COMBINATION TREATMENT
20240024515 ยท 2024-01-25
Inventors
- Eric Alton (London, GB)
- Jane Davies (London, GB)
- Uta Grisenbach (London, GB)
- Ana Sergijenko (London, GB)
- Deborah R. Gill (Oxford, GB)
- Stephen C. Hyde (Oxford, GB)
- Christopher Boyd (Edinburgh, GB)
- Gerry McLachlan (Midlothian, GB)
- Sebastian Kreuz (Ingelheim am Rhein, DE)
- Alena Moiseenko (Ingelheim am Rhein, DE)
Cpc classification
C12N2740/15043
CHEMISTRY; METALLURGY
A61K38/177
HUMAN NECESSITIES
A61K48/0066
HUMAN NECESSITIES
A61K31/443
HUMAN NECESSITIES
A61K31/4439
HUMAN NECESSITIES
C12N15/86
CHEMISTRY; METALLURGY
International classification
A61K48/00
HUMAN NECESSITIES
A61K31/4439
HUMAN NECESSITIES
A61K31/443
HUMAN NECESSITIES
Abstract
The present invention relates to combination treatments for cystic fibrosis, particularly combinations of modulators of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) and gene therapy.
Claims
1. A method of treating cystic fibrosis (CF) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of each of (i) a lentiviral vector pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, wherein said lentiviral vector comprises a cystic fibrosis transmembrane conductance regulator (CFTR) transgene and (ii) a CFTR modulator.
2. The method of treating CF according to claim 1, wherein the lentiviral vector is a SIV vector and the respiratory paramyxovirus is a Sendai virus.
3. The method of treating CF according to claim 1, wherein the transgene is: (i) operably linked to a promoter selected from the group consisting of a cytomegalovirus (CMV) promoter, elongation factor 1a (EF1a) promoter, and a hybrid human CMV enhancer/EF1a (hCEF) promoter; and/or (ii) a codon-optimised CFTR transgene, which optionally comprises or consist of a nucleotide sequence having at least 90% identity to SEQ ID NO: 1.
4. The method of treating CF according to claim 1, wherein the lentiviral vector comprises a hybrid human CMV enhancer/EF1a (hCEF) promoter, which optionally comprises or consist of a nucleotide sequence having at least 90% identity to SEQ ID NO: 2.
5. (canceled)
6. The method of treating CF according to claim 1, wherein the lentiviral vector is produced using codon-optimised plasmids wherein preferably the lentiviral vector is produced using (i) pGM691 and/or (ii) pGM830 or pGM326; and most preferably also using pGM299, pGM301 and/or pGM303.
7. (canceled)
8. The method of treating CF according to claim 1, wherein the lentiviral vector is vGM058, vGM195 or vGM244.
9. The method of treating CF according to claim 1, wherein the lentiviral vector is an SIV vector pseudotyped with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) proteins, wherein: said vector comprises a modified retroviral RNA sequence which comprises or consists of a nucleic acid sequence of SEQ ID NO: 16.
10. The method of treating CF according to claim 1, wherein the lentiviral vector: (i) comprises an F protein with a first subunit which comprises or consists of an amino acid sequence of SEQ ID NO: 19 and a second subunit which comprises or consists of an amino acid sequence of SEQ ID NO: 20; and/or (ii) further comprises: (a) a p17 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 22; (b) a p24 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 23; (c) a p8 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 24; (d) a protease comprising or consisting of an amino acid sequence of SEQ ID NO: 25; (e) a p51 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 26; (f) a p15 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 27, (g) a p31 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 28, (h) a Gag protein comprising or consisting of an amino acid sequence of SEQ ID NO: 29, and/or (i) a Pol protein comprising or consisting of an amino acid sequence of SEQ ID NO: 30; wherein optionally the vector comprises each of (a) to (g).
11. (canceled)
12. The method of treating CF according to claim 1, wherein the CFTR modulator is a CFTR potentiator and/or a CFTR corrector, preferably a CFTR potentiator and wherein the CFTR modulator selected from ivacaftor, tezacaftor, elexacaftor or lumacaftor, or a combination thereof, preferably ivacaftor.
13. (canceled)
14. (canceled)
15. A method of treating cystic fibrosis (CF) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of each of (A) an SIV vector pseudotyped with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) proteins, wherein: (a) said vector comprises a modified retroviral RNA sequence which comprises or consists of a nucleic acid sequence of SEQ ID NO: 16; and (b) the F protein comprises a first subunit which comprises or consists of an amino acid sequence of SEQ ID NO: 19 and a second subunit which comprises or consists of an amino acid sequence of SEQ ID NO: 20; and (B) ivacaftor.
16. The method of treating CF according to claim 1, wherein the vector further comprises one or more of: (a) a p17 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 22; (b) a p24 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 23; (c) p8 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 24; (d) a protease comprising or consisting of an amino acid sequence of SEQ ID NO: 25; (e) a p51 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 26; (f) a p15 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 27; (g) a p31 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 28; (h) a Gag protein comprising or consisting of an amino acid sequence of SEQ ID NO: 29; and/or (i) a Pol protein comprising or consisting of an amino acid sequence of SEQ ID NO: 30; wherein optionally the vector comprises each of (a) to (g).
17. The method of treating CF according to claim 1, wherein a patient to be treated has at least one class I, class II, class III, class IV, class V or class VI CFTR mutation and wherein preferably wherein the patient to be treated has at least one class I and/or class II CFTR mutation.
18. (canceled)
19. The method of treating CF according to claim 17, wherein: (a) the combination of a lentiviral vector and a CFTR modulator is suitable for use independent of the CFTR mutation of the patient; or (b) the patient to be treated has: i. at least one class I CFTR mutation selected from G542X, W1282X and/or R553C; and/or ii. at least one class II CFTR mutation selected from F508del, N1303K and/or I507del.
20. The method of treating CF according to claim 1, wherein the lentiviral vector and the CFTR modulator are administered simultaneously or sequentially.
21. The method of treating CF according to claim 1, wherein: (a) the lentiviral vector is administered by inhalation; and/or (b) the CFTR modulator is administered orally.
22. The method of treating CF according to claim 1, wherein: (a) the lentiviral vector is administered at a dose of between about 8.sup.8 to about 10.sup.14 transducing units (TU), preferably a dose of between about 10.sup.6 to about 10.sup.12 TU, wherein optionally the lentiviral vector is administered at a frequency of every 3 months, every 6 months, every 12 months, every 24 months, every 36 months or every 48 months; and/or (b) the CFTR modulator is administered at a concentration used for monotherapy of each modulator or lower.
23. The method of treating CF according to claim 1, wherein treatment restores CFTR activity to at least 10% of CFTR activity in a healthy control and wherein preferably the treatment restores CFTR activity to at least 50% of CFTR activity in a healthy control.
24. (canceled)
25. The method of treating CF according to claim 1, wherein treatment increases CFTR activity by at least 1.2 fold compared with treatment with the lentiviral vector alone and wherein preferably the treatment increases CFTR current by about 1.3 fold to about 3 fold or about 1.3 fold to about 1.8 fold compared with treatment with the lentiviral vector alone.
26. (canceled)
27. The method of treating CF according to claim 1, wherein the patient to be treated: (i) has a class I CFTR mutation and the treatment: (i) restores CFTR activity to at least 10% of CFTR activity in a healthy control; and/or (ii) increases CFTR current by about 1.3 fold to about 1.8 fold or about 1.3 fold to about 3 fold compared with treatment with the lentiviral vector alone; or (ii) has a class II CFTR mutation and the treatment: (i) restores CFTR activity to at least 10% of CFTR activity in a healthy control; and/or (ii) increases CFTR current by about 1.3 fold to about 3 fold or about 1.3 fold to about 1.8 fold compared with treatment with the lentiviral vector alone.
28. (canceled)
29. The method of treating CF according to claim 1, wherein a transduction rate of between about 10% to about 20%, preferably between about 14% to about 17% is sufficient to achieve a therapeutic effect on CFTR activity as defined in any one of claims 23 to 28.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0019]
[0020]
[0021]
[0022]
[0023]
[0024]
[0025]
[0026]
[0027]
[0028]
[0029]
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0030] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Singleton, et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20 ED., John Wiley and Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide the skilled person with a general dictionary of many of the terms used in this disclosure. The meaning and scope of the terms should be clear; however, in the event of any latent ambiguity, definitions provided herein take precedent over any dictionary or extrinsic definition.
[0031] It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such can vary. In particular, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of this disclosure.
[0032] The description of embodiments of the disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. While specific embodiments of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. For example, while method steps or functions are presented in a given order, alternative embodiments may perform functions in a different order, or functions may be performed substantially concurrently. The teachings of the disclosure provided herein can be applied to other procedures or methods as appropriate. The various embodiments described herein can be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above references and application to provide yet further embodiments of the disclosure. Moreover, due to biological functional equivalency considerations, some changes can be made in protein structure without affecting the biological or chemical action in kind or amount. These and other changes can be made to the disclosure in light of the detailed description. All such modifications are intended to be included within the scope of the appended claims.
[0033] Unless otherwise indicated, any nucleic acid sequences are written left to right in 5 to 3 orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
[0034] The headings provided herein are not limitations of the various aspects or embodiments of this disclosure.
[0035] As used herein, the term capable of when used with a verb, encompasses or means the action of the corresponding verb. For example, capable of interacting also means interacting, capable of cleaving also means cleaves, capable of binding also means binds and capable of specifically targeting . . . also means specifically targets.
[0036] Numeric ranges are inclusive of the numbers defining the range. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within this disclosure. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within this disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in this disclosure.
[0037] Amino acids are referred to herein using the name of the amino acid, the three letter abbreviation or the single letter abbreviation.
[0038] As used herein, the terms protein and polypeptide are used interchangeably herein to designate a series of amino acid residues, connected to each other by peptide bonds between the alpha-amino and carboxyl groups of adjacent residues. The terms protein, and polypeptide refer to a polymer of amino acids, including modified amino acids (e.g., phosphorylated, glycated, glycosylated, etc.) and amino acid analogues, regardless of its size or function. Protein and polypeptide are often used in reference to relatively large polypeptides, whereas the term peptide is often used in reference to small polypeptides, but usage of these terms in the art overlaps. The terms protein and polypeptide are used interchangeably herein when referring to a gene product and fragments thereof. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogues of the foregoing. In the present disclosure and claims, the conventional one-letter and three-letter codes for amino acid residues may be used. The 3-letter code for amino acids as defined in conformity with the IUPACIUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that a polypeptide may be coded for by more than one nucleotide sequence due to the degeneracy of the genetic code.
[0039] Minor variations in the amino acid sequences of the invention are contemplated as being encompassed by the present invention, providing that the variations in the amino acid sequence(s) maintain at least 60%, at least 70%, more preferably at least 80%, at least 85%, at least 90%, at least 95%, and most preferably at least 97% or at least 99% sequence identity to the amino acid sequence of the invention or a fragment thereof as defined anywhere herein. The term homology is used herein to mean identity. As such, the sequence of a variant or analogue sequence of an amino acid sequence of the invention may differ on the basis of substitution (typically conservative substitution) deletion or insertion. Proteins comprising such variations are referred to herein as variants.
[0040] Proteins of the invention may include variants in which amino acid residues from one species are substituted for the corresponding residue in another species, either at the conserved or non-conserved positions. Variants of protein molecules disclosed herein may be produced and used in the present invention. Following the lead of computational chemistry in applying multivariate data analysis techniques to the structure/property-activity relationships [see for example, Wold, et al. Multivariate data analysis in chemistry. Chemometrics-Mathematics and Statistics in Chemistry (Ed.: B. Kowalski); D. Reidel Publishing Company, Dordrecht, Holland, 1984 (ISBN 90-277-1846-6] quantitative activity-property relationships of proteins can be derived using well-known mathematical techniques, such as statistical regression, pattern recognition and classification [see for example Norman et al. Applied Regression Analysis. Wiley-Interscience; 3rd edition (April 1998) ISBN: 0471170828; Kandel, Abraham et al. Computer-Assisted Reasoning in Cluster Analysis. Prentice Hall PTR, (May 11, 1995), ISBN: 0133418847; Krzanowski, Wojtek. Principles of Multivariate Analysis: A User's Perspective (Oxford Statistical Science Series, No 22 (Paper)). Oxford University Press; (December 2000), ISBN: 0198507089; Witten, Ian H. et al Data Mining: Practical Machine Learning Tools and Techniques with Java Implementations. Morgan Kaufmann; (Oct. 11, 1999), ISBN:1558605525; Denison David G. T. (Editor) et al Bayesian Methods for Nonlinear Classification and Regression (Wiley Series in Probability and Statistics). John Wiley & Sons; (July 2002), ISBN: 0471490369; Ghose, Arup K. et al. Combinatorial Library Design and Evaluation Principles, Software, Tools, and Applications in Drug Discovery. ISBN: 0-8247-0487-8]. The properties of proteins can be derived from empirical and theoretical models (for example, analysis of likely contact residues or calculated physicochemical property) of proteins sequence, functional and three-dimensional structures and these properties can be considered individually and in combination.
[0041] Amino acid residues at non-conserved positions may be substituted with conservative or non-conservative residues. In particular, conservative amino acid replacements are contemplated.
[0042] A conservative amino acid substitution is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, or histidine), acidic side chains (e.g., aspartic acid or glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, or cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, or tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, or histidine). Thus, if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family, the amino acid substitution is considered to be conservative. The inclusion of conservatively modified variants in a protein of the invention does not exclude other forms of variant, for example polymorphic variants, interspecies homologs, and alleles.
[0043] Non-conservative amino acid substitutions include those in which (i) a residue having an electropositive side chain (e.g., Arg, His or Lys) is substituted for, or by, an electronegative residue (e.g., Glu or Asp), (ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by, a hydrophobic residue (e.g., Ala, Leu, Ile, Phe or Val), (iii) a cysteine or proline is substituted for, or by, any other residue, or (iv) a residue having a bulky hydrophobic or aromatic side chain (e.g., Val, His, Ile or Trp) is substituted for, or by, one having a smaller side chain (e.g., Ala or Ser) or no side chain (e.g., Gly).
[0044] Insertions or deletions are typically in the range of about 1, 2, or 3 amino acids. The variation allowed may be experimentally determined by systematically introducing insertions or deletions of amino acids in a protein using recombinant DNA techniques and assaying the resulting recombinant variants for activity. This does not require more than routine experiments for a skilled person.
[0045] A fragment of a polypeptide typically comprises at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 97% or more of the original polypeptide.
[0046] As used herein, the terms polynucleotides, nucleic acid and nucleic acid sequence refers to any molecule, preferably a polymeric molecule, incorporating units of ribonucleic acid, deoxyribonucleic acid or an analogue thereof. The nucleic acid can be either single-stranded or double-stranded. A single-stranded nucleic acid can be one nucleic acid strand of a denatured double-stranded DNA Alternatively, it can be a single-stranded nucleic acid not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including siRNA, shRNA, and antisense oligonucleotides. The terms transgene and gene are also used interchangeably and both terms encompass fragments or variants thereof encoding the target protein.
[0047] The transgenes of the present invention include nucleic acid sequences that have been removed from their naturally occurring environment, recombinant or cloned DNA isolates, and chemically synthesized analogues or analogues biologically synthesized by heterologous systems.
[0048] The polynucleotides of the present invention may be prepared by any means known in the art. For example, large amounts of the polynucleotides may be produced by replication in a suitable host cell. The natural or synthetic DNA fragments coding for a desired fragment will be incorporated into recombinant nucleic acid constructs, typically DNA constructs, capable of introduction into and replication in a prokaryotic or eukaryotic cell. Usually the DNA constructs will be suitable for autonomous replication in a unicellular host, such as yeast or bacteria, but may also be intended for introduction to and integration within the genome of a cultured insect, mammalian, plant or other eukaryotic cell lines.
[0049] The polynucleotides of the present invention may also be produced by chemical synthesis, e.g. by the phosphoramidite method or the tri-ester method, and may be performed on commercial automated oligonucleotide synthesizers. A double-stranded fragment may be obtained from the single stranded product of chemical synthesis either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence.
[0050] When applied to a nucleic acid sequence, the term isolated in the context of the present invention denotes that the polynucleotide sequence has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences (but may include naturally occurring 5 and 3 untranslated regions such as promoters and terminators), and is in a form suitable for use within genetically engineered protein production systems. Such isolated molecules are those that are separated from their natural environment.
[0051] In view of the degeneracy of the genetic code, considerable sequence variation is possible among the polynucleotides of the present invention. Degenerate codons encompassing all possible codons for a given amino acid are set forth below:
TABLE-US-00001 Amino Degenerate Acid Codons Codon Cys TGCTGT TGY Ser AGCAGTTCATCCTCGTCT WSN Thr ACAACCACGACT ACN Pro CCACCCCCGCCT CCN Ala GCAGCCGCGGCT GCN Gly GGAGGCGGGGGT GGN Asn AACAAT AAY Asp GACGAT GAY Glu GAAGAG GAR Gln CAACAG CAR His CACCAT CAY Arg AGAAGGCGACGCCGGCGT MGN Lys AAAAAG AAR Met ATG ATG Ile ATAATCATT ATH Leu CTACTCCTGCTTTTATTG YTN Val GTAGTCGTGGTT GTN Phe TTCTTT TTY Tyr TACTAT TAY Trp TGG TGG Ter TAATAGTGA TRR Asn/Asp RAY Glu/Gln SAR Any NNN
[0052] One of ordinary skill in the art will appreciate that flexibility exists when determining a degenerate codon, representative of all possible codons encoding each amino acid. For example, some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequences of the present invention.
[0053] A variant nucleic acid sequence has substantial homology or substantial similarity to a reference nucleic acid sequence (or a fragment thereof). A nucleic acid sequence or fragment thereof is substantially homologous (or substantially identical) to a reference sequence if, when optimally aligned (with appropriate nucleotide insertions or deletions) with the other nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 70%, 75%, 80%, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or more % of the nucleotide bases. Methods for homology determination of nucleic acid sequences are known in the art.
[0054] Alternatively, a variant nucleic acid sequence is substantially homologous with (or substantially identical to) a reference sequence (or a fragment thereof) if the variant and the reference sequence they are capable of hybridizing under stringent (e.g. highly stringent) hybridization conditions. Nucleic acid sequence hybridization will be affected by such conditions as salt concentration (e.g. NaCl), temperature, or organic solvents, in addition to the base composition, length of the complementary strands, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. Stringent temperature conditions are preferably employed, and generally include temperatures in excess of typically in excess of 37 C. and preferably in excess of 45 C. Stringent salt conditions will ordinarily be less than 1000 mM, typically less than 500 mM, and preferably less than 200 mM. The pH is typically between 7.0 and 8.3. The combination of parameters is much more important than any single parameter.
[0055] Methods of determining nucleic acid percentage sequence identity are known in the art. By way of example, when assessing nucleic acid sequence identity, a sequence having a defined number of contiguous nucleotides may be aligned with a nucleic acid sequence (having the same number of contiguous nucleotides) from the corresponding portion of a nucleic acid sequence of the present invention. Tools known in the art for determining nucleic acid percentage sequence identity include Nucleotide BLAST (as described below).
[0056] One of ordinary skill in the art appreciates that different species exhibit preferential codon usage. As used herein, the term preferential codon usage refers to codons that are most frequently used in cells of a certain species, thus favouring one or a few representatives of the possible codons encoding each amino acid. For example, the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian host cells ACC is the most commonly used codon; in other species, different codons may be preferential. Preferential codons for a particular host cell species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Thus, according to the invention, in addition to the gag-pol genes any nucleic acid sequence may be codon-optimised for expression in a host or target cell. In particular, the vector genome (or corresponding plasmid), the REV gene (or corresponding plasmid), the fusion protein (F) gene (or correspond plasmid) and/or the hemagglutinin-neuraminidase (HN) gene (or corresponding plasmid, or any combination thereof may be codon-optimised.
[0057] A fragment of a polynucleotide of interest comprises a series of consecutive nucleotides from the sequence of said full-length polynucleotide. By way of example, a fragment of a polynucleotide of interest may comprise (or consist of) at least 30 consecutive nucleotides from the sequence of said polynucleotide (e.g. at least 35, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 850, 900, 950 or 1000 consecutive nucleic acid residues of said polynucleotide). A fragment may include at least one antigenic determinant and/or may encode at least one antigenic epitope of the corresponding polypeptide of interest. Typically, a fragment as defined herein retains the same function as the full-length polynucleotide.
[0058] The terms increased, increase, enhance, or activate are all used herein to mean an increase by a statically significant amount. The terms increased, increase, enhance, or activate can mean an increase of at least 10% as compared to a reference level, for example an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 10-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level. In the context of a yield or titre, an increase is an observable or statistically significant increase in such level.
[0059] The terms decrease, reduced, reduction, or inhibit are all used herein to mean a decrease by a statistically significant amount. The terms reduce, reduction or decrease or inhibit typically means a decrease by at least 10% as compared to a reference level (e.g. the absence of a given treatment) and can include, for example, a decrease by at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, or more. As used herein, reduction or inhibition encompasses a complete inhibition or reduction as compared to a reference level. Complete inhibition is a 100% inhibition (i.e. abrogation) as compared to a reference level.
[0060] It must be noted that as used herein and in the appended claims, the singular forms a, an, and the include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to a CFTR modulator includes a plurality of such agents and reference to the CFTR modulator includes reference to one or more CFTR modulators and equivalents thereof known to those skilled in the art, and so forth. Furthermore, the use of the term including, as well as other forms, such as includes and included, is not limiting.
[0061] About may generally mean an acceptable degree of error for the quantity measured given the nature or precision of the measurements. Exemplary degrees of error are within 20 percent (%), typically, within 10%, and more typically, within 5% of a given value or range of values. Preferably, the term about shall be understood herein as plus or minus () 5%, preferably 4%, 3%, 2%, 1%, 0.5%, 0.1%, of the numerical value of the number with which it is being used.
[0062] The term consisting of refers to compositions, methods, and respective components thereof as described herein, which are exclusive of any element not recited in that description of the invention.
[0063] As used herein the term consisting essentially of refers to those elements required for a given invention. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristic(s) of that invention (i.e. inactive or non-immunogenic ingredients).
[0064] Embodiments described herein as comprising one or more features may also be considered as disclosure of the corresponding embodiments consisting of and/or consisting essentially of such features.
[0065] Concentrations, amounts, volumes, percentages and other numerical values may be presented herein in a range format. It is also to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited.
[0066] As used herein, the terms vector, retroviral vector and retroviral F/HN vector are used interchangeably to mean a retroviral vector comprising a retroviral RNA sequence and pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, unless otherwise stated. The terms lentiviral vector and lentiviral F/HN vector are used interchangeably to mean a lentiviral vector pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, unless otherwise stated. All disclosure herein in relation to retroviral vectors of the invention applies equally and without reservation to lentiviral vectors of the invention and to SIV vectors that are pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus (also referred to herein as SIV F/HN or SIV-FHN).
[0067] As defined herein, the term retroviral RNA sequence refers to the nucleic acid molecule that is contained within a retroviral vector. A retroviral RNA sequence comprises long terminal repeat (LTR) elements, nucleic acid sequences necessary for incorporation of the retroviral RNA sequence into retroviral particles, and the transgene expression cassette. The transgene expression cassette is comprised of a suitable enhancer/promoter element, the transgene cDNA and a posttranscriptional regulatory element. The retroviral RNA sequence starts with a 5 LTR R sequence and ends with a 3 LTR R sequence. The 5 region retroviral RNA sequence typically comprises or consists of a retroviral LTR R sequence followed by a retroviral LTR U5 sequence (in 5 to 3 order). The 3 region retroviral RNA sequence typically comprises or consists of a retroviral LTR R sequence followed by a retroviral LTR U5 sequence (in 5 to 3 order).
[0068] The terms DNA provirus or DNA provirus sequence and DNA proviral sequence refer interchangeably to the DNA sequence which is integrated into the genome of cells transduced with the retrovirus. The DNA provirus sequence contains additional regions of nucleic acid that are not found within the retroviral RNA sequence, including a 5 LTR U3 sequence and a 3 LTR U5 sequence. Therefore, the sequences of the DNA provirus and the retroviral RNA sequence are not identical, but rather the sequence of the retroviral RNA sequence is shorter than the proviral DNA sequence from which it is derived. The precise 5 and 3 limits of the retroviral RNA sequence compared with the proviral DNA sequence from which it is derived cannot readily and reliably be determined by simple analysis of the proviral DNA sequence.
[0069] The terms individual, subject, and patient, are used interchangeably herein to refer to a mammalian subject for whom diagnosis, prognosis, disease monitoring, treatment, therapy, and/or therapy optimisation is desired. The mammal can be (without limitation) a human, non-human primate, mouse, rat, dog, cat, horse, or cow. In a preferred embodiment, the individual, subject, or patient is a human. An individual may be an adult, juvenile or infant. An individual may be male or female.
[0070] A subject in need of treatment for a particular condition can be an individual having that condition, diagnosed as having that condition, or at risk of developing that condition.
[0071] A subject can be one who has been previously diagnosed with or identified as suffering from or having a condition in need of treatment or one or more complications related to such a condition, and optionally, have already undergone treatment for a condition as defined herein or the one or more complications related to said condition. Alternatively, a subject can also be one who has not been previously diagnosed as having a condition as defined herein or one or more complications related to said condition. For example, an individual can be one who exhibits one or more risk factors for a condition, or one or more complications related to said condition or a subject who does not exhibit risk factors.
[0072] As used herein, the term healthy individual refers to an individual or group of individuals who are in a healthy state, e.g. individuals who have not shown any symptoms of the disease, have not been diagnosed with the disease and/or are not likely to develop the disease e.g. cystic fibrosis (CF) or any other disease described herein). Preferably said healthy individual(s) is not on medication affecting CF and has not been diagnosed with any other disease. The one or more healthy individuals may have a similar sex, age, and/or body mass index (BMI) as compared with the test individual. Application of standard statistical methods used in medicine permits determination of normal levels of expression in healthy individuals, and significant deviations from such normal levels.
[0073] Herein the terms control and reference population are used interchangeably.
[0074] The term pharmaceutically acceptable as used herein means approved by a regulatory agency of the Federal or a state government, or listed in the U.S. Pharmacopeia, European Pharmacopeia or other generally recognized pharmacopeia.
[0075] Other definitions of terms may appear throughout the specification. Before the exemplary embodiments are described in more detail, it is to be understood that this disclosure is not limited to particular embodiments described, and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be defined only by the appended claims.
[0076] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that such publications constitute prior art to the claims appended hereto. All references cited in this specification are herewith incorporated by reference with respect to their entire disclosure content and the disclosure content specifically mentioned in this specification.
[0077] Disclosure related to the various methods of the invention are intended to be applied equally to other methods, therapeutic uses or methods, and vice versa.
Retroviral and Lentiviral Vectors
[0078] The invention relates to combination therapies comprising a retroviral/lentiviral (e.g. SIV) construct. The term retrovirus refers to any member of the Retroviridae family of RNA viruses that encode the enzyme reverse transcriptase. The term lentivirus refers to a family of retroviruses. Examples of retroviruses suitable for use in the present invention include gammaretroviruses such as murine leukaemia virus (MLV) and feline leukaemia virus (FLV). Examples of lentiviruses suitable for use in the present invention include Simian immunodeficiency virus (SIV), Human immunodeficiency virus (HIV), Feline immunodeficiency virus (FIV), Equine infectious anaemia virus (EIAV), and Visna/maedi virus. Typically the invention relates to combination therapies comprising lentiviral vectors, particularly combination therapies comprising an SIV vector (including all strains and subtypes), such as a SIV-AGM (originally isolated from African green monkeys, Cercopithecus aethiops).
[0079] The retroviral/lentiviral (e.g. SIV) vectors of the present invention are pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus. Preferably the respiratory paramyxovirus is a Sendai virus (murine parainfluenza virus type 1).
[0080] The F protein may be a truncated F protein, typically one in which the cytoplasmic domain is truncated. Preferably the truncated F protein is Fct4, in which 38 amino acids have been truncated from the C-terminus of the F protein, with 4 amino acids of the F protein cytoplasmic domain being retained. Thus, the F protein may comprise or consist of an Fct4 amino acid sequence having at least 70%, at least 80%, 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%, at least 99.5%, at least 99.9%, or more, up to 100% identity to SEQ ID NO: 17 or 18. Preferably the F protein may comprise or consist of an Fct4 amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 17 or 18.
[0081] The full length F protein, or C-terminally truncated form thereof (e.g. Fct4) is typically fusion inactive. The fusion inactive form of the F protein may be cleaved to produce two subunits, a first subunit, (also known as F.sub.2) and a second subunit (also known as F.sub.1).
[0082] The first subunit of the F protein may comprise or consist of an amino acid sequence having at least 70%, at least 80%, 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%, at least 99.5%, at least 99.9%, or more, up to 100% identity to SEQ ID NO: 19. Preferably the first subunit may be a subunit which may comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 19. SEQ ID NO: 19 is the first subunit of Fct4.
[0083] Alternatively or in addition, preferably in addition, the second subunit of the F protein may comprise or consist of an amino acid sequence having at least 70%, at least 80%, 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%, at least 99.5%, at least 99.9%, or more, up to 100% identity to SEQ ID NO: 20. Preferably the second subunit may be a subunit which may comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 20. SEQ ID NO: 20 is the second subunit of Fct4.
[0084] The F protein (e.g. Fct4) may comprise an N-terminal signal peptide. Alternatively, the F protein may lack such a signal peptide. The F protein signal peptide may comprise or consist of an amino acid sequence having at least 70%, at least 80%, 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%, at least 99.5%, at least 99.9%, or more, up to 100% identity to SEQ ID NO: 21. This signal peptide may be cleaved to form the mature F protein. The signal peptide of Fct4 is SEQ ID NO: 21, which forms amino acid residues 1-25 of SEQ ID NO: 18. Thus, the mature form of Fct4 may comprise or consist of an amino acid sequence having at least 70%, at least 80%, 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%, at least 99.5%, at least 99.9%, or more, up to 100% identity to amino acid residues 26-527 of SEQ ID NO: 18.
[0085] The HN protein may be a truncated and/or chimeric HN protein, typically one in which the cytoplasmic domain is truncated or substituted. Preferably, the HN protein is a chimeric HN protein in which (i) the cytoplasmic domain of the HN is replaced by the cytoplasmic domain of the transmembrane (TMP) protein; or (ii) the cytoplasmic domain of the TMP is added to the cytoplasmic domain of the HN protein. The HN protein may be as described in Kobayashi et al. (J. Virol. (2003) 77(4):2607-2614), which is herein incorporated by reference in its entirety.
[0086] The retroviral/lentiviral (e.g. SIV) vectors of the invention may comprise a codon-optimised Gag protein, a codon-optimised Pol protein, a codon-optimised GagPol polyprotein, or a combination thereof. Accordingly, the invention provides a retroviral/lentiviral (e.g. SIV) vector comprising a codon-optimised Gag protein comprising or consisting of an amino acid sequence having at least 70%, at least 80%, 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%, at least 99.5%, at least 99.9%, or more, up to 100% sequence identity to SEQ ID NO: 29. Preferably, the invention provides a retroviral vector comprising a codon-optimised Gag protein comprising or consisting of an amino acid sequence having at least 90%, at least 95%, or at least 99% identity to SEQ ID NO: 29. The invention provides a retroviral vector comprising a codon-optimised Pol protein comprising or consisting of an amino acid sequence having at least 70%, at least 80%, 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%, at least 99.5%, at least 99.9%, or more, up to 100% sequence identity to SEQ ID NO: 30. Preferably, the invention provides a retroviral vector comprising a codon-optimised Pol protein comprising or consisting of an amino acid sequence having a at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 30.
[0087] GagPol is expressed as polyprotein which is processed to produce a number of smaller proteins within viral particles. The extent of processing, and hence the presence and/or concentration of GagPol or any of the constituent proteins within a retroviral/lentiviral (e.g. SIV) vector of the invention may vary with time.
[0088] Accordingly, a retroviral/lentiviral (e.g. SIV) vector of the invention may comprise one or more of a p17 protein, a p27 protein, a p8 protein, a protease, a p51 protein, a p15 protein and a p31 protein. One or more of these proteins may be present in combination with Gag, Pol and/or GagPol. Preferably, the invention provides a retroviral vector comprising a p17 protein, a p27 protein, a p8 protein, a protease, a p51 protein, a p15 protein and a p31 protein. Again, these proteins may be present in combination with Gag, Pol and/or GagPol.
[0089] The p17 protein may comprise or consist of an amino acid sequence having at least 70%, at least 80%, 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%, at least 99.5%, at least 99.9%, or more, up to 100% sequence identity to SEQ ID NO: 22. Preferably, the p17 protein comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 22.
[0090] The p24 protein may comprise or consist of an amino acid sequence having at least 70%, at least 80%, 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%, at least 99.5%, at least 99.9%, or more, up to 100% sequence identity to SEQ ID NO: 23. Preferably, the p24 protein comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 23.
[0091] The p8 protein may comprise or consist of an amino acid sequence having at least 70%, at least 80%, 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%, at least 99.5%, at least 99.9%, or more, up to 100% sequence identity to SEQ ID NO: 24. Preferably, the p8 protein comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 24.
[0092] The protease may comprise or consist of an amino acid sequence having at least 70%, at least 80%, 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%, at least 99.5%, at least 99.9%, or more, up to 100% sequence identity to SEQ ID NO: 25. Preferably, the protease comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 25.
[0093] The p51 protein may comprise or consist of an amino acid sequence having at least 70%, at least 80%, 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%, at least 99.5%, at least 99.9%, or more, up to 100% sequence identity to SEQ ID NO: 26. Preferably, the p51 protein comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 26.
[0094] The p15 protein may comprise or consist of an amino acid sequence having at least 70%, at least 80%, 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%, at least 99.5%, at least 99.9%, or more, up to 100% sequence identity to SEQ ID NO: 27. Preferably, the p15 protein comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 27.
[0095] The p31 protein may comprise or consist of an amino acid sequence having at least 70%, at least 80%, 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%, at least 99.5%, at least 99.9%, or more, up to 100% sequence identity to SEQ ID NO: 28. Preferably, the p31 protein comprises or consists of an amino acid sequence having at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 28.
[0096] Retroviral/lentiviral (e.g. SIV) vectors of the invention may comprise a p17 protein comprising or consisting of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 22 (as described above), a p24 protein comprising or consisting of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 23 (as described above), a p8 protein comprising or consisting of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 24 (as described above), a protease comprising or consisting of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 25 (as described above), a p51 protein comprising or consisting of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 26 (as described above), a p15 protein comprising or consisting of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 27 (as described above), and a p31 protein comprising or consisting of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 28 (as described above).
[0097] A retroviral/lentiviral (e.g. SIV) vector produced according to the invention may be integrase-competent (IC). Alternatively, the lentiviral (e.g. SIV) vector may be integrase-deficient (ID).
[0098] Retroviral/Lentiviral vectors, such as those used in combination therapies according to the invention, can integrate into the genome of transduced cells and lead to long-lasting expression, making them suitable for transduction of stem/progenitor cells. In the lung, several cell types with regenerative capacity have been identified as responsible for maintaining specific cell lineages in the conducting airways and alveoli. These include basal cells and submucosal gland duct cells in the upper airways; ciliated, goblet, club cells (SCGB1A1+) and neuroendocrine cells in the bronchiolar airways; bronchioalveolar stem cells in the terminal bronchioles; and type II pneumocytes in the alveoli. Therefore, and without being bound by theory, it is believed that said retroviral/lentiviral (e.g. SIV) vectors bring about long term gene expression of the transgene of interest by introducing the transgene into one or more cell type of the airway epithelium, such as those listed above. It is predicted that cells of the airway epithelium have a lifespan of many months, such that transfection of these cells facilitates expression for the lifespan of the cells, having a long-term therapeutic effect.
[0099] Accordingly, the retroviral/lentiviral (e.g. SIV) vectors used in combination therapies according to the invention typically transduce one or more cell types or cell lineages within the airway epithelium. These cells may (or may not) have regenerative potential, but rather prolonged expression results from the long lifespan of the transduced cells. For example, the retroviral/lentiviral (e.g. SIV) vectors may transduce one or more cell type selected from: (i) basal cells and/or submucosal gland duct cells in the upper airways; (ii) ciliated, goblet, club cells (SCGB1A1+) and/or neuroendocrine cells in the bronchiolar airways; (iii) bronchioalveolar stem cells in the terminal bronchioles; and/or (iv) type II pneumocytes in the alveoli; or any combination thereof.
[0100] Alternatively or in addition, the retroviral/lentiviral (e.g. SIV) vectors used in combination therapies according to the invention may transduce one or more cell types or cell lineages with regenerative potential within the lung (including the airways and respiratory tract) to achieve long term gene expression. For example, the retroviral/lentiviral (e.g. SIV) vectors may transduce basal cells, such as those in the upper airways/respiratory tract. Basal cells have a central role in processes of epithelial maintenance and repair following injury. In addition, basal cells are widely distributed along the human respiratory epithelium, with a relative distribution ranging from 30% (larger airways) to 6% (smaller airways).
