Combination therapy

Abstract

The present invention relates to the combination treatment of cystic fibrosis, in particular the combination of modulators of cystic fibrosis transmembrane conductance regulatory protein (CFTR) and gene therapy.

Description

Technical Field

[0001] The present invention relates to combination treatments for cystic fibrosis, particularly the combination of modulators of the cystic fibrosis transmembrane conductance regulator (CFTR) and gene therapy. Background Art

[0002] Cystic fibrosis (CF) is a serious genetic disease caused by mutations in the CF transmembrane conductance regulator (CFTR) gene. These mutations lead to the production of faulty CFTR proteins, whose dysfunction affects the balance of salts and fluids inside and outside cells. This imbalance causes thick, sticky mucus in the lungs, pancreas, and other organs.

[0003] Current treatments for CF include CFTR modulator therapies, which attempt to correct CFTR dysfunction. These modulator drugs can enhance or even restore the functional expression of specific mutations that cause CF. These CFTR modulator drugs are divided into five categories based on their effect on the CFTR mutation: enhancers, correctors, stabilizers, read-through agents, and expanders. To date, four CFTR modulators have been commercially available, namely Kalydeco ® (ivacaftor), Orkambi ® (lumacaftor / ivacaftor), Symdeko ® (tezacaftor / ivacaftor) and Trikafta ® (elexacaftor / tesacaftor / ivacaftor).

[0004] CFTR modulators provide significant improvement for many CF patients, but approximately 10% remain modulator-insensitive or intolerant. Specifically, despite the recent success of CFTR channel modulators, there remains an unmet need for those patients who cannot tolerate the side effects of ion channel modulator therapy, or for subsets that still lack disease-modifying treatment options, such as those affected by homozygous class I mutations.

[0005] The main cause of CF morbidity and mortality is lung disease. CFTRGenes have been selected, and gene therapy has received significant attention as a possible treatment for CF. However, gene transfer efficiency to the airway epithelium is generally poor, at least in part because the individual receptors for many viral vectors appear to be primarily located on the basolateral surface of the airway epithelium. These vectors may also have difficulty overcoming the body's host defenses and still have difficulty producing efficient expression after re-administration. Due to these difficulties, although several gene therapy approaches for CF have been studied in clinical trials to date, including adenoviral vectors, adeno-associated virus (AAV) vectors, and plasmid-based vectors, none have yet progressed to the market authorization stage, largely due to issues with their limited efficiency. In addition, the ability to repeatedly administer conventional viral vectors (essential for lifelong treatment of self-renewing epithelium) is limited by the patient's adaptive immune response, which prevents successful repeated administration.

[0006] Therefore, there is a need for novel and effective therapies for CF, particularly for patients who are insensitive or intolerant to CF modulators or who lack disease-modifying treatment options. Specifically, one goal of the present invention is to provide novel therapies that can combine existing CF modulators, particularly CFTR enhancers, with CF gene therapy, which has the potential to maximize the benefits associated with CF gene therapy. Combination therapies can also potentially address some of the shortcomings associated with current treatments, including modulator insensitivity / tolerance and / or poor gene transfer efficiency of CF gene therapy vectors. Summary of the Invention

[0007] The present inventors have now demonstrated that the combination of CFTR modulators, particularly CFTR enhancers, and lentiviral gene therapy vectors can not only drive CTFR expression, but also improve CTFR function and restore airway cell function. Specifically, using air-liquid interface (ALI) cultures of cells with two different CFTR mutation backgrounds (class I and class II), the present inventors have demonstrated that (i) ivacaftor or (ii) a combination containing ivacaftor and containing CFTRThe combination of a transgenic SIV vector (rSIV.F / HN) pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxovirus is able to drive CFTR expression in an ALI model of both class I and class II CFTR mutations, thereby restoring CFTR chloride currents and increasing ciliary beat frequency. The inventors have demonstrated for the first time that the combination of CFTR modulators and CF gene therapy, particularly using the rSIV.F / HN vector, can achieve functional correction in a class I CFTR mutation model, where class I knockout mutations result in the complete absence of full-length CFTR protein and are therefore generally not amenable to functional correction using CFTR modulators alone. Thus, the inventors have demonstrated a beneficial and unexpected interaction between CFTR modulators, specifically potentiators, and rSIV.F / HN for at least class I and class II CFTR mutations. Even more unexpectedly, the present inventors have demonstrated that CFTR modulators, particularly CFTR enhancers, such as those including ivacaftor, achieve greater than expected enhancement of the CFTR transgene expressed by rSIV.F / HN. Specifically, the effect of a CFTR modulator, particularly a CFTR enhancer, and rSIV.F / HN-CFTR combination is greater than the additive effect of the individual effects of the CFTR modulator / enhancer and rSIV.F / HN-mediated CFTR expression. This is exemplified herein by the CFTR enhancer ivacaftor and rSIV.F / HN-CFTR, which achieve a cumulative effect greater than the individual effects of ivacaftor and rSIV.F / HN-mediated CFTR expression. Thus, the present inventors have demonstrated for the first time the favorable therapeutic potential of a combination of CFTR modulators and rSIV.F / HN-based CF gene therapy, particularly for patients with Class I CFTR mutations, or for patients who are otherwise insensitive, intolerant, or respond poorly to CFTR modulators.

[0008] Therefore, the present invention provides a combination of: (i) a lentiviral vector pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxovirus, wherein the lentiviral vector comprises a cystic fibrosis transmembrane conductance regulator (CFTR) transgene; and (ii) a CFTR modulator, for use in a method for treating cystic fibrosis (CF).

[0009] The lentiviral vector may be an SIV vector and the respiratory paramyxovirus may be Sendai virus. The transgene may be operably linked to a promoter selected from the group consisting of a cytomegalovirus (CMV) promoter, an elongation factor 1a (EF1a) promoter, and a hybrid human CMV enhancer / EF1a (hCEF) promoter. The lentiviral vector may comprise a hybrid human CMV enhancer / EF1a (hCEF) promoter, which may comprise or consist of a nucleotide sequence having at least 90% identity to SEQ ID NO: 2. The CFTR transgene may be a codon-optimized CFTR transgene, which may comprise or consist of a nucleotide sequence having at least 90% identity to SEQ ID NO: 1. The lentiviral vector may be produced using codon-optimized plasmids. The lentiviral vector can be produced using (i) pGM691 (SEQ ID NO: 7) and / or (ii) pGM830 (SEQ ID NO: 9) or pGM326 (SEQ ID NO: 8); and preferably also using pGM299 (SEQ ID NO: 11), pGM301 (SEQ ID NO: 12), and / or pGM303 (SEQ ID NO: 13). The lentiviral vector can be vGM058, vGM195, or vGM244. The lentiviral vector can be an SIV vector pseudotyped with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) proteins, wherein: the vector comprises a modified retroviral RNA sequence comprising or consisting of the nucleic acid sequence of SEQ ID NO: 16. The lentiviral vector may comprise an F protein having a first subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 19 and a second subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 20.The lentiviral vector may further comprise: (a) a p17 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 22; (b) a p24 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 23; (c) a p8 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 24; (d) a protease comprising or consisting of the amino acid sequence of SEQ ID NO: 25; (e) a p51 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 26; (f) a p15 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 27; (g) a p31 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 28; (h) a Gag protein comprising or consisting of the amino acid sequence of SEQ ID NO: 29; and / or (i) a Pol protein comprising or consisting of the amino acid sequence of SEQ ID NO: 30; wherein preferably, the vector comprises each of (a) to (g).

[0010] The CFTR modulator can be a CFTR enhancer and / or a CFTR corrector, preferably a CFTR enhancer. The CFTR modulator can be selected from ivacaftor, tesacaftor, elexacaftor, or lumacaftor, or a combination thereof. Preferably, the CFTR modulator is ivacaftor.

[0011] The present invention provides the following combination: (A) an SIV vector pseudotyped with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) proteins, wherein: (a) the vector comprises a modified retroviral RNA sequence comprising or consisting of the nucleic acid sequence of SEQ ID NO: 16; and (b) the F protein comprises a first subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 19 and a second subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 20; and (B) ivacaftor; for use in a method of treating cystic fibrosis (CF). In the combination, the vector may further comprise one or more of the following: (a) p17 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 22; (b) p24 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 23; (c) p8 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 24; (d) protease comprising or consisting of the amino acid sequence of SEQ ID NO: 25; (e) p51 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 26; (f) p15 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 27; (g) p31 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 28; (h) Gag protein comprising or consisting of the amino acid sequence of SEQ ID NO: 29; and / or (i) a protein comprising or consisting of the amino acid sequence of SEQ ID NO: 30 or a Pol protein consisting of the amino acid sequence; wherein preferably the vector comprises each of (a) to (g).

[0012] The patient to be treated may have at least one Class I, Class II, Class III, Class IV, Class V, and / or Class VI CFTR mutation. The patient to be treated may have at least one Class I and / or Class II CFTR mutation. The combination therapy of the present invention may be used independently of the patient's CFTR mutation. The patient to be treated may have: (a) at least one Class I CFTR mutation selected from G542X, W1282X, and / or R553C; and / or (b) at least one Class II CFTR mutation selected from F508del, N1303K, and / or I507del.

[0013] The lentiviral vector and the CFTR modulator can be administered simultaneously or sequentially. The lentiviral vector can be administered by inhalation; and / or the CFTR modulator can be administered orally. 8 to about 10 14The dose is between 10 transduction units (TU), preferably about 10 6 to about 10 12 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 the CFTR modulator can be administered at a concentration used as monotherapy for each modulator or a lower concentration.

[0014] Treatment can restore CFTR activity to at least 10% of the CFTR activity of healthy controls. Treatment can restore CFTR activity to at least 50% of the CFTR activity of healthy controls. Treatment can increase CFTR activity by at least 1.2-fold compared to treatment with a single lentiviral vector. Treatment can increase CFTR current by about 1.3-fold to about 3-fold or about 1.3-fold to about 1.8-fold compared to treatment with a single lentiviral vector. The patient to be treated may have a Class I CFTR mutation and the treatment can: (i) restore CFTR activity to at least 10% of the CFTR activity of a healthy control; and / or (ii) increase CFTR current by about 1.3-fold to about 1.8-fold or about 1.3-fold to about 3-fold compared to treatment with a single lentiviral vector. The patient to be treated may have a class II CFTR mutation and the treatment may: (i) restore CFTR activity to at least 10% of the CFTR activity of a healthy control; and / or (ii) increase CFTR current by about 1.3-fold to about 3-fold or about 1.3-fold to about 1.8-fold compared to treatment with a lentiviral vector alone. A transduction rate of between about 10% and about 20%, preferably between about 14% and about 17%, may be sufficient to achieve a therapeutic effect on CFTR activity as defined herein.

[0015] The present invention also provides a method of treating 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 respiratory paramyxovirus, wherein the lentiviral vector comprises a cystic fibrosis transmembrane conductance regulator (CFTR) transgene; and (ii) a CFTR modulator.

[0016] The present invention further provides the use of a lentiviral vector pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxovirus, wherein the lentiviral vector comprises a cystic fibrosis transmembrane conductance regulator (CFTR) transgene, for use in the manufacture of a medicament for use in a method for treating CF, wherein the method further comprises administering a CFTR modulator. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1A-1G Schematic diagram showing exemplary plasmids used to generate vectors of the invention.

[0018] Figure 2 Transduction efficiency and transduced cell types in HBEC ALI. (A) Experimental setup. (B) Quantification of transduced GFP+ cells at day 21 post-transduction. (C) Immunofluorescence of ciliated (ACTUB), basal (KRT5), rod-shaped (SCGB1A1), and goblet-shaped (MUC5AC) cells and colocalization with transduced GFP+ cells. Arrows indicate colocalization. (D) Flow cytometric quantification of the percentage of transduced cells in specific epithelial cell populations. (E) qPCR analysis of vector copy number (VCN) in bulk samples transduced with rSIV.F / HN expressing GFP or CFTR. (F) Transgenic gene mRNA expression in sorted single cells (RT-ddPCR). MOI = multiplicity of infection; WPRE = woodchuck hepatitis posttranscriptional regulatory element. Scale bar in C = 20 µm. Differences in VCN were analyzed using two-way analysis of variance (*** p < 0.001, **** p < 0.0001).

[0019] Figure 3Functional data demonstrating that rSIV.F / HN (vGM058) restores CFTR chloride currents in primary CF HBECs (class II). (A) Gene expression of the codon-optimized CFTR transgene demonstrates higher transgene expression in CFTR-transduced cells but not in negative control GFP-transduced cells. (B) Endogenous human CFTR gene expression. (C) Gene expression ratio between transgenic coCFTR and endogenous hCFTR. (D) Representative scheme of Ussing chamber measurements. (E) Ussing chamber data are expressed as a percentage of WT CFTR current; the difference between the forskolin peak current and the CFTR-inhibited trough current was calculated. (F) Ussing chamber data are expressed as a percentage of WT CFTR current; the difference between the forskolin plateau current and the CFTR-inhibited trough current was calculated. (G) Correlation between the percentage of cells expressing CFTR and restoration of CFTR chloride currents in primary CF HBECs. (H) Analysis of mean ciliary beat frequency in primary non-CF, CF, and transduced CF HBECs at day 28 after resuscitation. ΔIsc - change in short-circuit current, UC - Ussing chamber, Amil - amlodipine, Fsk - forskolin, Iva - ivacaftor, Luma - lumacaftor, Teza - tesacaftor, Elexa - elexacaftor, Hz - hertz. Differences in ΔIsc current and mean ciliary beat frequency were analyzed using one-way ANOVA (*p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001). For Ussing chamber experiments, N = 16-26 were used for most conditions except for MOI 30 and 90 (N = 4). Stars at the top of the graph indicate statistical significance compared to CF MOI 0 controls.

[0020] Figure 4Generation of CFTR KO (CFTR knockout, class I) hSABCi cell lines and evaluation of transduction with rSIV.F / HN-GFP (vGM107) and rSIV.F / HN-CFTR (vGM058). (A) CFTR protein expression in hSBACi (originally described in Wang et al., Respir. Res. (2019) 20:196) and CFTR KO cells from clone 5, which has the highest editing efficiency. (B) Flow cytometry quantification of transduced GFP+ CFTR KO cells at day 21 post-transduction. (C) qPCR analysis of vector copy number (VCN) in bulk CFTR KO samples transduced with vectors expressing GFP and CFTR. (D) Immunofluorescence of ciliary (ACTUB), basal (KRT5), rod-shaped (SCGB1A1), and goblet-shaped (MUC5AC) cells and colocalization with transduced GFP+ cells. Arrows indicate colocalization. (E) Flow cytometric quantification of the percentage of transduced cells in specific epithelial cell populations. MOI - multiplicity of infection, FITC-A - fluorescent isothiocyanate area, SSC-A - side scatter area. Scale bar in E - 20 µm. Differences in VCN were analyzed using two-way ANOVA (**p < 0.01, ****p < 0.0001).

[0021] Figure 5Functional data demonstrating that rSIV.F / HN (vGM058) restores CFTR chloride currents in CFTR KO cells (Class I) compared to modulators. (A) Gene expression of the codon-optimized CFTR transgene demonstrates higher transgene expression in CFTR-transduced cells but not in GFP-transduced cells. (B) Ussing chamber data are expressed as a percentage of WT CFTR currents; the difference between the forskolin peak and CFTR-inhibited trough currents was calculated. Notably, there was no activation of CFTR chloride currents following treatment with the modulators (Luma+Iva, Teza+Iva, and Elexa+Teza+Iva). (C) Ussing chamber data are expressed as a percentage of WT CFTR currents; the difference between the forskolin plateau and CFTR-inhibited trough currents was calculated. Notably, there was no activation of CFTR chloride currents following treatment with the modulators (Luma+Iva, Teza+Iva, and Elexa+Teza+Iva). (D) Correlation between the percentage of cells expressing CFTR and the recovery of CFTR chloride current in transduced CFTR KO cells. ΔIsc - short-circuit current change, Iva - ivacaftor, Luma - lumacaftor, Teza - tesakaftor, Elexa - elexacaftor. Differences in ΔIsc were analyzed using single-factor ANOVA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). For Ussing chamber experiments, N = 5-11. The star at the top of the figure indicates statistical significance compared to the CF MOI 0 control.

[0022] Figure 6 Comparison of vGM058 and vGM244: VCN, coCFTR expression, and functional correction levels. (A) ddPCR analysis of vector copy number (VCN) in bulk samples transduced with vGM107, vGM058, or vGM244 rSIV.F / HN. (B) Gene expression of the codon-optimized CFTR transgene in samples transduced with vGM107, vGM058, or vGM244 rSIV.F / HN. (C) Ussing chamber data are expressed as a percentage of WT CFTR current; the difference between the maximal forskolin peak current and the trough current after CFTR inhibition was calculated. (D) Ussing chamber data are expressed as a percentage of WT CFTR current; the difference between the forskolin plateau and the trough current after CFTR inhibition was calculated. WPRE - Woodchuck hepatitis posttranscriptional regulatory element. ddPCR data were analyzed using the Mann-Whitney test, and two-way analysis of variance was used to analyze Ussing chamber differences (*p<0.05).

[0023] Figure 7DNA vector copy number and coCFTR RNA expression in class II cells transduced with vGM0244 and vGM107. (A) qPCR analysis of vector copy number (VCN) in bulk samples transduced with rSIV.F / HN expressing GFP or CFTR. (B) Gene expression of the codon-optimized CFTR transgene demonstrates higher transgene expression in CFTR-transduced cells but not in GFP-transduced cells. WPRE - Woodchuck hepatitis posttranscriptional regulatory element. Differences were analyzed using two-way analysis of variance (****p < 0.0001).

[0024] Figure 8 Functional data demonstrating that rSIV.F / HN (vGM244) restores CFTR chloride currents in primary CF HBEC (class II). (A) Ussing chamber data are expressed as a percentage of WT CFTR current; the difference between the maximal forskolin peak current and the trough current after CFTR inhibition was calculated. (B) Ussing chamber data are expressed as a percentage of WT CFTR current; the difference between the forskolin plateau and the trough current after CFTR inhibition was calculated. Teza - Tezaca, Elexa - Elexa. Differences were analyzed using one-way ANOVA (**p<0.01, ***p<0.001, ****p<0.0001). For Ussing chamber experiments, N=25-36 were used for most conditions except MOI 90 (N=12).

[0025] Figure 9 Transduction with vGM244 induces restoration of ciliary beat frequency in type II CF HBEC. Average ciliary beat frequency was analyzed in primary non-CF, CF, and transduced CF HBEC at day 28 after resuscitation. Hz - Hertz. Differences were analyzed using one-way analysis of variance (*p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001).

[0026] Figure 10 DNA vector copy number and coCFTR RNA expression in class I cells transduced with vGM244 and vGM107. (A) qPCR analysis of vector copy number (VCN) in bulk samples transduced with rSIV.F / HN expressing GFP or CFTR. (B) Gene expression of the codon-optimized CFTR transgene demonstrates higher transgene expression in CFTR-transduced cells but not in GFP-transduced cells. WPRE - Woodchuck hepatitis posttranscriptional regulatory element. Differences were analyzed using two-way analysis of variance (*p < 0.05, **p < 0.005, ****p < 0.0001).

[0027] Figure 11Functional data demonstrating that rSIV.F / HN (vGM244) restores CFTR chloride currents in primary CF HBEC (class II). (A) Ussing chamber data are expressed as a percentage of WT CFTR current; the difference between the maximal forskolin peak current and the trough current after CFTR inhibition was calculated. (B) Ussing chamber data are expressed as a percentage of WT CFTR current; the difference between the forskolin plateau and the trough current after CFTR inhibition was calculated. Teza - Tezaca, Elexa - Elexa. Differences were analyzed using one-way ANOVA (*p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001). For Ussing chamber experiments, N = 10-18 were used for most conditions except MOI 90 (N = 4). DETAILED DESCRIPTION

[0028] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art in the art to which the present invention belongs. Singleton et al., DICTIONARY OF MICROBIOLOGY AND MOLECULAR BIOLOGY, 20th ed., John Wiley and Sons, New York (1994) and Hale and Marham, THE HARPER COLLINS DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide general dictionaries of many of the terms used in the present invention for those skilled in the art. The meaning and scope of the terms should be clear; however, in the event of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions.

[0029] It should be understood that the present invention is not limited to the specific methods, protocols, reagents, etc. described herein and can vary as such. In particular, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the present invention.

[0030] The description of the embodiments of the present invention is not intended to be exhaustive or to limit the disclosure to the exact form disclosed. Although specific embodiments and examples of the present invention are described herein for illustrative purposes, those skilled in the art in the relevant art will recognize that various equivalent modifications can be made within the scope of the present invention. For example, although the method steps or functions are presented in a specified order, alternative embodiments can perform functions in a different order, or can perform functions substantially simultaneously. The teachings of the present invention provided herein can be applied to other programs or methods as needed. The various embodiments described herein can be combined to provide other embodiments. Aspects of the present invention can be modified (if necessary) to adopt the compositions, functions and concepts of the above references and applications to obtain other embodiments of the present invention. In addition, due to biological function equivalence considerations, some changes can be made to the protein structure without affecting the type or amount of biological or chemical effects. These and other changes can be made to the present invention in view of the embodiments. All such modifications are intended to be included in the scope of the appended claims.

[0031] Unless otherwise indicated, any nucleic acid sequence is written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxyl orientation, respectively.

[0032] The headings provided herein are not limitations of the various aspects or embodiments of the invention.

[0033] As used herein, when used with a verb, the term "capable of" encompasses or means the action of the corresponding verb. For example, "capable of interacting" also means interacting, "capable of cleaving" also means cleaving, "capable of binding" also means binding, and "capable of specifically targeting..." also means specifically targeting.

[0034] Numerical ranges include the numbers that define the range. Where a range of values ​​is provided, it will be understood that unless the context clearly dictates otherwise, each intervening value between the upper and lower limits of the range is also specifically disclosed, accurate to the tenths of the unit of the lower limit. The present invention encompasses each smaller range between any stated value or intervening value in the stated range and any other stated value or intervening value in the stated range. The upper and lower limits of these smaller ranges may be independently included or excluded within the range, and each range where any limit, no limit, or both limits are included in the smaller range is also encompassed within the present invention, subject to any specifically excluded limits in the stated range. Where the range includes one or both of the limits, ranges that do not include either or both of those included limits are also encompassed within the present invention.

