derivedfromtheWT GFSTVQKEAVLVRVDSSSGLGDYLELHIHQGKIGV sequence, KFNVGTDDIAIEESNAIINDGKYHVVRFTRSGGNA responsiblefor TLQVDSWPVIERYPAGRQLTAFNSQATIIIGGKEQ dockingtheCAR- GQPFQGQLSGLYYNGLKVLNMAAENDANIAIVGN Tregintoa VRLVGEVP neuroligin- expressinglocus- tunedaffinitymutant 5 ASSLGAHHIHHFHGSSKHHSVPIAIYRSPASLRGG N212A Neurexin-segment HAGTTYIFSKGGGQITYKWPPNDRPSTRADRLAI derivedfromtheWT GFSTVQKEAVLVRVDSSSGLGDYLELHIHQGKIGV sequence, KFNVGTDDIAIEESNAIINDGKYHVVRFTRSGGNA responsiblefor TLQVDSWPVIERYPAGRQLTIFASQATIIIGGKEQG dockingtheCAR- QPFQGQLSGLYYNGLKVLNMAAENDANIAIVGNV Tregintoa RLVGEVP neuroligin- expressinglocus- tunedaffinitymutant 6 ASSLGAHHIHHFHGSSKHHSVPIAIYRSPASLRGG D141A/ Neurexin-segment HAGTTYIFSKGGGQITYKWPPNDRPSTRADRLAI I210A derivedfromtheWT GFSTVQKEAVLVRVDSSSGLGAYLELHIHQGKIGV sequence, KFNVGTDDIAIEESNAIINDGKYHVVRFTRSGGNA responsiblefor TLQVDSWPVIERYPAGRQLTAFNSQATIIIGGKEQ dockingtheCAR- GQPFQGQLSGLYYNGLKVLNMAAENDANIAIVGN Treginto VRLVGEVP a neuroligin- expressinglocus- tunedaffinitymutant 7 MALPVTALLLPLALLLHAARPEQKLISEEDLAAADI scFv Sequenceof VLTQTPLSLPVSLGDQASISCRSSQNIVHSNGNTY neuroligin- LEWYLQKPGQSPKLLIYKVSNRFSGVPDRFSGSG recognising SGTDFTLKISRVEAEDLGVYFCFQGSLLPWTFGG receptorinscFv GTKLEIKRADAAPTVSGSTSGSGKPGSGEGSEFT form,followedby SPASSSEVKLMESGGGSVKPGGSLKLSCVASGF synNotchandusing TFSDYYMYWVRQTPEKRLEWVATISDGGNYPYY Gal4/VP64asa PDSVKGRFTISRDNARNNLYLQMTSLKSEDTAMY transcriptionfactor. YCARGGAYSRSDGRFYYAMDYWGQGTSVTVSS ThisisthefullORF ARILDYSFTGGAGRDIPPPQIEEACELPECQVDAG oftheconstruct. NKVCNLQCNNHACGWDGGDCSLNFNDPWKNCT QSLQCWKYFSDGHCDSQCNSAGCLFDGFDCQL TEGQCNPLYDQYCKDHFSDGHCDQGCNSAECE WDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNS FHFLRELSHVLHTNVVFKRDAQGQQMIFPYYGHE EELRKHPIKRSTVGWATSSLLPGTSGGRQRRELD PMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVAAF LGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMY VAAAAFVLLFFVGCGVLLSRKRRRMKLLSSIEQAC DICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMIL KMDSLQDIKALLTGLFVQDNVNKDAVTDRLASVE TDMPLTLRQHRISATSSSEESSNKGQRQLTVSAA AGGSGGSGGSDALDDFDLDMLGSDALDDFDLDM LGSDALDDFDLDMLGSDALDDFDLDMLGS 8 PECQFPASSPCLGGNPCYNQGTCEPTSESPFYR Human Thisisaprotein CLCPAKFNGLLCHILDYSFGGGAGRDIPPPLIEEA notch sequenceofhuman CELPECQEDAGNKVCSLQCNNHACGWDGGDCS Notch,thatcanbe LNFNDPWKNCTQSLQCWKYFSDGHCDSQpcvgC usedinsteadof NSAGCLFDGFDCQRAEGQCNPLYDQYCKDHFSD mouseNotchinthe GHCDQGCNSAECEWDGLDCAEHVPERLAAGTLV synNotch. VVVLMPPEQLRNSSFHFLRELSRVLHTNVVFKRD AHGQQMIFPYYGREEELRKHPIKRAAEGWAAPDA LLGQVKASLLPGGSEGGRRRRELDPMDVRGSIVY LEIDNRQCVQASSQCFQSATDVAAFLGALASLGS LNIPYKIEAVQSETVEPPPPAQLHFMYVAAAAFVL LFFVGCGVLLSRKR 9 PPQIEEACELPECQVDAGNKVCNLQCNNHACGW Mouse Thisisaprotein DGGDCSLNFNDPWKNCTQSLQCWKYFSDGHCD Notch sequenceofmouse SQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDH Notch-1,thatcanbe FSDGHCDQGCNSAECEWDGLDCAEHVPERLAA usedinthe GTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVF synNotch. KRDAQGQQMIFPYYGHEEELRKHPIKRSTVGWAT SSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQC VQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEA VKSEPVEPPLPSQLHLMYVAAAAFVLLFFVGCGV LLS 10 PCVGSNPCYNQGTCEPTSENPFYRCLCPAKFNG Notch1 LLCHILDYSFTGGAGRDIPPPQIEEACELPECQVD betafrom AGNKVCNLQCNNHACGWDGGDCSLNFNDPWKN mice CTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDC QLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAE CEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRN NSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYG HEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRE LDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVA AFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHL MYVAAAAFVLLFFVGCGVLLS 11 PCVGSNPCYNQGTCEPTSENPFYRCLCPAKFNG Notch NotchFromour LLCHILDYSFTGGAGRDIPPPQIEEACELPECQVD Fromour construct AGNKVCNLQCNNHACGWDGGDCSLNFNDPWKN construct CTQSLQCWKYFSDGHCDSQCNSAGCLFDGFDC QLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAE CEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRN NSFHFLRELSHVLHTNVVFKRDAQGQQMIFPYYG HEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRE LDPMDIRGSIVYLEIDNRQCVQSSSQCFQSATDVA AFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHL MYVAAAAFVLLFFVGCGVLLSRKRRR 12 QLCIQKLMKLLSSIEQACDICRLKKLKCSKEKPKCA Gal4 Transcriptionfactor KCLKNNWECRYSPKTKRSPLTRAHLTEVESRLER Gal4,canbeused LEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQ fordrivinginducible DNVNKDAVTDRLASVETDMPLTLRQHRISATSSS expression EESSNKGQRQLTVSAAAGGSGGSGGSDALDDFD LDMLGSDALDDFDLDMLGSDALDDFDLDMLGSD ALDDFDLDMLGSGSGVKQTLNFDLLKLAGDVESN PGPVD 13 RPAKPMAPSLALGPSPGVLPSWKTAPKGSELLGT FoxP3 MouseFoxP3 RGSGGPFQGRDLRSGAHTSSSLNPLPPSQLQLP TVPLVMVAPSGARLGPSPHLQALLQDRPHFMHQ LSTVDAHAQTPVLQVRPLDNPAMISLPPPSAATG VFSLKARPGLPPGINVASLEWVSREPALLCTFPRS GTPRKDSNLLAAPQGSYPLLANGVCKWPGCEKV FEEPEEFLKHCQADHLLDEKGKAQCLLQREVVQS LEQQLELEKEKLGAMQAHLAGKMALAKAPSVAS MDKSSCCIVATSTQGSVLPAWSAPREAPDGGLFA VRRHLWGSHGNSSFPEFFHNMDYFKYHNMRPPF TYATLIRWAILEAPERQRTLNEIYHWFTRMFAYFR NHPATWKNAIRHNLSLHKCFVRVESEKGAVWTV DEFEFRKKRSQRPNKCSNPCP 14 MALPVTALLLPLALLLHAARPEQKLISEEDLMYQR Entire Entireconstruct MLRCGAELGSPGGGGGGGGGGGAGGRLALLWI construct ORFprotein VPLTLSGLLGVAWGASSLGAHHIHHFHGSSKHHS WT sequence, VPIAIYRSPASLRGGHAGTTYIFSKGGGQITYKWP containingSEQID: PNDRPSTRADRLAIGFSTVQKEAVLVRVDSSSGL 1WTneurexin GDYLELHIHQGKIGVKFNVGTDDIAIEESNAIINDG domain,followedby KYHVVRFTRSGGNATLQVDSWPVIERYPAGRQLT theGal4andFoxP3 IFNSQATIIIGGKEQGQPFQGQLSGLYYNGLKVLN afterP2A. MAAENDANIAIVGNVRLVGEVPGGGGSPCVGSN PCYNQGTCEPTSENPFYRCLCPAKFNGLLCHILD YSFTGGAGRDIPPPQIEEACELPECQVDAGNKVC NLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQ CWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQ CNPLYDQYCKDHFSDGHCDQGCNSAECEWDGL DCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFL RELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELR KHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDI RGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGA LASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAA AAFVLLFFVGCGVLLSRKRRRQLCIQKLMKLLSSI EQACDICRLKKLKCSKEKPKCAKCLKNNWECRYS PKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDL DMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLA SVETDMPLTLRQHRISATSSSEESSNKGQRQLTV SAAAGGSGGSGGSDALDDFDLDMLGSDALDDFD LDMLGSDALDDFDLDMLGSDALDDFDLDMLGSG SGVKQTLNFDLLKLAGDVESNPGPVDMPNPRPA KPMAPSLALGPSPGVLPSWKTAPKGSELLGTRGS GGPFQGRDLRSGAHTSSSLNPLPPSQLQLPTVPL VMVAPSGARLGPSPHLQALLQDRPHFMHQLSTV DAHAQTPVLQVRPLDNPAMISLPPPSAATGVFSL KARPGLPPGINVASLEWVSREPALLCTFPRSGTP RKDSNLLAAPQGSYPLLANGVCKWPGCEKVFEE PEEFLKHCQADHLLDEKGKAQCLLQREVVQSLEQ QLELEKEKLGAMQAHLAGKMALAKAPSVASMDK SSCCIVATSTQGSVLPAWSAPREAPDGGLFAVRR HLWGSHGNSSFPEFFHNMDYFKYHNMRPPFTYA TLIRWAILEAPERQRTLNEIYHWFTRMFAYFRNHP ATWKNAIRHNLSLHKCFVRVESEKGAVWTVDEFE FRKKRSQRPNKCSNPCP 15 MALPVTALLLPLALLLHAARPEQKLISEEDLMYQR Entireconstruct MLRCGAELGSPGGGGGGGGGGGAGGRLALLWI ORFprotein VPLTLSGLLGVAWGASSLGAHHIHHFHGSSKHHS sequence, VPIAIYRSPASLRGGHAGTTYIFSKGGGQITYKWP containingSEQID: PNDRPATRADRLAIGFSTVQKEAVLVRVDSSSGL 2WTneurexin GDYLELHIHQGKIGVKFNVGTDDIAIEESNAIINDG domain,followedby KYHVVRFTRSGGNATLQVDSWPVIERYPAGRQLT theGal4andFoxP3 IFNSQATIIIGGKEQGQPFQGQLSGLYYNGLKVLN afterP2A. MAAENDANIAIVGNVRLVGEVPGGGGSPCVGSN PCYNQGTCEPTSENPFYRCLCPAKFNGLLCHILD YSFTGGAGRDIPPPQIEEACELPECQVDAGNKVC NLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQ CWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQ CNPLYDQYCKDHFSDGHCDQGCNSAECEWDGL DCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFL RELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELR KHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDI RGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGA LASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAA AAFVLLFFVGCGVLLSRKRRRQLCIQKLMKLLSSI EQACDICRLKKLKCSKEKPKCAKCLKNNWECRYS PKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDL DMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLA SVETDMPLTLRQHRISATSSSEESSNKGQRQLTV SAAAGGSGGSGGSDALDDFDLDMLGSDALDDFD LDMLGSDALDDFDLDMLGSDALDDFDLDMLGSG SGVKQTLNFDLLKLAGDVESNPGPVDMPNPRPA KPMAPSLALGPSPGVLPSWKTAPKGSELLGTRGS GGPFQGRDLRSGAHTSSSLNPLPPSQLQLPTVPL VMVAPSGARLGPSPHLQALLQDRPHFMHQLSTV DAHAQTPVLQVRPLDNPAMISLPPPSAATGVFSL KARPGLPPGINVASLEWVSREPALLCTFPRSGTP RKDSNLLAAPQGSYPLLANGVCKWPGCEKVFEE PEEFLKHCQADHLLDEKGKAQCLLQREVVQSLEQ QLELEKEKLGAMQAHLAGKMALAKAPSVASMDK SSCCIVATSTQGSVLPAWSAPREAPDGGLFAVRR HLWGSHGNSSFPEFFHNMDYFKYHNMRPPFTYA TLIRWAILEAPERQRTLNEIYHWFTRMFAYFRNHP ATWKNAIRHNLSLHKCFVRVESEKGAVWTVDEFE FRKKRSQRPNKCSNPCP 16 MALPVTALLLPLALLLHAARPEQKLISEEDLMYQR Entireconstruct MLRCGAELGSPGGGGGGGGGGGAGGRLALLWI ORFprotein VPLTLSGLLGVAWGASSLGAHHIHHFHGSSKHHS sequence, VPIAIYRSPASLRGGHAGTTYIFSKGGGQITYKWP containingSEQID: PNDRPSTRADRLAIGFSTVQKEAVLVRVDSSSGL 3WTneurexin GDYLELHIHQGKIGVKFNVGTDAIAIEESNAIINDG domain,followedby KYHVVRFTRSGGNATLQVDSWPVIERYPAGRQLT theGal4andFoxP3 IFNSQATIIIGGKEQGQPFQGQLSGLYYNGLKVLN afterP2A. MAAENDANIAIVGNVRLVGEVPGGGGSPCVGSN PCYNQGTCEPTSENPFYRCLCPAKFNGLLCHILD YSFTGGAGRDIPPPQIEEACELPECQVDAGNKVC NLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQ CWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQ CNPLYDQYCKDHFSDGHCDQGCNSAECEWDGL DCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFL RELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELR KHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDI RGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGA LASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAA AAFVLLFFVGCGVLLSRKRRRQLCIQKLMKLLSSI EQACDICRLKKLKCSKEKPKCAKCLKNNWECRYS PKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDL DMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLA SVETDMPLTLRQHRISATSSSEESSNKGQRQLTV SAAAGGSGGSGGSDALDDFDLDMLGSDALDDFD LDMLGSDALDDFDLDMLGSDALDDFDLDMLGSG SGVKQTLNFDLLKLAGDVESNPGPVDMPNPRPA KPMAPSLALGPSPGVLPSWKTAPKGSELLGTRGS GGPFQGRDLRSGAHTSSSLNPLPPSQLQLPTVPL VMVAPSGARLGPSPHLQALLQDRPHFMHQLSTV DAHAQTPVLQVRPLDNPAMISLPPPSAATGVFSL KARPGLPPGINVASLEWVSREPALLCTFPRSGTP RKDSNLLAAPQGSYPLLANGVCKWPGCEKVFEE PEEFLKHCQADHLLDEKGKAQCLLQREVVQSLEQ QLELEKEKLGAMQAHLAGKMALAKAPSVASMDK SSCCIVATSTQGSVLPAWSAPREAPDGGLFAVRR HLWGSHGNSSFPEFFHNMDYFKYHNMRPPFTYA TLIRWAILEAPERQRTLNEIYHWFTRMFAYFRNHP ATWKNAIRHNLSLHKCFVRVESEKGAVWTVDEFE FRKKRSQRPNKCSNPCP 17 MALPVTALLLPLALLLHAARPEQKLISEEDLMYQR Entireconstruct MLRCGAELGSPGGGGGGGGGGGAGGRLALLWI ORFprotein VPLTLSGLLGVAWGASSLGAHHIHHFHGSSKHHS sequence, VPIAIYRSPASLRGGHAGTTYIFSKGGGQITYKWP containingSEQID: PNDRPSTRADRLAIGFSTVQKEAVLVRVDSSSGL 4WTneurexin GDYLELHIHQGKIGVKFNVGTDDIAIEESNAIINDG domain,followedby KYHVVRFTRSGGNATLQVDSWPVIERYPAGRQLT theGal4andFoxP3 AFNSQATIIIGGKEQGQPFQGQLSGLYYNGLKVLN afterP2A. MAAENDANIAIVGNVRLVGEVPGGGGSPCVGSN PCYNQGTCEPTSENPFYRCLCPAKFNGLLCHILD YSFTGGAGRDIPPPQIEEACELPECQVDAGNKVC NLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQ CWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQ CNPLYDQYCKDHFSDGHCDQGCNSAECEWDGL DCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFL RELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELR KHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDI RGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGA LASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAA AAFVLLFFVGCGVLLSRKRRRQLCIQKLMKLLSSI EQACDICRLKKLKCSKEKPKCAKCLKNNWECRYS PKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDL DMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLA SVETDMPLTLRQHRISATSSSEESSNKGQRQLTV SAAAGGSGGSGGSDALDDFDLDMLGSDALDDFD LDMLGSDALDDFDLDMLGSDALDDFDLDMLGSG SGVKQTLNFDLLKLAGDVESNPGPVDMPNPRPA KPMAPSLALGPSPGVLPSWKTAPKGSELLGTRGS GGPFQGRDLRSGAHTSSSLNPLPPSQLQLPTVPL VMVAPSGARLGPSPHLQALLQDRPHFMHQLSTV DAHAQTPVLQVRPLDNPAMISLPPPSAATGVFSL KARPGLPPGINVASLEWVSREPALLCTFPRSGTP RKDSNLLAAPQGSYPLLANGVCKWPGCEKVFEE PEEFLKHCQADHLLDEKGKAQCLLQREVVQSLEQ QLELEKEKLGAMQAHLAGKMALAKAPSVASMDK SSCCIVATSTQGSVLPAWSAPREAPDGGLFAVRR HLWGSHGNSSFPEFFHNMDYFKYHNMRPPFTYA TLIRWAILEAPERQRTLNEIYHWFTRMFAYFRNHP ATWKNAIRHNLSLHKCFVRVESEKGAVWTVDEFE FRKKRSQRPNKCSNPCP 18 MALPVTALLLPLALLLHAARPEQKLISEEDLMYQR Entireconstruct MLRCGAELGSPGGGGGGGGGGGGGRLALLWI ORFprotein VPLTLSGLLGVAWGASSLGAHHIHHFHGSSKHHS sequence, VPIAIYRSPASLRGGHAGTTYIFSKGGGQITYKWP containingSEQID: PNDRPSTRADRLAIGFSTVQKEAVLVRVDSSSGL 5WTneurexin GDYLELHIHQGKIGVKFNVGTDDIAIEESNAIINDG domain,followedby KYHVVRFTRSGGNATLQVDSWPVIERYPAGRQLT theGal4andFoxP3 IFASQATIIIGGKEQGQPFQGQLSGLYYNGLKVLN afterP2A. MAAENDANIAIVGNVRLVGEVPGGGGSPCVGSN PCYNQGTCEPTSENPFYRCLCPAKFNGLLCHILD YSFTGGAGRDIPPPQIEEACELPECQVDAGNKVC NLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQ CWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQ CNPLYDQYCKDHFSDGHCDQGCNSAECEWDGL DCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFL RELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELR KHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDI RGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGA LASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAA AAFVLLFFVGCGVLLSRKRRRQLCIQKLMKLLSSI EQACDICRLKKLKCSKEKPKCAKCLKNNWECRYS PKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDL DMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLA SVETDMPLTLRQHRISATSSSEESSNKGQRQLTV SAAAGGSGGSGGSDALDDFDLDMLGSDALDDFD LDMLGSDALDDFDLDMLGSDALDDFDLDMLGSG SGVKQTLNFDLLKLAGDVESNPGPVDMPNPRPA KPMAPSLALGPSPGVLPSWKTAPKGSELLGTRGS GGPFQGRDLRSGAHTSSSLNPLPPSQLQLPTVPL VMVAPSGARLGPSPHLQALLQDRPHFMHQLSTV DAHAQTPVLQVRPLDNPAMISLPPPSAATGVFSL KARPGLPPGINVASLEWVSREPALLCTFPRSGTP RKDSNLLAAPQGSYPLLANGVCKWPGCEKVFEE PEEFLKHCQADHLLDEKGKAQCLLQREVVQSLEQ QLELEKEKLGAMQAHLAGKMALAKAPSVASMDK SSCCIVATSTQGSVLPAWSAPREAPDGGLFAVRR HLWGSHGNSSFPEFFHNMDYFKYHNMRPPFTYA TLIRWAILEAPERQRTLNEIYHWFTRMFAYFRNHP ATWKNAIRHNLSLHKCFVRVESEKGAVWTVDEFE FRKKRSQRPNKCSNPCP 19 MALPVTALLLPLALLLHAARPEQKLISEEDLMYQR Entireconstruct MLRCGAELGSPGGGGGGGGGGGAGGRLALLWI ORFprotein VPLTLSGLLGVAWGASSLGAHHIHHFHGSSKHHS sequence, VPIAIYRSPASLRGGHAGTTYIFSKGGGQITYKWP containingSEQID: PNDRPSTRADRLAIGFSTVQKEAVLVRVDSSSGL 6WTneurexin GAYLELHIHQGKIGVKFNVGTDDIAIEESNAIINDG domain,followedby KYHVVRFTRSGGNATLQVDSWPVIERYPAGRQLT theGal4andFoxP3 AFNSQATIIIGGKEQGQPFQGQLSGLYYNGLKVLN afterP2A. MAAENDANIAIVGNVRLVGEVPGGGGSPCVGSN PCYNQGTCEPTSENPFYRCLCPAKFNGLLCHILD YSFTGGAGRDIPPPQIEEACELPECQVDAGNKVC NLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQ CWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQ CNPLYDQYCKDHFSDGHCDQGCNSAECEWDGL DCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFL RELSHVLHTNVVFKRDAQGQQMIFPYYGHEEELR KHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDI RGSIVYLEIDNRQCVQSSSQCFQSATDVAAFLGA LASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAA AAFVLLFFVGCGVLLSRKRRRQLCIQKLMKLLSSI EQACDICRLKKLKCSKEKPKCAKCLKNNWECRYS PKTKRSPLTRAHLTEVESRLERLEQLFLLIFPREDL DMILKMDSLQDIKALLTGLFVQDNVNKDAVTDRLA SVETDMPLTLRQHRISATSSSEESSNKGQRQLTV SAAAGGSGGSGGSDALDDFDLDMLGSDALDDFD LDMLGSDALDDFDLDMLGSDALDDFDLDMLGSG SGVKQTLNFDLLKLAGDVESNPGPVDMPNPRPA KPMAPSLALGPSPGVLPSWKTAPKGSELLGTRGS GGPFQGRDLRSGAHTSSSLNPLPPSQLQLPTVPL VMVAPSGARLGPSPHLQALLQDRPHFMHQLSTV DAHAQTPVLQVRPLDNPAMISLPPPSAATGVFSL KARPGLPPGINVASLEWVSREPALLCTFPRSGTP RKDSNLLAAPQGSYPLLANGVCKWPGCEKVFEE PEEFLKHCQADHLLDEKGKAQCLLQREVVQSLEQ QLELEKEKLGAMQAHLAGKMALAKAPSVASMDK SSCCIVATSTQGSVLPAWSAPREAPDGGLFAVRR HLWGSHGNSSFPEFFHNMDYFKYHNMRPPFTYA TLIRWAILEAPERQRTLNEIYHWFTRMFAYFRNHP ATWKNAIRHNLSLHKCFVRVESEKGAVWTVDEFE FRKKRSQRPNKCSNPCP 20 MALPVTALLLPLALLLHAARP signal-peptideon SynNotchconstruct 21 EQKLISEEDL myc-tag 22 MSMLFYTLITAFLIGIQA signalpeptidefrom tether 23 GGGGSGGGGSGGGGS Membraneanchor ontether 24 MSMLFYTLITAFLIGIQAEQKLISEEDLASSLGAHHI Tetheredneurexin HHFHGSSKHHSVPIAIYRSPASLRGGHAGTTYIFS fullsequence KGGGQITYKWPPNDRPSTRADRLAIGFSTVQKEA VLVRVDSSSGLGDYLELHIHQGKIGVKFNVGTDDI AIEESNAIINDGKYHVVRFTRSGGNATLQVDSWPV IERYPAGNNDNERLAIARQRIPYRLGRVVDEWLLD KGRQLTIFNSQATIIIGGKEQGQPFQGQLSGLYYN GLKVLNMAAENDANIAIVGNVRLVGEVPGGGGSG GGGSGGGGSTLVLFGAGFGAVITVVVIVVIIKCFC K 25 AGCGAGGAGGATCTGATGTACCAGAGGATGCT GAGGTGC 26 GCTGTAGTCCAGGATGGGCACCTCGCCCACCA GC 27 GCCGCCGCGATCGCCatggcattgcccgt 28 GCGGCCGGCCGTTTAtcatgatccgagcatgtccag 29 TGATTTATGCGTAACGCCATTTTGCAAGGCATG G 30 GATAAGCTTGATATCGAATTTTACTTGTACAGCT CGTCCATGCC 31 ATGGCGTTACGCATAAATCAATATTGGCTATTG GCCATTGC 32 ATGGCGTTACTTTATAGAGCTCATCCATCCCAA GTGTGA 33 GCTCTATAAAGTAACGCCATTTTGCAAGGCA 34 TCGACTCTAGAGTCGCGGCCGAGCATGTCCAG 35 TGATTTATGCGTAACGCCATTTTGCAAGGCATG G 36 GATAAGCTTGATATCGAATTTTACTTGTACAGCT CGTCCATGCC 37 ATGGCGTTACGCATAAATCAATATTGGCTATTG GCCATTGC 38 AATCAATGTCTTTATAGAGCTCATCCATCCCAAG TGTGA 39 GCTCTATAAAGACATTGATTATTGACTAGTTATT AATAGTAATCAATTACGGG 40 TCGACTCTAGAGTCGCGGCCGAGCATGTCCAG 41 DVQITQSPSSLSASLGERATINCRASKSINKYLAW Lightchainvariable YQQKPGKAPKLLIYSGSTLQSGIPARFSGSGSGT regionof DFTLTISSLEPEDFAMYYCQQHNEYPLTFGQGTK humanizedM1 LEIK 42 QVQLVQSGAELKKPGASVKVSCKSSGYHFTSYW Heavychain MHWVKQAPGQGLEWIGVIHPNSGSINYNEKFESR variableregionof VTITVDKSTSTAYMELSSLRSEDTAVYYCAGERDS humanizedM1 TEVLPMDYWGQGTTVTVSS

Additional Sequences Forming Part of the Application as Filed:

