Promoter Sequences for In Vitro and In Vivo Expression of Gene Therapy Products in CD3+ Cells

Abstract

Promoter sequences for use in expressing a transgene in CD3+ cells are provided. The promoter sequences can be inserted into a vector in a 5′ untranslated region proximal to a transgene. The promoters are selective for expression in CD3+ cells and contain binding sites for transcription factors found in CD3+ cells. The promoters can be integrated into vectors, including polymer-encapsulated lentiviral vector nanoparticles, used to transduce T-cells for genetic immunotherapy to treat cancer and infectious diseases. The T-cell selectivity of the promoters adds an improved safety factor to the use of viral vectors for immunotherapy in vitro and in vivo.

Claims

1. A promoter sequence for use in expression of a transgene under control of the promoter sequence in a CD3+ cell, the promoter sequence comprising nucleotides 1501-2000 of any of SEQ ID NOS:2-10 or 12-16, or a variant thereof having at least 90% identity to said sequence.

2. The promoter sequence of claim 1, wherein the promoter sequence comprises nucleotides 1001-2000 of any one of SEQ ID NOS:2-10 or 12-16 or a variant thereof having at least 90% identity to said sequence.

3. The promoter sequence of claim 2, wherein the promoter sequence comprises nucleotides 501-2000 of any one of SEQ ID NOS:2-10 or 12-16 or a variant thereof having at least 90% identity to said sequence.

4. The promoter sequence of claim 3, wherein the promoter sequence comprises the nucleotide sequence of any one of SEQ ID NOS:2-16 or a variant thereof having at least 90% identity to said sequence.

5. The promoter sequence of any of claims 1-4, wherein the promoter sequence comprises a binding sequence for one or more transcription factors selected from the group consisting of NF-kappaB, AP-1, STAT, GATA-3, and NFAT.

6. The promoter sequence of any of claims 1-5, wherein the promoter is capable of expressing the transgene at a higher level in CD3+ cells compared to CD3− cells.

7. The promoter sequence of claim 6, wherein the ratio of expression in CD3+ cells to CD3− cells is at least 2:1.

8. A promoter sequence for use in expression of a transgene under control of the promoter sequence in a CD3+ cell, the promoter sequence comprising SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:11, or SEQ ID NO:13.

9. A vector, plasmid, or nucleic acid molecule comprising the promoter sequence of any of claims 1-8.

10. The vector of claim 9 which is a viral vector.

11. The viral vector of claim 10 which is a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus, or a herpes simplex virus.

12. The viral vector of any of claims 9-11 which is incorporated into a nanoparticle.

13. The viral vector of any of claims 9-11 which is not incorporated into a nanoparticle.

14. The viral vector of any of claims 9-11 whose envelope lacks a fusion protein.

15. The vector of any of claims 9-14, wherein the vector comprises a transgene encoding a product selected from the group consisting of chimeric antigen receptors (CARs), checkpoint inhibitors, cytokines, chemokines, antibodies and antigen binding fragments and variants thereof, enzymes, structural proteins, and reporter genes.

16. The nucleic acid molecule of claim 9 which is an RNA molecule.

17. A cell comprising the vector, plasmid, or nucleic acid molecule of any of claims 9-16.

18. The cell of claim 17, wherein the cell comprises a genome-integrated viral vector.

19. The cell of claim 17, wherein the cell comprises an episomal form of the vector.

20. A nanoparticle comprising the vector of any of claims 9-15, wherein the nanoparticle is capable of delivery of the vector into a CD3+ cell.

21. The nanoparticle of claim 20, wherein the nanoparticle comprises a targeting moiety that promotes selective entry of the nanoparticle into CD3+ cells.

22. The nanoparticle of claim 20 or 21, wherein the nanoparticle is also capable of delivery of the vector into a CD3− cell.

23. The nanoparticle of any of claims 20-22, wherein the nanoparticle comprises a polymer.

24. The nanoparticle of claim 23, wherein the polymer is a poly(beta-amino ester).

25. A method of expressing a transgene in a CD3+ cell, the method comprising the steps of: (a) providing the vector of any of claims 9-15, or the nanoparticle of any of claims 20-24, and a CD3+ cell, wherein the vector comprises said transgene; (b) transducing or transfecting the cell with the vector; and (c) allowing the transgene to be expressed in the transduced or transfected cell.

26. The method of claim 25, wherein the vector is a lentiviral vector.

27. The method of claim 25, wherein the CD3+ cell is CD4+, CD4−, CD8+, or CD8−.

28. The method of claim 25, wherein step (b) comprises contacting the vector with a mixture of CD3+ and CD3− cells, and wherein the CD3+ cells are selectively transduced.

29. The method of any of claims 25-28, wherein step (b) is performed in vitro.

30. The method of any of claims 25-28, wherein step (b) is performed in vivo and comprises administration of the vector by intravenous, intratumoral, intramedullary, or intraperitoneal injection.

31. A method of making the vector of any one of claims 9-15, the method comprising adding the promoter sequence of any of claims 1-8 to a vector for use in transducing a CD3+ cell.

32. The method of claim 31, wherein said promoter sequence does not support expression of the transgene in packaging cells or producer cells used to make the vector.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0009] FIGS. 1A, 1B and 1C show in vitro results obtained with lentiviral vectors (LV) encoding green fluorescent protein (GFP) under the control of different human T-cell specific promoters. The transduction efficiency is given for each tested promoter at 72 h post-transduction of HEK293T cells (FIG. 1A), Ramos cells (FIG. 1B), and Jurkat cells (FIG. 1C) analyzed by flow cytometry. Control cells were not transduced (NT) but were kept in culture throughout the experiment and a positive control was obtained by cells transduced with LV carrying the ubiquitous CMV promoter to drive the expression of GFP. For each promoter, 3 different Multiple of Infection (MOI) were evaluated.

[0010] FIGS. 2A, 2B and 2C show in vitro results obtained after the transduction of human PBMCs with lentiviral vectors (LV) encoding GFP under the control of different human T-cell specific promoters. The transduction efficiency was measured for each tested promoter at 72 h post-transduction by flow cytometry either with total CD45+ cells (FIG. 2A), CD19+ cells (FIG. 2B) or CD3+ cells (FIG. 2C). Control cells (NT) were not transduced, but were activated and kept in culture throughout the experiment.

[0011] FIG. 3A shows the expression level of CD19 CAR on LV293 producing cells 72 h post-transfection with the packaging plasmid (pARA-pack) and the proviral plasmid (pARA-hUBC-CAR-CD19). CD19 CAR expression was measured by flow cytometry with CAR CD19 detection reagent and anti-biotin secondary antibody (α-biotin). Control cells were not transfected with the 2 plasmids and background staining was assessed with the anti-biotin antibody alone.

[0012] FIG. 3B shows the expression level of different transgenes (GFP and CD19 CAR) after transduction of PBMCs with PBAE encapsulated VSV-G− (“Bald”) LV or non-encapsulated VSV-G− (“Bald”) LV encoding for GFP or CD19 CAR under the control of ubiquitous promoters CMV and hUBC respectively.

DETAILED DESCRIPTION

[0013] The present technology provides promoter sequences for use in expressing any desired transgene in CD3+ cells. Each of the promoter sequences can be inserted into a vector for expression of a transgene just upstream of the transgene sequence, in the 5′ untranslated region most proximal to the transgene. The promoters are specific for expression in CD3+ cells, which adds an increased level of safety and specificity when using the transgene for gene transfer or immunotherapy, especially for in vivo therapy in which the vector is introduced into a patient.

[0014] One aspect of the present technology is a promoter sequence for use in expression of a transgene in a CD3+ cell. The promoter sequence includes at least nucleotides 1501-2000 of any of SEQ ID NOS:2-10 or 12-16 or a variant thereof. Alternatively, the promoter sequence can include nucleotides 1001-2000 of any one of SEQ ID NOS:2-10 or 12-16, nucleotides 501-2000 of any one of SEQ ID NOS:2-10 or 12-16, or nucleotides 1-2000 of any one of SEQ ID NOS:2-10 or 12-16, or a sequence variant of any of these. Further promoter sequences can be derived from any of SEQ ID NOS:2-16, or a variant thereof, by including one or more blocks of nucleotides starting from the 3′ end of the sequence and extending in a 5′ direction; such blocks of nucleotides can contain 200, 300, 400, 500, 600, 700, or more nucleotides, and extending up to a maximum of the first nucleotide of the sequence at the 5′ end. See, for example, SEQ ID NOS:19-29, described in Example 8. Sequence variants as described above can have at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, at least 98%, or at least 99% identity to the stated sequence or its complement.

[0015] Another aspect of a promoter sequence of the present technology is that it can contain a binding sequence for one or more transcription factors selected from the group consisting of NF-kappaB, AP-1, STAT, GATA-3, and NFAT, or another transcription factor. The transcription factor binding sites can occur in any combination, in any position, and in any order within the promoter sequence. An online tool for identifying transcription factor binding sites in promoter sequences or putative promoter sequences is available at http://alggen.lsi.upc.es/cgi-bin/promo_v3/promo/promoinit.cgi?dirDB=TF_8.3.

[0016] Yet another aspect of a promoter sequence of the present technology is that it is capable of selectively promoting the in vitro or in vivo expression of a transgene in CD3+ cells compared to CD3− cells. For example, the ratio of expression in CD3+ cells to CD3− cells can be at least 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, 20:1, or even 50:1 or 100:1, or higher. The promoter sequence can promote the expression of a transgene in CD3+ cells that are either CD4+ or CD4−, in CD3+ cells that are CD8+ or CD8− or in CD3+ cells that are CD4+ CD8+ or CD4− CD8−.

[0017] Still another aspect of the technology is a vector containing any of the above-described promoter sequences. The vector can be, for example, a viral vector, such as a lentiviral vector (LV). The vector can also be a “bald” LV, or other viral vector, which lacks a virus fusion protein on the surface of the vector particles, such as described in WO 2019/145796A2, which is incorporated by reference in its entirety herein. Other viral vectors such as retrovirus, adenovirus, adeno-associated virus (AAV), and herpes simplex virus used as gene-delivery vehicles can incorporate the above-described promoter sequences. The vector can be present in a cell, including cells used for immunotherapy or used to produce a recombinant protein. A promoter of the present technology also can be packaged into a plasmid or nucleic acid molecule, such as an RNA molecule, including an mRNA molecule, together with one or more transgenes under its control, for delivery to cells. The vector, plasmid, or nucleic acid molecule can be packaged in a nanoparticle, such as a polymer-containing nanoparticle, including nanoparticles comprising or consisting of polymer-encapsulated vector. Polymers of such nanoparticles can be poly(beta-amino ester)s (PBAEs), including PBAE polymers containing oligopeptide end caps. The vector contains a transgene for expression in a target cell. The transgene can be any gene, including genes intended for immunotherapy involving enhancing the activity of a T-cell or a sub-population of T-cells, including CD4+ cells, CD8+ cells, NK cells, Th cells, or Treg cells.

[0018] While the promoters of the present technology can be used to promote the expression of any transgene, the transgene can encode, for example, an anti-checkpoint protein or polypeptide, such as an inhibitor of CTLA-4, PD1, PDL1, LAG-3, TIM 3, B7-H3, ICOS, IDO, 4-1BB, or CD47. The transgene alternatively can encode a chimeric antigen receptor (CAR) having binding specificity for any desired antigen, such as a tumor antigen or an antigen on the surface of a pathogen such as a bacterium, virus, yeast, or parasite. The CAR can be a universal CAR, which binds to an adapter molecule having a domain for CAR binding as well as an antigen binding domain suitable for binding to an antigen such as a tumor antigen or an antigen on the surface of a pathogen such as a bacterium, virus, yeast, or parasite. The transgene can be a protein or a combination of proteins able to elicit an immune response and act as a vaccine. The elicited immune response can be prophylactic or therapeutic, and can stimulate an immune response against bacteria, viruses, other microbial pathogens, or cancer cells, or another undesired cell type found in the body of a subject.

[0019] Another aspect of the present technology is a method of expressing a transgene in a CD3+ cell. The method includes the steps of: (a) providing a vector or nanoparticle as described above and a CD3+ cell; (b) transducing the cell, either in vitro or in vivo, with the vector or nanoparticle; and (c) allowing the transgene to be expressed in the transduced cell. The vector or nanoparticles contains the transgene and a promoter as described above in the 5′ untranslated region proximal to the gene.

[0020] Even another aspect of the present technology is a method of making the vector described above. The method includes adding any of the above-described promoter sequences to a vector for use in transducing a CD3+ cell.

EXAMPLES

Example 1. Production of Lentiviral Vectors Containing Transgenes Under Control of T-Cell Promoters

[0021] Different batches of lentiviral vectors (LVs) were prepared and tested in vitro to investigate transgene expression. The LVs were made using the following materials and methods.

[0022] Selection of T-Cell Promoters

[0023] T lymphocytes represent a diverse population of CD3-positive immune cells, the main types being cytotoxic T-cells (CD8.sup.+, Tc cells), helper T-cells (CD4.sup.+, Th cells) and regulatory T-cells (Tregs). The hematopoietic lineage expressing the CD3 receptor also includes immature populations at various stages of differentiation (CD4.sup.+/CD8.sup.−, CD4.sup.−/CD8.sup.+, CD4+/CD8+) and innate lymphoid cells from which originate natural killer cells (NK cells).

[0024] Promoters for specific expression in CD3-positive cells were designed using orthogonal methods. A set of 15 promoters was selected for overexpression in CD3+ cells (SEQ ID NOS:2-16); these promoters are listed in Table 1. Among this group, 3 promoters (SEQ ID NOS:8, 12, and 15) have been described in the literature as partially repressed in Treg and B cells.

[0025] Molecular Cloning of T-Cell Promoters

[0026] The promoters were ordered as synthetic genes flanked by Mlul and BamHI restriction sites and subcloned into the transfer vector plasmid pARA-CMV-GFP upstream of the open reading frame (ORF) encoding Green Fluorescent Protein (GFP). CMV promoter (SEQ ID NO:1) was chosen as a control as it drives expression of transgenes at a high level in a ubiquitous manner. For SEQ ID NOS:3, 4, and 5, Mlul and BglII sites were chosen as flanking sites due to the presence of BamHI sites within the promoter regions.

[0027] The same strategy was followed to subclone the promoters with a T cell specific activity demonstrated in cell assays in a second transfer vector plasmid pARA-hUBC-CAR-CD19 upstream of the ORF encoding a chimeric antigen receptor (CAR) specific for the human CD19 antigen.