[0035] Amino acids are referred to herein using the amino acid name, three-letter abbreviation, or one-letter abbreviation.

[0036] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to refer to a series of amino acid residues connected to each other by peptide bonds between the α-amino and carboxyl groups of adjacent residues. The terms "protein" and "polypeptide" refer to polymers of amino acids (including modified amino acids (e.g., phosphorylated, glycosylated, glycosylated, etc.) and amino acid analogs), regardless of their size or function. "Protein" and "polypeptide" are typically used in relation to relatively large polypeptides, while the term "peptide" is typically used in relation to small polypeptides, but the usage of these terms overlaps in this technology. When referring to gene products and fragments thereof, the terms "protein" and "polypeptide" are used interchangeably herein. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs of the foregoing. In the present invention, conventional single-letter and three-letter codes for amino acid residues may be used. The 3-letter code for amino acids is as defined by the IUPAC IUB Joint Commission on Biochemical Nomenclature (JCBN). It is also understood that due to the degeneracy of the genetic code, a polypeptide may be encoded by more than one nucleotide sequence.

[0037] Slight variations of the amino acid sequences of the present invention are considered to be encompassed by the present invention, provided that the variations in the amino acid sequence 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 sequences of the present invention or fragments thereof as defined anywhere herein. The term homology is used herein to mean identity. Thus, variants or analogs of the amino acid sequences of the present invention may differ in sequence based on substitutions (usually conservative substitutions), deletions, or insertions. Proteins comprising such variations are referred to herein as variants.

[0038] The proteins of the present invention may include variants in which amino acid residues from one species are substituted with corresponding residues in another species at conserved or non-conserved positions. Variants of the protein molecules disclosed herein can be generated and used in the present invention. Following the application of multivariate data analysis techniques in computational chemistry to structure / property-activity relationships [see, e.g., 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 for proteins can be derived using well-known mathematical techniques, such as statistical regression, graphical recognition, and classification [see, e.g., Norman et al. Applied Regression Analysis. Wiley-Interscience; 3rd ed. (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 ofMultivariate Analysis: A User's Perspective (Oxford Statistical Science Series, Issue 22 (Paper)). Oxford University Press; (December 2000), ISBN: 0198507089; Witten, Ian H. et al. Data Mining: Practical Machine Learning Tools andTechniques with Java Implementations. Morgan Kaufmann; (October 11, 1999), ISBN: 1558605525; Denison David GT(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 a protein can be inferred from empirical and theoretical models of protein sequence, function, and three-dimensional structure (e.g., analysis of possible contact residues or calculated physicochemical properties), and these properties can be considered individually or in combination.

[0039] The amino acid residues at non-conserved positions can be substituted with conservative or non-conservative residues. In particular, conservative amino acid substitutions are contemplated.

[0040] A "conservative amino acid substitution" is a substitution in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include 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), non-polar 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, an amino acid substitution is considered conservative if an amino acid in a polypeptide is replaced with another amino acid from the same side chain family. The inclusion of conservatively modified variants in the proteins of the present invention does not exclude other forms of variants, such as polymorphic variants, interspecies homologs, and alleles.

[0041] "Non-conservative amino acid substitutions" include substitutions in which (i) a residue with a positive side chain (e.g., Arg, His, or Lys) replaces or is replaced by a negative residue (e.g., Glu or Asp); (ii) a hydrophilic residue (e.g., Ser or Thr) replaces or is replaced by a hydrophobic residue (e.g., Ala, Leu, Ile, Phe, or Val); (iii) cysteine ​​or proline replaces or is replaced by any other residue; or (iv) a residue with a bulky, hydrophobic or aromatic side chain (e.g., Val, His, Ile, or Trp) replaces or is replaced by a residue with a smaller side chain (e.g., Ala or Ser) or a residue without a side chain (e.g., Gly).

[0042] "Insertion" or "deletion" is usually within the range of about 1, 2 or 3 amino acids. The allowed variation can be determined experimentally by systematically introducing amino acid insertions or deletions into the protein using recombinant DNA technology and analyzing the activity of the resulting recombinant variants. For those skilled in the art, this requires no more than routine experimentation.

[0043] A "fragment" of a polypeptide typically contains 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.

[0044] As used herein, the terms "polynucleotide," "nucleic acid," and "nucleic acid sequence" refer to any molecule, preferably a polymeric molecule, that incorporates units of ribonucleic acid, deoxyribonucleic acid, or their analogs. Nucleic acids can be single-stranded or double-stranded. A single-stranded nucleic acid can be one strand of denatured double-stranded DNA. Alternatively, it can be a single-stranded nucleic acid that is 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" can also be used interchangeably and both terms encompass fragments or variants thereof that encode a protein of interest.

[0045] The transgenes of the present invention include nucleic acid sequences removed from their naturally occurring environment, recombinant or cloned DNA isolates, and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.

[0046] The polynucleotides of the present invention can be prepared by any means known in the art. For example, large quantities of polynucleotides can be produced by replication in suitable host cells. Natural or synthetic DNA fragments encoding the desired fragments will be incorporated into recombinant nucleic acid constructs, typically DNA constructs, that can be introduced into prokaryotic or eukaryotic cells and replicated therein. Typically, the DNA constructs will be suitable for autonomous replication in unicellular hosts such as yeast or bacteria, but may also be intended for introduction into and integration into the genome of cultured insect, mammalian, plant or other eukaryotic cell lines.

[0047] The polynucleotides of the present invention can also be produced by chemical synthesis, for example, by the phosphoamidite method or the triester method, and can be performed on commercial automated oligonucleotide synthesizers. Double-stranded fragments can be obtained from chemically synthesized single-stranded products by synthesizing complementary strands and ligating the strands together under appropriate conditions, or by adding complementary strands using a DNA polymerase in the presence of appropriate primer sequences.

[0048] When applied to nucleic acid sequences, in the context of the present invention, the term "isolated" means that the polynucleotide sequence has been removed from its natural genetic environment and is therefore free of other extraneous or undesired 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 in genetically engineered protein production systems. Such isolated molecules are molecules separated from their natural environment.

[0049] Given the degeneracy of the genetic code, considerable sequence variation is possible in the polynucleotides of the present invention. Degenerate codons encompassing all possible codons for a given amino acid are set forth below: amino acids codon Degenerate codons Cys TGC TGT TGY Ser AGC AGT TCA TCC TCG TCT WSN Thr ACA ACC ACG ACT ACN Pro CCA CCC CCG CCT CCN Ala GCA GCC GCG GCT GCN Gly GGA GGC GGG GGT GGN Asn AAC AAT AAY Asp GAC GAT GAY Glu GAA GAG GAR Gln CAA CAG CAR His CAC CAT CAY Arg AGA AGG CGA CGC CGG CGT MGN Lys AAA AAG AAR Met ATG ATG Ile ATA ATC ATT ATH Leu CTA CTC CTG CTT TTA TTG YTN Val GTA GTC GTG GTT GTN Phe TTC TTT TTY Tyr TAC TAT TAY Trp TGG TGG Ter TAA TAG TGA TRR Asn / Asp RAY Glu / Gln SAR Any NNN One of ordinary skill in the art will appreciate that there is flexibility in determining degenerate codons, which represent all possible codons encoding each amino acid. For example, some polynucleotides encompassed by a degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can readily identify such variant sequences by reference to the amino acid sequences of the present invention.

[0050] 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 a fragment thereof is "substantially homologous" (or "substantially identical") to a reference sequence if, when optimally aligned (with appropriate nucleotide insertions or deletions) with another 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 determining homology of nucleic acid sequences are known in the art.

[0051] Alternatively, a "variant" nucleic acid sequence is substantially homologous (or substantially identical) to a reference sequence (or fragment thereof) if the "variant" and the reference sequence are capable of hybridizing under stringent (e.g., highly stringent) hybridization conditions. As will be readily appreciated by those skilled in the art, nucleic acid sequence hybridization will also be affected by conditions such as salt concentration (e.g., NaCl), temperature, or organic solvent, in addition to base composition, the length of complementary strands, and the number of nucleotide base mismatches between hybrid nucleic acids. Stringent temperature conditions are preferably employed and generally include temperatures exceeding 30° C., typically exceeding 37° C., and preferably exceeding 45° C. Stringent salt conditions will typically 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.

[0052] Methods for determining percent identity of nucleic acid sequences are known in the art. For example, when assessing nucleic acid sequence identity, a sequence having a determined number of consecutive nucleotides can be compared with a nucleic acid sequence from the corresponding portion of the nucleic acid sequence of the present invention (having the same number of consecutive nucleotides). Tools known in the art for determining percent identity of nucleic acid sequences include Nucleotide BLAST (described below).

[0053] It will be understood by those skilled in the art that different species exhibit "preferred codon usage." As used herein, the term "preferred codon usage" refers to the codons most frequently used in the cells of a species, thereby favoring one or more representatives of the possible codons encoding each amino acid. For example, the amino acid threonine (Thr) can be encoded by ACA, ACC, ACG, or ACT, but ACC is the most commonly used codon in mammalian host cells; in other species, different codons may be preferred. Preferred 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. Introducing a preferred codon sequence into recombinant DNA can enhance protein production, for example, by making protein translation more efficient in a particular cell type or species. Thus, according to the present invention, any nucleic acid sequence, in addition to the gag-pol gene, can be codon-optimized for expression in a host or target cell. Specifically, the vector gene (or corresponding plasmid), REV gene (or corresponding plasmid), fusion protein (F) gene (or corresponding plasmid) and / or hemagglutinin-neuraminidase (HN) gene (or corresponding plasmid), or any combination thereof can be codon-optimized.

[0054] A "fragment" of a polynucleotide of interest comprises a series of contiguous nucleotides from the sequence of the full-length polynucleotide. For example, a "fragment" of a polynucleotide of interest may comprise (or consist of): at least 30 contiguous nucleotides from the sequence of the 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 contiguous nucleic acid residues of the polynucleotide). A fragment may comprise at least one antigenic determinant of a corresponding polypeptide of interest and / or may encode at least one antigenic epitope of a corresponding polypeptide of interest. Typically, a fragment as defined herein retains the same function as a full-length polynucleotide.

[0055] The terms "increased" or "increase," "enhance," or "activate" are all used herein to mean an increase by a statistically significant amount. The terms "increase," "enhance," or "activate" can mean an increase of at least 10% compared to a reference amount, such as 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% and 100% compared to a reference amount, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 10-fold, or any increase between 2-fold and 10-fold or greater compared to a reference amount. In the context of yield or titer, an "increase" is an observable or statistically significant increase in such levels.

[0056] The terms "decrease," "reduced," "reduction," or "inhibit" are all used herein to mean a reduction by a statistically significant amount. The terms "reduce" or "reduce" or "inhibit" generally mean a reduction of at least 10% compared to a reference amount (e.g., in the absence of a given treatment), and can include, for example, a reduction of 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, "reduce" or "inhibit" encompasses complete inhibition or reduction compared to a reference amount. "Complete inhibition" is 100% inhibition (i.e., elimination) compared to a reference amount.

[0057] It must be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references 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 known to those skilled in the art and equivalents thereof, and so forth. Furthermore, the use of the term "including," as well as other forms such as "includes" and "included," is not limiting.

[0058] "About" generally means an acceptable degree of error for the amount being measured, given the nature or precision of the value being measured. Exemplary degrees of error are within 20 percent (%) of a specified value or range of values, typically within 10%, and more typically within 5%. Preferably, the term "about" is understood herein to mean plus or minus (±) 5%, preferably ±4%, ±3%, ±2%, ±1%, ±0.5%, ±0.1%, of the numerical value with which it is used.

[0059] The term "consisting of refers to compositions, methods, and individual components thereof as described herein, to the exclusion of any element of the invention not enumerated in that description.

[0060] As used herein, the term "consisting essentially of" refers to those elements required for a given invention. The term allows for the presence of elements that do not materially affect the basic and novel or functional characteristics of the invention (i.e., inactive or non-immunogenic ingredients).

[0061] Embodiments described herein as “comprising” one or more features may also be considered disclosures of corresponding embodiments “consisting of” and / or “consisting essentially of” such features.

[0062] Concentrations, amounts, volumes, percentages and other numerical values ​​may be presented herein in a range format. It should also be understood that such range format is used only for convenience and brevity purposes and should be interpreted in a flexible manner to include not only the values ​​explicitly stated as the limits of the range, but also all individual values ​​or sub-ranges encompassed within the range, as if each value and sub-range were explicitly stated.

[0063] As used herein, unless otherwise stated, the terms "vector," "retroviral vector," and "retroviral F / HN vector" are used interchangeably to refer to a retroviral vector comprising a retroviral RNA sequence and pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxovirus. Unless otherwise stated, the terms "lentiviral vector" and "lentiviral F / HN vector" are used interchangeably to refer to a lentiviral vector pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxovirus. All disclosures herein regarding the retroviral vectors of the present invention apply equally and unreservedly to the lentiviral vectors of the present invention, and to SIV vectors pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxovirus (also referred to herein as SIV F / HN or SIV-FHN).

[0064] As defined herein, the term "retroviral RNA sequence" refers to a nucleic acid molecule contained within a retroviral vector. The retroviral RNA sequence comprises a long terminal repeat (LTR) component, a nucleic acid sequence required for incorporating the retroviral RNA sequence into a retroviral particle, and a transgenic gene expression cassette. The transgenic gene expression cassette comprises a suitable enhancer / promoter component, a transgenic gene cDNA, and a post-transcriptional regulatory component. The retroviral RNA sequence begins with a 5' LTR R sequence and ends with a 3' LTR R sequence. The 5' region retroviral RNA sequence typically comprises a retroviral LTR R sequence, followed by a retroviral LTR U5 sequence (in 5' to 3' order), or consists of such sequences. The 3' region retroviral RNA sequence typically comprises a retroviral LTR R sequence, followed by a retroviral LTR U5 sequence (in 5' to 3' order), or consists of such sequences.

[0065] The terms "DNA provirus," "DNA provirus sequence," and "DNA proviral sequence" interchangeably refer to a DNA sequence that is integrated into the genome of a cell transduced with a retrovirus. The DNA proviral sequence contains additional nucleic acid regions not found in the retroviral RNA sequence, including a 5' LTR U3 sequence and a 3' LTR U5 sequence. Thus, the sequence of the DNA provirus and the retroviral RNA sequence are not identical, but rather the retroviral RNA sequence is shorter than the proviral DNA sequence from which it is derived. In contrast to the proviral DNA sequence from which the retroviral RNA sequence is derived, the precise 5' and 3' limits of the retroviral RNA sequence cannot be easily and reliably determined by simple analysis of the proviral DNA sequence.

[0066] The terms "individual," "subject," and "patient" are used interchangeably herein to refer to a mammalian individual for whom diagnosis, prognosis, disease monitoring, treatment, therapy, and / or therapy optimization is desired. The mammal can be, but is not limited to, a human, non-human primate, mouse, rat, dog, cat, horse, or cow. In preferred embodiments, the individual, subject, or patient is a human. An "individual" can be an adult, adolescent, or infant. An "individual" can be male or female.

[0067] A "subject" in "need of" treatment for a particular condition can be one who has the condition, has been diagnosed with the condition, or is at risk of developing the condition.

[0068] The subject may 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 associated with such a condition, and optionally has been treated for the condition as defined herein or one or more complications associated with the condition. Alternatively, the subject may be one who has not been previously diagnosed with a condition as defined herein or one or more complications associated with the condition. For example, the subject may be one who exhibits one or more risk factors for the condition or one or more complications associated with the condition, or one who does not exhibit risk factors.

[0069] 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 yet displayed any symptoms of a disease, have not yet been diagnosed with a disease, and / or are unlikely to develop a disease (e.g., cystic fibrosis (CF) or any other disease described herein). Preferably, the healthy individual(s) are not taking medications that affect CF and have not been diagnosed with any other disease. One or more healthy individuals may be of similar sex, age, and / or body mass index (BMI) as compared to the test individual. Standard statistical methods used in application medicine allow for the determination of normal expression levels in healthy individuals, as well as significant deviations from such normal levels.

[0070] Herein, the terms "control" and "reference population" are used interchangeably.

[0071] As used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the US Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeia.

[0072] Other definitions of terms may be found throughout the specification. Before describing exemplary embodiments in greater detail, it should be understood that the present invention is not limited to the specific embodiments described and may vary accordingly. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting, as the scope of the present invention will be defined solely by the appended claims.

[0073] The publications discussed herein are provided solely as of the filing date of the present application. Nothing herein should be construed as an admission that such publications constitute prior art for the purposes of the claims appended hereto. All references cited in this specification are hereby incorporated by reference for their entire disclosures and for disclosures specifically mentioned in this specification.

[0074] It is intended that disclosure relating to various methods of the invention be equally applicable to other methods, therapeutic uses or approaches, and vice versa.

[0075] Retroviral and lentiviral vectors The present invention relates to combination therapies comprising retroviral / lentiviral (e.g., SIV) constructs. The term "retrovirus" refers to any member of the Retroviridae family of RNA viruses that encodes 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 leukemia virus (MLV) and feline leukemia 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 anemia virus (EIAV), and Visna / maedi virus. Generally, the present invention relates to combination therapies comprising lentiviral vectors, and in particular, SIV vectors (including all strains and subtypes), such as SIV-AGM (originally isolated from African green monkeys, also known as safari monkeys ( Cercopithecus aethiops )separate) combination therapy.

[0076] The retroviral / lentiviral (e.g., SIV) vectors of the present invention are pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxovirus. Preferably, the respiratory paramyxovirus is Sendai virus (murine parainfluenza virus type 1).

[0077] The F protein may be a truncated F protein, typically an F protein in which the cytoplasmic domain is truncated. Preferably, the truncated F protein is Fct4, ​​wherein 38 amino acids have been removed from the C-terminus of the F protein, wherein 4 amino acids of the F protein cytoplasmic domain are 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 with 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 with SEQ ID NO: 17 or 18.

[0078] The full-length F protein or its C-terminal truncated form (e.g., Fct4) is generally fusion-inactive. The fusion-inactive form of the F protein can be cleaved to produce two subunits: a first subunit (also referred to as F2) and a second subunit (also referred to as F1).

[0079] The first subunit of the F protein may comprise or consist of an amino acid sequence that is 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% identical to SEQ ID NO: 19. Preferably, the first subunit may be a subunit that may comprise or consist of an amino acid sequence that is at least 90%, at least 95% or at least 99% identical to SEQ ID NO: 19. SEQ ID NO: 19 is the first subunit of Fct4.

[0080] Alternatively or additionally, preferably additionally, 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 that may comprise or consist 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.

[0081] The F protein (e.g., Fct4) may include an N-terminal signal peptide. Alternatively, the F protein may lack such a signal peptide. The F protein signal peptide may comprise 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 or consist of the amino acid sequence. This signal peptide can be cleaved to form a 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 can comprise, or consist of, an amino acid sequence that is 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% identical to amino acid residues 26-527 of SEQ ID NO: 18.

[0082] The HN protein can be a truncated and / or chimeric HN protein, typically an HN protein in which the cytoplasmic domain is truncated or replaced. Preferably, the HN protein is a chimeric HN protein in which (i) the cytoplasmic domain of HN is replaced by the cytoplasmic domain of a transmembrane (TMP) protein; or (ii) the cytoplasmic domain of a TMP is added to the cytoplasmic domain of the HN protein. The HN protein can be as described in Kobayashi et al. (J. Virol. (2003) 77(4):2607-2614), which is incorporated herein by reference in its entirety.

[0083] The retroviral / lentiviral (e.g., SIV) vectors of the present invention may comprise a codon-optimized Gag protein, a codon-optimized Pol protein, a codon-optimized GagPol polyprotein, or a combination thereof. Thus, the present invention provides retroviral / lentiviral (e.g., SIV) vectors comprising a codon-optimized 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 present invention provides retroviral vectors comprising a codon-optimized 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 present invention provides a retroviral vector comprising a codon-optimized 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 present invention provides a retroviral vector comprising a codon-optimized Pol protein comprising or consisting of an amino acid sequence having at least 90%, at least 95% or at least 99% sequence identity to SEQ ID NO: 30.

[0084] GagPol appears as a polymeric protein that is processed to produce several smaller proteins within the virion.The extent of processing of GagPol or any of the constituent proteins within the retroviral / lentiviral (eg SIV) vectors of the invention, and therefore its presence and / or concentration, may vary over time.

[0085] Thus, the retroviral / lentiviral (e.g., SIV) vectors of the present invention may comprise one or more of p17 protein, p27 protein, p8 protein, protease, p51 protein, p15 protein, and p31 protein. One or more of these proteins may be present in combination with Gag, Pol, and / or GagPol. Preferably, the present invention provides a retroviral vector comprising p17 protein, p27 protein, p8 protein, protease, p51 protein, p15 protein, and p31 protein. Furthermore, these proteins may be present in combination with Gag, Pol, and / or GagPol.

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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.

[0092] 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.

[0093] The retroviral / lentiviral (e.g., SIV) vector of the present 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 (described above), a p24 protein comprising or consisting of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 23 (described above), a p8 protein comprising or consisting of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 24 (described above), a protease comprising or consisting of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 25 (described above), a p51 protein comprising or consisting of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 26 (described above), a p15 protein comprising or consisting of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 27 (described above), and a p24 protein comprising or consisting of an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 28 (described above). 28. A p31 protein (as described above) having an amino acid sequence having at least 90% sequence identity or consisting of said amino acid sequence.

[0094] The retroviral / lentiviral (eg SIV) vectors produced according to the present invention may be integrase competent (IC). Alternatively, the lentiviral (eg SIV) vectors may be integrase deficient (ID).

[0095] Retroviral / lentiviral vectors, such as those used in the combination therapy according to the present invention, can integrate into the genome of the transduced cells and cause long-lasting expression, making them suitable for transducing stem / precursor 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 cell types include basal cells and submucosal duct cells in the upper airways; ciliated, goblet, and club cells (SCGB1A1+) and neuroendocrine cells in the bronchiolar airways; bronchoalveolar stem cells in the terminal bronchioles; and type II pneumocytes in the alveoli. Therefore, and without being bound by theory, it is believed that these retroviral / lentiviral (e.g., SIV) vectors cause long-term gene expression of the relevant transgene by introducing the transgene into one or more airway epithelial cell types (such as those listed above). It is predicted that cells of the airway epithelium have a lifespan of many months, so that transfection of these cells promotes expression that persists for the lifespan of the cells, thereby having a long-term therapeutic effect.