TABLE-US-00002 SEQIDNO:43-Full(DNA)sequenceofpREF060(synNotchproducingGal4andGal4- inducibleCAR): tggaagggctaattcactcccaaagaagacaagatatccttgatctgtggatctaccacacacaaggctacttccctgattagcag aactacacaccagggccaggggtcagatatccactgacctttggatggtgctacaagctagtaccagttgagccagataaggta gaagaggccaataaaggagagaacaccagcttgttacaccctgtgagcctgcatgggatggatgacccggagagagaagtgt tagagtggaggtttgacagccgcctagcatttcatcacgtggcccgagagctgcatccggagtacttcaagaactgctgatatcga gcttgctacaagggactttccgctggggactttccagggaggcgtggcctggggggactggggagtggcgagccctcagatcc tgcatataagcagctgctttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacc cactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctca gacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctct ctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgac tagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaat tcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagtt aatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaaga acttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagac aagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccggccgctgatcttcagacctggaggagg agatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacccaccaagg caaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagcagcaggaag cactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcagaacaatttgctg agggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcctggctgtggaa agatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgccttggaatgctagtt ggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaagcttaa tacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtg gaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgct gtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccgacag gcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacggatctcgacggtatcg ccaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacata atagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcaga gatccagtttggatcgataagcttgatatcgaattGCATAAATCAATATTGGCTATTGGCCATTGCATACGT TGTATCTATATCATAAcggagcactgtcctccgaacgtcggagcactgtcctccgaacgtcggagcactgtcctccga acgtcggagcactgtcctccgaacggagcatgtcctccgaacgtcggagcactgtcctccgaacgactagttaggcgtgtacggt gggaggcctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaag acaccgggaccgatccagcctctcgacattcgttggGCCACCATGGGAACTAGTCTGCTGTGTTGGATG GCCCTCTGCTTGTTGGGCGCAGACCACGCTGACGGCCAAGTTCAATTGGTCCAGTCTG GTGCCGAACTGAAGAAGCCAGGAGCTAGCGTCAAGGTGAGTTGTAAGTCTTCCGGATA CCATTTCACGAGCTACTGGATGCATTGGGTAAAACAAGCCCCTGGACAAGGTCTCGAGT GGATAGGGGTAATACATCCTAACTCTGGGTCCATAAATTACAATGAAAAGTTCGAATCCA GGGTTACCATCACAGTCGATAAGTCCACTTCCACGGCCTATATGGAATTGAGCTCCCTC AGGAGCGAAGATACAGCTGTATACTACTGCGCTGGTGAACGAGACTCCACTGAGGTGT TGCCCATGGATTACTGGGGACAAGGCACCACTGTAACGGTCTCTAGCGGTGGGAGTAG TAGAAGCTCATCATCTGGCGGCGGCGGTTCAGGCGGTGGCGGCGACGTGCAGATCAC GCAAAGCCCGAGCTCACTCTCCGCCAGTCTGGGTGAACGTGCGACTATAAATTGTAGA GCCAGCAAGAGCATTAACAAGTATCTCGCCTGGTACCAGCAAAAGCCGGGCAAGGCTC CTAAACTCCTCATCTATTCCGGATCTACTCTCCAAAGCGGCATACCAGCGAGGTTCTCA GGAAGCGGATCAGGAACGGATTTTACGCTTACGATATCTAGCCTCGAGCCAGAGGATTT TGCCATGTACTATTGCCAACAACATAACGAATATCCCTTGACCTTCGGACAAGGGACGA AACTCGAGATCAAAGATTACAAAGACGATGACGACAAAACCACAACCCCAGCTCCTAGG CCCCCAACACCTGCACCTACCATCGCTAGCCAGCCTCTGAGCCTGCGTCCCGAGGCAT GTCGTCCTGCTGCAGGTGGGGCAGTCCACACAAGAGGTCTGGACTTTGCATGTGACTT TTGGGTCCTCGTAGTCGTCGGTGGCGTCCTGGCCTGTTATTCCCTTCTGGTTACGGTC GCCTTCATTATATTTTGGGTCAGGAGTAAACGGTCTAGGGGGGGGCACAGCGACTACA TGAATATGACGCCACGCCGCCCCGGGCCCACAAGAAAACACTACCAACCTTACGCTCC ACCCAGGGATTTTGCCGCGTACCGGAGCAGGGTAAAGTTCAGCCGCAGTGOGGATGC ACCAGCGTATCAACAAGGCCAGAATCAACTGTATAATGAGCTGAACTTGGGACGACGA GAGGAATATGATGTTCTGGATAAACGGCGCGGGAGAGATCCAGAGATGGGCGGAAAG CCTAGACGTAAGAACCCCCAAGAAGGCCTTTATAATGAGTTGCAGAAAGATAAGATGGC CGAAGCCTATTCTGAAATAGGCATGAAGGGAGAGCGGCGGCGTGGCAAGGGTCATGA CGGTCTTTACCAGGGCCTCTCTACCGCAACTAAAGATACCTATGATGCACTTCACATGC AAGCCCTGCCCCCTAGAGAGTTCGAAGGGTCAGCAGCAGCCGAGGGACGTGGCTCAT TGCTTACTTGCGGTGACGTTGAAGAAAACCCTGGCCCCAGCGGGATGGTAAGCAAGGG AGAAGAACTTTTCACGGGAGTAGTTCCTATTCTGGTGGAACTGGACGGGGATGTAAACG GGCATAAATTCTCTGTCAGCGGGGAAGGGGAAGGCGATGCAACATACGGGAAGCTTAC CCTCAAATTCATCTGTACGACAGGGAAACTTCCAGTGCCCTGGCCTACACTTGTGACAA CCCTGACTTATGGGGTCCAATGTTTCTCCAGGTACCCTGATCACATGAAACAGCATGAC TTCTTCAAGAGCGCAATGCCTGAAGGGTATGTCCAGGAACGCACGATTTTCTTCAAGGA CGATGGCAATTATAAAACTAGAGCCGAAGTAAAATTTGAGGGAGATACATTGGTAAACA GGATCGAGCTCAAAGGGATCGATTTCAAAGAGGATGGGAACATCCTGGGGCATAAGTT GGAGTATAACTATAACTCTCACAATGTCTATATTATGGCCGACAAGCAAAAGAACGGGA TAAAAGTCAACTTTAAAATAAGGCACAATATAGAGGACGGCAGTGTGCAATTGGCGGAC CACTATCAACAAAATACCCCGATCGGCGACGGTCCAGTGTTGCTCCCTGATAATCACTA CCTTTCAACACAAAGTGCACTTAGTAAAGATCCCAATGAGAAGCGGGATCACATGGTCC TGCTCGAATTTGTCACAGCAGCTGGCATCACACTTGGGATGGATGAGCTCTATAAATAA TAAACGGCCGGCCGCGGTCATAGCTGTTTCCTGAACAGATCCCGGGTGGCATCCCTGT GACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCACCAGCC TTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAATATTAT GGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTGTAGGG CCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCTCACTGC AATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTGTTGGGAT TCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACGGGGTTTC ACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCACCTTGGCC TCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCTTCTGATTT TAAAATAACTATACCAGCAGGAGGACGTCCAGACACAGCATAGGCTACCTGGCCATGC CCAACCGGTGGGACATTTGAGTTGCTTGCTTGGCACTGTCCTCTCATGCGTTGGGTCCA CTCAGTAGATGCCTGTTGAATTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTA CGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAAT GGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATG TTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGG TAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGA CGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGAC TTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTT TGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCC ACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAA TGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAG GTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGAATTTTGTAATACGACTCACTATA GGGCGGCCGGGAATTCGTCGACTGGATCCGGTACCGAGGAGATCTGCCGCCGCGATC GCCatggcattgcccgtgaccgccctgctgctgccactggccttgttgctccacgccgcgcggccagaacagaagCTGAT CAGCGAGGAGGATCTGATGTACCAGAGGATGCTGAGGTGCGGCGCCGAGCTGGGCAG CCCCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGGCGCCGGCGGCAGGCTGGC CCTGCTGTGGATCGTGCCCCTGACCCTGAGCGGCCTGCTGGGCGTGGCCTGGGGCGC CAGCAGCCTGGGCGCCCACCACATCCACCACTTCCACGGCAGCAGCAAGCACCACAG CGTGCCCATCGCCATCTACAGGAGCCCCGCCAGCCTGAGGGGCGGCCACGCCGGCAC CACCTACATCTTCAGCAAGGGCGGCGGCCAGATCACCTACAAGTGGCCCCCCAACGAC AGGCCCAGCACCAGGGCCGACAGGCTGGCCATCGGCTTCAGCACCGTGCAGAAGGAG GCCGTGCTGGTGAGGGTGGACAGCAGCAGCGGCCTGGGCGACTACCTGGAGCTGCAC ATCCACCAGGGCAAGATCGGCGTGAAGTTCAACGTGGGCACCGACGACATCGCCATCG AGGAGAGCAACGCCATCATCAACGACGGCAAGTACCACGTGGTGAGGTTCACCAGGAG CGGCGGCAACGCCACCCTGCAGGTGGACAGCTGGCCCGTGATCGAGAGGTACCCCGC CGGCAGGCAGCTGACCATCTTCAACAGCCAGGCCACCATCATCATCGGCGGCAAGGA GCAGGGCCAGCCCTTCCAGGGCCAGCTGAGCGGCCTGTACTACAACGGCCTGAAGGT GCTGAACATGGCCGCCGAGAACGACGCCAACATCGCCATCGTGGGCAACGTGAGGCT GGTGGGCGAGGTGCCCATCCTGGACTACAGCTTCACAGGtggcgctgggcgcgacattcccccacc gcagattgaggaggcctgtgagctgcctgagtgccaggtggatgcaggcaataaggtctgcaacctgcagtgtaataatcacgc atgtggctgggatggtggcgactgctccctcaacttcaatgacccctggaagaactgcacgcagtctctacagtgctggaagtattt tagcgacggccactgtgacagccagtgcaactcggccggctgcctctttgatggcttcgactgccagctcaccgagggacagtg caaccccctgtatgaccagtactgcaaggaccacttcagtgatggccactgcgaccagggctgtaacagtgccgaatgtgagtg ggatggcctagactgtgctgagcatgtacccgagcggctggcagccggcaccctggtgctggtggtgctgcttccacccgacca gctacggaacaactccttccactttctgcgggagctcagccacgtgctgcacaccaacgtggtcttcaagcgtgatgcgcaaggc cagcagatgatcttcccgtactatggccacgaggaagagctgcgcaagcacccaatcaagcgctctacagtgggttgggccac ctcttcactgcttcctggtaccagtggtgggcgccagcgcagggagctggaccccatggacatccgtggctccattgtctacctgg agatcgacaaccggcaatgtgtgcagtcatcctcgcagtgcttccagagtgccaccgatgtggctgccttcctaggtgctcttgcgt cacttggcagcctcaatattccttacaagattgaggccgtgaagagtgagccggtggagcctccgctgccctcgcagctgcacct catgtacgtggcagcggccgccttcgtgctcctgttctttgtgggctgtggggtgctgctgtcccgcaagcgccggcggatgaagct gctgagcagcatcgagcaggcctgtgacatctgccggctgaagaaactgaagtgcagcaaagaaaagcccaagtgcgccaa gtgcctgaagaacaactgggagtgccggtacagccccaagaccaagagaagccccctgaccagagcccacctgaccgagg tggaaagccggctggaaagactggaacagctgtttctgctgatcttcccacgcgaggacctggacatgatcctgaagatggaca gcctgcaggacatcaaggccctgctgaccggcctgttcgtgcaggacaacgtgaacaaggacgccgtgaccgacagactggc cagcgtggaaaccgacatgcccctgaccctgcggcagcacagaatcagcgccaccagcagcagcgaggaaagcagcaac aagggccagcggcagctgacagtgtctgctgctgcaggcggaagcggaggctctggcggatctgatgccctggacgacttcga cctggatatgctgggcagcgacgccctggatgattttgatctggacatgctgggatctgacgctctggacgatttcgatctcgacatg ttgggatcagatgcactggatgactttgacctggacatgctcggatcatgaTAAACGGCCGGCCGCGGTCATAGC TGTTTCCTGGGCCGCGACTCTAGAGTCGACCTGCAGGCATGCAAGCTTGATATCAAGC TTATCGATAATCAACCTCTGGATTACAAAATTTGtgAAAGATTGACTGGTATTCTTAACTAT GTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCT TCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAG GAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAA CCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTC CCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAG GGGCTCGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTT TCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTAC GTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCAATTCTACCGGGTAGGGGAGG CGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCG CTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCG GCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTT CCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCT CACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGG GGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGG GTGGGTCCGGGGGGGGCTCAGGGGGGGGCTCAGGGGGGGGGGGGCGCCCGAAG GTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTC TCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCAGCCCAAGCTTACCATGACCGAGTAC AAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCAGGGCCGTACGCACCCTC GCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACACCGTCGATCCGGACCGCCAC ATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCG GCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGGCGGTCTGGACCACGCCGGAG AGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAG CGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAAGGCCTCCTGGCGCCGCACCGGC CCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCGCCCGACCACCAGGGCA AGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCGGCCGAGCGCGCCGGG GTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCGGCTC GGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGCACCTGGTGCATG ACCCGCAAGCCCGGTGCCTGACTCGAGGGAATTAATTCGAGCTCGGTACCTTTAAGAC CAATGACTTACAAGGCAGCTGTAGATCTTAGCCACTTTTTAAAAGAAAAGGGGGGACTG GAAGGGCTAATTCACTCCCAACGAAGACAAGATCTGCTTTTTGCTTGTACTGGGTCTCT CTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAGGGAACCCACTGCTT AAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGA CTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAAAATCTCTAGCAgcatct agaattaattccgtgtattctatagtgtcacctaaatcgtatgtgtatgatacataaggttatgtattaattgtagccgcgttctaacgac aatatgtacaagcctaattgtgtagcatctggcttactgaagcagaccctatcatctctctcgtaaactgccgtcagagtcggtttggtt ggacgaaccttctgagtttctggtaacgccgtcccgcacccggaaatggtcagcgaaccaatcagcagggtcatcgctagccag atcctctacgccggacgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgccgacatcaccgat ggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggccgggggactgt tgggcgccatctccttgcatgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcagg agtcgcataagggagagcgtcgaatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccg ccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgc atgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatg ataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatat gtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgc ccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggt gcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgag cacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcag aatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataa ccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatggg ggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgt agcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggag gcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtggg tctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactat ggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatact ttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagtt ttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaac aaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagag cgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctct gctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataagg cgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctac agcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaaca ggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcg atttttgtgatgctcgtcaggggggggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggcctt ttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccg aacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggc cgattcattaatgcagctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagc atgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaat tagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctg actaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctag gcttttgcaaaaagcttggacacaagacaggcttgcgagatatgtttgagaataccactttatcccgcgtcagggagaggcagtg cgtaaaaagacgcggactcatgtgaaatactggtttttagtgcgccagatctctataatctcgcgcaacctattttcccctcgaacac tttttaagccgtagataaacaggctgggacacttcacatgagcgaaaaatacatcgtcacctgggacatgttgcagatccatgca cgtaaactcgcaagccgactgatgccttctgaacaatggaaaggcattattgccgtaagccgtggcggtctgtaccgggtgcgtta ctggcgcgtgaactgggtattcgtcatgtcgataccgtttgtatttccagctacgatcacgacaaccagcgcgagcttaaagtgctg aaacgcgcagaaggcgatggcgaaggcttcatcgttattgatgacctggtggataccggtggtactgcggttgcgattcgtgaaat gtatccaaaagcgcactttgtcaccatcttcgcaaaaccggctggtcgtccgctggttgatgactatgttgttgatatcccgcaagat acctggattgaacagccgtgggatatgggcgtcgtattcgtcccgccaatctccggtcgctaatcttttcaacgcctggcactgccg ggcgttgttctttttaacttcaggcgggttacaatagtttccagtaagtattctggaggctgcatccatgacacaggcaaacctgagc gaaaccctgttcaaaccccgctttaaacatcctgaaacctcgacgctagtccgccgctttaatcacggcgcacaaccgcctgtgc agtcggcccttgatggtaaaaccatccctcactggtatcgcatgattaaccgtctgatgtggatctggcgcggcattgacccacgc gaaatcctcgacgtccaggcacgtattgtgatgagcgatgccgaacgtaccgacgatgatttatacgatacggtgattggctacc gtggcggcaactggatttatgagtgggccccggatctttgtgaaggaaccttacttctgtggtgtgacataattggacaaactaccta cagagatttaaagctctaaggtaaatataaaatttttaagtgtataatgtgttaaactactgattctaattgtttgtgtattttagattccaa cctatggaactgatgaatgggagcagtggtggaatgcctttaatgaggaaaacctgttttgctcagaagaaatgccatctagtgat gatgaggctactgctgactctcaacattctactcctccaaaaaagaagagaaaggtagaagaccccaaggactttccttcagaat tgctaagttttttgagtcatgctgtgtttagtaatagaactcttgcttgctttgctatttacaccacaaaggaaaaagctgcactgctatac aagaaaattatggaaaaatattctgtaacctttataagtaggcataacagttataatcataacatactgttttttcttactccacacagg catagagtgtctgctattaataactatgctcaaaaattgtgtacctttagctttttaatttgtaaaggggttaataaggaatatttgatgtat agtgccttgactagagatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaac ctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttc acaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggatcaactggataactca agctaaccaaaatcatcccaaacttcccaccccataccctattaccactgccaattacctgtggtttcatttactctaaacctgtgattc ctctgaattattttcattttaaagaaattgtatttgttaaatatgtactacaaacttagtagt SEQIDNO:44-ProteinsequenceofsynNotch(receptor1)ofpREF060: MALPVTALLLPLALLLHAARPEQKLISEEDLMYQRMLRCGAELGSPGGGGGGGGGGGG GRLALLWIVPLTLSGLLGVAWGASSLGAHHIHHFHGSSKHHSVPIAIYRSPASLRGGHAGTT YIFSKGGGQITYKWPPNDRPSTRADRLAIGFSTVQKEAVLVRVDSSSGLGDYLELHIHQGKI GVKFNVGTDDIAIEESNAIINDGKYHVVRFTRSGGNATLQVDSWPVIERYPAGRQLTIFNSQ ATIIIGGKEQGQPFQGQLSGLYYNGLKVLNMAAENDANIAIVGNVRLVGEVPILDYSFTGGA GRDIPPPQIEEACELPECQVDAGNKVCNLQCNNHACGWDGGDCSLNFNDPWKNCTQSLQ CWKYFSDGHCDSQCNSAGCLFDGFDCQLTEGQCNPLYDQYCKDHFSDGHCDQGCNSAE CEWDGLDCAEHVPERLAAGTLVLVVLLPPDQLRNNSFHFLRELSHVLHTNVVFKRDAQGQ QMIFPYYGHEEELRKHPIKRSTVGWATSSLLPGTSGGRQRRELDPMDIRGSIVYLEIDNRQ CVQSSSQCFQSATDVAAFLGALASLGSLNIPYKIEAVKSEPVEPPLPSQLHLMYVAAAAFVL LFFVGCGVLLSRKRRRMKLLSSIEQACDICRLKKLKCSKEKPKCAKCLKNNWECRYSPKTK RSPLTRAHLTEVESRLERLEQLFLLIFPREDLDMILKMDSLQDIKALLTGLFVQDNVNKDAVT DRLASVETDMPLTLRQHRISATSSSEESSNKGQRQLTVSAAAGGSGGSGGSDALDDFDLD MLGSDALDDFDLDMLGSDALDDFDLDMLGSDALDDFDLDMLGS SEQIDNO:45-ProteinsequenceofCAR(receptor2)ofpREF060: MGTSLLCWMALCLLGADHADGQVQLVQSGAELKKPGASVKVSCKSSGYHFTSYWMHWV KQAPGQGLEWIGVIHPNSGSINYNEKFESRVTITVDKSTSTAYMELSSLRSEDTAVYYCAG ERDSTEVLPMDYWGQGTTVTVSSGGSSRSSSSGGGGSGGGGDVQITQSPSSLSASLGE RATINCRASKSINKYLAWYQQKPGKAPKLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEP EDFAMYYCQQHNEYPLTFGQGTKLEIKDYKDDDDKTTTPAPRPPTPAPTIASQPLSLRPEA CRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMN MTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPREFEGSAAAEGRGSLLTCGDVEENPGPSGMVSKGEELF TGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQ CFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDF KEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDG PVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK SEQIDNO:46-CARsequenceusedinexamples: MGTSLLCWMALCLLGADHADGQVQLVQSGAELKKPGASVKVSCKSSGYHFTSYWMHWV KQAPGQGLEWIGVIHPNSGSINYNEKFESRVTITVDKSTSTAYMELSSLRSEDTAVYYCAG ERDSTEVLPMDYWGQGTTVTVSSGGSSRSSSSGGGGSGGGGDVQITQSPSSLSASLGE RATINCRASKSINKYLAWYQQKPGKAPKLLIYSGSTLQSGIPARFSGSGSGTDFTLTISSLEP EDFAMYYCQQHNEYPLTFGQGTKLEIKDYKDDDDKTTTPAPRPPTPAPTIASQPLSLRPEA CRPAAGGAVHTRGLDFACDFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRGGHSDYMN MTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEY DVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLY QGLSTATKDTYDALHMQALPPR SEQIDNO:47-HumanNeurexinsequence>NP_620072.1neurexin-1isoformbeta1 [Homosapiens] MYQRMLRCGAELGSPGGGGGGGGGGGAGGRLALLWIVPLTLSGLLGVAWGASSLGAHH IHHFHGSSKHHSVPIAIYRSPASLRGGHAGTTYIFSKGGGQITYKWPPNDRPSTRADRLAIG FSTVQKEAVLVRVDSSSGLGDYLELHIHQGKIGVKFNVGTDDIAIEESNAIINDGKYHVVRFT RSGGNATLQVDSWPVIERYPAGRQLTIFNSQATIIIGGKEQGQPFQGQLSGLYYNGLKVLN MAAENDANIAIVGNVRLVGEVPSSMTTESTATAMQSEMSTSIMETTTTLATSTARRGKPPT KEPISQTTDDILVASAECPSDDEDIDPCEPSSGGLANPTRAGGREPYPGSAEVIRESSSTTG MVVGIVAAAALCILILLYAMYKYRNRDEGSYHVDESRNYISNSAQSNGAVVKEKQPSSAKS SNKNKKNKDKEYYV SEQIDNO:48-Tether.fwdarw.Cleavablepeptide.fwdarw.FoxP3-(NeurexintetherandFoxP3 linkedbytheP2A): MSMLFYTLITAFLIGIQAEQKLISEEDLASSLGAHHIHHFHGSSKHHSVPIAIYRSPASLRGGH AGTTYIFSKGGGQITYKWPPNDRPSTRADRLAIGFSTVQKEAVLVRVDSSSGLGDYLELHIH QGKIGVKFNVGTDDIAIEESNAIINDGKYHVVRFTRSGGNATLQVDSWPVIERYPAGNNDNE RLAIARQRIPYRLGRVVDEWLLDKGRQLTIFNSQATIIIGGKEQGQPFQGQLSGLYYNGLKV LNMAAENDANIAIVGNVRLVGEVPGGGGSGGGGSGGGGSTLVLFGAGFGAVITVVVIVVIIK CFCKGSGVKQTLNFDLLKLAGDVESNPGPVDMPNPRPAKPMAPSLALGPSPGVLPSWKT APKGSELLGTRGSGGPFQGRDLRSGAHTSSSLNPLPPSQLQLPTVPLVMVAPSGARLGPS PHLQALLQDRPHFMHQLSTVDAHAQTPVLQVRPLDNPAMISLPPPSAATGVFSLKARPGLP PGINVASLEWVSREPALLCTFPRSGTPRKDSNLLAAPQGSYPLLANGVCKWPGCEKVFEE PEEFLKHCQADHLLDEKGKAQCLLQREVVQSLEQQLELEKEKLGAMQAHLAGKMALAKAP SVASMDKSSCCIVATSTQGSVLPAWSAPREAPDGGLFAVRRHLWGSHGNSSFPEFFHNM DYFKYHNMRPPFTYATLIRWAILEAPERQRTLNEIYHWFTRMFAYFRNHPATWKNAIRHNL SLHKCFVRVESEKGAVWTVDEFEFRKKRSQRPNKCSNPCP SEQIDNO:49-pREF020sequence-lentiviralvectorwithsynNotchwithWTneurexin sequence AACAAAATATTAACGCTTACAATTTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAA GGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCT GCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGA CGGCCAGTGCCAAGCTGATCTATACATTGAATCAATATTGGCAATTAGCCATATTAGTC ATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATAT CATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTA TTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAG TTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCC GCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCAT TGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTA TCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCAT TATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGT CATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGG TTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTG GCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAA ATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACC GTCAGAATTTTGTAATACGACTCACTATAGGGCGGCCGGGAATTCGTCGACTGGATCC GGTACCGAGGAGATCTGCCGCCGCGATCGCCatggcattgcccgtgaccgccctgctgctgccactggc cttgttgctccacgccgcgcggccagaacagaagCTGATCAGCGAGGAGGATCTGATGTACCAGAGG ATGCTGAGGTGCGGCGCCGAGCTGGGCAGCCCCGGCGGCGGCGGCGGCGGCGGCG GCGGCGGCGGCGCCGGCGGCAGGCTGGCCCTGCTGTGGATCGTGCCCCTGACCCTG AGCGGCCTGCTGGGCGTGGCCTGGGGCGCCAGCAGCCTGGGCGCCCACCACATCCA CCACTTCCACGGCAGCAGCAAGCACCACAGCGTGCCCATCGCCATCTACAGGAGCCC CGCCAGCCTGAGGGGCGGCCACGCCGGCACCACCTACATCTTCAGCAAGGGCGGCG GCCAGATCACCTACAAGTGGCCCCCCAACGACAGGCCCAGCACCAGGGCCGACAGGC TGGCCATCGGCTTCAGCACCGTGCAGAAGGAGGCCGTGCTGGTGAGGGTGGACAGCA GCAGCGGCCTGGGCGACTACCTGGAGCTGCACATCCACCAGGGCAAGATCGGCGTGA AGTTCAACGTGGGCACCGACGACATCGCCATCGAGGAGAGCAACGCCATCATCAACG ACGGCAAGTACCACGTGGTGAGGTTCACCAGGAGCGGCGGCAACGCCACCCTGCAG GTGGACAGCTGGCCCGTGATCGAGAGGTACCCCGCCGGCAGGCAGCTGACCATCTTC AACAGCCAGGCCACCATCATCATCGGCGGCAAGGAGCAGGGCCAGCCCTTCCAGGGC CAGCTGAGCGGCCTGTACTACAACGGCCTGAAGGTGCTGAACATGGCCGCCGAGAAC GACGCCAACATCGCCATCGTGGGCAACGTGAGGCTGGTGGGCGAGGTGCCCATCCTG GACTACAGCTTCACAGGtggcgctgggcgcgacattcccccaccgcagattgaggaggcctgtgagctgcctgagt gccaggtggatgcaggcaataaggtctgcaacctgcagtgtaataatcacgcatgtggctgggatggtggcgactgctccctca acttcaatgacccctggaagaactgcacgcagtctctacagtgctggaagtattttagcgacggccactgtgacagccagtgca actcggccggctgcctctttgatggcttcgactgccagctcaccgagggacagtgcaaccccctgtatgaccagtactgcaagg accacttcagtgatggccactgcgaccagggctgtaacagtgccgaatgtgagtgggatggcctagactgtgctgagcatgtac ccgagcggctggcagccggcaccctggtgctggtggtgctgcttccacccgaccagctacggaacaactccttccactttctgcg ggagctcagccacgtgctgcacaccaacgtggtcttcaagcgtgatgcgcaaggccagcagatgatcttcccgtactatggcca cgaggaagagctgcgcaagcacccaatcaagcgctctacagtgggttgggccacctcttcactgcttcctggtaccagtggtgg gcgccagcgcagggagctggaccccatggacatccgtggctccattgtctacctggagatcgacaaccggcaatgtgtgcagt catcctcgcagtgcttccagagtgccaccgatgtggctgccttcctaggtgctcttgcgtcacttggcagcctcaatattccttacaa gattgaggccgtgaagagtgagccggtggagcctccgctgccctcgcagctgcacctcatgtacgtggcagcggccgccttcgt gctcctgttctttgtgggctgtggggtgctgctgtcccgcaagcgccggcggatgaagctgctgagcagcatcgagcaggcctgt gacatctgccggctgaagaaactgaagtgcagcaaagaaaagcccaagtgcgccaagtgcctgaagaacaactgggagtg ccggtacagccccaagaccaagagaagccccctgaccagagcccacctgaccgaggtggaaagccggctggaaagactg gaacagctgtttctgctgatcttcccacgcgaggacctggacatgatcctgaagatggacagcctgcaggacatcaaggccctg ctgaccggcctgttcgtgcaggacaacgtgaacaaggacgccgtgaccgacagactggccagcgtggaaaccgacatgccc ctgaccctgcggcagcacagaatcagcgccaccagcagcagcgaggaaagcagcaacaagggccagcggcagctgaca gtgtctgctgctgcaggcggaagcggaggctctggcggatctgatgccctggacgacttcgacctggatatgctgggcagcgac gccctggatgattttgatctggacatgctgggatctgacgctctggacgatttcgatctcgacatgttgggatcagatgcactggatg actttgacctggacatgctcggatcatgaTAAACGGCCGGCCGCGGTCATAGCTGTTTCCTGAACAG ATCCCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTG CCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACT AGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAG TTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAG TGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCC TCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGT TTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCT CAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTG CTCCCTTCCCTGTCCTTCTGATTTTAAAATAACTATACCAGCAGGAGGACGTCCAGACA CAGCATAGGCTACCTGGCCATGCCCAACCGGTGGGACATTTGAGTTGCTTGCTTGGCA CTGTCCTCTCATGCGTTGGGTCCACTCAGTAGATGCCTGTTGAATTGGGTACGCGGCC AGCTTGGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCA GGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCC CAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAT AGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCT CCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCT CTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAA GCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCG TTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGA GGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTT CCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGC CCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCG TTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATT GGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGT ATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCA TTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGT CTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTG TTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGC GATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTG TGGCCGGCTGGGTGTGGCCGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATAT TGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCC GCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGG GACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTC GATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCG GCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTT GTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAA AGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATOTTATCAT GTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCC TGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGT GTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACT GCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGC GCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCG CTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATA CGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGC AAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCC CCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGG ACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCG ACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTT CTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGG CTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGT CTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACA GGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAA CTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCT TCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTT TTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGA TCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTC ATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAAT CAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGG CACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTG TAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGC GAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGG CCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGC CGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTG CTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTC CCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCC TTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTAT GGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTG GTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTG CCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATC ATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCA GTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGC GTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGA CACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGG GTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGG TTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGCGGCGCATTAAG CGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGC GCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTC AAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGA CCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACG GTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACT GGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATT TCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTT SEQIDNO:50-LinearisedbyPCRbackbonesequence: ATCCTGGACTACAGCTTCACAGGtggcgctgggcgcgacattcccccaccgcagattgaggaggcctgtgagc tgcctgagtgccaggtggatgcaggcaataaggtctgcaacctgcagtgtaataatcacgcatgtggctgggatggtggcgact gctccctcaacttcaatgacccctggaagaactgcacgcagtctctacagtgctggaagtattttagcgacggccactgtgacag ccagtgcaactcggccggctgcctctttgatggcttcgactgccagctcaccgagggacagtgcaaccccctgtatgaccagta ctgcaaggaccacttcagtgatggccactgcgaccagggctgtaacagtgccgaatgtgagtgggatggcctagactgtgctg agcatgtacccgagcggctggcagccggcaccctggtgctggtggtgctgcttccacccgaccagctacggaacaactccttcc actttctgcgggagctcagccacgtgctgcacaccaacgtggtcttcaagcgtgatgcgcaaggccagcagatgatcttcccgta ctatggccacgaggaagagctgcgcaagcacccaatcaagcgctctacagtgggttgggccacctcttcactgcttcctggtac cagtggtgggcgccagcgcagggagctggaccccatggacatccgtggctccattgtctacctggagatcgacaaccggcaa tgtgtgcagtcatcctcgcagtgcttccagagtgccaccgatgtggctgccttcctaggtgctcttgcgtcacttggcagcctcaatat tccttacaagattgaggccgtgaagagtgagccggtggagcctccgctgccctcgcagctgcacctcatgtacgtggcagcggc cgccttcgtgctcctgttctttgtgggctgtggggtgctgctgtcccgcaagcgccggoggatgaagctgctgagcagcatcgagc aggcctgtgacatctgccggctgaagaaactgaagtgcagcaaagaaaagcccaagtgcgccaagtgcctgaagaacaac tgggagtgccggtacagccccaagaccaagagaagccccctgaccagagcccacctgaccgaggtggaaagccggctgg aaagactggaacagctgtttctgctgatcttcccacgcgaggacctggacatgatcctgaagatggacagcctgcaggacatca aggccctgctgaccggcctgttcgtgcaggacaacgtgaacaaggacgccgtgaccgacagactggccagcgtggaaacc gacatgcccctgaccctgcggcagcacagaatcagcgccaccagcagcagcgaggaaagcagcaacaagggccagcgg cagctgacagtgtctgctgctgcaggcggaagcggaggctctggcggatctgatgccctggacgacttcgacctggatatgctg ggcagcgacgccctggatgattttgatctggacatgctgggatctgacgctctggacgatttcgatctcgacatgttgggatcagat gcactggatgactttgacctggacatgctcggatcatgagatccttgacttgcggccgcaactcccacctgcaacatgcgtgactg actgaggccgcgactctagagtcgacctgcaggcatgcaagcttgatatcaagcttatcgataatcaacctctggattacaaaatt tgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttccc gtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgt gcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccct attgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgtt gtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcgg ccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtc ggatctccctttgggccgcctccccgcatcgataccgtcgacctcgagggaattaattcgagctcggtacctttaagaccaatgac ttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaag atctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaag cctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttag tcagtgtggaaaatctctagcagcatctagaattaattccgtgtattctatagtgtcacctaaatcgtatgtgtatgatacataaggtta tgtattaattgtagccgcgttctaacgacaatatgtacaagcctaattgtgtagcatctggcttactgaagcagaccctatcatctctct cgtaaactgccgtcagagtcggtttggttggacgaaccttctgagtttctggtaacgccgtcccgcacccggaaatggtcagcga accaatcagcagggtcatcgctagccagatcctctacgccggacgcatcgtggccggcatcaccggcgccacaggtgcggtt gctggcgcctatatcgccgacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggta tggtggcaggccccgtggccgggggactgttgggcgccatctccttgcatgcaccattccttgcggcggcggtgctcaacggcct caacctactactgggctgcttcctaatgcaggagtcgcataagggagagcgtcgaatggtgcactctcagtacaatctgctctgat gccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgctta cagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggc ctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcgga acccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaa ggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctg gtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgag agttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggca agagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggc atgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggagga ccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagcc ataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttac tctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggc tggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccg tatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgatt aagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaaga tcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatc ttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaag agctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggcc accacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgt gtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagccc agcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggaga aaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggt atctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaa cgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataa ccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagc ggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctgtggaatgtgtgtcagttagggt gtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtc cccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgccca tcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctc ggcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagcttggacacaagacaggcttgc gagatatgtttgagaataccactttatcccgcgtcagggagaggcagtgcgtaaaaagacgcggactcatgtgaaatactggttt ttagtgcgccagatctctataatctcgcgcaacctattttcccctcgaacactttttaagccgtagataaacaggctgggacacttca catgagcgaaaaatacatcgtcacctgggacatgttgcagatccatgcacgtaaactcgcaagccgactgatgccttctgaaca atggaaaggcattattgccgtaagccgtggcggtctgtaccgggtgcgttactggcgcgtgaactgggtattcgtcatgtcgatac cgtttgtatttccagctacgatcacgacaaccagcgcgagcttaaagtgctgaaacgcgcagaaggcgatggcgaaggcttcat cgttattgatgacctggtggataccggtggtactgcggttgcgattcgtgaaatgtatccaaaagcgcactttgtcaccatcttcgca aaaccggctggtcgtccgctggttgatgactatgttgttgatatcccgcaagatacctggattgaacagccgtgggatatgggcgt cgtattcgtcccgccaatctccggtcgctaatcttttcaacgcctggcactgccgggcgttgttctttttaacttcaggcgggttacaat agtttccagtaagtattctggaggctgcatccatgacacaggcaaacctgagcgaaaccctgttcaaaccccgctttaaacatcc tgaaacctcgacgctagtccgccgctttaatcacggcgcacaaccgcctgtgcagtcggcccttgatggtaaaaccatccctcac tggtatcgcatgattaaccgtctgatgtggatctggcgcggcattgacccacgcgaaatcctcgacgtccaggcacgtattgtgat gagcgatgccgaacgtaccgacgatgatttatacgatacggtgattggctaccgtggcggcaactggatttatgagtgggcccc ggatctttgtgaaggaaccttacttctgtggtgtgacataattggacaaactacctacagagatttaaagctctaaggtaaatataa aatttttaagtgtataatgtgttaaactactgattctaattgtttgtgtattttagattccaacctatggaactgatgaatgggagcagtgg tggaatgcctttaatgaggaaaacctgttttgctcagaagaaatgccatctagtgatgatgaggctactgctgactctcaacattcta ctcctccaaaaaagaagagaaaggtagaagaccccaaggactttccttcagaattgctaagttttttgagtcatgctgtgtttagta atagaactcttgcttgctttgctatttacaccacaaaggaaaaagctgcactgctatacaagaaaattatggaaaaatattctgtaa cctttataagtaggcataacagttataatcataacatactgttttttcttactccacacaggcatagagtgtctgctattaataactatgc tcaaaaattgtgtacctttagctttttaatttgtaaaggggttaataaggaatatttgatgtatagtgccttgactagagatcataatcag ccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgtt gttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcatt ctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggatcaactggataactcaagctaaccaaaatcatcccaaactt cccaccccataccctattaccactgccaattacctgtggtttcatttactctaaacctgtgattcctctgaattattttcattttaaagaaat tgtatttgttaaatatgtactacaaacttagtagttggaagggctaattcactcccaaagaagacaagatatccttgatctgtggatct accacacacaaggctacttccctgattagcagaactacacaccagggccaggggtcagatatccactgacctttggatggtgct acaagctagtaccagttgagccagataaggtagaagaggccaataaaggagagaacaccagcttgttacaccctgtgagcct gcatgggatggatgacccggagagagaagtgttagagtggaggtttgacagccgcctagcatttcatcacgtggcccgagagc tgcatccggagtacttcaagaactgctgatatcgagcttgctacaagggactttccgctggggactttccagggaggcgtggcctg ggcgggactggggagtggcgagccctcagatcctgcatataagcagctgctttttgcctgtactgggtctctctggttagaccagat ctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgc ccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagg gacttgaaagcgaaagggaaaccagaggagctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcg aggggcggcgactggtgagtacgccaaaaattttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagta ttaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatat agtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgg gacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaa aggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacag caagcggccggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagta gtaaaaattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaa taggagctttgttccttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagac aattattgtctggtatagtgcagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctgg ggcatcaagcagctccaggcaagaatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctct ggaaaactcatttgcaccactgctgtgccttggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctg gatggagtgggacagagaaattaacaattacacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaag aatgaacaagaattattggaattagataaatgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattat tcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccat tatcgtttcagacccacctcccaaccccgaggggacccgacaggcccgaaggaatagaagaagaaggtggagagagaga cagagacagatccattcgattagtgaacggatctcgacggtatcgccaaatggcagtattcatccacaattttaaaagaaaagg ggggattggggggtacagtgcaggggaaagaatagtagacataatagcaacagacatacaaactaaagaattacaaaaac aaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccagtttggatcgataagcttgatatcgaattgggta ggggaggcgcttttcccaaggcagtctggagcatgcgctttagcagccccgctgggcacttggcgctacacaagtggcctctgg cctcgcacacattccacatccaccggtaggcgccaaccggctccgttctttggtggccccttcgcgccaccttctactcctccccta gtcaggaagttcccccccgccccgcagctcgcgtcgtgcaggacgtgacaaatggaagtagcacgtctcactagtctcgtgca gatggacagcaccgctgagcaatggaagcgggtaggcctttggggcagcggccaatagcagctttgctccttcgctttctgggct cagaggctgggaaggggtgggtccggggggggctcaggggcgggctcaggggggggcgggcgcccgaaggtcctcc