[0101] The retroviral/lentiviral (e.g. SIV) vectors may be used to transduce isolated and expanded stem/progenitor cells ex vivo prior administration to a patient as part of a combination therapy as described herein. Preferably, the retroviral/lentiviral (e.g. SIV) vectors are used to transduce cells within the lung (or airways/respiratory tract) in vivo.
[0102] The retroviral/lentiviral (e.g. SIV) vectors of the invention demonstrate remarkable resistance to shear forces with only modest reduction in transduction ability when passaged through clinically-relevant delivery devices such as spray bottles and nebulisers. Other inhalative routes of administration, such as by bronchoscope, may similarly benefit from the retroviral/lentiviral (e.g. SIV) vectors of the invention shear force resistance.
[0103] A retroviral/lentiviral (e.g. SIV) vector of the invention may comprise one or more transgene that encodes a polypeptide or protein that is therapeutic for the treatment of CF. Preferably a retroviral/lentiviral (e.g. SIV) vector of the invention comprises a CFTR transgene, i.e. the transgene encodes a CFTR.
[0104] The transgene included in the vector of the invention may be modified to facilitate expression. For example, the transgene sequence may be in CpG-depleted (or CpG-fee) and/or codon-optimised form to facilitate gene expression. Standard techniques for modifying the transgene sequence in this way are known in the art.
[0105] Accordingly, an example of a CFTR cDNA is provided by SEQ ID NO: 1. Variants thereof (as described therein) are also included, particularly variants with at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to SEQ ID NO: 1. SEQ ID NO: 1 is a codon-optimized CpG depleted CFTR transgene previously designed by the present inventors to enhance translation in human cells. Variants of same sequence (as defined herein) which possess the same technical effect of enhancing translation compared with the unmodified (wild-type) CFTR gene sequence are also encompassed by the present invention.
[0106] The retroviral/lentiviral (e.g. SIV) vectors of the present invention enable high levels of transgene expression, resulting in high levels (therapeutic levels) of expression of a therapeutic protein. As such, the retroviral/lentiviral (e.g. SIV) vectors of the present invention may usefully provide high expression levels of a transgene when administered to a patient. The terms high expression and therapeutic expression are used interchangeably herein. Expression may be measured by any appropriate method (qualitative or quantitative, preferably quantitative), and concentrations given in any appropriate unit of measurement, for example ng/ml or nM.
[0107] Expression of a transgene of interest may be given relative to the expression of the corresponding endogenous (defective) gene in a patient. Expression may be measured in terms of DNA vector copy number (VCN), mRNA or protein expression. The expression of the transgene of the invention, such as a functional CFTR gene, may be quantified relative to the endogenous gene, such as the endogenous (dysfunctional) CFTR genes in terms of mRNA copies per cell or any other appropriate unit.
[0108] Expression levels of a CFTR transgene and/or the encoded CFTR protein of the invention may be measured in the lung tissue. A high and/or therapeutic expression level may therefore refer to the concentration in the lung, epithelial lining fluid and/or serum/plasma.
[0109] The retroviral/lentiviral (e.g. SIV) vectors of the invention exhibit efficient airway cell uptake, stable transgene expression, and suffer no loss of efficacy upon repeated administration. Accordingly, the retroviral/lentiviral (e.g. SIV) vectors of the invention are capable of producing long-lasting, repeatable, high-level expression in airway cells without inducing an undue immune response. An undue immune response may be defined as one extreme enough to preclude administration to a patient and/or to elicit a significant negative effect on vector transduction and/or CFTR expression.
[0110] The retroviral/lentiviral (e.g. SIV) vectors of the present invention enable long-term transgene expression, resulting in long-term expression of a therapeutic protein. As described herein, the phrases long-term expression, sustained expression, long-lasting expression and persistent expression are used interchangeably. Long-term expression according to the present invention means expression of a therapeutic gene and/or protein, preferably at therapeutic levels, for at least days, at least 60 days, at least 90 days, at least 120 days, at least 180 days, at least 250 days, at least 360 days, at least 450 days, at least 730 days or more. Preferably long-term expression means expression for at least 90 days, at least 120 days, at least 180 days, at least 250 days, at least 360 days, at least 450 days, at least 720 days or more, more preferably at least 360 days, at least 450 days, at least 720 days or more. This long-term expression may be achieved by repeated doses or by a single dose.
[0111] Repeated doses may be administered twice-daily, daily, twice-weekly, weekly, monthly, every two months, every three months, every four months, every six months, yearly, every two years, or more. Dosing may be continued for as long as required, for example, for at least six months, at least one year, two years, three years, four years, five years, ten years, fifteen years, twenty years, or more, up to for the lifetime of the patient to be treated.
[0112] The retroviral/lentiviral (e.g. SIV) vector comprises a promoter operably linked to a transgene, enabling expression of the transgene. Typically the promoter is a hybrid human CMV enhancer/EF1a (hCEF) promoter. This hCEF promoter may lack the intron corresponding to nucleotides 570-709 and the exon corresponding to nucleotides 728-733 of the hCEF promoter. A preferred example of an hCEF promoter sequence of the invention is provided by SEQ ID NO: 2. Thus, a hCEF promoter comprised in a retroviral/lentiviral (e.g. SIV) vector of the invention may comprise (or consist of) a nucleic acid sequence having at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the hCEF nucleic acid sequence of SEQ ID NO: 2. In a further embodiment, the hCEF may comprise (or consist of) a nucleic acid sequence having at least 95% (such as at least 95, 96, 97, 98, 99 or 100%) sequence identity to the hCEF nucleic acid sequence of SEQ ID NO: 2. Alternatively, the promoter may be a CMV promoter. An example of a CMV promoter sequence is provided by SEQ ID NO: 3. The promoter may be a human elongation factor 1a (EF1a) promoter. An example of a EF1a promoter is provided by SEQ ID NO: 4. Other promoters for transgene expression are known in the art and their suitability for the retroviral/lentiviral (e.g. SIV) vectors of the invention determined using routine techniques known in the art. Non-limiting examples of other promoters include UbC and UCOE. As described herein, the promoter may be modified to further regulate expression of the transgene of the invention.
[0113] The promoter included in the retroviral/lentiviral (e.g. SIV) vector of the invention may be specifically selected and/or modified to further refine regulation of expression of the therapeutic gene. Again, suitable promoters and standard techniques for their modification are known in the art. As a non-limiting example, a number of suitable (CpG-free) promoters suitable for use in the present invention are described in Pringle et al. (J. Mol. Med. Berl. 2012, 90(12): 1487-96), which is herein incorporated by reference in its entirety. Preferably, the retroviral/lentiviral vectors (particularly SIV F/HN vectors) of the invention comprise a hCEF promoter having low or no CpG dinucleotide content. The hCEF promoter may have all CG dinucleotides replaced with any one of AG, TG or GT. Thus, the hCEF promoter may be CpG-free. A preferred example of a CpG-free hCEF promoter sequence of the invention is provided by SEQ ID NO: 2. The absence of CpG dinucleotides further improves the performance of retroviral/lentiviral (e.g. SIV) vectors of the invention and in particular in situations where it is not desired to induce an immune response against an expressed antigen or an inflammatory response against the delivered expression construct. The elimination of CpG dinucleotides reduces the occurrence of flu-like symptoms and inflammation which may result from administration of constructs, particularly when administered to the airways.
[0114] The retroviral/lentiviral (e.g. SIV) vector of the invention may be modified to allow shut down of gene expression. Standard techniques for modifying the vector in this way are known in the art. As a non-limiting example, Tet-responsive promoters are widely used.
[0115] Thus, the invention relates to F/HN retroviral/lentiviral vectors comprising a promoter and a transgene, particularly SIV.F/HN vectors. The F/HN pseudotyping is particularly efficient at targeting cells in the airway epithelium, and as such, for therapeutic applications it is typically delivered to cells of the respiratory tract, including the cells of the airway epithelium. Accordingly, the retroviral/lentiviral (e.g. SIV) vectors of the invention are particularly suited for treatment of CF.
[0116] The retroviral/lentiviral (e.g. SIV) vector of the invention may have no intron positioned between the promoter and the transgene. Similarly, there may be no intron between the promoter and the transgene in the vector genome (pDNA1) plasmid (for example, pGM326 as described herein, illustrated in
[0117] Preferably, the retroviral/lentiviral (e.g. SIV) vector comprises a hCEF promoter and a CFTR transgene, including those described herein. Optionally said retroviral/lentiviral (e.g. SIV) vector may have no intron positioned between the promoter and the transgene. Such a retroviral/lentiviral (e.g. SIV) vector may be produced by the method described herein, using a genome plasmid carrying the CFTR transgene and a promoter. Particularly preferred is a SIV.F/HN vector with a hCEF promoter and a CFTR transgene, including those described herein.
[0118] The retroviral/lentiviral (e.g. SIV) vector as described herein comprises at least one transgene. The transgene comprises a nucleic acid sequence encoding a gene product, e.g., a protein, particularly a therapeutic protein, preferably said at least one transgene comprises or consists of CFTR.
[0119] For example, the nucleic acid sequence encoding a CFTR comprises (or consists of) a nucleic acid sequence having at least 90% (such as at least 90, 92, 94, 95, 96, 97, 98, 99 or 100%) sequence identity to the CFTR nucleic acid sequence respectively, examples of which are described herein. In a further embodiment, the nucleic acid sequence encoding CFTR comprises (or consists of) a nucleic acid sequence having at least 95% (such as at least 95, 96, 97, 98, 99 or 100%) sequence identity to the CFTR nucleic acid sequence respectively, examples of which are described herein. In one embodiment, the nucleic acid sequence encoding CFTR is provided by SEQ ID NO: 1, or variants thereof.
[0120] The amino acid sequence of the CFTR encoded by the CFTR transgene may comprise (or consist of) an amino acid sequence having at least 95% (such as at least 95, 96, 97, 98, 99 or 100%) sequence identity to the functional CFTR polypeptide sequence respectively.
[0121] The retroviral/lentiviral (e.g. SIV) vectors of the invention may comprise a central polypurine tract (cPPT) and/or the Woodchuck hepatitis virus posttranscriptional regulatory elements (WPRE). An exemplary WPRE sequence is provided by SEQ ID NO: 14.
[0122] The retroviral/lentiviral (e.g. SIV) vectors according to the invention may be as described in WO 2015/177501, International Application No. PCT/GB2022/050524 (which claims priority from UK Patent Application No. 2102832.9) and UK Patent Application No. 2212472.1, each of which is herein incorporated by reference in its entirety. Particularly preferred is a retroviral/lentiviral (e.g. SIV) vectors according to UK Patent Application No. 2212472.1.
[0123] Thus, particularly preferred is a retroviral/lentiviral (e.g. SIV) vector which is an SIV vector pseudotyped with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) proteins, wherein: (a) said vector comprises a modified retroviral RNA sequence which comprises or consists of a nucleic acid sequence of SEQ ID NO: 16 (which comprises a CFTR transgene), preferably wherein the modified retroviral RNA sequence consists of a nucleic acid sequence of SEQ ID NO: 16; and (b) the F protein comprises a first subunit which comprises or consists of an amino acid sequence of SEQ ID NO: 19 and a second subunit which comprises or consists of an amino acid sequence of SEQ ID NO: 20. Said vector may further comprise one or more of: (a) a p17 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 22; (b) a p24 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 23; (c) p8 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 24; (d) a protease comprising or consisting of an amino acid sequence of SEQ ID NO: 25; (e) a p51 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 26; (f) a p15 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 27; (g) a p31 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 28; (h) a Gag protein comprising or consisting of an amino acid sequence of SEQ ID NO: 29; and/or (i) a Pol protein comprising or consisting of an amino acid sequence of SEQ ID NO: 30; wherein optionally the vector comprises each of (a) to (g).
Methods of Making Retroviral/Lentiviral (e.g. SIV) Vectors
[0124] The retroviral/lentiviral (e.g. SIV) vectors of the invention may be produced by any appropriate method. Non-limiting examples of such methods are described in WO 2015/177501, International Application No. PCT/GB2022/050524 (which claims priority from UK Patent Application No. 2102832.9) and UK Patent Application No. 2212472.1, each of which is herein incorporated by reference in its entirety. Particularly preferred is a method as described in UK Patent Application No. 2212472.1.
[0125] The retroviral/lentiviral (e.g. SIV) vectors of the invention are typically produced by a scalable GMP-compatible method. Exemplary methods are described herein. The present invention encompasses combination therapies comprising the use of retroviral/lentiviral (e.g. SIV) vectors, particularly SIV.F/HN vectors obtained or obtainable by any method described herein.
[0126] The production of retroviral/lentiviral (e.g. SIV) vectors typically employs one or more plasmids which provide the elements needed for the production of the vector: the genome for the retroviral/lentiviral vector, the Gag-Pol, Rev, F and HN. Multiple elements can be provided on a single plasmid. Preferably each element is provided on a separate plasmid, such that there are five plasmids, one for each of the vector genome, the Gag-Pol, Rev, F and HN, respectively.
[0127] Alternatively, a single plasmid may provide the Gag-Pol and Rev elements, and may be referred to as a packaging plasmid (pDNA2). The remaining elements (genome, F and HN) may be provided by separate plasmids (pDNA1, pDNA3a, pDNA3b respectively), such that four plasmids are used for the production of a retroviral/lentiviral (e.g. SIV) vector according to the invention. In the four plasmid methods, pDNA1, pDNA3a and pDNA3b may be as described herein in the context of the five-plasmid method.
[0128] Any one of the plasmids used in the production of a retroviral/lentiviral (e.g. SIV) vectors of the invention may independently be codon optimised or at least partially codon optimised. Partial codon-optimisation encompasses at least 50%, at least 60%, at least 70%, at least 80%, at least 95%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more codon optimisation. Codon optimisation is a technique to maximise protein expression by increasing the translational efficiency of the encoding gene. Translational efficiency is increased by modification of the nucleic acid sequence. Codon optimisation is routine in the art, and it is within the routine practice of one of ordinary skill to devise a codon-optimised version of a given nucleic acid sequence. As described herein, the transgene and/or promoter may each independently be codon-optimised. Alternatively or in addition, the Gag-Pol genes may be codon-optimised. In particular, codon-optimisation of the Gag-Pol genes is preferred as can improve the safety profile of the resulting retroviral/lentiviral (e.g. SIV) vectors, particularly SIV.F/HN vectors, without negatively impacting the vector titre, and can even increase vector titre (as described in International Application No. PCT/GB2022/050524, which claims priority from UK Patent Application No. 2102832.9).
[0129] Accordingly, the retroviral/lentiviral (e.g. SIV) vectors of the invention, particularly those pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from a respiratory paramyxovirus, and comprising a promoter and a transgene, are produced by a method which comprises the use of codon-optimised gag-pol genes. Preferably the codon-optimised gag-pol genes used in the production methods of the invention are SIV gag-pol genes. Exemplary wild-type SIV gag-pol genes that may be modified to produce codon-optimised gag-pol genes are given in SEQ ID NO: 5. Exemplary codon-optimised gag-pol genes derived from SEQ ID NO: 5 are given in SEQ ID NO: 6. In addition to codon-optimisation, the codon-optimised gag-pol genes used in the production methods of the invention may comprise other modifications, such as a translational slip (which allows translation to slip from one region to another to allow the production of both Gag and Pol). Any suitable variation of codon usage may be used in the codon-optimised gag-pol genes of the invention, provided that (i) homology between the vector genome plasmid and GagPol plasmid is reduced to minimise the risk of RCL production and (ii) after codon optimisation there is production of sufficient GagPol without the inclusion of RRE (this further reduces homology and the risk of RCL production).
[0130] The codon-optimised gag-pol genes used in the production methods of the invention may be completely (100%) or partially codon-optimised. Partial codon-optimisation of the gag-pol genes encompasses at least 50%, at least 60%, at least 70%, at least 80%, at least 95%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more codon optimisation.
[0131] Preferably, the gag-pol genes themselves are completely codon-optimised, but may comprise non-contain regions of non-codon-optimised sequence (e.g. between the gag and pol genes). By way of non-limiting example, to maintain the translational slip of reading frames between the gag and pol genes, the region around the translational slip sequence may not be codon-optimised (e.g. in case the precise translational slip sequence is important for this function). A non-codon-optimised translational slip sequence within codon-optimised gag-pol genes is exemplified in SEQ ID NO: 6.
[0132] Preferably, the codon-optimised gag-pol genes used to produce a retroviral/lentiviral (e.g. SIV) of the invention comprise or consist of the nucleic acid sequence of SEQ ID NO: 6, or a variant thereof (as defined herein). In particular, the codon-optimised gag-pol genes may comprise or consist of a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 6. Preferably, the codon-optimised gag-pol genes used in a method of the invention comprise or consist of a nucleic acid sequence having at least 90%, more preferably at least 95%, even more preferably at least 98%, or more sequence identity to SEQ ID NO: 6. The codon-optimised gag-pol genes of SEQ ID NO: 6 comprise a translational slip, and so do not form a single conventional open reading frame.
[0133] Preferably, the codon-optimised gag-pol genes used in a method of the invention are comprised in a plasmid that comprises or consists of a nucleic acid sequence of SEQ ID NO: 7 (pGM691), or a variant thereof (as defined herein). In particular, the codon-optimised gag-pol genes are comprised in a plasmid that comprises or consists of a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more sequence identity to SEQ ID NO: 7. Preferably, the codon-optimised gag-pol genes are comprised in a plasmid that comprises or consists of a nucleic acid sequence having at least 90%, more preferably at least 95%, even more preferably at least 98%, or more sequence identity to SEQ ID NO: 7. In the plasmid of SEQ ID NO: 7 (or variants thereof): (i) the codon-optimised gag-pol genes of SEQ ID NO: 6 comprise a translational slip, and so do not form a single conventional open reading frame; and (ii) the codon-optimised gag-pol genes of SEQ ID NO: 6 are operably linked to a CAG promoter. An exemplary CAG promoter is set out in SEQ ID NO: 15.
[0134] In the preferred five plasmid method of the invention, the vector genome plasmid encodes all the genetic material that is packaged into final retroviral/lentiviral vector, including the transgene. Typically only a portion of the genetic material found in the vector genome plasmid ends up in the virus. The vector genome plasmid may be designated herein as pDNA1, and typically comprises the transgene and the transgene promoter.
[0135] The other four plasmids are manufacturing plasmids encoding the Gag-Pol, Rev, F and HN proteins. These plasmids may be designated pDNA2a, pDNA2b, pDNA3a and pDNA3b respectively.
[0136] Modifications may be made to the vector genome plasmid (pDNA1), particularly to further improve the safety profile of the vector. As exemplified herein, such modifications may comprise or consist of modifying the pDNA1 sequence to remove viral, particularly retroviral/lentiviral (e.g. SIV), ORFs from the pDNA1 sequence. Thus, the retroviral/lentiviral (e.g. SIV) vectors of the invention may be made using a modified pDNA1 which comprises a reduced number of non-transgene ORFs. Said modified pDNA1 may comprise modifications within any region of the plasmid sequence. In particular, a modified pDNA1 may comprise modifications to remove: (i) 5 to 3 ORFs; (ii) ORFs of 100 amino acids; and/or (iii) ORFs upstream of the transgene and/or the promoter operably linked to the transgene. Whilst a modified pDNA1 may comprise no ORFs other than the transgene, this is not essential. Rather, a modified pDNA1 may still comprise ORFs other than the transgene, but may comprise a reduced number of non-transgene ORFs compared to the unmodified pDNA1 from which it is derived. By way of non-limiting example, a modified pDNA1 may comprise at least 1, at least 2, at least 3, at least 4, at least 5 or more fewer non-transgene ORFs compared with the corresponding unmodified pDNA1. As a specific example, pGM830 (which is derived from pGM326) comprises 2 fewer non-transgene ORFs compared with pGM326. A modified pDNA1 may comprise at least 1, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, or more modifications (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15 or 20 modifications) compared with the corresponding unmodified pDNA1. By way of non-limiting example, a modified pDNA1 may comprise between about 1 to about 20, such as between about 5 to about 15, or between about 5 to about 10 modifications compared with the corresponding unmodified pDNA1. As a specific example, pGM830 (which is derived from pGM326) comprises 7 modifications compared with pGM326.
[0137] The use of modified pDNA1 (e.g. pGM830) as described herein has the potential to produce an improved SIV titre compared with a production method which uses an unmodified pDNA1 plasmid (e.g. pGM326), but in which all other plasmids and method parameters are kept constant.
[0138] The five plasmids may be characterised by
[0139] The pGM326 plasmid as defined in
[0140] In the five-plasmid method of the invention all five plasmids contribute to the formation of the final retroviral/lentiviral (e.g. SIV) vector. During manufacture of the retroviral/lentiviral (e.g. SIV) vector, the vector genome plasmid (pDNA1) provides the enhancer/promoter, Psi, RRE, cPPT, mWPRE, SIN LTR, SV40 polyA (see
[0141] For other retroviral/lentiviral (e.g. SIV) vectors of the invention, corresponding elements from the other vector genome plasmids (pDNA1) are required for manufacture (but not found in the final vector), or are present in the final retroviral/lentiviral (e.g. SIV) vector.
[0142] The F and HN proteins from pDNA3a and pDNA3b (preferably Sendai F and HN proteins) are important for infection of target cells with the final retroviral/lentiviral (e.g. SIV) vector, i.e. for entry of a patient's epithelial cells (typically lung, preferably airway epithelial cells, as described herein). The products of the pDNA2a and pDNA2b plasmids are important for virus transduction, i.e. for inserting the retroviral/lentiviral (e.g. SIV) DNA into the host's genome. The promoter, regulatory elements (such as WPRE) and transgene are important for transgene expression within the target cell(s).
[0143] A retroviral/lentiviral (e.g. SIV) vectors of the invention may be produced by a method comprising or consisting of the following steps: (a) growing cells in suspension; (b) transfecting the cells with one or more plasmids; (c) adding a nuclease; (d) harvesting the lentivirus (e.g. SIV); (e) adding trypsin; and (f) purification of the lentivirus (e.g. SIV).
[0144] This method may use the four- or five-plasmid system described herein. Thus, for the preferred five-plasmid method, the one or more plasmids may comprise or consist of: a vector genome plasmid pDNA1; a gag-pol plasmid, pDNA2a; a Rev plasmid, pDNA2b; a fusion (F) protein plasmid, pDNA3a; and a hemagglutinin-neuraminidase (HN) plasmid, pDNA3b. The pDNA1 may be selected from pGM326 and pGM830, preferably pGM830. The pDNA2a may be selected from pGM297 and pGM691, preferably pGM297. The pDNA2b may be pGM299. The pDNA3a may be pGM301. The pDNA3b may be pGM303. Any combination of pDNA1, pDNA2a, pDNA2b, pDNA3a and pDNA3b may be used. Preferably, the pDNA1 is pGM326 or pGM830 (pGM830 being particularly preferred); the pDNA2a is pGM297 or pGM691 (pGM691 being particularly preferred); the pDNA2b is pGM299; the pDNA3a is pGM301; and the pDNA3b is pGM303. A SIV vector produced using pGM830, pGM691, pGM299, pGM301, and pGM303 is designated vGM244. A SIV vector produced using pGM326, pGM691, pGM299, pGM301, and pGM303 is designated vGM195. vGM195 and vGM244 are preferred SIV.F/HN vectors for use in combination therapies according to the invention, with vGM244 being particularly preferred.
[0145] Any appropriate ratio of vector genome plasmid:co-gagpol plasmid:Rev plasmid:F plasmid:HN plasmid may be used to in the production of a retroviral/lentiviral (e.g. SIV).
[0146] Steps (a)-(f) of the method are typically carried out sequentially, starting at step (a) and continuing through to step (f). The method may include one or more additional step, such as additional purification steps, buffer exchange, concentration of the retroviral/lentiviral (e.g. SIV) vector after purification, and/or formulation of the retroviral/lentiviral (e.g. SIV) vector after purification (or concentration). Each of the steps may comprise one or more sub-steps. For example, harvesting may involve one or more steps or sub-steps, and/or purification may involve one or more steps or sub-steps.
[0147] Any appropriate cell type may be transfected with the one or more plasmids (e.g. the five-plasmids described herein) to produce a retroviral/lentiviral (e.g. SIV) vector of the invention. Typically mammalian cells, particularly human cell lines are used. Non-limiting examples of cells suitable for use in the methods of the invention are HEK293 cells (such as HEK293F or HEK293T cells) and 293T/17 cells. Commercial cell lines suitable for the production of virus are also readily available (e.g. Gibco Viral Production CellsCatalogue Number A35347 from ThermoFisher Scientific).
[0148] The cells may be grown as adherent or suspension culture in animal-component free media, including serum-free media. The cells may be grown in a media which contains human components. The cells may be grown in a defined media comprising or consisting of synthetically produced components.
[0149] Any appropriate transfection means may be used according to the invention. Selection of appropriate transfection means is within the routine practice of one of ordinary skill in the art. By way of non-limiting example, transfection may be carried out by the use of PEIPro, Lipofectamine2000, Lipofectamine3000 or calcium triphosphate.
[0150] Any appropriate nuclease may be used according to the invention. Selection of appropriate nuclease is within the routine practice of one of ordinary skill in the art. Typically the nuclease is an endonuclease. By way of non-limiting example, the nuclease may be Benzonase or Denarase. The addition of the nuclease may be at the pre-harvest stage or at the post-harvest stage, or between harvesting steps.
[0151] The trypsin activity may preferably be provided by an animal origin free, recombinant enzyme such as TrypLE Select. The addition of trypsin may be at the pre-harvest stage or at the post-harvest stage, or between harvesting steps.
[0152] Any appropriate purification means may be used to purify the retroviral/lentiviral (e.g. SIV) vector. Non-limiting examples of suitable purification steps include depth/end filtration, tangential flow filtration (TFF) and chromatography. The purification step typically comprises at least one chromatography step. Non-limiting examples of chromatography steps that may be used in accordance with the invention include mixed-mode size exclusion chromatography (SEC) and/or anion exchange chromatography. Elution may be carried out with or without the use of a salt gradient, preferably without.
[0153] This method may be used to produce the retroviral/lentiviral (e.g. SIV) vectors of the invention as described herein. Alternatively, the retroviral/lentiviral (e.g. SIV) vector of the invention comprises any of the above-mentioned genes, or the genes encoding the above-mentioned proteins.
[0154] A retroviral/lentiviral (e.g. SIV) vector of the invention may be produced by a method using any combination of one or more of the specific plasmid constructs provided by
CFTR Mutations
[0155] CF is caused by mutations in the CFTR gene. To-date, over 2000 different mutations have been identified within the CFTR gene. Some CFTR gene mutations result in no CFTR protein being produced. Others result in the production of a dysfunctional CFTR protein. Using the current conventional nomenclature, the different CF-causing mutations in the CFTR gene can be arranged into classes depending on the effect of the mutation on CFTR protein production, conformation or function.
[0156] Class I CFTR mutations are protein production mutations, which result in no functional CFTR protein being produced. Approximately 22% of CF patients have at least one class I CFTR mutation. Several nonsense and splice mutations fall within class I. Examples of class I CFTR mutations include G542X, W1282X and R553X.
[0157] Class II CFTR mutations are protein processing mutations. Class II CFTR mutations do not prevent CFTR protein being produced, but the translated CFTR protein is misfolded and cannot form the correct conformation. Typically CFTR protein with a class II mutation will not be transported to the cell membrane, or is transported at reduced levels compared with normal CFTR protein. Approximately 88% of CF patients have at least one class II CFTR mutation. Examples of class II CFTR mutations include F508del, N1303K and I507del. F508del is the most common CF-causing CFTR mutation
[0158] Class III CFTR mutations are gating mutations. Class III CFTR mutations do not prevent CFTR protein being produced or transported to the cell membrane. Rather, gating mutations force the CFTR protein to adopt a closed conformation, preventing or reducing chloride transport. Approximately 6% of CF patients have at least one class III CFTR mutation. Examples of class III CFTR mutations include G551D and S549N.
[0159] Class IV CFTR mutations are conduction mutations. Class IV CFTR mutations do not prevent CFTR protein being produced or transported to the cell membrane, nor do they hold the CFTR protein in a closed conformation. However, class IV mutations can affect the inner conformation of the chloride channel within the CFTR protein, reducing chloride transport. Approximately 6% of CF patients have at least one class IV CFTR mutation. Examples of class IV CFTR mutations include D1152H, R347P and R117H.
[0160] Class V CFTR mutations are termed insufficient protein mutations. Class V CFTR mutations result in a lower amount of CTFR protein being present at the cell membrane. This may occur because less CFTR protein is produced, only a small number of protein at the cell surface work correctly, or degradation of normal CFTR protein in the cell membrane occurs too quickly. Several missense and splice mutations fall within class V. Approximately 5% of CF patients have at least one class V CFTR mutation. Examples of class V CFTR mutations include 3849+10kbC.fwdarw.T, 2789+5G.fwdarw.A and A455E.
[0161] Class VI CFTR mutations destabilise the CFTR protein in post-endoplasmic reticulum (ER) compartments and/or at the cell membrane, by reducing conformational stability of the CFTR and/or by generating additional internalisation signals. These mutations consequently result in accelerated CFTR turnover at the cell membrane and reduced expression at the apical cell membrane.
[0162] The present invention relates to the treatment of CF caused by any combination of class I, II, III, IV, V and/or VI mutations. A patient to be treated according to the present invention may have CF caused by one or more class I mutation, one or more class II mutation, one or more class III mutation, one or more class IV mutation, one or more class V mutation and/or one or more class VI mutation2. A patient to be treated may have (i) one or more mutation in class I and one or more mutation in class II; (ii) one or more mutation in class I and one or more mutation in class III; (iii) one or more mutation in class I and one or more mutation in class IV; (iv) one or more mutation in class I and one or more mutation in class V; (v) one or more mutation in class I and one or more mutation in class VI; (vi) one or more mutation in class II and one or more mutation in class III; (vii) one or more mutation in class II and one or more mutation in class IV; (viii) one or more mutation in class II and one or more mutation in class V; (ix) one or more mutation in class II and one or more mutation in class VI; (x) one or more mutation in class III and one or more mutation in class IV; (xi) one or more mutation in class III and one or more mutation in class V; (xii) one or more mutation in class III and one or more mutation in class VI; (xiii) one or more mutation in class IV and one or more mutation in class V; (xiv) one or more mutation in class IV and one or more mutation in class VI; (xv) one or more mutation in class V and one or more mutation in class VI; (xvi) one or more mutation in class I, one or more mutation in class II and one or more mutation in class III; (xvii) one or more mutation in class I, one or more mutation in class II and one or more mutation in class IV; (xviii) one or more mutation in class I, one or more mutation in class II and one or more mutation in class V; (xix) one or more mutation in class I, one or more mutation in class II and one or more mutation in class VI; (xx) one or more mutation in class I, one or more mutation in class III and one or more mutation in class IV; (xxi) one or more mutation in class I, one or more mutation in class III and one or more mutation in class V; (xxii) one or more mutation in class I, one or more mutation in class III and one or more mutation in class VI; (xxiii) one or more mutation in class I, one or more mutation in class IV and one or more mutation in class V; (xxiv) one or more mutation in class I, one or more mutation in class IV and one or more mutation in class VI; (xxv) one or more mutation in class I, one or more mutation in class V and one or more mutation in class VI; (xxvi) one or more mutation in class II, one or more mutation in class III and one or more mutation in class IV; (xxvii) one or more mutation in class II, one or more mutation in class III and one or more mutation in class V; (xxviii) one or more mutation in class II, one or more mutation in class III and one or more mutation in class VI; (xxix) one or more mutation in class II, one or more mutation in class IV and one or more mutation in class V; (xxx) one or more mutation in class II, one or more mutation in class IV and one or more mutation in class VI; (xxxi) one or more mutation in class II, one or more mutation in class V and one or more mutation in class VI; (xxxii) one or more mutation in class III, one or more mutation in class IV and one or more mutation in class V; (xxxiii) one or more mutation in class III, one or more mutation in class IV and one or more mutation in class VI; (xxxiv) one or more mutation in class III, one or more mutation in class V and one or more mutation in class VI; (xxxv) one or more mutation in class IV, one or more mutation in class V and one or more mutation in class VI; (xxxvi) one or more mutation in class I, one or more mutation in class II, one or more mutation in class III and one or more mutation in class IV; (xxxvii) one or more mutation in class I, one or more mutation in class II, one or more mutation in class III and one or more mutation in class V; (xxxviii) one or more mutation in class I, one or more mutation in class II, one or more mutation in class III and one or more mutation in class VI; (xxxix) one or more mutation in class I, one or more mutation in class II, one or more mutation in class IV and one or more mutation in class V; (xl) one or more mutation in class I, one or more mutation in class II, one or more mutation in class IV and one or more mutation in class VI; (xli) one or more mutation in class I, one or more mutation in class II, one or more mutation in class V and one or more mutation in class VI; (xlii) one or more mutation in class I, one or more mutation in class III, one or more mutation in class IV and one or more mutation in class V; (xliii) one or more mutation in class I, one or more mutation in class III, one or more mutation in class IV and one or more mutation in class VI; (xliv) one or more mutation in class I, one or more mutation in class III, one or more mutation in class V and one or more mutation in class VI; (xlv) one or more mutation in class I, one or more mutation in class IV, one or more mutation in class V and one or more mutation in class VI; (xlvi) one or more mutation in class II, one or more mutation in class III, one or more mutation in class IV and one or more mutation in class V; (xlvii) one or more mutation in class II, one or more mutation in class III, one or more mutation in class IV and one or more mutation in class VI; (xlviii) one or more mutation in class II, one or more mutation in class III, one or more mutation in class V and one or more mutation in class VI; (xlix) one or more mutation in class II, one or more mutation in class IV, one or more mutation in class V and one or more mutation in class VI; (I) one or more mutation in class III, one or more mutation in class IV, one or more mutation in class V and one or more mutation in class VI; (Ii) one or more mutation in class I, one or more mutation in class II, one or more mutation in class III, one or more mutation in class IV and one or more mutation in class V; (hi) one or more mutation in class I, one or more mutation in class II, one or more mutation in class III, one or more mutation in class IV and one or more mutation in class VI; (liii) one or more mutation in class I, one or more mutation in class II, one or more mutation in class III, one or more mutation in class V and one or more mutation in class VI; (liv) one or more mutation in class I, one or more mutation in class II, one or more mutation in class IV, one or more mutation in class V and one or more mutation in class VI; (Iv) one or more mutation in class I, one or more mutation in class III, one or more mutation in class IV, one or more mutation in class V and one or more mutation in class VI; (Ivi) one or more mutation in class II, one or more mutation in class III, one or more mutation in class IV, one or more mutation in class V and one or more mutation in class VI; or (Ivii) one or more mutation in class I, one or more mutation in class II, one or more mutation in class III, one or more mutation in class IV, one or more mutation in class V and one or more mutation in class VI.
[0163] A patient to be treated according to the invention may have at least 1, at least 2, at least 3, at least 4, at least 5, at least 6 at least 7, at least 8, at least 9, at least 10 or more mutations in the CFTR gene, which may each be independently selected from a class I, class II, class III, class IV and/or class V mutation as described herein.
[0164] A patient to be treated according to the invention may have at least one class I and/or class II CFTR mutation, such as those described herein. A patient to be treated may have (i) at least one class I CFTR mutation that is optionally selected from G542X, W1282X and/or R553C; and/or (ii) at least one class II CFTR mutation that is optionally selected from F508del, N1303K and/or I507del.
[0165] As exemplified herein, a combination treatment of the invention successfully restored CFTR expression and function in a model of a class I CFTR mutation, and that the surprising effect of the combination was greater on the CFTR transgene expressed by the retro/lentiviral (e.g. SIV) vector than the endogenous CFTR gene. Accordingly, the combination therapy of the invention may be suitable for use independent of the CFTR mutation of the patient. In other words, and without being bound by theory, as the CFTR modulator may exert a greater therapeutic effect on the CFTR transgene, the nature of the CF-causing mutation in the endogenous CFTR gene may be irrelevant. The present invention therefore has the potential to treat patients with CF independent of the CF-causing mutation. This is advantageous, as the currently authorised CFTR modulator therapies are suitable only for patients with specific CFTR mutations.
CFTR Modulators
[0166] CFTR modulators are active pharmaceutical ingredients (APIs) designed to correct malfunctioning CFTR proteins. CFTR modulators are a specialised group of therapies, as different modulators are designed to address the underlying defect in the CFTR protein caused by a specific CFTR mutation or class of CFTR mutations.
[0167] There are three main types of CFTR modulators: CFTR potentiators, CFTR correctors and CFTR amplifiers.
[0168] As discussed herein, class III CFTR mutations, such as G551D, are gating mutations, which prevent normal opening of the CFTR protein to facilitate chloride transport. CFTR potentiators mitigate this defect by opening the CFTR protein gates and keeping them open longer to facilitate the smooth flow of chloride ions. Ivacaftor (Kalydeco) is an example of a CFTR potentiator developed by Vertex Pharmaceuticals. It is an oral medication approved by the U.S. Food and Drug Administration (FDA), the EU, and Health Canada for CF patients as young as 1 year with at least one mutation (such as G551D) that impairs chloride ion flow. Another example of a CFTR potentiator is the experimental treatment PTI-808 being developed by Proteostasis Therapeutics.