[0096] Thus, the retroviral / lentiviral (e.g., SIV) vectors used in the combination therapy according to the present 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 the prolonged performance results from the long lifespan of the transduced cells. For example, the retroviral / lentiviral (e.g., SIV) vectors may transduce one or more cell types selected from the group consisting of: (i) basal cells and / or submucosal duct cells in the upper airways; (ii) ciliated, goblet, club cells (SCGB1A1+), and / or neuroendocrine cells in the bronchiolar airways; (iii) bronchoalveolar stem cells in the terminal bronchioles; and / or (iv) type II pneumocytes in the alveoli; or any combination thereof.

[0097] Alternatively or in addition, the retroviral / lentiviral (e.g., SIV) vectors used in the combination therapy according to the present invention can transduce one or more cell types or cell lineages in the lungs (including the airways and respiratory tract) with regenerative potential to achieve long-term gene expression. For example, retroviral / lentiviral (e.g., SIV) vectors can transduce basal cells, such as basal cells in the upper airways / respiratory tracts. Basal cells play a central role in epithelial maintenance and repair after injury. In addition, basal cells are widely distributed along the human respiratory epithelium, with a relative distribution range of 30% (larger airways) to 6% (smaller airways).

[0098] Retroviral / lentiviral (e.g., SIV) vectors can be used to transduce isolated and expanded stem / precursor cells ex vivo and subsequently administered to a patient as part of a combination therapy as described herein. Preferably, retroviral / lentiviral (e.g., SIV) vectors are used to transduce cells in the lung (or airway / respiratory tract) in vivo.

[0099] The retroviral / lentiviral (e.g., SIV) vectors of the present invention exhibit remarkable resistance to shear forces, with only a moderate reduction in transduction capacity when delivered via clinically relevant delivery devices such as spray bottles and nebulizers. Other inhalation administration routes, such as via bronchoscope, may similarly benefit from the shear force resistance of the retroviral / lentiviral (e.g., SIV) vectors of the present invention.

[0100] The retroviral / lentiviral (e.g., SIV) vector of the present invention may comprise one or more transgenic genes encoding polypeptides or proteins that are therapeutic for the treatment of CF. Preferably, the retroviral / lentiviral (e.g., SIV) vector of the present invention comprises CFTR The transgenic gene encodes CFTR.

[0101] The transgenic genes included in the vectors of the present invention can be modified to promote expression. For example, the transgenic gene sequence can be CpG-depleted (or CpG-free) and / or codon-optimized to promote gene expression. Standard techniques for modifying transgenic gene sequences in this manner are known in the art.

[0102] therefore, CFTR An example of a cDNA is provided by SEQ ID NO: 1. Variants thereof (as described herein) are also included, particularly variants having 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 cDNA previously designed by the present inventors to enhance translation in human cells. CFTR The present invention also covers the transgenic gene compared to the unmodified (wild type) CFTR Variants of a gene sequence having the same sequence (as defined herein) have the same technical effect of enhancing translation.

[0103] The retroviral / lentiviral (e.g., SIV) vectors of the present invention are capable of achieving high transgene expression, resulting in high expression (therapeutic amount) of therapeutic proteins. Therefore, when administered to a patient, the retroviral / lentiviral (e.g., SIV) vectors of the present invention can effectively provide high transgene expression. The terms high expression and therapeutic expression are used interchangeably herein. Expression can be measured by any appropriate method (qualitative or quantitative, preferably quantitative), and the concentration is given in any appropriate measurement unit, such as ng / ml or nM.

[0104] The expression of the relevant transgenic gene can be given relative to the expression of the corresponding endogenous (defective) gene in the patient. Expression can be measured based on DNA vector copy number (VCN), mRNA or protein expression. The expression of the transgenic gene of the present invention (such as a functional CFTR gene) can be quantified relative to the endogenous gene, such as the endogenous (dysfunctional) CFTR gene based on the number of mRNA copies per cell or any other suitable unit.

[0105] The expression level of the CFTR transgene of the present invention and / or the encoded CFTR protein can be measured in lung tissue. Thus, "high level" and / or "therapeutic expression level" can refer to concentrations in the lung, epithelial lining fluid, and / or serum / plasma.

[0106] The retroviral / lentiviral (e.g., SIV) vectors of the present invention exhibit efficient airway cell uptake, stable transgene expression, and do not suffer from efficacy loss after repeated administration. Thus, the retroviral / lentiviral (e.g., SIV) vectors of the present invention are capable of producing sustained, reproducible, high-level expression in airway cells without inducing an inappropriate immune response. An inappropriate immune response can be defined as an immune response that is sufficiently extreme to prevent administration to a patient and / or causes a significant negative impact on vector transduction and / or CFTR expression.

[0107] The retroviral / lentiviral (e.g., SIV) vectors of the present invention are capable of achieving long-term transgene expression, resulting in long-term expression of therapeutic proteins. As described herein, the phrases "long-term expression," "sustained expression," "persistent expression," and "sustained expression" are used interchangeably. Long-term expression according to the present invention means expression of the therapeutic gene and / or protein, preferably in therapeutic amounts, for at least 45 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 longer. 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 longer, more preferably at least 360 days, at least 450 days, at least 720 days, or longer. This long-term expression can be achieved by repeated doses or by a single dose.

[0108] Repeated doses can be administered twice a day, daily, twice a week, weekly, monthly, every two months, every three months, every four months, every six months, yearly, every two years, or longer. Administration can continue for a desired period of time, 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 longer, up to the lifetime of the patient to be treated.

[0109] Retroviral / lentiviral (e.g., SIV) vectors contain 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 of the hCEF promoter and the exon corresponding to nucleotides 728-733 of the hCEF promoter. A preferred example of an hCEF promoter sequence of the present invention is provided by SEQ ID NO: 2. Thus, the hCEF promoter contained in a retroviral / lentiviral (e.g., SIV) vector of the present 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 another embodiment, 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 the human elongation factor 1a (EF1a) promoter. An example of an 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 present invention is determined using conventional techniques known in the art. Non-limiting examples of other promoters include UbC and UCOE. As described herein, promoters may be modified to further regulate the expression of the transgenes of the present invention.

[0110] The promoters included in the retroviral / lentiviral (e.g., SIV) vectors of the present invention may be specifically selected and / or modified to further optimize the regulation of the expression of the therapeutic gene. Similarly, suitable promoters and standard techniques for their modification are known in the art. As non-limiting examples, a variety 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 incorporated herein by reference in its entirety. Preferably, the retroviral / lentiviral vectors of the present invention (especially SIV F / HN vectors) comprise an hCEF promoter with low or no CpG dinucleotide content. The hCEF promoter may have all CG dinucleotides replaced with any 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 present invention is provided by SEQ ID NO: 2. The lack of CpG dinucleotides further improves the efficacy of the retroviral / lentiviral (e.g., SIV) vectors of the present invention, particularly when induction of an immune response to the expressed antigen or an inflammatory response to the delivered expression construct is not desired. Elimination of CpG dinucleotides reduces the occurrence of flu-like symptoms and inflammation that may result from administration of the construct, particularly when administered to the airways.

[0111] The retroviral / lentiviral (e.g., SIV) vectors of the present invention can be modified to allow for the cessation of gene expression. Standard techniques for modifying vectors in this manner are known in the art. As a non-limiting example, Tet-responsive promoters are widely used.

[0112] Therefore, the present invention relates to F / HN retroviral / lentiviral vectors, particularly SIV.F / HN vectors, comprising a promoter and a transgene. F / HN pseudotyping is particularly effective in targeting cells in the airway epithelium, and thus, for therapeutic applications, delivery is typically to cells of the respiratory tract, including those of the airway epithelium. Therefore, the retroviral / lentiviral (e.g., SIV) vectors of the present invention are particularly suitable for the treatment of CF.

[0113] The retroviral / lentiviral (e.g., SIV) vectors of the present invention may not have introns between the promoter and the transgenic gene. Similarly, the vector genome (pDNA1) plasmid (e.g., pGM326 as described herein, Figure 1A There may be no introns between the promoter in (depicted in FIG and having the sequence of SEQ ID NO: 3) and the transgenic gene.

[0114] Preferably, the retroviral / lentiviral (e.g., SIV) vector comprises the hCEF promoter and the CFTR transgene, including those described herein. Alternatively, the retroviral / lentiviral (e.g., SIV) vector may not have introns located between the promoter and the transgene. Such retroviral / lentiviral (e.g., SIV) vectors can be produced by the methods described herein using plasmids carrying the CFTR transgene and promoter. Particularly preferred are SIV.F / HN vectors having the hCEF promoter and the CFTR transgene, including those described herein.

[0115] The retroviral / lentiviral (e.g., SIV) vector 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 the at least one transgene comprises CFTR or by CFTR composition.

[0116] For example, encoding CFTR The nucleic acid sequences comprise CFTR A nucleic acid sequence having (or consisting of) at least 90% (such as at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity, examples of which are described herein. CFTR The nucleic acid sequences comprise CFTR The nucleic acid sequence has (or consists of) a nucleic acid sequence having at least 95% (such as at least 95%, 96%, 97%, 98%, 99% or 100%) sequence identity, examples of which are described herein. In one embodiment, the nucleic acid sequence encoding CFTR is provided by SEQ ID NO: 1 or a variant thereof.

[0117] Depend on CFTR The amino acid sequence of CFTR encoded by the transgene may comprise (or consist of) an amino acid sequence that has at least 95% (such as at least 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to a functional CFTR polypeptide sequence, respectively.

[0118] The retroviral / lentiviral (eg, SIV) vectors of the present invention may comprise a central polypurine tract (cPPT) and / or a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). An exemplary WPRE sequence is provided by SEQ ID NO: 14.

[0119] Retroviral / lentiviral (e.g., SIV) vectors according to the present invention may be 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 incorporated herein by reference in its entirety. Particularly preferred are retroviral / lentiviral (e.g., SIV) vectors according to UK Patent Application No. 2212472.1.

[0120] Therefore, particularly preferred are retroviral / lentiviral (e.g., SIV) vectors that are pseudotyped with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) proteins, wherein: (a) the vector comprises a modified retroviral RNA sequence comprising or consisting of the nucleic acid sequence of SEQ ID NO: 16 (the vector comprises a CFTR transgene), preferably wherein the modified retroviral RNA sequence consists of the nucleic acid sequence of SEQ ID NO: 16; and (b) the F protein comprises a first subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 19 and a second subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 20. The vector may further comprise one or more of the following: (a) p17 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 22; (b) p24 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 23; (c) p8 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 24; (d) protease comprising or consisting of the amino acid sequence of SEQ ID NO: 25; (e) p51 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 26; (f) p15 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 27; (g) p31 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 28; (h) Gag protein comprising or consisting of the amino acid sequence of SEQ ID NO: 29; and / or (i) a protein comprising or consisting of the amino acid sequence of SEQ ID NO: 30 or a Pol protein consisting of the amino acid sequence; wherein optionally, the vector comprises each of (a) to (g).

[0121] Methods for preparing retroviral / lentiviral (eg, SIV) vectors The retroviral / lentiviral (e.g., SIV) vectors of the present invention may be produced by any suitable 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 incorporated herein by reference in its entirety. Particularly preferred is the method described in UK Patent Application No. 2212472.1.

[0122] The retroviral / lentiviral (e.g., SIV) vectors of the present invention are typically produced by scalable GMP-compatible methods. 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 of the methods described herein.

[0123] Production of retroviral / lentiviral (e.g., SIV) vectors typically utilizes one or more plasmids that provide the components necessary for vector production: the retroviral / lentiviral vector genome, Gag-Pol, Rev, F, and HN. Multiple components can be provided on a single plasmid. Preferably, each component is provided on a separate plasmid, resulting in the use of five plasmids, each containing one of the vector genome, Gag-Pol, Rev, F, and HN.

[0124] Alternatively, a single plasmid can provide the Gag-Pol and Rev components and can be referred to as the packaging plasmid (pDNA2). The remaining components (genome, F, and HN) can be provided by separate plasmids (pDNA1, pDNA3a, and pDNA3b, respectively), allowing four plasmids to be used to produce retroviral / lentiviral (e.g., SIV) vectors according to the present invention. In the four-plasmid approach, pDNA1, pDNA3a, and pDNA3b can be as described herein in the context of the five-plasmid approach.

[0125] Any of the plasmids used to produce the retroviral / lentiviral (e.g., SIV) vectors of the present invention may be independently codon optimized or at least partially codon optimized. Partial codon optimization 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 optimization. Codon optimization is a technique for maximizing protein expression by improving the translation efficiency of coding genes. Translation efficiency is improved by modification of nucleic acid sequences. Codon optimization is conventional in the art, and designing a codon-optimized form of a given nucleic acid sequence is within the conventional practice of those generally skilled in the art. As described herein, the transgenic gene and / or promoter may be each independently codon optimized. Alternatively or in addition, the Gag-Pol gene may be codon optimized. In particular, codon optimization of the Gag-Pol gene is preferred because it can improve the safety profile of the resulting retroviral / lentiviral (e.g., SIV) vectors, especially SIV.F / HN vectors, without adversely affecting vector titer, and can even increase vector titer (as described in International Application No. PCT / GB2022 / 050524, which claims priority from UK Patent Application No. 2102832.9).

[0126] Thus, the retroviral / lentiviral (e.g., SIV) vectors of the present invention, particularly those pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxovirus and comprising a promoter and a transgene, are produced by a method comprising the use of a codon-optimized gag-pol gene. Preferably, the codon-optimized gag-pol gene used in the production methods of the present invention is the SIV gag-pol gene. An exemplary wild-type SIV gag-pol gene that can be modified to produce a codon-optimized gag-pol gene is set forth in SEQ ID NO: 5. An exemplary codon-optimized gag-pol gene derived from SEQ ID NO: 5 is set forth in SEQ ID NO: 6. In addition to codon optimization, the codon-optimized gag-pol gene used in the production methods of the present invention may comprise other modifications, such as translational slippage (which allows translation to slip from one region to another to allow production of both Gag and Pol). Any suitable changes in codon usage can be used in the codon-optimized gag-pol genes of the present invention, provided that (i) the homology between the vector gene plastid and the GagPol plastid is reduced to minimize the risk of RCL production, and (ii) after codon optimization, sufficient GagPol is produced without including the RRE (which further reduces homology and the risk of RCL production).

[0127] The codon-optimized gag-pol gene used in the production method of the present invention can be fully (100%) or partially codon-optimized. Partial codon optimization of the gag-pol gene 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 optimization.

[0128] Preferably, the gag-pol gene itself is fully codon-optimized, but may include non-codon-optimized regions (e.g., between the gag gene and the pol gene). As a non-limiting example, to maintain translational slippage of the reading frame between the gag gene and the pol gene, the region surrounding the translational slippery sequence may not be codon-optimized (e.g., where accurate translation of the slippery sequence is important for this function). A non-codon-optimized translational slippery sequence within the codon-optimized gag-pol gene is exemplified in SEQ ID NO: 6.

[0129] Preferably, the codon-optimized gag-pol gene used to generate the retrovirus / lentivirus (e.g., SIV) of the present invention comprises or consists of the nucleic acid sequence of SEQ ID NO: 6 or a variant thereof (as defined herein). In particular, the codon-optimized gag-pol gene 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 greater sequence identity to SEQ ID NO: 6. Preferably, the codon-optimized gag-pol gene used in the method of the present invention comprises or consists of a nucleic acid sequence having at least 90%, more preferably at least 95%, even more preferably at least 98% or greater sequence identity to SEQ ID NO: 6. The codon-optimized gag-pol gene of SEQ ID NO: 6 comprises a translational slip and therefore does not form a single conventional open reading frame.

[0130] Preferably, the codon-optimized gag-pol gene used in the method of the present invention is contained in a plastid comprising or consisting of a nucleic acid sequence of SEQ ID NO: 7 (pGM691) or a variant thereof (as defined herein). In particular, the codon-optimized gag-pol gene is contained in a plastid comprising or consisting 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 greater sequence identity to SEQ ID NO: 7. Preferably, the codon-optimized gag-pol gene is contained in a plastid comprising or consisting of a nucleic acid sequence having at least 90%, more preferably at least 95%, even more preferably at least 98% or greater sequence identity to SEQ ID NO: 7. In the plasmid of SEQ ID NO: 7 (or a variant thereof): (i) the codon-optimized gag-pol gene of SEQ ID NO: 6 comprises a translational slippage and thus does not form a single conventional open reading frame; and (ii) the codon-optimized gag-pol gene of SEQ ID NO: 6 is operably linked to a CAG promoter. An exemplary CAG promoter is set forth in SEQ ID NO: 15.

[0131] In the preferred five-plasmid method of the present invention, the vector gene plastid encodes all the genetic material, including the transgene, packaged into the final retroviral / lentiviral vector. Typically, only a portion of the genetic material found in the vector gene plastid ultimately becomes incorporated into the virus. The vector gene plastid may be designated herein as "pDNA1" and typically contains the transgene and its promoter.

[0132] The other four plasmids are production plasmids encoding Gag-Pol, Rev, F, and HN proteins. These plasmids can be designated as "pDNA2a," "pDNA2b," "pDNA3a," and "pDNA3b," respectively.

[0133] The vector gene plastid (pDNA1) can be modified, in particular 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 viruses, particularly retroviral / lentiviral (e.g., SIV) ORFs, from the pDNA1 sequence. Therefore, the retroviral / lentiviral (e.g., SIV) vector of the present invention can be prepared using a modified pDNA1 comprising a reduced number of non-transgenic gene ORFs. The modified pDNA1 may comprise modifications in any region of the plastid sequence. Specifically, the 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 transgenic gene and / or a promoter operably linked to the transgenic gene. Although the modified pDNA1 may not comprise ORFs other than the transgenic gene, this is not required. In practice, a modified pDNA1 may still contain ORFs other than the transgene, but may contain a reduced number of non-transgene ORFs compared to the unmodified pDNA1 from which it was derived. As a non-limiting example, a modified pDNA1 may contain at least one, at least two, at least three, at least four, at least five, or more fewer non-transgene ORFs than the corresponding unmodified pDNA1. As a specific example, pGM830 (derived from pGM326) contains two fewer non-transgene ORFs than pGM326. A modified pDNA1 may contain at least one, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least fifteen, at least twenty, or more modifications (e.g., one, two, three, four, five, six, seven, eight, nine, ten, fifteen, or twenty modifications) compared to the corresponding unmodified pDNA1. As a non-limiting example, the modified pDNA1 can comprise between about 1 and about 20 modifications compared to the corresponding unmodified pDNA1, such as between about 5 and about 15, or between about 5 and about 10. As a specific example, pGM830 (which is derived from pGM326) comprises 7 modifications compared to pGM326.

[0134] Use of a modified pDNA1 as described herein (eg, pGM830) has the potential to produce increased SIV titers compared to a production process using an unmodified pDNA1 plasmid (eg, pGM326) but in which all other plasmid and process parameters remain constant.

[0135] Five plastids can be Figure 1A-1G characterization, so pDNA1 is Figure 1A pGM326 plasmid or Figure 1B The pGM830 plasmid, pDNA2a is Figure 1C pGM691 plasmid or Figure 1D pGM297, pDNA2b Figure 1E The pGM299 plasmid, pDNA3a is Figure 1F The pGM301 plasmid and pDNA3b are Figure 1G

[00155] The pGM303 plasmid, or variants of any of these plasmids (as described herein), may be used. Using plasmids pGM326, pGM297, pGM299, pGM301, and pGM303, the final CFTR containing retroviral / lentiviral vector may be referred to as vGM058. Using plasmids pGM326, pGM691, pGM299, pGM301, and pGM303, the final CFTR containing retroviral / lentiviral vector may be referred to as vGM195. The pGM691 plasmid (usually using pGM326, pGM299, pGM301, and pGM303) and the vGM195 vector may be preferred. Using plasmids pGM830, pGM691, pGM299, pGM301, and pGM303, the final CFTR containing retroviral / lentiviral vector may be referred to as vGM244. The pGM691 and pGM830 plasmids (commonly used are pGM299, pGM301 and pGM303) and the vGM244 vector may be particularly preferred.

[0136] according to Figure 1A The pGM326 plasmid defined in the invention is represented by SEQ ID NO: 8; Figure 1B The pGM830 plasmid defined in the invention is represented by SEQ ID NO: 9; Figure 1C The pGM691 plasmid defined in the invention is represented by SEQ ID NO: 7; Figure 1D The pGM297 plasmid defined in the invention is represented by SEQ ID NO: 10; Figure 1E The pGM299 plasmid defined in the invention is represented by SEQ ID NO: 11; Figure 1F The pGM301 plasmid defined in the invention is represented by SEQ ID NO: 12; and Figure 1G The pGM303 plasmid as defined in SEQ ID NO: 13 is represented by SEQ ID NO: 13. Variants of these plasmids (as defined herein) are also encompassed by the present invention. Specifically, variants having at least 90% (such as at least 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%) sequence identity to any one of SEQ ID NOs: 7 to 13 are encompassed.

[0137] In the five-plasmid method of the present invention, all five plasmids contribute to the formation of the final retroviral / lentiviral (e.g., SIV) vector. During the manufacture of the retroviral / lentiviral (e.g., SIV) vector, the vector gene plasmid (pDNA1) provides the enhancer / promoter, Psi, RRE, cPPT, mWPRE, SIN LTR, SV40 polyA (see Figure 1A or Figure 1B ), which is important for viral production. Using pGM326 or pGM830 as non-limiting examples of pDNA1, the CMV enhancer / promoter, SV40 polyA, colE1 Ori, and KanR are involved in the production of the retroviral / lentiviral (e.g., SIV) vectors of the present invention (e.g., vGM195 or vGM244), but are not found in the final retroviral / lentiviral (e.g., SIV) vector. The RRE, central polypurine tract (cPPT), hCEF, soCFTR2 (transgene), and mWPRE from pGM326 or pGM830 are found in the final retroviral / lentiviral (e.g., SIV) vector. The long terminal repeats, SIN / IN self-inactivating (SINLTR), and packaging signal (Psi) can be found in the final retroviral / lentiviral (e.g., SIV) vector.