ggaggcccggcattctgcacgcttcaaaagcgcacgtctgccgcgctgttctcctcttcctcatctccgggcctttcgaattctcacg cgtcaagtggagcaaggcaggtggacagtggatcgccgccaccatggcattgcccgtgaccgccctgctgctgccactggcct tgttgctccacgccgcgcggccagaacagaagCTGATCAGCGAGGAGGATCTG SEQIDNO:51-PlasmidpREF061sequence-handlingvector,expressesGFP, lentiviralpackingvector tggaagggctaattcactcccaaagaagacaagatatccttgatctgtggatctaccacacacaaggctacttccctgattagca gaactacacaccagggccaggggtcagatatccactgacctttggatggtgctacaagctagtaccagttgagccagataagg tagaagaggccaataaaggagagaacaccagcttgttacaccctgtgagcctgcatgggatggatgacccggagagagaag tgttagagtggaggtttgacagccgcctagcatttcatcacgtggcccgagagctgcatccggagtacttcaagaactgctgatat cgagcttgctacaagggactttccgctggggactttccagggaggcgtggcctggggggactggggagtggcgagccctcag atcctgcatataagcagctgctttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactaggg aacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatc cctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagagg agctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaa attttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatggg aaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgat tcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggat cagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaa gctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccggccgctgatcttcagacct ggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacc caccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagca gcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcaga acaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcc tggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgcctt ggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaatt acacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaa tgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggttta agaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgag gggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacgg atctcgacggtatcgccaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaa gaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttatta cagggacagcagagatccagtttggatcgataagcttgatatcgaattcctgcagccccgataaaataaaagattttatttagtctc cagaaaaaggggggaatgaaagaccccacctgtaggtttggcaagctagctgcagtaacgccattttgcaaggcatggaaaa ataccaaaccaagaatagagaagttcagatcaagggcgggtacatgaaaatagctaacgttgggccaaacaggatatctgc ggtgagcagtttcggccccggcccggggccaagaacagatggtcaccgcagtttcggccccggcccgaggccaagaacag atggtccccagatatggcccaaccctcagcagtttcttaagacccatcagatgtttccaggctcccccaaggacctgaaatgacc ctgcgccttatttgaattaaccaatcagcctgcttctcgcttctgttcgcgcgcttctgcttcccgagctctataaaagagctcacaac ccctcactcggcgcgccagtcctccgacagactgagtcgcccgggggggatccaccggtcgccaccatggtgagcaagggc gaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcg agggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccac cctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgc catgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttc gagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagct ggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccg ccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgc tgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctgg agttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaagtaaagcggccgcgactctagagtcgacctgca ggcatgcaagcttgatatcaagcttatcgataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttg ctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttccccgtatggctttcattttctcctccttgtataaatcct ggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggt tggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggcggaactcategccgcctgcc ttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgct cgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccaattct accgggtaggggaggcgcttttcccaaggcagtctggagcatgcgctttagcagccccgctgggcacttggcgctacacaagt ggcctctggcctcgcacacattccacatccaccggtaggcgccaaccggctccgttctttggtggccccttcgcgccaccttctact cctcccctagtcaggaagttcccccccgccccgcagctcgcgtcgtgcaggacgtgacaaatggaagtagcacgtctcactagt ctcgtgcagatggacagcaccgctgagcaatggaagcgggtaggcctttggggcagcggccaatagcagctttgctccttcgct ttctgggctcagaggctgggaaggggtgggtccggggggggctcaggggcgggctcaggggggggggggcccgaa ggtcctccggaggcccggcattctgcacgcttcaaaagcgcacgtctgccgcgctgttctcctcttcctcatctccgggcctttcga cctgcagcccaagcttaccatgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacg caccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccga gctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtct ggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccgg ctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggc gtctcgcccgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggt gcccgccttcctggagacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggt gcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctgactcgagggaattaattcgagctcggtacctttaa gaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaac gaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaaccc actgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctca gacccttttagtcagtgtggaaaatctctagcagcatctagaattaattccgtgtattctatagtgtcacctaaatcgtatgtgtatgata cataaggttatgtattaattgtagccgcgttctaacgacaatatgtacaagcctaattgtgtagcatctggcttactgaagcagaccc tatcatctctctcgtaaactgccgtcagagtcggtttggttggacgaaccttctgagtttctggtaacgccgtcccgcacccggaaat ggtcagcgaaccaatcagcagggtcatcgctagccagatcctctacgccggacgcatcgtggccggcatcaccggcgccac aggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttc ggcgtgggtatggtggcaggccccgtggccgggggactgttgggcgccatctccttgcatgcaccattccttgcggcggcggtg ctcaacggcctcaacctactactgggctgcttcctaatgcaggagtcgcataagggagagcgtcgaatggtgcactctcagtac aatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctccc ggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgag acgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaa atgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaat aatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacc cagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggt aagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtatt gacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagca tcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgaca acgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggag ctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactg gcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggc ccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatg gtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagata ggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaag gatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaa aagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggttt gtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgta gccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgcca gtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggtt cgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgc ttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagg gggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcgg agcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatccc ctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcag tgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctgtggaat gtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaacc aggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgc ccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagag gccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagcttggac acaagacaggcttgcgagatatgtttgagaataccactttatcccgcgtcagggagaggcagtgcgtaaaaagacgcggactc atgtgaaatactggtttttagtgcgccagatctctataatctcgcgcaacctattttcccctcgaacactttttaagccgtagataaaca ggctgggacacttcacatgagcgaaaaatacatcgtcacctgggacatgttgcagatccatgcacgtaaactcgcaagccgac tgatgccttctgaacaatggaaaggcattattgccgtaagccgtggcggtctgtaccgggtgcgttactggcgcgtgaactgggta ttcgtcatgtcgataccgtttgtatttccagctacgatcacgacaaccagcgcgagcttaaagtgctgaaacgcgcagaaggcga tggcgaaggcttcatcgttattgatgacctggtggataccggtggtactgcggttgcgattcgtgaaatgtatccaaaagcgcacttt gtcaccatcttcgcaaaaccggctggtcgtccgctggttgatgactatgttgttgatatcccgcaagatacctggattgaacagccg tgggatatgggcgtcgtattcgtcccgccaatctccggtcgctaatcttttcaacgcctggcactgccgggcgttgttctttttaacttca ggcgggttacaatagtttccagtaagtattctggaggctgcatccatgacacaggcaaacctgagcgaaaccctgttcaaaccc cgctttaaacatcctgaaacctcgacgctagtccgccgctttaatcacggcgcacaaccgcctgtgcagtcggcccttgatggta aaaccatccctcactggtatcgcatgattaaccgtctgatgtggatctggcgcggcattgacccacgcgaaatcctcgacgtcca ggcacgtattgtgatgagcgatgccgaacgtaccgacgatgatttatacgatacggtgattggctaccgtggcggcaactggattt atgagtgggccccggatctttgtgaaggaaccttacttctgtggtgtgacataattggacaaactacctacagagatttaaagctct aaggtaaatataaaatttttaagtgtataatgtgttaaactactgattctaattgtttgtgtattttagattccaacctatggaactgatga atgggagcagtggtggaatgcctttaatgaggaaaacctgttttgctcagaagaaatgccatctagtgatgatgaggctactgctg actctcaacattctactcctccaaaaaagaagagaaaggtagaagaccccaaggactttccttcagaattgctaagttttttgagt catgctgtgtttagtaatagaactcttgcttgctttgctatttacaccacaaaggaaaaagctgcactgctatacaagaaaattatgg aaaaatattctgtaacctttataagtaggcataacagttataatcataacatactgttttttcttactccacacaggcatagagtgtctg ctattaataactatgctcaaaaattgtgtacctttagctttttaatttgtaaaggggttaataaggaatatttgatgtatagtgccttgact agagatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacata aaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaa gcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggatcaactggataactcaagctaacca aaatcatcccaaacttcccaccccataccctattaccactgccaattacctgtggtttcatttactctaaacctgtgattcctctgaatt attttcattttaaagaaattgtatttgttaaatatgtactacaaacttagtagt SEQIDNO:52-pREF0123sequence-TetherwithGal4underauto-cleavablepeptide tggaagggctaattcactcccaaagaagacaagatatccttgatctgtggatctaccacacacaaggctacttccctgattagca gaactacacaccagggccaggggtcagatatccactgacctttggatggtgctacaagctagtaccagttgagccagataagg tagaagaggccaataaaggagagaacaccagcttgttacaccctgtgagcctgcatgggatggatgacccggagagagaag tgttagagtggaggtttgacagccgcctagcatttcatcacgtggcccgagagctgcatccggagtacttcaagaactgctgatat cgagcttgctacaagggactttccgctggggactttccagggaggcgtggcctggggggactggggagtggcgagccctcag atcctgcatataagcagctgctttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactaggg aacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatc cctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagagg agctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaa attttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatggg aaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgat tcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggat cagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaa gctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccggccgctgatcttcagacct ggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacc caccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagca gcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcaga acaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcc tggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgcctt ggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaatt acacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaa tgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggttta agaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgag gggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacgg atctcgacggtatcgccaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaa gaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttatta cagggacagcagagatccagtttggatcgataagcttgatatcgaattgtaacgccattttgcaaggcatggaaaaataccaaa ccaagaatagagaagttcagatcaagggcgggtacatgaaaatagctaacgttgggccaaacaggatatctgcggtgagca gtttcggccccggcccggggccaagaacagatggtcaccgcagtttcggccccggcccgaggccaagaacagatggtcccc agatatggcccaaccctcagcagtttcttaagacccatcagatgtttccaggctcccccaaggacctgaaatgaccctgcgcctt atttgaattaaccaatcagcctgcttctcgcttctgttcgcgcgcttctgcttcccgagctctataaaagagctcacaacccctcactc ggcgcgccagtcctccgacagactgagtcgcccggggggGATCCGCCACCATGAGCATGCTGTTCTATA CCCTGATCACCGCCTTTCTGATCGGCATCCAGGCCGAGCAGAAGCTGATCTCCGAGGA AGATCTGGCCTCTAGCCTGGGCGCTCACCACATCCACCACTTTCACGGCAGCAGCAAG CACCACTCTGTGCCTATCGCCATCTACAGAAGCCCCGCTTCTCTGAGAGGCGGCCATG CTGGAACCACCTACATCTTCTCTAAAGGCGGCGGACAGATCACCTACAAGTGGCCTCC AAACGACAGACCCAGCACCAGAGCCGATAGACTGGCCATCGGCTTCAGCACCGTGCA GAAAGAAGCCGTGCTCGTCAGAGTGGACAGCAGCTCTGGACTGGGCGACTACCTGGA ACTGCACATCCATCAGGGCAAGATCGGCGTGAAGTTCAACGTGGGCACCGACGACATT GCCATCGAGGAAAGCAACGCCATCATCAACGACGGCAAGTACCACGTCGTGCGGTTC ACAAGAAGCGGCGGCAACGCTACACTGCAGGTCGACTCTTGGCCCGTGATCGAGAGA TACCCTGCCGGCAACAACGACAACGAGAGACTGGCTATCGCCAGACAGAGAATCCCCT ACAGACTGGGAAGAGTGGTGGACGAGTGGCTGCTGGATAAGGGCAGACAGCTGACCA TCTTCAATAGCCAGGCCACCATCATCATCGGCGGAAAAGAGCAGGGCCAGCCTTTCCA GGGACAGCTGAGCGGACTGTACTACAACGGCCTGAAGGTGCTGAACATGGCCGCTGA GAACGACGCCAATATCGCTATCGTGGGCAACGTGCGGCTCGTGGGAGAAGTTCCTGG CGGCGGAGGATCTGGCGGAGGTGGAAGTGGCGGAGGCGGATCTACACTGGTGCTGT TTGGCGCTGGCTTCGGCGCCGTGATTACAGTGGTGGTCATCGTCGTGATCATCAAGTG TTTCTGCAAAGAAGGACGCGGCTCCCTGCTTACGTGTGGCGATGTTGAAGAAAATCCT GGGCCTTCCGGCGCCATGAAGCTGCTGAGCAGCATCGAGCAGGCCTGCGACATCTGC AGGCTGAAGAAGCTGAAGTGCAGCAAGGAGAAGCCCAAGTGCGCCAAGTGCCTGAAG AACAACTGGGAGTGCAGGTACAGCCCCAAGACCAAGAGGAGCCCCCTGACCAGGGCC CACCTGACCGAGGTGGAGAGCAGGCTGGAGAGGCTGGAGCAGCTGTTCCTGCTGATC TTCCCCAGGGAGGACCTGGACATGATCCTGAAGATGGACAGCCTGCAGGACATCAAG GCCCTGCTGACCGGCCTGTTCGTGCAGGACAACGTGAACAAGGACGCCGTGACCGAC AGGCTGGCCAGCGTGGAGACCGACATGCCCCTGACCCTGAGGcagcacagaatcagcgccac cagcagcagcgaggaaagcagcaacaagggccagcggcagctgacagtgtctgctgctgcaggcggaagcggaggctct ggcggatctgatgccctggacgacttcgacctggatatgctgggcagcgacgccctggatgattttgatctggacatgctgggatc tgacgctctggacgatttcgatctcgacatgttgggatcagatgcactggatgactttgacctggacatgctcggatcatgaggcc gcgactctagagtcgacctgcaggcatgcaagcttgatatcaagcttatcgataatcaacctctggattacaaaatttgtgaaaga ttgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttccccgtatggcttt cattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgt ttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacg gcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcgggga aatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatc cagcggaccttccttcccaattctaccgggtaggggaggcgcttttcccaaggcagtctggagcatgcgctttagcagccccgct gggcacttggcgctacacaagtggcctctggcctcgcacacattccacatccaccggtaggcgccaaccggctccgttctttggt ggccccttcgcgccaccttctactcctcccctagtcaggaagttcccccccgccccgcagctcgcgtcgtgcaggacgtgacaa atggaagtagcacgtctcactagtctcgtgcagatggacagcaccgctgagcaatggaagcgggtaggcctttggggcagcg gccaatagcagctttgctccttcgctttctgggctcagaggctgggaaggggtgggtccggggggggctcaggggcgggctca ggggggggcgggcgcccgaaggtcctccggaggcccggcattctgcacgcttcaaaagcgcacgtctgccgcgctgttctc ctcttcctcatctccgggcctttcgacctgcagcccaagcttaccatgaccgagtacaagcccacggtgcgcctcgccacccgcg acgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacacegtcgatccggacc gccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcgga cgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgca tggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagccc gcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtgg aggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctccccttctacgagcggctcggcttc accgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctgactcgaggg aattaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggac tggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctggg agctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgt gactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagcatctagaattaattccgtgtattctatag tgtcacctaaatcgtatgtgtatgatacataaggttatgtattaattgtagccgcgttctaacgacaatatgtacaagcctaattgtgta gcatctggcttactgaagcagaccctatcatctctctcgtaaactgccgtcagagtcggtttggttggacgaaccttctgagtttctgg taacgccgtcccgcacccggaaatggtcagcgaaccaatcagcagggtcatcgctagccagatcctctacgccggacgcatc gtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggctcgcc acttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggccgggggactgttgggcgccatctccttgcat gcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagtcgcataagggagag cgtcgaatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgc gccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcac cgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttaga cgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagac aataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcgg cattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacat cgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgct atgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagt actcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataac actgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactc gccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaac aacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagtt gcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatc attgcagcactggggccagatggtaagccccccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacg aaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgat ttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttcca ctgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaa accaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcag ataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaa tcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgca gcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcg tgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacagga gagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgattt ttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttg ctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccga acgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggc cgattcattaatgcagctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaag catgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctca attagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatgg ctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcc taggcttttgcaaaaagcttggacacaagacaggcttgcgagatatgtttgagaataccactttatcccgcgtcagggagaggca gtgcgtaaaaagacgcggactcatgtgaaatactggtttttagtgcgccagatctctataatctcgcgcaacctattttcccctcgaa cactttttaagccgtagataaacaggctgggacacttcacatgagcgaaaaatacatcgtcacctgggacatgttgcagatccat gcacgtaaactcgcaagccgactgatgccttctgaacaatggaaaggcattattgccgtaagccgtggcggtctgtaccgggtg cgttactggcgcgtgaactgggtattcgtcatgtcgataccgtttgtatttccagctacgatcacgacaaccagcgcgagcttaaag