[0169] As discussed herein, class II CFTR mutations are protein processing mutations, which result in misfolding of the CFTR protein, which can affect transport of the misfolded CFTR to the cell surface. CFTR correctors assist the CFTR protein in folding correctly into its 3D conformation, allowing it to be successfully transported to the cell membrane so that it can function. Lumacaftor (VX-809) and tezacaftor (VX-661) are two therapies by Vertex Pharmaceuticals that function as correctors. Another CFTR corrector is elexacaftor. These correctors help the CFTR protein fold correctly and reach the cell surface, but fall short in alleviating CF symptoms by themselves. Therefore, they are not approved as a monotherapy for CF. Another CFTR corrector under development (by Proteostasis Therapeutics) is PTI-801.
[0170] As discussed herein, class V CFTR mutations result in reduced levels of CFTR protein being present at the cell surface, for example because they result in lower levels of CFTR protein being expressed, or increase the rate of degradation of CFTR protein. Amplifiers are a type of CFTR modulator that enhances the production of CFTR protein by the cells. PTI-428 is an investigational first-generation CFTR amplifier by Proteostasis Therapeutics, which is being tested as a single and combination therapy for CF.
[0171] CFTR potentiators, correctors and amplifiers are described in the art for use independently as single therapies. In addition, combinations of these CFTR modulators are also known, with three of the four currently approved CFTR modulator therapies being combination therapies. By way of non-limiting example, combining a potentiator with a corrector may improve CFTR activity by correcting the CFTR conformation and opening the gate to allow chloride transport. In particular, a combination of the potentiator ivacaftor and corrector lumacaftor is authorised as a combination therapy and is marketed as Orkambi by Vertex for use in the treatment for CF patients with two F508del CFTR mutations. Another example of an authorised combination treatment is the potentiator ivacaftor and corrector tezacaftor, which is marked by Vertex as Symdeko in the US and Symkevi in the EU.
[0172] Amplifiers can also be used in combination with other CFTR modulators. Combining CFTR amplifiers with other CFTR modulators may be advantageous as the CFTR amplifier can result in more CFTR protein being expressed, which can then be acted upon by the other CFTR modulator(s).
[0173] In addition to these first-generation CFTR modulators, so-called next-generation modulators may combine multiple CFTR correctors to produce combination therapies with three or more APIs. An example of an approved next-generation CFTR modulator is Trikafta, which is a combination of the potentiator ivacaftor and two correctors, tezacaftor and elexacaftor.
[0174] Any reference to a CFTR modulator herein encompasses any and all salts, derivatives and analogues of said CFTR modulator, unless expressly stated to the contrary. Thus, the invention relates to the combination of a known CFTR modulators such as those individualised herein (particularly ivacaftor) or a salt, derivative or analogue thereof. Salt forms preferably include pharmaceutically acceptable salts. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or with organic acids such as acetic, oxalic, tartaric, maleic, and the like. An analogue of a CFTR modulator, as the term is used herein, refers to a chemical structure that preserves substantial similarity with the CFTR modulator, although it may not be readily derived synthetically from the CFTR modulator. A related chemical structure that is readily derived synthetically from a CFTR modulator structure is referred to as a derivative. These are all within the scope of the invention. By way of non-limiting example, a reference to ivacaftor includes salt forms, analogues and derivatives of ivacaftor, such as deuterated ivacaftor (D-ivacaftor).
[0175] Accordingly, the invention relates to therapy comprising a gene therapy vector, particularly using a retro/lentiviral (e.g. SIV) vector as described herein, preferably a SIV.F/HN vector, in combination with one or more CFTR modulator, which may be selected from one or more CFTR potentiator, one or more CFTR corrector and/or one or more CFTR amplifier, or combination thereof. Combinations comprising one or more CFTR potentiator with a retro/lentiviral (e.g. SIV) vector are particularly preferred. As such, the invention may relate to the use of a gene therapy vector, particularly using a retro/lentiviral (e.g. SIV) vector as described herein, preferably a SIV.F/HN vector, in combination with one or more CFTR potentiator. One or more CFTR corrector and/or one or more CFTR amplifier may be used in addition to the combination of a gene therapy vector, particularly using a retro/lentiviral (e.g. SIV) vector as described herein, preferably a SIV.F/HN vector, and one or more CFTR potentiator.
[0176] Thus, the invention relates to a combination of a retro/lentiviral (e.g. SIV) vector as described herein, preferably a SIV.F/HN vector, and a CFTR modulator, which may be selected from one or more CFTR potentiator, one or more CFTR corrector and/or one or more CFTR amplifier, or combination thereof. Typically the invention relates to a combination of a retro/lentiviral (e.g. SIV) vector as described herein, preferably a SIV.F/HN vector, and a CFTR potentiator and/or CFTR corrector. Preferably, the invention relates to a combination of a retro/lentiviral (e.g. SIV) vector, as described herein, preferably a SIV.F/HN vector, and a CFTR potentiator, and optionally one or more CFTR corrector and/or one or more CFTR amplifier.
[0177] Any CFTR modulator may be used in combination with a retro/lentiviral (e.g. SIV) vector as described herein, preferably a SIV.F/HN vector, according to the present invention, such as those described herein. Accordingly, non-limiting examples of CFTR modulators that may be used in combination with a retro/lentiviral (e.g. SIV) vector as described herein, preferably a SIV.F/HN vector, according to the present invention include ivacaftor (Kalydeco), PTI-808, VX-809 (Lumacaftor) and VX-661 (tezacaftor), elexacaftor, PTI-801, PTI-428, as well as combinations such as ivacaftor+lumacaftor (Orkambi), ivacaftor+tezacaftor (Symdeko or Symkevi) and ivacaftor+tezacaftor+elexacaftor)(Trikafta.
[0178] Preferably the invention relates to the combination of a retro/lentiviral (e.g. SIV) vector as described herein, preferably a SIV.F/HN vector, with a CFTR modulator selected from ivacaftor, tezacaftor, elexacaftor or lumacaftor, of a combination thereof. The combination of a retro/lentiviral (e.g. SIV) vector as described herein, preferably a SIV.F/HN vector, with the CFTR modulator (specifically the CFTR potentiator) ivacaftor is particularly preferred.
[0179] Preferred embodiments relate to the use of (A) a SIV vector pseudotyped with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) proteins, wherein: (a) said vector comprises a modified retroviral RNA sequence which comprises or consists of a nucleic acid sequence of SEQ ID NO: 16 (which comprises a CFTR transgene), preferably wherein the modified retroviral RNA sequence consists of a nucleic acid sequence of SEQ ID NO: 16; and (b) the F protein comprises a first subunit which comprises or consists of an amino acid sequence of SEQ ID NO: 19 and a second subunit which comprises or consists of an amino acid sequence of SEQ ID NO: 20 in combination with (B) a CFTR modulator selected from ivacaftor, tezacaftor, elexacaftor or lumacaftor, of a combination thereof.
[0180] Particularly preferred embodiments relate to the use of (A) a SIV vector pseudotyped with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) proteins, wherein: (a) said vector comprises a modified retroviral RNA sequence which comprises or consists of a nucleic acid sequence of SEQ ID NO: 16 (which comprises a CFTR transgene), preferably wherein the modified retroviral RNA sequence consists of a nucleic acid sequence of SEQ ID NO: 16; and (b) the F protein comprises a first subunit which comprises or consists of an amino acid sequence of SEQ ID NO: 19 and a second subunit which comprises or consists of an amino acid sequence of SEQ ID NO: 20 in combination with (B) the CFTR modulator (specifically the CFTR potentiator) ivacaftor.
[0181] As described herein, in said particularly preferred embodiments, the SIV vector pseudotyped with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) proteins, wherein: (a) said vector comprises a modified retroviral RNA sequence which comprises or consists of a nucleic acid sequence of SEQ ID NO: 16 (which comprises a CFTR transgene), preferably wherein the modified retroviral RNA sequence consists of a nucleic acid sequence of SEQ ID NO: 16; and (b) the F protein comprises a first subunit which comprises or consists of an amino acid sequence of SEQ ID NO: 19 and a second subunit which comprises or consists of an amino acid sequence of SEQ ID NO: 20, wherein said vector further comprise one or more of: (a) a p17 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 22; (b) a p24 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 23; (c) p8 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 24; (d) a protease comprising or consisting of an amino acid sequence of SEQ ID NO: 25; (e) a p51 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 26; (f) a p15 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 27; (g) a p31 protein comprising or consisting of an amino acid sequence of SEQ ID NO: 28; (h) a Gag protein comprising or consisting of an amino acid sequence of SEQ ID NO: 29; and/or (i) a Pol protein comprising or consisting of an amino acid sequence of SEQ ID NO: 30; wherein optionally the vector comprises each of (a) to (g), and is combined with a CFTR modulator selected from ivacaftor, tezacaftor, elexacaftor or lumacaftor, of a combination thereof, particularly combined with the CFTR modulator (specifically the CFTR potentiator) ivacaftor.
Therapeutic Indications
[0182] The retroviral/lentiviral (e.g. SIV) vectors of the present invention enable higher and sustained gene expression through efficient gene transfer. The F/HN-pseudotyped retroviral/lentiviral (e.g. SIV) vectors of the invention are capable of: (i) airway transduction without disruption of epithelial integrity; (ii) persistent gene expression; (iii) lack of chronic toxicity; and (iv) efficient repeated administration. Long term/persistent stable gene expression, preferably at a therapeutically-effective level, may be achieved using repeated doses of a vector of the present invention. Alternatively, a single dose may be used to achieve the desired long-term expression. Advantageously, the retroviral/lentiviral (e.g. SIV) vectors of the present invention can be used in gene therapy for CF, by providing a functional copy of the CFTR gene to ameliorate or prevent lung disease in CF patients, independent of the underlying mutation.
[0183] CFTR modulators are breakthrough therapies that target the underlying cause of CF, rather than ameliorating symptoms of the disease. However, current CFTR modulators are only effective in patients with specific mutations.
[0184] Therefore, combining the use of gene therapy with CFTR modulators offers a potentially significant advance in the treatment of CF. The present inventors are the first to investigate the effects of combining retroviral/lentiviral (e.g. SIV) vectors with CFTR modulators. In particular, the present inventors have shown that gene therapy with a retroviral/lentiviral (e.g. SIV) vectors of the present invention produces a greater than expected therapeutic effect when combined with a CFTR modulator. As exemplified herein, the inventors have surprisingly demonstrated that the effect of a CFTR modulator, particularly a CFTR potentiator, and rSIV.F/HN-CFTR combination is greater than the additive effects of the separate effects of the CFTR modulator, particularly the CFTR potentiator, and rSIV.F/HN-mediated CFTR expression.
[0185] Accordingly, retroviral/lentiviral (e.g. SIV) vectors with a CFTR transgene according to the invention may be used in combination with one or more CFTR modulator to treat CF.
[0186] Preferred embodiments relate to the therapeutic use of (A) a SIV vector pseudotyped with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) proteins, wherein: (a) said vector comprises a modified retroviral RNA sequence which comprises or consists of a nucleic acid sequence of SEQ ID NO: 16 (which comprises a CFTR transgene), preferably wherein the modified retroviral RNA sequence consists of a nucleic acid sequence of SEQ ID NO: 16; and (b) the F protein comprises a first subunit which comprises or consists of an amino acid sequence of SEQ ID NO: 19 and a second subunit which comprises or consists of an amino acid sequence of SEQ ID NO: 20 in combination with (B) a CFTR modulator selected from ivacaftor, tezacaftor, elexacaftor or lumacaftor, of a combination thereof.
[0187] Particularly preferred embodiments relate to the therapeutic use of (A) a SIV vector pseudotyped with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) proteins, wherein: (a) said vector comprises a modified retroviral RNA sequence which comprises or consists of a nucleic acid sequence of SEQ ID NO: 16 (which comprises a CFTR transgene), preferably wherein the modified retroviral RNA sequence consists of a nucleic acid sequence of SEQ ID NO: 16; and (b) the F protein comprises a first subunit which comprises or consists of an amino acid sequence of SEQ ID NO: 19 and a second subunit which comprises or consists of an amino acid sequence of SEQ ID NO: 20 in combination with (B) the CFTR modulator (specifically the CFTR potentiator) ivacaftor.
[0188] A retroviral/lentiviral (e.g. SIV) vector and one or more CFTR modulator may be administered to a patient with CF who is exhibiting one or more symptom of CF. When administered to such a patient, a retroviral/lentiviral (e.g. SIV) vector and one or more CFTR modulator can cure, delay, reduce the severity of, or ameliorate one or more symptoms, and/or prolong the survival of a patient beyond that expected in the absence of such treatment and/or beyond that expected using a conventional CF treatment (e.g. a CFTR modulator, particularly a CFTR potentiator, alone). Thus, retroviral/lentiviral (e.g. SIV) vector and one or more CFTR modulator, particularly a CFTR potentiator, may be administered to a patient with CF to ameliorate the disease and/or prolong the survival of a patient with CF beyond that expected in the absence of such treatment and/or beyond that expected using a conventional CF treatment (e.g. a CFTR modulator or CFTR potentiator alone).
[0189] The retroviral/lentiviral (e.g. SIV) vector and one or more CFTR modulator are administered in combination. Administered in combination, encompasses both simultaneous (also referred to as concurrent) administration/delivery and sequential (also referred to as separate) administration/delivery.
[0190] For, simultaneous or concurrent delivery, the delivery of the retroviral/lentiviral (e.g. SIV) vector may still be occurring when the delivery of the CFTR modulator begins, or the delivery of CFTR modulator may still be occurring when the delivery of the retroviral/lentiviral (e.g. SIV) vector begins, so that there is overlap in terms of administration. Simultaneous delivery may encompass delivery of the retroviral/lentiviral (e.g. SIV) vector and CFTR modulator within weeks to months or even years of each other, typically so that the retroviral/lentiviral (e.g. SIV) vector delivery overlaps with the delivery of the CFTR modulator.
[0191] Alternatively, the delivery of the retroviral/lentiviral (e.g. SIV) vector may end before the delivery of the CFTR modulator begins, or the delivery of the CFTR modulator may end before delivery of the retroviral/lentiviral (e.g. SIV) vector begins. Sequential administration may involve the retroviral/lentiviral (e.g. SIV) vector and CFTR modulator being administered within 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 6 hours, 12 hours or 24 hours, 1 week, 2 weeks, 1 month, 2 months, or longer of each other.
[0192] The CFTR modulator may be administered at an interval of once every hour, once every 2 hours, once every 3 hours, once every 4 hours, once every 6 hours, once every 8 hours, once every 12 hours, daily, once every 2 days or more. Typically the CFTR modulator is administered every 12 hours.
[0193] The retroviral/lentiviral (e.g. SIV) vector may be administered once every month, once every 2 months, once every 3 months, once every 4 months, once every 6 months, once every 8 months, once every 12 months or more. As the frequency of administration of the retroviral/lentiviral (e.g. SIV) vector is lower than the frequency of administration of the CFTR modulator, administration of the combination therapy is typically by sequential administration.
[0194] Treatment with a retroviral/lentiviral (e.g. SIV) vector and/or CFTR modulator at the desired dosing frequency may be continued for as long as required, for example, for at least six months, at least one year, two years, three years, four years, five years, ten years, fifteen years, twenty years, or more, up to for the lifetime of the patient to be treated.
[0195] Typically the treatment is more effective because of combined administration. For example, treatment with the CFTR modulator may be more effective, e.g., an equivalent effect is seen with less of the CFTR modulator, or the CFTR modulator reduces symptoms to a greater extent, than would be seen if the CFTR modulator were administered in the absence of the retroviral/lentiviral (e.g. SIV) vector, or the analogous situation is seen with the retroviral/lentiviral (e.g. SIV) vector. Typically, delivery is such that the reduction in a symptom, or other parameter related to CF is greater than what would be observed with the CFTR modulator delivered in the absence of the retroviral/lentiviral (e.g. SIV) vector, or the analogous situation is seen with the retroviral/lentiviral (e.g. SIV) vector.
[0196] It will be appreciated that appropriate dosage of the retroviral/lentiviral (e.g. SIV) vector may and/or the CFTR modulator, will depend on the specific agent, and can also vary from patient to patient.
[0197] It will be appreciated that appropriate dosage of the retroviral/lentiviral (e.g. SIV) vector and/or CFTR modulator, will depend on the specific agent, and can also vary from patient to patient.
[0198] Determining the optimal dosage will generally involve the balancing of the level of therapeutic benefit against any risk or deleterious side effects of the treatments described herein. The selected dosage level will depend on a variety of factors including, but not limited to, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the compound, the duration of the treatment, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of compound and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve local concentrations at the site of action which achieve the desired effect without causing substantial harmful or deleterious side-effects. Non-limiting exemplary dosages and routes of administration are described herein.
[0199] Administration in vivo can be effected in one dose, continuously or intermittently (e.g. in divided doses at appropriate intervals) throughout the course of treatment. Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the treating physician.
[0200] The duration of action of a combination therapy according to the invention may be for at least 6 hours, at least 12 hours, at least 18 hours, at least 24 hours, at least 48 hours, at least 72 hours, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 6 weeks, at least 8 weeks, at least 12 weeks, at least six months, at least 1 year or more. Typically this is assessed relative to the last administration of the retroviral/lentiviral (e.g. SIV) vector and/or CFTR modulator, particularly the last administration of the retroviral/lentiviral (e.g. SIV) vector.
[0201] A retroviral/lentiviral (e.g. SIV) vector according to the invention is typically administered by inhalation. Accordingly, said retroviral/lentiviral (e.g. SIV) vector may be formulated for inhalation, as described herein. The one or more CFTR modulator may be administered by any appropriate route, and may be formulated accordingly. In particular, the one or more CFTR modulator may be administered orally, and may be formulated for oral administration.
[0202] Accordingly, the invention provides a method of treating CF in a subject in need thereof, said method comprising administering to the subject a therapeutically effective amount of each of (i) a retroviral/lentiviral (e.g. SIV) vector of the invention; and (ii) one or more CFTR modulator. Any retroviral/lentiviral (e.g. SIV) vector as described herein may be used in combination with any one or more CFTR modulator, such as those described and exemplified herein.
[0203] The combination therapies of the invention may restore CFTR expression and/or activity to a level which provides a therapeutic benefit. A combination therapy may restore (increase) CFTR expression and/or activity to a level which matches or exceeds CFTR expression and/or activity in a healthy control. However, restoration of healthy CFTR expression and/or activity is not essential to achieve a therapeutic benefit. Rather, the therapeutic threshold, i.e. the level above which a therapeutic benefit is achieved may be lower than the level of CFTR expression and/or activity in a healthy control. Indeed, patients, particularly those with a class I CFTR mutation resulting in null CFTR expression, may receive a therapeutic benefit from even relatively small increases in CFTR expression and/or activity (e.g. 5% or 10% CFTR expression and/or activity compared to healthy control expression levels).
[0204] A retroviral/lentiviral (e.g. SIV) vector as described herein, particularly in the context of a combination therapy of the invention may increase (restore) CFTR expression (particularly cellular CFTR expression levels and/or global expression in the lungs or respiratory tree), CFTR activity and/or CFTR current as described herein. Any combination of increase in CFTR expression (cellular and/or global), CFTR activity and/or CFTR current, including the quantified increases described below is encompassed by the present invention.
[0205] A retroviral/lentiviral (e.g. SIV) vector as described herein, particularly in the context of a combination therapy of the invention may increase (restore) CFTR expression (particularly cellular CFTR expression levels and/or global expression in the lungs or respiratory tree) to at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 120% or more of CFTR expression in a healthy control. Typically a retroviral/lentiviral (e.g. SIV) vector as described herein, particularly in the context of a combination therapy of the invention may increase (restore) CFTR expression, particularly CFTR cellular expression levels, to at least 20%, preferably at least 50%, more preferably at least 75% of CFTR expression in a healthy control. A retroviral/lentiviral (e.g. SIV) vector as described herein, particularly in the context of a combination therapy of the invention may increase (restore) CFTR expression, particularly global CFTR expression in the lungs, to at least 5%, at least 10%, preferably at least 20% of CFTR expression in a healthy control. Expression levels of a transgene and/or the encoded therapeutic protein of the invention may be measured in the lung tissue, A high and/or therapeutic expression level may therefore refer to the concentration in the lung. CFTR expression may be quantified using one or more of the following techniques: CFTR RNA expression in lung tissue, CFTR protein expression in lung tissue, PK assays (vector copy number, integration), DNA content in sputum (as surrogate for NETosis).
[0206] Other endpoints for efficacy assessment of treatment according to the invention include: improvement in FEV1 (lung function); MRI and/or CT for efficacy assessment; reduced expression of one or more biomarker of inflammation, such as IL-8, IL1beta, IL-6, TNFalpha (typically measured in sputum), calprotectin (typically measured in serum); differential cell count in sputum; reduced surfactant protein D (SP-D) in serum as marker for reduced epithelial injury; reduction of pulmonary exacerbations; improvement of Lung Clearance index, Quality of Life as patient reported outcome via CFQ-R respiratory domain (and/or other appropriate questionnaires as well), exploratory imaging endpoints (to show improved lung ventilation, mucus plugging and/or others, e.g. via Eichinger score of mRI).
[0207] Alternatively or in addition, a combination therapy of the invention may increase (restore) CFTR activity (particularly cellular CFTR activity and/or global activity in the lungs or respiratory tree) to at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 120% or more of CFTR activity in a healthy control. Typically a combination therapy of the invention may increase (restore) CFTR activity, particularly CFTR cellular activity, to at least 20%, preferably at least 50%, more preferably at least 75% of CFTR activity in a healthy control. A retroviral/lentiviral (e.g. SIV) vector as described herein, particularly in the context of a combination therapy of the invention may increase (restore) CFTR activity, particularly global CFTR expression in the lungs, to at least 5%, at least 10%, preferably at least 20% of CFTR activity in a healthy control.
[0208] As used herein, typically a healthy control is an equivalent individual or population who do not have CF. Preferably a healthy control is an equivalent individual or population who do not have CF and is otherwise in good health. A healthy control may be matched with the subject using standard clinical methodology (e.g. age/sex matching, or matching based on other criteria).
[0209] Other controls may be an equivalent individual with CF who has been treated with either the retroviral/lentiviral (e.g. SIV) vector or CFTR modulator alone.
[0210] CFTR activity may be defined in terms of the CFTR RNA expression in lung tissue; CFTR protein expression in lung tissue and/or activity of the CFTR channel itself (e.g. by electrophysiological measurements using patient's bronchial brushings).
[0211] A combination therapy of the invention may increase (restore) CFTR current by at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 2 fold or more compared with treatment with the retroviral/lentiviral (e.g. SIV) vector alone (i.e. compared with the increase in CFTR current achieved when treating with the retroviral/lentiviral (e.g. SIV) alone). A combination therapy of the invention may increase (restore) CFTR current by between about 1.3 fold to about 3 fold or between about 1.2 fold to about 2 fold compared with treatment with the retroviral/lentiviral (e.g. SIV) vector alone (i.e. compared with the increase in CFTR current achieved when treating with the retroviral/lentiviral (e.g. SIV) alone). Preferably, a combination therapy of the invention may increase (restore) CFTR current by at least about 1.2 fold, at least about 1.3 fold, at least about 1.5 fold, at least about 1.8 fold, such as by about 1.3 fold to about 1.8 fold compared with treatment with the retroviral/lentiviral (e.g. SIV) vector alone.
[0212] A combination therapy of the invention may increase (restore) CFTR current to at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 90%, at least 95%, at least 100%, at least 120% or more of CFTR current in a healthy control. Typically a combination therapy of the invention may increase (restore) CFTR cellular current to at least 20%, preferably at least 50%, more preferably at least 75% of CFTR current in a healthy control. A retroviral/lentiviral (e.g. SIV) vector as described herein, particularly in the context of a combination therapy of the invention may increase (restore) CFTR current, particularly globally in the lungs, to at least 5%, at least 10%, preferably at least 20% of CFTR current in a healthy control.
[0213] The invention provides a combination of a retroviral/lentiviral (e.g. SIV) and one or more CFTR modulator as described herein for use in treating CF, wherein the treatment: (i) restores cellular CFTR activity to at least 10% or at least 50% of the CFTR activity (e.g. at least 70%) in a healthy control; (ii) restores global CFTR activity in the lungs to at least 5% or at least 10% of the CFTR activity (e.g. at least 20%) in a healthy control; and/or (iii) increases CFTR current by at least about 1.3 fold (e.g. from about 1.3 fold to about 1.8 fold, from about 1.3 fold to about 3 fold, or about 1.3 fold) compared with treatment with the retroviral/lentiviral (e.g. SIV) vector alone. Typically the invention provides a combination of a retroviral/lentiviral (e.g. SIV) and one or more CFTR modulator as described herein for use in treating CF, wherein the treatment: (i) restores global CFTR activity in the lungs to at least 5% or at least 10% of the CFTR activity (e.g. at least 20%) in a healthy control; and/or (ii) increases CFTR current by at least about 1.3 fold (e.g. from about 1.3 fold to about 1.8 fold, from about 1.3 fold to about 3 fold, or about 1.3 fold) compared with treatment with the retroviral/lentiviral (e.g. SIV) vector alone.
[0214] The invention provides a combination of a retroviral/lentiviral (e.g. SIV) and one or more CFTR modulator as described herein for use in treating CF, wherein the patient to be treated has at least one class I CFTR mutation and the treatment: (i) restores cellular CFTR activity to at least 10% or at least 50% of the CFTR activity (e.g. at least 70%) in a healthy control; (ii) restores global CFTR activity in the lungs to at least 5% or at least 10% of the CFTR activity (e.g. at least 20%) in a healthy control; and/or (iii) increases CFTR current by at least about 1.3 fold (e.g. from about 1.3 fold to about 1.8 fold, from about 1.3 fold to about 3 fold, or about 1.3 fold) compared with treatment with the retroviral/lentiviral (e.g. SIV) vector alone. Typically the invention provides a combination of a retroviral/lentiviral (e.g. SIV) and one or more CFTR modulator as described herein for use in treating CF, wherein the patient to be treated has at least one class I CFTR mutation and the treatment: (i) restores global CFTR activity in the lungs to at least 5% or at least 10% of the CFTR activity (e.g. at least 20%) in a healthy control; and/or (ii) increases CFTR current by at least about 1.3 fold (e.g. from about 1.3 fold to about 1.8 fold, from about 1.3 fold to about 3 fold, or about 1.3 fold) compared with treatment with the retroviral/lentiviral (e.g. SIV) vector alone.
[0215] The invention provides a combination of a retroviral/lentiviral (e.g. SIV) and one or more CFTR modulator as described herein for use in treating CF, wherein the patient to be treated has at least one class II CFTR mutation and the treatment: (i) restores cellular CFTR activity to at least 10% or at least 50% of the CFTR activity (e.g. at least 70%) in a healthy control; (ii) restores global CFTR activity in the lungs to at least 5% or at least 10% of the CFTR activity (e.g. at least 20%) in a healthy control; and/or (ii) increases CFTR current by at least about 1.3 fold (e.g. from about 1.3 fold to about 1.8 fold, from about 1.3 fold to about 3 fold, or about 1.3 fold) compared with treatment with the retroviral/lentiviral (e.g. SIV) vector alone. Typically the invention provides a combination of a retroviral/lentiviral (e.g. SIV) and one or more CFTR modulator as described herein for use in treating CF, wherein the patient to be treated has at least one class II CFTR mutation and the treatment: (i) restores global CFTR activity in the lungs to at least 5% or at least 10% of the CFTR activity (e.g. at least 20%) in a healthy control; and/or (ii) increases CFTR current by at least about 1.3 fold (e.g. from about 1.3 fold to about 1.8 fold, from about 1.3 fold to about 3 fold, or about 1.3 fold) compared with treatment with the retroviral/lentiviral (e.g. SIV) vector alone.
[0216] A retroviral/lentiviral (e.g. SIV) described herein, typically as part of a combination therapy of the invention, may transduce of airway epithelial cells with an transduction rate sufficient to achieve a therapeutic effect on CFTR expression and/or activity. Typically, a retroviral/lentiviral (e.g. SIV) described herein, typically as part of a combination therapy of the invention, may transduce airway epithelial cells at a transduction rate of at least about 5%, at least about 7%, at least about 10%, at least about 15%, at least about 20% or more, such as from about 10% to about 20% (i.e. about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or 20%). Preferably a retroviral/lentiviral (e.g. SIV) described herein, typically as part of a combination therapy of the invention, may transduce of airway epithelial cells at a transduction rate of from about 1% to about 50% (i.e. about 14%, 15%, 17%, 18%, 20%, 25%, 30%, 35%, 40% or 45%). As defined herein, the term airway epithelial cell encompasses any cell found within the airway epithelium, as described herein, including but not limited to basal cells and submucosal gland duct cells in the upper airways, goblet, club cells and neuroendocrine cells in the bronchiolar airways, bronchioalveolar stem cells in the terminal bronchioles and type II pneumocytes in the alveoli, and any combination thereof.
[0217] A retroviral/lentiviral (e.g. SIV) vector as described herein, particularly in the context of a combination therapy of the invention may achieve a VCN of at least 1 copy/cell, 2 copies/cell, 3 copies/cell, 4 copies/cell, 5 copies/cell, 6 copies/cell, 7 copies/cell, 8, copies per cell, 9 copies per cell, at least 10 copies/cell or more.
[0218] In some embodiments, the invention relates to the use of retroviral/lentiviral (e.g. SIV) vectors with a CFTR transgene according to the invention used in combination with one or more CFTR modulator, wherein said combination does not further comprise a LasB inhibitor. In other words, the invention relates to the treatment of CF using a retroviral/lentiviral (e.g. SIV) vector with a CFTR transgene according to the invention used in combination with one or more CFTR modulator, with the proviso that a LasB inhibitor is not used in said method. Particularly, in such embodiments the use of indanes as a LasB inhibitor is disclaimed, such as those disclosed in WO 2021/191240.
[0219] The invention also provides a combination of a retroviral/lentiviral (e.g. SIV) vector as described herein and one or more CFTR modulator for use in a method of treating CF. Any retroviral/lentiviral (e.g. SIV) vector as described herein may be used in combination with any one or more CFTR modulator, such as those described and exemplified herein.
[0220] The invention also provides the use of a retroviral/lentiviral (e.g. SIV) vector as described herein in the manufacture of a medicament for use in a method of treating CF, wherein said method of treatment further comprises the administration of one or more CFTR modulator. The invention also provides the use of a CFTR modulator as described herein in the manufacture of a medicament for use in a method of treating CF, wherein said method of treatment further comprises the administration of a retroviral/lentiviral (e.g. SIV) vector. Any retroviral/lentiviral (e.g. SIV) vector as described herein may be used in combination with any one or more CFTR modulator, such as those described and exemplified herein.
Formulation and Administration
[0221] The retroviral/lentiviral (e.g. SIV) vectors and the CFTR modulators of the invention may each independently be administered in any dosage appropriate for achieving the desired therapeutic effect. Appropriate dosages may be determined by a clinician or other medical practitioner using standard techniques and within the normal course of their work.
[0222] Non-limiting examples of suitable dosages of the retroviral/lentiviral (e.g. SIV) vectors include from about 110.sup.6 (which may also be written as 10.sup.6) transducing units (TU) to about 110.sup.14 (which may also be written as 10.sup.14) TU, preferably from between about 10.sup.6 TU to about 10.sup.12 TU, such as about 10.sup.6 TU, 1.510.sup.6 TU, 10.sup.7 TU, 1.510.sup.7 TU, 10.sup.8 TU, 1.510.sup.8 TU, 510.sup.8 TU, 810.sup.8 TU, 10.sup.9 TU, 1.510.sup.9 TU, 10.sup.10 TU, 1.510.sup.10 TU, 10.sup.11 TU, 1.510.sup.11 TU or more. Preferred dose ranges include between about 8.sup.8 to about 10.sup.14 TU, or between about 10.sup.6 to about 10.sup.12 TU, These doses may be administered at any dosing interval determined by the treating clinician, such as those dosing intervals described herein (e.g. at a frequency of every 3 months, every 6 months, every 12 months, every 24 months, every 36 months or every 48 months). By way of non-limiting example, a dose of about 10.sup.6 TU may be administered once every 6 months. By way of a further non-limiting example, a dose of about 10.sup.10 TU may be administered every 12 months.
[0223] Each CFTR modulator may be administered at the standard dose indicated for single therapy of CF with said CFTR modulator, i.e. at an approved or standard dose/concentration for monotherapy with said modulator. Each CFTR modulator may be administered as part of combination therapy according to the invention at a dose lower than the standard dose indicated for single therapy with said CFTR modulator, i.e. at concentration lower than an approved or standard dose/concentration for monotherapy with said modulator. A CFTR modulator may be administered at a dose of between about 5 mg to about 200 mg, such as between about 5 mg to about 150 mg, between about 25 mg to about 150 mg, or between about 75 mg to about 150 mg. These doses may be administered at any dosing interval determined by the treating clinician, such as those dosing intervals described herein (e.g. at a frequency of every 4 hours, every 8 hours, or every 12 hours, preferably every 12 hours).
[0224] By way of non-limiting example, ivacaftor may be administered at a dose of from about 5 mg to about 150 mg, preferably from about 25 mg to about 150 mg, such as about 150 mg every 12 hours. By way of a further non-limiting example, for paediatric dosing ivacaftor may be administered at a dose of about 75 mg every 12 hours.
[0225] By way of a further non-limiting example, Trikafta (elexacaftor+tezacaftor+ivacaftor) may be administered every 12 hours, with a first (typically morning) dose of Trikafta typically comprising about 200 mg elexacaftor, about 100 mg tezacaftor and about 150 mg ivacaftor (e.g. in the form of 2 tablets each containing elexacaftor+tezacaftor+ivacaftor), and a second (typically evening) dose of about 150 mg ivacaftor (e.g. in the form of 2 tablets).
[0226] By way of a further non-limiting example, Orkambi (lumacaftor+ivacaftor) may be administered every 12 hours, with each dose of Orkambi typically comprising about 400 mg lumacaftor and about 250 mg ivacaftor (e.g. in the form of 2 tablets each containing lumacaftor+ivacaftor). By way of a further non-limiting example, for paediatric dosing Orkambi may be administered every 12 hours, with each dose of Orkambi typically comprising about 200 mg lumacaftor and about 250 mg ivacaftor (e.g. in the form of 2 tablets each containing lumacaftor+ivacaftor).
[0227] By way of a further non-limiting example, Symdeko (tezacaftor+ivacaftor) may be administered every 12 hours, with a first (typically morning) dose of Symdeko typically comprising about 100 mg tezacaftor and about 150 mg ivacaftor (e.g. in the form of a single tablet containing tezacaftor+ivacaftor), and a second (typically evening) dose of about 150 mg ivacaftor (e.g. in the form of 1 tablet).
[0228] Combination therapy according to the invention may use compositions comprising the retroviral/lentiviral (e.g. SIV) vectors described above, and a pharmaceutically-acceptable carrier. Typically said compositions are formulated for administration by inhalation.
[0229] The CFTR modulators used in the combination therapies of the invention may be appropriately formulated in a composition comprising a pharmaceutically-acceptable carrier. The CFTR modulators are typically formulated for administration orally.
[0230] Administration of CFTR modulators is generally by conventional routes e.g. oral, intravenous, subcutaneous, intraperitoneal, or mucosal routes. The administration may be by parenteral injection, for example, a subcutaneous, intradermal or intramuscular injection. For example, CFTR modulators may be particularly suited to administration orally. Administration of small molecule CFTR modulators may be injection, such as intravenously, intramuscularly, intradermally, or subcutaneously, or preferably by oral administration (small molecules with molecule weight of less than 500 Da typically exhibiting oral bioavailability).
[0231] CFTR modulators may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid prior to injection may alternatively be prepared. The preparation may also be emulsified, or the peptide encapsulated in liposomes or microcapsules.
[0232] Preferably CFTR modulators are prepared as for oral administration. A CFTR modulator may be encapsulated within an oral dosage form. Oral formulations include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders.
[0233] The active ingredients (such as the CFTR modulators used in the combination therapies of the invention) are often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof. In addition, if desired, the composition may contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and/or adjuvants which enhance the effectiveness of the CFTR modulators.
[0234] Non-limiting examples of pharmaceutically acceptable carriers include water, saline, and phosphate-buffered saline. In some embodiments, however, the composition is in lyophilized form, in which case it may include a stabilizer, such as bovine serum albumin (BSA). In some embodiments, it may be desirable to formulate the composition with a preservative, such as thiomersal or sodium azide, to facilitate long-term storage.
[0235] Preferably the CFTR modulators used in combination therapies of the invention are formulated for oral administration and are administered orally. Thus, the invention typically relates to combination therapies using two separate formulations, one comprising the retroviral/lentiviral (e.g. SIV) vectors (typically for inhalative administration) and one comprising the one or more CFTR modulator (typically for oral administration).