[0138] For other retroviral / lentiviral (e.g., SIV) vectors of the invention, corresponding components from other vector gene 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.

[0139] The F and HN proteins from pDNA3a and pDNA3b (preferably Sendai virus F and HN proteins) are important for infection of target cells with the final retroviral / lentiviral (e.g., SIV) vector, i.e., for entry into the patient's epithelial cells (typically the lungs, preferably airway epithelial cells, as described herein). The products of the pDNA2a and pDNA2b plasmids are important for viral transduction, i.e., for insertion of retroviral / lentiviral (e.g., SIV) DNA into the host genome. Promoters, regulatory elements (such as WPRE), and the transgene are important for transgene expression within the target cells.

[0140] The retroviral / lentiviral (e.g., SIV) vectors of the present invention can 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 nucleases; (d) harvesting the lentivirus (e.g., SIV); (e) adding trypsin; and (f) purifying the lentivirus (e.g., SIV).

[0141] This method can utilize either the four-plastid or five-plastid systems described herein. Thus, for the preferred five-plastid method, one or more plasmids may comprise or consist of: a vector gene plasmid, pDNA1; a gag-pol plasmid, pDNA2a; a Rev plasmid, pDNA2b; a fusion (F) protein plasmid, pDNA3a; and a hemagglutinin-neuraminidase (HN) plasmid, pDNA3b. pDNA1 can be selected from pGM326 and pGM830, preferably pGM830. pDNA2a can be selected from pGM297 and pGM691, preferably pGM297. pDNA2b can be pGM299. pDNA3a can be pGM301. pDNA3b can be pGM303. Any combination of pDNA1, pDNA2a, pDNA2b, pDNA3a, and pDNA3b can be used. Preferably, pDNA1 is pGM326 or pGM830 (pGM830 is particularly preferred); pDNA2a is pGM297 or pGM691 (pGM691 is particularly preferred); pDNA2b is pGM299; pDNA3a is pGM301; and pDNA3b is pGM303. The SIV vector generated using pGM830, pGM691, pGM299, pGM301, and pGM303 is designated vGM244. The SIV vector generated 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 present invention, with vGM244 being particularly preferred.

[0142] Any suitable ratio of vector gene plasmid: co-gagpol plasmid: Rev plasmid: F plasmid: HN plasmid can be used to produce retrovirus / lentivirus (eg, SIV).

[0143] Steps (a)-(f) of the method are typically performed sequentially, starting at step (a) and continuing through step (f). The method may include one or more additional steps, 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 these steps may comprise one or more substeps. For example, harvesting may involve one or more steps or substeps, and / or purification may involve one or more steps or substeps.

[0144] Any suitable cell type can be transfected with one or more plasmids (e.g., the five plasmids described herein) to produce the retroviral / lentiviral (e.g., SIV) vectors of the present invention. Typically, mammalian cells, particularly human cell lines, are used. Non-limiting examples of cells suitable for the methods of the present invention are HEK293 cells (such as HEK293F or HEK293T cells) and 293T / 17 cells. Commercial cell lines suitable for virus production are also readily available (e.g., Gibco Virus Producer Cells - Catalog No. A35347, from ThermoFisher Scientific).

[0145] Cells can be grown in animal component-free media, including serum-free media, as adherent or suspension cultures. Cells can be grown in media containing human components. Cells can be grown in defined media containing or consisting of synthetically produced components.

[0146] Any suitable transfection means may be used in accordance with the present invention. The selection of suitable transfection means is within the routine practice of those skilled in the art. As a non-limiting example, transfection may be performed using PEIPro TM 、Lipofectamine2000 TM 、Lipofectamine3000 TM or calcium triphosphate.

[0147] Any suitable nuclease can be used in accordance with the present invention. The selection of a suitable nuclease is within the routine practice of one of ordinary skill in the art. Typically, the nuclease is an endonuclease. As non-limiting examples, the nuclease can be Benzonase® or Denarase®. The nuclease can be added during the pre-harvest stage or during the post-harvest stage, or between harvesting steps.

[0148] Trypsin activity can preferably be provided by a recombinant enzyme free of animal origin, such as TrypLE Select™. Addition of trypsin can be done in the pre-harvest stage or in the post-harvest stage, or between harvesting steps.

[0149] Any suitable purification means can be used to purify retroviral / lentiviral (e.g., SIV) vectors. 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 can be used according to the present invention include mixed-mode size exclusion chromatography (SEC) and / or anion exchange chromatography. Elution can be performed with or without a salt gradient, preferably without a salt gradient.

[0150] This method can be used to generate the retroviral / lentiviral (eg, SIV) vectors of the invention as described herein. Alternatively, the retroviral / lentiviral (eg, SIV) vectors of the invention comprise any of the above genes or genes encoding the above proteins.

[0151] The retroviral / lentiviral (eg SIV) vectors of the present invention can be delivered by using Figure 1A-1G Any combination of one or more specific plastid constructs is produced by the method.

[0152] mutation CF is caused by mutations in the CFTR gene. To date, over 2,000 different mutations have been identified within the CFTR gene. Some CFTR gene mutations result in no CFTR protein production. Others result in the production of dysfunctional CFTR protein. Using current conventional nomenclature, the different CF-causing mutations in the CFTR gene can be arranged into several categories, depending on the effect the mutation has on CFTR protein production, conformation, or function.

[0153] Category I CFTR Mutations are protein-producing mutations that do not result in the production of a functional CFTR protein. Approximately 22% of people with CF have at least one Class I CFTR Mutations. Several nonsense and splicing mutations belong to Class I. Class I CFTR Examples of mutations include G542X, W1282X, and R553X.

[0154] Category II CFTR Mutations are protein processing mutations. Class II CFTR The mutation does not prevent the production of CFTR protein, 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 will be transported at reduced levels compared to normal CFTR protein. Approximately 88% of CF patients have at least one Class II mutation. CFTR Mutation. Class II CFTR Examples of mutations include F508del, N1303K, and I507del. F508del is the most common CF-causing CFTR mutation Category III CFTR Mutations are gated mutations. Class III CFTR The mutation does not prevent the CFTR protein from being produced or transported to the cell membrane. Instead, the gating mutation forces the CFTR protein to adopt a closed conformation, preventing or reducing chloride ion transport. Approximately 6% of CF patients have at least one Class III CFTR Mutation. Class III CFTR Examples of mutations include G551D and S549N.

[0155] Category IV CFTR The mutation is a conduction mutation. CFTR The mutation does not prevent the CFTR protein from being produced or transported to the cell membrane, nor does it keep the CFTR protein in a closed conformation. However, class IV mutations may affect the internal conformation of the chloride channel within the CFTR protein, reducing chloride transport. Approximately 6% of people with CF have at least one class IV mutation. CFTR Mutation. Class IV CFTR Examples of mutations include D1152H, R347P, and R117H.

[0156] Category V CFTR Mutations are called "inadequate protein mutations". CFTR The mutation results in lower amounts of CTFR protein being present at the cell membrane. This can occur because less CFTR protein is produced, only a small amount of protein at the cell surface functions properly, or degradation of the normal CFTR protein in the cell membrane occurs too quickly. Several missense and splicing mutations fall into Class V. Approximately 5% of CF patients have at least one Class V mutation. CFTR Mutation. Class V CFTR Examples of mutations include 3849+10kbC→T, 2789+5G→A, and A455E.

[0157] Class VI CFTR Mutations destabilize the CFTR protein in the post-endoplasmic reticulum (ER) compartment and / or at the cell membrane by reducing its conformational stability and / or by generating additional internalization signals. These mutations thus cause accelerated CFTR turnover at the cell membrane and reduced expression at the apical cell membrane.

[0158] 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 mutations, one or more Class II mutations, one or more Class III mutations, one or more Class IV mutations, one or more Class V mutations and / or one or more Class VI mutations. The patient to be treated may have (i) one or more mutations in Class I and one or more mutations in Class II; (ii) one or more mutations in Class I and one or more mutations in Class III; (iii) one or more mutations in Class I and one or more mutations in Class IV; (iv) one or more mutations in Class I and one or more mutations in Class V; (v) one or more mutations in Class I and one or more mutations in Class VI; (vi) one or more mutations in Class II and one or more mutations in Class III; (vii) one or more mutations in Class II and one or more mutations in Class IV; (viii) one or more mutations in Class II and one or more mutations in Class V; (ix) one or more mutations in Class II and one or more mutations in Class VI; (x) one or more mutations in Class III and one or more mutations in Class IV; (xi) one or more mutations in Class III and one or more mutations in Class V; (xii) one or more mutations in Class III and one or more mutations in Class VI; (xiii) One or more mutations in Class IV and one or more mutations in Class V; (xiv) one or more mutations in Class IV and one or more mutations in Class VI; (xv) one or more mutations in Class V and one or more mutations in Class VI; (xvi) one or more mutations in Class I, one or more mutations in Class II, and one or more mutations in Class III; (xvii) one or more mutations in Class I, one or more mutations in Class II, and one or more mutations in Class IV; (xviii) one or more mutations in Class I, one or more mutations in Class II, and one or more mutations in Class V; (xix) one or more mutations in Class I, one or more mutations in Class II, and one or more mutations in Class VI; (xx) one or more mutations in Class I, one or more mutations in Class III, and one or more mutations in Class IV; (xxi) one or more mutations in Class I, one or more mutations in Class III, and one or more mutations in Class V; (xxii) One or more mutations in class I, one or more mutations in class III, and one or more mutations in class VI; (xxiii) one or more mutations in class I, one or more mutations in class IV, and one or more mutations in class V; (xxiv) one or more mutations in class I, one or more mutations in class IV, and one or more mutations in class VI; (xxv) one or more mutations in class I, one or more mutations in class V, and one or more mutations in class VI;(xxvi) one or more mutations in Class II, one or more mutations in Class III, and one or more mutations in Class IV; (xxvii) one or more mutations in Class II, one or more mutations in Class III, and one or more mutations in Class V; (xxviii) one or more mutations in Class II, one or more mutations in Class III, and one or more mutations in Class VI; (xxix) one or more mutations in Class II, one or more mutations in Class IV, and one or more mutations in Class V; (xxx) one or more mutations in Class II, one or more mutations in Class IV, and one or more mutations in Class VI; (xxxi) one or more mutations in Class II, one or more mutations in Class V, and one or more mutations in Class VI; (xxxii) one or more mutations in Class III, one or more mutations in Class IV, and one or more mutations in Class V; (xxxiii) one or more mutations in Class III, one or more mutations in Class IV, and one or more mutations in Class VI; (xxxiv) One or more mutations in Class III, one or more mutations in Class V, and one or more mutations in Class VI; (xxxv) one or more mutations in Class IV, one or more mutations in Class V, and one or more mutations in Class VI; (xxxvi) one or more mutations in Class I, one or more mutations in Class II, one or more mutations in Class III, and one or more mutations in Class IV; (xxxvii) one or more mutations in Class I, one or more mutations in Class II, one or more mutations in Class III, and one or more mutations in Class V; (xxxviii) one or more mutations in Class I, one or more mutations in Class II, one or more mutations in Class III, and one or more mutations in Class VI; (xxxix) one or more mutations in Class I, one or more mutations in Class II, one or more mutations in Class IV, and one or more mutations in Class V; (xl) one or more mutations in Class I, one or more mutations in Class II, one or more mutations in Class IV, and one or more mutations in Class VI; (xli) one or more mutations in class I, one or more mutations in class II, one or more mutations in class V, and one or more mutations in class VI; (xlii) one or more mutations in class I, one or more mutations in class III, one or more mutations in class IV, and one or more mutations in class V; (xliii) one or more mutations in class I, one or more mutations in class III, one or more mutations in class IV, and one or more mutations in class VI; (xliv) one or more mutations in class I, one or more mutations in class III, one or more mutations in class V, and one or more mutations in class VI; (xlv) one or more mutations in class I, one or more mutations in class IV, one or more mutations in class V, and one or more mutations in class VI;(xlvi) one or more mutations in Class II, one or more mutations in Class III, one or more mutations in Class IV, and one or more mutations in Class V; (xlvii) one or more mutations in Class II, one or more mutations in Class III, one or more mutations in Class IV, and one or more mutations in Class VI; (xlviii) one or more mutations in Class II, one or more mutations in Class III, one or more mutations in Class V, and one or more mutations in Class VI; (xlix) one or more mutations in Class II, one or more mutations in Class IV, one or more mutations in Class V, and one or more mutations in Class VI; (l) one or more mutations in Class III, one or more mutations in Class IV, one or more mutations in Class V, and one or more mutations in Class VI; (li) one or more mutations in Class I, one or more mutations in Class II, one or more mutations in Class III, one or more mutations in Class IV, and one or more mutations in Class V; (lii) one or more mutations in class I, one or more mutations in class II, one or more mutations in class III, one or more mutations in class IV, and one or more mutations in class VI; (liii) one or more mutations in class I, one or more mutations in class II, one or more mutations in class III, one or more mutations in class V, and one or more mutations in class VI; (liv) one or more mutations in class I, one or more mutations in class II, one or more mutations in class IV, one or more mutations in class V, and one or more mutations in class VI; (lv) one or more mutations in class I, one or more mutations in class III, one or more mutations in class IV, one or more mutations in class V, and one or more mutations in class VI; (lvi) one or more mutations in class II, one or more mutations in class III, one or more mutations in class IV, one or more mutations in class V, and one or more mutations in class VI; or (lvii) One or more mutations in class I, one or more mutations in class II, one or more mutations in class III, one or more mutations in class IV, one or more mutations in class V, and one or more mutations in class VI.

[0159] Patients to be treated according to the present invention may be CFTR The gene has 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, each of which can be independently selected from Class I, Class II, Class III, Class IV and / or Class V mutations described herein.

[0160] Patients to be treated according to the present invention may have at least one Class I and / or Class II CFTRMutations, such as those described herein. The patient to be treated may have (i) at least one Class I mutation, optionally selected from G542X, W1282X and / or R553C CFTR mutation; and / or (ii) at least one Class II mutation optionally selected from F508del, N1303K and / or I507del CFTR mutation.

[0161] As exemplified herein, the combination therapy of the present invention successfully restored Class I CFTR CFTR expression and function in mutant models, and the unexpected effect of this combination on the expression of CFTR by retroviral / lentiviral (e.g., SIV) vectors. CFTR Comparison of transgenic genes with endogenous genes CFTR The gene is larger. Therefore, the use of the combination therapy of the present invention can be independent of the patient's CFTR mutation. In other words, and without being bound by theory, since the CFTR modulator can Transgenic genes exert a greater therapeutic effect, endogenous CFTR The nature of the mutation in the gene that causes CF may not be relevant. Therefore, the present invention has the potential to treat CF patients independently of the mutation that causes CF. This is advantageous because currently licensed CFTR modulator therapies are only available for patients with specific CFTR patients with mutations.

[0162] Conditioner CFTR modulators are active pharmaceutical ingredients (APIs) designed to correct a dysfunctional CFTR protein. CFTR modulators are a specialized class of therapeutics because different modulators are designed to address specific CFTR Mutations or specific categories CFTR Mutations cause underlying defects in the CFTR protein.

[0163] There are three main types of CFTR modulators: CFTR enhancers, CFTR correctors, and CFTR amplifiers.

[0164] As discussed in this article, Class III CFTRMutations such as G551D are gate mutations that prevent the CFTR protein from opening normally to promote chloride ion transport. CFTR enhancers alleviate this defect by opening the CFTR protein gate and keeping it open longer to promote the smooth flow of chloride ions. Ivacaftor (Kalydeco®) is an example of a CFTR enhancer developed by Vertex Pharmaceuticals. It is an oral medication approved by the U.S. Food and Drug Administration (FDA), EU, and Health Canada for CF patients as young as 1 year old with at least one mutation that weakens chloride ion flow (such as G551D). Another example of a CFTR enhancer is the experimental therapeutic agent PTI-808 being developed by Proteostasis Therapeutics.

[0165] According to this article, Class II CFTR The mutation is a protein processing mutation that causes the misfolding of the CFTR protein, which may affect the delivery of the misfolded CFTR to the cell surface. CFTR correctors assist the CFTR protein in correctly folding into its 3D configuration, allowing it to be successfully delivered to the cell membrane so that it can function. Lumacato (VX-809) and Tesakato (VX-661) are two therapies that act as correctors according to Vertex Pharmaceuticals. Another CFTR corrector is Erezato. These correctors help the CFTR protein to fold correctly and reach the cell surface, but are insufficient in alleviating CF symptoms themselves. Therefore, it is not approved as a single-party therapy for CF. Another CFTR corrector in development (according to Proteostasis Therapeutics) is PTI-801.

[0166] As discussed herein, Class V CFTR mutations cause a decrease in the amount of CFTR protein present at the cell surface, for example, by causing a decrease in the amount of expressed CFTR protein or an increase in the rate of degradation of the CFTR protein. Amplifiers are a type of CFTR modulator that enhances the production of CFTR protein by cells. PTI-428 is an investigational first-generation CFTR amplifier by Proteostasis Therapeutics that is being tested as a monotherapy and in combination with other therapies for CF.

[0167] CFTR enhancers, correctors, and amplifiers are described in the art for use independently as monotherapy. In addition, combinations of these CFTR modulators are also known, with three of the four currently approved CFTR modulator therapies being combination therapies. As a non-limiting example, combining enhancers with correctors can improve CFTR activity by correcting CFTR conformation and opening the gate to allow chloride ion transport. Specifically, the combination of enhancer ivacaftor and corrector lumacaftor is authorized as a combination therapy and sold as Orkambi® by Vertex for the treatment of CF patients with two F508del CFTR mutations. Another example of an authorized combination therapy is enhancer ivacaftor and corrector tesacaftor, which are labeled Symdeko® by Vertex in the United States and Symkevi® in the EU.

[0168] Amplifiers can also be used in combination with other CFTR modulators. Combining a CFTR amplifier with other CFTR modulators can be advantageous because the CFTR amplifier can cause more CFTR protein to be expressed, which can then be acted upon by other CFTR modulators.

[0169] In addition to these first-generation CFTR modulators, so-called next-generation modulators can combine multiple CFTR correctors to create 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 with two correctors, tesacaftor and elexacaftor.

[0170] Unless expressly stated to the contrary, any reference to a CFTR modulator herein encompasses any and all salts, derivatives, and analogs of that CFTR modulator. Thus, the present invention relates to combinations of known CFTR modulators, such as the individualized CFTR modulators herein (particularly ivacaftor) or salts, derivatives, or analogs thereof. Salt forms preferably include pharmaceutically acceptable salts. Pharmaceutically acceptable salts include acid addition salts formed with inorganic acids (such as, for example, hydrochloric acid or phosphoric acid) or with organic acids (such as acetic acid, oxalic acid, tartaric acid, maleic acid, and the like). As used herein, an "analog" of a CFTR modulator refers to a chemical structure that retains substantial similarity to a CFTR modulator but that may not be readily synthetically derived from the CFTR modulator. Related chemical structures that are readily synthetically derived from CFTR modulators are referred to as "derivatives." These are all within the scope of the present invention. As a non-limiting example, reference to "ivacator" includes salt forms, analogs, and derivatives of ivacaftor, such as deuterated ivacaftor (D-ivacator).

[0171] Therefore, the present invention relates to a therapy comprising a gene therapy vector, in particular, a reverse transcription / lentiviral (e.g., SIV) vector as described herein, preferably a SIV.F / HN vector, in combination with one or more CFTR modulators, which can be selected from one or more CFTR enhancers, one or more CFTR correctors, and / or one or more CFTR amplifiers, or a combination thereof. Combinations comprising one or more CFTR enhancers and a reverse transcription / lentiviral (e.g., SIV) vector are particularly preferred. Therefore, the present invention may relate to the use of a gene therapy vector, in particular, a reverse transcription / lentiviral (e.g., SIV) vector as described herein, preferably a SIV.F / HN vector, in combination with one or more CFTR enhancers. In addition to a gene therapy vector, in particular, a reverse transcription / lentiviral (e.g., SIV) vector as described herein, preferably a SIV.F / HN vector, and one or more CFTR enhancers, one or more CFTR correctors and / or one or more CFTR amplifiers may also be used.

[0172] Thus, the present invention relates to a combination of a reverse transcription / 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 enhancers, one or more CFTR correctors, and / or one or more CFTR amplifiers, or a combination thereof. Generally, the present invention relates to a combination of a reverse transcription / lentiviral (e.g., SIV) vector as described herein, preferably a SIV.F / HN vector, and a CFTR enhancer and / or a CFTR corrector. Preferably, the present invention relates to a combination of a reverse transcription / lentiviral (e.g., SIV) vector as described herein, preferably a SIV.F / HN vector, and a CFTR enhancer and, optionally, one or more CFTR correctors and / or one or more CFTR amplifiers.

[0173] Any CFTR modulator can be used in combination with a retroviral / lentiviral (e.g., SIV) vector as described herein, preferably a SIV.F / HN vector according to the present invention (such as those described herein). Thus, non-limiting examples of CFTR modulators that can be used in combination with a retroviral / 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 (tesakaftor), eresakaftor, PTI-801, PTI-428, and combinations such as ivacaftor + lumacaftor (Orkambi®), ivacaftor + tesakaftor (Symdeko® or Symkevi®), and ivacaftor + tesakaftor + eresakaftor (Trikafta®).

[0174] Preferably, the present invention relates to a combination of a retroviral / lentiviral (e.g., SIV) vector as described herein, preferably a SIV.F / HN vector, and a CFTR modulator selected from ivacaftor, tesacaftor, elexacaftor, or lumacaftor, or a combination thereof. The combination of a retroviral / lentiviral (e.g., SIV) vector as described herein, preferably a SIV.F / HN vector, and the CFTR modulator (particularly the CFTR enhancer) ivacaftor is particularly preferred.