TABLE-US-00001 TABLE 1 Summary of 15 human promoters selected for expression in CD3-positive cells. SEQ ID NO:, gene function and predicted expression pattern in the CD3.sup.+ lymphoid lineage are given. Unknown: data not available in browsed databases. Predicted expression pattern within CD3.sup.+ lymphoid lineage SEQ Innate ID lymphoid NO: Promoter Gene function CD4.sup.+ CD8.sup.+ Treg CD4.sup.+/CD8.sup.− CD4.sup.−/CD8.sup.+ CD4.sup.+/CD8.sup.+ cells 2 LAIR2.sup.1 Leucocyte-associated + + + Unknown Unknown Unknown + Ig-like receptor 2 3 TNFSF8 Tumor necrosis factor + + + + + + − ligand superfamily member 8 4 UBASH3A Ubiquitin- associated + + + + + + + and SH3 domain containing A 5 ZAP70 Zeta chain- associated + + + + + + + protein kinase 70 6 BCL11B Zinc finger protein Unknown Unknown Unknown Unknown Unknown Unknown Unknown 7 CTLA4 cytotoxic T- + − + + − − − lymphocyte-associated protein 4 8 TCF T-cell specific + + − + + + − transcription factor, HMG-box 9 TIGIT T cell immunoreceptor + + + − − − + with Ig and ITIM 10 GIMAP7 GTPase, IMAP family + + + + + − + member 7 11 TCR.sup.2 T cell receptor Vβ 8.1 Unknown Unknown Unknown Unknown Unknown Unknown Unknown 12 EOME.sup.3 Eomesodermin + + − − − − + 13 ICOS Inducible T-cell co- + + + + + − + stimulator 14 LCK Lymphocyte specific Unknown Unknown Unknown Unknown Unknown Unknown Unknown protein tyrosine kinase 15 ITK.sup.4 IL-2 inducible T-cell + + − + + + + kinase 16 IL2RA IL2 receptor alpha − − + − − − −

[0028] Materials

[0029] The transfer vector plasmids were pARA-XXX-GFP and pARA-XXX-CAR-CD19. The kanamycin-resistant plasmids coding for the provirus (a non-pathogenic and non-replicative recombinant proviral DNA derived from HIV-1, strain NL4-3), in which expression cassettes encoding GFP or CAR CD19 were cloned. The insert contained the transgene, the promoter for transgene expression and sequences added to increase the transgene expression and to allow the lentiviral vector to transduce all cell types including non-mitotic ones. The promoters were the human T-cell promoters or the CMV promoter devoid of any enhancer sequence. The non-coding sequences and expression signals corresponded to Long Terminal Repeat sequences (LTR) with the whole cis-active elements for the 5′LTR (U3-R-U5) and the deleted one for the 3′LTR, hence lacking the promoter region (ΔU3-R-U5). For the transcription and integration experiments, encapsidation sequences (SD and 5′Gag), the central PolyPurine Tract/Central Termination Site for the nuclear translocation of the vectors, and the BGH polyadenylation site were added.

[0030] The packaging plasmid was pARA-Pack. The kanamycin resistant plasmid encoded for the structural lentiviral proteins (GAG, POL, TAT and REV) used in trans for the encapsidation of the lentiviral provirus. The coding sequences corresponded to a polycistronic gene gag-pol-tat-rev, coding for the structural (Matrix MA, Capsid CA and Nucleocapside NC), enzymatic (Protease PR, Integrase IN and Reverse Transcriptase RT) and regulatory (TAT and REV) proteins. The non-coding sequences and expression signals corresponded to a minimal promoter from CMV for transcription initiation, a polyadenylation signal from the insulin gene for transcription termination, and an HIV-1 Rev Responsive Element (RRE) participating for the nuclear export of the packaging RNA.

[0031] The envelope plasmid, when used, was pENV1. This kanamycin-resistant plasmid encoded glycoprotein G from the Vesicular Stomatitis Virus (VSV-G) Indiana strain, used for the pseudotyping of some of the lentiviral vectors. The VSV-G genes were codon optimized for expression in human cells, and the gene was cloned into pVAX1 plasmid (Invitrogen). The coding sequences corresponded to codon-optimized VSV-G gene, and the noncoding sequences and expression signals corresponded to a minimal promoter from CMV for transcription initiation, and the BGH polyadenylation site to stabilize the RNA.

[0032] Production of VSV-G.sup.− (“Bald”) Lentiviral Vector Particles

[0033] LV293 cells were seeded at 5×10.sup.5 cells/mL in 2×3000 mL Erlenmeyer flasks (Corning) in 1000 mL of LV-Max Production Medium (Gibco Invitrogen). The two Erlenmeyers were incubated at 37° C., 65 rpm under humidified 8% CO.sub.2. The day after seeding, the transient transfection was performed. PEIPro transfectant reagent (PolyPlus Transfection, Illkirch, France) was mixed with transfer vector plasmids (pARA-CMV-GFP or pARA-XXX-GFP or pARA-XXX-CAR-CD19) and packaging plasmid (pARA-Pack). After incubation at room temperature, the mix PEIPro/Plasmid was added dropwise to the cell line and incubated at 37° C., 65 rpm under humidified 8% CO.sub.2. At day 3, the lentivector production was stimulated by sodium butyrate at 5 mM final concentration. The bulk mixture was incubated at 37° C., 65 rpm under humidified 8% CO.sub.2 for 24 hours. After clarification by deep filtration at 5 and 0.5 μm (Pall Corporation), the clarified bulk mixture was incubated 1 hour at room temperature for DNase treatment.

[0034] Lentivector purification was performed by chromatography on a Q mustang membrane (Pall Corporation) and eluted by NaCl gradient. Tangential flow filtration was performed on a 100 kDa HYDROSORT membrane (Sartorius), which allowed to reduce the volume and to formulate in specific buffer at pH 7, ensuring at least 2 years of stability. After sterile filtration at 0.22 μm (Millipore), the bulk drug product was filled in 2 mL glass vials with aliquots less than 1 ml, then labelled, frozen and stored at <−70° C.

[0035] The bald LV number was evaluated by physical titer quantification with two methods: p24 ELISA and qRT-PCR. The p24 ELISA was performed by detection and quantitation of the lentivirus associated HIV-1 p24 core protein only (Cell Biolabs Inc.). A pre-treatment of the samples allows to distinguish the free p24 from destroyed Lentivectors. The qRT-PCR was performed by purification of lentiviral RNA with Nucleospin RNA virus kit (Macherey Nagel) and quantitation with Lenti-X qRT-PCR titration kit (Takara). For each LV batch, physical titer, particle size and size distribution were measured by Nanoparticle Tracking Analyzer and Dynamic Light Scattering (Viewsizer 3000 and NanoPartica SZ-100V2 instruments respectively, Horiba Instruments Inc., USA) Analyses were performed at room temperature after dilution of LV in formulation buffer (10-fold for DLS and 300-fold for NTA) but without filtration not to impact biophysical properties of the samples. The results were determined using the control Horiba softwares of the equipments.

[0036] Production of VSV-G.sup.+ (“Pseudotyped”) Lentiviral Vector Particles

[0037] The same above-described method was used except that PEIPro transfectant reagent (PolyPlus, 115-010) was mixed with transfer vector plasmid (pARA-CMV-GFP or pARA-XXX-GFP or pAra-XXX-CAR-CD19), packaging plasmid (pARA-Pack) and the envelope plasmid (pENV1). Titration for the pseudotyped Lentivectors was performed by quantitative PCR three days post-transduction of HEK293T cells (8×10.sup.5 cells/well) with clarified bulk obtained during the bioproduction.

[0038] As shown in Table 2, production yields were generally as effective as those obtained with the CMV promoter despite the fact that these promoter regions were significantly longer. For all constructs, titers were at least in the 10.sup.7 TU/mL range and were consistent among different production runs. The lowest titers were obtained with promoters based on SEQ ID 4 and 8. Yields of production are a criteria for the selection of the promoters, as a low yield implies future difficulties for industrialization. In any case, no issue that would impact industrial bioproduction was observed.

TABLE-US-00002 TABLE 2 Summary of production yields obtained for pseudotyped LV coding for GFP under the control of 14 human promoters identified by a bioinformatics approach to be expressed in CD3-positive cells. Size of promoter Gene of Infectious titer SEQ ID Promoter region (bp) interest qPCR (TU/mL) 1 CMV 776 GFP 2.38 × 10.sup.7 2 LAIR2 2000 GFP 2.61 × 10.sup.7 3 TNFSF8 2000 GFP 2.63 × 10.sup.7 4 UBASH3A 2000 GFP 1.05 × 10.sup.7 5 ZAP70 2000 GFP 3.93 × 10.sup.7 6 BCL11B 2000 GFP 2.99 × 10.sup.7 7 CTLA4 2000 GFP 1.73 × 10.sup.7 8 TCF 2000 GFP 1.18 × 10.sup.7 9 TIGIT 2000 GFP 4.44 × 10.sup.7 10 GIMAP7 2000 GFP 4.49 × 10.sup.7 11 TCR 781 GFP 3.54 × 10.sup.7 12 EOME 2000 GFP 1.56 × 10.sup.7 13 ICOS 2000 GFP 2.16 × 10.sup.7 14 LCK 2000 GFP 6.00 × 10.sup.7 15 ITK 2000 GFP 1.72 × 10.sup.7

Example 2. Transduction of HEK293T, Ramos, and Jurkat Cells by Lentiviral Vectors Containing Transgenes Under Control of T-Cell Promoters

[0039] In order to investigate the CD3-specific activity of the 15 promoters identified by bioinformatic tools, we used pseudotyped Lentiviral Vectors carrying a green fluorescent protein (GFP) transgene under the control of a given promoter to transduce different human cell types and analyze GFP expression. These studies were performed on CD3-positive Jurkat (Acute T Cell Leukemia Human Cell Line—ATCC TIB-152), CD3-negative Ramos (Burkitt's Lymphoma Human Cell Line—ATCC CRL-1596) and non-lymphocyte HEK293T (Human Embryonic Kidney Cell Line—ATCC CRL-1573).

[0040] HEKT293 cells were seeded in 24-well plates at a density of 8×10.sup.4 cells per well in DMEM medium (Gibco Invitrogen) supplemented with 10% FBS (Gibco Invitrogen), 1 penicillin/streptomycin and incubated for 4 h to adhere. Cells were then transduced by replacing the medium with 300 μL of Lentiviral Vector (at a MOI of 1, 5 or 10) in culture medium or culture medium (NT controls), followed by incubation at 37° C., 5% CO.sub.2 for 2 h. After adsorption, 1 mL of complete medium was added to each well. At 72 h post transduction, the cells were trypsinized and resuspended in 200 μL of Cellfix 1×, and the percentage of cells expressing GFP was determined with an Attune N×T flow cytometer (ThermoFisher) using the BL1 channel.

[0041] Jurkat and Ramos cells were seeded in 24-well plates at a density of 8×10.sup.4 cells per well in RPMI-1640 medium (Gibco Invitrogen) supplemented with 10% FBS (Gibco Invitrogen), 1% penicillin/streptomycin. Cells were then transduced by replacing the medium with 300 μL of Lentiviral Vector (at a Multiple of Infection (MOI) of 10, 30 or 50) in culture medium or culture medium alone (NT controls). After 2 h incubation at 37° C., 5% CO.sub.2, 500 μL of fresh complete medium was added to each well. The percentage of cells expressing GFP transgene was determined 72 h post-transduction with an Attune N×T flow cytometer using the BL1 channel.

[0042] The percentages of viable and GFP-positive cells were determined by gating on debris excluded/viable/single cells. Graphed data represent means of triplicates of a representative experiment.

[0043] The results presented in FIG. 1A (HEK293T cells), 1B (Ramos CD3− cells) and 1C (Jurkat CD3+ cells) show that, for all cell types, differences in expression patterns were observed between the promoters. Although lower than levels observed with ubiquitous CMV promoter, significant GFP expression (above 10%) was measured in human CD3-negative lymphocytes (Ramos cells) and non-lymphocyte cells (HEK293T) at the 3 tested MOI with promoters ZAP70 (SEQ ID NO: 5), BCL11B (SEQ ID NO: 6), TCF7 (SEQ ID NO: 8), TIGIT (SEQ ID NO: 9), GIMAP7 (SEQ ID NO: 10) and EOMES (SEQ ID NO: 12) ruling out any specificity for CD3-positive cells.

[0044] Promoters TNFS8 (SEQ ID NO: 3), UBASH3 (SEQ ID NO: 4), CTLA4 (SEQ ID NO: 7), ICOS (SEQ ID NO: 13) and LCK (SEQ ID NO: 14) drove the highest GFP expression (above 40% at MOI 10) in CD3-positive Jurkat cells at levels comparable with CMV promoter. However, background GFP expression (5-10% GFP-positive cells) was detected in CD3-negative cells with these promoters. A dose-dependent and strictly restricted to CD3-positive Jurkat cells GFP expression was achieved with promoters LAIR2 (SEQ ID NO: 2), TCR (SEQ ID NO: 11) and ITK (SEQ ID NO: 15). The CD3-specific activity of these last three promoters resulted in weaker GFP expression with maximal values of 45% reached at MOI of 50.

[0045] Of note, none of the tested promoters had an impact on viability of transduced cells (data not shown).

Example 3. Transduction of Human PBMCs Using Lentiviral Vectors Containing Transgenes Under Control of T-Cell Promoters

[0046] Purification of Human PBMCs

[0047] Peripheral Blood Mononuclear Cells (PBMCs) were isolated from buffy coats obtained from healthy donors (Etablissement Francais du Sang, Division Rhones-Alpes). After diluting the blood with DPBS, the PBMCs were separated over a FICOLL density gradient (GE Healthcare) and washed twice with DPBS. Then, residual Red Blood Cells were lysed during a 5 min incubation of PBMCs in ACK lysis buffer (Gibco) and an additional DPBS washing. PBMCs were frozen at a density of 20×10.sup.6 cells/mL in 10% DMSO (Sigma), 90% FBS (Gibco) and stored at −150° C. until use.