[0236] The retroviral/lentiviral (e.g. SIV) vectors of the invention and/or the one or more CFTR modulator may each independently be administered by any appropriate route. It may be desired to direct the compositions of the present invention (as described above) to the respiratory system of a subject. Efficient transmission of a therapeutic/prophylactic composition or medicament to the site of infection in the respiratory tract may be achieved by oral administration or by inhalation, for example, as aerosols, or by catheters. Typically the retroviral/lentiviral (e.g. SIV) vectors of the invention are administered by inhalation, and as such are preferably stable in clinically relevant nebulisers, inhalers (including metered dose inhalers), catheters and aerosols, etc. Typically the one or more CFTR modulator is administered orally.
[0237] Formulations for inhalative administration may be in the form of droplets, and may be administered by nebulisation using a suitable device. A formulation for inhalative administration may comprise droplets having approximate mass median aerodynamic diameters (MMADs) in the range of 0.1-50 m, such as 1-25 m, 1-10 m, or 1-5 m, particularly 1-10 m. Alternatively, in terms of volume, the droplets may be in the range of about 0.001-100 l, such as 0.1-50 l or 1.0-25 l, or such as 0.001-1 l.
[0238] The aerosol formulation may take the form of a powder, suspension or solution. The size of aerosol droplets is relevant to the delivery capability of an aerosol. Smaller droplets may travel further down the respiratory airway towards the alveoli than would larger particles. In one embodiment, the aerosol droplets have a diameter distribution to facilitate delivery along the entire length of the trachea, bronchi and bronchioles i.e. the conducting airways. Alternatively, the droplet size distribution may be selected to target a particular section of the respiratory airway, for example the bronchioles or alveoli. In the case of aerosol delivery of the medicament, the droplets may have diameters in the approximate range of 0.1-50 m, preferably 1-25 m, more preferably 1-10 m or 1-5 m.
[0239] Aerosol particles may be for delivery using a nebulizer (e.g. via the mouth) or nasal spray. An aerosol formulation may optionally contain a propellant and/or surfactant.
[0240] The formulation of pharmaceutical aerosols is routine to those skilled in the art, see for example, Sciarra, J. in Remington's Pharmaceutical Sciences (supra). The agents may be formulated as solution aerosols, dispersion or suspension aerosols of dry powders, emulsions or semisolid preparations. The aerosol may be delivered using any propellant system known to those skilled in the art. The aerosols may be applied to the upper respiratory tract, for example by nasal inhalation, or to the lower respiratory tract or to both. The part of the lung that the medicament is delivered to may be determined by the disorder. Compositions comprising a vector of the invention, in particular where intranasal delivery is to be used, may comprise a humectant. This may help reduce or prevent drying of the mucus membrane and to prevent irritation of the membranes. Suitable humectants include, for instance, sorbitol, mineral oil, vegetable oil and glycerol; soothing agents; membrane conditioners; sweeteners; and combinations thereof. The compositions may comprise a surfactant. Suitable surfactants include non-ionic, anionic and cationic surfactants. Examples of surfactants that may be used include, for example, polyoxyethylene derivatives of fatty acid partial esters of sorbitol anhydrides, such as for example, Tween 80, Polyoxyl 40 Stearate, Polyoxy ethylene 50 Stearate, fusieates, bile salts and Octoxynol.
[0241] As described herein, in some cases after an initial administration a subsequent administration of a retroviral/lentiviral (e.g. SIV) vector may be performed. The administration may, for instance, be at least a week, two weeks, a month, two months, three months, four months, six months, a year or more after the initial administration. In some instances, retroviral/lentiviral (e.g. SIV) vector of the invention may be administered at least once a week, once a fortnight, once a month, every two months, every six months, annually or at longer intervals. Preferably, administration is every six months, more preferably annually. The retroviral/lentiviral (e.g. SIV) vectors may, for instance, be administered at intervals dictated by when the effects of the previous administration are decreasing. Administration of the retroviral/lentiviral (e.g. SIV) vectors at the desired frequency may continue for the life of the patient.
[0242] Also as described herein, the CFTR modulator is typically administered on an on-going basis at a frequency as described herein. The CFTR modulator may, for instance, be administered at intervals dictated by when the effects of the previous administration are decreasing. Administration of the CFTR modulator at the desired frequency may continue for the life of the patient.
[0243] The combination therapy of the present invention may be combined with one or more additional treatment for CF, including one or more additional CF modulator, bronchodilators, steroids, agents which thin or clear mucus or other pulmonary secretions, antibiotics and/or airway clearance techniques such as active cycle of breathing techniques (ACBT) and autogenic drainage. The one or more additional treatment for CF may be administered sequentially or simultaneously (as defined herein) with the combination therapy of the invention.
Sequence Homology
[0244] Any of a variety of sequence alignment methods can be used to determine percent identity, including, without limitation, global methods, local methods and hybrid methods, such as, e.g., segment approach methods. Protocols to determine percent identity are routine procedures within the scope of one skilled in the art. Global methods align sequences from the beginning to the end of the molecule and determine the best alignment by adding up scores of individual residue pairs and by imposing gap penalties. Non-limiting methods include, e.g., CLUSTAL W, see, e.g., Julie D. Thompson et al., CLUSTAL W: Improving the Sensitivity of Progressive Multiple Sequence Alignment Through Sequence Weighting, Position-Specific Gap Penalties and Weight Matrix Choice, 22(22) Nucleic Acids Research 4673-4680 (1994); and iterative refinement, see, e.g., Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264(4) J. Mol. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all of the input sequences. Non-limiting methods include, e.g., Match-box, see, e.g., Eric Depiereux and Ernest Feytmans, Match-Box: A Fundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbs sampling, see, e.g., C. E. Lawrence et al., Detecting Subtle Sequence Signals: A Gibbs Sampling Strategy for Multiple Alignment, 262(5131) Science 208-214 (1993); Align-M, see, e.g., Ivo Van Wa Ile et al., Align-MA New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics:1428-1435 (2004).
[0245] Thus, percent sequence identity is determined by conventional methods. See, for example, Altschul et al., Bull. Math. Bio. 48: 603-16, 1986 and Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915-19, 1992. Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the blosum 62 scoring matrix of Henikoff and Henikoff (ibid.) as shown below (amino acids are indicated by the standard one-letter codes).
[0246] The percent sequence identity between two or more nucleic acid or amino acid sequences is a function of the number of identical positions shared by the sequences. Thus, % identity may be calculated as the number of identical nucleotides/amino acids divided by the total number of nucleotides/amino acids, multiplied by 100. Calculations of % sequence identity may also take into account the number of gaps, and the length of each gap that needs to be introduced to optimize alignment of two or more sequences. Sequence comparisons and the determination of percent identity between two or more sequences can be carried out using specific mathematical algorithms, such as BLAST, which will be familiar to a skilled person.
TABLE-US-00002 ALIGNMENT SCORES FOR DETERMINING SEQUENCE IDENTITY A R N D C Q E G H I L K M F P S T W Y V A 4 R 1 5 N 2 0 6 D 2 2 1 6 C 0 3 3 3 9 Q 1 1 0 0 3 5 E 1 0 0 2 4 2 5 G 0 2 0 1 3 2 2 6 H 2 0 1 1 3 0 0 2 8 I 1 3 3 3 1 3 3 4 3 4 L 1 2 3 4 1 2 3 4 3 2 4 K 1 2 0 1 3 1 1 2 1 3 2 5 M 1 1 2 3 1 0 2 3 2 1 2 1 5 F 2 3 3 3 2 3 3 3 1 0 0 3 0 6 P 1 2 2 1 3 1 1 2 2 3 3 1 2 4 7 S 1 1 1 0 1 0 0 0 1 2 2 0 1 2 1 4 T 0 1 0 1 1 1 1 2 2 1 1 1 1 2 1 1 5 W 3 3 4 4 2 2 3 2 2 3 2 3 1 1 4 3 2 11 Y 2 2 2 3 2 1 2 3 2 1 1 2 1 3 3 2 2 2 7 V 0 3 3 3 1 2 2 3 3 3 1 2 1 1 2 2 0 3 1 4
The percent identity is then calculated as:
[0247] Substantially homologous polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (as described herein) and other substitutions that do not significantly affect the folding or activity of the polypeptide; small deletions, typically of one to about amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.
[0248] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyl lysine, 2-aminoisobutyric acid, isovaline and -methyl serine) may be substituted for amino acid residues of the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, and unnatural amino acids may be substituted for polypeptide amino acid residues. The polypeptides of the present invention can also comprise non-naturally occurring amino acid residues.
[0249] Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2,4-methano-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, pipecolic acid, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods are known in the art for incorporating non-naturally occurring amino acid residues into proteins. For example, an in vitro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs. Methods for synthesizing amino acids and aminoacylating tRNA are known in the art. Transcription and translation of plasmids containing nonsense mutations is carried out in a cell free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J. Am. Chem. Soc. 113:2722, 1991; Ellman et al., Methods Enzymol. 202:301, 1991; Chung et al., Science 259:806-9, 1993; and Chung et al., Proc. Natl. Acad. Sci. USA 90:10145-9, 1993). In a second method, translation is carried out in Xenopus oocytes by microinjection of mutated mRNA and chemically aminoacylated suppressor tRNAs (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). Within a third method, E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine). The non-naturally occurring amino acid is incorporated into the polypeptide in place of its natural counterpart. See, Koide et al., Biochem. 33:7470-6, 1994. Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).
[0250] A limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for amino acid residues of polypeptides of the present invention.
[0251] Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081-5, 1989). Sites of biological interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labelling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al., J. Mol. Biol. 224:899-904, 1992; Wlodaver et al., FEBS Lett. 309:59-64, 1992. The identities of essential amino acids can also be inferred from analysis of homologies with related components (e.g. the translocation or protease components) of the polypeptides of the present invention.
[0252] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).
[0253] Multiple amino acid substitutions can be made and tested using known methods of mutagenesis and screening, such as those disclosed by Reidhaar-Olson and Sauer (Science 241:53-7, 1988) or Bowie and Sauer (Proc. Natl. Acad. Sci. USA 86:2152-6, 1989). Briefly, these authors disclose methods for simultaneously randomizing two or more positions in a polypeptide, selecting for functional polypeptide, and then sequencing the mutagenized polypeptides to determine the spectrum of allowable substitutions at each position. Other methods that can be used include phage display (e.g., Lowman et al., Biochem. 30:10832-7, 1991; Ladner et al., U.S. Pat. No. 5,223,409; Huse, WIPO Publication WO 92/06204) and region-directed mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).
Sequence Information
Key to Sequences
[0254] SEQ ID NO: 1 Exemplified CFTR transgene (soCFTR2)
SEQ ID NO: 2 Exemplified hCEF promoter
SEQ ID NO: 3 Exemplified CMV promoter
SEQ ID NO: 4 Exemplified EF1a promoter
SEQ ID NO: 5 wild-type SIV gag-pol nucleic acid sequence
SEQ ID NO: 6 codon-optimised SIV gal-pol nucleic acid sequence
SEQ ID NO: 7 Plasmid as defined in
SEQ ID NO: 8 Plasmid as defined in
SEQ ID NO: 9 Plasmid as defined in
SEQ ID NO: 10 Plasmid as defined in
SEQ ID NO: 11 Plasmid as defined in
SEQ ID NO: 12 Plasmid as defined in
SEQ ID NO: 13 Plasmid as defined in
SEQ ID NO: 14 Exemplified WPRE component (mWPRE)
SEQ ID NO: 15 Exemplary CAG promoter
SEQ ID NO: 16 modified SIV/CFTR RNA sequence
SEQ ID NO: 17 Fct4 protein
SEQ ID NO: 18 Fct4 protein (including signal sequence)
SEQ ID NO: 19 Fct4 protein (fragment 1)
SEQ ID NO: 20 Fct4 protein (fragment 2)
SEQ ID NO: 21 Fct4 protein signal sequence
SEQ ID NO: 22 p17 protein sequence
SEQ ID NO: 23 p24 protein sequence
SEQ ID NO: 24 p8 protein sequence
SEQ ID NO: 25 Protease sequence
SEQ ID NO: 26 p51 protein sequence
SEQ ID NO: 27 p15 protein sequence
SEQ ID NO: 28 p31 protein sequence
SEQ ID NO: 29 Gag protein
SEQ ID NO: 30 Pol protein
Sequences
[0255]
TABLE-US-00003 SEQIDNO:1ExemplifiedCFTRtransgene(soCFTR2) 1 GCTAGCCACCATGCAGAGAAGCCCTCTGGAGAAGGCCTCTGTGGTGAGCAAGCTGTTCTT 61 CAGCTGGACCAGGCCCATCCTGAGGAAGGGCTACAGGCAGAGACTGGAGCTGTCTGACAT 121 CTACCAGATCCCCTCTGTGGACTCTGCTGACAACCTGTCTGAGAAGCTGGAGAGGGAGTG 181 GGATAGAGAGCTGGCCAGCAAGAAGAACCCCAAGCTGATCAATGCCCTGAGGAGATGCTT 241 CTTCTGGAGATTCATGTTCTATGGCATCTTCCTGTACCTGGGGGAAGTGACCAAGGCTGT 301 GCAGCCTCTGCTGCTGGGCAGAATCATTGCCAGCTATGACCCTGACAACAAGGAGGAGAG 361 GAGCATTGCCATCTACCTGGGCATTGGCCTGTGCCTGCTGTTCATTGTGAGGACCCTGCT 421 GCTGCACCCTGCCATCTTTGGCCTGCACCACATTGGCATGCAGATGAGGATTGCCATGTT 481 CAGCCTGATCTACAAGAAAACCCTGAAGCTGTCCAGCAGAGTGCTGGACAAGATCAGCAT 541 TGGCCAGCTGGTGAGCCTGCTGAGCAACAACCTGAACAAGTTTGATGAGGGCCTGGCCCT 601 GGCCCACTTTGTGTGGATTGCCCCTCTGCAGGTGGCCCTGCTGATGGGCCTGATTTGGGA 661 GCTGCTGCAGGCCTCTGCCTTTTGTGGCCTGGGCTTCCTGATTGTGCTGGCCCTGTTTCA 721 GGCTGGCCTGGGCAGGATGATGATGAAGTACAGGGACCAGAGGGCAGGCAAGATCAGTGA 781 GAGGCTGGTGATCACCTCTGAGATGATTGAGAACATCCAGTCTGTGAAGGCCTACTGTTG 841 GGAGGAAGCTATGGAGAAGATGATTGAAAACCTGAGGCAGACAGAGCTGAAGCTGACCAG 901 GAAGGCTGCCTATGTGAGATACTTCAACAGCTCTGCCTTCTTCTTCTCTGGCTTCTTTGT 961 GGTGTTCCTGTCTGTGCTGCCCTATGCCCTGATCAAGGGGATCATCCTGAGAAAGATTTT 1021 CACCACCATCAGCTTCTGCATTGTGCTGAGGATGGCTGTGACCAGACAGTTCCCCTGGGC 1081 TGTGCAGACCTGGTATGACAGCCTGGGGGCCATCAACAAGATCCAGGACTTCCTGCAGAA 1141 GCAGGAGTACAAGACCCTGGAGTACAACCTGACCACCACAGAAGTGGTGATGGAGAATGT 1201 GACAGCCTTCTGGGAGGAGGGCTTTGGGGAGCTGTTTGAGAAGGCCAAGCAGAACAACAA 1261 CAACAGAAAGACCAGCAATGGGGATGACTCCCTGTTCTTCTCCAACTTCTCCCTGCTGGG 1321 CACACCTGTGCTGAAGGACATCAACTTCAAGATTGAGAGGGGGCAGCTGCTGGCTGTGGC 1381 TGGATCTACAGGGGCTGGCAAGACCAGCCTGCTGATGATGATCATGGGGGAGCTGGAGCC 1441 TTCTGAGGGCAAGATCAAGCACTCTGGCAGGATCAGCTTTTGCAGCCAGTTCAGCTGGAT 1501 CATGCCTGGCACCATCAAGGAGAACATCATCTTTGGAGTGAGCTATGATGAGTACAGATA 1561 CAGGAGTGTGATCAAGGCCTGCCAGCTGGAGGAGGACATCAGCAAGTTTGCTGAGAAGGA 1621 CAACATTGTGCTGGGGGAGGGAGGCATTACACTGTCTGGGGGCCAGAGAGCCAGAATCAG 1681 CCTGGCCAGGGCTGTGTACAAGGATGCTGACCTGTACCTGCTGGACTCCCCCTTTGGCTA 1741 CCTGGATGTGCTGACAGAGAAGGAGATTTTTGAGAGCTGTGTGTGCAAGCTGATGGCCAA 1801 CAAGACCAGAATCCTGGTGACCAGCAAGATGGAGCACCTGAAGAAGGCTGACAAGATCCT 1861 GATCCTGCATGAGGGCAGCAGCTACTTCTATGGGACCTTCTCTGAGCTGCAGAACCTGCA 1921 GCCTGACTTCAGCTCTAAGCTGATGGGCTGTGACAGCTTTGACCAGTTCTCTGCTGAGAG 1981 GAGGAACAGCATCCTGACAGAGACCCTGCACAGATTCAGCCTGGAGGGAGATGCCCCTGT 2041 GAGCTGGACAGAGACCAAGAAGCAGAGCTTCAAGCAGACAGGGGAGTTTGGGGAGAAGAG 2101 GAAGAACTCCATCCTGAACCCCATCAACAGCATCAGGAAGTTCAGCATTGTGCAGAAAAC 2161 CCCCCTGCAGATGAATGGCATTGAGGAAGATTCTGATGAGCCCCTGGAGAGGAGACTGAG 2221 CCTGGTGCCTGATTCTGAGCAGGGAGAGGCCATCCTGCCTAGGATCTCTGTGATCAGCAC 2281 AGGCCCTACACTGCAGGCCAGAAGGAGGCAGTCTGTGCTGAACCTGATGACCCACTCTGT 2341 GAACCAGGGCCAGAACATCCACAGGAAAACCACAGCCTCCACCAGGAAAGTGAGCCTGGC 2401 CCCTCAGGCCAATCTGACAGAGCTGGACATCTACAGCAGGAGGCTGTCTCAGGAGACAGG 2461 CCTGGAGATTTCTGAGGAGATCAATGAGGAGGACCTGAAAGAGTGCTTCTTTGATGACAT 2521 GGAGAGCATCCCTGCTGTGACCACCTGGAACACCTACCTGAGATACATCACAGTGCACAA 2581 GAGCCTGATCTTTGTGCTGATCTGGTGCCTGGTGATCTTCCTGGCTGAAGTGGCTGCCTC 2641 TCTGGTGGTGCTGTGGCTGCTGGGAAACACCCCACTGCAGGACAAGGGCAACAGCACCCA 2701 CAGCAGGAACAACAGCTATGCTGTGATCATCACCTCCACCTCCAGCTACTATGTGTTCTA 2761 CATCTATGTGGGAGTGGCTGATACCCTGCTGGCTATGGGCTTCTTTAGAGGCCTGCCCCT 2821 GGTGCACACACTGATCACAGTGAGCAAGATCCTCCACCACAAGATGCTGCACTCTGTGCT 2881 GCAGGCTCCTATGAGCACCCTGAATACCCTGAAGGCTGGGGGCATCCTGAACAGATTCTC 2941 CAAGGATATTGCCATCCTGGATGACCTGCTGCCTCTCACCATCTTTGACTTCATCCAGCT 3001 GCTGCTGATTGTGATTGGGGCCATTGCTGTGGTGGCAGTGCTGCAGCCCTACATCTTTGT 3061 GGCCACAGTGCCTGTGATTGTGGCCTTCATCATGCTGAGGGCCTACTTTCTGCAGACCTC 3121 CCAGCAGCTGAAGCAGCTGGAGTCTGAGGGCAGAAGCCCCATCTTCACCCACCTGGTGAC 3181 AAGCCTGAAGGGCCTGTGGACCCTGAGAGCCTTTGGCAGGCAGCCCTACTTTGAGACCCT 3241 GTTCCACAAGGCCCTGAACCTGCACACAGCCAACTGGTTCCTCTACCTGTCCACCCTGAG 3301 ATGGTTCCAGATGAGAATTGAGATGATCTTTGTCATCTTCTTCATTGCTGTGACCTTCAT 3361 CAGCATTCTGACCACAGGAGAGGGAGAGGGCAGAGTGGGCATTATCCTGACCCTGGCCAT 3421 GAACATCATGAGCACACTGCAGTGGGCAGTGAACAGCAGCATTGATGTGGACAGCCTGAT 3481 GAGGAGTGTGAGCAGAGTGTTCAAGTTCATTGATATGCCCACAGAGGGCAAGCCTACCAA 3541 GAGCACCAAGCCCTACAAGAATGGCCAGCTGAGCAAAGTGATGATCATTGAGAACAGCCA 3601 TGTGAAGAAGGATGATATCTGGCCCAGTGGAGGCCAGATGACAGTGAAGGACCTGACAGC 3661 CAAGTACACAGAGGGGGGCAATGCTATCCTGGAGAACATCTCCTTCAGCATCTCCCCTGG 3721 CCAGAGAGTGGGACTGCTGGGAAGAACAGGCTCTGGCAAGTCTACCCTGCTGTCTGCCTT 3781 CCTGAGGCTGCTGAACACAGAGGGAGAGATCCAGATTGATGGAGTGTCCTGGGACAGCAT 3841 CACACTGCAGCAGTGGAGGAAGGCCTTTGGTGTGATCCCCCAGAAAGTGTTCATCTTCAG 3901 TGGCACCTTCAGGAAGAACCTGGACCCCTATGAGCAGTGGTCTGACCAGGAGATTTGGAA 3961 AGTGGCTGATGAAGTGGGCCTGAGAAGTGTGATTGAGCAGTTCCCTGGCAAGCTGGACTT 4021 TGTCCTGGTGGATGGGGGCTGTGTGCTGAGCCATGGCCACAAGCAGCTGATGTGCCTGGC 4081 CAGATCAGTGCTGAGCAAGGCCAAGATCCTGCTGCTGGATGAGCCTTCTGCCCACCTGGA 4141 TCCTGTGACCTACCAGATCATCAGGAGGACCCTCAAGCAGGCCTTTGCTGACTGCACAGT 4201 CATCCTGTGTGAGCACAGGATTGAGGCCATGCTGGAGTGCCAGCAGTTCCTGGTGATTGA 4261 GGAGAACAAAGTGAGGCAGTATGACAGCATCCAGAAGCTGCTGAATGAGAGGAGCCTGTT 4321 CAGGCAGGCCATCAGCCCCTCTGATAGAGTGAAGCTGTTCCCCCACAGGAACAGCTCCAA 4381 GTGCAAGAGCAAGCCCCAGATTGCTGCCCTGAAGGAGGAGACAGAGGAGGAAGTGCAGGA 4441 CACCAGGCTGTGAGGGCCC SEQIDNO:2ExemplifiedhCEFpromoter 1 AGATCTGTTACATAACTTATGGTAAATGGCCTGCCTGGCTGACTGCCCAATGACCCCTGC 61 CCAATGATGTCAATAATGATGTATGTTCCCATGTAATGCCAATAGGGACTTTCCATTGAT 121 GTCAATGGGTGGAGTATTTATGGTAACTGCCCACTTGGCAGTACATCAAGTGTATCATAT 181 GCCAAGTATGCCCCCTATTGATGTCAATGATGGTAAATGGCCTGCCTGGCATTATGCCCA 241 GTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTATGTATTAGTCATTGCTAT 301 TACCATGGGAATTCACTAGTGGAGAAGAGCATGCTTGAGGGCTGAGTGCCCCTCAGTGGG 361 CAGAGAGCACATGGCCCACAGTCCCTGAGAAGTTGGGGGGAGGGGTGGGCAATTGAACTG 421 GTGCCTAGAGAAGGTGGGGCTTGGGTAAACTGGGAAAGTGATGTGGTGTACTGGCTCCAC 481 CTTTTTCCCCAGGGTGGGGGAGAACCATATATAAGTGCAGTAGTCTCTGTGAACATTCAA 541 GCTTCTGCCTTCTCCCTCCTGTGAGTTTGCTAGC SEQIDNO:3ExemplifiedCMVpromoter CCGCGGAGATCTCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCT ATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACC GCCATGTTGGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCA TATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCC CATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGT GGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATT GACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTT GGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCG TGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGG CACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGC GTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCACTAGAAGCTTTATTGC GGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGCTGC AGAAGTTGGTCGTGAGGCACTGGGCAGGCTAGC SEQIDNO:4ExemplifiedEF1apromoter AGATCCATATCCGCGGCAATTTTAAAAGAAAGGGAGGAATAGGGGGACAGACTTCAGCAGAGAGACTAATTAATA TAATAACAACACAATTAGAAATACAACATTTACAAACCAAAATTCAAAAAATTTTAAATTTTAGAGCCGCGGAGA TCCCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGG TCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCC TTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTG CCGCCAGAACACAGGCTAGC SEQIDNO:5wild-typeSIVgag-polnucleicacidsequence(frompGM297) ATGGGGGCGGCTACCTCAGCACTAAATAGGAGACAATTAGACCAATTTGAGAAAATACGACTTCGCCCGAACGGA AAGAAAAAGTACCAAATTAAACATTTAATATGGGCAGGCAAGGAGATGGAGCGCTTCGGCCTCCATGAGAGGTTG TTGGAGACAGAGGAGGGGTGTAAAAGAATCATAGAAGTCCTCTACCCCCTAGAACCAACAGGATCGGAGGGCTTA AAAAGTCTGTTCAATCTTGTGTGCGTACTATATTGCTTGCACAAGGAACAGAAAGTGAAAGACACAGAGGAAGCA GTAGCAACAGTAAGACAACACTGCCATCTAGTGGAAAAAGAAAAAAGTGCAACAGAGACATCTAGTGGACAAAAG AAAAATGACAAGGGAATAGCAGCGCCACCTGGTGGCAGTCAGAATTTTCCAGCGCAACAACAAGGAAATGCCTGG GTACATGTACCCTTGTCACCGCGCACCTTAAATGCGTGGGTAAAAGCAGTAGAGGAGAAAAAATTTGGAGCAGAA ATAGTACCCATGTTTCAAGCCCTATCAGAAGGCTGCACACCCTATGACATTAATCAGATGCTTAATGTGCTAGGA GATCATCAAGGGGCATTACAAATAGTGAAAGAGATCATTAATGAAGAAGCAGCCCAGTGGGATGTAACACACCCA CTACCCGCAGGACCCCTACCAGCAGGACAGCTCAGGGACCCTCGCGGCTCAGATATAGCAGGGACCACCAGCTCA GTACAAGAACAGTTAGAATGGATCTATACTGCTAACCCCCGGGTAGATGTAGGTGCCATCTACCGGAGATGGATT ATTCTAGGACTTCAAAAGTGTGTCAAAATGTACAACCCAGTATCAGTCCTAGACATTAGGCAGGGACCTAAAGAG CCCTTCAAGGATTATGTGGACAGATTTTACAAGGCAATTAGAGCAGAACAAGCCTCAGGGGAAGTGAAACAATGG ATGACAGAATCATTACTCATTCAAAATGCTAATCCAGATTGTAAGGTCATCCTGAAGGGCCTAGGAATGCACCCC ACCCTTGAAGAAATGTTAACGGCTTGTCAGGGGGTAGGAGGCCCAAGCTACAAAGCAAAAGTAATGGCAGAAATG ATGCAGACCATGCAAAATCAAAACATGGTGCAGCAGGGAGGTCCAAAAAGACAAAGACCCCCACTAAGATGTTAT AATTGTGGAAAATTTGGCCATATGCAAAGACAATGTCCGGAACCAAGGAAAACAAAATGTCTAAAGTGTGGAAAA TTGGGACACCTAGCAAAAGACTGCAGGGGACAGGTGAATTTTTTAGGGTATGGACGGTGGATGGGGGCAAAACCG AGAAATTTTCCCGCCGCTACTCTTGGAGCGGAACCGAGTGCGCCTCCTCCACCGAGCGGCACCACCCCATACGAC CCAGCAAAGAAGCTCCTGCAGCAATATGCAGAGAAAGGGAAACAACTGAGGGAGCAAAAGAGGAATCCACCGGCA ATGAATCCGGATTGGACCGAGGGATATTCTTTGAACTCCCTCTTTGGAGAAGACCAATAAAGACAGTGTATATAG AAGGGGTCCCCATTAAGGCACTGCTAGACACAGGGGCAGATGACACCATAATTAAAGAAAATGATTTACAATTAT CAGGTCCATGGAGACCCAAAATTATAGGGGGCATAGGAGGAGGCCTTAATGTAAAAGAATATAACGACAGGGAAG TAAAAATAGAAGATAAAATTTTGAGAGGAACAATATTGTTAGGAGCAACTCCCATTAATATAATAGGTAGAAATT TGCTGGCCCCGGCAGGTGCCCGGTTAGTAATGGGACAATTATCAGAAAAAATTCCTGTCACACCTGTCAAATTGA AGGAAGGGGCTCGGGGACCCTGTGTAAGACAATGGCCTCTCTCTAAAGAGAAGATTGAAGCTTTACAGGAAATAT GTTCCCAATTAGAGCAGGAAGGAAAAATCAGTAGAGTAGGAGGAGAAAATGCATACAATACCCCAATATTTTGCA TAAAGAAGAAGGACAAATCCCAGTGGAGGATGCTAGTAGACTTTAGAGAGTTAAATAAGGCAACCCAAGATTTCT TTGAAGTGCAATTAGGGATACCCCACCCAGCAGGATTAAGAAAGATGAGACAGATAACAGTTTTAGATGTAGGAG ACGCCTATTATTCCATACCATTGGATCCAAATTTTAGGAAATATACTGCTTTTACTATTCCCACAGTGAATAATC AGGGACCCGGGATTAGGTATCAATTCAACTGTCTCCCGCAAGGGTGGAAAGGATCTCCTACAATCTTCCAAAATA CAGCAGCATCCATTTTGGAGGAGATAAAAAGAAACTTGCCAGCACTAACCATTGTACAATACATGGATGATTTAT GGGTAGGTTCTCAAGAAAATGAACACACCCATGACAAATTAGTAGAACAGTTAAGAACAAAATTACAAGCCTGGG GCTTAGAAACCCCAGAAAAGAAGGTGCAAAAAGAACCACCTTATGAGTGGATGGGATACAAACTTTGGCCTCACA AATGGGAACTAAGCAGAATACAACTGGAGGAAAAAGATGAATGGACTGTCAATGACATCCAGAAGTTAGTTGGGA AACTAAATTGGGCAGCACAATTGTATCCAGGTCTTAGGACCAAGAATATATGCAAGTTAATTAGAGGAAAGAAAA ATCTGTTAGAGCTAGTGACTTGGACACCTGAGGCAGAAGCTGAATATGCAGAAAATGCAGAGATTCTTAAAACAG AACAGGAAGGAACCTATTACAAACCAGGAATACCTATTAGGGCAGCAGTACAGAAATTGGAAGGAGGACAGTGGA GTTACCAATTCAAACAAGAAGGACAAGTCTTGAAAGTAGGAAAATACACCAAGCAAAAGAACACCCATACAAATG AACTTCGCACATTAGCTGGTTTAGTGCAGAAGATTTGCAAAGAAGCTCTAGTTATTTGGGGGATATTACCAGTTC TAGAACTCCCGATAGAAAGAGAGGTATGGGAACAATGGTGGGCGGATTACTGGCAGGTAAGCTGGATTCCCGAAT GGGATTTTGTCAGCACCCCACCTTTGCTCAAACTATGGTACACATTAACAAAAGAACCCATACCCAAGGAGGACG TTTACTATGTAGATGGAGCATGCAACAGAAATTCAAAAGAAGGAAAAGCAGGATACATCTCACAATACGGAAAAC AGAGAGTAGAAACATTAGAAAACACTACCAATCAGCAAGCAGAATTAACAGCTATAAAAATGGCTTTGGAAGACA GTGGGCCTAATGTGAACATAGTAACAGACTCTCAATATGCAATGGGAATTTTGACAGCACAACCCACACAAAGTG ATTCACCATTAGTAGAGCAAATTATAGCCTTAATGATACAAAAGCAACAAATATATTTGCAGTGGGTACCAGCAC ATAAAGGAATAGGAGGAAATGAGGAGATAGATAAATTAGTGAGTAAAGGCATTAGAAGAGTTTTATTCTTAGAAA AAATAGAAGAAGCTCAAGAAGAGCATGAAAGATATCATAATAATTGGAAAAACCTAGCAGATACATATGGGCTTC CACAAATAGTAGCAAAAGAGATAGTGGCCATGTGTCCAAAATGTCAGATAAAGGGAGAACCAGTGCATGGACAAG TGGATGCCTCACCTGGAACATGGCAGATGGATTGTACTCATCTAGAAGGAAAAGTAGTCATAGTTGCGGTCCATG TAGCCAGTGGATTCATAGAAGCAGAAGTCATACCTAGGGAAACAGGAAAAGAAACGGCAAAGTTTCTATTAAAAA TACTGAGTAGATGGCCTATAACACAGTTACACACAGACAATGGGCCTAACTTTACCTCCCAAGAAGTGGCAGCAA TATGTTGGTGGGGAAAAATTGAACATACAACAGGTATACCATATAACCCCCAATCTCAAGGATCAATAGAAAGCA TGAACAAACAATTAAAAGAGATAATTGGGAAAATAAGAGATGATTGCCAATATACAGAGACAGCAGTACTGATGG CTTGCCATATTCACAATTTTAAAAGAAAGGGAGGAATAGGGGGACAGACTTCAGCAGAGAGACTAATTAATATAA TAACAACACAATTAGAAATACAACATTTACAAACCAAAATTCAAAAAATTTTAAATTTTAGAGTCTACTACAGAG AAGGGAGAGACCCTGTGTGGAAAGGACCAGCACAATTAATCTGGAAAGGGGAAGGAGCAGTGGTCCTCAAGGACG GAAGTGACCTAAAGGTTGTACCAAGAAGGAAAGCTAAAATTATTAAGGATTATGAACCCAAACAAAGAGTGGGTA ATGAGGGTGACGTGGAAGGTACCAGGGGATCTGATAACTAA SEQIDNO:6 codon-optimisedSIVgal-polnucleicacidsequence(frompGM691) ATGGGAGCTGCCACATCTGCCCTGAATAGACGGCAGCTGGACCAGTTCGAGAAGATCAGACTGCGGCCCAACGGC AAGAAGAAGTACCAGATCAAGCACCTGATCTGGGCCGGCAAAGAGATGGAAAGATTCGGCCTGCACGAGCGGCTG CTGGAAACCGAGGAAGGCTGCAAGAGAATTATCGAGGTGCTGTACCCTCTGGAACCTACCGGCTCTGAGGGCCTG AAGTCCCTGTTCAATCTCGTGTGCGTGCTGTACTGCCTGCACAAAGAACAGAAAGTGAAGGACACCGAAGAGGCC GTGGCCACAGTTAGACAGCACTGCCACCTGGTGGAAAAAGAGAAGTCCGCCACAGAGACAAGCAGCGGCCAGAAG AAGAACGACAAGGGAATTGCTGCCCCTCCTGGCGGCAGCCAGAATTTTCCTGCTCAGCAGCAGGGAAACGCCTGG GTGCACGTTCCACTGAGCCCTAGAACACTGAATGCCTGGGTCAAAGCCGTGGAAGAGAAGAAGTTTGGCGCCGAG ATCGTGCCCATGTTCCAGGCTCTGTCTGAGGGCTGCACCCCTTACGACATCAACCAGATGCTGAACGTGCTGGGA GATCACCAGGGCGCTCTGCAGATCGTGAAAGAGATCATCAACGAAGAGGCTGCCCAGTGGGACGTGACACATCCA TTGCCTGCTGGACCTCTGCCAGCCGGACAACTGAGAGATCCTAGAGGCTCTGATATCGCCGGCACCACCAGCTCT GTGCAAGAGCAGCTGGAATGGATCTACACCGCCAATCCTAGAGTGGACGTGGGCGCCATCTACAGAAGATGGATC ATCCTGGGCCTGCAGAAATGCGTGAAGATGTACAACCCCGTGTCCGTGCTGGACATCAGACAGGGACCCAAAGAG CCCTTCAAGGACTACGTGGACCGGTTCTATAAGGCCATTAGAGCCGAGCAGGCCAGCGGCGAAGTGAAGCAGTGG ATGACAGAGAGCCTGCTGATCCAGAACGCCAATCCAGACTGCAAAGTGATCCTGAAAGGCCTGGGCATGCACCCC ACACTGGAAGAGATGCTGACAGCCTGTCAAGGCGTTGGCGGCCCTTCTTACAAAGCCAAAGTGATGGCCGAGATG ATGCAGACCATGCAGAACCAGAACATGGTGCAGCAAGGCGGCCCTAAGAGACAGAGGCCTCCTCTGAGATGCTAC AACTGCGGCAAGTTCGGCCACATGCAGAGACAGTGTCCTGAGCCTAGGAAAACAAAATGTCTAAAGTGTGGAAAA TTGGGACACCTAGCAAAAGACTGCAGGGGACAGGTGAATTTTTTAGGGTATGGACGGTGGATGGGGGCAAAACCG AGAAATTTTCCCGCCGCTACTCTTGGAGCGGAACCGAGTGCGCCTCCTCCACCGAGCGGCACCACCCCATACGAC CCAGCAAAGAAGCTCCTGCAGCAATATGCAGAGAAAGGGAAACAACTGAGGGAGCAAAAGAGGAATCCACCGGCA ATGAATCCGGATTGGACCGAGGGATATTCTTTGAACTCCCTCTTTGGAGAAGACCAATAAAGACCGTGTACATCG AGGGCGTGCCCATCAAGGCTCTGCTGGATACAGGCGCCGACGACACCATCATCAAAGAGAACGACCTGCAGCTGA GCGGCCCTTGGAGGCCTAAGATCATTGGAGGAATCGGCGGAGGCCTGAACGTCAAAGAGTACAACGACCGGGAAG TGAAGATCGAGGACAAGATCCTGAGGGGCACAATCCTGCTGGGCGCCACACCTATCAACATCATCGGCAGAAATC TGCTGGCCCCTGCCGGCGCTAGACTGGTTATGGGACAGCTCTCTGAGAAGATCCCCGTGACACCCGTGAAGCTGA AAGAAGGCGCTAGAGGACCTTGTGTGCGACAGTGGCCTCTGAGCAAAGAGAAGATTGAGGCCCTGCAAGAAATCT GTAGCCAGCTGGAACAAGAGGGCAAGATCAGCAGAGTTGGCGGCGAGAACGCCTACAATACCCCTATCTTCTGCA TCAAGAAAAAGGACAAGAGCCAGTGGCGGATGCTGGTGGACTTTAGAGAGCTGAACAAGGCTACCCAGGACTTCT TCGAGGTGCAGCTGGGAATTCCTCATCCTGCCGGCCTGCGGAAGATGAGACAGATCACAGTGCTGGATGTGGGCG ACGCCTACTACAGCATCCCTCTGGACCCCAACTTCAGAAAGTACACCGCCTTCACAATCCCCACCGTGAACAATC AAGGCCCTGGCATCAGATACCAGTTCAACTGCCTGCCTCAAGGCTGGAAGGGCAGCCCCACCATTTTTCAGAATA