[0175] Preferred embodiments relate to the use of the following combination: (A) an SIV vector pseudotyped with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) proteins, wherein: (a) the vector comprises a modified retroviral RNA sequence comprising or consisting of the nucleic acid sequence of SEQ ID NO: 16 (the vector comprises a CFTR transgene), preferably, wherein the modified retroviral RNA sequence consists of the nucleic acid sequence of SEQ ID NO: 16; and (b) the F protein comprises a first subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 19 and a second subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 20, and (B) a CFTR modulator selected from ivacaftor, tesacaftor, elexacaftor, or lumacaftor, or a combination thereof.

[0176] Particularly preferred embodiments relate to the use of the following combination: (A) an SIV vector pseudotyped with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) proteins, wherein: (a) the vector comprises a modified retroviral RNA sequence comprising or consisting of the nucleic acid sequence of SEQ ID NO: 16 (the vector comprises a CFTR transgene), preferably, wherein the modified retroviral RNA sequence consists of the nucleic acid sequence of SEQ ID NO: 16; and (b) the F protein comprises a first subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 19 and a second subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 20, and (B) the CFTR modulator (particularly the CFTR enhancer) ivacaftor.

[0177] As described herein, in particularly preferred embodiments, the SIV vector is pseudotyped with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) proteins, wherein: (a) the vector comprises a modified retroviral RNA sequence comprising or consisting of the nucleic acid sequence of SEQ ID NO: 16 (the vector comprises a CFTR transgene), preferably, wherein the modified retroviral RNA sequence consists of the nucleic acid sequence of SEQ ID NO: 16; and (b) the F protein comprises a first subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 19 and a second subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 20, wherein the vector further comprises one or more of the following: (a) p17 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 22; (b) p24 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 23; (c) a p25 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 24. (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, tesacaftor, elexacaftor or lumacaftor or a combination thereof, in particular, in combination with the CFTR modulator (in particular, the CFTR enhancer) ivacaftor.

[0178] Treatment indications The retroviral / lentiviral (e.g., SIV) vectors of the present invention are capable of achieving high and sustained gene expression through efficient gene transfer. The F / HN pseudotyped retroviral / lentiviral (e.g., SIV) vectors of the present invention are capable of: (i) airway transduction without disrupting epithelial integrity; (ii) sustained gene expression; (iii) lack of chronic toxicity; and (iv) efficient repeated administration. Long-term / sustained stable gene expression, preferably in therapeutically effective amounts, can be achieved using repeated doses of the vectors of the present invention. Alternatively, a single dose can be used to achieve the desired long-term expression. Advantageously, the retroviral / lentiviral (e.g., SIV) vectors of the present invention can be provided by CFTR Functional copies of genes can ameliorate or prevent lung disease in CF patients, independent of the underlying mutation, for use in gene therapy for CF.

[0179] CFTR modulators are breakthrough therapies that target the underlying cause of CF rather than improving the symptoms of the disease. However, current CFTR modulators are only effective in patients with specific mutations.

[0180] Therefore, the combination of gene therapy and CFTR modulators provides a possible significant improvement in CF treatment. The present inventors first studied the effect of combining retroviral / lentiviral (e.g., SIV) vectors with CFTR modulators. Specifically, the present inventors have demonstrated that, when combined with CFTR modulators, gene therapy using retroviral / lentiviral (e.g., SIV) vectors of the present invention produces a therapeutic effect exceeding expectations. As exemplified herein, the present inventors have unexpectedly demonstrated that the effect of CFTR modulators, particularly CFTR enhancers and rSIV.F / HN-CFTR combinations is greater than the cumulative effect of the individual effects of CFTR modulators, particularly CFTR enhancers and rSIV.F / HN-mediated CFTR performance.

[0181] Therefore, according to the present invention, CFTR Retroviral / lentiviral (eg, SIV) vectors that transfer genes can be used in combination with one or more CFTR modulators to treat CF.

[0182] Preferred embodiments relate to the medical use of the following combination: (A) an SIV vector pseudotyped with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) proteins, wherein: (a) the vector comprises a modified retroviral RNA sequence comprising or consisting of the nucleic acid sequence of SEQ ID NO: 16 (the vector comprises a CFTR transgene), preferably wherein the modified retroviral RNA sequence consists of the nucleic acid sequence of SEQ ID NO: 16; and (b) the F protein comprises a first subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 19 and a second subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 20, and (B) a CFTR modulator selected from ivacaftor, tesacaftor, elexacaftor, or lumacaftor, or a combination thereof.

[0183] Particularly preferred embodiments relate to the medical use of the following combination: (A) an SIV vector pseudotyped with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) proteins, wherein: (a) the vector comprises a modified retroviral RNA sequence comprising or consisting of the nucleic acid sequence of SEQ ID NO: 16 (the vector comprises a CFTR transgene), preferably wherein the modified retroviral RNA sequence consists of the nucleic acid sequence of SEQ ID NO: 16; and (b) the F protein comprises a first subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 19 and a second subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 20, and (B) the CFTR modulator (specifically, the CFTR enhancer) ivacaftor.

[0184] Retroviral / lentiviral (e.g., SIV) vectors and one or more CFTR modulators can be administered to patients with CF who exhibit one or more symptoms of CF. When administered to such patients, retroviral / lentiviral (e.g., SIV) vectors and one or more CFTR modulators can cure, delay, reduce the severity of, or alleviate one or more symptoms of one or more symptoms, and / or prolong patient survival beyond the expected survival in the absence of such treatment and / or beyond the expected survival using conventional CF treatments (e.g., CFTR modulators alone, particularly CFTR enhancers). Thus, retroviral / lentiviral (e.g., SIV) vectors and one or more CFTR modulators, particularly CFTR enhancers, can be administered to patients with CF to alleviate the disease and / or prolong the survival of patients with CF beyond the expected survival in the absence of such treatment and / or beyond the expected survival using conventional CF treatments (e.g., CFTR modulators alone or CFTR enhancers).

[0185] The retroviral / lentiviral (eg, SIV) vector and one or more CFTR modulators are administered in combination. "Combined" administration encompasses both simultaneous (also referred to as concurrent) administration / delivery and sequential (also referred to as separate) administration / delivery.

[0186] For "simultaneous" or "concurrent delivery," delivery of the retroviral / lentiviral (e.g., SIV) vector can still occur at the start of delivery of the CFTR modulator, or delivery of the CFTR modulator can still occur at the start of delivery of the retroviral / lentiviral (e.g., SIV) vector, so that there is overlap in administration. Concurrent delivery can encompass delivery of the retroviral / lentiviral (e.g., SIV) vector and the CFTR modulator within weeks to months or even years of each other, typically resulting in overlap of retroviral / lentiviral (e.g., SIV) vector delivery with CFTR modulator delivery.

[0187] Alternatively, delivery of the retroviral / lentiviral (e.g., SIV) vector can be completed before delivery of the CFTR modulator begins, or delivery of the CFTR modulator can be completed before delivery of the retroviral / lentiviral (e.g., SIV) vector begins. Sequential administration can involve administering the retroviral / lentiviral (e.g., SIV) vector and the CFTR modulator 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 more of each other.

[0188] The CFTR modulator can be administered hourly, every 2 hours, every 3 hours, every 4 hours, every 6 hours, every 8 hours, every 12 hours, every day, every 2 days, or longer. Typically, the CFTR modulator is administered every 12 hours.

[0189] Retroviral / lentiviral (e.g., SIV) vectors can be administered monthly, every 2 months, every 3 months, every 4 months, every 6 months, every 8 months, every 12 months, or longer. Since the administration frequency of retroviral / lentiviral (e.g., SIV) vectors is lower than that of CFTR modulators, the combination therapy is typically administered sequentially.

[0190] Treatment with a retroviral / lentiviral (e.g., SIV) vector and / or a CFTR modulator can continue for as long as desired, at a desired dosing frequency, 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 the lifespan of the patient being treated.

[0191] Typically, the treatment is more effective due to combined administration. For example, treatment with a CFTR modulator may be more effective than when a CFTR modulator is administered in the absence of a retroviral / slow viral (e.g., SIV) vector, e.g., an equivalent effect is seen in the absence of a CFTR modulator, or a CFTR modulator alleviates symptoms to a greater extent; or in the case of a retroviral / slow viral (e.g., SIV) vector, similar situations are seen. Typically, delivery results in symptom alleviation, or other parameters associated with CF are greater than those observed when a CFTR modulator is delivered in the absence of a retroviral / slow viral (e.g., SIV) vector; or in the case of a retroviral / slow viral (e.g., SIV) vector, similar situations are seen.

[0192] It will be appreciated that appropriate dosages of the retroviral / lentiviral (eg, SIV) vector and / or CFTR modulator will depend on the particular agent and may also vary from patient to patient.

[0193] It will be appreciated that appropriate dosages of the retroviral / lentiviral (eg, SIV) vector and / or CFTR modulator will depend on the particular agent and may also vary from patient to patient.

[0194] Determining the optimal dose will generally involve balancing the degree of therapeutic benefit against any risks or adverse side effects of the treatments described herein. The selected dose concentration will depend on various factors, including but not limited to the activity of the particular compound, the route of administration, the time of administration, the rate of elimination of the compound, the duration of treatment, other drugs, compounds and / or materials used in combination; and the patient's age, sex, weight, condition, overall health and previous medical history. The amount of compound and the route of administration will ultimately be at the discretion of the physician, but in general, the dose will achieve a local concentration at the site of action that achieves the desired effect without causing substantial harmful or adverse side effects. Non-limiting exemplary doses and routes of administration are described herein.

[0195] In vivo administration can be achieved in one dose continuously or intermittently (e.g., divided doses at appropriate intervals) throughout the course of treatment. Methods for determining the most effective mode of administration and dosage are well known to those skilled in the art and will vary with the formulation used for therapy, the purpose of therapy, the target cells being treated, and the individual being treated. Single or multiple administrations can be performed, and the dose concentration and pattern are selected by the treating physician.

[0196] The duration of action of the combination therapy according to the present invention can be sustained 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 longer. Typically, this is assessed relative to the last administration of a retroviral / lentiviral (e.g., SIV) vector and / or CFTR modulator, particularly the last administration of a retroviral / lentiviral (e.g., SIV) vector.

[0197] Retroviral / lentiviral (e.g., SIV) vectors according to the present invention are typically administered by inhalation. Thus, the retroviral / lentiviral (e.g., SIV) vectors can be formulated for inhalation as described herein. One or more CFTR modulators can be administered by any suitable route and can be formulated accordingly. Specifically, one or more CFTR modulators can be administered orally and can be formulated for oral administration.

[0198] Thus, the present invention provides a method of treating CF in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of each of: (i) a retroviral / lentiviral (e.g., SIV) vector of the present invention; and (ii) one or more CFTR modulators. Any of the retroviral / lentiviral (e.g., SIV) vectors described herein can be used in combination with any one or more CFTR modulators, such as those described and exemplified herein.

[0199] The combination therapy of the present invention can restore CFTR expression and / or activity to a degree that provides a therapeutic benefit. The combination therapy can restore (increase) CFTR expression and / or activity to a degree that matches or exceeds that of CFTR expression and / or activity in healthy controls. However, restoring healthy CFTR expression and / or activity is not necessary for obtaining a therapeutic benefit. In fact, the treatment threshold (that is, the level above which a therapeutic benefit is achieved) can be lower than the degree of CFTR expression and / or activity in healthy controls. In fact, patients, especially those with Class I CFTR mutations that result in ineffective CFTR expression, can receive a therapeutic benefit from even a relatively small increase in CFTR expression and / or activity (e.g., 5% or 10% CFTR expression and / or activity compared to the amount expressed in healthy controls).

[0200] The retroviral / lentiviral (e.g., SIV) vectors described herein, particularly in the context of the combination therapies of the present invention, can increase (restore) CFTR expression (particularly cellular CFTR expression and / or global expression in the lung or respiratory tree), CFTR activity, and / or CFTR currents as described herein. The present invention encompasses any combination of increases in CFTR expression (cellular and / or global), CFTR activity, and / or CFTR currents, including the quantitative increases described below.

[0201] The retroviral / lentiviral (e.g., SIV) vectors described herein, particularly in the context of the combination therapies of the present invention, can increase (restore) CFTR expression (particularly cellular CFTR expression and / or overall expression in the lung 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 healthy controls. Typically, the retroviral / lentiviral (e.g., SIV) vectors described herein, particularly in the context of the combination therapies of the present invention, can increase (restore) CFTR expression, particularly cellular CFTR expression, to at least 20%, preferably at least 50%, and more preferably at least 75% of CFTR expression in healthy controls. According to the retroviral / lentiviral (e.g., SIV) vectors described herein, CFTR expression, especially overall CFTR expression in the lungs, can be increased (restored) to at least 5%, at least 10%, and preferably at least 20% of CFTR expression in healthy controls, particularly in the context of the combination therapy of the present invention. The expression of the transgenic gene and / or encoded therapeutic protein of the present invention can be measured in lung tissue, so high and / or therapeutic expression can refer to concentrations in the lungs. CFTR expression can be quantified using one or more of the following techniques: CFTR RNA expression in lung tissue, CFTR protein expression in lung tissue, PK analysis (vector copy number, integration), DNA content in sputum (as a surrogate for NETosis).

[0202] Other endpoints for efficacy assessment of treatment according to the present invention include: improvement in FEV1 (lung function); MRI and / or CT for efficacy assessment; reduction in expression of one or more biomarkers of inflammation, such as IL-8, IL1β, IL-6, TNFα (usually measured in sputum), calprotectin (usually measured in serum); differentiated cell counts in sputum; reduction in surfactant protein D (SP-D) in serum as a marker of reduced epithelial damage; reduction in lung exacerbations; improvement in lung clearance index, quality of life as a patient-reported outcome via the CFQ-R respiratory domain (and / or other appropriate questionnaires), exploratory imaging endpoints (to demonstrate improved lung ventilation, mucus plugging and / or other symptoms, such as the Eichinger score via mRI).

[0203] Alternatively or additionally, the combination therapy of the present invention can increase (restore) CFTR activity (particularly cellular CFTR activity and / or overall activity in the lung 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 the CFTR activity in healthy controls. Typically, the combination therapy of the present invention can increase (restore) CFTR activity (particularly cellular CFTR activity) to at least 20%, preferably at least 50%, and more preferably at least 75% of the CFTR activity in healthy controls. In particular, the combination therapy of the present invention, using the retroviral / lentiviral (e.g., SIV) vectors described herein, can increase (restore) CFTR activity, particularly overall CFTR expression in the lung, to at least 5%, at least 10%, and preferably at least 20% of the CFTR activity in healthy controls.

[0204] As used herein, a healthy control is generally an equivalent individual or population that does not have CF. Preferably, a healthy control is an equivalent individual or population that does not have CF and is otherwise in good health. Healthy controls can be matched to individuals using standard clinical methods (e.g., age / sex matching, or matching based on other criteria).

[0205] Additional controls can be equivalent individuals with CF who have been treated with either the retroviral / lentiviral (eg, SIV) vector or the CFTR modulator alone.

[0206] CFTR activity can be defined by: CFTR RNA expression in lung tissue; CFTR protein expression in lung tissue and / or activity of the CFTR channel itself (e.g., as measured electrophysiologically using bronchial brushings from patients).

[0207] The combination therapies of the present invention can 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 to treatment with a retroviral / lentiviral (e.g., SIV) vector alone (i.e., compared to the increase in CFTR current achieved with retroviral / lentiviral (e.g., SIV) treatment alone). The combination therapies of the present invention can increase (restore) CFTR current by between about 1.3-fold and about 3-fold, or between about 1.2-fold and about 2-fold, compared to treatment with a retroviral / lentiviral (e.g., SIV) vector alone (i.e., compared to the increase in CFTR current achieved with retroviral / lentiviral (e.g., SIV) treatment alone). Preferably, the combination therapies of the present invention can 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 about 1.3-fold to about 1.8-fold, compared to treatment with a retroviral / lentiviral (e.g., SIV) vector alone.

[0208] The combination therapy of the present invention can increase (restore) the 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 the CFTR current in healthy controls. Typically, the combination therapy of the present invention can increase (restore) the CFTR cell current to at least 20%, preferably at least 50%, and more preferably at least 75% of the CFTR current in healthy controls. According to the retroviral / lentiviral (e.g., SIV) vectors described herein, in particular, in the context of the combination therapy of the present invention, the CFTR current, especially the overall current in the lung, can be increased (restored) to at least 5%, at least 10%, and preferably at least 20% of the CFTR current in healthy controls.

[0209] The present invention provides a combination of a retroviral / lentiviral vector (e.g., SIV) as described herein and one or more CFTR modulators for use in treating CF, wherein the treatment: (i) restores cellular CFTR activity to at least 10% or at least 50% (e.g., at least 70%) of the CFTR activity in healthy controls; (ii) restores global CFTR activity in the lung to at least 5% or at least 10% (e.g., at least 20%) of the CFTR activity in healthy controls; and / or (iii) increases CFTR current by at least about 1.3-fold (e.g., about 1.3-fold to about 1.8-fold, about 1.3-fold to about 3-fold, or about 1.3-fold), compared to treatment with the retroviral / lentiviral vector (e.g., SIV) alone. Generally, the present invention provides a combination of a retroviral / lentiviral vector (e.g., SIV) as described herein and one or more CFTR modulators for use in treating CF, wherein the treatment: (i) restores overall CFTR activity in the lung to at least 5% or at least 10% (e.g., at least 20%) of the CFTR activity in healthy controls; and / or (ii) increases CFTR current by at least about 1.3-fold (e.g., about 1.3-fold to about 1.8-fold, about 1.3-fold to about 3-fold, or about 1.3-fold), compared to treatment with the retroviral / lentiviral vector (e.g., SIV) vector alone.

[0210] The present invention provides a combination of a retrovirus / lentivirus (e.g., SIV) as described herein and one or more CFTR modulators 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% (e.g., at least 70%) of the CFTR activity in healthy controls; (ii) restores global CFTR activity in the lung to at least 5% or at least 10% (e.g., at least 20%) of the CFTR activity in healthy controls; and / or (iii) increases CFTR current by at least about 1.3-fold (e.g., about 1.3-fold to about 1.8-fold, about 1.3-fold to about 3-fold, or about 1.3-fold), compared to treatment with the retrovirus / lentivirus (e.g., SIV) vector alone. Generally, the present invention provides a combination of a retrovirus / lentivirus (e.g., SIV) as described herein and one or more CFTR modulators 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 lung to at least 5% or at least 10% (e.g., at least 20%) of the CFTR activity in healthy controls; and / or (ii) increases CFTR current by at least about 1.3-fold (e.g., about 1.3-fold to about 1.8-fold, about 1.3-fold to about 3-fold, or about 1.3-fold), compared to treatment with the retroviral / lentiviral (e.g., SIV) vector alone.

[0211] The present invention provides a combination of a retrovirus / lentivirus (e.g., SIV) as described herein and one or more CFTR modulators 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% (e.g., at least 70%) of the CFTR activity in healthy controls; (ii) restores global CFTR activity in the lung to at least 5% or at least 10% (e.g., at least 20%) of the CFTR activity in healthy controls; and / or (iii) increases CFTR current by at least about 1.3-fold (e.g., about 1.3-fold to about 1.8-fold, about 1.3-fold to about 3-fold, or about 1.3-fold), compared to treatment with the retrovirus / lentivirus (e.g., SIV) vector alone. Generally, the present invention provides a combination of a retrovirus / lentivirus (e.g., SIV) as described herein and one or more CFTR modulators for use in treating CF, wherein the patient to be treated has at least one class II CFTR mutation and the treatment: (i) restores overall CFTR activity in the lung to at least 5% or at least 10% (e.g., at least 20%) of the CFTR activity in healthy controls; and / or (ii) increases CFTR current by at least about 1.3-fold (e.g., about 1.3-fold to about 1.8-fold, about 1.3-fold to about 3-fold, or about 1.3-fold), compared to treatment with the retroviral / lentiviral (e.g., SIV) vector alone.

[0212] The retroviruses / lentiviruses described herein (e.g., SIV), typically as part of the combination therapies of the present invention, can transduce airway epithelial cells at a transduction rate sufficient to achieve a therapeutic effect on CFTR expression and / or activity. Typically, the retroviruses / lentiviruses described herein (e.g., SIV), typically as part of the combination therapies of the present invention, can 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 about 10% to about 20% (i.e., about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%). Preferably, the retroviruses / lentiviruses described herein (e.g., SIV), typically as part of the combination therapies of the present invention, can transduce airway epithelial cells at a transduction rate of 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 duct cells in the upper airways, goblet cells, club cells, and neuroendocrine cells in the bronchiolar airways, bronchoalveolar stem cells in the terminal bronchioles, and type II pneumocytes in the alveoli, and any combination thereof.

[0213] With the retroviral / lentiviral (e.g., SIV) vectors described herein, particularly in the context of the combination therapy of the present invention, VCNs 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 / cell, 9 copies / cell, at least 10 copies / cell, or more can be achieved.

[0214] In some embodiments, the present invention relates to the use of a retroviral / lentiviral (e.g., SIV) vector having a CFTR transgene according to the present invention in combination with one or more CFTR modulators, wherein the combination does not further comprise a LasB inhibitor. In other words, the present invention relates to the treatment of CF using a retroviral / lentiviral (e.g., SIV) vector having a CFTR transgene according to the present invention used in combination with one or more CFTR modulators, with the proviso that a LasB inhibitor is not used in the method. Specifically, in such embodiments, the use of indanes as LasB inhibitors, such as those disclosed in WO 2021 / 191240, is rejected.

[0215] The present invention also provides combinations of retroviral / lentiviral (e.g., SIV) vectors as described herein and one or more CFTR modulators for use in methods of treating CF. Any retroviral / lentiviral (e.g., SIV) vector described herein can be used in combination with any one or more CFTR modulators, such as those described and exemplified herein.

[0216] The present invention also provides the use of a retroviral / lentiviral (e.g., SIV) vector as described herein for the manufacture of a medicament for use in a method for treating CF, wherein the treatment method further comprises administering one or more CFTR modulators. The present invention also provides the use of a CFTR modulator as described herein for the manufacture of a medicament for use in a method for treating CF, wherein the treatment method further comprises administering a retroviral / lentiviral (e.g., SIV) vector. Any retroviral / lentiviral (e.g., SIV) vector as described herein can be used in combination with any one or more CFTR modulators, such as those described and exemplified herein.