[0048] Activation and Transduction of PBMCs with VSV-G.sup.+ (“Pseudotyped”) Lentiviral Vector

[0049] The CD3-specific activity of the six promoters screened in Example 2 was evaluated in human quiescent cells with LV carrying a GFP transgene under the control of a given promoter to transduce previously frozen human PBMCs. Thawed human PBMCs were seeded in 24-well plates at a density of 1×10.sup.6 cells per well in RPMI medium containing 10% FBS (Gibco) and 1% penicillin/streptomycin (Gibco), activated in presence of CD3-CD28 Dynabeads (Gibco) and incubated for 72 h at 37° C. and 5% CO.sub.2. Activated PMBCs were then pooled and transduced with encapsulated pseudotyped LV (at a MOI of 20, 50 or 100) in culture medium or culture medium alone (NT controls) in 24-well plates at a density of 1×10.sup.5 cells. After 2 h incubation at 37° C., 5% CO.sub.2, 500 μL of fresh culture medium (Gibco Invitrogen) was added to each well and incubated for 72h. The percentage of cells expressing GFP was determined 72 h post-transduction with an Attune N×T flow cytometer. The phenotype of transduced cells expressing GFP transgene was determined by flow cytometry staining with antibodies specific for the following cell types following manufacturer's instructions (Biolegend): CD3-AF700, CD14-PE-Cy7, CD16-BV711, CD19-BV605, CD45-BV510, CD56-BV421 and Zombie NIR for live/dead discrimination. After 30 min incubation at 4° C., cells were centrifuged at 500×g for 2 min and fixed with CelIFix solution (BD Biosciences). Fluorescence-positive cells were counted by flow cytometry (AttuneNXT; Invitrogen, Inc.) on BL1 (GFP), RL2 (AF700 dye), RL3 (Zombie NIR), VL1 (BV421 dye), VL2 (BV510 dye), VL3 (BV605 dye), VL4 (BV711 dye) and YL4 (PE-Cy7 dye) channels. Cell phenotypes were defined for CD45+, viable and single cells as follows: T lymphocytes (CD3.sup.pos-CD19.sup.neg), B lymphocytes (CD3.sup.neg-CD19.sup.pos), NK cells (CD3.sup.neg-CD19.sup.neg-CD56.sup.pos), monocytes (CD14.sup.pos) and granulocytes (SSC.sup.high-CD16.sup.pos).

[0050] The results presented in FIG. 2A (whole CD45+ PBMCs), 2B (CD19+ cells among PBMCs) and 2C (CD3+ cells among PBMCs) show the differences in the GFP expression patterns obtained with the tested promoters. The highest GFP expression (above 40%) was measured on total CD45+ PBMCs and among gated CD3+ T cells at the 2 tested MOI with promoters CTLA4 (SEQ ID NO: 7) and ICOS (SEQ ID NO: 13). However, GFP expression was weaker in CD3+ primary cells than on CD3+ cell lines for LAIR2 (SEQ ID NO: 2), LCK (SEQ ID NO: 14), TCR (SEQ ID NO: 11) and TNFS8 (SEQ ID NO: 3) (below 20%). In addition, only LCK (SEQ ID NO: 14) at MOI 100 was shown to induce GFP expression in CD19+ cells at the same level as for CMV. The other selected promoters resulted in weaker GFP expression than CMV, which confirms their CD3+ selectivity of both cell lines and primary samples. Finally, none of the tested promoters had an impact on viability of transduced primary cells (data not shown).

Example 4. Transduction of Primary Lymphocytes and PBMCs Using PBAE-Encapsulated VSV-G− (“Bald”) LV

[0051] The CD3-specific activity of the promoters screened in Examples 2 and 3 is evaluated in human quiescent cells with PBAE-encapsulated bald LV carrying a GFP transgene under the control of a given promoter to transduce human PBMCs. PBMCs are isolated from buffy coats obtained from healthy donors (Etablissement Francais du Sang, Division Rhones-Alpes) or from blood samples from lymphoma patients as purified and frozen cells available at Lonza and CALYM Network (Centre Hospitalier Lyon-Sud, France) Biobanks.

[0052] Purification of Human PBMCs

[0053] After diluting the fresh blood with DPBS, the PBMCs are separated over a FICOLL density gradient (GE Healthcare) and washed twice with DPBS. Then, residual red blood cells are lysed during a 5 min incubation of PBMCs in ACK lysis buffer (Gibco) and an additional DPBS washing. PBMCs are frozen at density of 20×10.sup.6 cells/mL in 10% DMSO (Sigma), 90% FBS (Gibco) and stored at −150° C. until use.

[0054] Transduction of PBAE-Encapsulation of VSV-G− (“Bald”) Lentiviral Vector

[0055] Because non-dividing cells are generally difficult to transduce with LV (without cytokine and CD3-CD28 activation), oligopeptide-modified poly(beta-amino ester) (OM-PBAE) polymers are used as transfection agents instead. OM-PBAEs have already been described as transfection agents that form polymer-encapsulated vehicles able to deliver genetic material (plasmids or other nucleic acid molecules) to eukaryotic cells (US2016/0145348A1, Mangraviti et al. 2015, Anderson et al. 2004, WO2016/116887). OM-PBAEs have been successfully used to coat transduction-deficient lentiviral vectors and engineer human cells to stably express various transgenes including reporter genes (Green Fluorescent Protein -GFP and mCherry) and CARs (see WO2019/145796). The polymers used in the following encapsulation experiments are poly(beta-amino esters) (PBAEs) conjugated to charged peptides. Polymer PBAE-CR3 refers to PBAE conjugated to the peptide CRRR (SEQ ID NO:17 (same peptide at both ends). PBAE-CH3 polymer refers to PBAE conjugated to the peptide CHHH (SEQ ID NO:18). Mixtures of these OM-PBAEs are tested at a 60/40 molar ratio.

[0056] Human PBMCs are seeded in 24-well plates at a density of 1×10.sup.5 cells per well in RPMI medium containing 10% FBS and 1% penicillin/streptomycin. Cells are then transduced by replacing the medium with 100 to 300 μL of encapsulated pseudotyped LV in culture medium or culture medium alone (NT controls). After 2 h incubation at 37° C., 5% CO.sub.2, 600 μL of fresh complete medium are added to each well. The percentage of cells expressing GFP is determined 72 h post-transduction with an Attune N×T flow cytometer using the BL1 channel. The phenotype of transduced cells expressing GFP transgene is determined by flow cytometry staining with antibodies specific for the following cell types following manufacturer's instructions (Biolegend): CD3-AF700, CD14-PE-Cy7, CD16-BV711, CD19-BV605, CD45-BV510, CD56-BV421 and Zombie NIR for live/dead discrimination. After 30 min incubation at 4° C., cells are centrifuged at 500×g for 2 min and fixed with CelIFix solution (BD Biosciences). Fluorescence-positive cells are counted by flow cytometry (AttuneNXT; Invitrogen, Inc.) on BL1 (GFP), RL2 (AF700 dye), RL3 (Zombie NIR), VL1 (BV421 dye), VL2 (BV510 dye), VL3 (BV605 dye), VL4 (BV711 dye) and YL4 (PE-Cy7 dye) channels. Cell phenotypes are defined for CD45+, viable and single cells as follows: T lymphocytes (CD3pos-CD19neg), B lymphocytes (CD3 neg-CD19pos), NK cells (CD3neg-CD19neg-CD56pos), monocytes (CD14pos) and granulocytes (SSChigh-CD16pos).

[0057] Finally, this set of experiments is carried out with the promoters that showed the most stringent CD3-specific activity for the expression of intracellular GFP but in the context of transgenes expressed on the surface of lymphocytes, LV without the VSV-G envelope and carrying an anti-CD19 CAR under the control of a given promoter are used to transduce PBMCs from healthy donors and lymphoma patients. The percentage of cells expressing CAR CD19 is determined 72 h post-transduction by flow cytometry with a human CD19 detection reagent (Miltenyi) and an anti-biotin-BB515 antibody (Miltenyi) according to manufacturer instructions using the BL1 channel.

Example 5. In Vivo Transduction of Peripheral Blood Mononuclear Cells with VSV-G.SUP.− (“Bald”) Lentiviral Vector Particles Encapsulated in OM-PBAEs

[0058] The CD3-specific activity of the 2 best promoters confirmed in Example 4 to trigger the expression of GFP in lymphocytes is evaluated in vivo in immunodeficient NSG mice engrafted with human PBMCs from healthy donors or PBMCs from lymphoma patients. Another mouse model which can be used is NSG mice engrafted with human CD34-positive hematopoeitic stem cells that exhibit multi-lineage engraftment of human immune cell populations. Nanoparticles described in Example 4 consisting in LV carrying a GFP transgene under the control of a given promoter and formulated with and without PBAEs covalently linked with CD3 targeting agents are repeatedly injected intravenously in mice that have previously been injected with PBMCs or CD34-positive hematopoietic stem cells. Control animals are injected with vehicle or transduction-deficient lentiviral vectors lacking the VSV-G envelope and not encapsulated in PBAE polymers.

[0059] In vivo biodistribution of expressed GFP transgene is evaluated weekly over 24 to 90 days on whole blood cells by flow cytometry staining with specific antibody panels purchased from Biolegend: general panel (CD3-AF700, CD11b-APC, CD11c-PE, CD14-PE-Cy7, CD16-BV711, CD19-BV605, CD45-BV510, CD56-BV421, CD66b-PerCP-Cy5.5, HLA-DR PE-Dazzle594, and Zombie NIR) and T cell panel (CD3-AF700, CD4-PerCP-Cy5.5, CD8-BV605, CD25-PE, CD45-BV510, CD45RA-BV711, CD69-PE-Dazzle594, CD127-APC, TCRg/d-PE-Cy7, CCR7 (CD197)-BV421 and Zombie NIR).

[0060] At sacrifice, the phenotype of GFP-expressing cells is determined on blood cells and cell suspension prepared from collected spleens and bone marrows. Tissue biodistribution of genome-integrated lentiviral vector is analyzed by duplex quantitative PCR on genomic DNA extracted from blood and organs collected at sacrifice. Treatment toxicity is evaluated by determining blood cell counts (flow cytometry as already described in Example 2), ALT/AST hepatic enzymes (enzyme activity kits) and cytokine levels (Th1/Th2 Cytometry Bead Array) in blood samples collected pre-treatment and at weekly post-treatment intervals. Behavior of animals, body weight, water and food consumption are recorded 3 times a week as additional read-outs for treatment safety and tolerance.

[0061] Finally, this set of animal experiments is carried out with the promoters selected in Example 4 but in the context of an anti-CD19 CAR transgene expressed on the surface of lymphocytes. The only difference with the above-described animal procedures lies in the evaluation of the phenotype of CD19 CAR-expressing cells that is determined by flow cytometry with the human CD19 detection reagent (Miltenyi) and an anti-biotin-BB515 antibody (Miltenyi) according to manufacturer instructions already described in Example 4.

Example 6. Efficacy of In Vivo Transduction of Peripheral Blood Mononuclear Cells with VSV-G.SUP.− (“Bald”) Lentiviral Vector Particles Encapsulated in OM-PBAEs

[0062] The CD3-specific activity of the 2 best promoters screened in Example 4 to trigger the expression of CAR CD19 in lymphocytes is evaluated in vivo in immunodeficient NSG mice bearing Ramos cancer cells modified to constitutively express the Luciferase reporter gene. Different NSG mice models are used: NSG mice engrafted with human PBMCs from healthy donors or PBMCs from lymphoma patients and finally NSG mice engrafted with human CD34-positive hematopoeitic stem cells that exhibit multi-lineage engraftment of human immune cell populations.

[0063] Nanoparticles described in Example 4 consisting of LV carrying a CAR CD19 transgene under the control of a given promoter and formulated with and without PBAEs covalently linked with CD3 targeting agents are repeatedly injected intravenously in mice that have previously been injected with Ramos-Luc cells. Control animals are injected with vehicle or transduction-deficient LV lacking the VSV-G envelope and not encapsulated in PBAE polymers.

[0064] In vivo efficacy is evaluated weekly over 24 to 90 days by whole animal bioluminescence imaging to measure the tumor growth and rate associated with circulating

[0065] Ramos-Luc cells and survival in the treated groups versus controls. Treatment toxicity is evaluated by determining blood cell counts (flow cytometry as already described in Example 5), ALT/AST hepatic enzymes (enzyme activity kits) and cytokine levels (Cytometry Bead Array) in blood samples collected pre-treatment and at weekly post-treatment intervals. Behavior of animals, body weight, water and food consumption are recorded 3 times a week as additional read-outs for treatment safety and tolerance.

Example 7. Design of T-Cell Specific Chimeric Promoters

[0066] The promoter sequences described above contain some transcription factor binding sites, including NFκB; AP1, Stats, GATA and NFAT. Table 3 summarizes the presence of those binding sequences inside each promoter sequence. Transcription factor binding sites are depicted in the sequences as well.

TABLE-US-00003 TABLE 3 Mapping of the promoter sequences with T-cell specific transcription factor binding sites. Indicated in italics are the promoters driving an unspecific expression of the transgene (according to the experiments described in FIG. 1). Promoter NFκB AP-1 STATS GATA-3 NFAT EOMES 3 4 13 3 13 LCK 2 6 6 2 6 BCL11B 3 3 6 2 4 ZAP70 3 5 6 1 7 ICOS 5 7 8 4 10 UBASH3A 7 4 15 7 0 GIMAP7 2 4 4 5 12 LAIR2 1 7 8 1 5 CTLA4 3 4 5 2 6 ICR2A 8 1 7 1 5 TCR 2 1 3 1 2 TIGIT 5 5 6 4 6 TCF7 1 6 6 1 1 TNFS8 2 4 9 6 7 ITK 2 4 12 8 8 The CD3+ specific promoters (LAIR2, TNFS8, TCR and ITK) present less than 2 NFκB sites, less than 8 NFAT boxes and less than 8 NFκB + AP1 sites.

Example 8: Optimization and Shortening of Promoter Sequences

[0067] To define the significant regions involved in inducible gene expression and increased transgene expression level.sup.5,6,7, 5′ deletion constructs were designed by shortening of 2 kb-promoters.

[0068] Shortening and Molecular Cloning of Tcell Promoters

[0069] Among the set of 15 promoters, CTLA4 (SEQ ID NO: 7) and ICOS (SEQ ID NO: 13) were selected for overexpression in CD3-expressing cell lines and primary cells, and low or no expression in non-CD3-expressing cells. The promoters were shortened from 5′ to 3′ by PCR using the synthetic gene as template and Phusion HF high fidelity DNA polymerase (Thermofisher). Forward primers, containing a Mul restriction site, were specific of 5′ regions of CTLA4 and ICOS and reverse primers, containing a BglII site (for SEQ ID NOS: 24-25) or a BamHI site (for SEQ ID NOS: 19-23, 26-29) were specific to the 3′ end of CTLA4 or ICOS. PCR fragments were then subcloned in the transfer vector plasmid pARA-CMV-GFP upstream of the open reading frame (ORF) encoding GFP and instead of the CMV promoter.