CCGCCGCCAGCATCCTGGAAGAAATCAAGAGAAACCTGCCTGCTCTGACCATCGTGCAGTACATGGACGATCTGT GGGTCGGAAGCCAAGAGAATGAGCACACCCACGACAAGCTGGTGGAACAGCTGAGAACAAAGCTGCAGGCCTGGG GCCTCGAAACCCCTGAGAAGAAGGTGCAGAAAGAACCTCCTTACGAGTGGATGGGCTACAAGCTGTGGCCTCACA AGTGGGAGCTGAGCCGGATTCAGCTCGAAGAGAAGGACGAGTGGACCGTGAACGACATCCAGAAACTCGTGGGCA AGCTGAATTGGGCAGCCCAGCTGTATCCCGGCCTGAGGACCAAGAACATCTGCAAGCTGATCCGGGGAAAGAAGA ACCTGCTGGAACTGGTCACATGGACACCTGAGGCCGAGGCCGAATATGCCGAGAATGCCGAAATCCTGAAAACCG AGCAAGAGGGGACCTACTACAAGCCTGGCATTCCAATCAGAGCTGCCGTGCAGAAACTGGAAGGCGGCCAGTGGT CCTACCAGTTTAAGCAAGAAGGCCAGGTCCTGAAAGTGGGCAAGTACACCAAGCAGAAGAACACCCACACCAACG AGCTGAGGACACTGGCTGGCCTGGTCCAGAAAATCTGCAAAGAGGCCCTGGTCATTTGGGGCATCCTGCCTGTTC TGGAACTGCCCATTGAGCGGGAAGTGTGGGAACAGTGGTGGGCCGATTACTGGCAAGTGTCTTGGATCCCCGAGT GGGACTTCGTGTCTACCCCTCCTCTGCTGAAACTGTGGTACACCCTGACAAAAGAGCCCATTCCTAAAGAGGACG TCTACTACGTTGACGGCGCCTGCAACCGGAACTCCAAAGAAGGCAAGGCCGGCTACATCAGCCAGTACGGCAAGC AGAGAGTGGAAACCCTGGAAAACACCACCAACCAGCAGGCCGAGCTGACCGCCATTAAGATGGCCCTGGAAGATA GCGGCCCCAATGTGAACATCGTGACCGACTCTCAGTACGCCATGGGAATCCTGACAGCCCAGCCTACACAGAGCG ATAGCCCTCTGGTTGAGCAGATCATTGCCCTGATGATTCAGAAGCAGCAAATCTACCTGCAGTGGGTGCCCGCTC ACAAAGGCATCGGCGGAAACGAAGAGATCGATAAGCTGGTGTCCAAGGGAATCAGACGGGTGCTGTTCCTGGAAA AGATTGAAGAGGCCCAAGAGGAACACGAGCGCTACCACAACAACTGGAAGAATCTGGCCGACACCTACGGACTGC CCCAGATCGTGGCCAAAGAAATCGTGGCTATGTGCCCCAAGTGTCAGATCAAGGGCGAACCTGTGCACGGCCAAG TGGATGCTTCTCCTGGCACATGGCAGATGGACTGTACCCACCTGGAAGGCAAAGTGGTCATCGTGGCTGTGCACG TGGCCTCCGGCTTTATTGAGGCCGAAGTGATCCCCAGAGAGACAGGCAAAGAAACCGCCAAGTTCCTGCTGAAGA TCCTGTCCAGATGGCCCATCACACAGCTGCACACCGACAACGGCCCTAACTTCACATCTCAAGAGGTGGCCGCCA TCTGTTGGTGGGGAAAGATTGAGCACACAACCGGCATTCCCTACAATCCACAGAGCCAGGGCAGCATCGAGTCCA TGAACAAGCAGCTCAAAGAGATTATCGGCAAGATCCGGGACGACTGCCAGTACACAGAAACAGCCGTGCTGATGG CCTGTCACATCCACAACTTCAAGCGGAAAGGCGGCATCGGAGGACAGACATCTGCCGAGAGACTGATCAATATCA TCACCACTCAGCTGGAAATCCAGCACCTCCAGACCAAGATCCAGAAGATTCTGAACTTCCGGGTGTACTACCGCG AGGGCAGAGATCCTGTTTGGAAAGGCCCAGCACAGCTGATCTGGAAAGGCGAAGGTGCCGTGGTGCTGAAGGATG GCTCTGATCTGAAGGTGGTGCCCAGACGGAAGGCCAAGATTATCAAGGATTACGAGCCCAAACAGCGCGTGGGCA ATGAAGGCGACGTTGAGGGCACAAGAGGCAGCGACAATTGA SEQIDNO:7PlasmidasdefinedinFigure1C(pDNA2apGM691) ATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCG TTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGT ATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACT TGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCA TGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTAT TTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGGGGG GCGAGGGGGGGGGGGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTT TTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCC TTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGG TGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGT GGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGT GTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGC TTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGG GGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAAC CCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCG CGGGGCTCGCCGTGCCGGGGGGGGGGGGCGGCAGGTGGGGGTGCCGGGCGGGGGGGGGCCGCCTCGGGCCGGGG AGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCC TTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGC GCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGGGGGGAGGGCC TTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTC GGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTC ATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAAT TGCTCGAGCCACCATGGGAGCTGCCACATCTGCCCTGAATAGACGGCAGCTGGACCAGTTCGAGAAGATCAGACT GCGGCCCAACGGCAAGAAGAAGTACCAGATCAAGCACCTGATCTGGGCCGGCAAAGAGATGGAAAGATTCGGCCT GCACGAGCGGCTGCTGGAAACCGAGGAAGGCTGCAAGAGAATTATCGAGGTGCTGTACCCTCTGGAACCTACCGG CTCTGAGGGCCTGAAGTCCCTGTTCAATCTCGTGTGCGTGCTGTACTGCCTGCACAAAGAACAGAAAGTGAAGGA CACCGAAGAGGCCGTGGCCACAGTTAGACAGCACTGCCACCTGGTGGAAAAAGAGAAGTCCGCCACAGAGACAAG CAGCGGCCAGAAGAAGAACGACAAGGGAATTGCTGCCCCTCCTGGCGGCAGCCAGAATTTTCCTGCTCAGCAGCA GGGAAACGCCTGGGTGCACGTTCCACTGAGCCCTAGAACACTGAATGCCTGGGTCAAAGCCGTGGAAGAGAAGAA GTTTGGCGCCGAGATCGTGCCCATGTTCCAGGCTCTGTCTGAGGGCTGCACCCCTTACGACATCAACCAGATGCT GAACGTGCTGGGAGATCACCAGGGCGCTCTGCAGATCGTGAAAGAGATCATCAACGAAGAGGCTGCCCAGTGGGA CGTGACACATCCATTGCCTGCTGGACCTCTGCCAGCCGGACAACTGAGAGATCCTAGAGGCTCTGATATCGCCGG CACCACCAGCTCTGTGCAAGAGCAGCTGGAATGGATCTACACCGCCAATCCTAGAGTGGACGTGGGCGCCATCTA CAGAAGATGGATCATCCTGGGCCTGCAGAAATGCGTGAAGATGTACAACCCCGTGTCCGTGCTGGACATCAGACA GGGACCCAAAGAGCCCTTCAAGGACTACGTGGACCGGTTCTATAAGGCCATTAGAGCCGAGCAGGCCAGCGGCGA AGTGAAGCAGTGGATGACAGAGAGCCTGCTGATCCAGAACGCCAATCCAGACTGCAAAGTGATCCTGAAAGGCCT GGGCATGCACCCCACACTGGAAGAGATGCTGACAGCCTGTCAAGGCGTTGGCGGCCCTTCTTACAAAGCCAAAGT GATGGCCGAGATGATGCAGACCATGCAGAACCAGAACATGGTGCAGCAAGGCGGCCCTAAGAGACAGAGGCCTCC TCTGAGATGCTACAACTGCGGCAAGTTCGGCCACATGCAGAGACAGTGTCCTGAGCCTAGGAAAACAAAATGTCT AAAGTGTGGAAAATTGGGACACCTAGCAAAAGACTGCAGGGGACAGGTGAATTTTTTAGGGTATGGACGGTGGAT GGGGGCAAAACCGAGAAATTTTCCCGCCGCTACTCTTGGAGCGGAACCGAGTGCGCCTCCTCCACCGAGCGGCAC CACCCCATACGACCCAGCAAAGAAGCTCCTGCAGCAATATGCAGAGAAAGGGAAACAACTGAGGGAGCAAAAGAG GAATCCACCGGCAATGAATCCGGATTGGACCGAGGGATATTCTTTGAACTCCCTCTTTGGAGAAGACCAATAAAG ACCGTGTACATCGAGGGCGTGCCCATCAAGGCTCTGCTGGATACAGGCGCCGACGACACCATCATCAAAGAGAAC GACCTGCAGCTGAGCGGCCCTTGGAGGCCTAAGATCATTGGAGGAATCGGCGGAGGCCTGAACGTCAAAGAGTAC AACGACCGGGAAGTGAAGATCGAGGACAAGATCCTGAGGGGCACAATCCTGCTGGGCGCCACACCTATCAACATC ATCGGCAGAAATCTGCTGGCCCCTGCCGGCGCTAGACTGGTTATGGGACAGCTCTCTGAGAAGATCCCCGTGACA CCCGTGAAGCTGAAAGAAGGCGCTAGAGGACCTTGTGTGCGACAGTGGCCTCTGAGCAAAGAGAAGATTGAGGCC CTGCAAGAAATCTGTAGCCAGCTGGAACAAGAGGGCAAGATCAGCAGAGTTGGCGGCGAGAACGCCTACAATACC CCTATCTTCTGCATCAAGAAAAAGGACAAGAGCCAGTGGCGGATGCTGGTGGACTTTAGAGAGCTGAACAAGGCT ACCCAGGACTTCTTCGAGGTGCAGCTGGGAATTCCTCATCCTGCCGGCCTGCGGAAGATGAGACAGATCACAGTG CTGGATGTGGGCGACGCCTACTACAGCATCCCTCTGGACCCCAACTTCAGAAAGTACACCGCCTTCACAATCCCC ACCGTGAACAATCAAGGCCCTGGCATCAGATACCAGTTCAACTGCCTGCCTCAAGGCTGGAAGGGCAGCCCCACC ATTTTTCAGAATACCGCCGCCAGCATCCTGGAAGAAATCAAGAGAAACCTGCCTGCTCTGACCATCGTGCAGTAC ATGGACGATCTGTGGGTCGGAAGCCAAGAGAATGAGCACACCCACGACAAGCTGGTGGAACAGCTGAGAACAAAG CTGCAGGCCTGGGGCCTCGAAACCCCTGAGAAGAAGGTGCAGAAAGAACCTCCTTACGAGTGGATGGGCTACAAG CTGTGGCCTCACAAGTGGGAGCTGAGCCGGATTCAGCTCGAAGAGAAGGACGAGTGGACCGTGAACGACATCCAG AAACTCGTGGGCAAGCTGAATTGGGCAGCCCAGCTGTATCCCGGCCTGAGGACCAAGAACATCTGCAAGCTGATC CGGGGAAAGAAGAACCTGCTGGAACTGGTCACATGGACACCTGAGGCCGAGGCCGAATATGCCGAGAATGCCGAA ATCCTGAAAACCGAGCAAGAGGGGACCTACTACAAGCCTGGCATTCCAATCAGAGCTGCCGTGCAGAAACTGGAA GGCGGCCAGTGGTCCTACCAGTTTAAGCAAGAAGGCCAGGTCCTGAAAGTGGGCAAGTACACCAAGCAGAAGAAC ACCCACACCAACGAGCTGAGGACACTGGCTGGCCTGGTCCAGAAAATCTGCAAAGAGGCCCTGGTCATTTGGGGC ATCCTGCCTGTTCTGGAACTGCCCATTGAGCGGGAAGTGTGGGAACAGTGGTGGGCCGATTACTGGCAAGTGTCT TGGATCCCCGAGTGGGACTTCGTGTCTACCCCTCCTCTGCTGAAACTGTGGTACACCCTGACAAAAGAGCCCATT CCTAAAGAGGACGTCTACTACGTTGACGGCGCCTGCAACCGGAACTCCAAAGAAGGCAAGGCCGGCTACATCAGC CAGTACGGCAAGCAGAGAGTGGAAACCCTGGAAAACACCACCAACCAGCAGGCCGAGCTGACCGCCATTAAGATG GCCCTGGAAGATAGCGGCCCCAATGTGAACATCGTGACCGACTCTCAGTACGCCATGGGAATCCTGACAGCCCAG CCTACACAGAGCGATAGCCCTCTGGTTGAGCAGATCATTGCCCTGATGATTCAGAAGCAGCAAATCTACCTGCAG TGGGTGCCCGCTCACAAAGGCATCGGCGGAAACGAAGAGATCGATAAGCTGGTGTCCAAGGGAATCAGACGGGTG CTGTTCCTGGAAAAGATTGAAGAGGCCCAAGAGGAACACGAGCGCTACCACAACAACTGGAAGAATCTGGCCGAC ACCTACGGACTGCCCCAGATCGTGGCCAAAGAAATCGTGGCTATGTGCCCCAAGTGTCAGATCAAGGGCGAACCT GTGCACGGCCAAGTGGATGCTTCTCCTGGCACATGGCAGATGGACTGTACCCACCTGGAAGGCAAAGTGGTCATC GTGGCTGTGCACGTGGCCTCCGGCTTTATTGAGGCCGAAGTGATCCCCAGAGAGACAGGCAAAGAAACCGCCAAG TTCCTGCTGAAGATCCTGTCCAGATGGCCCATCACACAGCTGCACACCGACAACGGCCCTAACTTCACATCTCAA GAGGTGGCCGCCATCTGTTGGTGGGGAAAGATTGAGCACACAACCGGCATTCCCTACAATCCACAGAGCCAGGGC AGCATCGAGTCCATGAACAAGCAGCTCAAAGAGATTATCGGCAAGATCCGGGACGACTGCCAGTACACAGAAACA GCCGTGCTGATGGCCTGTCACATCCACAACTTCAAGCGGAAAGGCGGCATCGGAGGACAGACATCTGCCGAGAGA CTGATCAATATCATCACCACTCAGCTGGAAATCCAGCACCTCCAGACCAAGATCCAGAAGATTCTGAACTTCCGG GTGTACTACCGCGAGGGCAGAGATCCTGTTTGGAAAGGCCCAGCACAGCTGATCTGGAAAGGCGAAGGTGCCGTG GTGCTGAAGGATGGCTCTGATCTGAAGGTGGTGCCCAGACGGAAGGCCAAGATTATCAAGGATTACGAGCCCAAA CAGCGCGTGGGCAATGAAGGCGACGTTGAGGGCACAAGAGGCAGCGACAATTGAAATTCACTCCTCAGGTGCAGG CTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTG CCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTG CAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGA ATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAG GTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTT TTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGAT TTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATCCCTCGACCTGCAGCCCA AGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGA GCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTG CCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCGGATCCGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCC TAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTA TTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAG GCTTTTGCAAAAAGCTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCAC AAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTCC GCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTA ATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAAC CGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCA AGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCT CCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGC TCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAG CCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCA GCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAAC TACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGT AGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGA AAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAA GGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCA ATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTA TTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAG TTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTC CCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAACAGC TTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAA CCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGA ATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAAT ACCTGGAATGCTGTTTTTCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTG ATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTA CCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGC CCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAA GACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCAT GATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACAATTGGTCGAC SEQIDNO:8PlasmidasdefinedinFIG.1A(pDNA1pGM326) GGTACCTCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATT GCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTG ATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTAC ATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGT TCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGC AGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTA TGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGT GATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCAT TGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTGCGATCGCCC GCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGCTGGCTTGTAACT CAGTCTCTTACTAGGAGACCAGCTTGAGCCTGGGTGTTCGCTGGTTAGCCTAACCTGGTTGGCCACCAGGGGTAA GGACTCCTTGGCTTAGAAAGCTAATAAACTTGCCTGCATTAGAGCTTATCTGAGTCAAGTGTCCTCATTGACGCC TCACTCTCTTGAACGGGAATCTTCCTTACTGGGTTCTCTCTCTGACCCAGGCGAGAGAAACTCCAGCAGTGGCGC CCGAACAGGGACTTGAGTGAGAGTGTAGGCACGTACAGCTGAGAAGGCGTCGGACGCGAAGGAAGCGCGGGGTGC GACGCGACCAAGAAGGAGACTTGGTGAGTAGGCTTCTCGAGTGCCGGGAAAAAGCTCGAGCCTAGTTAGAGGACT AGGAGAGGCCGTAGCCGTAACTACTCTGGGCAAGTAGGGCAGGCGGTGGGTACGCAATGGGGGCGGCTACCTCAG CACTAAATAGGAGACAATTAGACCAATTTGAGAAAATACGACTTCGCCCGAACGGAAAGAAAAAGTACCAAATTA AACATTTAATATGGGCAGGCAAGGAGATGGAGCGCTTCGGCCTCCATGAGAGGTTGTTGGAGACAGAGGAGGGGT GTAAAAGAATCATAGAAGTCCTCTACCCCCTAGAACCAACAGGATCGGAGGGCTTAAAAAGTCTGTTCAATCTTG TGTGCGTGCTATATTGCTTGCACAAGGAACAGAAAGTGAAAGACACAGAGGAAGCAGTAGCAACAGTAAGACAAC ACTGCCATCTAGTGGAAAAAGAAAAAAGTGCAACAGAGACATCTAGTGGACAAAAGAAAAATGACAAGGGAATAG CAGCGCCACCTGGTGGCAGTCAGAATTTTCCAGCGCAACAACAAGGAAATGCCTGGGTACATGTACCCTTGTCAC CGCGCACCTTAAATGCGTGGGTAAAAGCAGTAGAGGAGAAAAAATTTGGAGCAGAAATAGTACCCATGTTTCAAG CCCTATCGAATTCCCGTTTGTGCTAGGGTTCTTAGGCTTCTTGGGGGCTGCTGGAACTGCAATGGGAGCAGCGGC GACAGCCCTGACGGTCCAGTCTCAGCATTTGCTTGCTGGGATACTGCAGCAGCAGAAGAATCTGCTGGCGGCTGT GGAGGCTCAACAGCAGATGTTGAAGCTGACCATTTGGGGTGTTAAAAACCTCAATGCCCGCGTCACAGCCCTTGA GAAGTACCTAGAGGATCAGGCACGACTAAACTCCTGGGGGTGCGCATGGAAACAAGTATGTCATACCACAGTGGA GTGGCCCTGGACAAATCGGACTCCGGATTGGCAAAATATGACTTGGTTGGAGTGGGAAAGACAAATAGCTGATTT GGAAAGCAACATTACGAGACAATTAGTGAAGGCTAGAGAACAAGAGGAAAAGAATCTAGATGCCTATCAGAAGTT AACTAGTTGGTCAGATTTCTGGTCTTGGTTCGATTTCTCAAAATGGCTTAACATTTTAAAAATGGGATTTTTAGT AATAGTAGGAATAATAGGGTTAAGATTACTTTACACAGTATATGGATGTATAGTGAGGGTTAGGCAGGGATATGT TCCTCTATCTCCACAGATCCATATCCGCGGCAATTTTAAAAGAAAGGGAGGAATAGGGGGACAGACTTCAGCAGA GAGACTAATTAATATAATAACAACACAATTAGAAATACAACATTTACAAACCAAAATTCAAAAAATTTTAAATTT TAGAGCCGCGGAGATCTGTTACATAACTTATGGTAAATGGCCTGCCTGGCTGACTGCCCAATGACCCCTGCCCAA TGATGTCAATAATGATGTATGTTCCCATGTAATGCCAATAGGGACTTTCCATTGATGTCAATGGGTGGAGTATTT ATGGTAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTATGCCCCCTATTGATGTCAATGATGGT AAATGGCCTGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTATGTATTA GTCATTGCTATTACCATGGGAATTCACTAGTGGAGAAGAGCATGCTTGAGGGCTGAGTGCCCCTCAGTGGGCAGA GAGCACATGGCCCACAGTCCCTGAGAAGTTGGGGGGAGGGGTGGGCAATTGAACTGGTGCCTAGAGAAGGTGGGG CTTGGGTAAACTGGGAAAGTGATGTGGTGTACTGGCTCCACCTTTTTCCCCAGGGTGGGGGAGAACCATATATAA GTGCAGTAGTCTCTGTGAACATTCAAGCTTCTGCCTTCTCCCTCCTGTGAGTTTGCTAGCCACCATGCAGAGAAG CCCTCTGGAGAAGGCCTCTGTGGTGAGCAAGCTGTTCTTCAGCTGGACCAGGCCCATCCTGAGGAAGGGCTACAG GCAGAGACTGGAGCTGTCTGACATCTACCAGATCCCCTCTGTGGACTCTGCTGACAACCTGTCTGAGAAGCTGGA GAGGGAGTGGGATAGAGAGCTGGCCAGCAAGAAGAACCCCAAGCTGATCAATGCCCTGAGGAGATGCTTCTTCTG GAGATTCATGTTCTATGGCATCTTCCTGTACCTGGGGGAAGTGACCAAGGCTGTGCAGCCTCTGCTGCTGGGCAG AATCATTGCCAGCTATGACCCTGACAACAAGGAGGAGAGGAGCATTGCCATCTACCTGGGCATTGGCCTGTGCCT GCTGTTCATTGTGAGGACCCTGCTGCTGCACCCTGCCATCTTTGGCCTGCACCACATTGGCATGCAGATGAGGAT TGCCATGTTCAGCCTGATCTACAAGAAAACCCTGAAGCTGTCCAGCAGAGTGCTGGACAAGATCAGCATTGGCCA GCTGGTGAGCCTGCTGAGCAACAACCTGAACAAGTTTGATGAGGGCCTGGCCCTGGCCCACTTTGTGTGGATTGC CCCTCTGCAGGTGGCCCTGCTGATGGGCCTGATTTGGGAGCTGCTGCAGGCCTCTGCCTTTTGTGGCCTGGGCTT CCTGATTGTGCTGGCCCTGTTTCAGGCTGGCCTGGGCAGGATGATGATGAAGTACAGGGACCAGAGGGCAGGCAA GATCAGTGAGAGGCTGGTGATCACCTCTGAGATGATTGAGAACATCCAGTCTGTGAAGGCCTACTGTTGGGAGGA AGCTATGGAGAAGATGATTGAAAACCTGAGGCAGACAGAGCTGAAGCTGACCAGGAAGGCTGCCTATGTGAGATA CTTCAACAGCTCTGCCTTCTTCTTCTCTGGCTTCTTTGTGGTGTTCCTGTCTGTGCTGCCCTATGCCCTGATCAA GGGGATCATCCTGAGAAAGATTTTCACCACCATCAGCTTCTGCATTGTGCTGAGGATGGCTGTGACCAGACAGTT CCCCTGGGCTGTGCAGACCTGGTATGACAGCCTGGGGGCCATCAACAAGATCCAGGACTTCCTGCAGAAGCAGGA GTACAAGACCCTGGAGTACAACCTGACCACCACAGAAGTGGTGATGGAGAATGTGACAGCCTTCTGGGAGGAGGG CTTTGGGGAGCTGTTTGAGAAGGCCAAGCAGAACAACAACAACAGAAAGACCAGCAATGGGGATGACTCCCTGTT CTTCTCCAACTTCTCCCTGCTGGGCACACCTGTGCTGAAGGACATCAACTTCAAGATTGAGAGGGGGCAGCTGCT GGCTGTGGCTGGATCTACAGGGGCTGGCAAGACCAGCCTGCTGATGATGATCATGGGGGAGCTGGAGCCTTCTGA GGGCAAGATCAAGCACTCTGGCAGGATCAGCTTTTGCAGCCAGTTCAGCTGGATCATGCCTGGCACCATCAAGGA GAACATCATCTTTGGAGTGAGCTATGATGAGTACAGATACAGGAGTGTGATCAAGGCCTGCCAGCTGGAGGAGGA CATCAGCAAGTTTGCTGAGAAGGACAACATTGTGCTGGGGGAGGGAGGCATTACACTGTCTGGGGGCCAGAGAGC CAGAATCAGCCTGGCCAGGGCTGTGTACAAGGATGCTGACCTGTACCTGCTGGACTCCCCCTTTGGCTACCTGGA TGTGCTGACAGAGAAGGAGATTTTTGAGAGCTGTGTGTGCAAGCTGATGGCCAACAAGACCAGAATCCTGGTGAC CAGCAAGATGGAGCACCTGAAGAAGGCTGACAAGATCCTGATCCTGCATGAGGGCAGCAGCTACTTCTATGGGAC CTTCTCTGAGCTGCAGAACCTGCAGCCTGACTTCAGCTCTAAGCTGATGGGCTGTGACAGCTTTGACCAGTTCTC TGCTGAGAGGAGGAACAGCATCCTGACAGAGACCCTGCACAGATTCAGCCTGGAGGGAGATGCCCCTGTGAGCTG GACAGAGACCAAGAAGCAGAGCTTCAAGCAGACAGGGGAGTTTGGGGAGAAGAGGAAGAACTCCATCCTGAACCC CATCAACAGCATCAGGAAGTTCAGCATTGTGCAGAAAACCCCCCTGCAGATGAATGGCATTGAGGAAGATTCTGA TGAGCCCCTGGAGAGGAGACTGAGCCTGGTGCCTGATTCTGAGCAGGGAGAGGCCATCCTGCCTAGGATCTCTGT GATCAGCACAGGCCCTACACTGCAGGCCAGAAGGAGGCAGTCTGTGCTGAACCTGATGACCCACTCTGTGAACCA GGGCCAGAACATCCACAGGAAAACCACAGCCTCCACCAGGAAAGTGAGCCTGGCCCCTCAGGCCAATCTGACAGA GCTGGACATCTACAGCAGGAGGCTGTCTCAGGAGACAGGCCTGGAGATTTCTGAGGAGATCAATGAGGAGGACCT GAAAGAGTGCTTCTTTGATGACATGGAGAGCATCCCTGCTGTGACCACCTGGAACACCTACCTGAGATACATCAC AGTGCACAAGAGCCTGATCTTTGTGCTGATCTGGTGCCTGGTGATCTTCCTGGCTGAAGTGGCTGCCTCTCTGGT GGTGCTGTGGCTGCTGGGAAACACCCCACTGCAGGACAAGGGCAACAGCACCCACAGCAGGAACAACAGCTATGC TGTGATCATCACCTCCACCTCCAGCTACTATGTGTTCTACATCTATGTGGGAGTGGCTGATACCCTGCTGGCTAT GGGCTTCTTTAGAGGCCTGCCCCTGGTGCACACACTGATCACAGTGAGCAAGATCCTCCACCACAAGATGCTGCA CTCTGTGCTGCAGGCTCCTATGAGCACCCTGAATACCCTGAAGGCTGGGGGCATCCTGAACAGATTCTCCAAGGA TATTGCCATCCTGGATGACCTGCTGCCTCTCACCATCTTTGACTTCATCCAGCTGCTGCTGATTGTGATTGGGGC CATTGCTGTGGTGGCAGTGCTGCAGCCCTACATCTTTGTGGCCACAGTGCCTGTGATTGTGGCCTTCATCATGCT GAGGGCCTACTTTCTGCAGACCTCCCAGCAGCTGAAGCAGCTGGAGTCTGAGGGCAGAAGCCCCATCTTCACCCA CCTGGTGACAAGCCTGAAGGGCCTGTGGACCCTGAGAGCCTTTGGCAGGCAGCCCTACTTTGAGACCCTGTTCCA CAAGGCCCTGAACCTGCACACAGCCAACTGGTTCCTCTACCTGTCCACCCTGAGATGGTTCCAGATGAGAATTGA GATGATCTTTGTCATCTTCTTCATTGCTGTGACCTTCATCAGCATTCTGACCACAGGAGAGGGAGAGGGCAGAGT GGGCATTATCCTGACCCTGGCCATGAACATCATGAGCACACTGCAGTGGGCAGTGAACAGCAGCATTGATGTGGA CAGCCTGATGAGGAGTGTGAGCAGAGTGTTCAAGTTCATTGATATGCCCACAGAGGGCAAGCCTACCAAGAGCAC CAAGCCCTACAAGAATGGCCAGCTGAGCAAAGTGATGATCATTGAGAACAGCCATGTGAAGAAGGATGATATCTG GCCCAGTGGAGGCCAGATGACAGTGAAGGACCTGACAGCCAAGTACACAGAGGGGGGCAATGCTATCCTGGAGAA CATCTCCTTCAGCATCTCCCCTGGCCAGAGAGTGGGACTGCTGGGAAGAACAGGCTCTGGCAAGTCTACCCTGCT GTCTGCCTTCCTGAGGCTGCTGAACACAGAGGGAGAGATCCAGATTGATGGAGTGTCCTGGGACAGCATCACACT GCAGCAGTGGAGGAAGGCCTTTGGTGTGATCCCCCAGAAAGTGTTCATCTTCAGTGGCACCTTCAGGAAGAACCT GGACCCCTATGAGCAGTGGTCTGACCAGGAGATTTGGAAAGTGGCTGATGAAGTGGGCCTGAGAAGTGTGATTGA GCAGTTCCCTGGCAAGCTGGACTTTGTCCTGGTGGATGGGGGCTGTGTGCTGAGCCATGGCCACAAGCAGCTGAT GTGCCTGGCCAGATCAGTGCTGAGCAAGGCCAAGATCCTGCTGCTGGATGAGCCTTCTGCCCACCTGGATCCTGT GACCTACCAGATCATCAGGAGGACCCTCAAGCAGGCCTTTGCTGACTGCACAGTCATCCTGTGTGAGCACAGGAT TGAGGCCATGCTGGAGTGCCAGCAGTTCCTGGTGATTGAGGAGAACAAAGTGAGGCAGTATGACAGCATCCAGAA GCTGCTGAATGAGAGGAGCCTGTTCAGGCAGGCCATCAGCCCCTCTGATAGAGTGAAGCTGTTCCCCCACAGGAA CAGCTCCAAGTGCAAGAGCAAGCCCCAGATTGCTGCCCTGAAGGAGGAGACAGAGGAGGAAGTGCAGGACACCAG GCTGTGAGGGCCCAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCC TTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCATTTTCTC CTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTG CACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGC TTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTT GGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTG GATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCT GCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCC GCAAGCTTCGCACTTTTTAAAAGAAAAGGGAGGACTGGATGGGATTTATTACTCCGATAGGACGCTGGCTTGTAA CTCAGTCTCTTACTAGGAGACCAGCTTGAGCCTGGGTGTTCGCTGGTTAGCCTAACCTGGTTGGCCACCAGGGGT AAGGACTCCTTGGCTTAGAAAGCTAATAAACTTGCCTGCATTAGAGCTCTTACGCGTCCCGGGCTCGAGATCCGC ATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCC ATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCC AGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTAACTTGTTTATTGCAGCTTATAATGG TTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTC CAAACTCATCAATGTATCTTATCATGTCTGTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTG CGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAAC ATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGC CCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAG GCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTT CTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCC AAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCC AACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGC GGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTG CTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGT TTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGG TCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAG ATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTAGAAA AACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGT TTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCG ACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGA GTGACGACTGAATCCGGTGAGAATGGCAACAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTA CGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACG CGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACA ATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTTCCGGGGATCGCAGTGGTGAGTAAC CATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTG ACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTC CCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCA TCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTA CTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTT TGAGACACAACAATTGGTCGACGGATCC SEQIDNO:9PlasmidasdefinedinFIG.1B(pDNA1pGM830) GGTACCTCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATT GCATACGTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTG ATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTAC ATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGT TCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGC AGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTA TGCCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGT GATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCAT TGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTGCGATCGCCC GCCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGCTGGCTTGTAACT CAGTCTCTTACTAGGAGACCAGCTTGAGCCTGGGTGTTCGCTGGTTAGCCTAACCTGGTTGGCCACCAGGGGTAA GGACTCCTTGGCTTAGAAAGCTAATAAACTTGCCTGCATTAGAGCTTATCTGAGTCAAGTGTCCTCATTGACGCC TCACTCTCTTGAACGGGAATCTTCCTTACTGGGTTCTCTCTCTGACCCAGGCGAGAGAAACTCCAGCAGTGGCGC CCGAACAGGGACTTGAGTGAGAGTGTAGGCACGTACAGCTGAGAAGGCGTCGGACGCGAAGGAAGCGCGGGGTGC GACGCGACCAAGAAGGAGACTTGGTGAGTAGGCTTCTCGAGTGCCGGGAAAAAGCTCGAGCCTAGTTAGAGGACT AGGAGAGGCCGTAGCCGTAACTACTCTGGGCAAGTAGGGCAGGCGGTGGGTACGCAATTGGGGGCGGCTACCTCA GCACTAAATAGGAGACAATTAGACCAATTTGAGAAAATACGACTTCGCCCGAACGGAAAGAAAAAGTACCAAATT AAACATTTAATATTGGGCAGGCAAGGAGATTGGAGCGCTTCGGCCTCCATGAGAGGTTGTTGGAGACAGAGGAGG GGTGTAAAAGAATCATAGAAGTCCTCTACCCCCTAGAACCAACAGGATCGGAGGGCTTAAAAAGTCTGTTCAATC TTGTGTGCGTGCTATATTGCTTGCACAAGGAACAGAAAGTGAAAGACACAGAGGAAGCAGTAGCAACAGTAAGAC AACACTGCCATCTAGTGGAAAAAGAAAAAAGTGCAACAGAGACATCTAGTGGACAAAAGAAAAATGACAAGGGAA TAGCAGCGCCACCTGGTGGCAGTCAGAATTTTCCAGCGCAACAACAAGGAAATTGCCTGGGTACATGTACCCTTG TCACCGCGCACCTTAAATGCGTGGGTAAAAGCAGTAGAGGAGAAAAAATTTGGAGCAGAAATAGTACCCATGTTT CAAGCCCTATCGCCTGCAGGCCGTTTGTGCTAGGGTTCTTAGGCTTCTTGGGGGCTGCTGGAACTGCATTGGGAG CAGCGGCGACAGCCCTGACGGTCCAGTCTCAGCATTTGCTTGCTGGGATACTGCAGCAGCAGAAGAATCTGCTGG CGGCTGTGGAGGCTCAACAGCAGATGTTGAAGCTGACCATTTGGGGTGTTAAAAACCTCAATGCCCGCGTCACAG CCCTTGAGAAGTACCTAGAGGATCAGGCACGACTAAACTCCTGGGGGTGCGCATGGAAACAAGTATGTCATACCA CAGTGGAGTGGCCCTGGACAAATCGGACTCCGGATTGGCAAAATAAGACTTGGTTGGAGTGGGAAAGACAAATAG CTGATTTGGAAAGCAACATTACGAGACAATTAGTGAAGGCTAGAGAACAAGAGGAAAAGAATCTAGATGCCTATC AGAAGTTAACTAGTTGGTCAGATTTCTGGTCTTGGTTCGATTTCTCAAAATGGCTTAACATTTTAAAAAAGGGAT TTTTAGTAATAGTAGGAATAATAGGGTTAAGATTACTTTACACAGTATATGGATGTATAGTGAGGGTTAGGCAGG GATATGTTCCTCTATCTCCACAGATCCATATAAAGCGGCAATTTTAAAAGAAAGGGAGGAATAGGGGGACAGACT TCAGCAGAGAGACTAATTAATATAATAACAACACAATTAGAAATACAACATTTACAAACCAAAATTCAAAAAATT TTAAATTTTAGAGCCGCGGAGATCTGTTACATAACTTATGGTAAATGGCCTGCCTGGCTGACTGCCCAATGACCC CTGCCCAATGATGTCAATAATGATGTATGTTCCCATGTAATGCCAATAGGGACTTTCCATTGATGTCAATGGGTG GAGTATTTATGGTAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTATGCCCCCTATTGATGTCA ATGATGGTAAATGGCCTGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCT ATGTATTAGTCATTGCTATTACCATGGGAATTCACTAGTGGAGAAGAGCATGCTTGAGGGCTGAGTGCCCCTCAG TGGGCAGAGAGCACATGGCCCACAGTCCCTGAGAAGTTGGGGGGAGGGGTGGGCAATTGAACTGGTGCCTAGAGA AGGTGGGGCTTGGGTAAACTGGGAAAGTGATGTGGTGTACTGGCTCCACCTTTTTCCCCAGGGTGGGGGAGAACC ATATATAAGTGCAGTAGTCTCTGTGAACATTCAAGCTTCTGCCTTCTCCCTCCTGTGAGTTTGCTAGCCACCATG CAGAGAAGCCCTCTGGAGAAGGCCTCTGTGGTGAGCAAGCTGTTCTTCAGCTGGACCAGGCCCATCCTGAGGAAG GGCTACAGGCAGAGACTGGAGCTGTCTGACATCTACCAGATCCCCTCTGTGGACTCTGCTGACAACCTGTCTGAG AAGCTGGAGAGGGAGTGGGATAGAGAGCTGGCCAGCAAGAAGAACCCCAAGCTGATCAATGCCCTGAGGAGATGC TTCTTCTGGAGATTCATGTTCTATGGCATCTTCCTGTACCTGGGGGAAGTGACCAAGGCTGTGCAGCCTCTGCTG CTGGGCAGAATCATTGCCAGCTATGACCCTGACAACAAGGAGGAGAGGAGCATTGCCATCTACCTGGGCATTGGC CTGTGCCTGCTGTTCATTGTGAGGACCCTGCTGCTGCACCCTGCCATCTTTGGCCTGCACCACATTGGCATGCAG ATGAGGATTGCCATGTTCAGCCTGATCTACAAGAAAACCCTGAAGCTGTCCAGCAGAGTGCTGGACAAGATCAGC ATTGGCCAGCTGGTGAGCCTGCTGAGCAACAACCTGAACAAGTTTGATGAGGGCCTGGCCCTGGCCCACTTTGTG TGGATTGCCCCTCTGCAGGTGGCCCTGCTGATGGGCCTGATTTGGGAGCTGCTGCAGGCCTCTGCCTTTTGTGGC CTGGGCTTCCTGATTGTGCTGGCCCTGTTTCAGGCTGGCCTGGGCAGGATGATGATGAAGTACAGGGACCAGAGG GCAGGCAAGATCAGTGAGAGGCTGGTGATCACCTCTGAGATGATTGAGAACATCCAGTCTGTGAAGGCCTACTGT TGGGAGGAAGCTATGGAGAAGATGATTGAAAACCTGAGGCAGACAGAGCTGAAGCTGACCAGGAAGGCTGCCTAT GTGAGATACTTCAACAGCTCTGCCTTCTTCTTCTCTGGCTTCTTTGTGGTGTTCCTGTCTGTGCTGCCCTATGCC CTGATCAAGGGGATCATCCTGAGAAAGATTTTCACCACCATCAGCTTCTGCATTGTGCTGAGGATGGCTGTGACC AGACAGTTCCCCTGGGCTGTGCAGACCTGGTATGACAGCCTGGGGGCCATCAACAAGATCCAGGACTTCCTGCAG AAGCAGGAGTACAAGACCCTGGAGTACAACCTGACCACCACAGAAGTGGTGATGGAGAATGTGACAGCCTTCTGG GAGGAGGGCTTTGGGGAGCTGTTTGAGAAGGCCAAGCAGAACAACAACAACAGAAAGACCAGCAATGGGGATGAC TCCCTGTTCTTCTCCAACTTCTCCCTGCTGGGCACACCTGTGCTGAAGGACATCAACTTCAAGATTGAGAGGGGG CAGCTGCTGGCTGTGGCTGGATCTACAGGGGCTGGCAAGACCAGCCTGCTGATGATGATCATGGGGGAGCTGGAG CCTTCTGAGGGCAAGATCAAGCACTCTGGCAGGATCAGCTTTTGCAGCCAGTTCAGCTGGATCATGCCTGGCACC ATCAAGGAGAACATCATCTTTGGAGTGAGCTATGATGAGTACAGATACAGGAGTGTGATCAAGGCCTGCCAGCTG GAGGAGGACATCAGCAAGTTTGCTGAGAAGGACAACATTGTGCTGGGGGAGGGAGGCATTACACTGTCTGGGGGC CAGAGAGCCAGAATCAGCCTGGCCAGGGCTGTGTACAAGGATGCTGACCTGTACCTGCTGGACTCCCCCTTTGGC TACCTGGATGTGCTGACAGAGAAGGAGATTTTTGAGAGCTGTGTGTGCAAGCTGATGGCCAACAAGACCAGAATC CTGGTGACCAGCAAGATGGAGCACCTGAAGAAGGCTGACAAGATCCTGATCCTGCATGAGGGCAGCAGCTACTTC TATGGGACCTTCTCTGAGCTGCAGAACCTGCAGCCTGACTTCAGCTCTAAGCTGATGGGCTGTGACAGCTTTGAC CAGTTCTCTGCTGAGAGGAGGAACAGCATCCTGACAGAGACCCTGCACAGATTCAGCCTGGAGGGAGATGCCCCT GTGAGCTGGACAGAGACCAAGAAGCAGAGCTTCAAGCAGACAGGGGAGTTTGGGGAGAAGAGGAAGAACTCCATC CTGAACCCCATCAACAGCATCAGGAAGTTCAGCATTGTGCAGAAAACCCCCCTGCAGATGAATGGCATTGAGGAA GATTCTGATGAGCCCCTGGAGAGGAGACTGAGCCTGGTGCCTGATTCTGAGCAGGGAGAGGCCATCCTGCCTAGG ATCTCTGTGATCAGCACAGGCCCTACACTGCAGGCCAGAAGGAGGCAGTCTGTGCTGAACCTGATGACCCACTCT GTGAACCAGGGCCAGAACATCCACAGGAAAACCACAGCCTCCACCAGGAAAGTGAGCCTGGCCCCTCAGGCCAAT CTGACAGAGCTGGACATCTACAGCAGGAGGCTGTCTCAGGAGACAGGCCTGGAGATTTCTGAGGAGATCAATGAG GAGGACCTGAAAGAGTGCTTCTTTGATGACATGGAGAGCATCCCTGCTGTGACCACCTGGAACACCTACCTGAGA TACATCACAGTGCACAAGAGCCTGATCTTTGTGCTGATCTGGTGCCTGGTGATCTTCCTGGCTGAAGTGGCTGCC TCTCTGGTGGTGCTGTGGCTGCTGGGAAACACCCCACTGCAGGACAAGGGCAACAGCACCCACAGCAGGAACAAC AGCTATGCTGTGATCATCACCTCCACCTCCAGCTACTATGTGTTCTACATCTATGTGGGAGTGGCTGATACCCTG CTGGCTATGGGCTTCTTTAGAGGCCTGCCCCTGGTGCACACACTGATCACAGTGAGCAAGATCCTCCACCACAAG ATGCTGCACTCTGTGCTGCAGGCTCCTATGAGCACCCTGAATACCCTGAAGGCTGGGGGCATCCTGAACAGATTC TCCAAGGATATTGCCATCCTGGATGACCTGCTGCCTCTCACCATCTTTGACTTCATCCAGCTGCTGCTGATTGTG ATTGGGGCCATTGCTGTGGTGGCAGTGCTGCAGCCCTACATCTTTGTGGCCACAGTGCCTGTGATTGTGGCCTTC ATCATGCTGAGGGCCTACTTTCTGCAGACCTCCCAGCAGCTGAAGCAGCTGGAGTCTGAGGGCAGAAGCCCCATC TTCACCCACCTGGTGACAAGCCTGAAGGGCCTGTGGACCCTGAGAGCCTTTGGCAGGCAGCCCTACTTTGAGACC CTGTTCCACAAGGCCCTGAACCTGCACACAGCCAACTGGTTCCTCTACCTGTCCACCCTGAGATGGTTCCAGATG AGAATTGAGATGATCTTTGTCATCTTCTTCATTGCTGTGACCTTCATCAGCATTCTGACCACAGGAGAGGGAGAG GGCAGAGTGGGCATTATCCTGACCCTGGCCATGAACATCATGAGCACACTGCAGTGGGCAGTGAACAGCAGCATT GATGTGGACAGCCTGATGAGGAGTGTGAGCAGAGTGTTCAAGTTCATTGATATGCCCACAGAGGGCAAGCCTACC AAGAGCACCAAGCCCTACAAGAATGGCCAGCTGAGCAAAGTGATGATCATTGAGAACAGCCATGTGAAGAAGGAT GATATCTGGCCCAGTGGAGGCCAGATGACAGTGAAGGACCTGACAGCCAAGTACACAGAGGGGGGCAATGCTATC CTGGAGAACATCTCCTTCAGCATCTCCCCTGGCCAGAGAGTGGGACTGCTGGGAAGAACAGGCTCTGGCAAGTCT