tgctgaaacgcgcagaaggcgatggcgaaggcttcatcgttattgatgacctggtggataccggtggtactgcggttgcgattcgt gaaatgtatccaaaagcgcactttgtcaccatcttcgcaaaaccggctggtcgtccgctggttgatgactatgttgttgatatcccgc aagatacctggattgaacagccgtgggatatgggcgtcgtattcgtcccgccaatctccggtcgctaatcttttcaacgcctggca ctgccgggcgttgttctttttaacttcaggcgggttacaatagtttccagtaagtattctggaggctgcatccatgacacaggcaaac ctgagcgaaaccctgttcaaaccccgctttaaacatcctgaaacctcgacgctagtccgccgctttaatcacggcgcacaaccg cctgtgcagtcggcccttgatggtaaaaccatccctcactggtatcgcatgattaaccgtctgatgtggatctggcgcggcattgac ccacgcgaaatcctcgacgtccaggcacgtattgtgatgagcgatgccgaacgtaccgacgatgatttatacgatacggtgattg gctaccgtggcggcaactggatttatgagtgggccccggatctttgtgaaggaaccttacttctgtggtgtgacataattggacaaa ctacctacagagatttaaagctctaaggtaaatataaaatttttaagtgtataatgtgttaaactactgattctaattgtttgtgtattttag attccaacctatggaactgatgaatgggagcagtggtggaatgcctttaatgaggaaaacctgttttgctcagaagaaatgccat ctagtgatgatgaggctactgctgactctcaacattctactcctccaaaaaagaagagaaaggtagaagaccccaaggactttc cttcagaattgctaagttttttgagtcatgctgtgtttagtaatagaactcttgcttgctttgctatttacaccacaaaggaaaaagctgc actgctatacaagaaaattatggaaaaatattctgtaacctttataagtaggcataacagttataatcataacatactgttttttcttact ccacacaggcatagagtgtctgctattaataactatgctcaaaaattgtgtacctttagctttttaatttgtaaaggggttaataagga atatttgatgtatagtgccttgactagagatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacac ctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatag catcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggatca actggataactcaagctaaccaaaatcatcccaaacttcccaccccataccctattaccactgccaattacctgtggtttcatttact ctaaacctgtgattcctctgaattattttcattttaaagaaattgtatttgttaaatatgtactacaaacttagtagt SEQINNO:53-pREF044sequence-Gal4-inducibleCAR AACAAAATATTAACGCTTACAATTTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAA GGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCT GCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGA CGGCCAGTGCCAAGCTGATCTATACATTGAATCAATATTGGCAATTAGCCATATTAGTC ATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATAT CATAAcggagcactgtcctccgaacgtcggagcactgtcctccgaacgtcggagcactgtcctccgaacgtcggagcactgt cctccgaacggagcatgtcctccgaacgtcggagcactgtcctccgaacgactagttaggcgtgtacggtgggaggcctatata agcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgctgttttgacctccatagaagacaccgggaccg atccagcctctcgacattcgttggGCCACCATGGGAACTAGTCTGCTGTGTTGGATGGCCCTCTGC TTGTTGGGCGCAGACCACGCTGACGGCCAAGTTCAATTGGTCCAGTCTGGTGCCGAAC TGAAGAAGCCAGGAGCTAGCGTCAAGGTGAGTTGTAAGTCTTCCGGATACCATTTCAC GAGCTACTGGATGCATTGGGTAAAACAAGCCCCTGGACAAGGTCTCGAGTGGATAGG GGTAATACATCCTAACTCTGGGTCCATAAATTACAATGAAAAGTTCGAATCCAGGGTTA CCATCACAGTCGATAAGTCCACTTCCACGGCCTATATGGAATTGAGCTCCCTCAGGAG CGAAGATACAGCTGTATACTACTGCGCTGGTGAACGAGACTCCACTGAGGTGTTGCCC ATGGATTACTGGGGACAAGGCACCACTGTAACGGTCTCTAGCGGTGGGAGTAGTAGAA GCTCATCATCTGGCGGCGGCGGTTCAGGCGGTGGCGGCGACGTGCAGATCACGCAAA GCCCGAGCTCACTCTCCGCCAGTCTGGGTGAACGTGCGACTATAAATTGTAGAGCCAG CAAGAGCATTAACAAGTATCTCGCCTGGTACCAGCAAAAGCCGGGCAAGGCTCCTAAA CTCCTCATCTATTCCGGATCTACTCTCCAAAGCGGCATACCAGCGAGGTTCTCAGGAA GCGGATCAGGAACGGATTTTACGCTTACGATATCTAGCCTCGAGCCAGAGGATTTTGC CATGTACTATTGCCAACAACATAACGAATATCCCTTGACCTTCGGACAAGGGACGAAAC TCGAGATCAAAGATTACAAAGACGATGACGACAAAACCACAACCCCAGCTCCTAGGCC CCCAACACCTGCACCTACCATCGCTAGCCAGCCTCTGAGCCTGCGTCCCGAGGCATGT CGTCCTGCTGCAGGTGGGGCAGTCCACACAAGAGGTCTGGACTTTGCATGTGACTTTT GGGTCCTCGTAGTCGTCGGTGGCGTCCTGGCCTGTTATTCCCTTCTGGTTACGGTCGC CTTCATTATATTTTGGGTCAGGAGTAAACGGTCTAGGGGGGGGCACAGCGACTACATG AATATGACGCCACGCCGCCCCGGGCCCACAAGAAAACACTACCAACCTTACGCTCCAC CCAGGGATTTTGCCGCGTACCGGAGCAGGGTAAAGTTCAGCCGCAGTGCGGATGCAC CAGCGTATCAACAAGGCCAGAATCAACTGTATAATGAGCTGAACTTGGGACGACGAGA GGAATATGATGTTCTGGATAAACGGCGCGGGAGAGATCCAGAGATGGGCGGAAAGCC TAGACGTAAGAACCCCCAAGAAGGCCTTTATAATGAGTTGCAGAAAGATAAGATGGCC GAAGCCTATTCTGAAATAGGCATGAAGGGAGAGCGGCGGCGTGGCAAGGGTCATGAC GGTCTTTACCAGGGCCTCTCTACCGCAACTAAAGATACCTATGATGCACTTCACATGCA AGCCCTGCCCCCTAGAGAGTTCGAAGGGTCAGCAGCAGCCGAGGGACGTGGCTCATT GCTTACTTGCGGTGACGTTGAAGAAAACCCTGGCCCCAGCGGGATGGTAAGCAAGGG AGAAGAACTTTTCACGGGAGTAGTTCCTATTCTGGTGGAACTGGACGGGGATGTAAAC GGGCATAAATTCTCTGTCAGCGGGGAAGGGGAAGGCGATGCAACATACGGGAAGCTT ACCCTCAAATTCATCTGTACGACAGGGAAACTTCCAGTGCCCTGGCCTACACTTGTGAC AACCCTGACTTATGGGGTCCAATGTTTCTCCAGGTACCCTGATCACATGAAACAGCATG ACTTCTTCAAGAGCGCAATGCCTGAAGGGTATGTCCAGGAACGCACGATTTTCTTCAAG GACGATGGCAATTATAAAACTAGAGCCGAAGTAAAATTTGAGGGAGATACATTGGTAAA CAGGATCGAGCTCAAAGGGATCGATTTCAAAGAGGATGGGAACATCCTGGGGCATAAG TTGGAGTATAACTATAACTCTCACAATGTCTATATTATGGCCGACAAGCAAAAGAACGG GATAAAAGTCAACTTTAAAATAAGGCACAATATAGAGGACGGCAGTGTGCAATTGGCG GACCACTATCAACAAAATACCCCGATCGGCGACGGTCCAGTGTTGCTCCCTGATAATC ACTACCTTTCAACACAAAGTGCACTTAGTAAAGATCCCAATGAGAAGCGGGATCACATG GTCCTGCTCGAATTTGTCACAGCAGCTGGCATCACACTTGGGATGGATGAGCTCTATA AATAATAAACGGCCGGCCGCGGTCATAGCTGTTTCCTGAACAGATCCCGGGTGGCATC CCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCACTCCAGTGCCCAC CAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAGGTGTCCTTCTATAA TATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTGGGAAGACAACCTG TAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGGCACAATCTTGGCT CACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCAGCCTCCCGAGTTG TTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTTTTTGGTAGAGACG GGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCAGGTGATCTACCCAC CTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCTCCCTTCCCTGTCCT TCTGATTTTAAAATAACTATACCAGCAGGAGGACGTCCAGACACAGCATAGGCTACCTG GCCATGCCCAACCGGTGGGACATTTGAGTTGCTTGCTTGGCACTGTCCTCTCATGCGT TGGGTCCACTCAGTAGATGCCTGTTGAATTGGGTACGCGGCCAGCTTGGCTGTGGAAT GTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAA AGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAG GCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACT CCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGA CTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAA GTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGT ATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACA AGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGA CTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCA GGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAG GACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGT GCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGG GCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGAT GCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCG AAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATG ATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGG CGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGA ATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGT GGCCGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGC GGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAG CGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGA AATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCC TTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCC AGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTAT AATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTG CATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCG ACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTA TCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGT GCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTC GGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGG TTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTT CGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAAT CAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACC GTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCA CAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAG GCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCG GATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTG TAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCC CCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGG TAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAG GTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGA AGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGG TAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGC AGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCT GACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAG GATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATAT GAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGAT CTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATAC GGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCAC CGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTG GTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTA AGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGT GTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGA GTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGT TGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATT CTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAG TCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGG ATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCG GGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTC GTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAA ACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATAC TCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCG GATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCC GAAAAGTGCCACCTGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTG GTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTT TCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGG GCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATT AGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGAC GTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACC CTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAA AAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTT SEQIDNO:54-pREF031-transientexpressionvectorforthefirstreceptor(synNotch WTneurexinsequence) AACAAAATATTAACGCTTACAATTTCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAA GGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCT GCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGA CGGCCAGTGCCAAGCTGATCTATACATTGAATCAATATTGGCAATTAGCCATATTAGTC ATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATAT CATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGACATTGATTA TTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAG TTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCC GCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCAT TGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTA TCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCAT TATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGT CATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGG TTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTG GCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAA ATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACC GTCAGAATTTTGTAATACGACTCACTATAGGGCGGCCGGGAATTCGTCGACTGGATCC GGTACCGAGGAGATCTGCCGCCGCGATCGCCatggcattgcccgtgaccgccctgctgctgccactggc cttgttgctccacgccgcgcggccagaacagaagCTGATCAGCGAGGAGGATCTGATGTACCAGAGG ATGCTGAGGTGCGGCGCCGAGCTGGGCAGCCCCGGGGGGGGGGGGCGGCGGCG GCGGCGGCGCCGGCGGCAGGCTGGCCCTGCTGTGGATCGTGCCCCTGACCCTGAGC GGCCTGCTGGGCGTGGCCTGGGGCGCCAGCAGCCTGGGCGCCCACCACATCCACCA CTTCCACGGCAGCAGCAAGCACCACAGCGTGCCCATCGCCATCTACAGGAGCCCCGC CAGCCTGAGGGGCGGCCACGCCGGCACCACCTACATCTTCAGCAAGGGCGGCGGCC AGATCACCTACAAGTGGCCCCCCAACGACAGGCCCAGCACCAGGGCCGACAGGCTGG CCATCGGCTTCAGCACCGTGCAGAAGGAGGCCGTGCTGGTGAGGGTGGACAGCAGCA GCGGCCTGGGCGACTACCTGGAGCTGCACATCCACCAGGGCAAGATCGGCGTGAAGT TCAACGTGGGCACCGACGACATCGCCATCGAGGAGAGCAACGCCATCATCAACGACG GCAAGTACCACGTGGTGAGGTTCACCAGGAGCGGCGGCAACGCCACCCTGCAGGTG GACAGCTGGCCCGTGATCGAGAGGTACCCCGCCGGCAGGCAGCTGACCATCTTCAAC AGCCAGGCCACCATCATCATCGGCGGCAAGGAGCAGGGCCAGCCCTTCCAGGGCCA GCTGAGCGGCCTGTACTACAACGGCCTGAAGGTGCTGAACATGGCCGCCGAGAACGA CGCCAACATCGCCATCGTGGGCAACGTGAGGCTGGTGGGCGAGGTGCCCATCCTGGA CTACAGCTTCACAGGtggcgctgggcgcgacattcccccaccgcagattgaggaggcctgtgagctgcctgagtgcc aggtggatgcaggcaataaggtctgcaacctgcagtgtaataatcacgcatgtggctgggatggtggcgactgctccctcaactt caatgacccctggaagaactgcacgcagtctctacagtgctggaagtattttagcgacggccactgtgacagccagtgcaactc ggccggctgcctctttgatggcttcgactgccagctcaccgagggacagtgcaaccccctgtatgaccagtactgcaaggacca cttcagtgatggccactgcgaccagggctgtaacagtgccgaatgtgagtgggatggcctagactgtgctgagcatgtacccga gcggctggcagccggcaccctggtgctggtggtgctgcttccacccgaccagctacggaacaactccttccactttctgcggga gctcagccacgtgctgcacaccaacgtggtcttcaagcgtgatgcgcaaggccagcagatgatcttcccgtactatggccacga ggaagagctgcgcaagcacccaatcaagcgctctacagtgggttgggccacctcttcactgcttcctggtaccagtggtgggcg ccagcgcagggagctggaccccatggacatccgtggctccattgtctacctggagatcgacaaccggcaatgtgtgcagtcatc ctcgcagtgcttccagagtgccaccgatgtggctgccttcctaggtgctcttgcgtcacttggcagcctcaatattccttacaagattg aggccgtgaagagtgagccggtggagcctccgctgccctcgcagctgcacctcatgtacgtggcagcggccgccttcgtgctc ctgttctttgtgggctgtggggtgctgctgtcccgcaagcgccggcggatgaagctgctgagcagcatcgagcaggcctgtgaca tctgccggctgaagaaactgaagtgcagcaaagaaaagcccaagtgcgccaagtgcctgaagaacaactgggagtgccgg tacagccccaagaccaagagaagccccctgaccagagcccacctgaccgaggtggaaagccggctggaaagactggaac agctgtttctgctgatcttcccacgcgaggacctggacatgatcctgaagatggacagcctgcaggacatcaaggccctgctga ccggcctgttcgtgcaggacaacgtgaacaaggacgccgtgaccgacagactggccagcgtggaaaccgacatgcccctga ccctgcggcagcacagaatcagcgccaccagcagcagcgaggaaagcagcaacaagggccagcggcagctgacagtgt ctgctgctgcaggcggaagcggaggctctggcggatctgatgccctggacgacttcgacctggatatgctgggcagcgacgcc ctggatgattttgatctggacatgctgggatctgacgctctggacgatttcgatctcgacatgttgggatcagatgcactggatgactt tgacctggacatgctcggatcatgaTAAACGGCCGGCCGCGGTCATAGCTGTTTCCTGAACAGATC CCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTGGAAGTTGCCA CTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTTGTCTGACTAG GTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAGGGGCAAGTTG GGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGGAGTGCAGTGG CACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTCTCCTGCCTCA GCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTAATTTTTGTTTT TTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACTCCTAATCTCA GGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTGAACCACTGCT CCCTTCCCTGTCCTTCTGATTTTAAAATAACTATACCAGCAGGAGGACGTCCAGACACA GCATAGGCTACCTGGCCATGCCCAACCGGTGGGACATTTGAGTTGCTTGCTTGGCACT GTCCTCTCATGCGTTGGGTCCACTCAGTAGATGCCTGTTGAATTGGGTACGCGGCCAG CTTGGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAG GCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCC AGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATA GTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTC CGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTC TGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAG CTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTT TCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAG GCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTC CGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCC CTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTT CCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTG GGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTAT CCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATT CGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCT TGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTT CGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCG ATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGT GGCCGGCTGGGTGTGGCCGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATT GCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCC GCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGG GACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTC GATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCG GCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTT GTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAA AGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCAT GTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCC TGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGT GTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACT GCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGC GCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCG CTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATA CGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGC AAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCC CCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGG ACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCG ACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTT CTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGG CTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGT CTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACA GGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAA CTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCT TCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTT TTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGA TCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTC ATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAAT CAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGG CACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTG TAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGC GAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGG CCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGC CGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTG CTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTC CCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCC TTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTAT GGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTG GTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTG CCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATC ATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCA GTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGC GTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGA CACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGG GTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGG TTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGCGCCCTGTAGCGGCGCATTAAG CGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGC GCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTC AAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGA CCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACG GTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACT GGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATT TCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTT SEQIDNO:55-ConstructpREF043sequence-constitutivelyexpressedGal4andCAR undercontrolofGal4-inducibleelement tggaagggctaattcactcccaaagaagacaagatatccttgatctgtggatctaccacacacaaggctacttccctgattagca gaactacacaccagggccaggggtcagatatccactgacctttggatggtgctacaagctagtaccagttgagccagataagg tagaagaggccaataaaggagagaacaccagcttgttacaccctgtgagcctgcatgggatggatgacccggagagagaag tgttagagtggaggtttgacagccgcctagcatttcatcacgtggcccgagagctgcatccggagtacttcaagaactgctgatat cgagcttgctacaagggactttccgctggggactttccagggaggcgtggcctggggggactggggagtggcgagccctcag atcctgcatataagcagctgctttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactaggg aacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatc cctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagagg agctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaa attttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatggg aaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgat tcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggat cagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaa gctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccggccgctgatcttcagacct ggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacc caccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagca gcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcaga acaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcc tggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgcctt ggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaatt acacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaa tgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggttta agaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgag gggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacgg atctcgacggtatcgccaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaa gaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttatta cagggacagcagagatccagtttggatcgataagcttgatatcgaattGCATAAATCAATATTGGCTATTGGCC ATTGCATACGTTGTATCTATATCATAAcggagcactgtcctccgaacgtcggagcactgtcctccgaacgtcg gagcactgtcctccgaacgtcggagcactgtcctccgaacggagcatgtcctccgaacgtcggagcactgtcctccgaacgac tagttaggcgtgtacggtgggaggcctatataagcagagctcgtttagtgaaccgtcagatcgcctggagacgccatccacgct gttttgacctccatagaagacaccgggaccgatccagcctctcgacattcgttggGCCACCATGGGAACTAGTCTG CTGTGTTGGATGGCCCTCTGCTTGTTGGGCGCAGACCACGCTGACGGCCAAGTTCAAT TGGTCCAGTCTGGTGCCGAACTGAAGAAGCCAGGAGCTAGCGTCAAGGTGAGTTGTAA GTCTTCCGGATACCATTTCACGAGCTACTGGATGCATTGGGTAAAACAAGCCCCTGGA CAAGGTCTCGAGTGGATAGGGGTAATACATCCTAACTCTGGGTCCATAAATTACAATGA AAAGTTCGAATCCAGGGTTACCATCACAGTCGATAAGTCCACTTCCACGGCCTATATGG AATTGAGCTCCCTCAGGAGCGAAGATACAGCTGTATACTACTGCGCTGGTGAACGAGA CTCCACTGAGGTGTTGCCCATGGATTACTGGGGACAAGGCACCACTGTAACGGTCTCT AGCGGTGGGAGTAGTAGAAGCTCATCATCTGGCGGCGGCGGTTCAGGCGGTGGCGG CGACGTGCAGATCACGCAAAGCCCGAGCTCACTCTCCGCCAGTCTGGGTGAACGTGC GACTATAAATTGTAGAGCCAGCAAGAGCATTAACAAGTATCTCGCCTGGTACCAGCAAA AGCCGGGCAAGGCTCCTAAACTCCTCATCTATTCCGGATCTACTCTCCAAAGCGGCAT ACCAGCGAGGTTCTCAGGAAGCGGATCAGGAACGGATTTTACGCTTACGATATCTAGC CTCGAGCCAGAGGATTTTGCCATGTACTATTGCCAACAACATAACGAATATCCCTTGAC CTTCGGACAAGGGACGAAACTCGAGATCAAAGATTACAAAGACGATGACGACAAAACC ACAACCCCAGCTCCTAGGCCCCCAACACCTGCACCTACCATCGCTAGCCAGCCTCTGA GCCTGCGTCCCGAGGCATGTCGTCCTGCTGCAGGTGGGGCAGTCCACACAAGAGGTC TGGACTTTGCATGTGACTTTTGGGTCCTCGTAGTCGTCGGTGGCGTCCTGGCCTGTTA TTCCCTTCTGGTTACGGTCGCCTTCATTATATTTTGGGTCAGGAGTAAACGGTCTAGGG GCGGGCACAGCGACTACATGAATATGACGCCACGCCGCCCCGGGCCCACAAGAAAAC ACTACCAACCTTACGCTCCACCCAGGGATTTTGCCGCGTACCGGAGCAGGGTAAAGTT CAGCCGCAGTGCGGATGCACCAGCGTATCAACAAGGCCAGAATCAACTGTATAATGAG CTGAACTTGGGACGACGAGAGGAATATGATGTTCTGGATAAACGGCGCGGGAGAGAT CCAGAGATGGGCGGAAAGCCTAGACGTAAGAACCCCCAAGAAGGCCTTTATAATGAGT TGCAGAAAGATAAGATGGCCGAAGCCTATTCTGAAATAGGCATGAAGGGAGAGCGGC GGCGTGGCAAGGGTCATGACGGTCTTTACCAGGGCCTCTCTACCGCAACTAAAGATAC CTATGATGCACTTCACATGCAAGCCCTGCCCCCTAGAGAGTTCGAAGGGTCAGCAGCA GCCGAGGGACGTGGCTCATTGCTTACTTGCGGTGACGTTGAAGAAAACCCTGGCCCC AGCGGGATGGTAAGCAAGGGAGAAGAACTTTTCACGGGAGTAGTTCCTATTCTGGTGG AACTGGACGGGGATGTAAACGGGCATAAATTCTCTGTCAGCGGGGAAGGGGAAGGCG ATGCAACATACGGGAAGCTTACCCTCAAATTCATCTGTACGACAGGGAAACTTCCAGTG CCCTGGCCTACACTTGTGACAACCCTGACTTATGGGGTCCAATGTTTCTCCAGGTACC CTGATCACATGAAACAGCATGACTTCTTCAAGAGCGCAATGCCTGAAGGGTATGTCCA GGAACGCACGATTTTCTTCAAGGACGATGGCAATTATAAAACTAGAGCCGAAGTAAAAT TTGAGGGAGATACATTGGTAAACAGGATCGAGCTCAAAGGGATCGATTTCAAAGAGGA TGGGAACATCCTGGGGCATAAGTTGGAGTATAACTATAACTCTCACAATGTCTATATTAT GGCCGACAAGCAAAAGAACGGGATAAAAGTCAACTTTAAAATAAGGCACAATATAGAG GACGGCAGTGTGCAATTGGCGGACCACTATCAACAAAATACCCCGATCGGCGACGGT CCAGTGTTGCTCCCTGATAATCACTACCTTTCAACACAAAGTGCACTTAGTAAAGATCC CAATGAGAAGCGGGATCACATGGTCCTGCTCGAATTTGTCACAGCAGCTGGCATCACA CTTGGGATGGATGAGCTCTATAAATAATAAACGGCCGGCCGCGGTCATAGCTGTTTCC TGAACAGATCCCGGGTGGCATCCCTGTGACCCCTCCCCAGTGCCTCTCCTGGCCCTG GAAGTTGCCACTCCAGTGCCCACCAGCCTTGTCCTAATAAAATTAAGTTGCATCATTTT GTCTGACTAGGTGTCCTTCTATAATATTATGGGGTGGAGGGGGGTGGTATGGAGCAAG GGGCAAGTTGGGAAGACAACCTGTAGGGCCTGCGGGGTCTATTGGGAACCAAGCTGG AGTGCAGTGGCACAATCTTGGCTCACTGCAATCTCCGCCTCCTGGGTTCAAGCGATTC TCCTGCCTCAGCCTCCCGAGTTGTTGGGATTCCAGGCATGCATGACCAGGCTCAGCTA ATTTTTGTTTTTTTGGTAGAGACGGGGTTTCACCATATTGGCCAGGCTGGTCTCCAACT CCTAATCTCAGGTGATCTACCCACCTTGGCCTCCCAAATTGCTGGGATTACAGGCGTG AACCACTGCTCCCTTCCCTGTCCTTCTGATTTTAAAATAACTATACCAGCAGGAGGACG TCCAGACACAGCATAGGCTACCTGGCCATGCCCAACCGGTGGGACATTTGAGTTGCTT GCTTGGCACTGTCCTCTCATGCGTTGGGTCCACTCAGTAGATGCCTGTTGAATTGACAT TGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATAT ATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACG ACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACT TTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCA AGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCC TGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGT ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGAT AGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTT GTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGA CGCAAATGGGGGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGT GAACCGTCAGAATTTTGTAATACGACTCACTATAGGGCGGCCGGGAATTCGTCGACTG GATCCGGTACCGAGGAGATCTGCCGCCGCGATCGCCatgaagctgctgagcagcatcgagcaggc ctgtgacatctgccggctgaagaaactgaagtgcagcaaagaaaagcccaagtgcgccaagtgcctgaagaacaactggg agtgccggtacagccccaagaccaagagaagccccctgaccagagcccacctgaccgaggtggaaagccggctggaaag actggaacagctgtttctgctgatcttcccacgcgaggacctggacatgatcctgaagatggacagcctgcaggacatcaaggc cctgctgaccggcctgttcgtgcaggacaacgtgaacaaggacgccgtgaccgacagactggccagcgtggaaaccgacat gcccctgaccctgcggcagcacagaatcagcgccaccagcagcagcgaggaaagcagcaacaagggccagcggcagct gacagtgtctgctgctgcaggcggaagcggaggctctggcggatctgatgccctggacgacttcgacctggatatgctgggcag cgacgccctggatgattttgatctggacatgctgggatctgacgctctggacgatttcgatctcgacatgttgggatcagatgcact ggatgactttgacctggacatgctcggatcatgaTAAACGGCCGGCCGCGGTCATAGCTGTTTCCTGGG CCGCGACTCTAGAgtcgacctgcaggcatgcaagcttgatatcaagcttatcgataatcaacctctggattacaaaattt gtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttcccc gtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgt gcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccct attgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgtt gtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcgg ccctcaatccagcggaccttccttcccaattctaccgggtaggggaggcgcttttcccaaggcagtctggagcatgcgctttagca gccccgctgggcacttggcgctacacaagtggcctctggcctcgcacacattccacatccaccggtaggcgccaaccggctcc gttctttggtggccccttcgcgccaccttctactcctcccctagtcaggaagttcccccccgccccgcagctcgcgtcgtgcaggac gtgacaaatggaagtagcacgtctcactagtctcgtgcagatggacagcaccgctgagcaatggaagcgggtaggcctttggg gcagcggccaatagcagctttgctccttcgctttctgggctcagaggctgggaaggggtgggtccggggggggctcaggggc gggctcaggggggggcgggcgcccgaaggtcctccggaggcccggcattctgcacgcttcaaaagcgcacgtctgccgcg ctgttctcctcttcctcatctccgggcctttcgacctgcagcccaagcttaccatgaccgagtacaagcccacggtgcgcctcgcca cccgcgacgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatc cggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggt cgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggc ccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaa ggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctgggcagegccgtcgtgctcccc ggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctccccttctacgagcgg ctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctgac tcgagggaattaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaag gggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctga gcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgt ctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagcatctagaattaattccgtgt attctatagtgtcacctaaatcgtatgtgtatgatacataaggttatgtattaattgtagccgcgttctaacgacaatatgtacaagcct aattgtgtagcatctggcttactgaagcagaccctatcatctctctcgtaaactgccgtcagagtcggtttggttggacgaaccttctg agtttctggtaacgccgtcccgcacccggaaatggtcagcgaaccaatcagcagggtcatcgctagccagatcctctacgccg gacgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagatcg ggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggccgggggactgttgggcgccatc tccttgcatgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagtcgcataa gggagagcgtcgaatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacaccc gctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcaga ggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatgg tttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctc atgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattccct tttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtg ggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaa gttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttg gttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagt gataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcat gtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaat ggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcgga taaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcg cggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggat gaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttag attgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttc gttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaaca aaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagag cgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctc tgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataag gcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagataccta cagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaa caggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcg tcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgg ccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgca gccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgc gttggccgattcattaatgcagctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatg caaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgc atctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccc catggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttgg aggcctaggcttttgcaaaaagcttggacacaagacaggcttgcgagatatgtttgagaataccactttatcccgcgtcagggag aggcagtgcgtaaaaagacgcggactcatgtgaaatactggtttttagtgcgccagatctctataatctcgcgcaacctattttccc ctcgaacactttttaagccgtagataaacaggctgggacacttcacatgagcgaaaaatacatcgtcacctgggacatgttgca gatccatgcacgtaaactcgcaagccgactgatgccttctgaacaatggaaaggcattattgccgtaagccgtggcggtctgtac cgggtgcgttactggcgcgtgaactgggtattcgtcatgtcgataccgtttgtatttccagctacgatcacgacaaccagcgcgag cttaaagtgctgaaacgcgcagaaggcgatggcgaaggcttcatcgttattgatgacctggtggataccggtggtactgcggttg cgattcgtgaaatgtatccaaaagcgcactttgtcaccatcttcgcaaaaccggctggtcgtccgctggttgatgactatgttgttgat atcccgcaagatacctggattgaacagccgtgggatatgggcgtcgtattcgtcccgccaatctccggtcgctaatcttttcaacgc ctggcactgccgggcgttgttctttttaacttcaggcgggttacaatagtttccagtaagtattctggaggctgcatccatgacacag gcaaacctgagcgaaaccctgttcaaaccccgctttaaacatcctgaaacctcgacgctagtccgccgctttaatcacggcgca caaccgcctgtgcagtcggcccttgatggtaaaaccatccctcactggtatcgcatgattaaccgtctgatgtggatctggcgcgg cattgacccacgcgaaatcctcgacgtccaggcacgtattgtgatgagcgatgccgaacgtaccgacgatgatttatacgatac ggtgattggctaccgtggcggcaactggatttatgagtgggccccggatctttgtgaaggaaccttacttctgtggtgtgacataatt ggacaaactacctacagagatttaaagctctaaggtaaatataaaatttttaagtgtataatgtgttaaactactgattctaattgtttg tgtattttagattccaacctatggaactgatgaatgggagcagtggtggaatgcctttaatgaggaaaacctgttttgctcagaaga aatgccatctagtgatgatgaggctactgctgactctcaacattctactcctccaaaaaagaagagaaaggtagaagacccca aggactttccttcagaattgctaagttttttgagtcatgctgtgtttagtaatagaactcttgcttgctttgctatttacaccacaaaggaa aaagctgcactgctatacaagaaaattatggaaaaatattctgtaacctttataagtaggcataacagttataatcataacatactg ttttttcttactccacacaggcatagagtgtctgctattaataactatgctcaaaaattgtgtacctttagctttttaatttgtaaaggggtt aataaggaatatttgatgtatagtgccttgactagagatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacc tcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataa agcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgt ctggatcaactggataactcaagctaaccaaaatcatcccaaacttcccaccccataccctattaccactgccaattacctgtggt ttcatttactctaaacctgtgattcctctgaattattttcattttaaagaaattgtatttgttaaatatgtactacaaacttagtagt SEQIDNO:56-pREF097-FoxP3-inducibleCAR tggaagggctaattcactcccaaagaagacaagatatccttgatctgtggatctaccacacacaaggctacttccctgattagca gaactacacaccagggccaggggtcagatatccactgacctttggatggtgctacaagctagtaccagttgagccagataagg tagaagaggccaataaaggagagaacaccagcttgttacaccctgtgagcctgcatgggatggatgacccggagagagaag tgttagagtggaggtttgacagccgcctagcatttcatcacgtggcccgagagctgcatccggagtacttcaagaactgctgatat cgagcttgctacaagggactttccgctggggactttccagggaggcgtggcctggggggactggggagtggcgagccctcag atcctgcatataagcagctgctttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactaggg aacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatc cctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagagg agctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaa attttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatggg aaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgat tcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggat cagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaa gctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccggccgctgatcttcagacct ggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacc caccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagca gcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcaga acaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcc tggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgcctt ggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaatt acacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaa tgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggttta agaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgag gggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacgg atctcgacggtatcgccaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaa gaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttatta cagggacagcagagatccagtttggatcgataagcttgatatcgaattcgtagctgaagaaaggattcataaatgaagttcaatc cttctcatcaaccccagcccacacctccagcaattgaacttgaaaaaaaaaacctggtttgaaaaattaccgcaaactatattgt catcaaaaaaaaaaaaaaaaaaaaacacttcctatatttgagatgagagaagagagtgctaggcagtttcctggctgaacac gccagcccaatacttaaagagagcaactcctgactccgatagagactggatggacccacaagggtgacagcccaggcgga ccgatcttcccatcccacatcctccggcgcgatgccaaaaagaggctgacggcaactgggccttctgcagagaaagacctcc gcttcactgccccggctggtcccaagggtcaggaagGCCACCATGGGAACTAGTCTGCTGTGTTGGATG GCCCTCTGCTTGTTGGGCGCAGACCACGCTGACGGCCAAGTTCAATTGGTCCAGTCTG GTGCCGAACTGAAGAAGCCAGGAGCTAGCGTCAAGGTGAGTTGTAAGTCTTCCGGATA CCATTTCACGAGCTACTGGATGCATTGGGTAAAACAAGCCCCTGGACAAGGTCTCGAG TGGATAGGGGTAATACATCCTAACTCTGGGTCCATAAATTACAATGAAAAGTTCGAATC CAGGGTTACCATCACAGTCGATAAGTCCACTTCCACGGCCTATATGGAATTGAGCTCC CTCAGGAGCGAAGATACAGCTGTATACTACTGCGCTGGTGAACGAGACTCCACTGAGG TGTTGCCCATGGATTACTGGGGACAAGGCACCACTGTAACGGTCTCTAGCGGTGGGA GTAGTAGAAGCTCATCATCTGGCGGCGGCGGTTCAGGCGGTGGCGGCGACGTGCAGA TCACGCAAAGCCCGAGCTCACTCTCCGCCAGTCTGGGTGAACGTGCGACTATAAATTG TAGAGCCAGCAAGAGCATTAACAAGTATCTCGCCTGGTACCAGCAAAAGCCGGGCAAG GCTCCTAAACTCCTCATCTATTCCGGATCTACTCTCCAAAGCGGCATACCAGCGAGGTT CTCAGGAAGCGGATCAGGAACGGATTTTACGCTTACGATATCTAGCCTCGAGCCAGAG GATTTTGCCATGTACTATTGCCAACAACATAACGAATATCCCTTGACCTTCGGACAAGG GACGAAACTCGAGATCAAAGATTACAAAGACGATGACGACAAAACCACAACCCCAGCT CCTAGGCCCCCAACACCTGCACCTACCATCGCTAGCCAGCCTCTGAGCCTGCGTCCC GAGGCATGTCGTCCTGCTGCAGGTGGGGCAGTCCACACAAGAGGTCTGGACTTTGCA TGTGACTTTTGGGTCCTCGTAGTCGTCGGTGGCGTCCTGGCCTGTTATTCCCTTCTGG TTACGGTCGCCTTCATTATATTTTGGGTCAGGAGTAAACGGTCTAGGGGGGGGCACAG CGACTACATGAATATGACGCCACGCCGCCCCGGGCCCACAAGAAAACACTACCAACCT TACGCTCCACCCAGGGATTTTGCCGCGTACCGGAGCAGGGTAAAGTTCAGCCGCAGT GCGGATGCACCAGCGTATCAACAAGGCCAGAATCAACTGTATAATGAGCTGAACTTGG GACGACGAGAGGAATATGATGTTCTGGATAAACGGCGCGGGAGAGATCCAGAGATGG GCGGAAAGCCTAGACGTAAGAACCCCCAAGAAGGCCTTTATAATGAGTTGCAGAAAGA TAAGATGGCCGAAGCCTATTCTGAAATAGGCATGAAGGGAGAGCGGCGGCGTGGCAA GGGTCATGACGGTCTTTACCAGGGCCTCTCTACCGCAACTAAAGATACCTATGATGCA CTTCACATGCAAGCCCTGCCCCCTAGAGAGTTCGAAGGGTCAGCAGCAGCCTGAggccg cgactctagagtcgacctgcaggcatgcaagcttgatatcaagcttatcgataatcaacctctggattacaaaatttgtgaaagatt gactggtattcttaactatgttgctccttttacgctatgtggatacgctgctttaatgcctttgtatcatgctattgcttccccgtatggctttc attttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgttt gctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacgg cggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaa atcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatcc agcggaccttccttcccaattctaccgggtaggggaggcgcttttcccaaggcagtctggagcatgcgctttagcagccccgctg ggcacttggcgctacacaagtggcctctggcctcgcacacattccacatccaccggtaggcgccaaccggctccgttctttggtg gccccttcgcgccaccttctactcctcccctagtcaggaagttcccccccgccccgcagctcgcgtcgtgcaggacgtgacaaat ggaagtagcacgtctcactagtctcgtgcagatggacagcaccgctgagcaatggaagcgggtaggcctttggggcagcggc caatagcagctttgctccttcgctttctgggctcagaggctgggaaggggtgggtccggggggggctcaggggcgggctcag gggcggggcgggcgcccgaaggtcctccggaggcccggcattctgcacgcttcaaaagcgcacgtctgccgcgctgttctcct