[0070] The same strategy was followed to subclone the truncated promoters in a second transfer vector plasmid pARA-hUBC-CAR-CD19 upstream of the ORF encoding a chimeric antigen receptor (CAR) specific for the human CD19 antigen. Sequence identity of truncated promoters and in frame insertion upstream of the transgene were verified by Sanger sequencing. Resulting optimized promoter sequences are described in Table 4.

TABLE-US-00004 TABLE 4 Shortened CTLA4 and ICOS Promoters. Size of promoter SEQ ID Promoter region (bp) 19 CTLA4_1 1844 20 CTLA4_2 1237 21 CTLA4_3 1048 22 CTLA4_4 745 23 CTLA4_5 344 24 ICOS_1 1703 25 ICOS_2 1443 26 ICOS_3 1219 27 ICOS_4 1000 28 ICOS_5 837 29 ICOS_6 550

[0071] LV Production and Transduction of Human Cell Lines and Primary Cells

[0072] Transfer plasmids encoding GFP and a CD19-targeted CAR were used to produce LV and transduced cell lines and primary cells as described in Examples 3 and 4. GFP or CD19 CAR transgene expression were measured by flow cytometry.

Example 9: “Pseudotyping” of Lentiviral Vectors with CARs

[0073] The experiments of Examples 3 and 4 showed that bald LV packaged into nanoparticles, such as by OM-PBAE encapsulation, have the ability to transduce PBMCs, including T lymphocytes, and direct the expression of a transgene. Since the goal of CAR T cell therapy is to introduce a CAR into T cells and express the CAR on the surface of such T cells, the question arises whether the presence of any expressed CAR on the surface of LV intended for use in producing CAR T cells can serve to pseudotype the LV, and possibly direct the LV to transduce cells that were intended for attack by CAR T cells. The experiments described below were designed to address this question.

[0074] Production of Lentiviral Vector Particles Coding for CD19 CAR

[0075] Production of bald LV particles was performed as described in Example 1. Briefly, LV293 cells were transiently transfected the day after seeding. PEIPro transfectant reagent (PolyPlus Transfection, Illkirch, France) was mixed with the transfer vector plasmid pARA-hUBC-CAR-CD19 and the packaging plasmid pARA-Pack. Bald LV particles encapsulated in OM-PBAEs were prepared as described in Example 3 and used to transduce LV293 cells or human PBMCs.

[0076] CD19 CAR Detection

[0077] After a DPBS washing step, cells (LV293 transfected and untransfected or LV-transduced PBMCs) were stained with human CD19 detection reagent (Miltenyi) and an anti-biotin-BB515 antibody (Miltenyi) according to manufacturer instructions as already described in Example 4.

[0078] The results presented in FIG. 3A show CD19 CAR expression on the surface of LV293 cells used for the production of LVs (transfected with pARA-hUBC-CAR-CD19 and pARA-Pack plasmids). Untransfected cells were used as control. A strong expression of the CD19 CAR was detected only on the surface of LV293-producing cells after transfection.

[0079] In addition, results presented in FIG. 3B show expression obtained on human PBMCs transduced with VSV-G− (“Bald”) LV encoding CD19 CAR under the control of hUBC promoter or GFP under the control of CMV promoter. While no expression of GFP was observed in PBMCs exposed to bald LVs, their encapsulation in OM-PBAE polymers restored their transduction efficiency. When VSV-G− (“Bald”) LV encoding CD19 CAR were used to transduce PBMCs, similar expression levels of the chimeric receptor were observed in presence or absence of the MO-PBAE polymers.

[0080] Altogether, these results suggest that the use of a ubiquitous promoter to control the expression of CD19 CARs results in the expression of chimeric receptor on the surface of the LV293 cell line that produces LVs (bald or pseudotyped) decorated with membrane proteins. This pseudotyping with CD19 CARs must be sufficient to allow the bald vector to bind to cells and generate an artefactual CD19-CAR signal, because the bald LV are deficient for transduction by nature, even though the CD19-CAR is not an efficient pseudotyping protein, as it does not allow endosomal escape of the LV). This “pseudotyping” may lead to safety issues, as the regular LV (with VSV-G) will directly target CD19+B lymphocytes both in vitro and in vivo. The results obtained here indicate that use of a T-cell specific promoter as described herein will abrogate the expression of CD19 CAR at the surface of LV293, and no pseudotyping of LV will be observed. This will increase the safety margin of LV designed for producing CAR T cells.

Sequences

[0081] Transcription factor binding sites are depicted in each promoter sequence as follows: NFkB (bold), AP-1 (underlined), STATS (italics), GATA-3 (double-underlined) and NFAT 5 (boxed). Of note, some binding sites are overlapping.