ACCCTGCTGTCTGCCTTCCTGAGGCTGCTGAACACAGAGGGAGAGATCCAGATTGATGGAGTGTCCTGGGACAGC ATCACACTGCAGCAGTGGAGGAAGGCCTTTGGTGTGATCCCCCAGAAAGTGTTCATCTTCAGTGGCACCTTCAGG AAGAACCTGGACCCCTATGAGCAGTGGTCTGACCAGGAGATTTGGAAAGTGGCTGATGAAGTGGGCCTGAGAAGT GTGATTGAGCAGTTCCCTGGCAAGCTGGACTTTGTCCTGGTGGATGGGGGCTGTGTGCTGAGCCATGGCCACAAG CAGCTGATGTGCCTGGCCAGATCAGTGCTGAGCAAGGCCAAGATCCTGCTGCTGGATGAGCCTTCTGCCCACCTG GATCCTGTGACCTACCAGATCATCAGGAGGACCCTCAAGCAGGCCTTTGCTGACTGCACAGTCATCCTGTGTGAG CACAGGATTGAGGCCATGCTGGAGTGCCAGCAGTTCCTGGTGATTGAGGAGAACAAAGTGAGGCAGTATGACAGC ATCCAGAAGCTGCTGAATGAGAGGAGCCTGTTCAGGCAGGCCATCAGCCCCTCTGATAGAGTGAAGCTGTTCCCC CACAGGAACAGCTCCAAGTGCAAGAGCAAGCCCCAGATTGCTGCCCTGAAGGAGGAGACAGAGGAGGAAGTGCAG GACACCAGGCTGTGAGGGCCCAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTAT GTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTC ATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGC GTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGG ACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCT CGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTT GCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCTCAATCCAGCGGACCTTCCTTCCCGC GGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCC GCCTCCCCGCAAGCTTCGCACTTTTTAAAAGAAAAGGGAGGACTGGATGGGATTTATTACTCCGATAGGACGCTG GCTTGTAACTCAGTCTCTTACTAGGAGACCAGCTTGAGCCTGGGTGTTCGCTGGTTAGCCTAACCTGGTTGGCCA CCAGGGGTAAGGACTCCTTGGCTTAGAAAGCTAATAAACTTGCCTGCATTAGAGCTCTTACGCGTCCCGGGCTCG AGATCCGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAG TTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGA GCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTAACTTGTTTATTGCAGCT TATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGT GGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCG TTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAG GAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATA GGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAA GATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGT CCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCG TTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTC TTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGT ATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCT GCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTA GCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTT CTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCT TCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACA GTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAA AAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTG CGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAAT CACCATGAGTGACGACTGAATCCGGTGAGAATGGCAACAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCC AGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGAC GAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCG CATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTTCCGGGGATCGCAGTGG TGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGT TTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCAT CGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATA AATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCC TTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATC AGAGATTTTGAGACACAACAATTGGTCGACGGATCC SEQIDNO:10PlasmidasdefinedinFIG.1D(pDNA2apGM297) ATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCG TTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGT ATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACT TGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCA TGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTAT TTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGGGGG GCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTT TTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCC TTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGG TGAGCGGGGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGT GGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGT GTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGC TTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGG GGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAAC CCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCG CGGGGCTCGCCGTGCCGGGGGGGGGGTGGCGGCAGGTGGGGGTGCCGGGGGGGGGGGGGCCGCCTCGGGCCGGGG AGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCC TTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGC GCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGGGGGGAGGGCC TTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTC GGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTC ATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAAT TGCTCGAGACTAGTGACTTGGTGAGTAGGCTTCGAGCCTAGTTAGAGGACTAGGAGAGGCCGTAGCCGTAACTAC TCTGGGCAAGTAGGGCAGGCGGTGGGTACGCAATGGGGGCGGCTACCTCAGCACTAAATAGGAGACAATTAGACC AATTTGAGAAAATACGACTTCGCCCGAACGGAAAGAAAAAGTACCAAATTAAACATTTAATATGGGCAGGCAAGG AGATGGAGCGCTTCGGCCTCCATGAGAGGTTGTTGGAGACAGAGGAGGGGTGTAAAAGAATCATAGAAGTCCTCT ACCCCCTAGAACCAACAGGATCGGAGGGCTTAAAAAGTCTGTTCAATCTTGTGTGCGTACTATATTGCTTGCACA AGGAACAGAAAGTGAAAGACACAGAGGAAGCAGTAGCAACAGTAAGACAACACTGCCATCTAGTGGAAAAAGAAA AAAGTGCAACAGAGACATCTAGTGGACAAAAGAAAAATGACAAGGGAATAGCAGCGCCACCTGGTGGCAGTCAGA ATTTTCCAGCGCAACAACAAGGAAATGCCTGGGTACATGTACCCTTGTCACCGCGCACCTTAAATGCGTGGGTAA AAGCAGTAGAGGAGAAAAAATTTGGAGCAGAAATAGTACCCATGTTTCAAGCCCTATCAGAAGGCTGCACACCCT ATGACATTAATCAGATGCTTAATGTGCTAGGAGATCATCAAGGGGCATTACAAATAGTGAAAGAGATCATTAATG AAGAAGCAGCCCAGTGGGATGTAACACACCCACTACCCGCAGGACCCCTACCAGCAGGACAGCTCAGGGACCCTC GCGGCTCAGATATAGCAGGGACCACCAGCTCAGTACAAGAACAGTTAGAATGGATCTATACTGCTAACCCCCGGG TAGATGTAGGTGCCATCTACCGGAGATGGATTATTCTAGGACTTCAAAAGTGTGTCAAAATGTACAACCCAGTAT CAGTCCTAGACATTAGGCAGGGACCTAAAGAGCCCTTCAAGGATTATGTGGACAGATTTTACAAGGCAATTAGAG CAGAACAAGCCTCAGGGGAAGTGAAACAATGGATGACAGAATCATTACTCATTCAAAATGCTAATCCAGATTGTA AGGTCATCCTGAAGGGCCTAGGAATGCACCCCACCCTTGAAGAAATGTTAACGGCTTGTCAGGGGGTAGGAGGCC CAAGCTACAAAGCAAAAGTAATGGCAGAAATGATGCAGACCATGCAAAATCAAAACATGGTGCAGCAGGGAGGTC CAAAAAGACAAAGACCCCCACTAAGATGTTATAATTGTGGAAAATTTGGCCATATGCAAAGACAATGTCCGGAAC CAAGGAAAACAAAATGTCTAAAGTGTGGAAAATTGGGACACCTAGCAAAAGACTGCAGGGGACAGGTGAATTTTT TAGGGTATGGACGGTGGATGGGGGCAAAACCGAGAAATTTTCCCGCCGCTACTCTTGGAGCGGAACCGAGTGCGC CTCCTCCACCGAGCGGCACCACCCCATACGACCCAGCAAAGAAGCTCCTGCAGCAATATGCAGAGAAAGGGAAAC AACTGAGGGAGCAAAAGAGGAATCCACCGGCAATGAATCCGGATTGGACCGAGGGATATTCTTTGAACTCCCTCT TTGGAGAAGACCAATAAAGACAGTGTATATAGAAGGGGTCCCCATTAAGGCACTGCTAGACACAGGGGCAGATGA CACCATAATTAAAGAAAATGATTTACAATTATCAGGTCCATGGAGACCCAAAATTATAGGGGGCATAGGAGGAGG CCTTAATGTAAAAGAATATAACGACAGGGAAGTAAAAATAGAAGATAAAATTTTGAGAGGAACAATATTGTTAGG AGCAACTCCCATTAATATAATAGGTAGAAATTTGCTGGCCCCGGCAGGTGCCCGGTTAGTAATGGGACAATTATC AGAAAAAATTCCTGTCACACCTGTCAAATTGAAGGAAGGGGCTCGGGGACCCTGTGTAAGACAATGGCCTCTCTC TAAAGAGAAGATTGAAGCTTTACAGGAAATATGTTCCCAATTAGAGCAGGAAGGAAAAATCAGTAGAGTAGGAGG AGAAAATGCATACAATACCCCAATATTTTGCATAAAGAAGAAGGACAAATCCCAGTGGAGGATGCTAGTAGACTT TAGAGAGTTAAATAAGGCAACCCAAGATTTCTTTGAAGTGCAATTAGGGATACCCCACCCAGCAGGATTAAGAAA GATGAGACAGATAACAGTTTTAGATGTAGGAGACGCCTATTATTCCATACCATTGGATCCAAATTTTAGGAAATA TACTGCTTTTACTATTCCCACAGTGAATAATCAGGGACCCGGGATTAGGTATCAATTCAACTGTCTCCCGCAAGG GTGGAAAGGATCTCCTACAATCTTCCAAAATACAGCAGCATCCATTTTGGAGGAGATAAAAAGAAACTTGCCAGC ACTAACCATTGTACAATACATGGATGATTTATGGGTAGGTTCTCAAGAAAATGAACACACCCATGACAAATTAGT AGAACAGTTAAGAACAAAATTACAAGCCTGGGGCTTAGAAACCCCAGAAAAGAAGGTGCAAAAAGAACCACCTTA TGAGTGGATGGGATACAAACTTTGGCCTCACAAATGGGAACTAAGCAGAATACAACTGGAGGAAAAAGATGAATG GACTGTCAATGACATCCAGAAGTTAGTTGGGAAACTAAATTGGGCAGCACAATTGTATCCAGGTCTTAGGACCAA GAATATATGCAAGTTAATTAGAGGAAAGAAAAATCTGTTAGAGCTAGTGACTTGGACACCTGAGGCAGAAGCTGA ATATGCAGAAAATGCAGAGATTCTTAAAACAGAACAGGAAGGAACCTATTACAAACCAGGAATACCTATTAGGGC AGCAGTACAGAAATTGGAAGGAGGACAGTGGAGTTACCAATTCAAACAAGAAGGACAAGTCTTGAAAGTAGGAAA ATACACCAAGCAAAAGAACACCCATACAAATGAACTTCGCACATTAGCTGGTTTAGTGCAGAAGATTTGCAAAGA AGCTCTAGTTATTTGGGGGATATTACCAGTTCTAGAACTCCCGATAGAAAGAGAGGTATGGGAACAATGGTGGGC GGATTACTGGCAGGTAAGCTGGATTCCCGAATGGGATTTTGTCAGCACCCCACCTTTGCTCAAACTATGGTACAC ATTAACAAAAGAACCCATACCCAAGGAGGACGTTTACTATGTAGATGGAGCATGCAACAGAAATTCAAAAGAAGG AAAAGCAGGATACATCTCACAATACGGAAAACAGAGAGTAGAAACATTAGAAAACACTACCAATCAGCAAGCAGA ATTAACAGCTATAAAAATGGCTTTGGAAGACAGTGGGCCTAATGTGAACATAGTAACAGACTCTCAATATGCAAT GGGAATTTTGACAGCACAACCCACACAAAGTGATTCACCATTAGTAGAGCAAATTATAGCCTTAATGATACAAAA GCAACAAATATATTTGCAGTGGGTACCAGCACATAAAGGAATAGGAGGAAATGAGGAGATAGATAAATTAGTGAG TAAAGGCATTAGAAGAGTTTTATTCTTAGAAAAAATAGAAGAAGCTCAAGAAGAGCATGAAAGATATCATAATAA TTGGAAAAACCTAGCAGATACATATGGGCTTCCACAAATAGTAGCAAAAGAGATAGTGGCCATGTGTCCAAAATG TCAGATAAAGGGAGAACCAGTGCATGGACAAGTGGATGCCTCACCTGGAACATGGCAGATGGATTGTACTCATCT AGAAGGAAAAGTAGTCATAGTTGCGGTCCATGTAGCCAGTGGATTCATAGAAGCAGAAGTCATACCTAGGGAAAC AGGAAAAGAAACGGCAAAGTTTCTATTAAAAATACTGAGTAGATGGCCTATAACACAGTTACACACAGACAATGG GCCTAACTTTACCTCCCAAGAAGTGGCAGCAATATGTTGGTGGGGAAAAATTGAACATACAACAGGTATACCATA TAACCCCCAATCTCAAGGATCAATAGAAAGCATGAACAAACAATTAAAAGAGATAATTGGGAAAATAAGAGATGA TTGCCAATATACAGAGACAGCAGTACTGATGGCTTGCCATATTCACAATTTTAAAAGAAAGGGAGGAATAGGGGG ACAGACTTCAGCAGAGAGACTAATTAATATAATAACAACACAATTAGAAATACAACATTTACAAACCAAAATTCA AAAAATTTTAAATTTTAGAGTCTACTACAGAGAAGGGAGAGACCCTGTGTGGAAAGGACCAGCACAATTAATCTG GAAAGGGGAAGGAGCAGTGGTCCTCAAGGACGGAAGTGACCTAAAGGTTGTACCAAGAAGGAAAGCTAAAATTAT TAAGGATTATGAACCCAAACAAAGAGTGGGTAATGAGGGTGACGTGGAAGGTACCAGGGGATCTGATAACTAAAT GGCAGGGAATAGTCAGATATTGGATGAGACAAAGAAATTTGAAATGGAACTATTATATGCATCAGCTGGCGGCCG CGAATTCACTAGTGATTCCCGTTTGTGCTAGGGTTCTTAGGCTTCTTGGGGGCTGCTGGAACTGCAATGGGAGCA GCGGCGACAGCCCTGACGGTCCAGTCTCAGCATTTGCTTGCTGGGATACTGCAGCAGCAGAAGAATCTGCTGGCG GCTGTGGAGGCTCAACAGCAGATGTTGAAGCTGACCATTTGGGGTGTTAAAAACCTCAATGCCCGCGTCACAGCC CTTGAGAAGTACCTAGAGGATCAGGCACGACTAAACTCCTGGGGGTGCGCATGGAAACAAGTATGTCATACCACA GTGGAGTGGCCCTGGACAAATCGGACTCCGGATTGGCAAAATATGACTTGGTTGGAGTGGGAAAGACAAATAGCT GATTTGGAAAGCAACATTACGAGACAATTAGTGAAGGCTAGAGAACAAGAGGAAAAGAATCTAGATGCCTATCAG AAGTTAACTAGTTGGTCAGATTTCTGGTCTTGGTTCGATTTCTCAAAATGGCTTAACATTTTAAAAATGGGATTT TTAGTAATAGTAGGAATAATAGGGTTAAGATTACTTTACACAGTATATGGATGTATAGTGAGGGTTAGGCAGGGA TATGTTCCTCTATCTCCACAGATCCATATCCAATCGAATTCCCGCGGCCGCAATTCACTCCTCAGGTGCAGGCTG CCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCA AAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAA TAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATG AGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTC ATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTT TTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTT TCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATCCCTCGACCTGCAGCCCAAGC TTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCC GGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCC GCTTTCCAGTCGGGAAACCTGTCGTGCCAGCGGATCCGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAA CTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTT ATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCT TTTGCAAAAAGCTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAA TAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTCCGCT TCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATA CGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGT AAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGT CAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCT GTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCA CGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCC GACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCA GCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTAC GGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGC TCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAA AAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGG ATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATC TAAAGTATATATGAGTAAACTTGGTCTGACAGTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTC ATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTC CATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCC TCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAACAGCTTA TGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCG TTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATC GAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACC TGGAATGCTGTTTTTCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATG GTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCT TTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCG ACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGAC GTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGAT GATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACAATTGGTCGAC SEQIDNO:11PlasmidasdefinedinFIG.1E(pDNA2bpGM299) TCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATAC GTTGTATCTATATCATAATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATT GACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACT TACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCAT AGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACA TCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCA GTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCG GTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGT CAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCA AATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCACTAGAAG CTTTATTGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAG CTGCAGAAGTTGGTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAA ACTGGGCTTGTCGAGACAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGC CTTTCTCTCCACAGGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTACTTAATACGACTCACTATA GGCTAGCCTCGAGAATTCGATTATGCCCCTAGGACCAGAAGAAAGAAGATTGCTTCGCTTGATTTGGCTCCTTTA CAGCACCAATCCATATCCACCAAGTGGGGAAGGGACGGCCAGACAACGCCGACGAGCCAGGAGAAGGTGGAGACA ACAGCAGGATCAAATTAGAGTCTTGGTAGAAAGACTCCAAGAGCAGGTGTATGCAGTTGACCGCCTGGCTGACGA GGCTCAACACTTGGCTATACAACAGTTGCCTGACCCTCCTCATTCAGCTTAGAATCACTAGTGAATTCACGCGTG GTACCTCTAGAGTCGACCCGGGCGGCCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCAC AACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAG CTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTT TTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAGGATCCGTCGACCAATTGTTGTGTCTCAAAATC TCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATA CAAGGGGTGTTATGAGCCATATTCAACGGGAAACGTCTTGCTCTAGGCCGCGATTAAATTCCAACATGGATGCTG ATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCAGGTGCGACAATCTATCGATTGTATGGGAAGC CCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGAC TAAACTGGCTGACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTAC TCACCACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATCCTGATTCAGGTGAAAATATTGTTG ATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTAT TTCGTCTCGCTCAGGCGCAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATG GCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTGTTGCCATTCTCACCGGATTCAGTCGTCACTCATG GTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAA TCGCAGACCGATACCAGGATCTTGCCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGC TTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGATGAGTTTTTCT AACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGG TGAAGATCCTTTTTGATAATCTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCG TAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCAC CGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAG CGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTA CATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACT CAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGC GAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGG CGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGT ATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGA GCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGGCTC GACAGATCT SEQIDNO:12PlasmidasdefinedinFIG.1F(pDNA3apGM301) ATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCG TTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGT ATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACT TGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCA TGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTAT TTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGG GCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTT TTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCC TTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGG TGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGT GGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGT GTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGC TTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGG GGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAAC CCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCG CGGGGCTCGCCGTGCCGGGCGGGGGGGGGCGGCAGGTGGGGGTGCCGGGGGGGGCGGGGCCGCCTCGGGCCGGGG AGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCC TTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGC GCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGGGGGGAGGGCC TTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTC GGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTC ATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAAT TCGATTGCCATGGCAACATATATCCAGAGAGTACAGTGCATCTCAACATCACTACTGGTTGTTCTCACCACATTG GTCTCGTGTCAGATTCCCAGGGATAGGCTCTCTAACATAGGGGTCATAGTCGATGAAGGGAAATCACTGAAGATA GCTGGATCCCACGAATCGAGGTACATAGTACTGAGTCTAGTTCCGGGGGTAGACTTTGAGAATGGGTGCGGAACA GCCCAGGTTATCCAGTACAAGAGCCTACTGAACAGGCTGTTAATCCCATTGAGGGATGCCTTAGATCTTCAGGAG GCTCTGATAACTGTCACCAATGATACGACACAAAATGCCGGTGCTCCCCAGTCGAGATTCTTCGGTGCTGTGATT GGTACTATCGCACTTGGAGTGGCGACATCAGCACAAATCACCGCAGGGATTGCACTAGCCGAAGCGAGGGAGGCC AAAAGAGACATAGCGCTCATCAAAGAATCGATGACAAAAACACACAAGTCTATAGAACTGCTGCAAAACGCTGTG GGGGAACAAATTCTTGCTCTAAAGACACTCCAGGATTTCGTGAATGATGAGATCAAACCCGCAATAAGCGAATTA GGCTGTGAGACTGCTGCCTTAAGACTGGGTATAAAATTGACACAGCATTACTCCGAGCTGTTAACTGCGTTCGGC TCGAATTTCGGAACCATCGGAGAGAAGAGCCTCACGCTGCAGGCGCTGTCTTCACTTTACTCTGCTAACATTACT GAGATTATGACCACAATCAGGACAGGGCAGTCTAACATCTATGATGTCATTTATACAGAACAGATCAAAGGAACG GTGATAGATGTGGATCTAGAGAGATACATGGTCACCCTGTCTGTGAAGATCCCTATTCTTTCTGAAGTCCCAGGT GTGCTCATACACAAGGCATCATCTATTTCTTACAACATAGACGGGGAGGAATGGTATGTGACTGTCCCCAGCCAT ATACTCAGTCGTGCTTCTTTCTTAGGGGGTGCAGACATAACCGATTGTGTTGAGTCCAGATTGACCTATATATGC CCCAGGGATCCCGCACAACTGATACCTGACAGCCAGCAAAAGTGTATCCTGGGGGACACAACAAGGTGTCCTGTC ACAAAAGTTGTGGACAGCCTTATCCCCAAGTTTGCTTTTGTGAATGGGGGCGTTGTTGCTAACTGCATAGCATCC ACATGTACCTGCGGGACAGGCCGAAGACCAATCAGTCAGGATCGCTCTAAAGGTGTAGTATTCCTAACCCATGAC AACTGTGGTCTTATAGGTGTCAATGGGGTAGAATTGTATGCTAACCGGAGAGGGCACGATGCCACTTGGGGGGTC CAGAACTTGACAGTCGGTCCTGCAATTGCTATCAGACCCGTTGATATTTCTCTCAACCTTGCTGATGCTACGAAT TTCTTGCAAGACTCTAAGGCTGAGCTTGAGAAAGCACGGAAAATCCTCTCGGAGGTAGGTAGATGGTACAACTCA AGAGAGACTGTGATTACGATCATAGTAGTTATGGTCGTAATATTGGTGGTCATTATAGTGATCATCATCGTGCTT TATAGACTCAGAAGGTGAAATCACTAGTGAATTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGG TGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAA GCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTG TCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGC AACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCC TGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATT TTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCC AGTCATAGCTGTCCCTCTTCTCTTATGGAGATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGGTCATAGC TGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCT GGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGT CGTGCCAGCGGATCCGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAAC TCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTC GGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTAACTTGTTT ATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCAT TCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTCCGCTTCCTCGCTCACTGACTCGCTGC GCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGG ATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGT TTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAG GACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCG GATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGG TGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTA ACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCA GAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTAT TTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCA CCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTT TGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAA AAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTT GGTCTGACAGTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCAT ATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGT ATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAA GTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAACAGCTTATGCATTTCTTTCCAGACTTGTT CAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCT GAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACA CTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTTCCGGGGA TCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCG TCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACT CTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTAT ACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCA TAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAA TGTAACATCAGAGATTTTGAGACACAACAATTGGTCGAC SEQIDNO:13PlasmidasdefinedinFIG.1G(pDNA3bpGM303) ATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCG TTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGT ATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACT TGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCA TGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTAT TTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGGGGG GCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTT TTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCC TTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGG TGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGT GGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGT GTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGC TTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGG GGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAAC CCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCG CGGGGCTCGCCGTGCCGGGCGGGGGGGGGCGGCAGGTGGGGGTGCCGGGGGGGGCGGGGCCGCCTCGGGCCGGGG AGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCC TTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGC GCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGGGGGGAGGGCC TTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGGGCAGGGC GGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCT ACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCCTCGAGCATGTGGTCTG AGTTAAAAATCAGGAGCAACGACGGAGGTGAAGGACCAGAGGACGCCAACGACCCCCGGGGAAAGGGGGTGCAAC ACATCCATATCCAGCCATCTCTACCTGTTTATGGACAGAGGGTTAGGGATGGTGATAGGGGCAAACGTGACTCGT ACTGGTCTACTTCTCCTAGTGGTAGCACCACAAAACCAGCATCAGGTTGGGAGAGGTCAAGTAAAGCCGACACAT GGTTGCTGATTCTCTCATTCACCCAGTGGGCTTTGTCAATTGCCACAGTGATCATCTGTATCATAATTTCTGCTA GACAAGGGTATAGTATGAAAGAGTACTCAATGACTGTAGAGGCATTGAACATGAGCAGCAGGGAGGTGAAAGAGT CACTTACCAGTCTAATAAGGCAAGAGGTTATAGCAAGGGCTGTCAACATTCAGAGCTCTGTGCAAACCGGAATCC CAGTCTTGTTGAACAAAAACAGCAGGGATGTCATCCAGATGATTGATAAGTCGTGCAGCAGACAAGAGCTCACTC AGCACTGTGAGAGTACGATCGCAGTCCACCATGCCGATGGAATTGCCCCACTTGAGCCACATAGTTTCTGGAGAT GCCCTGTCGGAGAACCGTATCTTAGCTCAGATCCTGAAATCTCATTGCTGCCTGGTCCGAGCTTGTTATCTGGTT CTACAACGATCTCTGGATGTGTTAGGCTCCCTTCACTCTCAATTGGCGAGGCAATCTATGCCTATTCATCAAATC TCATTACACAAGGTTGTGCTGACATAGGGAAATCATATCAGGTCCTGCAGCTAGGGTACATATCACTCAATTCAG ATATGTTCCCTGATCTTAACCCCGTAGTGTCCCACACTTATGACATCAACGACAATCGGAAATCATGCTCTGTGG TGGCAACCGGGACTAGGGGTTATCAGCTTTGCTCCATGCCGACTGTAGACGAAAGAACCGACTACTCTAGTGATG GTATTGAGGATCTGGTCCTTGATGTCCTGGATCTCAAAGGGAGAACTAAGTCTCACCGGTATCGCAACAGCGAGG TAGATCTTGATCACCCGTTCTCTGCACTATACCCCAGTGTAGGCAACGGCATTGCAACAGAAGGCTCATTGATAT TTCTTGGGTATGGTGGACTAACCACCCCTCTGCAGGGTGATACAAAATGTAGGACCCAAGGATGCCAACAGGTGT CGCAAGACACATGCAATGAGGCTCTGAAAATTACATGGCTAGGAGGGAAACAGGTGGTCAGCGTGATCATCCAGG TCAATGACTATCTCTCAGAGAGGCCAAAGATAAGAGTCACAACCATTCCAATCACTCAAAACTATCTCGGGGCGG AAGGTAGATTATTAAAATTGGGTGATCGGGTGTACATCTATACAAGATCATCAGGCTGGCACTCTCAACTGCAGA TAGGAGTACTTGATGTCAGCCACCCTTTGACTATCAACTGGACACCTCATGAAGCCTTGTCTAGACCAGGAAATA AAGAGTGCAATTGGTACAATAAGTGTCCGAAGGAATGCATATCAGGCGTATACACTGATGCTTATCCATTGTCCC CTGATGCAGCTAACGTCGCTACCGTCACGCTATATGCCAATACATCGCGTGTCAACCCAACAATCATGTATTCTA ACACTACTAACATTATAAATATGTTAAGGATAAAGGATGTTCAATTAGAGGCTGCATATACCACGACATCGTGTA TCACGCATTTTGGTAAAGGCTACTGCTTTCACATCATCGAGATCAATCAGAAGAGCCTGAATACCTTACAGCCGA TGCTCTTTAAGACTAGCATCCCTAAATTATGCAAGGCCGAGTCTTAAGCGGCCGCGCATGCGAATTCACTCCTCA GGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTT TCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTAT TTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAA ACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCT ATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCTATTCCTTATTCCATAGAAAAGCCTTGACTTGAGG TTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACT AGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATCCCTCGACCT GCAGCCCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACA ACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGC GCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCGGATCCGCATCTCAATTAGTCAGCAACCATAGT CCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAAT TTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGG AGGCCTAGGCTTTTGCAAAAAGCTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACA AATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCAT GTCTGTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAA AGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGG CCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATC GACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCG TGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTT CTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCC CCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGC CACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGT GGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAA GAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTA CGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACT CACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTT TTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTAGAAAAACTCATCGAGCATCAAATGAAACT GCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCAC CGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTA TTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATG GCAACAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCAT CAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTAC AAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATT CTTCTAATACCTGGAATGCTGTTTTTCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAA AATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGG CAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCAC CTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCC TAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTA TTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACAATTGGTCGAC SEQIDNO:14ExemplifiedWPREcomponent(mWPRE) 1 GGGCCCAATCAACCTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTAT 61 GTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCT 121 TCCCGTATGGCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAG 181 GAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACC 241 CCCACTGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCC 301 CTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCT 361 CGGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGG 421 CTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCG 481 GCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCG 541 CGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCAAGCT SEQIDNO:15ExemplaryCAGpromoter ATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCG TTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGT ATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACT TGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGC ATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCA TGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTAT TTATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGG GCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGCCAATCAGAGCGGCGCGCTCCGAAAGTTTCCTT TTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCC TTCGCCCCGTGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGG TGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTTTAATGACGGCTTGTTTCTTTTCTGT GGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGT GTGTGTGCGTGGGGAGCGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGC TTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGTGCGGGGGGGGCTGCGAGG GGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAAC CCCCCCTGCACCCCCCTCCCCGAGTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCG CGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCCTCGGGCCGGGG AGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCC TTTTATGGTAATCGTGCGAGAGGGCGCAGGGACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGC GCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGGGGGGAGGGCC TTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTC GGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTC ATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAAT TGCTCGAGCCACC SEQIDNO:16ModifiedSIV/CFTRRNAsequence ucucuuacuaggagaccagcuugagccuggguguucgcugguuagccuaaccugguuggc 60 caccagggguaaggacuccuuggcuuagaaagcuaauaaacuugccugcauuagagcuua 120 ucugagucaaguguccucauugacgccucacucucuugaacgggaaucuuccuuacuggg 180 uucucucucugacccaggcgagagaaacuccagcaguggcgcccgaacagggacuugagu 240 gagaguguaggcacguacagcugagaaggcgucggacgcgaaggaagcgcggggugcgac 300 gcgaccaagaaggagacuuggugaguaggcuucucgagugccgggaaaaagcucgagccu 360 aguuagaggacuaggagaggccguagccguaacuacucugggcaaguagggcaggcggug 420 gguacgcaauugggggcggcuaccucagcacuaaauaggagacaauuagaccaauuugag 480 aaaauacgacuucgcccgaacggaaagaaaaaguaccaaauuaaacauuuaauauugggc 540 aggcaaggagauuggagcgcuucggccuccaugagagguuguuggagacagaggaggggu 600 guaaaagaaucauagaaguccucuacccccuagaaccaacaggaucggagggcuuaaaaa 660 gucuguucaaucuugugugcgugcuauauugcuugcacaaggaacagaaagugaaagaca 720 cagaggaagcaguagcaacaguaagacaacacugccaucuaguggaaaaagaaaaaagug 780 caacagagacaucuaguggacaaaagaaaaaugacaagggaauagcagcgccaccuggug 840 gcagucagaauuuuccagcgcaacaacaaggaaauugccuggguacauguacccuuguca 900 ccgcgcaccuuaaaugcguggguaaaagcaguagaggagaaaaaauuuggagcagaaaua 960 guacccauguuucaagcccuaucgccugcaggccguuugugcuaggguucuuaggcuucu 1020 ugggggcugcuggaacugcauugggagcagcggcgacagcccugacgguccagucucagc 1080 auuugcuugcugggauacugcagcagcagaagaaucugcuggcggcuguggaggcucaac 1140 agcagauguugaagcugaccauuugggguguuaaaaaccucaaugcccgcgucacagccc 1200 uugagaaguaccuagaggaucaggcacgacuaaacuccugggggugcgcauggaaacaag 1260 uaugucauaccacaguggaguggcccuggacaaaucggacuccggauuggcaaaauaaga 1320 cuugguuggagugggaaagacaaauagcugauuuggaaagcaacauuacgagacaauuag 1380 ugaaggcuagagaacaagaggaaaagaaucuagaugccuaucagaaguuaacuaguuggu 1440 cagauuucuggucuugguucgauuucucaaaauggcuuaacauuuuaaaaaagggauuuu 1500 uaguaauaguaggaauaauaggguuaagauuacuuuacacaguauauggauguauaguga 1560 ggguuaggcagggauauguuccucuaucuccacagauccauauaaagcggcaauuuuaaa 1620 agaaagggaggaauagggggacagacuucagcagagagacuaauuaauauaauaacaaca 1680 caauuagaaauacaacauuuacaaaccaaaauucaaaaaauuuuaaauuuuagagccgcg 1740 gagaucuguuacauaacuuaugguaaauggccugccuggcugacugcccaaugaccccug 1800 cccaaugaugucaauaaugauguauguucccauguaaugccaauagggacuuuccauuga 1860 ugucaauggguggaguauuuaugguaacugcccacuuggcaguacaucaaguguaucaua 1920 ugccaaguaugcccccuauugaugucaaugaugguaaauggccugccuggcauuaugccc 1980 aguacaugaccuuaugggacuuuccuacuuggcaguacaucuauguauuagucauugcua 2040 uuaccaugggaauucacuaguggagaagagcaugcuugagggcugagugccccucagugg 2100 gcagagagcacauggcccacagucccugagaaguuggggggaggggugggcaauugaacu 2160 ggugccuagagaagguggggcuuggguaaacugggaaagugaugugguguacuggcucca 2220 ccuuuuuccccagggugggggagaaccauauauaagugcaguagucucugugaacauuca 2280 agcuucugccuucucccuccugugaguuugcuagccaccaugcagagaagcccucuggag 2340 aaggccucuguggugagcaagcuguucuucagcuggaccaggcccauccugaggaagggc 2400 uacaggcagagacuggagcugucugacaucuaccagauccccucuguggacucugcugac 2460 aaccugucugagaagcuggagagggagugggauagagagcuggccagcaagaagaacccc 2520 aagcugaucaaugcccugaggagaugcuucuucuggagauucauguucuauggcaucuuc 2580 cuguaccugggggaagugaccaaggcugugcagccucugcugcugggcagaaucauugcc 2640 agcuaugacccugacaacaaggaggagaggagcauugccaucuaccugggcauuggccug 2700 ugccugcuguucauugugaggacccugcugcugcacccugccaucuuuggccugcaccac 2760 auuggcaugcagaugaggauugccauguucagccugaucuacaagaaaacccugaagcug 2820 uccagcagagugcuggacaagaucagcauuggccagcuggugagccugcugagcaacaac 2880 cugaacaaguuugaugagggccuggcccuggcccacuuuguguggauugccccucugcag 2940 guggcccugcugaugggccugauuugggagcugcugcaggccucugccuuuuguggccug 3000 ggcuuccugauugugcuggcccuguuucaggcuggccugggcaggaugaugaugaaguac 3060 agggaccagagggcaggcaagaucagugagaggcuggugaucaccucugagaugauugag 3120 aacauccagucugugaaggccuacuguugggaggaagcuauggagaagaugauugaaaac 3180 cugaggcagacagagcugaagcugaccaggaaggcugccuaugugagauacuucaacagc 3240 ucugccuucuucuucucuggcuucuuugugguguuccugucugugcugcccuaugcccug 3300 aucaaggggaucauccugagaaagauuuucaccaccaucagcuucugcauugugcugagg 3360 auggcugugaccagacaguuccccugggcugugcagaccugguaugacagccugggggcc 3420 aucaacaagauccaggacuuccugcagaagcaggaguacaagacccuggaguacaaccug 3480 accaccacagaaguggugauggagaaugugacagccuucugggaggagggcuuuggggag 3540 cuguuugagaaggccaagcagaacaacaacaacagaaagaccagcaauggggaugacucc 3600 cuguucuucuccaacuucucccugcugggcacaccugugcugaaggacaucaacuucaag 3660 auugagagggggcagcugcuggcuguggcuggaucuacaggggcuggcaagaccagccug 3720 cugaugaugaucaugggggagcuggagccuucugagggcaagaucaagcacucuggcagg 3780 aucagcuuuugcagccaguucagcuggaucaugccuggcaccaucaaggagaacaucauc 3840 uuuggagugagcuaugaugaguacagauacaggagugugaucaaggccugccagcuggag 3900 gaggacaucagcaaguuugcugagaaggacaacauugugcugggggagggaggcauuaca 3960 cugucugggggccagagagccagaaucagccuggccagggcuguguacaaggaugcugac 4020 cuguaccugcuggacucccccuuuggcuaccuggaugugcugacagagaaggagauuuuu 4080 gagagcugugugugcaagcugauggccaacaagaccagaauccuggugaccagcaagaug 4140 gagcaccugaagaaggcugacaagauccugauccugcaugagggcagcagcuacuucuau 4200 gggaccuucucugagcugcagaaccugcagccugacuucagcucuaagcugaugggcugu 4260 gacagcuuugaccaguucucugcugagaggaggaacagcauccugacagagacccugcac 4320 agauucagccuggagggagaugccccugugagcuggacagagaccaagaagcagagcuuc 4380 aagcagacaggggaguuuggggagaagaggaagaacuccauccugaaccccaucaacagc 4440 aucaggaaguucagcauugugcagaaaaccccccugcagaugaauggcauugaggaagau 4500 ucugaugagccccuggagaggagacugagccuggugccugauucugagcagggagaggcc 4560 auccugccuaggaucucugugaucagcacaggcccuacacugcaggccagaaggaggcag 4620 ucugugcugaaccugaugacccacucugugaaccagggccagaacauccacaggaaaacc 4680 acagccuccaccaggaaagugagccuggccccucaggccaaucugacagagcuggacauc 4740 uacagcaggaggcugucucaggagacaggccuggagauuucugaggagaucaaugaggag 4800 gaccugaaagagugcuucuuugaugacauggagagcaucccugcugugaccaccuggaac 4860 accuaccugagauacaucacagugcacaagagccugaucuuugugcugaucuggugccug 4920 gugaucuuccuggcugaaguggcugccucucugguggugcuguggcugcugggaaacacc 4980 ccacugcaggacaagggcaacagcacccacagcaggaacaacagcuaugcugugaucauc 5040 accuccaccuccagcuacuauguguucuacaucuaugugggaguggcugauacccugcug 5100 gcuaugggcuucuuuagaggccugccccuggugcacacacugaucacagugagcaagauc 5160 cuccaccacaagaugcugcacucugugcugcaggcuccuaugagcacccugaauacccug 5220 aaggcugggggcauccugaacagauucuccaaggauauugccauccuggaugaccugcug 5280 ccucucaccaucuuugacuucauccagcugcugcugauugugauuggggccauugcugug 5340 guggcagugcugcagcccuacaucuuuguggccacagugccugugauuguggccuucauc 5400 augcugagggccuacuuucugcagaccucccagcagcugaagcagcuggagucugagggc 5460 agaagccccaucuucacccaccuggugacaagccugaagggccuguggacccugagagcc 5520 uuuggcaggcagcccuacuuugagacccuguuccacaaggcccugaaccugcacacagcc 5580 aacugguuccucuaccuguccacccugagaugguuccagaugagaauugagaugaucuuu 5640 gucaucuucuucauugcugugaccuucaucagcauucugaccacaggagagggagagggc 5700 agagugggcauuauccugacccuggccaugaacaucaugagcacacugcagugggcagug 5760 aacagcagcauugauguggacagccugaugaggagugugagcagaguguucaaguucauu 5820 gauaugcccacagagggcaagccuaccaagagcaccaagcccuacaagaauggccagcug 5880 agcaaagugaugaucauugagaacagccaugugaagaaggaugauaucuggcccagugga 5940 ggccagaugacagugaaggaccugacagccaaguacacagaggggggcaaugcuauccug 6000 gagaacaucuccuucagcaucuccccuggccagagagugggacugcugggaagaacaggc 6060 ucuggcaagucuacccugcugucugccuuccugaggcugcugaacacagagggagagauc 6120 cagauugauggaguguccugggacagcaucacacugcagcaguggaggaaggccuuuggu 6180 gugaucccccagaaaguguucaucuucaguggcaccuucaggaagaaccuggaccccuau 6240 gagcaguggucugaccaggagauuuggaaaguggcugaugaagugggccugagaagugug 6300 auugagcaguucccuggcaagcuggacuuuguccugguggaugggggcugugugcugagc 6360 cauggccacaagcagcugaugugccuggccagaucagugcugagcaaggccaagauccug 6420 cugcuggaugagccuucugcccaccuggauccugugaccuaccagaucaucaggaggacc 6480 cucaagcaggccuuugcugacugcacagucauccugugugagcacaggauugaggccaug 6540 cuggagugccagcaguuccuggugauugaggagaacaaagugaggcaguaugacagcauc 6600 cagaagcugcugaaugagaggagccuguucaggcaggccaucagccccucugauagagug 6660 aagcuguucccccacaggaacagcuccaagugcaagagcaagccccagauugcugcccug 6720 aaggaggagacagaggaggaagugcaggacaccaggcugugagggcccaaucaaccucug 6780 gauuacaaaauuugugaaagauugacugguauucuuaacuauguugcuccuuuuacgcua 6840 uguggauacgcugcuuuaaugccuuuguaucaugcuauugcuucccguauggcuuucauu 6900 uucuccuccuuguauaaauccugguugcugucucuuuaugaggaguuguggcccguuguc 6960 aggcaacguggcguggugugcacuguguuugcugacgcaacccccacugguuggggcauu 7020 gccaccaccugucagcuccuuuccgggacuuucgcuuucccccucccuauugccacggcg 7080 gaacucaucgccgccugccuugcccgcugcuggacaggggcucggcuguugggcacugac 7140 aauuccgugguguugucggggaaaucaucguccuuuccuuggcugcucgccuguguugcc 7200 accuggauucugcgcgggacguccuucugcuacgucccuucggcccucaauccagcggac 7260 cuuccuucccgcggccugcugccggcucugcggccucuuccgcgucuucgccuucgcccu 7320 cagacgagucggaucucccuuugggccgccuccccgcaagcuucgcacuuuuuaaaagaa 7380 aagggaggacuggaugggauuuauuacuccgauaggacgcuggcuuguaacucagucucu 7440 uacuaggagaccagcuugagccuggguguucgcugguuagccuaaccugguuggccacca 7500 gggguaaggacuccuuggcuuagaaagcuaauaaacuugccugcauuagagcu 7553 SEQIDNO:17Fct4protein GlnIleProArgAspArgLeuSerAsnIleGlyValIleValAspGlu 151015 GlyLysSerLeuLysIleAlaGlySerHisGluSerArgTyrIleVal 202530 LeuSerLeuValProGlyValAspPheGluAsnGlyCysGlyThrAla 354045 GlnValIleGlnTyrLysSerLeuLeuAsnArgLeuLeuIleProLeu 505560 ArgAspAlaLeuAspLeuGlnGluAlaLeuIleThrValThrAsnAsp 65707580 ThrThrGlnAsnAlaGlyAlaProGlnSerArgPhePheGlyAlaVal 859095 IleGlyThrIleAlaLeuGlyValAlaThrSerAlaGlnIleThrAla 100105110 GlyIleAlaLeuAlaGluAlaArgGluAlaLysArgAspIleAlaLeu 115120125 IleLysGluSerMetThrLysThrHisLysSerIleGluLeuLeuGln 130135140 AsnAlaValGlyGluGlnIleLeuAlaLeuLysThrLeuGlnAspPhe 145150155160 ValAsnAspGluIleLysProAlaIleSerGluLeuGlyCysGluThr 165170175 AlaAlaLeuArgLeuGlyIleLysLeuThrGlnHisTyrSerGluLeu 180185190 LeuThrAlaPheGlySerAsnPheGlyThrIleGlyGluLysSerLeu 195200205 ThrLeuGlnAlaLeuSerSerLeuTyrSerAlaAsnIleThrGluIle 210215220 MetThrThrIleArgThrGlyGlnSerAsnIleTyrAspValIleTyr 225230235240 ThrGluGlnIleLysGlyThrValIleAspValAspLeuGluArgTyr 245250255 MetValThrLeuSerValLysIleProIleLeuSerGluValProGly 260265270 ValLeuIleHisLysAlaSerSerIleSerTyrAsnIleAspGlyGlu 275280285 GluTrpTyrValThrValProSerHisIleLeuSerArgAlaSerPhe 290295300 LeuGlyGlyAlaAspIleThrAspCysValGluSerArgLeuThrTyr 305310315320 IleCysProArgAspProAlaGlnLeuIleProAspSerGlnGlnLys 325330335 CysIleLeuGlyAspThrThrArgCysProValThrLysValValAsp 340345350 SerLeuIleProLysPheAlaPheValAsnGlyGlyValValAlaAsn 355360365 CysIleAlaSerThrCysThrCysGlyThrGlyArgArgProIleSer 370375380 GlnAspArgSerLysGlyValValPheLeuThrHisAspAsnCysGly 385390395400 LeuIleGlyValAsnGlyValGluLeuTyrAlaAsnArgArgGlyHis 405410415 AspAlaThrTrpGlyValGlnAsnLeuThrValGlyProAlaIleAla 420425430 IleArgProValAspIleSerLeuAsnLeuAlaAspAlaThrAsnPhe 435440445 LeuGlnAspSerLysAlaGluLeuGluLysAlaArgLysIleLeuSer 450455460 GluValGlyArgTrpTyrAsnSerArgGluThrValIleThrIleIle 465470475480 ValValMetValValIleLeuValValIleIleValIleIleIleVal 485490495 LeuTyrArgLeuArgArg SEQIDNO:18Fct4protein(includingsignalsequence) MetAlaThrTyrIleGlnArgValGlnCysIleSerThrSerLeuLeu 151015 ValValLeuThrThrLeuValSerCysGlnIleProArgAspArgLeu 202530 SerAsnIleGlyValIleValAspGluGlyLysSerLeuLysIleAla 354045 GlySerHisGluSerArgTyrIleValLeuSerLeuValProGlyVal 505560 AspPheGluAsnGlyCysGlyThrAlaGlnValIleGlnTyrLysSer 65707580 LeuLeuAsnArgLeuLeuIleProLeuArgAspAlaLeuAspLeuGln 859095 GluAlaLeuIleThrValThrAsnAspThrThrGlnAsnAlaGlyAla 100105110 ProGlnSerArgPhePheGlyAlaValIleGlyThrIleAlaLeuGly 115120125 Val130ThrSerAlaGln135ThrAlaGlyIle140LeuAlaGluAla 130135140 ArgGluAlaLysArgAspIleAlaLeuIleLysGluSerMetThrLys 145150155160 ThrHisLysSerIleGluLeuLeuGlnAsnAlaValGlyGluGlnIle 165170175 LeuAlaLeuLysThrLeuGlnAspPheValAsnAspGluIleLysPro 180185190 AlaIleSerGluLeuGlyCysGluThrAlaAlaLeuArgLeuGlyIle 195200205 LysLeuThrGlnHisTyrSerGluLeuLeuThrAlaPheGlySerAsn 210215220 PheGlyThrIleGlyGluLysSerLeuThrLeuGlnAlaLeuSerSer 225230235240 LeuTyrSerAlaAsnIleThrGluIleMetThrThrIleArgThrGly 245250255 GlnSerAsnIleTyrAspValIleTyrThrGluGlnIleLysGlyThr 260265270 ValIleAspValAspLeuGluArgTyrMetValThrLeuSerValLys 275280285 IleProIleLeuSerGluValProGlyValLeuIleHisLysAlaSer 290295300 SerIleSerTyrAsnIleAspGlyGluGluTrpTyrValThrValPro 305310315320 SerHisIleLeuSerArgAlaSerPheLeuGlyGlyAlaAspIleThr 325330335 AspCysValGluSerArgLeuThrTyrIleCysProArgAspProAla 340345350 GlnLeuIleProAspSerGlnGlnLysCysIleLeuGlyAspThrThr 355360365 ArgCysProValThrLysValValAspSerLeuIleProLysPheAla 370375380 PheValAsnGlyGlyValValAlaAsnCysIleAlaSerThrCysThr 385390395400 CysGlyThrGlyArgArgProIleSerGlnAspArgSerLysGlyVal 405410415 ValPheLeuThrHisAspAsnCysGlyLeuIleGlyValAsnGlyVal 420425430 GluLeuTyrAlaAsnArgArgGlyHisAspAlaThrTrpGlyValGln 435440445 AsnLeuThrValGlyProAlaIleAlaIleArgProValAspIleSer 450455460 LeuAsnLeuAlaAspAlaThrAsnPheLeuGlnAspSerLysAlaGlu 465470475480 LeuGluLysAlaArgLysIleLeuSerGluValGlyArgTrpTyrAsn 485490495 SerArgGluThrValIleThrIleIleValValMetValValIleLeu 500505510 ValValIleIleValIleIleIleValLeuTyrArgLeuArgArg 515520525 SEQIDNO:19Fct4protein(fragment1) PhePheGlyAlaValIleGlyThrIleAlaLeuGlyValAlaThrSer 151015 AlaGlnIleThrAlaGlyIleAlaLeuAlaGluAlaArgGluAlaLys 202530 ArgAspIleAlaLeuIleLysGluSerMetThrLysThrHisLysSer 354045 IleGluLeuLeuGlnAsnAlaValGlyGluGlnIleLeuAlaLeuLys 505560 ThrLeuGlnAspPheValAsnAspGluIleLysProAlaIleSerGlu 65707580 LeuGlyCysGluThrAlaAlaLeuArgLeuGlyIleLysLeuThrGln 859095 HisTyrSerGluLeuLeuThrAlaPheGlySerAsnPheGlyThrIle 100105110 GlyGluLysSerLeuThrLeuGlnAlaLeuSerSerLeuTyrSerAla 115120125 AsnIleThrGluIleMetThrThrIleArgThrGlyGlnSerAsnIle 130135140 TyrAspValIleTyrThrGluGlnIleLysGlyThrValIleAspVal 145150155160 AspLeuGluArgTyrMetValThrLeuSerValLysIleProIleLeu 165170175 SerGluValProGlyValLeuIleHisLysAlaSerSerIleSerTyr 180185190 AsnIleAspGlyGluGluTrpTyrValThrValProSerHisIleLeu 195200205 SerArgAlaSerPheLeuGlyGlyAlaAspIleThrAspCysValGlu 210215220 SerArgLeuThrTyrIleCysProArgAspProAlaGlnLeuIlePro 225230235240 AspSerGlnGlnLysCysIleLeuGlyAspThrThrArgCysProVal 245250255 ThrLysValValAspSerLeuIleProLysPheAlaPheValAsnGly 260265270 GlyValValAlaAsnCysIleAlaSerThrCysThrCysGlyThrGly 275280285 ArgArgProIleSerGlnAspArgSerLysGlyValValPheLeuThr 290295300 HisAspAsnCysGlyLeuIleGlyValAsnGlyValGluLeuTyrAla 305310315320 AsnArgArgGlyHisAspAlaThrTrpGlyValGlnAsnLeuThrVal 325330335 GlyProAlaIleAlaIleArgProValAspIleSerLeuAsnLeuAla 340345350 AspAlaThrAsnPheLeuGlnAspSerLysAlaGluLeuGluLysAla 355360365 ArgLysIleLeuSerGluValGlyArgTrpTyrAsnSerArgGluThr 370375380 ValIleThrIleIleValValMetValValIleLeuValValIleIle 385390395400 ValIleIleIleValLeuTyrArgLeuArgArg 405410 SEQIDNO:20Fct4protein(fragment2) GlnIleProArgAspArgLeuSerAsnIleGlyValIleValAspGlu 151015 GlyLysSerLeuLysIleAlaGlySerHisGluSerArgTyrIleVal 202530 LeuSerLeuValProGlyValAspPheGluAsnGlyCysGlyThrAla 354045 Gln50IleGlnTyrLysSerLeuLeuAsnArgLeuLeuIleProLeu 505560 ArgAspAlaLeuAspLeuGlnGluAlaLeuIleThrValThrAsnAsp 65707580 ThrThrGlnAsnAlaGlyAlaProGlnSerArg 8590 SEQIDNO:21Fct4proteinsignalsequence MATYIQRVOCISTSLLVVLTTLVSC 25 SEQIDNO:22p17proteinsequence GlyAlaAlaThrSerAlaLeuAsnArgArgGlnLeuAspGlnPheGlu 151015 LysIleArgLeuArgProAsnGlyLysLysLysTyrGlnIleLysHis 202530 LeuIleTrpAlaGlyLysGluMetGluArgPheGlyLeuHisGluArg 354045 LeuLeuGluThrGluGluGlyCysLysArgIleIleGluValLeuTyr 505560 ProLeuGluProThrGlySerGluGlyLeuLysSerLeuPheAsnLeu 65707580 ValCysValLeuTyrCysLeuHisLysGluGlnLysValLysAspThr 859095 GluGluAlaValAlaThrValArgGlnHisCysHisLeuValGluLys 100105110 GluLysSerAlaThrGluThrSerSerGlyGlnLysLysAsnAspLys 115120125 GlyIleAlaAlaProProGlyGlySerGlnAsnPhe 130135140 SEQIDNO:23p24proteinsequence ProAlaGlnGlnGlnGlyAsnAlaTrpValHisValProLeuSerPro 151015 ArgThrLeuAsnAlaTrpValLysAlaValGluGluLysLysPheGly 202530 AlaGluIleValProMetPheGlnAlaLeuSerGluGlyCysThrPro 354045 TyrAspIleAsnGlnMetLeuAsnValLeuGlyAspHisGlnGlyAla 505560 LeuGlnIleValLysGluIleIleAsnGluGluAlaAlaGlnTrpAsp 65707580 ValThrHisProLeuProAlaGlyProLeuProAlaGlyGlnLeuArg 859095 AspProArgGlySerAspIleAlaGlyThrThrSerSerValGlnGlu 100105110 GlnLeuGluTrpIleTyrThrAlaAsnProArgValAspValGlyAla 115120125 IleTyrArgArgTrpIleIleLeuGlyLeuGlnLysCysValLysMet 130135140 TyrAsnProValSerValLeuAspIleArgGlnGlyProLysGluPro 145150155160 PheLysAspTyrValAspArgPheTyrLysAlaIleArgAlaGluGln 165170175 AlaSerGlyGluValLysGlnTrpMetThrGluSerLeuLeuIleGln 180185190 AsnAlaAsnProAspCysLysValIleLeuLysGlyLeuGlyMetHis 195200205 ProThrLeuGluGluMetLeuThrAlaCysGlnGlyValGlyGlyPro 210215220 SerTyrLysAlaLysValMet 225230 SEQIDNO:24p8proteinsequence ValGlnGlnGlyGlyProLysArgGlnArgProProLeuArgCysTyr 151015 AsnCysGlyLysPheGlyHisMetGlnArgGlnCysProGluProArg 202530 LysThrLysCysLeuLysCysGlyLysLeuGlyHisLeuAlaLysAsp 354045 CysArgGlyGlnValAsn 50 SEQIDNO:25Proteasesequence PheGluLeuProLeuTrpArgArgProIleLysThrValTyrIleGlu 151015 GlyValProIleLysAlaLeuLeuAspThrGlyAlaAspAspThrIle 202530 IleLysGluAsnAspLeuGlnLeuSerGlyProTrpArgProLysIle 354045 IleGlyGlyIleGlyGlyGlyLeuAsnValLysGluTyrAsnAspArg 505560 GluValLysIleGluAspLysIleLeuArgGlyThrIleLeuLeuGly 65707580 AlaThrProIleAsnIleIleGlyArgAsnLeuLeuAlaProAlaGly 859095 AlaArgLeuValMet 100 SEQIDNO:26p51proteinsequence GlyGlnLeuSerGluLysIleProValThrProValLysLeuLysGlu 151015 GlyAlaArgGlyProCysValArgGlnTrpProLeuSerLysGluLys 202530 IleGluAlaLeuGlnGluIleCysSerGlnLeuGluGlnGluGlyLys 354045 IleSerArgValGlyGlyGluAsnAlaTyrAsnThrProIlePheCys 505560 IleLysLysLysAspLysSerGlnTrpArgMetLeuValAspPheArg 65707580 GluLeuAsnLysAlaThrGlnAspPhePheGluValGlnLeuGlyIle 859095 ProHisProAlaGlyLeuArgLysMetArgGlnIleThrValLeuAsp 100105110 ValGlyAspAlaTyrTyrSerIleProLeuAspProAsnPheArgLys 115120125 TyrThrAlaPheThrIleProThrValAsnAsnGlnGlyProGlyIle 130135140 ArgTyrGlnPheAsnCysLeuProGlnGlyTrpLysGlySerProThr 145150155160 IlePheGlnAsnThrAlaAlaSerIleLeuGluGluIleLysArgAsn 165170175 LeuProAlaLeuThrIleValGlnTyrMetAspAspLeuTrpValGly 180185190 SerGlnGluAsnGluHisThrHisAspLysLeuValGluGlnLeuArg 195200205 ThrLysLeuGlnAlaTrpGlyLeuGluThrProGluLysLysValGln 210215220 LysGluProProTyrGluTrpMetGlyTyrLysLeuTrpProHisLys 225230235240 TrpGluLeuSerArgIleGlnLeuGluGluLysAspGluTrpThrVal 245250255 AsnAspIleGlnLysLeuValGlyLysLeuAsnTrpAlaAlaGlnLeu 260265270 TyrProGlyLeuArgThrLysAsnIleCysLysLeuIleArgGlyLys 275280285 LysAsnLeuLeuGluLeuValThrTrpThrProGluAlaGluAlaGlu 290295300 TyrAlaGluAsnAlaGluIleLeuLysThrGluGlnGluGlyThrTyr 305310315320 TyrLysProGlyIleProIleArgAlaAlaValGlnLysLeuGluGly 325330335 GlyGlnTrpSerTyrGlnPheLysGlnGluGlyGlnValLeuLysVal 340345350 GlyLysTyrThrLysGlnLysAsnThrHisThrAsnGluLeuArgThr 355360365 LeuAlaGlyLeuValGlnLysIleCysLysGluAlaLeuValIleTrp 370375380 GlyIleLeuProValLeuGluLeuProIleGluArgGluValTrpGlu 385390395400 GlnTrpTrpAlaAspTyrTrpGlnValSerTrpIleProGluTrpAsp 405410415 PheValSerThrProProLeuLeuLysLeuTrpTyrThrLeuThrLys 420425430 GluProIleProLysGluAspValTyr 435440 SEQIDNO:27p15proteinsequence TyrValAspGlyAlaCysAsnArgAsnSerLysGluGlyLysAlaGly 151015 TyrIleSerGlnTyrGlyLysGlnArgValGluThrLeuGluAsnThr 202530 ThrAsnGlnGlnAlaGluLeuThrAlaIleLysMetAlaLeuGluAsp 354045 SerGlyProAsnValAsnIleValThrAspSerGlnTyrAlaMetGly 505560 IleLeuThrAlaGlnProThrGlnSerAspSerProLeuValGluGln 65707580 IleIleAlaLeuMetIleGlnLysGlnGlnIleTyrLeuGlnTrpVal 859095 ProAlaHisLysGlyIleGlyGlyAsnGluGluIleAspLysLeuVal 100105110 SerLysGlyIleArgArgValLeu 115120 SEQIDNO:28p31proteinsequence PheLeuGluLysIleGluGluAlaGlnGluGluHisGluArgTyrHis 151015 AsnAsnTrpLysAsnLeuAlaAspThrTyrGlyLeuProGlnIleVal 202530 AlaLysGluIleValAlaMetCysProLysCysGlnIleLysGlyGlu 354045 ProValHisGlyGlnValAspAlaSerProGlyThrTrpGlnMetAsp 505560 CysThrHisLeuGluGlyLysValValIleValAlaValHisValAla 65707580 SerGlyPheIleGluAlaGluValIleProArgGluThrGlyLysGlu 859095 ThrAlaLysPheLeuLeuLysIleLeuSerArgTrpProIleThrGln 100105110 LeuHisThrAspAsnGlyProAsnPheThrSerGlnGluValAlaAla 115120125 IleCysTrpTrpGlyLysIleGluHisThrThrGlyIleProTyrAsn 130135140 ProGlnSerGlnGlySerIleGluSerMetAsnLysGlnLeuLysGlu 145150155160 IleIleGlyLysIleArgAspAspCysGlnTyrThrGluThrAlaVal 165170175 LeuMetAlaCysHisIleHisAsnPheLysArgLysGlyGlyIleGly 180185190 GlyGlnThrSerAlaGluArgLeuIleAsnIleIleThrThrGlnLeu 195200205 GluIleGlnHisLeuGlnThrLysIleGlnLysIleLeuAsnPheArg 210215220 ValTyrTyrArgGluGlyArgAspProValTrpLysGlyProAlaGln 225230235240 LeuIleTrpLysGlyGluGlyAlaValValLeuLysAspGlySerAsp 245250255 LeuLysValValProArgArgLysAlaLysIleIleLysAspTyrGlu 260265270 ProLysGlnArgValGlyAsnGluGlyAspValGluGlyThrArgGly 275280285 SerAspAsn 290 SEQIDNO:29Gagprotein MetGlyAlaAlaThrSerAlaLeuAsnArgArgGlnLeuAspGlnPhe 151015 GluLysIleArgLeuArgProAsnGlyLysLysLysTyrGlnIleLys 202530 HisLeuIleTrpAlaGlyLysGluMetGluArgPheGlyLeuHisGlu 354045 ArgLeuLeuGluThrGluGluGlyCysLysArgIleIleGluValLeu 505560 TyrProLeuGluProThrGlySerGluGlyLeuLysSerLeuPheAsn 65707580 LeuValCysValLeuTyrCysLeuHisLysGluGlnLysValLysAsp 859095 ThrGluGluAlaValAlaThrValArgGlnHisCysHisLeuValGlu 100105110 LysGluLysSerAlaThrGluThrSerSerGlyGlnLysLysAsnAsp 115120125 LysGlyIleAlaAlaProProGlyGlySerGlnAsnPheProAlaGln 130135140 GlnGlnGlyAsnAlaTrpValHisValProLeuSerProArgThrLeu 145150155160 AsnAlaTrpValLysAlaValGluGluLysLysPheGlyAlaGluIle 165170175 ValProMetPheGlnAlaLeuSerGluGlyCysThrProTyrAspIle 180185190 AsnGlnMetLeuAsnValLeuGlyAspHisGlnGlyAlaLeuGlnIle 195200205 ValLysGluIleIleAsnGluGluAlaAlaGlnTrpAspValThrHis 210215220 ProLeuProAlaGlyProLeuProAlaGlyGlnLeuArgAspProArg 225230235240 GlySerAspIleAlaGlyThrThrSerSerValGlnGluGlnLeuGlu 245250255 TrpIleTyrThrAlaAsnProArgValAspValGlyAlaIleTyrArg 260265270 ArgTrpIleIleLeuGlyLeuGlnLysCysValLysMettyrAsnPro 275280285 ValSerValLeuAspIleArgGlnGlyProLysGluProPheLysAsp 290295300 TyrValAspArgPheTyrLysAlaIleArgAlaGluGlnAlaSerGly 305310315320 GluValLysGlnTrpMetThrGluSerLeuLeuIleGlnAsnAlaAsn 325330335 ProAspCysLysValIleLeuLysGlyLeuGlyMetHisProThrLeu 340345350 GluGluMetLeuThrAlaCysGlnGlyValGlyGlyProSerTyrLys 355360365 AlaLysValMetAlaGluMetMetGlnThrMetGlnAsnGlnAsnMet 370375380 ValGlnGlnGlyGlyProLysArgGlnArgProProLeuArgCysTyr 385390395400 AsnCysGlyLysPheGlyHisMetGlnArgGlnCysProGluProArg 405410415 LysThrLysCysLeuLysCysGlyLysLeuGlyHisLeuAlaLysAsp 420425430 CysArgGlyGlnValAsnPheLeuGlyTyrGlyArgTrpMetGlyAla 435440445 LysProArgAsnPheProAlaAlaThrLeuGlyAlaGluProSerAla 450455460 ProProProProSerGlyThrThrProTyrAspProAlaLysLysLeu 465470475480 LeuGlnGlnTyrAlaGluLysGlyLysGlnLeuArgGluGlnLysArg 485490495 AsnProProAlaMetAsnProAspTrpThrGluGlyTyrSerLeuAsn 500505510 SerLeuPheGlyGluAspGln 515 SEQIDNO:30Polprotein MetSerLysValTrpLysIleGlyThrproSerLysArgLeuglnGly 151015 ThrGlyGluPhePheArgValTrpThrValAspGlyglyLysThrGlu 202530 LysPheSerArgArgTyrSerTrpSerGlyThrGluCysAlaSerSer 354045 ThrGluArgHisHisProIleArgProSerLysGluAlaProAlaAla 505560 IleCysArgGluArgGluThrThrGluGlyAlaLysGluGluSerThr 65707580 GlyAsnGluSerGlyLeuAspArgGlyIlePhePheGluLeuProLeu 859095 TrpArgArgProIleLysThrValTyrIleGluGlyValProIleLys 100105110 AlaLeuLeuAspThrGlyAlaAspAspThrIleIleLysGluAsnAsp 115120125 LeuGlnLeuSerGlyProTrpArgProLysIleIleGlyGlyIleGly 130135140 GlyGlyLeuAsnValLysGluTyrAsnAspArgGluValLysIleGlu 145150155160 AspLysIleLeuArgGlyThrIleLeuLeuGlyAlaThrProIleAsn 165170175 IleIleGlyArgAsnLeuLeuAlaProAlaGlyAlaArgLeuValMet 180185190 GlyGlnLeuSerGluLysIleProValThrProValLysLeuLysGlu 195200205 GlyAlaArgGlyProCysValArgGlnTrpProLeuSerLysGluLys 210215220 IleGluAlaLeuGlnGluIleCysSerGlnLeuGluGlnGluGlyLys 225230235240 IleSerArgValGlyGlyGluAsnAlaTyrAsnThrProIlePheCys 245250255 IleLysLysLysAspLysSerGlnTrpArgMetLeuValAspPheArg 260265270 GluLeuAsnLysAlaThrGlnAspPhePheGluValGlnLeuGlyIle 275280285 ProHisProAlaGlyLeuArgLysMetArgGlnIleThrValLeuAsp 290295300 ValGlyAspAlaTyrTyrSerIleProLeuAspProAsnPheArgLys 305310315320 TyrThrAlaPheThrIleProThrValAsnAsnGlnGlyProGlyIle 325330335 ArgTyrGlnPheAsnCysLeuProGlnGlyTrpLysGlySerProThr 340345350 IlePheGlnAsnThrAlaAlaSerIleLeuGluGluIleLysArgAsn 355360365 LeuProAlaLeuThrIleValGlnTyrMetAspAspLeuTrpValGly 370375380 SerGlnGluAsnGluHisThrHisAspLysLeuValGluGlnLeuArg 385390395400 ThrLysLeuGlnAlaTrpGlyLeuGluthrProGluLysLysValGln 405410415 LysGluProProTyrGluTrpMetGlyTyrLysLeuTrpProHisLys 420425430 TrpGluLeuSerArgIleGlnLeuGluGluLysAspGluTrpThrVal 435440445 AsnAspIleGlnLysLeuValGlyLysLeuAsnTrpAlaAlaGlnLeu 450455460 TyrProGlyLeuArgThrLysAsnIleCysLysLeuIleArgGlyLys 465470475480 LysAsnLeuLeuGluLeuValThrTrpThrProGluAlaGluAlaGlu 485490495 TyrAlaGluAsnAlaGluIleLeuLysThrGluGlnGluGlyThrTyr 500505510 TyrLysProGlyIleProIleArgAlaAlaValGlnLysLeuGluGly 515520525 GlyGlnTrpSerTyrGlnPheLysGlnGluGlyGlnValLeuLysVal 530535540 GlyLysTyrThrLysGlnLysAsnThrHisThrAsnGluLeuArgThr 545550555560 LeuAlaGlyLeuValGlnLysIleCysLysGluAlaLeuValIleTrp 565570575 GlyIleLeuProValLeuGluLeuProIleGluArgGluValTrpGlu 580585590 GlnTrpTrpAlaAspTyrTrpGlnValSerTrpIleProGluTrpAsp 595600605 PheValSerThrProProLeuLeuLysLeuTrpTyrThrLeuThrLys 610615620 GluProIleProLysGluAspValTyrTyrValAspGlyAlaCysAsn 625630635640 ArgAsnSerLysGluGlyLysAlaGlyTyrIleSerGlnTyrGlyLys 645650655 GlnArgValGluThrLeuGluAsnThrThrAsnGlnGlnAlaGluLeu 660665670 ThrAlaIleLysMetAlaLeuGluAspSerGlyProAsnValAsnIle 675680685 ValThrAspSerGlnTyrAlaMetGlyIleLeuThrAlaGlnProThr 690695700 GlnSerAspSerProLeuValGluGlnIleIleAlaLeuMetIleGln 705710715720 LysGlnGlnIleTyrLeuGlnTrpValProAlaHisLysGlyIleGly 725730735 GlyAsnGluGluIleAspLysLeuValSerLysGlyIleArgArgVal 740745750 LeuPheLeuGluLysIleGluGluAlaglnGluGluHisGluArgTyr 755760765 HisAsnAsnTrpLysAsnLeuAlaAspThrTyrGlyLeuProGlnIle 770775780 ValAlaLysGluIleValAlaMetCysProLysCysGlnIleLysGly 785790795800 GluProValHisGlyGlnValAspAlaSerProGlyThrTrpGlnMet 805810815 AspCysThrHisLeuGluGlyLysValValIleValAlaValHisVal 820825830 AlaSerGlyPheIleGluAlaGluValIleProArgGluThrGlyLys 835840845 GluThrAlaLysPheLeuLeuLysIleLeuSerArgTrpproIleThr 850855860 GlnLeuHisThrAspAsnGlyProAsnPheThrSerGlnGluValAla 865870875880 AlaIleCysTrpTrpGlyLysIleGluHisThrThrGlyIleProTyr 885890895 AsnProGlnSerGlnGlySerIleGluSerMetAsnLysGlnLeuLys 900905910 GluIleIleGlyLysIleArgAspAspCysGlnTyrThrGluThrAla 915920925 ValLeuMetAlaCysHisIleHisAsnPheLysArgLysGlyGlyIle 930935940 GlyGlyGlnThrSerAlaGluArgLeuIleAsnIleIleThrThrGln 945950955960 LeuGluIleGlnHisLeuGlnThrLysIleGlnLYsIleLeuAsnPhe 965970975 ArgValTyrTyrArgGluGlyArgAspProValTrpLysGluProAla 980985990 GlnLeuIleTrpLysGlyGluGlyAlaValValLeuLysAspGlySer 99510001005 AspLeuLysValValProArgArgLysAlaLysIleIleLysAsp 101010151020 TyrGluProLysGlnArgValGlyAsnGluGlyAspValGluGly 102510301035 ThrArgGlySerAspAsn 1040
EXAMPLES
[0256] The invention is now described with reference to the Examples below. These are not limiting on the scope of the invention, and a person skilled in the art would be appreciate that suitable equivalents could be used within the scope of the present invention. Thus, the Examples may be considered component parts of the invention, and the individual aspects described therein may be considered as disclosed independently, or in any combination.