[0217] Formulation and administration The retroviral / lentiviral (e.g., SIV) vectors and CFTR modulators of the present invention can each be administered independently at any dose suitable to achieve the desired therapeutic effect. Appropriate dosages can be determined by a clinician or other medical practitioner using standard techniques and in the normal course of their practice.

[0218] Non-limiting examples of suitable dosages for retroviral / lentiviral (e.g., SIV) vectors include about 1×10 6 (It can also be written as 10 6 ) transduction units (TU) to approximately 1×10 14 (It can also be written as 10 14 ) TUs, preferably about 10 6 TU to about 10 12 TUs, such as about 10 6 TU, 1.5×10 6 TUs, 10 7 TU, 1.5×10 7 TUs, 10 8 TU, 1.5×10 8 TU, 5×10 8 TU, 8×10 8 TUs, 10 9 TU, 1.5×10 9 TUs, 10 10 TU, 1.5×10 10 TUs, 10 11 TU, 1.5×10 11 TU or more. The preferred dosage range is included in about 8 8 to about 10 14 TUs, or between about 10 6 to about 10 12 These doses may be administered at any dosing interval determined by the treating clinician, such as those 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). As a non-limiting example, about 10 6 A dose of TU may be administered once every 6 months. As another non-limiting example, approximately 10 10 A dose of TU may be administered every 12 months.

[0219] Each CFTR modulator can be used with the standard dose indicated for the monotherapy of CF of the CFTR modulator, that is, with the approval or standard dose / concentration of the monotherapy with the modulator.Each CFTR modulator can be used as a part of the combination therapy according to the present invention, with a dosage lower than the standard dose indicated by the monotherapy with the CFTR modulator, that is, with a concentration lower than the approval or standard dose / concentration of the monotherapy with the modulator.CFTR modulator can be used at a dosage 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 dosages can be used at any dosing time interval determined by the treatment clinician, such as those described herein (for example, with a frequency of every 4 hours, every 8 hours or every 12 hours, preferably every 12 hours).

[0220] As a non-limiting example, ivacaftor can be administered at a dose of about 5 mg to about 150 mg, preferably about 25 mg to about 150 mg, such as about 150 mg every 12 hours. As another non-limiting example, for pediatric administration, ivacaftor can be administered at a dose of about 75 mg every 12 hours.

[0221] As another non-limiting example, Trikafta® (elexacator + tesacator + ivacaftor) can be administered every 12 hours, with the first (typically in the morning) dose of Trikafta® typically comprising about 200 mg elexacator, about 100 mg tesacator, and about 150 mg ivacaftor (e.g., in the form of 2 tablets, each containing elexacator + tesacator + ivacaftor), and the second (typically in the evening) dose being about 150 mg ivacaftor (e.g., in the form of 2 tablets).

[0222] As another non-limiting example, Orkambi® (lumacator + ivacaftor) can be administered every 12 hours, wherein each dose of Orkambi® typically comprises about 400 mg of lumacaftor and about 250 mg of ivacaftor (e.g., in the form of 2 tablets, each containing lumacaftor + ivacaftor). As another non-limiting example, for pediatric dosing, Orkambi® can be administered every 12 hours, wherein each dose of Orkambi® typically comprises about 200 mg of lumacaftor and about 250 mg of ivacaftor (e.g., in the form of 2 tablets, each containing lumacaftor + ivacaftor).

[0223] As another non-limiting example, Symdeko® (tesakator + ivacaftor) can be administered every 12 hours, with the first (typically in the morning) dose of Symdeko® typically comprising about 100 mg tesakator and about 150 mg ivacaftor (e.g., in the form of a single tablet containing tesakator + ivacaftor), and the second (typically in the evening) dose being about 150 mg ivacaftor (e.g., in the form of 1 tablet).

[0224] Combination therapies according to the present invention may employ compositions comprising the retroviral / lentiviral (eg, SIV) vectors described above and a pharmaceutically acceptable carrier. Typically, such compositions are formulated for administration by inhalation.

[0225] The CFTR modulators used in the combination therapies of the present invention may be suitably formulated in the form of a composition comprising a pharmaceutically acceptable carrier. CFTR modulators are typically formulated for oral administration.

[0226] The administration of CFTR modulators is generally carried out by conventional routes, such as oral, intravenous, subcutaneous, intraperitoneal or mucosal routes. It can be administered by parenteral injection, such as subcutaneous, intradermal or intramuscular injection. For example, CFTR modulators may be particularly suitable for oral administration. The administration of small molecule CFTR modulators may be injection, such as intravenous, intramuscular, intradermal or subcutaneous injection, or preferably by oral administration (small molecules with a molecular weight of less than 500 Da generally exhibit oral bioavailability).

[0227] CFTR modulators can be prepared as injectables in the form of liquid solutions or suspensions. Alternatively, solid forms suitable for dissolution or suspension in liquids prior to injection can be prepared. The formulation can also be emulsified, or the peptide can be encapsulated in liposomes or microcapsules.

[0228] Preferably, the CFTR modulator is prepared for oral administration. The CFTR modulator can be encapsulated in an oral dosage form. Oral formulations include conventional excipients such as, for example, pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, and the like. These compositions are in the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders.

[0229] The active ingredient (such as the CFTR modulator used in the combination therapy of the present invention) is generally mixed with a pharmaceutically acceptable excipient that is compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if necessary, the composition may contain small amounts of auxiliary substances, such as wetting agents or emulsifiers, pH buffers, and / or adjuvants that enhance the effect of the CFTR modulator.

[0230] Non-limiting examples of pharmaceutically acceptable carriers include water, saline, and phosphate-buffered saline. However, in some embodiments, 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 thimerosal or sodium azide to facilitate long-term storage.

[0231] Preferably, the CFTR modulators used in the combination therapies of the present invention are formulated for oral administration and are administered orally. Thus, the present invention generally relates to combination therapies using two separate formulations, one comprising a retroviral / lentiviral (e.g., SIV) vector (typically for inhaled administration) and one comprising one or more CFTR modulators (typically for oral administration).

[0232] The retroviral / lentiviral (e.g., SIV) vectors of the present invention and / or one or more CFTR modulators can each be administered independently by any appropriate route. It may be desirable to direct the composition of the present invention (as described above) to the respiratory system of the individual. Effective delivery of the therapeutic / prophylactic composition or drug to the site of infection in the respiratory tract can be achieved by oral administration or by inhalation (e.g., as an aerosol) or by catheter. Typically, the retroviral / lentiviral (e.g., SIV) vectors of the present invention are administered by inhalation and are therefore preferably stable in clinically relevant nebulizers, inhalers (including metered dose inhalers), catheters, and aerosols, etc. Typically, one or more CFTR modulators are administered orally.

[0233] Formulations for inhalation administration may be in the form of droplets and may be administered by atomization using a suitable device. Formulations for inhalation administration may comprise droplets having a mass median aerodynamic diameter (MMAD) of approximately 0.1-50 μm, such as 1-25 μm, 1-10 μm, or 1-5 μm, particularly 1-10 μm. Alternatively, in terms of volume, the droplets may be in the range of about 0.001-100 μl, such as 0.1-50 μl or 1.0-25 μl, or such as 0.001-1 μl.

[0234] Aerosol formulations can be in the form of powders, suspensions, or solutions. The size of the aerosol droplets is related to the delivery capacity of the aerosol. Smaller droplets can travel farther along the respiratory airways toward the alveoli than larger particles. In one embodiment, the aerosol droplets have a diameter distribution that facilitates delivery along the entire length of the trachea, bronchi, and bronchioles (i.e., the conducting airways). Alternatively, the droplet size distribution can be selected to target a specific portion of the respiratory airways, such as the bronchioles or alveoli. In the case of aerosol delivery of the drug, the diameter of the droplets can be in the following approximate range: 0.1-50 μm, preferably 1-25 μm, more preferably 1-10 μm or 1-5 μm.

[0235] Aerosol particles may be used for delivery using a nebulizer (eg, via the mouth) or a nasal spray.Aerosol formulations may alternatively contain a propellant and / or a surfactant.

[0236] The formulation of pharmaceutical aerosols is routine to those skilled in the art, see, for example, Sciarra, J. in Remington's Pharmaceutical Sciences. (Same as above) The medicament can be formulated as a solution aerosol, dispersion or suspension aerosol of a dry powder, emulsion or semisolid preparation. The aerosol can be delivered using any propellant system known to those skilled in the art. The aerosol can be applied to the upper respiratory tract, or to the lower respiratory tract, or to both, for example, by nasal inhalation. The part of the lung to which the drug is delivered can be determined by the condition. The composition comprising the carrier of the present invention, particularly when intranasal delivery is to be used, may comprise a moisturizer. This can help reduce or prevent dryness of the mucous membranes and prevent irritation to the membranes. Suitable moisturizers include, for example, sorbitol, mineral oil, vegetable oil and glycerin; soothing agents; membrane regulators; sweeteners; and combinations thereof. The composition may comprise a surfactant. Suitable surfactants include nonionic, anionic and cationic surfactants. Examples of surfactants that can be used include, for example, polyoxyethylene derivatives of fatty acid partial esters of sorbitan, such as, for example, Tween 80, polyethylene glycol 40 stearate, polyoxyethylene 50 stearate, fusieate, bile salts and octoxynol.

[0237] As described herein, in some cases, subsequent administration of a retroviral / lentiviral (e.g., SIV) vector may be performed after the initial administration. The administration may, for example, be at least one week, two weeks, one month, two months, three months, four months, six months, one year, or longer after the initial administration. In some cases, the retroviral / lentiviral (e.g., SIV) vector of the present invention may be administered at least once a week, once every two weeks, once a month, every two months, every six months, annually, or at longer intervals. Preferably, administration is performed every six months, more preferably annually. The retroviral / lentiviral (e.g., SIV) vector may be administered, for example, at intervals specified when the effect of the previous administration diminishes. Administration of the retroviral / lentiviral (e.g., SIV) vector may continue at a desired frequency for the life of the patient.

[0238] Also as described herein, CFTR modulators are typically administered continuously at a frequency as described herein. CFTR modulators can be administered, for example, at prescribed intervals as the effect of the previous administration diminishes. CFTR modulators can continue to be administered at a desired frequency for the life of the patient.

[0239] The combination therapy of the present invention may be combined with one or more additional treatments for CF, including one or more additional CF regulating agents, bronchodilators, steroids, agents that thin or clear mucus or other lung secretions, antibiotics, and / or airway clearance techniques, such as active breathing cycle technique (ACBT) and autologous drainage. The one or more additional treatments for CF may be administered sequentially or simultaneously (as defined herein) with the combination therapy of the present invention.

[0240] Sequence homology Any of a variety of sequence alignment methods can be used to determine percent identity, including but not limited to global methods, local methods, and hybrid methods, such as, for example, segmented methods. Protocols for determining percent identity are routine procedures within the skill of the art. Global methods align sequences from the beginning to the end of the molecule and determine the optimal alignment by accumulating the scores for individual residue pairs and by imposing gap penalties. Non-limiting methods include, for example, CLUSTAL W, see, for example, 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, for example, Osamu Gotoh, Significant Improvement in Accuracy of Multiple Protein. Sequence Alignments by Iterative Refinement as Assessed by Reference to Structural Alignments, 264 (4) J. MoI. Biol. 823-838 (1996). Local methods align sequences by identifying one or more conserved motifs shared by all input sequences.Non-limiting methods include, for example, Match-box, see, for example, Eric Depiereux and Ernest Feytmans, Match-Box: AFundamentally New Algorithm for the Simultaneous Alignment of Several Protein Sequences, 8(5) CABIOS 501-509 (1992); Gibbs sampling, see, for example, 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, for example, Ivo Van Walle et al., Align-M - A New Algorithm for Multiple Alignment of Highly Divergent Sequences, 20(9) Bioinformatics: 1428-1435 (2004).

[0241] Therefore, the 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 using a gap opening penalty of 10, a gap extension penalty of 1, and the "blosum 62" scoring matrix of Henikoff and Henikoff (supra) as shown below to optimize the alignment score (amino acids are indicated by the standard single-letter code).

[0242] The "percentage of 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 can be calculated as the number of identical nucleotides / amino acids divided by the total number of nucleotides / amino acids multiplied by 100. The calculation of % sequence identity can also take into account the number of gaps and the length of each gap that need to be introduced to optimize the alignment of two or more sequences. Sequence comparisons between two or more sequences and determination of percent identity can be performed using specific mathematical algorithms, such as BLAST, which will be familiar to those skilled in the art.

[0243] Alignment score used to determine 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: (Total number of identical matches / length of the longer sequence plus the number of gaps introduced into the longer sequence to align the two sequences) × 100 Substantially homologous polypeptides are characterized by having one or more amino acid substitutions, deletions, or additions. These changes are preferably of a minor nature, i.e., 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 one to about 30 amino acid deletions; 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.

[0244] In addition to the 20 standard amino acids, non-standard amino acids (such as 4-hydroxyproline, 6-N-methyllysine, 2-aminoisobutyric acid, isovaline, and α-methylserine) can be substituted for amino acid residues in the polypeptides of the present invention. A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, and unnatural amino acids can be substituted for polypeptide amino acid residues. The polypeptides of the present invention may also contain non-naturally occurring amino acid residues.

[0245] Non-naturally occurring amino acids include, but are not limited to, trans-3-methylproline, 2,4-methylene-proline, cis-4-hydroxyproline, trans-4-hydroxy-proline, N-methylglycine, allo-threonine, methyl-threonine, hydroxy-ethylcysteine, hydroxyethylhomo-cysteine, nitro-glutamine, homoglutamine, 2-piperidinic acid, tertiary leucine, norvaline, 2-azaphenylalanine, 3-azaphenyl-alanine, 4-azaphenyl-alanine, and 4-fluorophenylalanine. Several methods for incorporating non-naturally occurring amino acid residues into proteins are known in the art. For example, an in vitro system can be employed in which nonsense mutations are suppressed using chemically amine-acylated suppressor tRNAs. Methods for synthesizing amino acids and amine-acylated tRNAs are known in the art. Transcription and translation of plastids containing nonsense mutations are performed in a cell-free system containing Escherichia coli (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 approach, translation is performed in Xenopus oocytes by microinjection of mutant mRNA and chemically aminoacylated suppressor tRNA (Turcatti et al., J. Biol. Chem. 271:19991-8, 1996). In the third approach, E. coli cells are cultured in the absence of the natural amino acid to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid (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 scope of substitution (Wynn and Richards, Protein Sci. 2:395-403, 1993).

[0246] A limited number of non-conservative amino acids, amino acids not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids can be substituted for amino acid residues in the polypeptides of the invention.

[0247] Essential amino acids in the polypeptides of the 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 the structure, as determined by techniques such as nuclear magnetic resonance, crystallography, electron diffraction, or photoaffinity labeling, combined 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 identity of essential amino acids can also be inferred from homology analysis with related components of the polypeptides of the invention (e.g., translocation or protease components).

[0248] 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 functional polypeptides, and then sequencing the mutagenized polypeptides to determine the spectrum of permissible 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. Patent No. 5,223,409; Huse, WIPO Publication WO 92 / 06204) and localized mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0249] 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 functional polypeptides, and then sequencing the mutagenized polypeptides to determine the spectrum of permissible 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. Patent No. 5,223,409; Huse, WIPO Publication WO 92 / 06204) and localized mutagenesis (Derbyshire et al., Gene 46:145, 1986; Ner et al., DNA 7:127, 1988).