TABLE-US-00005 SEQ ID NO: 1 GGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCC GCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGA CGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATAT GCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGT ACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACC ATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATT TCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACT TTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGG GAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATC SEQ ID NO: 2 CCCACTTGCTCCCTGGGCTTGGAGCACAACCACTCAAACAGAACTGGCTTTTGGTCAGTAAG GAAGAAGTGAGCAACGGCTGCGGTGTAGACCCTCGTCAATGCCTGCGACGGTTACACCTGGA GACAAGCTCCCCAGTGTCCTCAGGAGCAGCGGAGATGAGAATCCATGATAGGGTGGGCTCTG TCCCCCTCAGCTCCGTGATGCCGAAATGCACTGCTGGTCCTGGTCCTGCTCCTCATTCCACA CCCGGCTGAGTGCCCATCTGACCCCAGACCTCAACGCGAGGTTCTAAGCACTGTCTCCTGAC [00001] [00002] GTGTGTGTGAATGTGTGTGTGTATTATACATAATATATATAATTACAACATTGTTAATGGGG CCGGGCGCGGTGGCTCACACCTGTAATCACAGCACTTTGGGAGGCTGAGATGGGCAGATCAC ATGAGGTCAGGAGTTCAAGACCAGCCTGGCCAACATGGTGAAACCCCGTCTCTACTAAAATA CAAAAATTAGCCGGGCATGGTGGCGTGCGCCTGTAGTCCCGGCTACTCAGCAGGCTGAGGCA AGAGAATTGCTTGAGCCTGGGAGGCGGAGGTTGCAGTGAGCCAAGATTGCACCACTGCACTC CAGCCTGGGCAACAGAGTACGACTCCATCTCCACACACACATACACACACACACACACACAC ACAAATTGTTAATTGTATATGTATAATTATAATAGTTTATATATATTAACAATTATATATAA ACTTGCATATATATAGTATTTGCTGTATTATTATATATAAACAATTATATATGTAATGACTG TATAAAATAGATAAACAATTTTAACTAATAATATTATATTAATTATATTATTATTAGATGTA ATAATTATAATTATTTATATATAATATTTATTATTATGTATCATTGTTAGAACACTTAACGT GAGCTCTGTCCTCTTAACAAATTTCAAGTGAACAAGACGTTATTGCTGACGATGGGTCGTAT [00003] [00004] GTGACATCATGCAGTACTTGGTCTCTGCCTCTGCGTCGCTTGGCGTGATGTCCTCAGGTTTC GTCCGTGTTGTCGCCCATGGCAGAATTTTCTTCCTTGTTTAAGGCTGAATAGTATTCCCCTG TGTGTGCACCACATTTTCTATATCAATTCTTCTATCAATGGACATTTAGATGATTTTCACGT [00005] [00006] TTAGTTTTTTGAGGAACCTCCCCACTGTTCTCTATAGTGAGTGCACAATTTTTCAGCCTCCC AAAGTGCTGGGATCACAGGCGTGAGCCACCGCGCCCGGCGCACTGTAGGATCTTTTTTAATG CATTATATACCTTGCTGAGATTTTAGCAGAGATCACAATATTAAAAACTTGGGGAAGGATTT [00007] GAGCGGTGGTCGTTAATGTGTGGAGTTGGGATGACATCCAAGTCAGTTGGTTGCACGACCTT TATTCTGTCTTGTCCCATAGATTTAGAAAGAGGCTGACACATCGGGTAACTAGTTTAAGGTC ATCTGATCATGCGGGTAAGCGACATTTTTCAGAAACCAAGGCCCTCCCTCTCATCTCACTAG TGGGAAGGGTGGAAAG SEQ ID NO: 3 TTTTCCACTGCCCTTCTCAGCCCCACAGGCCTTCAGGAAGACCCGAACTTCAAGCAAAGCCT [00008] AGGCTGAACAAGAGGGCCAAGGGAGCAGCTGGTTCTCAGCAACTCTGGGCAGACCACAGAGC CCTTGCTACCCACTCACTGTCCGTGCCCACCAGAGGACACAGCCTTCTCCCCAAATCAGCCA GGTACATGCCCCAGAAAACACTGGCTTGCCTCGTTCCCACCTTAATTACCGGACCAAACGAA CGTGAACACACTGTTTTCAAAACCAAACTCAATTGGGATCACGGGGGCCTCGGTTTCCTTGA TTGTAAAATGGGTATATCGCCCCCCACTCTATTTAGCCCAAGAAAACAATTTATCTCTTGAT [00009] ATCCTAAAGCCTGATCTAATTTGGGAGATGCTCAGAAGTTTTGGTTCTATGCAAGAACAGCA GTGGTAATAATCCAAGCTTGGCTTTAGACACAGGACGTTTCCTTAGGGGCATCTGGGATCTC [00010] AGAATGAGCTTCTTCCACGGTGATACCAAATGGAGCTTTCAAAGGCCCACATCTGGAGGCAG CAGCTATGCAGTGATTAACATTTTAAACGGTATTTTGAAATGGAGATCATTAGTAACCACAG ATGTGATCTGACTCTGTCCCCCAGGTAATCTGTCTATTGTATCTAAATTCCAGACTTAGCCC AGTAGACAGCTTGGGATGTTTAACAGGAATTGTCCAACACCATCCCCAAATCTATTTTTATT CATGGAGTACTCTGACATCATCTCGCTTGGTCTTCCTGATGACTGTAATGCAGATTGGGAAC [00011] ACTTCAGGTTTTAGCTTTCAGGTGCAGTGAAGATTGAATGACTTAGCACGAGCTTTCAGCCA GGCAGGCTGCAAAGTGCACCCAAGTTCTTTCAGTGATCGACACTTGCGACTTAGGTTGAGAT [00012] GAGGGTGGAGGTGGAAGTGGAATGAAGCCACATGTAACTCCTAGCGCTGTGCCTGAGACAGT AGAGGTTCAATTATAGTAGTCACATACACACACAACACATACATACACAAGACACAACACAC CACACACAACACATACATACACACCACACACAACACTTACATACGCACCACACTCGGCATGC ATTGCAGATCACAAATGCACACCACACACACACTGCATACATATACCATAGAGAACACACAA CACCTACACATAACCCCATACCACAAATACACACACACTACATAGTACACCACGCAGAACAC ACACAGCCCCCATATTCCACACCACACCATCTCACTGCCAATTCCTTCCCCTCTTCATGAGT TTTACGGCAGGTCCAGGTTCATCTGCCAGTTTAACAGATCCCAAAACTTCTGCAGGAGTGTT [00013] [00014] [00015] ATTACACTGCAGTCCGAGGGATCCGGCCTCCCTGAGACCCAGCAAGGACTCATTATCTGGGG AGGTCTTCTGAGCCACAGGCCTCGCTGAAAGAAGGTGCAGCTTCTTGAACAGGAAGGCGTTT TGTGGCAGAGTCTAAA SEQ ID NO: 4 CAGGATAGCCCTTTCAATGCGGAAGTAGATGAAAGTTTACCCCTACCTCACACCATACACAG AATGAATTCAAATGGATCATAGAGCTAAATATGAAGAGCAAAAGTAGCAAAACTTCTAGAAC AAAACATAGATGATCTTCATCGATGTTGGATTAACAACAATCTCTTGAACAGGATGCAGAAA [00016] [00017] [00018] CTACACACCACCCCCACCACAGAGTGGCTAAAATTAGGGACTGATAATATTAAGTGTGCATG AGGATATAGAGCTTTGTACAACTGCTTGGCAGTTCCTAATGGAGTTTCACGTGCTCTGTGAT CCAACAACCCCACTCCTAGATGCACACCTAACAGAAATGCATGCACAAGGCACTCAAAGACA CATACAAGAACGTCCTCAGCAACATTAGTTACAATAGCAAAACAACCGAAAACTTCCTGAAT GCATATCAACAGAGAAATGGATAAATGAATTATGTTATATTTATTTTATTTATTTATTTTTG AGCTGGAGTCTCACTCTGTCCCAGGCTGGAGTGCAGTGATGCAATCTTTGCTCACTGTAACC TCCACCTCCTGGGTTCAAATGATTCTCCTGCTTCACCCTCCTGAGTAGCTGGGATTACAGGC ATGCACCACCATGCCCAACTAATTTTTGTATTTTTTAGTAGAGACAGGGTTTCAACATGTTG GCCAGGCTGGTCTCGAACTCCTGACCTCAGGTGATCCACCCCCCCCTTGGCCTCCCAAAGTC CCAAAGTGCTGGGATTACAGGCGTGAGCCACCACGCCTGGCTGGTATATTTATTAATCAAAT ACTACACAGCAATAACAGTGAACTAACTGTGGCTACAAGCAATAATGTGGATAAATTTTACA GGCATAATTTGAGGAAAAAGAAATGAGATATGATAGAGAATATGCTATATGGTTCTATTTAC ATAAGGCTCAACCACAGGCACAATTCATATACAAGTGATACAGCAGTTTACTTTCTGGGGGA GGGAGGAGATAACTGGAGGAGGCTGAGCTGGGGGCAGAGGTGCATCTGAGGTTCTGAGAATG TTCCAGAGCAGGCTATCTCCACCTTGGCACTAGTGAAATTTTGTTGGGGAGACTGTCCTGGG [00019] ACCACCCCCGCCTCATTTTGGAAACAAAAATGTCTCCAGACATTTCCAAATGTCCCCTTGGG TGGAGGAGGTACAAAATTCCCCTGGTTGCCCTGTTTGATTTTGGGTCGTGGGGGAATTAGGG GTGTGGGCTCATTTATGGAAATTCCTTGAGCTATACACATATGATTCATCAACTTTTCTGTA CACATATAAAGCTTTTAAAGTTTTTAAAATAAAAAAACAAGTCAATGAATGACTGAAAGAAC CAGTAAGTTTTCCAGCAATCCGAGACAATAGATAGAGTGGGGTTTAATGTCTTGTTCACAGA [00020] TAGCCAAGTCCCTACAGAGGCTGATCCCGTGCAGGGGCTCGGTTGGACCTTTAGCCTCAAAC TCCCGTGGTGGAAACAGTTAGGATTGGTGGACGCTGCACCCAGCATCCTGCAAACAGAATTC TGAAAACTGATATCTCATGCAAACAGGATATCAACTTTTGTCAGAAAGATCAAAAAGAGCTT GAGGTTTTTCTTCACA SEQ ID NO: 5 CATGTGCGTACAGCAATGTCTGGCACATAATCAGTGCGACTCTCATTATTGTAGTATGAATG [00021] AAGAAATGAGCGTCCAGCATGGATGTGAAGGGATGCTTGCTGCACCTGGAGTTTCAAGATGC [00022] AGTAGCCTAGAGCCGGAACTCAGATTTTCTAAAGGGGATTCAGGCGGCCAGAGGATAGTAGA GGGCCAGAAAGGAGGTCCCCAAGGAGGCTTCGGGGTAGGGTGTGAGGGGATCCTCAGGCAGA CTGGGGAGCTGAGCCTGTTGTAACCGAGCGAGTTACAGAGAAACGCCGCACTTTGAGACGAA TTCAGGGGTCCTTTATTAGCTGGCGACTGAGAGACAGCTAGTGCTCAAAATTCTCTCAGCCC CAAAGAAGGGGCTTGATTTTCTTTTATACTTTGGTTTAGAAAGGACAGGTGGGGGTCTAAAA [00023] AGTTCCAGGTGCAGGGGCTTAAAATCTATCACAAGGTGATAGACACGGGGCTTTGGGCGTTA TCAACCGGACACAAACGCCGGGGCTCTGGGTGCTATTAACCGGGCGAATTCCTGGGAACTGC GGATATAGCTTGCCACAGTATCTTATCAGTTAATTGCATTCTTGGATGTGCTGGGAGTCAGC TTGCACAAATTAAGTCCTTGAGGAAGCGGGGTGGGTAAGGGGCTGCAAATGAAAGAGCCAAG ATGGAGTCTGTCTGGCTCTCTTAGCTAAGGGAGAGTCAATTCAGGTTAAAACAAGGTAGGGT ATCACAAGCCCACTTAACAAGGGCAGCAGGACCCCAAGAAGAAAAGCTTTAGGAGTCTCCAC AGTGGGCCCAGGGCAGTTCCACACAGGTCTCTGAGGCCCCACAGACAGGAGAGCTGTGACGA CTCCCTTAGTGCCCAAGAAAGCAAGGAGGTGGTGGGCAAGGGGCTCCCAGAGGCCTTGGGGC ACTAGAGGGGAGATGGAGCCGAGGGAGTGGCTCTGCAGGCCCCTCTCTGAGAGAGTGCATGA GGTGTGGCCCCAGGCCCAAGGGTAGGGGGTGCACAGGGTGAGGGAAGGGAGGAGAGGAAGAG GAGGAGGAGGTGGTGGCCACAGCGGGATGGCACTCAGCCAGGTTCAGCTTCGTGGGAAAGGT CCCAGGTGGGCCGGGCTAGCACTGGGGATGCCCTGGCTCTGTGTCTCGGTTGGGTGGCTGAC TCCTCCTGGAACGCATCCCTGACATCCTCCAGGCTGGCTTCCCCATACTCTGGGGACACACA GTGCCCAGGCTTCCGGCCTCCCAGCCCTGGCTCCCTGTGGACTCAGCTGCCCCCAACAAGCC TGCGAGCCACTGGAGAGCAAGGGCAGGCTGTCCTTGCCATCCAGATGCCTGGCACAGAAGGG GTTCTCACCTTCGGGGTATGGGCTGACTCAGTGGGTTTCTCCTCCCTCCAGCTCTAACCTGC CTGTGGAATAAACAAATCACTCCTCTGGGTTGTCTAGCTGGTGCAGGCCCCAGGGCGACGCC [00024] CCACTGCTGCCTACCCTCCGGTTCCAGGTATGCAGGCTTCCTCCCTTCTGACGGTTCCTGCT GCTGGAGTCGTCCTTCCTGAAACCCTGCCTTTGCTTAGCCTCATTCCCATCTCTCAGTCCCA TCCTGCAGCTGGGCAGGCAGTGCTGGGCCCCGGAAATGCCCTCTGCCTCCCTGGAGCACGTG [00025] [00026] SEQ ID NO: 6 GCTTCACATCGCTAGTTCTCAGAAAGAGAGCGCGAGTTGATCTTTCAAATTTAGAGTCCGGG CGAGTAGGGATGGCCAGTTTATGAATGTCTTAAAGAAGTTTATTAAAGGTGCGTGTGGGCAT [00027] GCCCTACTAGACTTTGGACCACGTGGAGGTGGGGTGTTGGGGGTGGGCAGCCGGGTGCAGCC TGCCCGGCCAGGGCGAGGAGGTGCAAGTCCGCGCCCCGCCCCCGGTCTCCATCCGCTCGGCC TCGCGTCCGCCCACCGTGGAGCCCACAGTTTACAGAACTGCAAGCCAGGGCACATTTTATTG TTATTATTTTTTAGGGTATGGGACTGGAAGGGATCTCTTTCTGTTCTCGCTCACGGACTCTG GGCGCTGTTAAATTGTTGGTGCCATCGCCCCACCCAAGACCTTCGGGACGAAAACAAAAACC CCCGTGTGCCCCCCGAGGAAAAGGCCCCGAGGGAGGCGGCGCTGAGGCGTCACGGCCTCGCC ATGCGCCCGGGTGAAGCCGCCGGAGCCAGGCCTGGGGGCGGAGTGGGGTGGGTGCAGCTGGA CTCGCCCGCGAAGCCTAGCCGGAGCCAGGCCCCGACCCGCCCTGGCCGCCTCCAGAGAAACC CTGAGAAGGGAGAGGAGGCCAGCCGCGTGGCGCGCCCAGCCCTGGAGGCCAGGTCATCAAAT TGACAAAGTTGGCCACAGCCTGGGGAATGGGGGGTGTCCACGGGGGCCGTGGTCTGGCCTTG TCCCCGGGCCACGGAGAGGCCCCACAACTCCTCTTGTCCTCGCCGGTTGGAGATCAAACGGC TGACAAGGCAGCTGCGTCCTTCCCAGTCTGACAGGCATTTCCAAGTCCTACCCTGGGCCGAG GAGCGCGGGGGGCGGGGTGTGACTTCCAGGTTGCTGTGATTGTCAGGGGCGCCTCCTGGAGT