Example 1Transduction Efficiency in Human Bronchial Epithelial Cells (HBEC, F508del/F508del, Class II) Grown at an Air Liquid Interface (ALI) Culture
[0257] To analyse the transduction efficiency of HBECs (basal cells) subsequently grown at an ALI culture, cells were transduced in submerged culture with rSIV.F/HN, expressing GFP (vFM107, rSIV.F/HN-GFP) at different multiplicities of infection (MOI) of 3, 10, 30 and 90, followed by airlift at 2 days post transduction (
[0258] Next, the cellular profile of the ALIs derived from the transduced basal cells was examined by applying immunofluorescence staining for different epithelial cell markers at day 28 post transduction. Co-localisation of GFP with ACTUB (ciliated cells), KRT5 (basal cells), SCGB1A1 (club cells) and MUC5AC (goblet cells) was detectable, confirming that rSIV.F/HN produced expression in multiple cell types subsequent to basal cell transduction (
[0259] Additionally, average integrations in genome (vector copy numbers (VCN)) were analysed in DNA samples from ALI cultures, which were independently transduced with either GFP- (vGM107) or CFTR- (vGM058) expressing rSIV.F/HN. Bulk DNA analysis showed a dose related increase in VCN for both GFP- and CFTR-expressing rSIV.F/HN (30.05.2/35.84.0; 60.311.9/58.66.3; 124.225.2/87.210.5 copies/ng DNA in cells transduced at MOI 3, 10, 90 with GFP/CFTR-expressing rSIV.F/HN). No difference in VCN was observed between GFP- and CFTR-expressing rSIV.F/HN at any of the MOIs analysed (
[0260] Vector-derived Woodchuck hepatitis post-transcriptional regulatory element (WPRE) mRNA expression was also analyzed on sorted single cells of ALIs transduced at MOI 10 and again showed no difference in WPRE expression between cells transduced with GFP- (vGM107) or CFTR-expressing (vGM058) rSIV.F/HN (
[0261] Based on these data, transduction rates of GFP-expressing rSIV.F/HN were used as a surrogate readout to estimate transduction levels of CFTR-expressing rSIV.F/HN, thereby enabling correlative analyses between transduction levels and the degree of functional CFTR restoration.
Example 2Transduction of CF ALI Cultures (F508deI/F508deI, Class II) with rSIV.F/HN-CFTR (vGM058) Results in Transgenic CFTR Expression, Restoration of CFTR Chloride Current and Increased Ciliary Beat Frequency
[0262] To determine whether rSIV.F/HN-CFTR transduced CF ALIs can efficiently produce vector-derived codon optimised CFTR mRNA (coCFTR), quantitative ddPCR analysis was performed. Dose-dependent coCFTR expression was observed in cells transduced with rSIV.F/HN-CFTR (vGM058) at MOI 3 and 10, while no expression was observed in cells transduced with rSIV.F/HN-GFP (vGM107) (
[0263] To analyse the functionality of rSIV.F/HN-CFTR-expressed channels, CFTR-mediated chloride current was measured in an Ussing chamber using rSIV.F/HN-CFTR (vGM058) transduced ALI cultures. First, sodium channels (ENaC) were blocked using amiloride, followed by stimulation of CFTR using forskolin and the change in short circuit current (lsc) was calculated (both peak and plateau values). In some experiments, ivacaftor, a small molecule CFTR potentiator which increases channel open probability, was added for additional stimulation of CFTR current. Finally, the chloride current was blocked with CFTR-Inhibitor 172 (
[0264] As expected, non-transduced cells (MOI 0), or cells transduced with rSIV.F/HN-GFP did not respond to forskolin or the CFTR-inhibitor confirming the absence of functional CFTR channels. In contrast, a dose-related increase in CFTR chloride current was observed in ALIs transduced with rSIV.F/HN-CFTR. At an MOI of 3, there was restoration of 496% (peak, p<0.0001) and 384% (plateau, p<0.01) of the non-CF chloride current. When the rSIV.F/HN-CFTR (MOI 3) was combined with the potentiator ivacaftor, there was a significant increase in stimulation of chloride current (614% for both peak and plateau). With an increased MOI of 10, we observed significantly higher restoration (9411% peak, 666% plateau, p<0.0001) of the non-CF chloride current; a combination of MOI 10 and ivacaftor led to further increase in restoration (12111% for both peak and plateau). Higher MOIs (30 and 90) resulted in further significant increase: 14427 and 16249% of restoration (peak, p<0.0001) and 10137 and 11436% (plateau, p<0.0001) respectively.
[0265] Thus, rSIV.F/HN-CFTR is able to completely restore the CFTR-related chloride current to non-CF values and a combination of rSIV.F/HN-CFTR with ivacaftor amplifies this effect of gene therapy by 1.3-1.8-fold. These data compare favourably with current approved modulator therapies which were also assessed. Thus, treatment with lumacaftor+ivacaftor and tezacaftor+ivacaftor produced 34% and 21% of CFTR current restoration respectively, whilst treatment with elexacaftor+tezacaftor+ivacaftor resulted in 81% of restoration (
TABLE-US-00004 TABLE 1 Ussing chamber results for Class II F508del/F508del CF ALI cultures transduced with rSIV.F/HN (vGM058) Dose CFTR- CFTR- % of % of Treatment of LV Fsk.sub.peak Fsk.sub.plateau inh172.sub.peak inh172.sub.plateau WT.sub.(peak) WT.sub.(plateau) Non-CF 32.3 3.5 31.8 3.2 34.3 3.4 33.6 3.4 100 100 (WT) CF 0.003 0.1 0.003 0.1 0.4 0.1 0.4 0.1 rSIV.F/HN-GFP MOI 90 0.6 0.07 0.6 0.07 0.7 0.1 0.7 0.1 rSIV.F/HN-CFTR MOI 3 14.1 1.9 9.7 1.2 16.8 2.1 12.9 1.5 49 38 MOI 10 30.1 3.6 18.1 1.6 32.4 3.6 22.5 1.9 94 67 MOI 30 38.2 6.1 23.3 6.7 49.4 9.3 34.6 12.8 144 103 MOI 90 47.3 14.6 33.9 11.4 55.5 16.8 39.1 12.4 162 116 Iva 0.1 0.3 0.1 0.3 2.1 0.3 2.1 0.3 rSIV.F/HN- MOI 3 20.4 1.6 20.4 1.6 21.0 1.5 21.0 1.5 61 62 CFTR + Iva MOI 10 35.3 3.3 35.3 3.3 41.4 3.8 41.4 3.8 121 123 Luma + Iva 12.1 1.1 12.1 1.1 11.6 1.1 11.6 1.1 34 34 Teza + Iva 7.4 0.5 7.4 0.5 7.1 0.6 7.1 0.6 21 21 Elexa + 33.5 0.5 33.5 0.5 27.9 3.4 27.9 3.4 81 83 Teza + Iva LVlentivirus, Fskforskolin, CFTR-inh172CFTR inhibitor 172, Ivaivacaftor, Lumalumacaftor, Tezatezacaftor, Elexaelexacaftor
[0266] Since the transduction efficiency of rSIV.F/HN-GFP and rSIV.F/HN-CFTR was shown to be similar (
[0267] used in combination with ivacaftor full restoration was achieved with approximately 14% transduced cells.
[0268] To investigate the downstream functional consequences of coCFTR expression, ciliary beat frequency (CBF) was measured as a surrogate readout of mucociliary clearance. A significant reduction in CBF in the CF ALIs (6.90.8 Hz in comparison to 8.21 Hz in non-CF ALIs, p<0.05) was demonstrated. Transduction with rSIV.F/HN-CFTR was able to restore CBF to non-CF values (9.31.5, 9.72.6 and 9.52.2 Hz for MOI 3, 10 and 30 respectively, all p>0.0001) (
Example 3Generation of CFTR Knockout hSABCi Cell Line as a CF Translational Class I Mutation Model and Transduction of CFTR Knockout Cells with rSIV.F/HN (vGM107, vGM058)
[0269] Class I CFTR null mutations result in complete absence of full length CFTR protein and are thus not amenable to functional correction by current CFTR modulators. Gene therapy provides an opportunity to establish a disease-modifying treatment for patients with all mutation types, including those homozygous for these null mutations. Primary airway epithelial cells derived from the small number of patients with Class I mutations are difficult to obtain. Thus, in order to enable functional characterization of rSIV.F/HN-CFTR in a Class I mutation background, a mutation model was generated via a CRISPR/Cas9 mediated bi-allelic CFTR knockout (KO) in exon 4 of the CFTR gene using the previously described immortalized human small airway basal cell line hSABCi-NS1.1 (hSABCi). This cell line maintains basal cells features (TP63+, KRT5+) after more than 200 cell division cycles and 70 passages.
[0270] When cultured in air liquid interface conditions, hSABCi cells consistently form tight junctions and differentiate into ciliated (ARL13B+), club (SCGB1A1+), goblet (MUC5AC+, MUC5B+), neuroendocrine (CHGA+), ionocyte (FOXI1+) and surfactant protein positive cells (SFTPA+, SFTPB+, SFTPD+). Additionally, this cell line has been validated for the presence of ENaC and CFTR channel activity. hSABCi cells were transfected with a complex of synthetic tracr:cr RNA and recombinant Cas9. To analyse editing efficiency, the edited locus was amplified by PCR and then subjected to Sanger sequencing. Editing efficiency was determined for selected single clones using an established bioinformatic procedure to generate inference of CRISPR editing (ICE) metrics (data not shown). The clone (clone 5) with the highest out-of-frame editing/knock out efficiency of 99.4% (data not shown) was chosen for further experiments.
[0271] To characterize further the CFTR-KO phenotype, CFTR protein levels were analysed by Western blotting using monoclonal hCFTR antibody (R&D systems, Minneapolis, MN, US). A weak signal for the mature CFTR in the non-edited hSABCi cell line was observed, whereas no signal for the mature CFTR could be detected in CFTR KO cells (
[0272] Next, the efficiency of lentiviral transduction in CFTR KO cells grown in ALI cultures was analysed. CFTR KO cells were transduced and analysed as for primary F508del/F508del cells. Flow cytometry analysis revealed that transduction with rSIV.F/HN-GFP (vGM107) at MOIs 3, 10, 30 and 90 resulted in average mean of 12.21.7 (p<0.0001), 26.91.2 (p<0.0001), 36.31.1 (p<0.01) and 32.62.4% of GFP+ cells respectively (
Example 4Transduction of CFTR KO ALI Cultures (Class I) with rSIV.F/HN-CFTR (vGM058) Results in Expression of coCFTR mRNA and CFTR Current Restoration, while Ion Channel Modulators Fail to Restore CFTR Current
[0273] Codon optimised CFTR mRNA expression was analyzed in CFTR KO ALIs transduced with rSIV.F/HN-CFTR (vGM058). Dose-dependent increase in coCFTR expression was observed in cells transduced with rSIV.F/HN-CFTR in comparison to non-transduced cells, while no coCFTR expression was observed in cells transduced with rSIV.F/HN-GFP at all MOIs as expected (
[0274] Functional analysis via Ussing chamber measurements showed a dose-related increase in CFTR current in CFTR KO ALIs (class I) transduced with rSIV.F/HN-CFTR (vGM058) (
[0275] Since the transduction efficiencies of rSIV.F/HN-GFP and rSIV.F/HN-CFTR are similar (
Example 5Comparison of Pre-Clinical Candidate vGM058 and Clinical Candidate vGM244 (BI 3720931
[0276] To compare pre-clinical candidate virus (vGM058) with the clinical candidate virus (vGM244) class II HBECs were transduced with rSIV.F/HN, expressing GFP (vGM107) and rSIV.F/HN, expressing CFTR (vGM058 and vGM244) with MOIs of 3 and 10. At day 21 after airlift cells were collected and average integrations in genome (vector copy numbers (VCN)) were analysed in DNA samples from ALI cultures transduced at MOI 10.
[0277] No difference in VCN levels was observed between vGM107 and vGM058. However, cells transduced with vGM244 had significantly (p<0.05) lower VCN levels in comparison to cells transduced with vGM058 (
[0278] To compare the functionality of vGM058 and vGM244, CFTR-mediated chloride current was measured in an Ussing chamber using class II ALI cultures transduced with both vGM058 and vGM244. For both MOI 3 and 10, levels of functional correction for the vGM244 virus were lower, however non-significant, than for the vGM058 virus (
[0279] Any difference in VCN, coCFTR and Ussing chamber functional data between vGM058 and vGM244 may be attributed to titer variability of the virus due to different protocols of titer measurement between Oxford Biomedica (source of vGM244) and Oxford University (source of vGM058).
TABLE-US-00005 TABLE 2 Ussing chamber results for Class I CFTR KO ALI cultures transduced with rSIV.F/HN (vGM058) Dose CFTR- CFTR- % of % of Treatment of LV Fsk.sub.peak Fsk .sub.plateau inh172.sub.peak inh172.sub.plateau WT.sub.(peak) WT.sub.(plateau) Non-CF 33.6 0.7 33.6 0.7 31.7 1.1 31.7 1.1 100 100 (WT) CF 0.5 0.1 0.5 0.1 1.1 0.2 1.1 0.2 rSIV.F/HN-GFP MOI 90 .sup.1 0.7 .sup.1 0.7 3.0 0.3 3.0 0.3 rSIV.F/HN-CFTR MOI 3 6.8 0.4 5.7 0.4 14.3 0.4 13.1 0.5 45 41 MOI 10 12.4 0.6 10.3 0.3 27.5 1.5 25.0 1.4 87 79 MOI 30 15.2 1.3 13.1 1.6 34.9 2.6 32.8 2.2 110 103 MOI 90 20.9 1.3 19.6 1.1 37.5 3.0 36.2 2.7 118 114 Iva 1.9 0.1 1.9 0.1 3.0 0.2 3.0 0.2 rSIV.F/HN- MOI 3 11.3 0.7 11.3 0.7 18.9 1.0 18.9 1.0 60 60 CFTR + Iva MOI 10 19.0 0.9 19.0 0.9 33.7 2.5 33.7 2.5 106 106 MOI 30 27.7 3.1 27.7 3.1 44.8 3.3 44.8 3.3 141 141 MOI 90 26.8 3.1 26.8 3.1 39.3 2.5 39.3 2.5 124 124 Luma + Iva 0.1 1.2 0.1 1.2 0.5 1.1 0.5 1.1 Teza + Iva .sup.1 0.2 .sup.1 0.2 0.3 0.1 0.3 0.1 Elexa + 0.9 0.1 0.9 0.1 1.1 0.6 1.1 0.6 Teza + Iva LVlentivirus, Fskforskolin, CFTR-inh172CFTR inhibitor 172, Ivaivacaftor, Lumalumacaftor, Tezatezacaftor, Elexaelexacaftor
Example 6Analysis of Vector Copy Number and coCFTR Expression in Human Bronchial Epithelial Cells (HBEC, F508del/F508del, Class II) Transduced with vGM244
[0280] For experiments with clinical candidate virus vGM244 class II HBECs were transduced with both rSIV.F/HN, expressing GFP (vGM107, rSIV.F/HN-GFP) and rSIV.F/HN, expressing CFTR (vGM244, rSIV.F/HN-CFTR) with MOIs of 1, 3, 10, 30 and 90.
[0281] Average integrations in genome (vector copy numbers (VCN)) were analysed in DNA samples from ALI cultures and showed a dose related increase in VCN for both GFP- and CFTR-expressing rSIV.F/HN (3.80.6/8.30.8, 11.81.4/18.51.6; 25.13.2/21.12.1; 47.95.5/26.32.2; 81.019.8/17.92.4 copies/ng DNA in cells transduced at MOI 1, 3, 10, 30 and 90 with GFP/CFTR-expressing rSIV.F/HN). No difference in VCN was observed between GFP- and CFTR-expressing rSIV.F/HN at MOIs 1, 3 and 10, however at MOIs 30 and 90 the difference in VCN between GFP- and CFTR-transduced cells was significant (p<0.0001) (
[0282] Codon optimized CFTR mRNA expression was analysed in class II ALIs transduced with rSIV.F/HN-CFTR (vGM244). Dose-dependent increase in coCFTR expression was observed in cells transduced with rSIV.F/HN-CFTR in comparison to non-transduced cells, while no coCFTR expression was observed in cells transduced with rSIV.F/HN-GFP at all MOIs as expected (
Example 7Transduction of CF ALI Cultures (F508del/F508del, Class II) with SIV.F/HN-CFTR (vGM244) Results in Restoration of CFTR Chloride Current and Increased Ciliary Beat Frequency
[0283] To analyse the functionality of rSIV.F/HN-CFTR-expressed channels, CFTR-mediated chloride current was measured in an Ussing chamber using rSIV.F/HN-CFTR (vGM244) transduced class II ALI cultures (Table 3). As expected, non-transduced cells (MOI 0), or cells transduced with rSIV.F/HN-GFP did not respond to forskolin or the CFTR-inhibitor confirming the absence of functional CFTR channels (
TABLE-US-00006 TABLE 3 Using chamber results for Class II F508del/F508del CF ALI cultures transduced with rSIV.F/HN (vGM244) Dose CFTR- CFTR- % of % of Treatment of LV Fsk.sub.peak Fsk.sub.plateau inh172.sub.peak inh172.sub.plateau WT.sub.(peak) WT.sub.(plateau) Non-CF 31.2 1.4 29.4 1.3 41.8 1.9 39.9 1.7 100 100 (WT) CF 0.4 0.1 0.3 0.2 0.2 0.2 0.2 0.1 rSIV.F/HN-GFP MOI 90 0.3 0.1 0.4 0.1 0.3 0.1 0.3 0.1 rSIV.F/HN-CFTR MOI 1 5.5 0.4 4.9 0.3 6.3 0.6 5.8 0.5 15 14 MOI 3 10.1 0.8 8.3 0.7 14.3 0.7 12.5 0.6 34 31 MOI 10 22.3 1.8 12.4 1.0 30.5 2.1 20.6 1.3 73 52 MOI 30 23.0 1.3 12.7 0.7 31.3 1.6 21.1 1.1 75 53 MOI 90 25.1 1.7 13.0 0.8 31.9 2.6 19.9 1.9 76 50 Iva 1.0 0.3 1.0 0.3 1.6 0.1 1.6 0.1 rSIV.F/HN-CFTR + MOI 1 10.7 1.0 10.7 1.0 11.1 1.0 11.1 1.0 26 28 Iva MOI 3 17.3 2.1 17.3 2.1 19.8 1.5 19.8 1.5 47 50 MOI 10 28.4 2.2 28.4 2.2 35.7 1.9 35.7 1.9 85 90 MOI 30 30.1 1.8 30.1 1.8 36.6 1.8 36.6 1.8 87 92 MOI 90 30.2 2.8 30.2 2.8 32.7 3.8 32.7 3.8 78 82 Elexa + Teza 20.6 0.8 20.5 0.8 23.8 1.6 23.7 1.5 57 59 Elexa + Teza + 26.9 1.0 26.9 1.0 30.1 1.3 30.1 1.3 72 75 Iva (Trikafta) rSIV.F/HN-CFTR + MOI 1 27.9 1.7 27.9 1.7 33.7 2.1 33.4 2.1 81 84 Trikafta MOI 3 26.5 1.4 26.5 1.4 40.3 1.9 40.3 1.9 96 101 MOI 10 29.3 1.1 29.3 1.1 50.0 1.9 50.0 2.0 119 125 MOI 30 31.3 1.8 31.3 1.8 53.7 1.9 53.9 1.9 128 135 MOI 90 32.1 1.6 32.1 1.6 47.3 2.8 47.3 2.8 113 119 LVlentivirus, Fskforskolin, CFTR-inh172CFTR inhibitor 172, Ivaivacaftor, Lumalumacaftor, Tezatezacaftor, Elexaelexacaftor
[0284] To investigate the downstream functional consequences of coCFTR expression, ciliary beat frequency (CBF) was measured as a surrogate readout of mucociliary clearance. A significant reduction in CBF in the CF ALIs (5.20.2 Hz in comparison to 8.00.2 Hz in non-CF ALIs, p<0.0001) was demonstrated. Transduction with rSIV.F/HN-CFTR was able to restore CBF to non-CF values (8.20.5, 7.50.5, 8.80.4 and 9.70.5 Hz for MOI 1, 3, 10 and 30 respectively, p<0.001, p<0.0001) (
Example 8 Analysis of Vector Copy Number and coCFTR Expression in CFTR KO Cells (Class I) Transduced with vGM244
[0285] For experiments with clinical candidate virus vGM244 class I cells were transduced with both rSIV.F/HN, expressing GFP (vGM107, rSIV.F/HN-GFP) and rSIV.F/HN, expressing CFTR (vGM244, rSIV.F/HN-CFTR) with MOIs of 1, 3, 10, 30 and 90. Average integrations in genome (vector copy numbers (VCN)) were analysed in DNA samples from ALI cultures and showed a dose related increase in VCN for both GFP- and CFTR-expressing rSIV.F/HN (4.00.4/10.91.1, 14.22.9/24.12.2; 47.77.9/38.52.2; 81.312.8/57.26.2; 83.28.5/49.93.6 copies/ng DNA in cells transduced at MOI 1, 3, 10, 30 and 90 with GFP/CFTR-expressing rSIV.F/HN). No difference in VCN was observed between GFP- and CFTR-expressing rSIV.F/HN at MOIs 1, 3, 10 and 90, however at MOIs 30 the difference in VCN between GFP- and CFTR-transduced cells was significant (p<0.05) (
Example 9Transduction of CFTR KO ALI Cultures (Class I) with SIV.F/HN-CFTR (vGM244) Results in Restoration of CFTR Chloride
[0286] Functional analysis via Ussing chamber measurements showed a dose-related increase in CFTR current in CFTR KO ALIs (class I) transduced with rSIV.F/HN-CFTR (vGM244) (
[0287] Thus, rSIV.F/HN-CFTR is able to completely restore the CFTR-related chloride current to non-CF values and a combination of rSIV.F/HN-CFTR with ivacaftor amplifies this effect of gene therapy by 1.2-2.0-fold. Additionally, treatment with Trikafta (Elexacaftor+Tezacaftor+Ivacaftor) was also analysed and did not give any restoration effect for Class I cells as expected. Apart from that a combination of rSIV.F/HN-CFTR (vGM244) with Trikafta was analysed and showed restoration effect similar to the combination of rSIV.F/HN-CFTR (vGM244) with Ivacaftor. Thus, again suggesting that not only ivacaftor could give a therapeutic benefit but also ivacaftor-containing modulator therapies (
Discussion
[0288] These examples provide an in-depth functional characterization of rSIV.F/HN to further prepare rSIV.F/HN vector for pre-clinical and clinical development. Specifically, these examples provide evidence for the first time in human bronchial tissues that the vector is capable of completely correcting the CF chloride defect.
TABLE-US-00007 TABLE 4 Ussing chamber results for Class I CFTR KO ALI cultures transduced with rSIV.F/HN (vGM244) Dose CFTR- CFTR- % of % of Treatment of LV Fsk.sub.peak Fsk.sub.plateau inh172.sub.peak inh172.sub.plateau WT.sub.(peak) WT.sub.(plateau) Non-CF 26.2 1.4 26.0 1.4 24.5 1.4 24.3 1.5 100 100 (WT) CF 0.3 0.1 0.4 0.1 0.9 0.1 0.8 0.1 rSIV.F/HN-GFP MOI 90 0.2 0.2 0.1 0.3 1.0 0.2 0.7 0.2 rSIV.F/HN-CFTR MOI 1 3.7 0.3 2.3 0.5 5.2 0.2 3.8 0.1 21 16 MOI 3 9.1 0.5 6.3 0.6 10.4 0.7 7.7 0.3 43 32 MOI 10 16.5 1.5 10.9 1.0 19.5 2.3 13.8 0.7 80 57 MOI 30 22.0 1.5 16.0 1.6 24.9 2.7 21.3 1.2 102 84 MOI 90 36.4 3.1 17.6 1.9 46.4 3.6 31.4 2.9 190 129 Iva 0.7 0.2 0.7 0.2 0.2 0.2 0.2 0.2 rSIV.F/HN-CFTR + MOI 1 3.5 0.6 3.5 0.6 4.0 0.6 4.0 0.6 16 16 Iva MOI 3 13.9 0.9 13.9 0.9 14.8 0.9 14.8 0.9 61 61 MOI 10 27.3 2.3 27.3 2.3 25.0 3.2 25.0 3.2 102 103 MOI 30 44.0 3.6 44.0 3.6 34.2 3.7 34.2 3.7 140 141 MOI 90 52.2 10.1 52.2 10.1 47.3 8.9 47.3 8.9 193 195 Elexa + Teza + 1.0 0.2 1.0 0.2 0.7 0.1 0.7 0.1 Iva (Trikafta) rSIV.F/HN-CFTR + MOI 1 2.1 0.4 10.2 2.7 5.8 0.8 5.8 0.8 24 24 Trikafta MOI 3 11.3 1.0 11.3 1.0 15.7 1.4 15.7 1.4 64 65 MOI 10 28.5 2.8 28.5 2.8 28.7 2.5 28.7 2.5 117 118 MOI 30 33.6 2.6 33.6 2.6 32.5 1.3 32.5 1.3 133 134 MOI 90 24.0 6.9 24.0 6.9 32.1 8.3 32.1 8.3 131 132 LVlentivirus, Fskforskolin, CFTR-inh172CFTR inhibitor 172, Ivaivacaftor, Lumalumacaftor, Tezatezacaftor, Elexaelexacaftor
[0289] Remarkably, these experiments demonstrate that CFTR modulators, particularly CFTR potentiators, achieve a greater than expected potentiation of the CFTR transgene expressed by rSIV.F/HN. In particular, the effect of the CFTR modulator, particularly the CFTR potentiator, and rSIV.F/HN-CFTR combination is greater than the additive effects of the separate effects of the CFTR modulator, particularly the CFTR potentiator, and rSIV.F/HN-mediated CFTR expression.
[0290] A relationship between transduced cell number and degree of correction has been established, and the vector integration profile of such a self-inactivating lentiviral vector in a relevant cell type for lung gene therapy assessed. These data provide further support for the translation of this vector into first-in-man trials.
[0291] Previous studies have suggested that a range of 5-25% corrected cells should be sufficient to restore the CFTR chloride current to normal values. This is also supported by previous study that show that CF individuals with certain mild mutations that retain 10% of normal CFTR expression per cell do generally not suffer from disease. Therefore, without being bound by the theory, the present work indicates that even a low percentage of transduced cells, combined with a strong promotor driving CFTR expression is sufficient to provide significant restoration of the CFTR related chloride current.
[0292] As exemplified herein for the first time, the effect of gene therapy can be further enhanced with the clinically approved CFTR potentiator ivacaftor and ivacaftor containing products like TRIKAFTA.
[0293] Ivacaftor and other CFTR potentiators, as well as products containing ivacaftor (e.g. TRIKAFTA) or other potentiators, act by increasing channel open probability. Given the typical open probability of wild-type CFTR chloride channels of approximately 0.4, this effect is potentially clinically important. The combination of gene therapy plus the potentiator reduced the number of transduced cells needed to achieve full restoration of the chloride current and decreased the needed MOI by approximately 1.2-2.0 fold. This has significant potential therapeutic and economic importance, and surprising given the greater than expected potentiation of the CFTR transgene expressed by rSIV.F/HN. There are potentially benefits in terms of efficacy, cost-of-goods and applicability to a broad swathe of the CF population given that ivacaftor is a constituent of currently used modulators.
[0294] Whilst the optimal airway cell type to target for successful CF gene therapy is unclear, recent studies have provided indications. Utilizing single-cell RNA-seq technologies it has been shown that apart from the ionocytes, which is a rare cell type, CFTR is mainly expressed by SCGB1A1+ club cells and to a lesser extent by basal cells, collectively accounting for .sup.80% of all CFTR+ cells. The rSIV.F/HN vector is able to transduce all these relevant types of cells in the murine lung in vivo, and here further confirmation for sufficient transduction efficiency in human bronchial epithelial cells has been obtained.
[0295] Apart from providing a novel and sustainable treatment option for a broad range of CFTR patients, including patients affected by the most common F508del/F508del mutation, gene therapy is especially attractive for those carrying Class I mutations, which result in complete absence of CFTR protein and for whom there is currently no modulator treatment available. In order to analyse the functional effects of rSIV.F/HN in Class I mutation cells, a novel CFTR knock-out cell line was generated. Transduction of these cells with rSIV.F/HN again resulted in robust transgene expression in all relevant cell types and resulted in restoration of forskolin-stimulated CFTR current to wild-type levels. As expected, none of the modulators produced an effect on the CFTR chloride current. In contrast rSIV.F/HN was able to fully restore CFTR function in a dose-dependent manner, as was the case for the F508del/F508del cells. Further, similarly to Class II mutation cells, we observed an unexpected increase in effect with ivacaftor and TRIKAFTA. These data suggest that rSIV.F/H N is a viable contender for the treatment of patients carrying null mutations, or those patients who are insensitive to, or unable to tolerate modulators. Where such patients can tolerate modulators, there is the further opportunity to improve or optimise treatment.
[0296] The downstream consequences of chloride current restoration through assessment of Ciliary beat frequency (CBF) as a surrogate readout for mucociliary clearance (MCC) was also assessed. Clinical data suggest that MMC becomes impaired with increasing disease severity, likely related to lack of adequate hydration of both the mucus and the underlying airway surface liquid. The CF ALIs used in this study demonstrated a reduced ciliary beat frequency (CBF), likely secondary to these two parameters and rSIV.F/HN CFTR restored CBF in F508del/F508del ALIs to wildtype levels. These findings provide a further indication that rSIV.F/HN CFTR could improve lung physiology in CF patients in vivo, through an effect on MCC.
[0297] In conclusion, this work demonstrates the high transduction efficiency and consequent complete functional correction of the CF chloride defect in human ALIs, both in primary HBE cells from F508del/F508del patients as well as in a novel CFTR KO hSABCi cell line as a model of Class I homozygous null mutations. The potentiator ivacaftor showed a surprising and greater than expected effect when combined with rSIV.F/HN. These data suggest that this lentiviral vector is a leading candidate for the treatment of CF patients independent of mutation class and, that combination therapy using this vector with one or more CFTR modulators, particularly a potentiator such as ivacaftor, or a product containing a potentiator such as ivacaftor (e.g. TRIKAFTA) is an attractive prospect for treating CF, with an initial focus on modulator insensitive patients.