[0250] Sequence information Sequence key table SEQ ID NO: 1 Exemplary CFTR transgene ( soCFTR2 ) SEQ ID NO: 2 Exemplary hCEF promoter SEQ ID NO: 3 Exemplary CMV promoter SEQ ID NO: 4 Exemplary EF1a promoter SEQ ID NO: 5 Wild-type SIV gag-pol nucleic acid sequence SEQ ID NO: 6 Codon-optimized SIV gal-pol nucleic acid sequence SEQ ID NO: 7 according to Figure 1C The plasmid defined in (pDNA2a pGM691) SEQ ID NO: 8 Figure 1A The plasmid defined in (pDNA1 pGM326) SEQ ID NO: 9 according to Figure 1B The plasmid defined in (pDNA1 pGM830) SEQ ID NO: 10 Figure 1D The plasmid defined in (pDNA2a pGM297) SEQ ID NO: 11 Figure 1E The plasmid defined in (pDNA2b pGM299) SEQ ID NO: 12 Figure 1F The plasmid defined in (pDNA3a pGM301) SEQ ID NO: 13 Figure 1G The plasmid defined in (pDNA3b pGM303) SEQ ID NO: 14 Exemplary 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 sequence SEQ ID NO: 1 Exemplary CFTR transgene (soCFTR2) 1 GCTAGCCACC ATGCAGAGAA GCCCTCTGGA GAAGGCCTCT GTGGTGAGCA AGCTGTTCTT 61 CAGCTGGACC AGGCCCATCC TGAGGAAGGG CTACAGGCAG AGACTGGAGC TGTCTGACAT 121 CTACCAGATC CCCTCTGTGG ACTCTGCTGA CAACCTGTCT GAGAAGCTGG AGAGGGAGTG 181 GGATAGAGAG CTGGCCAGCA AGAAGAACCC CAAGCTGATC AATGCCCTGA GGAGATGCTT 241 CTTCTGGAGA TTCATGTTCT ATGGCATCTT CCTGTACCTG GGGGAAGTGA CCAAGGCTGT 301 GCAGCCTCTG CTGCTGGGCA GAATCATTGC CAGCTATGAC CCTGACAACA AGGAGGAGAG 361 GAGCATTGCC ATCTACCTGG GCATTGGCCT GTGCCTGCTG TTCATTGTGA GGACCCTGCT 421 GCTGCACCCT GCCATCTTTG GCCTGCACCA CATTGGCATG CAGATGAGGA TTGCCATGTT 481 CAGCCTGATC TACAAGAAAA CCCTGAAGCT GTCCAGCAGA GTGCTGGACA AGATCAGCAT 541 TGGCCAGCTG GTGAGCCTGC TGAGCAACAA CCTGAACAAG TTTGATGAGG GCCTGGCCCT 601 GGCCCACTTT GTGTGGATTG CCCCTCTGCA GGTGGCCCTG CTGATGGGCC TGATTTGGGA 661 GCTGCTGCAG GCCTCTGCCT TTTGTGGCCT GGGCTTCCTG ATTGTGCTGG CCCTGTTTCA 721 GGCTGGCCTG GGCAGGATGA TGATGAAGTA CAGGGACCAG AGGGCAGGCA AGATCAGTGA 781 GAGGCTGGTG ATCACCTCTG AGATGATTGA GAACATCCAG TCTGTGAAGG CCTACTGTTG 841 GGAGGAAGCT ATGGAGAAGA TGATTGAAAA CCTGAGGCAG ACAGAGCTGA AGCTGACCAG 901 GAAGGCTGCC TATGTGAGAT ACTTCAACAG CTCTGCCTTC TTCTTCTCTG GCTTCTTTGT 961 GGTGTTCCTG TCTGTGCTGC CCTATGCCCT GATCAAGGGG ATCATCCTGA GAAAGATTTT 1021 CACCACCATC AGCTTCTGCA TTGTGCTGAG GATGGCTGTG ACCAGACAGT TCCCCTGGGC 1081 TGTGCAGACC TGGTATGACA GCCTGGGGGC CATCAACAAG ATCCAGGACT TCCTGCAGAA 1141 GCAGGAGTAC AAGACCCTGG AGTACAACCT GACCACCACA GAAGTGGTGA TGGAGAATGT 1201 GACAGCCTTC TGGGAGGAGG GCTTTGGGGA GCTGTTTGAG AAGGCCAAGC AGAACAACAA 1261 CAACAGAAAG ACCAGCAATG GGGATGACTC CCTGTTCTTC TCCAACTTCT CCCTGCTGGG 1321 CACACCTGTG CTGAAGGACA TCAACTTCAA GATTGAGAGG GGGCAGCTGC TGGCTGTGGC 1381 TGGATCTACA GGGGCTGGCA AGACCAGCCT GCTGATGATG ATCATGGGGG AGCTGGAGCC 1441 TTCTGAGGGC AAGATCAAGC ACTCTGGCAG GATCAGCTTT TGCAGCCAGT TCAGCTGGAT 1501 CATGCCTGGC ACCATCAAGG AGAACATCAT CTTTGGAGTG AGCTATGATG AGTACAGATA 1561 CAGGAGTGTG ATCAAGGCCT GCCAGCTGGA GGAGGACATC AGCAAGTTTG CTGAGAAGGA 1621 CAACATTGTG CTGGGGGAGG GAGGCATTAC ACTGTCTGGG GGCCAGAGAG CCAGAATCAG 1681 CCTGGCCAGG GCTGTGTACA AGGATGCTGA CCTGTACCTG CTGGACTCCC CCTTTGGCTA 1741 CCTGGATGTG CTGACAGAGA AGGAGATTTT TGAGAGCTGT GTGTGCAAGC TGATGGCCAA 1801 CAAGACCAGA ATCCTGGTGA CCAGCAAGAT GGAGCACCTG AAGAAGGCTG ACAAGATCCT 1861 GATCCTGCAT GAGGGCAGCA GCTACTTCTA TGGGACCTTC TCTGAGCTGC AGAACCTGCA 1921 GCCTGACTTC AGCTCTAAGC TGATGGGCTG TGACAGCTTT GACCAGTTCT CTGCTGAGAG 1981 GAGGAACAGC ATCCTGACAG AGACCCTGCA CAGATTCAGC CTGGAGGGAG ATGCCCCTGT 2041 GAGCTGGACA GAGACCAAGA AGCAGAGCTT CAAGCAGACA GGGGAGTTTG GGGAGAAGAG 2101 GAAGAACTCC ATCCTGAACC CCATCAACAG CATCAGGAAG TTCAGCATTG TGCAGAAAAC 2161 CCCCCTGCAG ATGAATGGCA TTGAGGAAGA TTCTGATGAG CCCCTGGAGA GGAGACTGAG 2221 CCTGGTGCCT GATTCTGAGC AGGGAGAGGC CATCCTGCCT AGGATCTCTG TGATCAGCAC 2281 AGGCCCTACA CTGCAGGCCA GAAGGAGGCA GTCTGTGCTG AACCTGATGA CCCACTCTGT 2341 GAACCAGGGC CAGAACATCC ACAGGAAAAC CACAGCCTCC ACCAGGAAAG TGAGCCTGGC 2401 CCCTCAGGCC AATCTGACAG AGCTGGACAT CTACAGCAGG AGGCTGTCTC AGGAGACAGG 2461 CCTGGAGATT TCTGAGGAGA TCAATGAGGA GGACCTGAAA GAGTGCTTCT TTGATGACAT 2521 GGAGAGCATC CCTGCTGTGA CCACCTGGAA CACCTACCTG AGATACATCA CAGTGCACAA 2581 GAGCCTGATC TTTGTGCTGA TCTGGTGCCT GGTGATCTTC CTGGCTGAAG TGGCTGCCTC 2641 TCTGGTGGTG CTGTGGCTGC TGGGAAACAC CCCACTGCAG GACAAGGGCA ACAGCACCCA 2701 CAGCAGGAAC AACAGCTATG CTGTGATCAT CACCTCCACC TCCAGCTACT ATGTGTTCTA 2761 CATCTATGTG GGAGTGGCTG ATACCCTGCT GGCTATGGGC TTCTTTAGAG GCCTGCCCCT 2821 GGTGCACACA CTGATCACAG TGAGCAAGAT CCTCCACCAC AAGATGCTGC ACTCTGTGCT 2881 GCAGGCTCCT ATGAGCACCC TGAATACCCT GAAGGCTGGG GGCATCCTGA ACAGATTCTC 2941 CAAGGATATT GCCATCCTGG ATGACCTGCT GCCTCTCACC ATCTTTGACT TCATCCAGCT 3001 GCTGCTGATT GTGATTGGGG CCATTGCTGT GGTGGCAGTG CTGCAGCCCT ACATCTTTGT 3061 GGCCACAGTG CCTGTGATTG TGGCCTTCAT CATGCTGAGG GCCTACTTTC TGCAGACCTC 3121 CCAGCAGCTG AAGCAGCTGG AGTCTGAGGG CAGAAGCCCC ATCTTCACCC ACCTGGTGAC 3181 AAGCCTGAAG GGCCTGTGGA CCCTGAGAGC CTTTGGCAGG CAGCCCTACT TTGAGACCCT 3241 GTTCCACAAG GCCCTGAACC TGCACACAGC CAACTGGTTC CTCTACCTGT CCACCCTGAG 3301 ATGGTTCCAG ATGAGAATTG AGATGATCTT TGTCATCTTC TTCATTGCTG TGACCTTCAT 3361 CAGCATTCTG ACCACAGGAG AGGGAGAGGG CAGAGTGGGC ATTATCCTGA CCCTGGCCAT 3421 GAACATCATG AGCACACTGC AGTGGGCAGT GAACAGCAGC ATTGATGTGG ACAGCCTGAT 3481 GAGGAGTGTG AGCAGAGTGT TCAAGTTCAT TGATATGCCC ACAGAGGGCA AGCCTACCAA 3541 GAGCACCAAG CCCTACAAGA ATGGCCAGCT GAGCAAAGTG ATGATCATTG AGAACAGCCA 3601 TGTGAAGAAG GATGATATCT GGCCCAGTGG AGGCCAGATG ACAGTGAAGG ACCTGACAGC 3661 CAAGTACACA GAGGGGGGCA ATGCTATCCT GGAGAACATC TCCTTCAGCA TCTCCCCTGG 3721 CCAGAGAGTG GGACTGCTGG GAAGAACAGG CTCTGGCAAG TCTACCCTGC TGTCTGCCTT 3781 CCTGAGGCTG CTGAACACAG AGGGAGAGAT CCAGATTGAT GGAGTGTCCT GGGACAGCAT 3841 CACACTGCAG CAGTGGAGGA AGGCCTTTGG TGTGATCCCC CAGAAAGTGT TCATCTTCAG 3901 TGGCACCTTC AGGAAGAACC TGGACCCCTA TGAGCAGTGG TCTGACCAGG AGATTTGGAA 3961 AGTGGCTGAT GAAGTGGGCC TGAGAAGTGT GATTGAGCAG TTCCCTGGCA AGCTGGACTT 4021 TGTCCTGGTG GATGGGGGCT GTGTGCTGAG CCATGGCCAC AAGCAGCTGA TGTGCCTGGC 4081 CAGATCAGTG CTGAGCAAGG CCAAGATCCT GCTGCTGGATGAGCCTTCTG CCCACCTGGA 4141 TCCTGTGACC TACCAGATCA TCAGGAGGAC CCTCAAGCAG GCCTTTGCTG ACTGCACAGT 4201 CATCCTGTGT GAGCACAGGA TTGAGGCCAT GCTGGAGTGC CAGCAGTTCC TGGTGATTGA 4261 GGAGAACAAA GTGAGGCAGT ATGACAGCAT CCAGAAGCTG CTGAATGAGA GGAGCCTGTT 4321 CAGGCAGGCC ATCAGCCCCT CTGATAGAGT GAAGCTGTTC CCCCACAGGA ACAGCTCCAA 4381 GTGCAAGAGC AAGCCCCAGA TTGCTGCCCT GAAGGAGGAG ACAGAGGAGG AAGTGCAGGA 4441 CACCAGGCTG TGAGGGCCC SEQ ID NO: 2 Exemplary hCEF promoter 1 AGATCTGTTA CATAACTTAT GGTAAATGGC CTGCCTGGCT GACTGCCCAA TGACCCCTGC 61 CCAATGATGT CAATAATGAT GTATGTTCCC ATGTAATGCC AATAGGGACT TTCCATTGAT 121 GTCAATGGGT GGAGTATTTA TGGTAACTGC CCACTTGGCA GTACATCAAG TGTATCATAT 181 GCCAAGTATG CCCCCTATTG ATGTCAATGA TGGTAAATGG CCTGCCTGGC ATTATGCCCA 241 GTACATGACC TTATGGGACT TTCCTACTTG GCAGTACATC TATGTATTAG TCATTGCTAT 301 TACCATGGGA ATTCACTAGT GGAGAAGAGC ATGCTTGAGG GCTGAGTGCC CCTCAGTGGG 361 CAGAGAGCAC ATGGCCCACA GTCCCTGAGA AGTTGGGGGG AGGGGTGGGC AATTGAACTG 421 GTGCCTAGAG AAGGTGGGGC TTGGGTAAAC TGGGAAAGTG ATGTGGTGTA CTGGCTCCAC 481 CTTTTTCCCC AGGGTGGGGG AGAACCATAT ATAAGTGCAG TAGTCTCTGT GAACATTCAA 541 GCTTCTGCCT TCTCCCTCCT GTGAGTTTGC TAGC SEQ ID NO: 3 Exemplary CMV promoter CCGCGGAGATCTCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCT ATTGGCCATTGCATACGTTGTATCTATATCAT AATATGTACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCA TATATG GAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTT TCCATTGACGTCAATGGGT GGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCG CCTGGCATTATGCCCAGTACATGACCTTACGGGACTTTCCTACTT GGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGC GTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGG CACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCG CCCCGTTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCACTAGAAGCTTTATTGC GGTAGTTTATCACAGTT AAATTGCTAACGCAGTCAGTGCTTCTGACACAACAGTCTCGAACTTAAGCTGCAGAAGTTGGTCGTGAGGCACTGGGCAGGCTAGC SEQ ID NO: 4 Exemplary EF1a promoter AGATCCATATCCGCGGCAATTTTAAAAGAAAGGGAGGAATAGGGGGACAGACTTCAGCAGAGAGACTAATTAATATAATAACAACACAATTAGAAATACAACAT TTACAAACCAAAATTCAAAAAATTTTAAATTTTAGAGCCGCGGAGATCCCGTGAGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGG AGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTG CAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGCTAGC SEQ ID NO: 5 Wild-type SIV gag-pol nucleic acid sequence (from pGM297) ATGGGGGCGGCTACCTCAGCACTAAATAGGAGACAATTAGACCAATTTGAGAAAATACGACTTCGCCCGAACGGAAAGAAAAAGTACCAAATTAAACATTTAAT ATGGGCAGGCAAGGAGATGGAGCGCTTCGGCCTCCATGAGAGGTTGTTGGAGACAGAGGAGGGGTGTAAAAGAATCATAGAAGTCCTCTACCCCCTAGAACCAACAGGATCGGAGG GCTTAAAAAGTCTGTTCAATCTTGTGTGCGTACTATATTGCTTGCACAAGGAACAGAAAGTGAAAGACACAGAGGAAGCAGTAGCAACAGTAAGACAACACTGCCATCTAGTGGAA AAAGAAAAAAGTGCAACAGAGACATCTAGTGGACAAAAGAAAAATGACAAGGGAATAGCAGCGCCACCTGGTGGCAGTCAGAATTTTCCAGCGCAACAACAAGGAAATGCCTGGGT ACATGTACCCTTGTCACCGCGCACCTTAAATGCGTGGGTAAAAGCAGTAGAGGAGAAAAAATTTGGAGCAGAAATAGTACCCATGTTTCAAGCCCTATCAGAAGGCTGCACACCCT ATGACATTAATCAGATGCTTAATGTGCTAGGAGATCATCAAGGGGCATTACAAATAGTGAAAGAGATCATTAATGAAGAAGCAGCCCAGTGGGATGTAACACACCCACTACCCGCA GGACCCCTACCAGCAGGACAGCTCAGGGACCCTCGCGGCTCAGATATAGCAGGGACCACCAGCTCAGTACAAGAACAGTTAGAATGGATCTATACTGCTAACCCCCGGGTAGATGT AGGTGCCATCTACCGGAGATGGATTATTCTAGGACTTCAAAAGTGTGTCAAAATGTACAACCCAGTATCAGTCCTAGACATTAGGCAGGGACCTAAAGAGCCCTTCAAGGATTATG TGGACAGATTTTACAAGGCAATTAGAGCAGAACAAGCCTCAGGGGAAGTGAAACAATGGATGACAGAATCATTACTCATTCAAAATGCTAATCCAGATTGTAAGGTCATCCTGAAG GGCCTAGGAATGCACCCCACCCTTGAAGAAATGTTAACGGCTTGTCAGGGGGTAGGAGGCCCAAGCTACAAAGCAAAAGTAATGGCAGAAATGATGCAGACCATGCAAAATCAAAA CATGGTGCAGCAGGGAGGTCCAAAAAGACAAAGACCCCCACTAAGATGTTATAATTGTGGAAAATTTGGCCATATGCAAAGACAATGTCCGGAACCAAGGAAAACAAAATGTCTAA AGTGTGGAAAATTGGGACACCTAGCAAAAGACTGCAGGGGACAGGTGAATTTTTTAGGGTATGGACGGTGGATGGGGGCAAAACCGAGAAATTTTCCCGCCGCTACTCTTGGAGCG GAACCGAGTGCGCCTCCTCCACCGAGCGGCACCACCCCATACGACCCAGCAAAGAAGCTCCTGCAGCAATATGCAGAGAAAGGGAAACAACTGAGGGAGCAAAAGAGGAATCCACC GGCAATGAATCCGGATTGGACCGAGGGATATTCTTTGAACTCCCTCTTTGGAGAAGACCAATAAAGACAGTGTATATAGAAGGGGTCCCCATTAAGGCACTGCTAGACACAGGGGC AGATGACACCATAATTAAAGAAAATGATTTACAATTATCAGGTCCATGGAGACCCAAAATTATAGGGGGCATAGGAGGAGGCCTTAATGTAAAAGAATATAACGACAGGGAAGTAA AAATAGAAGATAAAATTTTGAGAGGAACAATATTGTTAGGAGCAACTCCCATTAATATAATAGGTAGAAATTTGCTGGCCCCGGCAGGTGCCCGGTTAGTAATGGGACAATTATCA GAAAAAATTCCTGTCACACCTGTCAAATTGAAGGAAGGGGCTCGGGGACCCTGTGTAAGACAATGGCCTCTCTCTAAAGAGAAGATTGAAGCTTTACAGGAAATATGTTCCCAATT AGAGCAGGAAGGAAAAATCAGTAGAGTAGGAGGAGAAAATGCATACAATACCCCAATATTTTGCATAAAGAAGAAGGACAAATCCCAGTGGAGGATGCTAGTAGACTTTAGAGAGT TAAATAAGGCAACCCAAGATTTCTTTGAAGTGCAATTAGGGATACCCCACCCAGCAGGATTAAGAAAGATGAGACAGATAACAGTTTTAGATGTAGGAGACGCCTATTATTCCATA CCATTGGATCCAAATTTTAGGAAATATACTGCTTTTACTATTCCCACAGTGAATAATCAGGGACCCGGGATTAGGTATCAATTCAACTGTCTCCCGCAAGGGTGGAAAGGATCTCC TACAATCTTCCAAAATACAGCAGCATCCATTTTGGAGGAGATAAAAAGAAACTTGCCAGCACTAACCATTGTACAATACATGGATGATTTATGGGTAGGTTCTCAAGAAAATGAAC ACACCCATGACAAATTAGTAGAACAGTTAAGAACAAAATTACAAGCCTGGGGCTTAGAAACCCCAGAAAAGAAGGTGCAAAAAGAACCACCTTATGAGTGGATGGGATACAAACTT TGGCCTCACAAATGGGAACTAAGCAGAATACAACTGGAGGAAAAAGATGAATGGACTGTCAATGACATCCAGAAGTTAGTTGGGAAACTAAATTGGGCAGCACAATTGTATCCAGG TCTTAGGACCAAGAATATATGCAAGTTAATTAGAGGAAAGAAAAATCTGTTAGAGCTAGTGACTTGGACACCTGAGGCAGAAGCTGAATATGCAGAAAATGCAGAGATTCTTAAAA CAGAACAGGAAGGAACCTATTACAAACCAGGAATACCTATTAGGGCAGCAGTACAGAAATTGGAAGGAGGACAGTGGAGTTACCAATTCAAACAAGAAGGACAAGTCTTGAAAGTA GGAAAATACACCAAGCAAAAGAACACCCATACAAATGAACTTCGCACATTAGCTGGTTTAGTGCAGAAGATTTGCAAAGAAGCTCTAGTTATTTGGGGGATATTACCAGTTCTAGA ACTCCCGATAGAAAGAGAGGTATGGGAACAATGGTGGGCGGATTACTGGCAGGTAAGCTGGATTCCCGAATGGGATTTTGTCAGCACCCCACCTTTGCTCAAACTATGGTACACAT TAACAAAAGAACCCATACCCAAGGAGGACGTTTACTATGTAGATGGAGCATGCAACAGAAATTCAAAAGAAGGAAAAGCAGGATACATCTCACAATACGGAAAACAGAGAGTAGAA ACATTAGAAAACACTACCAATCAGCAAGCAGAATTAACAGCTATAAAAATGGCTTTGGAAGACAGTGGGCCTAATGTGAACATAGTAACAGACTCTCAATATGCAATGGGAATTTT GACAGCACAACCCACACAAAGTGATTCACCATTAGTAGAGCAAATTATAGCCTTAATGATACAAAAGCAACAAATATATTTGCAGTGGGTACCAGCACATAAAGGAATAGGAGGAA ATGAGGAGATAGATAAATTAGTGAGTAAAGGCATTAGAAGAGTTTTATTCTTAGAAAAAATAGAAGAAGCTCAAGAAGAGCATGAAAGATATCATAATAATTGGAAAAACCTAGCA GATACATATGGGCTTCCACAAATAGTAGCAAAAGAGATAGTGGCCATGTGTCCAAAATGTCAGATAAAGGGAGAACCAGTGCATGGACAAGTGGATGCCTCACCTGGAACATGGCA GATGGATTGTACTCATCTAGAAGGAAAAGTAGTCATAGTTGCGGTCCATGTAGCCAGTGGATTCATAGAAGCAGAAGTCATACCTAGGGAAACAGGAAAAGAAACGGCAAAGTTTC TATTAAAAATACTGAGTAGATGGCCTATAACACAGTTACACACAGACAATGGGCCTAACTTTACCTCCCAAGAAGTGGCAGCAATATGTTGGTGGGGAAAAATTGAACATACAACA GGTATACCATATAACCCCCAATCTCAAGGATCAATAGAAAGCATGAACAAACAATTAAAAGAGATAATTGGGAAAATAAGAGATGATTGCCAATATACAGAGACAGCAGTACTGAT GGCTTGCCATATTCACAATTTTAAAAGAAAGGGAGGAATAGGGGGACAGACTTCAGCAGAGAGACTAATTAATATAATAACAACACAATTAGAAATACAACATTTACAAACCAAAA TTCAAAAAATTTTAAATTTTAGAGTCTACTACAGAGAAGGGAGAGACCCTGTGTGGAAAGGACCAGCACAATTAATCTGGAAAGGGGAAGGAGCAGTGGTCCTCAAGGACGGAAGT GACCTAAAGGTTGTACCAAGAAGGAAAGCTAAAATTATTAAGGATTATGAACCCAAACAAAGAGTGGGTAATGAGGGTGACGTGGAAGGTACCAGGGGATCTGATAACTAA SEQ ID NO: 6 Codon-optimized SIV gal-pol nucleic acid sequence (from pGM691) ATGGGAGCTGCCACATCTGCCCTGAATAGACGGCAGCTGGACCAGTTCGAGAAGATCAGACTGCGGCCCAACGGCAAGAAGAAGTACCAGATCAAGCACCTGAT CTGGGCCGGCAAAGAGATGGAAAGATTCGGCCTGCACGAGCGGCTGCTGGAAACCGAGGAAGGCTGCAAGAGAATTATCGAGGTGCTGTACCCTCTGGAACCTACCGGCTCTGAGG GCCTGAAGTCCCTGTTCAATCTCGTGTGCGTGCTGTACTGCCTGCACAAAGAACAGAAAGTGAAGGACACCGAAGAGGCCGTGGCCACAGTTAGACAGCACTGCCACCTGGTGGAA AAAGAGAAGTCCGCCACAGAGACAAGCAGCGGCCAGAAGAAGAACGACAAGGGAATTGCTGCCCCTCCTGGCGGCAGCCAGAATTTTCCTGCTCAGCAGCAGGGAAACGCCTGGGT GCACGTTCCACTGAGCCCTAGAACACTGAATGCCTGGGTCAAAGCCGTGGAAGAGAAGAAGTTTGGCGCCGAGATCGTGCCCATGTTCCAGGCTCTGTCTGAGGGCTGCACCCCTT