CACCCACCCGAGTATCTGCGGAGATTTGGCCACACCTACTCCTGGGCTGGCAAGTCCAAGTC CCTCTCCCACGTGGAGCGCCCCGGTCCCTTTACCCTCTCGCTTGCCATAGACCCAGTTTAGG AGGTGGGGTCGTGTTTGACCCCAGGAGTTAATCGGGGTAAGGTGAGGGGAGGGGACAAAGAT [00028] ACGTGCTCTTGATTCTGTTGTCTTAGTGTCGTTTATTTTGTCATCCTCACCCTCCCAGAGTC TGTAATACCAGGATTTATATGGAGACACTTTTTGGAAGTTGAACTCCAACATCTACTCCCCT [00029] [00030] GCCCCCACCCCCAAAGTCCAAGTCCGAAAGAGCTTTTGGGTGGGTGGAGACTTGCATTGATT CAATTTAGTTCACTCACACAGCACCCCGCCCCCCTTCTCAGGGGGTCCTGCTCCCAGGATGG AGGAGATATAAGGACGATTTTTCTTTTATTTTAAAATAAGCTGCCCAGTGGCCCCCCCCAAC CCCCTCCCGCTGTTGCGCAGCCGGGGCTCGGGGGAGATGAGCGCACAAAAACGCGGTTTGCA CGTGTGTCCGGCTTGGGCTGCGGGTGTGCGCAACTGGCGACTGTGTGTGTGTGTGTGTGAGT GTGCGCGCGCGCGCGAGTGTGTCTCTGTGTGTGCTTTCTTGTTCTCTTACAGGGTACAATGT TAAAAAGCCACCGCTAGTCGCCCCCAGTGCTCCGACTCTCTGGGTCTTTTTGTCTCTAGTGC AGATTAAACGTCACGTCCGCACTTGAACTTGAATTTTATCCCATTGTACAGAGGCAGCCCCA GCCATAGAGAGACCGA SEQ ID NO: 7 TAGAAAGCTTTCCAAATCCTGGTGCCTGGCGTATTCCAATAGTCTTCTTCCCAGTCTTCCTG [00031] TCATACCAGTTTGCTCCTTATGTTTCATTGGCCCTTGCTGCTAAGAGCATCCGCTTGCACCT [00032] CTTTAGCCCATGTTATTCTTCTTGTCTGAATATCCACCCTTTTCTCTGTTCTCAATAATAAG TTCAGGCTTTTCGTCTTCTGAGAAGCCCTTTCTGACTTCCACAGGCTGAACCACTGGCTTCT GCTCCTCTACATAATACTTCAATTCCAGCATTGATCTCACTCTATCATGATCATGGGTTTAG CTGTCTGTCCCTGCCACTGCTGTGTGTTCCTCTTGAGGGCAGGAACATTTGTTTTTCACTTT TTAAAAAACCTCTGTTGCCCAGTCTGGCATTAGGAAGTGCCCATTAGGTTGTTATTGCTTGT TGGCGCTTGAGCTGGGGCTTGAAGGTTTCTATAATGTGTAGCAGTGTATAGAAAACAGGCAG GTCAGAAAAGGCTTCTGTGCATCACACCAACATGGCACATGTATACATATGTAACAAATCTG CATGTTGTGCACATGTACCCTAAAACTTAAAGTATAATAATAATAAAATTTTAAAAAAAAAA AGAAGAGGCTTCCTGGAGGAGATGACAGCTGAGCTAAGTCCTGGAGGATGAGAAGGAGTATA AAATAAGATAATAGGAGAAAAAAGGCAGTAGGAACAGCATGGGTAAAGGTGATGAGGCCTGA [00033] AGGCCAAAGAGGGAGGCATTTGGTGAGTATTCTGCAGAGTCTCCTCTGCTGTGCTGAGGTGT GGACAATGGGAAACCATGGACGGACTGGAGTAGGCAAATGTCATATTCCCTGTTACAACTGT CTGTTTGCATGTCAGCCTTCTAGAAGCCCCTTAAGGTATCAACTATGTTTTTGTTTTGTCAT CATTCAATCCTAAGTGCACAGAATTCCGGGCATATTACAGGTTCCCCATGAATGTTTCTTTC TTTATTAAAATGTATGAAAACTCTCCAGATTTAAGGAAGGTCCTCAATGTTTCAAATTCTTT TTGTTAGATCATTGGTCCTGTCTACAGCTGTCACAAATTTAAGGACTCTGGTTATATTTAAT CTTCACTTTTGAATTTTCTGCTTGAAAAATTTGTATTAGAAAAAAAAGTCTATCCTTTTATG GACGGCTCTAATCTCTTGAATCATTTGGGTTGGCTTTTCTTTGGACCTTCTTCAACTCTGTT [00034] [00035] AAATGCTAAATGGATTTAGGAGAAATAAACTTATTTGTAAAGCTGTCAAGGGACCATTAGAA GGATGGTGCTTCACAGATAGAATACAGTTTTTATTAATGATGCCTAGACAAATCCTGCCATT AGCCCAAGGGCTCAGAAAGTTAGCAGCCTAGTAGTTTTGGAGTTGTCAATGAAATGAATTGG ACTGGATGGTTAAGGATGCCCAGAAGATTGAATAAAATTGGGATTTAGGAGGACCCTTGTAC [00036] [00037] AGGTTGTCTTTTCGACGTAACAGCTAAACCCACGGCTTCCTTTCTCGTAAAACCAAAACAAA AAGGCTTTCTATTCAA SEQ ID NO: 8 TGAGGCGTGAGCGTCTACAGTGAACCCAGCACAGAAACCTGCTAGGGGAGCTGCTGTTGACT GCATCGCGATCCAAAGGACCGGCGTCTTTTGTAGATGCAGGGGCTGAGCCAGGCCAAGCGCG [00038] [00039] AGGCCAGGGTCAACTTCAGAGTCATGGGCTCCCTAAATGCGACTTCTAGGGTTGAGTTGCTG TGGACGAGCGACCCATGTCGGAATCCCGCGCCCACGTGGCTGCCCAAAGTTCCGAGTCTCCG GGCTGCAGGTTCTAGTCACGGAACCGAGTTGGGAGAGTCATAGGGGCTGGGACTTGGAGGAT CGGCTGAGGTCCGGTGCTCTTGGCTGTGTTCGCGGCTCGGAGCCGTCGCCTGACTGAGGGGC CCGTCACAGATGTGTGATGTATAAGCTCTGCACGCAACAGGAGCTCAATAAATGTGCGAAGG GGGGTATACTTATGTTCGCACTGTATGCAGGCGGCCTAGAAGGAAGTCCCTGATTGGCACAG GGATGGAGGATGGGGCAAGAGCCGCAACAGCGCCGCGGAGTTCCAACGCTGCCGGTTCCCTG GGGTACGAGCACAGCCTCAAGCAGCCTCAAGCCCTAGGAAGCCCCCAGTTCAAAGCACAGGG CGCATTGGAGCCTGGGCACGATACAGTTCACACCACGGCTGCGATGGTAAGCCACGCCCAAG TCCCAAGGGCCTAGGGGACCCCCGCCCTCCACAGCCGGAGGAGAAACCTGGGCGCAGAAAGC AGGGGGAATATCTGGTTGTAGGTGAGTAAGCGGGGTCAGGAGTTCCCGTTAGAGTCTCTGCG TTTCGGGAGAAGGGTGATCATTCCCAGGCTTGTCCGACGTCTCTCTCAGGGTGCGCTCCGGA AGAGCGAGCCCTTTAAGGCTATGCCGAGTGGGCGCGTCCCGGCCTCTCCCGGGAGAGGAGAG GCGGGGCGGACCTGTGTCCCGCCCCCGGCCCGGCCCGCCCCCAGTGCCCGCCCCGCCCCCGG CACTCGGCCGGCGGCGCCTTTGATGTTCCGACCCGCCAGCTCGCGGAGCCGCTCTGCCCCGC GCCCTAGCCCGCGCCTGCAGCCCGCCCAGGCGGAGTCAGCCCGCGCTCCGCCCGCCGCGATC CGAGCTCGGAGGTTCGGACTCCGGGCTCGCCGCCCCCCGGGCCGGCTCCGCGCCCCGCACTC CCGGCGCCCAGCGCCCCGCGCCCCGGCGGGCGGAGCGCACCATGCCGCAGCTGGACTCCGGC GGGGGCGGCGCGGGCGGCGGCGACGACCTCGGCGCGCCGGACGAGCTGCTGGCCTTCCAGGA TGAAGGCGAGGAGCAGGACGACAAGAGCCGCGACAGCGCCGCCGGTCCCGAGCGCGACCTGG CCGAGCTCAAGTCGTCGCTCGTGAACGAGTCCGAGGGCGCGGCCGGCGGCGCAGGGATCCCG GGGGTCCCGGGGGCCGGCGCCGGGGCCCGCGGCGAGGCCGAGGTGAGCCCCCGCCGGCGCCG GCTCCTCCCCCGCGGTCGCCGCGCCGCGCCGCCCCAGTTGCGCGCGGCCCTCGGGGTCTCCA GCGCGCAGAGCGTCCCTGCCCCGGCGTCGGCCCCGACCCCCGCGGTCCCACCGCCCCTCACT CCCCTCCGGTTCTCCCTCCAGGCTCTCGGGCGGGAACACGCTGCGCAGAGACTCTTCCCGGA CAAACTTCCAGAGCCCCTGGAGGACGGTGAGTTTCTGCCCGGCCCGGCTTCCCTTCGTCGCG CTCAGGCCCTGGCCTCGGTGGGACGGGGACGCCAAGGACCGCGGGGAGCCGGGTGCCTCCCC CACCGCAGCTCAGGAGGCGGCAGAACCCAGGGGTGGAGAGTGGGGGGCGGGCTTCCCGGGCG CCGCCGGGTCGAGTCA SEQ ID NO: 9 GACCAATATGGTGAAACCCCATCTCTACTAAACACAAAAAATTAGCTGGGCGTGGTGGTGCA TGCCTGTACTTGGGAGGCTGAGGCAGGAGAATCGCTTGAACCCGGGAGGTGGGAGTTGCAGT GAGCCAAGACTGTGCCATTGTATTCCAGCCTGGGCAACAAGAGAGAAACTCCATCAAATAAA TAAATAAATAAATAGGTCATGGGGATTGATTTCATCAGATTGCTTATCAGAATCAAAGGAAA TGATTATAGGCTTTGAAAAATATAGACCTTATATTTATTATTAGTGGATTTTGCAATATTGA ATCATTCTTGCTACCCTGGAATAAACCCTACCTGGTGATTATGTATTTTGTTTAATATATTA [00040] TGGTGTTCTTTAATTGTGTGCAATCTTAATCACTTTAATATCAGTTTTGAATTCATATCATA AAACTAATGAAGAAGGCTTTATGTTCTATGTCTGTATATTTTAAGTAACATTGAAATTATCT GCTCTTCAGAAGTTTGTTGTAGTCTCTTTGTGAAATCTTCTAGGTATGGCATTCTTTTTGCT GGGGACAGGAGGAGCTCTTGATGCTTGAGGGAGTCTTGGAATGATATGTTCACGTGTACTTC TATCCCAGTCCTAGTTGTCTTGGATGACTGAGGGCTCTAGGTAAGAGGAAGAAGGAAAGAAG [00041] AGGACTGAGAGTAACAATGATTCTTTGCTTGACCAAACTTTGTCAGCAAAGTTTGTCCTGAA CCTTCTCCTAGGCCCATCCATGCACTTCCTTGTAAAATCCATTTTTTAGCCAAGAACAAGGA TAAATCAGTTTTGCAAGAACCCCTATCCCCCTGTATCTGATCACCCTTGATATCTTATCAGG TTTCTCATCCTCCACCATTTCTCAGTTCATGTCTGATCTACCCTGGCCTGTCTTTAGCAAGA [00042] [00043] AATTGAGCCCAATCTCTCCCCCTCTGCAAAATCCCATTGCCTTGGTCTCTATACCTATTGTG ATGGTCCTGAATAAAGGCTGCCTTACCTTTAAGAAGTGTCATTGCCTAAGTTGTCTAATTAT ACGATAGTTTAATCCCATCTAGAATAGAGGAGGAGAAGGAGGGAAGCAATGTGGGTGTGTAG GAGGGTTGTGAAGAGAGGCGGCAGGAGAGGCCTAAGCTGAGAGGATCAGCCGCCTCTTCCCA [00044] [00045] GGAGATAGATCCTCAGAGCCACCTCACGGGAGGGAGTTTTGACAAACAGAAGTCAGCGGTCC CACTCCCAAGTTCTCCTCATTTGTATTTGCCAGTGTCAGCCAAGTCCCATGGCTTGTTAATA AGCCTCAGATCACGGGCAGTCCTTTCGCGTGGAGTCTGATTTTCTGAGGCAGCTCTCTCCAC [00046] GAGACAGCTGGGGGTGATGGGACACAGGGCGGGCCCTTCCTGGTGCCTGGGGCGGCCAGGTC CCCTTGTCCCCCCTGTGTCAGGGCTCCCGTGCTTCCTCGCCCTTCTCTCCTAACATCTAAGG CAGGAGGCAGGGCCTTCTCGCCCCCCGCCATAATATAACCTGCCACCAGGAGGCGCTGTTCT ACAGGATGGAGAGTGG SEQ ID NO: 10 TGGTCACTATTTACTGTGTGAGTTCCACTGGAGACAAGCACACTTGCTCCAATCCCAGAGTC TACACCTGACCGGGAATTTTTCATTTTGGATTCTGATTGAGGGAAAATACTGATATCCAATG GGCTATAAGGGGGACCTTTCAGTTTCTGTTAACAGCATCTGAGTTCTATGCACGTCCTGGAA AGGCACCTGCCCACTGGCTAATTCACATGTTGTAATCCCAACACTGTAAATATCTGACTTCA CATTATACCCATGTAAATCCTGTCTCAGTAGTTCTGGACTCAGCCATGGCTGCACTAATGTG [00047] [00048] [00049] [00050] [00051] [00052] [00053] AGGAGTATGCCGTGCTAGATGGACAGAAGTCAACTTGTCAAATCCTCTTCCTATTTCTACTT GGAGCTCGTAGTGAGAAACGTTGGCGGAACATACTACTTCACTGGCTCTAGTGGATGGAGGC [00054] CATGTTTGAGTGATTCAACTTGTGTTCTTGAACTAAAGCTCACAGAAAGTGACAAAAATCCA AATATTCCAAAGAAGGCTTTTGTTTTATCTTCCTTTGAAGATGCAGATATATCCTACTTAGC GGCGACGACGGCCAGCGACTCCCTACAGGCTGCCAGTGGTGCGGACCTGGGCGGGTGCCGGA GCCCGGATCACAGAGGGGCAAGGCGAGCGCTCTAGGCCCACTCCTGGCGTGGGGCGCTGGAG GGTGGCAAGGCCGCGACAGGGGCTGTGGGCTTGGAACTCTAAACATAGTATTCTTACGTAGC CAACTGGAATATCTGGTCAACCGTGAGAGCTTTGGGAACCTGCCTCTCACCTCAAGAAACCT CCAGAGCAGATTGGAACCATCCACTGGGTAAATTCTGGAAAATCTGTCTCTGCCTGAGACTG GGGTAGGAAGATGTGGGGTAGAGAGGAAGGTGGGGAAGGGATGTAGGATCTATTCCTTGGGT [00055] [00056] GGTTCCTAATGATTTAGACTCTGCCTTTGAGCCTCAGTGCTGTTAACTTTTGAATGAGTAAC TTGAGGCCTTCCTGAAAGACTATAATAAAAAACCCATATTATACAATCACTAAGAACCCCCA [00057] GCCTCCCACTCCACCCTGGGAAGACAATCAGGTCCTGTGCCTTGTTTTCTAGGCAATATTTG [00058] [00059] ACCAGATTATGGAGCCGTATTTCTGTTTCTTTAAAGACAAACATTTTTGTGTGTGTGGCTCC ACCCAGCCTGAGCTTC SEQ ID NO: 11 [00060] GGGAAAAAGCCAGGTGTTAATGATGAAAAAACATTCAACTTTTCTACCGCTACTAATAACAT TTAATTCAAGTACTGAGAACATTTACCTCAAATCTTCAAGAATAAGACAATATTATCCCCTT TCTCTTTTATTGTCGGACTAGAGAATGTGAGAGAGGTTACATTCCATGGGCTTTGGGAATTT AATATGGTTCAAGGATAAACACACCCAGGTTTTTTCACTCCAGAGAAGAGCTTCAAATATAAT CCAGTTTTCAGGTCATCAGCTCAGCTCTTGTATCCCTAACAATGCGGTTGACATACCGTCTT CTCACATAGTCTAAACTCCTAAACTCACTAAGCCATACTTTAAAGTACATATAAAGGACTAG AAGCACCAAGCTACCAGTGAGACGAAGAGGAGAGTTTCCACAGAAGCTGGCTTCAAATAAGA CAATGAGTTCATCTTTAAATACTTGCCATTTGAGGTGCAGATGGATATAGTTGGCAGGCTCC TATCTAACGCATGTTATGCACAAGCTACCGTGAATTGATAATATCAAAACAAATATCCAGCG AGCCTCTGCAAGTGTGCATCTCTATTTCACACCAATTATAGTTGACTTAATTCCTCCCTCAT TCATCTCCCAGAGATGCAGCCTCCTCTTAAAGAAGTTGCGGCTGGTGGCCCATTCAGTGATG [00061] SEQ ID NO: 12 TAGCTCCAAATAACGTGAGATTAGGTCTCACATTAGGGAGATTTCTCATCCCTACTTTTGCC