cttcctcatctccgggcctttcgacctgcagcccaagcttaccatgaccgagtacaagcccacggtgcgcctcgccacccgcga cgacgtccccagggccgtacgcaccctcgccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccg ccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggac gacggcgccgcggtggcggtctggaccacgccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcat ggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccg cgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtgga ggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctccccttctacgagcggctcggcttca ccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctgactcgaggga attaattcgagctcggtacctttaagaccaatgacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggact ggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctggga gctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtg actctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagcatctagaattaattccgtgtattctatagt gtcacctaaatcgtatgtgtatgatacataaggttatgtattaattgtagccgcgttctaacgacaatatgtacaagcctaattgtgta gcatctggcttactgaagcagaccctatcatctctctcgtaaactgccgtcagagtcggtttggttggacgaaccttctgagtttctgg taacgccgtcccgcacccggaaatggtcagcgaaccaatcagcagggtcatcgctagccagatcctctacgccggacgcatc gtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgccgacatcaccgatggggaagategggctcgcc acttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtggccgggggactgttgggcgccatctccttgcat gcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggctgcttcctaatgcaggagtcgcataagggagag cgtcgaatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgc gccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcac cgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttaga cgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagac aataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcgg cattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacat cgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgct atgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagt actcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataac actgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactc gccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaac aacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagtt gcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatc attgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacg aaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgat ttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttcca ctgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaa accaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcag ataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaa tcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgca gcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcg tgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacagga gagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgattt ttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttg ctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccga acgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggc cgattcattaatgcagctgtggaatgtgtgtcagttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaag catgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctca attagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatgg ctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcc taggcttttgcaaaaagcttggacacaagacaggcttgcgagatatgtttgagaataccactttatcccgcgtcagggagaggca gtgcgtaaaaagacgcggactcatgtgaaatactggtttttagtgcgccagatctctataatctcgcgcaacctattttcccctcgaa cactttttaagccgtagataaacaggctgggacacttcacatgagcgaaaaatacatcgtcacctgggacatgttgcagatccat gcacgtaaactcgcaagccgactgatgccttctgaacaatggaaaggcattattgccgtaagccgtggcggtctgtaccgggtg cgttactggcgcgtgaactgggtattcgtcatgtcgataccgtttgtatttccagctacgatcacgacaaccagcgcgagcttaaag tgctgaaacgcgcagaaggcgatggcgaaggcttcatcgttattgatgacctggtggataccggtggtactgcggttgcgattcgt gaaatgtatccaaaagcgcactttgtcaccatcttcgcaaaaccggctggtcgtccgctggttgatgactatgttgttgatatcccgc aagatacctggattgaacagccgtgggatatgggcgtcgtattcgtcccgccaatctccggtcgctaatcttttcaacgcctggca ctgccgggcgttgttctttttaacttcaggcgggttacaatagtttccagtaagtattctggaggctgcatccatgacacaggcaaac ctgagcgaaaccctgttcaaaccccgctttaaacatcctgaaacctcgacgctagtccgccgctttaatcacggcgcacaaccg cctgtgcagtcggcccttgatggtaaaaccatccctcactggtatcgcatgattaaccgtctgatgtggatctggcgcggcattgac ccacgcgaaatcctcgacgtccaggcacgtattgtgatgagcgatgccgaacgtaccgacgatgatttatacgatacggtgattg gctaccgtggcggcaactggatttatgagtgggccccggatctttgtgaaggaaccttacttctgtggtgtgacataattggacaaa ctacctacagagatttaaagctctaaggtaaatataaaatttttaagtgtataatgtgttaaactactgattctaattgtttgtgtattttag attccaacctatggaactgatgaatgggagcagtggtggaatgcctttaatgaggaaaacctgttttgctcagaagaaatgccat ctagtgatgatgaggctactgctgactctcaacattctactcctccaaaaaagaagagaaaggtagaagaccccaaggactttc cttcagaattgctaagttttttgagtcatgctgtgtttagtaatagaactcttgcttgctttgctatttacaccacaaaggaaaaagctgc actgctatacaagaaaattatggaaaaatattctgtaacctttataagtaggcataacagttataatcataacatactgttttttcttact ccacacaggcatagagtgtctgctattaataactatgctcaaaaattgtgtacctttagctttttaatttgtaaaggggttaataagga atatttgatgtatagtgccttgactagagatcataatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacac ctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatag catcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggatca actggataactcaagctaaccaaaatcatcccaaacttcccaccccataccctattaccactgccaattacctgtggtttcatttact ctaaacctgtgattcctctgaattattttcattttaaagaaattgtatttgttaaatatgtactacaaacttagtagt SEQIDNO:57-pREF014-CAR-encodingplasmid,canexpressconstitutivelyCAR underSFFVpromoter CTCGAGCTTATTCCAGATGCGTGCGGATGGAATTCGAGCTCGGTACCATGCCAAAAGC AAAGCGCTATCGCGCCTTACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTG CGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGG AAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAG GAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAG ACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAG GTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCC CAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCCCCTCAAGCG TATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCT GGGGCCTCGGTGCACATGCTTTTCATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCC CCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATAACATGACCG AGTACAAGCCCACGGTGCGCCTCGCCACCCGCGACGACGTCCCCAGGGCCGTACGC ACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACACCGTCGATCCGGAC CGCCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCACGCGCGTCGGGCTC GACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCGGTGGCGGTCTGGACCAC GCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCG AGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAACAGATGGAGGGCCTCCTGGCGCCG CACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCCACCGTCGGCGTCTCGCCCGACCA CCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCCGCCGAGC GCGCCGGGGTGCCCGCCTTCCTGGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACG AGCGGCTCGGCTTCACCGTCACCGCCGACGTCGAGGTGCCCGAAGGACCGCGCACC TGGTGCATGACCCGCAAGCCCGGTGCCTAGGCTAGCTTGACTGACTGAGTCGACAATC AACCTTTTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCT TTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATG GCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGG CCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACT GGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCC CTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTC GGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAGCTGACGTCCTTTCCATG GCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCT TCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCT CTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCC CGCCTGGAATTCGAGCTCGGTACCTTTAAGACCAATGACTTACAAGGCAGCTGTAGAT CTTAGCCACTTTTTAAAAGAAAAGGGGGGACTGGAAGGGCTAATTCACTCCCAACGAA GACAAGATCTGCTTTTTGCTTGTACTGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGG GAGCTCTCTGGCTAACTAGGGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGT GCTTCAAGTAGTGTGTGCCCGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGA CCCTTTTAGTCAGTGTGGAAAATCTCTAGCAGTCCTGGCCAACGTGAGCACCGTGCTG ACCTCCAAATATCGTTAAGCTGGAGCCTGGGAGCCGGCCTGGCCCTCCGCCCCCCCC ACCCCCGCAGCCCACCCCTGGTCTTTGAATAAAGTCTGAGTGAGTGGCCGACAGTGCC CGTGGAGTTCTCGTGACCTGAGGTGCAGGGCCGGCGCTAGGGACACGTCCGTGCAC GTGCCGAGGCCCCCTGTGCAGCTGCAAGGGACAGGCCTAGCCCTGCAGGCCTAACTC CGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCTCATGGCTGACT AATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGT AGTGAGGACGCTTTTTTGGAGGCCGAGGCTTTTGCAAAGATCGAACAAGAGACAGGAC CTGCAGGTTAATTAAATTTAAATCATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACC GTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCA CAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAG GCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCG GATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTG TAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCC CCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGG TAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAG GTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGA AGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGG TAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGC AGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGG TCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAA AAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATA TATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGC GATCTGTCTATTTCGTTCATCCATAGTTGCATTTAAATGGCCGGCCTGGCGCGCCGTTT AAACCTAGATATTGATAGTCTGATCGGTCAACGTATAATCGAGTCCTAGCTTTTGCAAA CATCTATCAAGAGACAGGATCAGCAGGAGGCTTTCGCATGAGTATTCAACATTTCCGTG TCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACCCAGAAACGC TGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCGCGAGTGGGTTACATCGAACT GGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGCTTTCCAATG ATGAGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCA AGAGCAACTCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTATTCACCAG TCACAGAAAAGCATCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATA ACCATGAGTGATAACACTGCGGCCAACTTACTTCTGACAACGATTGGAGGACCGAAGG AGCTAACCGCTTTTTTGCACAACATGGGGGATCATGTAACTCGCCTTGATCGTTGGGAA CCGGAGCTGAATGAAGCCATACCAAACGACGAGCGTGACACCACGATGCCTGTAGCA ATGGCAACAACCTTGCGTAAACTATTAACTGGCGAACTACTTACTCTAGCTTCCCGGCA ACAGTTGATAGACTGGATGGAGGCGGATAAAGTTGCAGGACCACTTCTGCGCTCGGCC CTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCGTGGGTCTCGCG GTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATCTACAC GACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGC CTCACTGATTAAGCATTGGTAACCGATTCTAGGTGCATTGGCGCAGAAAAAAATGCCTG ATGCGACGCTGCGCGTCTTATACTCCCACATATGCCAGATTCAGCAACGGATACGGCT TCCCCAACTTGCCCACTTCCATACGTGTCCTCCTTACCAGAAATTTATCCTTAAGATCCC GAATCGTTTAAACGCGATCGCAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATAT ATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACG ACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACT TTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCA AGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCC TGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGT ATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGAT AGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTT GTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGA CGCAAATGGGGGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGT GAACCGGGGTCTCTCTGGTTAGACCAGATCTGAGCCTGGGAGCTCTCTGGCTAACTAG GGAACCCACTGCTTAAGCCTCAATAAAGCTTGCCTTGAGTGCTTCAAGTAGTGTGTGCC CGTCTGTTGTGTGACTCTGGTAACTAGAGATCCCTCAGACCCTTTTAGTCAGTGTGGAA AATCTCTAGCAGTGGCGCCCGAACAGGGACCTGAAAGCGAAAGGGAAACCAGAGCTC TCTCGACGCAGGACTCGGCTTGCTGAAGCGCGCACGGCAAGAGGCGAGGGGCGGCG ACTGGTGAGTACGCCAAAAATTTTGACTAGCGGAGGCTAGAAGGAGAGAGATGGGTGC GAGAGCGTCAGTATTAAGCGGGGGAGAATTAGATCGCGATGGGAAAAAATTCGGTTAA GGCCAGGGGGAAAGAAAAAATATAAATTAAAACATATAGTATGGGCAAGCAGGGAGCT AGAACGATTCGCAGTTAATCCTGGCCTGTTAGAAACATCAGAAGGCTGTAGACAAATAC TGGGACAGCTACAACCATCCCTTCAGACAGGATCAGAAGAACTTAGATCATTATATAAT ACAGTAGCAACCCTCTATTGTGTGCATCAAAGGATAGAGATAAAAGACACCAAGGAAG CTTTAGACAAGATAGAGGAAGAGCAAAACAAAAGTAAGACCACCGCACAGCAAGCGGC CGCTGATCTTCAGACCTGGAGGAGGAGATATGAGGGACAATTGGAGAAGTGAATTATA TAAATATAAAGTAGTAAAAATTGAACCATTAGGAGTAGCACCCACCAAGGCAAAGAGAA GAGTGGTGCAGAGAGAAAAAAGAGCAGTGGGAATAGGAGCTTTGTTCCTTGGGTTCTT GGGAGCAGCAGGAAGCACTATGGGCGCAGCCTCAATGACGCTGACGGTACAGGCCAG ACAATTATTGTCTGGTATAGTGCAGCAGCAGAACAATTTGCTGAGGGCTATTGAGGCG CAACAGCATCTGTTGCAACTCACAGTCTGGGGCATCAAGCAGCTCCAGGCAAGAATCC TGGCTGTGGAAAGATACCTAAAGGATCAACAGCTCCTGGGGATTTGGGGTTGCTCTGG AAAACTCATTTGCACCACTGCTGTGCCTTGGAATGCTAGTTGGAGTAATAAATCTCTGG AACAGATTTGGAATCACACGACCTGGATGGAGTGGGACAGAGAAATTAACAATTACACA AGCTTAATACACTCCTTAATTGAAGAATCGCAAAACCAGCAAGAAAAGAATGAACAAGA ATTATTGGAATTAGATAAATGGGCAAGTTTGTGGAATTGGTTTAACATAACAAATTGGCT GTGGTATATAAAATTATTCATAATGATAGTAGGAGGCTTGGTAGGTTTAAGAATAGTTTT TGCTGTACTTTCTATAGTGAATAGAGTTAGGCAGGGATATTCACCATTATCGTTTCAGAC CCACCTCCCAACCCCGAGGGGACCCGACAGGCCCGAAGGAATAGAAGAAGAAGGTGG AGAGAGAGACAGAGACAGATCCATTCGATTAGTGAACGGATCTCGACGGTATCGGTTA ACTTTTAAAAGAAAAGGGGGGATTGGGGGGTACAGTGCAGGGGAAAGAATAGTAGACA TAATAGCAACAGACATACAAACTAAAGAATTACAAAAACAAATTACAAAAATTCAAAATTT TGGCTCCCGATCGTTGCGTTACACACACAATTACTGCTGATCGAGTGTAGCCTTCGAAT GAAAGACCCCACCTGTAGGTTTGGCAAGATAGCTGCAGTAACGCCATTTTGCAAGGCA TGGAAAAATACCAAACCAAGAATAGAGAAGTTCAGATCAAGGGCGGGTACATGAAAAT AGCTAACGTTGGGCCAAACAGGATATCTGCGGTGAGCAGTTTCGGCCCCGGCCCGGG GCCAAGAACAGATGGTCACCGCAGTTTCGGCCCCGGCCCGAGGCCAAGAACAGATGG TCCCCAGATATGGCCCAACCCTCAGCAGTTTCTTAAGACCCATCAGATGTTTCCAGGCT CCCCCAAGGACCTGAAATGACCCTGCGCCTTATTTGAATTAACCAATCAGCCTGCTTCT CGCTTCTGTTCGCGCGCTTCTGCTTCCCGAGCTCTATAAAAGAGCTCACAACCCCTCA CTCGGCGCGCCAGTCCTCCGATTGACTGAGTCGCCCTGATCATTGTCGATCCTACCAT CCACTCGACACACCCGCCAGGGCCCGCATCCACCATCGCAGACTTATCATGGATCCG CCACCATGGGAACTAGTCTGCTGTGTTGGATGGCCCTCTGCTTGTTGGGCGCAGACCA CGCTGACGGCCAAGTTCAATTGGTCCAGTCTGGTGCCGAACTGAAGAAGCCAGGAGC TAGCGTCAAGGTGAGTTGTAAGTCTTCCGGATACCATTTCACGAGCTACTGGATGCATT GGGTAAAACAAGCCCCTGGACAAGGTCTCGAGTGGATAGGGGTAATACATCCTAACTC TGGGTCCATAAATTACAATGAAAAGTTCGAATCCAGGGTTACCATCACAGTCGATAAGT CCACTTCCACGGCCTATATGGAATTGAGCTCCCTCAGGAGCGAAGATACAGCTGTATA CTACTGCGCTGGTGAACGAGACTCCACTGAGGTGTTGCCCATGGATTACTGGGGACAA GGCACCACTGTAACGGTCTCTAGCGGTGGGAGTAGTAGAAGCTCATCATCTGGCGGC GGCGGTTCAGGCGGTGGCGGCGACGTGCAGATCACGCAAAGCCCGAGCTCACTCTCC GCCAGTCTGGGTGAACGTGCGACTATAAATTGTAGAGCCAGCAAGAGCATTAACAAGT ATCTCGCCTGGTACCAGCAAAAGCCGGGCAAGGCTCCTAAACTCCTCATCTATTCCGG ATCTACTCTCCAAAGCGGCATACCAGCGAGGTTCTCAGGAAGCGGATCAGGAACGGAT TTTACGCTTACGATATCTAGCCTCGAGCCAGAGGATTTTGCCATGTACTATTGCCAACA ACATAACGAATATCCCTTGACCTTCGGACAAGGGACGAAACTCGAGATCAAAACCACAA CCCCAGCTCCTAGGCCCCCAACACCTGCACCTACCATCGCTAGCCAGCCTCTGAGCCT GCGTCCCGAGGCATGTCGTCCTGCTGCAGGTGGGGCAGTCCACACAAGAGGTCTGGA CTTTGCATGTGACTTTTGGGTCCTCGTAGTCGTCGGTGGCGTCCTGGCCTGTTATTCC CTTCTGGTTACGGTCGCCTTCATTATATTTTGGGTCAGGAGTAAACGGTCTAGGGGCG GGCACAGCGACTACATGAATATGACGCCACGCCGCCCCGGGCCCACAAGAAAACACT ACCAACCTTACGCTCCACCCAGGGATTTTGCCGCGTACCGGAGCAGGGTAAAGTTCAG CCGCAGTGCGGATGCACCAGCGTATCAACAAGGCCAGAATCAACTGTATAATGAGCTG AACTTGGGACGACGAGAGGAATATGATGTTCTGGATAAACGGCGCGGGAGAGATCCA GAGATGGGCGGAAAGCCTAGACGTAAGAACCCCCAAGAAGGCCTTTATAATGAGTTGC AGAAAGATAAGATGGCCGAAGCCTATTCTGAAATAGGCATGAAGGGAGAGCGGCGGC GTGGCAAGGGTCATGACGGTCTTTACCAGGGCCTCTCTACCGCAACTAAAGATACCTA TGATGCACTTCACATGCAAGCCCTGCCCCCTAGAGAGTTCGAAGGGTCAGCAGCAGCC GAGGGACGTGGCTCATTGCTTACTTGCGGTGACGTTGAAGAAAACCCTGGCCCCAGC GGGATGGTAAGCAAGGGAGAAGAACTTTTCACGGGAGTAGTTCCTATTCTGGTGGAAC TGGACGGGGATGTAAACGGGCATAAATTCTCTGTCAGCGGGGAAGGGGAAGGCGATG CAACATACGGGAAGCTTACCCTCAAATTCATCTGTACGACAGGGAAACTTCCAGTGCC CTGGCCTACACTTGTGACAACCCTGACTTATGGGGTCCAATGTTTCTCCAGGTACCCTG ATCACATGAAACAGCATGACTTCTTCAAGAGCGCAATGCCTGAAGGGTATGTCCAGGA ACGCACGATTTTCTTCAAGGACGATGGCAATTATAAAACTAGAGCCGAAGTAAAATTTG AGGGAGATACATTGGTAAACAGGATCGAGCTCAAAGGGATCGATTTCAAAGAGGATGG GAACATCCTGGGGCATAAGTTGGAGTATAACTATAACTCTCACAATGTCTATATTATGG CCGACAAGCAAAAGAACGGGATAAAAGTCAACTTTAAAATAAGGCACAATATAGAGGAC GGCAGTGTGCAATTGGCGGACCACTATCAACAAAATACCCCGATCGGCGACGGTCCA GTGTTGCTCCCTGATAATCACTACCTTTCAACACAAAGTGCACTTAGTAAAGATCCCAAT GAGAAGCGGGATCACATGGTCCTGCTCGAATTTGTCACAGCAGCTGGCATCACACTTG GGATGGATGAGCTCTATAAATAA SEQIDNO:58-pREF091DNAsequenceofTether-containingplasmid tggaagggctaattcactcccaaagaagacaagatatccttgatctgtggatctaccacacacaaggctacttccctgattagca gaactacacaccagggccaggggtcagatatccactgacctttggatggtgctacaagctagtaccagttgagccagataagg tagaagaggccaataaaggagagaacaccagcttgttacaccctgtgagcctgcatgggatggatgacccggagagagaag tgttagagtggaggtttgacagccgcctagcatttcatcacgtggcccgagagctgcatccggagtacttcaagaactgctgatat cgagcttgctacaagggactttccgctggggactttccagggaggcgtggcctggggggactggggagtggcgagccctcag atcctgcatataagcagctgctttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactaggg aacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatc cctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagagg agctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaa attttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatggg aaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgat tcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggat cagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaa gctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccggccgctgatcttcagacct ggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacc caccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagca gcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcaga acaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcc tggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgcctt ggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaatt acacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaa tgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggttta agaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgag gggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacgg atctcgacggtatcgccaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaa gaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttatta cagggacagcagagatccagtttggatcgataagcttgatatcgaattcctgcagccccgataaaataaaagattttatttagtctc cagaaaaaggggggaatgaaagaccccacctgtaggtttggcaagctagctgcagtaacgccattttgcaaggcatggaaaa ataccaaaccaagaatagagaagttcagatcaagggcgggtacatgaaaatagctaacgttgggccaaacaggatatctgc ggtgagcagtttcggccccggcccggggccaagaacagatggtcaccgcagtttcggccccggcccgaggccaagaacag