TTAGATATCTTGCAACTCAGGTAAAGGATAATGTGGAGGATCCTACGAATAGTAATCAGGAC [00062] [00063] [00064] GAAGGCATGGACAATGTAGTGATCAAAACAGCCGTGCCAAACACTAACTATACTGATGACAC AAAACGCAGGAAGCGCAGGGCAGAACGCGCTCAGTACCAGCTTTCTGGCGATCTGCTCCTGG CCACTCTGTCCCAGGACCTACAAGGCCACCGCCCCAGCCCACTAGTCTCAACTTGCCCCCTC CCCTGTGGCTGTGCCTTGACCCTGAAAATCTTCGCGCACTCCTGGGTTTCACCACCACACAC ACGCGAGCGCGCGAGCGCGCGAACACACACATCAAAAAAGAAAAAAAAAACCGTGATAAATG CCCTGGAGAGTCTAAGTGCATTAAGGACCTCGTTCTCGGAGACCCCAGACATGCCGATGTTG [00065] [00066] [00067] GCGGCGTCTGTAATTGCTTATTAACAGCGAATATTCAGGCTTCTCCTTATCCGCAACGAAAC GTGCCCCCCGCTTCCGTAATAATGAAACGATAAAATATGACGGCCCCGCTCTTGAATCTATC [00068] ACTAATTATTCTACCTTCTGTATTTGCCGCAGAGCAGAGGCGCAGGGAATCCTAACTGTGGG [00069] [00070] CTCCCCGAAAGCAGTTGTGTTGGCGTGAGTATGAAGTTGGTGTTAGCTCGCTTAGTTTCACT CTTGTCCTGTCCCCAGTACCCTGTCCAAGCTTCTGATTTAGAAGCTGAGAGCTTAGGTCCTC CAGGATGGCAATGTGCTCTGGCCGTCCCCGGAATCCCAAAAGCTTAGCGCGAGTTTCCTCTC [00071] [00072] [00073] CTTCCCTGCAAGCTATCAGCTTGAAGAGTCTTTCTCTCTCTGGCCTCTAAAATCTGCTCCAG CCATCTCATTCCTCCAGCGGAGGGCTGAGATGACTAATGCGCCAAGGGTGGACACCGCTATG [00074] [00075] CAGGCGACTTGATCCAATTAGTAAATGCCTAATTGAATTAGTGTCACCTCCACCAGCCAATA GGAGGGTCTCCGGGCTGTGAGGCCGGGAAGCCCCTCCCTGGCCGCAAATATATAAAGCAGCT AGTGACAGCCCATAAG SEQ ID NO: 13 ATAATTTCTAGCCAAATAAATGAGGATAAGTGTGTGAGTGTGGTGGGGTAGTTAAGAGTTTG GAATCCAGAGTTTCACTGTCTGGATTTGAACAGGCTCCGTTGCTCATCAGCTCTGTGACTCC GGGCAAATGACTGAATCTCTCTAACTCTCAGTTTTCTCATCTATGAAATGAGAATAAGTAAC AGACTCTCCCTCGCTGAGTTGTTTGAGAACGGGATCAGACAATGCACGTGGAGCCCCGTGTC TGGCACACACTATGTCCTCATTGCATATTTATTATTAGGTTTCTGCTTTGTATTTTTCTCTG [00076] TACTCAGTTCCAGAGCTAGGCCTCACCTAGGTTGGAAGAAGTCAATTGTTCTCCACTGCCTG [00077] GCTACGGGGAGTACAGTGAAAAGGGAGGAGATTGGGGTAGCAAGGTCCCAGGGGTCCTGGCA [00078] [00079] AGGATCAGCCTCCTCCTTACCTTGCAGACCCCTTGGCTGTGGTCTGGCATTCCTAGTGGCTG [00080] [00081] [00082] TGGTATTGGTGGTTCTACCTGCTTCGGTTAAGAGTGAGGACTCTGGAGCCAGACTCTCTAAA TCATAGCACTGACACTTACTATATGACTTGGGCCAGGTTCGTTACCTGTCTGTGCCTCAGTT [00083] [00084] TGCATATAAAACATAATATAAATGTAATATTAGCTATATTCATATATAAAAATAGGTATATT TTATATATAAAATATATTACATTTAATTTTTTACATTTTGCATTAAGATATATTATTTCATA TAAAACATTTAAGTACATATACATATAATTATCTATGTAATTATTATTGTTATTGTTGTTAT TGTCTACTTTACCTCCCTGCAGGGATGGGGAGACCACGGCAGGCTTTTAAACATTCTTGGTC TCACTGAGCCGTAGGTGAGGAATCCCTGGTTCCTACAGACTCTGCTGTAATATGAGGAGCAG [00085] CATAAATATCATTTCTCAGATTCCTATGATGCTCTTCTTTCAGATCTTTTCACTTCAATTTC [00086] [00087] TTAACCCCCATAACTATTGATTCAGAGAAGTAGGGTGGTTCTGAAAAATACAGGCATAATCT [00088] AATAATCCATGCATTGTTTAACTCATGTCATAAGCAATAATGCCTTTCATATAGCCATTGGC [00089] [00090] SEQ ID NO: 14 AAAAATACAAAAGTTAGCCAAGCGTGATGGTGCATGCCTGTAATCCCAACTACTCAGGAGGC [00091] [00092] ACTGGGTTCGGTGGTGCATGCCTATAATCCTAGCATTTGGGGAGGCCAAGGCAGGAGGATTG CTTGAGCCCAGGAGTTCGAGATCAGACTGGGCAACATAGTGAGACCTCATCTCTACAAAAAA ATAATCAGCTGGGCATGATGGTGTGCACCTTTAGTCTCAGCTACTGGGGAGGTTGAGGTGGG AAGATTGCTTGAGCCCAGGAGGTCTAAGGGGATTGCGCCACTGCACTTCAGCCTGGGAGAGG [00093] AGAGAAAGAGAGAGAGAGAGAGAGAGAGAGAGCGAGAGAGCGCACGCATTTCAAAGCAGGGA [00094] TAGTTCAGAACACAGGCTGTGTGTGTGGGGGTGTGTGGGGGTGCCTGTGTGTGGGTGTTTGT GTGTGTGTGAGTGTGGGTGTGTGGGGGTATCTATGGAGGGGATGCCTGTGTGTGTGTGTAAG GTGAGTGTGTGGATGTGTGCATGGGTGTGGGTGAGTGTGTGGAGGTGCCTGTGTGTGTGTAT GAATGTGTGTTGGGGAGGGGAGAATGTGTGTGTGTGGGGTGAGTGCGTGTGTGTGGGGGTGA ATGCGTGTGTGTGTGTATGTGGGTGTGACTGTGTGGGTGTGTGGACGAGTGTGTGTGTTCCT GGGTGTGGGAGCCTGTGTGTGTAGGGGGAGTATGTATAGGGTGTGTGCATGAATGTGTGTGT GGGTACGTGTGTGAGAGTGGGTGCCTGTGTGTGGGGGGTGAGTGTGTGTGTGGGGGGGCACT TGTGGAGGGTGAGTGTATGTGTTTACTGAGTGTGAGTGTGGGTGCCTGTGTGTGGGAGGGTG AGTCTGTGTGTGAGTGTGTGGGGGAGTACCTGTGAGGGGTGAGTGTGTGTGTTTATGTGAAA GTGTGTGTGTGTGGATGCCTCTGTGGAGGTGGGATAGGGGGTGCCTCTGTGTGTGTGTGTGA GAGTGTGTGTGTGTAGGGTGTGTATATGTATAGGGTGTGTGTGAGTGTGTGTGTGTGAGAGA GTGTGTGTGTGGCAGAATAGACTGCGGAGGTGGATTTCATCTTGATATGAAAGGTCTGGAAT [00095] AGGAGGAGCTGCAGGGACTCCGGGGGCTTCAAAGTGAGGGCCCCACTCTGCTTCAGGCAAAA [00096] CACTGCTAAAACAGGCAAATAAACAAAAAAAAAGTTATGGCCAACAGAGTCACTGGAGGGTT TTCTGCTGGGGAGAAGCAAGCCCGTGTTTGAAGGAACCCTGTGAGATGACTGTGGGCTGTGT GAGGGGAACAGCGGGGGCTTGATGGTGGACTTCGGGAGCAGAAGCCTCTTTCTCAGCCTCCT [00097] [00098] CCAACACAGGGTACTGGCAGAGGGAGAGGGAGGGGGCAGAGGCAGGAAGTGGGTAACTAGAC TAACAAAGGTGCCTGTGGCGGTTTGCCCATCCCAGGTGGGAGGGTGGGGCTAGGGCTCAGGG GCCGTGTGTGAATTTA SEQ ID NO: 15 [00099] [00100] AAAGATGAGTCTCAGTTAGTGGGTTTGATGGCTCTATGAGAAAGAGAATAAAATTACTTACG ATGACACTCCATGTAGACTGAGCAAACGAAAGACTCCTAAGTATTGCAACAGCTACTGATGA [00101] CAGGTGTAAAATGTACTTTCCTCAGAGGAGTTTGTAGTCTAGTTTGAGAAGATAGCACTGTA TTATCTAGGTTTGCTGGTAGTGACAGAAACCTCAAAATAACTGAGACTTAGAAAATAGATAT TAAAAAATGTAATCCAGGGCTGGCATGGAAGATCCATGGTCACCAGGGTCCCAGGCTCTTTC [00102] [00103] [00104] TGACCAGAAGACAGTCATACAACTACACCTAGCTACAAGGGAGGCTAAAATTTGTAGTCTTT ATTCTTGCTGGCCAGATGCCCAGATAACTGTCAGGGATTCTGTTACTCTAGTAAGAAGGAGA GAGTAGCTATTTGGGAATAGTAGCAATCCCTACCTCAAGCCCATACTCAGAAAAAAAAAATA [00105] AATTTAAAATATACCAGTACCGTACCCTCCTGGGTGTCTTGATTATGCTGGGGCATCCAGTA TATGAAATAAAATTAGGCTGTTGTCTGGTAATTTACATTTAAAAATAAACATTGGAGAATTT [00106] ATCTGCTTACACTAAAATCTCTTGATTATTAACATAGTAGAGGTAACTGTCACACATTATAC TTCAACTATGTACTAACTCTAAGGTGACATGTATGACTAGCACCTACCATTCATAAGATGTA TGGGATTCAGTCCTCCAAAGGACCCTCACAAGGAAGCTAAGGATCAGAGAAGTCATGGCTTG CTCAAAGAGAAACAGCATAGTAAGTGACAGAGCTGGGAATCCAACACAAGTTTGTCTGTTTT [00107] CCACAAAACGTCAGGACAGAAAGGCAGTCATCTATATGTAGCATTGAGAAATTTCTATGGGC TTTAATTTAAATCTTAAAAACTAATAAACTTCAGCAACATATTTCTTCACTTTAATGTGTGA [00108] AATTTTTCTTGAACATCTGTTTTATGTGTCAGGTACTGTGATGGTTGCTGAGTATATCATGG TAAGCAAAACCACACATTGTCCTTACCCCTTGTGCTACATGATTTAACTGCTGGATGGATTA AACCAGGAACTATAAAGATTAAACTGTAATCCTTACCACAATTTTGAGTTTACAGTTTTTCA AATATCATTTCAAGCCATTAGCAAAGGCTGTATGTATTTTTTACATATGCCTCCTCGTTTTG TGAATTTTGAAAGGATGTGGTTTCGGCCTTTGACATCAGAGGAGAAGCTCAGCTATGTTGGC TGAACGTTGATAGAAAGATAACGTTGAAGGCAAGTTGCCCTTGAGCAGCTCTCTGAAGATCA ACTGCCTCCACATTGC SEQ ID NO: 16 [00109] CATCAGCAAACGACGACAGAGCGTTCATCCGTAAGGTGAACCAGAAAAGCCAGTTCAATGAC TTGTTTAACCATGGTCCATCTCAGAACCAAGAGTTGGGCCTCTTATTTACCAGAAAAATTGT GGGGGCTTTGTGATATGGCTTTAAAAAAATCTTGTAATTGCCAGGCGTGGTGGCTCACACCT GTAATCCCAGCACTTTGGGAGGCCGAGGTGGGTGAATCGCCTAAGGTCAGGAGTTCGAGACC AGCCTGACCAACATGGTGAAACTCCGTCTCTACTAAAAATACAAAAACTAGCTGGATGTGGT GACGCGTGCCTGTAATCCTAGCTACTCAGGAGGCTGACGCAGGAGAATCACTTGAACCTGGG AGGCAGAGGTTGCAGTGAGCCAAGATTGTGCCATTGCGCTCCAAAAAAAAAAAAAAAAAGAC [00110] [00111] [00112] [00113] GATGGAAGTCTCACTCTGTCGCCCAGGCTGGAGTGCAGTGGCGCAATCTCAGCTCACTGCAG CCTGCACCTCCTCGGTTCCAGCTATTCTCTTGTCTCAGCCTCCTGAGTAACTGGGATTACAG GCGCCCGCCACTACGCCTGGCTAATTTTTGTATTTTTAGTAGAAATGGGGTTTTACCATGTT GGCCAGACTGGTCTCAAACTCCCGACCTCAGGTGATCTGCCTGCCTCAGCCTCCCAAAGTGC TGGAATTACAGGCGTGTGCCACTGCGCCTGGCTAATTTTTTTTTTTTTTTTTTTTTTTTTTT TTTTTTAGTAGAGACGGTGGTTTCACCATGTCATCCAGGCTGGTCTCAAACTCCTGACCTCA GGTGATCCACCCACCTTGGTCTACCAAAGTGCTCGGATTACAGGCATGAGCCACCAGGCCCA GTCAACGTGATGTGTTTTGGAACCCTGAATTCCTTGGCTTGCCCGGAGGGTTTTCTTTTTGT TAATATCTTTGCTTGCTTTCTAGTATTTAAAAAATTGTGTTTTGCTCTAACTATGCAATGGC [00114] GAGAGCTCTGATCAATAAATAAGCAAGACTGAATTTTACAAAATAATCCAAAGTTTAAAACC AAAGCCCACTTTTTGCATGATCCTTTAAGAGAAAGAAATCTGGAAGCAAAACACCTTATAAA ATGACAATGCACTTTCAGGAGCCCAGGGCACTGTGGTGAAATGATGATGGCTAGTACAGGTT ATAAGCCTTGGGGAATTATTTATGAATTCTCAGGATCCTTCAGTTCGCCGCATCCTTCTCCA TTATTTGAATATTGGAGGCTGCCTGACCAGAATCTTGTCAGGACTTTGCTCCTTCATCCCAG GTGGTCCCGGCTGACTCCTGAGGACGTTACAGCCCTGAGGGGAGGACTCAGCTTATGAAGTG CTGGGTGAGACCACTGCCAAGAAGTGCTTGCTCACCCTACCTTCAACGGCAGGGGAATCTCC CTCTCCTTTTATGGGCGTAGCTGAAGAAAGGATTCATAAATGAAGTTCAATCCTTCTCATCA [00115] CAAACTATATTGTCATCAAAAAAAAAAAAAAAAAAAACACTTCCTATATTTGAGATGAGAG AAGAGAGTGCTAGGCA SEQ ID NO: 17 Cys-Arg-Arg-Arg SEQ ID NO: 18 Cys-His-His-His SEQ ID NO: 19 CCTTGCTGCTAAGAGCATCCGCTTGCACCTTCTGCTCATCCCCAGACAAGCTTTGTCCTGTG [00116] TCCACCCTTTTCTCTGTTCTCAATAATAAGTTCAGGCTTTTCGTCTTCTGAGAAGCCCTTTC TGACTTCCACAGGCTGAACCACTGGCTTCTGCTCCTCTACATAATACTTCAATTCCAGCATT GATCTCACTCTATCATGATCATGGGTTTAGCTGTCTGTCCCTGCCACTGCTGTGTGTTCCTC TTGAGGGCAGGAACATTTGTTTTTCACTTTTTAAAAAACCTCTGTTGCCCAGTCTGGCATTA GGAAGTGCCCATTAGGTTGTTATTGCTTGTTGGCGCTTGAGCTGGGGCTTGAAGGTTTCTAT AATGTGTAGCAGTGTATAGAAAACAGGCAGGTCAGAAAAGGCTTCTGTGCATCACACCAACA TGGCACATGTATACATATGTAACAAATCTGCATGTTGTGCACATGTACCCTAAAACTTAAAG TATAATAATAATAAAATTTTAAAAAAAAAAAGAAGAGGCTTCCTGGAGGAGATGACAGCTGA GCTAAGTCCTGGAGGATGAGAAGGAGTATAAAATAAGATAATAGGAGAAAAAAGGCAGTAGG [00117] AAGGGGGAATGGGAGGGAATGCTGGGGTACAGGCCAAAGAGGGAGGCATTTGGTGAGTATTC TGCAGAGTCTCCTCTGCTGTGCTGAGGTGTGGACAATGGGAAACCATGGACGGACTGGAGTA GGCAAATGTCATATTCCCTGTTACAACTGTCTGTTTGCATGTCAGCCTTCTAGAAGCCCCTT AAGGTATCAACTATGTTTTTGTTTTGTCATCATTCAATCCTAAGTGCACAGAATTCCGGGCA TATTACAGGTTCCCCATGAATGTTTCTTTCTTTATTAAAATGTATGAAAACTCTCCAGATTT AAGGAAGGTCCTCAATGTTTCAAATTCTTTTTGTTAGATCATTGGTCCTGTCTACAGCTGTC ACAAATTTAAGGACTCTGGTTATATTTAATCTTCACTTTTGAATTTTCTGCTTGAAAAATTT GTATTAGAAAAAAAAGTCTATCCTTTTATGGACGGCTCTAATCTCTTGAATCATTTGGGTTG GCTTTTCTTTGGACCTTCTTCAACTCTGTTTTGTCTCTGTTGAGTTAAGGCTTTTAAGAACA [00118] TCTTGTGATTTTAGTTTTTTTCTCTTAACCAAATGCTAAATGGATTTAGGAGAAATAAACTT ATTTGTAAAGCTGTCAAGGGACCATTAGAAGGATGGTGCTTCACAGATAGAATACAGTTTTT ATTAATGATGCCTAGACAAATCCTGCCATTAGCCCAAGGGCTCAGAAAGTTAGCAGCCTAGT AGTTTTGGAGTTGTCAATGAAATGAATTGGACTGGATGGTTAAGGATGCCCAGAAGATTGAA TAAAATTGGGATTTAGGAGGACCCTTGTACTCCAGGAAATTCTCCAAGTCTCCACTTAGTTA [00119] ATTGGCTTGTTTTGTTCAGTTGAGTGCTTGAGGTTGTCTTTTCGACGTAACAGCTAAACCCA CGGCTTCCTTTCTCGTAAAACCAAAACAAAAAGGCTTTCTATTCAA SEQ ID NO: 20 AGATGACAGCTGAGCTAAGTCCTGGAGGATGAGAAGGAGTATAAAATAAGATAATAGGAGAA [00120] [00121] TTGGTGAGTATTCTGCAGAGTCTCCTCTGCTGTGCTGAGGTGTGGACAATGGGAAACCATGG ACGGACTGGAGTAGGCAAATGTCATATTCCCTGTTACAACTGTCTGTTTGCATGTCAGCCTT CTAGAAGCCCCTTAAGGTATCAACTATGTTTTTGTTTTGTCATCATTCAATCCTAAGTGCAC AGAATTCCGGGCATATTACAGGTTCCCCATGAATGTTTCTTTCTTTATTAAAATGTATGAAA ACTCTCCAGATTTAAGGAAGGTCCTCAATGTTTCAAATTCTTTTTGTTAGATCATTGGTCCT GTCTACAGCTGTCACAAATTTAAGGACTCTGGTTATATTTAATCTTCACTTTTGAATTTTCT GCTTGAAAAATTTGTATTAGAAAAAAAAGTCTATCCTTTTATGGACGGCTCTAATCTCTTGA ATCATTTGGGTTGGCTTTTCTTTGGACCTTCTTCAACTCTGTTTTGTCTCTGTTGAGTTAAG [00122] TTTTCAGTTTATTTCTTGTGATTTTAGTTTTTTTCTCTTAACCAAATGCTAAATGGATTTAG GAGAAATAAACTTATTTGTAAAGCTGTCAAGGGACCATTAGAAGGATGGTGCTTCACAGATA GAATACAGTTTTTATTAATGATGCCTAGACAAATCCTGCCATTAGCCCAAGGGCTCAGAAAG TTAGCAGCCTAGTAGTTTTGGAGTTGTCAATGAAATGAATTGGACTGGATGGTTAAGGATGC CCAGAAGATTGAATAAAATTGGGATTTAGGAGGACCCTTGTACTCCAGGAAATTCTCCAAGT [00123] [00124] ACAGCTAAACCCACGGCTTCCTTTCTCGTAAAACCAAAACAAAAAGGCTTTCTATTCAA SEQ ID NO: 21 GTGAGTATTCTGCAGAGTCTCCTCTGCTGTGCTGAGGTGTGGACAATGGGAAACCATGGACG GACTGGAGTAGGCAAATGTCATATTCCCTGTTACAACTGTCTGTTTGCATGTCAGCCTTCTA GAAGCCCCTTAAGGTATCAACTATGTTTTTGTTTTGTCATCATTCAATCCTAAGTGCACAGA ATTCCGGGCATATTACAGGTTCCCCATGAATGTTTCTTTCTTTATTAAAATGTATGAAAACT CTCCAGATTTAAGGAAGGTCCTCAATGTTTCAAATTCTTTTTGTTAGATCATTGGTCCTGTC TACAGCTGTCACAAATTTAAGGACTCTGGTTATATTTAATCTTCACTTTTGAATTTTCTGCT TGAAAAATTTGTATTAGAAAAAAAAGTCTATCCTTTTATGGACGGCTCTAATCTCTTGAATC ATTTGGGTTGGCTTTTCTTTGGACCTTCTTCAACTCTGTTTTGTCTCTGTTGAGTTAAGGCT [00125] TCAGTTTATTTCTTGTGATTTTAGTTTTTTTCTCTTAACCAAATGCTAAATGGATTTAGGAG AAATAAACTTATTTGTAAAGCTGTCAAGGGACCATTAGAAGGATGGTGCTTCACAGATAGAA TACAGTTTTTATTAATGATGCCTAGACAAATCCTGCCATTAGCCCAAGGGCTCAGAAAGTTA GCAGCCTAGTAGTTTTGGAGTTGTCAATGAAATGAATTGGACTGGATGGTTAAGGATGCCCA GAAGATTGAATAAAATTGGGATTTAGGAGGACCCTTGTACTCCAGGAAATTCTCCAAGTCTC [00126] [00127] GCTAAACCCACGGCTTCCTTTCTCGTAAAACCAAAACAAAAAGGCTTTCTATTCAA SEQ ID NO: 22 TCCTGTCTACAGCTGTCACAAATTTAAGGACTCTGGTTATATTTAATCTTCACTTTTGAATT TTCTGCTTGAAAAATTTGTATTAGAAAAAAAAGTCTATCCTTTTATGGACGGCTCTAATCTC TTGAATCATTTGGGTTGGCTTTTCTTTGGACCTTCTTCAACTCTGTTTTGTCTCTGTTGAGT [00128] CCTATTTTCAGTTTATTTCTTGTGATTTTAGTTTTTTTCTCTTAACCAAATGCTAAATGGAT TTAGGAGAAATAAACTTATTTGTAAAGCTGTCAAGGGACCATTAGAAGGATGGTGCTTCACA GATAGAATACAGTTTTTATTAATGATGCCTAGACAAATCCTGCCATTAGCCCAAGGGCTCAG AAAGTTAGCAGCCTAGTAGTTTTGGAGTTGTCAATGAAATGAATTGGACTGGATGGTTAAGG ATGCCCAGAAGATTGAATAAAATTGGGATTTAGGAGGACCCTTGTACTCCAGGAAATTCTCC [00129] [00130] CGTAACAGCTAAACCCACGGCTTCCTTTCTCGTAAAACCAAAACAAAAAGGCTTTCTATTCA A SEQ ID NO: 23 TAGACAAATCCTGCCATTAGCCCAAGGGCTCAGAAAGTTAGCAGCCTAGTAGTTTTGGAGTT GTCAATGAAATGAATTGGACTGGATGGTTAAGGATGCCCAGAAGATTGAATAAAATTGGGAT [00131] [00132] TGTTCAGTTGAGTGCTTGAGGTTGTCTTTTCGACGTAACAGCTAAACCCACGGCTTCCTTTC TCGTAAAACCAAAACAAAAAGGCTTTCTATTCAA SEQ ID NO: 24 GTATTTTTCTCTGTGCAACAGCCCTGAGCTCTAGAACTTATGACAGCTTTTCTCAAGTGAAG [00133] [00134] CTTTCCTCATGATGCTACGGGGAGTACAGTGAAAAGGGAGGAGATTGGGGTAGCAAGGTCCC [00135] [00136] GGGGGATCCCTGCAGGATCAGCCTCCTCCTTACCTTGCAGACCCCTTGGCTGTGGTCTGGCA TTCCTAGTGGCTGGCCTAAAGCCCCAAACCTGGAATCTGTTTCAGAGCCTGGACCTTTTGGA [00137] [00138] GATCAGCAGGCCTTGGTATTGGTGGTTCTACCTGCTTCGGTTAAGAGTGAGGACTCTGGAGC CAGACTCTCTAAATCATAGCACTGACACTTACTATATGACTTGGGCCAGGTTCGTTACCTGT CTGTGCCTCAGTTTTCCTCATCTGTAAAATGGGAGATTAGTGGTCACCATTGGGATGATTAA [00139] GTAACCATTTACATGCATATAAAACATAATATAAATGTAATATTAGCTATATTCATATATAA AAATAGGTATATTTTATATATAAAATATATTACATTTAATTTTTTACATTTTGCATTAAGAT ATATTATTTCATATAAAACATTTAAGTACATATACATATAATTATCTATGTAATTATTATTG TTATTGTTGTTATTGTCTACTTTACCTCCCTGCAGGGATGGGGAGACCACGGCAGGCTTTTA AACATTCTTGGTCTCACTGAGCCGTAGGTGAGGAATCCCTGGTTCCTACAGACTCTGCTGTA [00140] ACAATGAATGCCACATAAATATCATTTCTCAGATTCCTATGATGCTCTTCTTTCAGATCTTT [00141] [00142] TTTTTAGGTCCTGTTAACCCCCATAACTATTGATTCAGAGAAGTAGGGTGGTTCTGAAAAAT [00143] [00144] [00145] [00146] SEQ ID NO: 25 [00147] [00148] CAGGATCAGCCTCCTCCTTACCTTGCAGACCCCTTGGCTGTGGTCTGGCATTCCTAGTGGCT [00149] [00150] [00151] TTGGTATTGGTGGTTCTACCTGCTTCGGTTAAGAGTGAGGACTCTGGAGCCAGACTCTCTAA ATCATAGCACTGACACTTACTATATGACTTGGGCCAGGTTCGTTACCTGTCTGTGCCTCAGT [00152] [00153] ATGCATATAAAACATAATATAAATGTAATATTAGCTATATTCATATATAAAAATAGGTATAT TTTATATATAAAATATATTACATTTAATTTTTTACATTTTGCATTAAGATATATTATTTCAT ATAAAACATTTAAGTACATATACATATAATTATCTATGTAATTATTATTGTTATTGTTGTTA TTGTCTACTTTACCTCCCTGCAGGGATGGGGAGACCACGGCAGGCTTTTAAACATTCTTGGT CTCACTGAGCCGTAGGTGAGGAATCCCTGGTTCCTACAGACTCTGCTGTAATATGAGGAGCA [00154] ACATAAATATCATTTCTCAGATTCCTATGATGCTCTTCTTTCAGATCTTTTCACTTCAATTT [00155] [00156] GTTAACCCCCATAACTATTGATTCAGAGAAGTAGGGTGGTTCTGAAAAATACAGGCATAATC [00157] CAATAATCCATGCATTGTTTAACTCATGTCATAAGCAATAATGCCTTTCATATAGCCATTGG [00158] [00159] SEQ ID NO: 26 [00160] [00161] TCTGGATGGAGGGATCAGCAGGCCTTGGTATTGGTGGTTCTACCTGCTTCGGTTAAGAGTGA GGACTCTGGAGCCAGACTCTCTAAATCATAGCACTGACACTTACTATATGACTTGGGCCAGG TTCGTTACCTGTCTGTGCCTCAGTTTTCCTCATCTGTAAAATGGGAGATTAGTGGTCACCAT [00162] TATATACAAACAGTAACCATTTACATGCATATAAAACATAATATAAATGTAATATTAGCTAT ATTCATATATAAAAATAGGTATATTTTATATATAAAATATATTACATTTAATTTTTTACATT TTGCATTAAGATATATTATTTCATATAAAACATTTAAGTACATATACATATAATTATCTATG TAATTATTATTGTTATTGTTGTTATTGTCTACTTTACCTCCCTGCAGGGATGGGGAGACCAC GGCAGGCTTTTAAACATTCTTGGTCTCACTGAGCCGTAGGTGAGGAATCCCTGGTTCCTACA [00163] ACATTGCAATCTACAATGAATGCCACATAAATATCATTTCTCAGATTCCTATGATGCTCTTC [00164] [00165] ATCATGTGAGTATTTTTAGGTCCTGTTAACCCCCATAACTATTGATTCAGAGAAGTAGGGTG [00166] [00167] [00168] [00169] SEQ ID NO: 27 CTGACACTTACTATATGACTTGGGCCAGGTTCGTTACCTGTCTGTGCCTCAGTTTTCCTCAT [00170] ATGTATAAACTATAGTTATAATGTAAATATATATACAAACAGTAACCATTTACATGCATATA AAACATAATATAAATGTAATATTAGCTATATTCATATATAAAAATAGGTATATTTTATATAT AAAATATATTACATTTAATTTTTTACATTTTGCATTAAGATATATTATTTCATATAAAACAT TTAAGTACATATACATATAATTATCTATGTAATTATTATTGTTATTGTTGTTATTGTCTACT TTACCTCCCTGCAGGGATGGGGAGACCACGGCAGGCTTTTAAACATTCTTGGTCTCACTGAG CCGTAGGTGAGGAATCCCTGGTTCCTACAGACTCTGCTGTAATATGAGGAGCAGGGTTTAAG [00171] TCATTTCTCAGATTCCTATGATGCTCTTCTTTCAGATCTTTTCACTTCAATTTCTATAATAA [00172] [00173] CATAACTATTGATTCAGAGAAGTAGGGTGGTTCTGAAAAATACAGGCATAATCTCTTTAACT [00174] ATGCATTGTTTAACTCATGTCATAAGCAATAATGCCTTTCATATAGCCATTGGCATCAAAGA [00175] [00176] SEQ ID NO: 28 CAGTAACCATTTACATGCATATAAAACATAATATAAATGTAATATTAGCTATATTCATATAT AAAAATAGGTATATTTTATATATAAAATATATTACATTTAATTTTTTACATTTTGCATTAAG ATATATTATTTCATATAAAACATTTAAGTACATATACATATAATTATCTATGTAATTATTAT TGTTATTGTTGTTATTGTCTACTTTACCTCCCTGCAGGGATGGGGAGACCACGGCAGGCTTT TAAACATTCTTGGTCTCACTGAGCCGTAGGTGAGGAATCCCTGGTTCCTACAGACTCTGCTG [00177] CTACAATGAATGCCACATAAATATCATTTCTCAGATTCCTATGATGCTCTTCTTTCAGATCT [00178] [00179] TATTTTTAGGTCCTGTTAACCCCCATAACTATTGATTCAGAGAAGTAGGGTGGTTCTGAAAA [00180] [00181] [00182] [00183] SEQ ID NO: 29 CCTGGTTCCTACAGACTCTGCTGTAATATGAGGAGCAGGGTTTAAGTTAGTTTAAAACTGAC [00184] TATGATGCTCTTCTTTCAGATCTTTTCACTTCAATTTCTATAATAATTTTGTTTGTTTCTTG [00185] [00186] GAGAAGTAGGGTGGTTCTGAAAAATACAGGCATAATCTCTTTAACTTGTTTTATAGGAACCA [00187] [00188] [00189]

[0082] As used herein, “consisting essentially of” allows the inclusion of materials or steps that do not materially affect the basic and novel characteristics of the claim. Any recitation herein of the term “comprising”, particularly in a description of components of a composition or in a description of elements of a device, can be exchanged with the alternative expressions “consisting essentially of” or “consisting of”.

[0083] While the present invention has been described in conjunction with certain preferred embodiments, one of ordinary skill, after reading the foregoing specification, will be able to effect various changes, substitutions of equivalents, and other alterations to the compositions and methods set forth herein.

REFERENCES

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