atggtccccagatatggcccaaccctcagcagtttcttaagacccatcagatgtttccaggctcccccaaggacctgaaatgacc ctgcgccttatttgaattaaccaatcagcctgcttctcgcttctgttcgcgcgcttctgcttcccgagctctataaaagagctcacaac ccctcactcggcgcgccagtcctccgacagactgagtcgcccggggggGATCCGCCACCATGAGCATGCTGT TCTATACCCTGATCACCGCCTTTCTGATCGGCATCCAGGCCGAGCAGAAGCTGATCTC CGAGGAAGATCTGGCCTCTAGCCTGGGCGCTCACCACATCCACCACTTTCACGGCAG CAGCAAGCACCACTCTGTGCCTATCGCCATCTACAGAAGCCCCGCTTCTCTGAGAGGC GGCCATGCTGGAACCACCTACATCTTCTCTAAAGGGGGGGGACAGATCACCTACAAGT GGCCTCCAAACGACAGACCCAGCACCAGAGCCGATAGACTGGCCATCGGCTTCAGCA CCGTGCAGAAAGAAGCCGTGCTCGTCAGAGTGGACAGCAGCTCTGGACTGGGCGACT ACCTGGAACTGCACATCCATCAGGGCAAGATCGGCGTGAAGTTCAACGTGGGCACCG ACGACATTGCCATCGAGGAAAGCAACGCCATCATCAACGACGGCAAGTACCACGTCGT GCGGTTCACAAGAAGCGGCGGCAACGCTACACTGCAGGTCGACTCTTGGCCCGTGAT CGAGAGATACCCTGCCGGCAACAACGACAACGAGAGACTGGCTATCGCCAGACAGAG AATCCCCTACAGACTGGGAAGAGTGGTGGACGAGTGGCTGCTGGATAAGGGCAGACA GCTGACCATCTTCAATAGCCAGGCCACCATCATCATCGGCGGAAAAGAGCAGGGCCA GCCTTTCCAGGGACAGCTGAGCGGACTGTACTACAACGGCCTGAAGGTGCTGAACAT GGCCGCTGAGAACGACGCCAATATCGCTATCGTGGGCAACGTGCGGCTCGTGGGAGA AGTTCCTGGCGGCGGAGGATCTGGCGGAGGTGGAAGTGGCGGAGGCGGATCTACAC TGGTGCTGTTTGGCGCTGGCTTCGGCGCCGTGATTACAGTGGTGGTCATCGTCGTGAT CATCAAGTGTTTCTGCAAATGAGCggccgcgactctagagtcgacctgcaggcatgcaagcttgatatcaagct tatcgataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctg ctttaatgcctttgtatcatgctattgcttccccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgt ggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcag ctcctttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctc ggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgc gcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccaattctaccgggtaggggaggcgcttttcc caaggcagtctggagcatgcgctttagcagccccgctgggcacttggcgctacacaagtggcctctggcctcgcacacattcca catccaccggtaggcgccaaccggctccgttctttggtggccccttcgcgccaccttctactcctcccctagtcaggaagttccccc ccgccccgcagctcgcgtcgtgcaggacgtgacaaatggaagtagcacgtctcactagtctcgtgcagatggacagcaccgct gagcaatggaagcgggtaggcctttggggcagcggccaatagcagctttgctccttcgctttctgggctcagaggctgggaagg ggtgggtccggggggggctcaggggcgggctcaggggggggcgggcgcccgaaggtcctccggaggcccggcattctg cacgcttcaaaagcgcacgtctgccgcgctgttctcctcttcctcatctccgggcctttcgacctgcagcccaagcttaccatgacc gagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcaccctcgccgccgegttcgccgac taccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgcgcg tcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaa gcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaa ggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcccgaccaccagggcaag ggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgc cccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgc atgacccgcaagcccggtgcctgactcgagggaattaattcgagctcggtacctttaagaccaatgacttacaaggcagctgta gatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtact gggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgcc ttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctc tagcagcatctagaattaattccgtgtattctatagtgtcacctaaatcgtatgtgtatgatacataaggttatgtattaattgtagccgc gttctaacgacaatatgtacaagcctaattgtgtagcatctggcttactgaagcagaccctatcatctctctcgtaaactgccgtcag agtcggtttggttggacgaaccttctgagtttctggtaacgccgtcccgcacccggaaatggtcagcgaaccaatcagcagggtc atcgctagccagatcctctacgccggacgcatcgtggccggcatcaccggcgccacaggtgcggttgctggcgcctatatcgcc gacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtgggtatggtggcaggccccgtg gccgggggactgttgggcgccatctccttgcatgcaccattccttgcggcggcggtgctcaacggcctcaacctactactgggct gcttcctaatgcaggagtcgcataagggagagcgtcgaatggtgcactctcagtacaatctgctctgatgccgcatagttaagcc agccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgacc gtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctattttt ataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttct aaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtatt caacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatg ctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaaga acgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgc cgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaat tatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaacc gcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgag cgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaac aattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaat ctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacac gacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgt cagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctca tgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatccttttttt ctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactcttttt ccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaact ctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttgga ctcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaac gacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggt atccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgt cgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcg gcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgccttt gagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgccca atacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctgtggaatgtgtgtcagttagggtgtggaaagtcccca ggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtggaaagtccccaggctccccag caggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaactccgcccatcccgcccctaactc cgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgaggccgcctcggcctctgagctattc cagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagcttggacacaagacaggcttgcgagatatgtttgaga ataccactttatcccgcgtcagggagaggcagtgcgtaaaaagacgcggactcatgtgaaatactggtttttagtgcgccagatc tctataatctcgcgcaacctattttcccctcgaacactttttaagccgtagataaacaggctgggacacttcacatgagcgaaaaat acatcgtcacctgggacatgttgcagatccatgcacgtaaactcgcaagccgactgatgccttctgaacaatggaaaggcattat tgccgtaagccgtggcggtctgtaccgggtgcgttactggcgcgtgaactgggtattcgtcatgtcgataccgtttgtatttccagcta cgatcacgacaaccagcgcgagcttaaagtgctgaaacgcgcagaaggcgatggcgaaggcttcatcgttattgatgacctg gtggataccggtggtactgcggttgcgattcgtgaaatgtatccaaaagcgcactttgtcaccatcttcgcaaaaccggctggtcgt ccgctggttgatgactatgttgttgatatcccgcaagatacctggattgaacagccgtgggatatgggcgtcgtattcgtcccgcca atctccggtcgctaatcttttcaacgcctggcactgccgggcgttgttctttttaacttcaggcgggttacaatagtttccagtaagtatt ctggaggctgcatccatgacacaggcaaacctgagcgaaaccctgttcaaaccccgctttaaacatcctgaaacctcgacgct agtccgccgctttaatcacggcgcacaaccgcctgtgcagtcggcccttgatggtaaaaccatccctcactggtatcgcatgatta accgtctgatgtggatctggcgcggcattgacccacgcgaaatcctcgacgtccaggcacgtattgtgatgagcgatgccgaac gtaccgacgatgatttatacgatacggtgattggctaccgtggcggcaactggatttatgagtgggccccggatctttgtgaagga accttacttctgtggtgtgacataattggacaaactacctacagagatttaaagctctaaggtaaatataaaatttttaagtgtataat gtgttaaactactgattctaattgtttgtgtattttagattccaacctatggaactgatgaatgggagcagtggtggaatgcctttaatga ggaaaacctgttttgctcagaagaaatgccatctagtgatgatgaggctactgctgactctcaacattctactcctccaaaaaaga agagaaaggtagaagaccccaaggactttccttcagaattgctaagttttttgagtcatgctgtgtttagtaatagaactcttgcttgc tttgctatttacaccacaaaggaaaaagctgcactgctatacaagaaaattatggaaaaatattctgtaacctttataagtaggcat aacagttataatcataacatactgttttttcttactccacacaggcatagagtgtctgctattaataactatgctcaaaaattgtgtacct ttagctttttaatttgtaaaggggttaataaggaatatttgatgtatagtgccttgactagagatcataatcagccataccacatttgtag aggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatgcaattgttgttgttaacttgtttattgc agcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaa actcatcaatgtatcttatcatgtctggatcaactggataactcaagctaaccaaaatcatcccaaacttcccaccccataccctatt accactgccaattacctgtggtttcatttactctaaacctgtgattcctctgaattattttcattttaaagaaattgtatttgttaaatatgtac tacaaacttagtagt SEQIDNO:59-pREF096sequence(GFPunderFoxP3-induciblepromoter) tggaagggctaattcactcccaaagaagacaagatatccttgatctgtggatctaccacacacaaggctacttccctgattagca gaactacacaccagggccaggggtcagatatccactgacctttggatggtgctacaagctagtaccagttgagccagataagg tagaagaggccaataaaggagagaacaccagcttgttacaccctgtgagcctgcatgggatggatgacccggagagagaag tgttagagtggaggtttgacagccgcctagcatttcatcacgtggcccgagagctgcatccggagtacttcaagaactgctgatat cgagcttgctacaagggactttccgctggggactttccagggaggcgtggcctggggggactggggagtggcgagccctcag atcctgcatataagcagctgctttttgcctgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactaggg aacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatc cctcagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagagg agctctctcgacgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaa attttgactagcggaggctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatggg aaaaaattcggttaaggccagggggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgat tcgcagttaatcctggcctgttagaaacatcagaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggat cagaagaacttagatcattatataatacagtagcaaccctctattgtgtgcatcaaaggatagagataaaagacaccaaggaa gctttagacaagatagaggaagagcaaaacaaaagtaagaccaccgcacagcaagcggccggccgctgatcttcagacct ggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaaaattgaaccattaggagtagcacc caccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttccttgggttcttgggagca gcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtgcagcagcaga acaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaagaatcc tggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgcctt ggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaatt acacaagcttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaa tgggcaagtttgtggaattggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggttta agaatagtttttgctgtactttctatagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgag gggacccgacaggcccgaaggaatagaagaagaaggtggagagagagacagagacagatccattcgattagtgaacgg atctcgacggtatcgccaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaa gaatagtagacataatagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttatta cagggacagcagagatccagtttggatcgataagcttgatatcgaattcgtagctgaagaaaggattcataaatgaagttcaatc cttctcatcaaccccagcccacacctccagcaattgaacttgaaaaaaaaaacctggtttgaaaaattaccgcaaactatattgt catcaaaaaaaaaaaaaaaaaaaaacacttcctatatttgagatgagagaagagagtgctaggcagtttcctggctgaacac gccagcccaatacttaaagagagcaactcctgactccgatagagactggatggacccacaagggtgacagcccaggcgga ccgatcttcccatcccacatcctccggcgcgatgccaaaaagaggctgacggcaactgggccttctgcagagaaagacctcc gcttcactgccccggctggtcccaagggtcaggaagATGGTAAGCAAGGGAGAAGAACTTTTCACGGGA GTAGTTCCTATTCTGGTGGAACTGGACGGGGATGTAAACGGGCATAAATTCTCTGTCAG CGGGGAAGGGGAAGGCGATGCAACATACGGGAAGCTTACCCTCAAATTCATCTGTACG ACAGGGAAACTTCCAGTGCCCTGGCCTACACTTGTGACAACCCTGACTTATGGGGTCC AATGTTTCTCCAGGTACCCTGATCACATGAAACAGCATGACTTCTTCAAGAGCGCAATGC CTGAAGGGTATGTCCAGGAACGCACGATTTTCTTCAAGGACGATGGCAATTATAAAACTA GAGCCGAAGTAAAATTTGAGGGAGATACATTGGTAAACAGGATCGAGCTCAAAGGGATC GATTTCAAAGAGGATGGGAACATCCTGGGGCATAAGTTGGAGTATAACTATAACTCTCAC AATGTCTATATTATGGCCGACAAGCAAAAGAACGGGATAAAAGTCAACTTTAAAATAAGG CACAATATAGAGGACGGCAGTGTGCAATTGGCGGACCACTATCAACAAAATACCCCGAT CGGCGACGGTCCAGTGTTGCTCCCTGATAATCACTACCTTTCAACACAAAGTGCACTTA GTAAAGATCCCAATGAGAAGCGGGATCACATGGTCCTGCTCGAATTTGTCACAGCAGCT GGCATCACACTTGGGATGGATGAGCTCTATAAATAAggccgcgactctagagtcgacctgcaggcatgc aagcttgatatcaagcttatcgataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttac gctatgtggatacgctgctttaatgcctttgtatcatgctattgcttccccgtatggctttcattttctcctccttgtataaatcctggttgctgt ctctttatgaggagttgtggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcat tgccaccacctgtcagctcctttccgggactttcgctttccccctccctattgccacggeggaactcatcgccgcctgccttgcccgc tgctggacaggggctcggctgttgggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgt tgccacctggattctgcgcgggacgtccttctgctacgtcccttcggccctcaatccagcggaccttccttcccaattctaccgggta ggggaggcgcttttcccaaggcagtctggagcatgcgctttagcagccccgctgggcacttggcgctacacaagtggcctctgg cctcgcacacattccacatccaccggtaggcgccaaccggctccgttctttggtggccccttcgcgccaccttctactcctccccta gtcaggaagttcccccccgccccgcagctcgcgtcgtgcaggacgtgacaaatggaagtagcacgtctcactagtctcgtgca gatggacagcaccgctgagcaatggaagcgggtaggcctttggggcagcggccaatagcagctttgctccttcgctttctgggct cagaggctgggaaggggtgggtccggggggggctcaggggcgggctcaggggggggggggcccgaaggtcctcc ggaggcccggcattctgcacgcttcaaaagcgcacgtctgccgcgctgttctcctcttcctcatctccgggcctttcgacctgcagc ccaagcttaccatgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcaccctcg ccgccgcgttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagctgcaag aactcttcctcacgcgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccac gccggagagcgtcgaagcgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgc gcagcaacagatggaaggcctcctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggcgtctcgcc cgaccaccagggcaagggtctgggcagcgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgcct tcctggagacctccgcgccccgcaacctccccttctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaa ggaccgcgcacctggtgcatgacccgcaagcccggtgcctgactcgagggaattaattcgagctcggtacctttaagaccaat gacttacaaggcagctgtagatcttagccactttttaaaagaaaaggggggactggaagggctaattcactcccaacgaagac aagatctgctttttgcttgtactgggtctctctggttagaccagatctgagcctgggagctctctggctaactagggaacccactgctt aagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccctcagaccctt ttagtcagtgtggaaaatctctagcagcatctagaattaattccgtgtattctatagtgtcacctaaatcgtatgtgtatgatacataag gttatgtattaattgtagccgcgttctaacgacaatatgtacaagcctaattgtgtagcatctggcttactgaagcagaccctatcatc tctctcgtaaactgccgtcagagtcggtttggttggacgaaccttctgagtttctggtaacgccgtcccgcacccggaaatggtcag cgaaccaatcagcagggtcatcgctagccagatcctctacgccggacgcatcgtggccggcatcaccggcgccacaggtgc ggttgctggcgcctatatcgccgacatcaccgatggggaagatcgggctcgccacttcgggctcatgagcgcttgtttcggcgtg ggtatggtggcaggccccgtggccgggggactgttgggcgccatctccttgcatgcaccattccttgcggcggcggtgctcaacg gcctcaacctactactgggctgcttcctaatgcaggagtcgcataagggagagcgtcgaatggtgcactctcagtacaatctgct ctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatcc gcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaa gggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcg cggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattga aaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaac gctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatcct tgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccg ggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacgg atggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcg gaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatg aagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaact acttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgcteggcccttccg gctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagcc ctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctc actgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctagg tgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaa aggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccgg atcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagtt aggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgata agtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcaca cagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaa gggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaa cgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatg gaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctg tggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcga ggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctgtggaatgtgtgtca gttagggtgtggaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccaggtgtg gaaagtccccaggctccccagcaggcagaagtatgcaaagcatgcatctcaattagtcagcaaccatagtcccgcccctaact ccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggccgagg ccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctaggcttttgcaaaaagcttggacacaagac aggcttgcgagatatgtttgagaataccactttatcccgcgtcagggagaggcagtgcgtaaaaagacgcggactcatgtgaaa tactggtttttagtgcgccagatctctataatctcgcgcaacctattttcccctcgaacactttttaagccgtagataaacaggctggg acacttcacatgagcgaaaaatacatcgtcacctgggacatgttgcagatccatgcacgtaaactcgcaagccgactgatgcct tctgaacaatggaaaggcattattgccgtaagccgtggcggtctgtaccgggtgcgttactggcgcgtgaactgggtattcgtcat gtcgataccgtttgtatttccagctacgatcacgacaaccagcgcgagcttaaagtgctgaaacgcgcagaaggcgatggcga aggcttcatcgttattgatgacctggtggataccggtggtactgcggttgcgattcgtgaaatgtatccaaaagcgcactttgtcacc atcttcgcaaaaccggctggtcgtccgctggttgatgactatgttgttgatatcccgcaagatacctggattgaacagccgtgggat atgggcgtcgtattcgtcccgccaatctccggtcgctaatcttttcaacgcctggcactgccgggcgttgttctttttaacttcaggcgg gttacaatagtttccagtaagtattctggaggctgcatccatgacacaggcaaacctgagcgaaaccctgttcaaaccccgcttta aacatcctgaaacctcgacgctagtccgccgctttaatcacggcgcacaaccgcctgtgcagtcggcccttgatggtaaaacca tccctcactggtatcgcatgattaaccgtctgatgtggatctggcgcggcattgacccacgcgaaatcctcgacgtccaggcacgt attgtgatgagcgatgccgaacgtaccgacgatgatttatacgatacggtgattggctaccgtggcggcaactggatttatgagtg ggccccggatctttgtgaaggaaccttacttctgtggtgtgacataattggacaaactacctacagagatttaaagctctaaggtaa atataaaatttttaagtgtataatgtgttaaactactgattctaattgtttgtgtattttagattccaacctatggaactgatgaatgggag cagtggtggaatgcctttaatgaggaaaacctgttttgctcagaagaaatgccatctagtgatgatgaggctactgctgactctcaa cattctactcctccaaaaaagaagagaaaggtagaagaccccaaggactttccttcagaattgctaagttttttgagtcatgctgtg tttagtaatagaactcttgcttgctttgctatttacaccacaaaggaaaaagctgcactgctatacaagaaaattatggaaaaatatt ctgtaacctttataagtaggcataacagttataatcataacatactgttttttcttactccacacaggcatagagtgtctgctattaataa ctatgctcaaaaattgtgtacctttagctttttaatttgtaaaggggttaataaggaatatttgatgtatagtgccttgactagagatcat aatcagccataccacatttgtagaggttttacttgctttaaaaaacctcccacacctccccctgaacctgaaacataaaatgaatg caattgttgttgttaacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttca ctgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggatcaactggataactcaagctaaccaaaatcatccc aaacttcccaccccataccctattaccactgccaattacctgtggtttcatttactctaaacctgtgattcctctgaattattttcattttaa agaaattgtatttgttaaatatgtactacaaacttagtagt SEQIDNO:60-hypervariableregionoflightchainvariableregionofhumanizedM1- HM1HVR-L1 RASKSINKYLA SEQIDNO:61-hypervariableregionoflightchainvariableregionofhumanizedM1- HM1HVR-L2 SGSTLQS SEQIDNO:62-hypervariableregionoflightchainvariableregionofhumanizedM1- HM1HVR-L3 QQHNEYPLT SEQIDNO:63-hypervariableregionofheavychainvariableregionofhumanizedM1- HM1HVR-H1 GYHFTSYWMH SEQIDNO:64-hypervariableregionofheavychainvariableregionofhumanizedM1- HM1HVR-H2 VIHPNSGSINYNEKFES SEQIDNO:65-hypervariableregionofheavychainvariableregionofhumanizedM1- HM1HVR-H3 ERDSTEVLPMDY