ACGACATCAACCAGATGCTGAACGTGCTGGGAGATCACCAGGGCGCTCTGCAGATCGTGAAAGAGATCATCAACGAAGAGGCTGCCCAGTGGGACGTGACACATCCATTGCCTGCTGGACCTCTGCCAGCCGGACAACTGAGAGATCCTAGAGGCTCTGATATCGCCGGCACCACCAGCTCTGTGCAAGAGCAGCTGGAATGGATCTACACCGCCAATCCTAGAGTGGACGT GGGCGCCATCTACAGAAGATGGATCATCCTGGGCCTGCAGAAATGCGTGAAGATGTACAACCCCGTGTCCGTGCTGGACATCAGACAGGGACCCAAAGAGCCCTTCAAGGACTACG TGGACCGGTTCTATAAGGCCATTAGAGCCGAGCAGGCCAGCGGCGAAGTGAAGCAGTGGATGACAGAGAGCCTGCTGATCCAGAACGCCAATCCAGACTGCAAAGTGATCCTGAAA GGCCTGGGCATGCACCCCACACTGGAAGAGATGCTGACAGCCTGTCAAGGCGTTGGCGGCCCTTCTTACAAAGCCAAAGTGATGGCCGAGATGATGCAGACCATGCAGAACCAGAA CATGGTGCAGCAAGGCGGCCCTAAGAGACAGAGGCCTCCTCTGAGATGCTACAACTGCGGCAAGTTCGGCCACATGCAGAGACAGTGTCCTGAGCCTAGGAAAACAAAATGTCTAA AGTGTGGAAAATTGGGACACCTAGCAAAAGACTGCAGGGGACAGGTGAATTTTTTAGGGTATGGACGGTGGATGGGGGCAAAACCGAGAAATTTTCCCGCCGCTACTCTTGGAGCG GAACCGAGTGCGCCTCCTCCACCGAGCGGCACCACCCCATACGACCCAGCAAAGAAGCTCCTGCAGCAATATGCAGAGAAAGGGAAACAACTGAGGGAGCAAAAGAGGAATCCACC GGCAATGAATCCGGATTGGACCGAGGGATATTCTTTGAACTCCCTCTTTGGAGAAGACCAATAAAGACCGTGTACATCGAGGGCGTGCCCATCAAGGCTCTGCTGGATACAGGCGC CGACGACACCATCATCAAAGAGAACGACCTGCAGCTGAGCGGCCCTTGGAGGCCTAAGATCATTGGAGGAATCGGCGGAGGCCTGAACGTCAAAGAGTACAACGACCGGGAAGTGA AGATCGAGGACAAGATCCTGAGGGGCACAATCCTGCTGGGCGCCACACCTATCAACATCATCGGCAGAAATCTGCTGGCCCCTGCCGGCGCTAGACTGGTTATGGGACAGCTCTCT GAGAAGATCCCCGTGACACCCGTGAAGCTGAAAGAAGGCGCTAGAGGACCTTGTGTGCGACAGTGGCCTCTGAGCAAAGAGAAGATTGAGGCCCTGCAAGAAATCTGTAGCCAGCT GGAACAAGAGGGCAAGATCAGCAGAGTTGGCGGCGAGAACGCCTACAATACCCCTATCTTCTGCATCAAGAAAAAGGACAAGAGCCAGTGGCGGATGCTGGTGGACTTTAGAGAGC TGAACAAGGCTACCCAGGACTTCTTCGAGGTGCAGCTGGGAATTCCTCATCCTGCCGGCCTGCGGAAGATGAGACAGATCACAGTGCTGGATGTGGGCGACGCCTACTACAGCATC CCTCTGGACCCCAACTTCAGAAAGTACACCGCCTTCACAATCCCCACCGTGAACAATCAAGGCCCTGGCATCAGATACCAGTTCAACTGCCTGCCTCAAGGCTGGAAGGGCAGCCC CACCATTTTTCAGAATACCGCCGCCAGCATCCTGGAAGAAATCAAGAGAAACCTGCCTGCTCTGACCATCGTGCAGTACATGGACGATCTGTGGGTCGGAAGCCAAGAGAATGAGC ACACCCACGACAAGCTGGTGGAACAGCTGAGAACAAAGCTGCAGGCCTGGGGCCTCGAAACCCCTGAGAAGAAGGTGCAGAAAGAACCTCCTTACGAGTGGATGGGCTACAAGCTG TGGCCTCACAAGTGGGAGCTGAGCCGGATTCAGCTCGAAGAGAAGGACGAGTGGACCGTGAACGACATCCAGAAACTCGTGGGCAAGCTGAATTGGGCAGCCCAGCTGTATCCCGG CCTGAGGACCAAGAACATCTGCAAGCTGATCCGGGGAAAGAAGAACCTGCTGGAACTGGTCACATGGACACCTGAGGCCGAGGCCGAATATGCCGAGAATGCCGAAATCCTGAAAA CCGAGCAAGAGGGGACCTACTACAAGCCTGGCATTCCAATCAGAGCTGCCGTGCAGAAACTGGAAGGCGGCCAGTGGTCCTACCAGTTTAAGCAAGAAGGCCAGGTCCTGAAAGTG GGCAAGTACACCAAGCAGAAGAACACCCACACCAACGAGCTGAGGACACTGGCTGGCCTGGTCCAGAAAATCTGCAAAGAGGCCCTGGTCATTTGGGGCATCCTGCCTGTTCTGGA ACTGCCCATTGAGCGGGAAGTGTGGGAACAGTGGTGGGCCGATTACTGGCAAGTGTCTTGGATCCCCGAGTGGGACTTCGTGTCTACCCCTCCTCTGCTGAAACTGTGGTACACCC TGACAAAAGAGCCCATTCCTAAAGAGGACGTCTACTACGTTGACGGCGCCTGCAACCGGAACTCCAAAGAAGGCAAGGCCGGCTACATCAGCCAGTACGGCAAGCAGAGAGTGGAA ACCCTGGAAAACACCACCAACCAGCAGGCCGAGCTGACCGCCATTAAGATGGCCCTGGAAGATAGCGGCCCCAATGTGAACATCGTGACCGACTCTCAGTACGCCATGGGAATCCT GACAGCCCAGCCTACACAGAGCGATAGCCCTCTGGTTGAGCAGATCATTGCCCTGATGATTCAGAAGCAGCAAATCTACCTGCAGTGGGTGCCCGCTCACAAAGGCATCGGCGGAA ACGAAGAGATCGATAAGCTGGTGTCCAAGGGAATCAGACGGGTGCTGTTCCTGGAAAAGATTGAAGAGGCCCAAGAGGAACACGAGCGCTACCACAACAACTGGAAGAATCTGGCC GACACCTACGGACTGCCCCAGATCGTGGCCAAAGAAATCGTGGCTATGTGCCCCAAGTGTCAGATCAAGGGCGAACCTGTGCACGGCCAAGTGGATGCTTCTCCTGGCACATGGCA GATGGACTGTACCCACCTGGAAGGCAAAGTGGTCATCGTGGCTGTGCACGTGGCCTCCGGCTTTATTGAGGCCGAAGTGATCCCCAGAGAGACAGGCAAAGAAACCGCCAAGTTCC TGCTGAAGATCCTGTCCAGATGGCCCATCACACAGCTGCACACCGACAACGGCCCTAACTTCACATCTCAAGAGGTGGCCGCCATCTGTTGGTGGGGAAAGATTGAGCACACAACC GGCATTCCCTACAATCCACAGAGCCAGGGCAGCATCGAGTCCATGAACAAGCAGCTCAAAGAGATTATCGGCAAGATCCGGGACGACTGCCAGTACACAGAAACAGCCGTGCTGAT GGCCTGTCACATCCACAACTTCAAGCGGAAAGGCGGCATCGGAGGACAGACATCTGCCGAGAGACTGATCAATATCATCACCACTCAGCTGGAAATCCAGCACCTCCAGACCAAGA TCCAGAAGATTCTGAACTTCCGGGTGTACTACCGCGAGGGCAGAGATCCTGTTTGGAAAGGCCCAGCACAGCTGATCTGGAAAGGCGAAGGTGCCGTGGTGCTGAAGGATGGCTCT GATCTGAAGGTGGTGCCCAGACGGAAGGCCAAGATTATCAAGGATTACGAGCCCAAACAGCGCGTGGGCAATGAAGGCGACGTTGAGGGCACAAGAGGCAGCGACAATTGA SEQ ID NO: 7 according to Figure 1C the plastid (pDNA2a pGM691) defined in ATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCT GGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTA CTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTT ATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGC CAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCG TGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTT TAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAG CGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGT GCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGA GTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCC TCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGG GACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAG GGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCG TGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTGC TCGAGCCACCATGGGAGCTGCCACATCTGCCCTGAATAGACGGCAGCTGGACCAGTTCGAGAAGATCAGACTGCGGCCCAACGGCAAGAAGAAGTACCAGATCAAGCACCTGATCT GGGCCGGCAAAGAGATGGAAAGATTCGGCCTGCACGAGCGGCTGCTGGAAACCGAGGAAGGCTGCAAGAGAATTATCGAGGTGCTGTACCCTCTGGAACCTACCGGCTCTGAGGGC CTGAAGTCCCTGTTCAATCTCGTGTGCGTGCTGTACTGCCTGCACAAAGAACAGAAAGTGAAGGACACCGAAGAGGCCGTGGCCACAGTTAGACAGCACTGCCACCTGGTGGAAAA AGAGAAGTCCGCCACAGAGACAAGCAGCGGCCAGAAGAAGAACGACAAGGGAATTGCTGCCCCTCCTGGCGGCAGCCAGAATTTTCCTGCTCAGCAGCAGGGAAACGCCTGGGTGC ACGTTCCACTGAGCCCTAGAACACTGAATGCCTGGGTCAAAGCCGTGGAAGAGAAGAAGTTTGGCGCCGAGATCGTGCCCATGTTCCAGGCTCTGTCTGAGGGCTGCACCCCTTAC GACATCAACCAGATGCTGAACGTGCTGGGAGATCACCAGGGCGCTCTGCAGATCGTGAAAGAGATCATCAACGAAGAGGCTGCCCAGTGGGACGTGACACATCCATTGCCTGCTGG ACCTCTGCCAGCCGGACAACTGAGAGATCCTAGAGGCTCTGATATCGCCGGCACCACCAGCTCTGTGCAAGAGCAGCTGGAATGGATCTACACCGCCAATCCTAGAGTGGACGTGG GCGCCATCTACAGAAGATGGATCATCCTGGGCCTGCAGAAATGCGTGAAGATGTACAACCCCGTGTCCGTGCTGGACATCAGACAGGGACCCAAAGAGCCCTTCAAGGACTACGTG GACCGGTTCTATAAGGCCATTAGAGCCGAGCAGGCCAGCGGCGAAGTGAAGCAGTGGATGACAGAGAGCCTGCTGATCCAGAACGCCAATCCAGACTGCAAAGTGATCCTGAAAGG CCTGGGCATGCACCCCACACTGGAAGAGATGCTGACAGCCTGTCAAGGCGTTGGCGGCCCTTCTTACAAAGCCAAAGTGATGGCCGAGATGATGCAGACCATGCAGAACCAGAACA TGGTGCAGCAAGGCGGCCCTAAGAGACAGAGGCCTCCTCTGAGATGCTACAACTGCGGCAAGTTCGGCCACATGCAGAGACAGTGTCCTGAGCCTAGGAAAACAAAATGTCTAAAG TGTGGAAAATTGGGACACCTAGCAAAAGACTGCAGGGGACAGGTGAATTTTTTAGGGTATGGACGGTGGATGGGGGCAAAACCGAGAAATTTTCCCGCCGCTACTCTTGGAGCGGA ACCGAGTGCGCCTCCTCCACCGAGCGGCACCACCCCATACGACCCAGCAAAGAAGCTCCTGCAGCAATATGCAGAGAAAGGGAAACAACTGAGGGAGCAAAAGAGGAATCCACCGG CAATGAATCCGGATTGGACCGAGGGATATTCTTTGAACTCCCTCTTTGGAGAAGACCAATAAAGACCGTGTACATCGAGGGCGTGCCCATCAAGGCTCTGCTGGATACAGGCGCCG ACGACACCATCATCAAAGAGAACGACCTGCAGCTGAGCGGCCCTTGGAGGCCTAAGATCATTGGAGGAATCGGCGGAGGCCTGAACGTCAAAGAGTACAACGACCGGGAAGTGAAG ATCGAGGACAAGATCCTGAGGGGCACAATCCTGCTGGGCGCCACACCTATCAACATCATCGGCAGAAATCTGCTGGCCCCTGCCGGCGCTAGACTGGTTATGGGACAGCTCTCTGA GAAGATCCCCGTGACACCCGTGAAGCTGAAAGAAGGCGCTAGAGGACCTTGTGTGCGACAGTGGCCTCTGAGCAAAGAGAAGATTGAGGCCCTGCAAGAAATCTGTAGCCAGCTGG AACAAGAGGGCAAGATCAGCAGAGTTGGCGGCGAGAACGCCTACAATACCCCTATCTTCTGCATCAAGAAAAAGGACAAGAGCCAGTGGCGGATGCTGGTGGACTTTAGAGAGCTG AACAAGGCTACCCAGGACTTCTTCGAGGTGCAGCTGGGAATTCCTCATCCTGCCGGCCTGCGGAAGATGAGACAGATCACAGTGCTGGATGTGGGCGACGCCTACTACAGCATCCC TCTGGACCCCAACTTCAGAAAGTACACCGCCTTCACAATCCCCACCGTGAACAATCAAGGCCCTGGCATCAGATACCAGTTCAACTGCCTGCCTCAAGGCTGGAAGGGCAGCCCCA CCATTTTTCAGAATACCGCCGCCAGCATCCTGGAAGAAATCAAGAGAAACCTGCCTGCTCTGACCATCGTGCAGTACATGGACGATCTGTGGGTCGGAAGCCAAGAGAATGAGCAC ACCCACGACAAGCTGGTGGAACAGCTGAGAACAAAGCTGCAGGCCTGGGGCCTCGAAACCCCTGAGAAGAAGGTGCAGAAAGAACCTCCTTACGAGTGGATGGGCTACAAGCTGTG GCCTCACAAGTGGGAGCTGAGCCGGATTCAGCTCGAAGAGAAGGACGAGTGGACCGTGAACGACATCCAGAAACTCGTGGGCAAGCTGAATTGGGCAGCCCAGCTGTATCCCGGCC TGAGGACCAAGAACATCTGCAAGCTGATCCGGGGAAAGAAGAACCTGCTGGAACTGGTCACATGGACACCTGAGGCCGAGGCCGAATATGCCGAGAATGCCGAAATCCTGAAAACCGAGCAAGAGGGGACCTACTACAAGCCTGGCATTCCAATCAGAGCTGCCGTGCAGAAACTGGAAGGCGGCCAGTGGTCCTACCAGTTTAAGCAAGAAGGCCAGGTCCTGAAAGTGGG CAAGTACACCAAGCAGAAGAACACCCACACCAACGAGCTGAGGACACTGGCTGGCCTGGTCCAGAAAATCTGCAAAGAGGCCCTGGTCATTTGGGGCATCCTGCCTGTTCTGGAAC TGCCCATTGAGCGGGAAGTGTGGGAACAGTGGTGGGCCGATTACTGGCAAGTGTCTTGGATCCCCGAGTGGGACTTCGTGTCTACCCCTCCTCTGCTGAAACTGTGGTACACCCTG ACAAAAGAGCCCATTCCTAAAGAGGACGTCTACTACGTTGACGGCGCCTGCAACCGGAACTCCAAAGAAGGCAAGGCCGGCTACATCAGCCAGTACGGCAAGCAGAGAGTGGAAAC CCTGGAAAACACCACCAACCAGCAGGCCGAGCTGACCGCCATTAAGATGGCCCTGGAAGATAGCGGCCCCAATGTGAACATCGTGACCGACTCTCAGTACGCCATGGGAATCCTGA CAGCCCAGCCTACACAGAGCGATAGCCCTCTGGTTGAGCAGATCATTGCCCTGATGATTCAGAAGCAGCAAATCTACCTGCAGTGGGTGCCCGCTCACAAAGGCATCGGCGGAAAC GAAGAGATCGATAAGCTGGTGTCCAAGGGAATCAGACGGGTGCTGTTCCTGGAAAAGATTGAAGAGGCCCAAGAGGAACACGAGCGCTACCACAACAACTGGAAGAATCTGGCCGA CACCTACGGACTGCCCCAGATCGTGGCCAAAGAAATCGTGGCTATGTGCCCCAAGTGTCAGATCAAGGGCGAACCTGTGCACGGCCAAGTGGATGCTTCTCCTGGCACATGGCAGA TGGACTGTACCCACCTGGAAGGCAAAGTGGTCATCGTGGCTGTGCACGTGGCCTCCGGCTTTATTGAGGCCGAAGTGATCCCCAGAGAGACAGGCAAAGAAACCGCCAAGTTCCTG CTGAAGATCCTGTCCAGATGGCCCATCACACAGCTGCACACCGACAACGGCCCTAACTTCACATCTCAAGAGGTGGCCGCCATCTGTTGGTGGGGAAAGATTGAGCACACAACCGG CATTCCCTACAATCCACAGAGCCAGGGCAGCATCGAGTCCATGAACAAGCAGCTCAAAGAGATTATCGGCAAGATCCGGGACGACTGCCAGTACACAGAAACAGCCGTGCTGATGG CCTGTCACATCCACAACTTCAAGCGGAAAGGCGGCATCGGAGGACAGACATCTGCCGAGAGACTGATCAATATCATCACCACTCAGCTGGAAATCCAGCACCTCCAGACCAAGATC CAGAAGATTCTGAACTTCCGGGTGTACTACCGCGAGGGCAGAGATCCTGTTTGGAAAGGCCCAGCACAGCTGATCTGGAAAGGCGAAGGTGCCGTGGTGCTGAAGGATGGCTCTGA TCTGAAGGTGGTGCCCAGACGGAAGGCCAAGATTATCAAGGATTACGAGCCCAAACAGCGCGTGGGCAATGAAGGCGACGTTGAGGGCACAAGAGGCAGCGACAATTGAAATTCAC TCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGC CCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAA CATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCA TTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGAT TTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAA TTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTT <000027​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​AGATCAGCATTGGCCAGCTGGTGAGCCTGCTGAGCAACAACCTGAACAAGTTTGATGAGGGCCTGGCCCTGGCCCACTTTGTGTGGATTGCCCCTCTGCAGGTGGCCCTGCTGATG GGCCTGATTTGGGAGCTGCTGCAGGCCTCTGCCTTTTGTGGCCTGGGCTTCCTGATTGTGCTGGCCCTGTTTCAGGCTGGCCTGGGCAGGATGATGATGAAGTACAGGGACCAGAG GGCAGGCAAGATCAGTGAGAGGCTGGTGATCACCTCTGAGATGATTGAGAACATCCAGTCTGTGAAGGCCTACTGTTGGGAGGAAGCTATGGAGAAGATGATTGAAAACCTGAGGC AGACAGAGCTGAAGCTGACCAGGAAGGCTGCCTATGTGAGATACTTCAACAGCTCTGCCTTCTTCTTCTCTGGCTTCTTTGTGGTGTTCCTGTCTGTGCTGCCCTATGCCCTGATC AAGGGGATCATCCTGAGAAAGATTTTCACCACCATCAGCTTCTGCATTGTGCTGAGGATGGCTGTGACCAGACAGTTCCCCTGGGCTGTGCAGACCTGGTATGACAGCCTGGGGGC CATCAACAAGATCCAGGACTTCCTGCAGAAGCAGGAGTACAAGACCCTGGAGTACAACCTGACCACCACAGAAGTGGTGATGGAGAATGTGACAGCCTTCTGGGAGGAGGGCTTTG GGGAGCTGTTTGAGAAGGCCAAGCAGAACAACAACAACAGAAAGACCAGCAATGGGGATGACTCCCTGTTCTTCTCCAACTTCTCCCTGCTGGGCACACCTGTGCTGAAGGACATC AACTTCAAGATTGAGAGGGGGCAGCTGCTGGCTGTGGCTGGATCTACAGGGGCTGGCAAGACCAGCCTGCTGATGATGATCATGGGGGAGCTGGAGCCTTCTGAGGGCAAGATCAA GCACTCTGGCAGGATCAGCTTTTGCAGCCAGTTCAGCTGGATCATGCCTGGCACCATCAAGGAGAACATCATCTTTGGAGTGAGCTATGATGAGTACAGATACAGGAGTGTGATCA AGGCCTGCCAGCTGGAGGAGGACATCAGCAAGTTTGCTGAGAAGGACAACATTGTGCTGGGGGAGGGAGGCATTACACTGTCTGGGGGCCAGAGAGCCAGAATCAGCCTGGCCAGG GCTGTGTACAAGGATGCTGACCTGTACCTGCTGGACTCCCCCTTTGGCTACCTGGATGTGCTGACAGAGAAGGAGATTTTTGAGAGCTGTGTGTGCAAGCTGATGGCCAACAAGAC CAGAATCCTGGTGACCAGCAAGATGGAGCACCTGAAGAAGGCTGACAAGATCCTGATCCTGCATGAGGGCAGCAGCTACTTCTATGGGACCTTCTCTGAGCTGCAGAACCTGCAGC CTGACTTCAGCTCTAAGCTGATGGGCTGTGACAGCTTTGACCAGTTCTCTGCTGAGAGGAGGAACAGCATCCTGACAGAGACCCTGCACAGATTCAGCCTGGAGGGAGATGCCCCT GTGAGCTGGACAGAGACCAAGAAGCAGAGCTTCAAGCAGACAGGGGAGTTTGGGGAGAAGAGGAAGAACTCCATCCTGAACCCCATCAACAGCATCAGGAAGTTCAGCATTGTGCA GAAAACCCCCCTGCAGATGAATGGCATTGAGGAAGATTCTGATGAGCCCCTGGAGAGGAGACTGAGCCTGGTGCCTGATTCTGAGCAGGGAGAGGCCATCCTGCCTAGGATCTCTG TGATCAGCACAGGCCCTACACTGCAGGCCAGAAGGAGGCAGTCTGTGCTGAACCTGATGACCCACTCTGTGAACCAGGGCCAGAACATCCACAGGAAAACCACAGCCTCCACCAGG AAAGTGAGCCTGGCCCCTCAGGCCAATCTGACAGAGCTGGACATCTACAGCAGGAGGCTGTCTCAGGAGACAGGCCTGGAGATTTCTGAGGAGATCAATGAGGAGGACCTGAAAGA GTGCTTCTTTGATGACATGGAGAGCATCCCTGCTGTGACCACCTGGAACACCTACCTGAGATACATCACAGTGCACAAGAGCCTGATCTTTGTGCTGATCTGGTGCCTGGTGATCT TCCTGGCTGAAGTGGCTGCCTCTCTGGTGGTGCTGTGGCTGCTGGGAAACACCCCACTGCAGGACAAGGGCAACAGCACCCACAGCAGGAACAACAGCTATGCTGTGATCATCACC TCCACCTCCAGCTACTATGTGTTCTACATCTATGTGGGAGTGGCTGATACCCTGCTGGCTATGGGCTTCTTTAGAGGCCTGCCCCTGGTGCACACACTGATCACAGTGAGCAAGAT CCTCCACCACAAGATGCTGCACTCTGTGCTGCAGGCTCCTATGAGCACCCTGAATACCCTGAAGGCTGGGGGCATCCTGAACAGATTCTCCAAGGATATTGCCATCCTGGATGACC TGCTGCCTCTCACCATCTTTGACTTCATCCAGCTGCTGCTGATTGTGATTGGGGCCATTGCTGTGGTGGCAGTGCTGCAGCCCTACATCTTTGTGGCCACAGTGCCTGTGATTGTG GCCTTCATCATGCTGAGGGCCTACTTTCTGCAGACCTCCCAGCAGCTGAAGCAGCTGGAGTCTGAGGGCAGAAGCCCCATCTTCACCCACCTGGTGACAAGCCTGAAGGGCCTGTG GACCCTGAGAGCCTTTGGCAGGCAGCCCTACTTTGAGACCCTGTTCCACAAGGCCCTGAACCTGCACACAGCCAACTGGTTCCTCTACCTGTCCACCCTGAGATGGTTCCAGATGA GAATTGAGATGATCTTTGTCATCTTCTTCATTGCTGTGACCTTCATCAGCATTCTGACCACAGGAGAGGGAGAGGGCAGAGTGGGCATTATCCTGACCCTGGCCATGAACATCATG AGCACACTGCAGTGGGCAGTGAACAGCAGCATTGATGTGGACAGCCTGATGAGGAGTGTGAGCAGAGTGTTCAAGTTCATTGATATGCCCACAGAGGGCAAGCCTACCAAGAGCAC CAAGCCCTACAAGAATGGCCAGCTGAGCAAAGTGATGATCATTGAGAACAGCCATGTGAAGAAGGATGATATCTGGCCCAGTGGAGGCCAGATGACAGTGAAGGACCTGACAGCCA AGTACACAGAGGGGGGCAATGCTATCCTGGAGAACATCTCCTTCAGCATCTCCCCTGGCCAGAGAGTGGGACTGCTGGGAAGAACAGGCTCTGGCAAGTCTACCCTGCTGTCTGCC TTCCTGAGGCTGCTGAACACAGAGGGAGAGATCCAGATTGATGGAGTGTCCTGGGACAGCATCACACTGCAGCAGTGGAGGAAGGCCTTTGGTGTGATCCCCCAGAAAGTGTTCAT CTTCAGTGGCACCTTCAGGAAGAACCTGGACCCCTATGAGCAGTGGTCTGACCAGGAGATTTGGAAAGTGGCTGATGAAGTGGGCCTGAGAAGTGTGATTGAGCAGTTCCCTGGCA AGCTGGACTTTGTCCTGGTGGATGGGGGCTGTGTGCTGAGCCATGGCCACAAGCAGCTGATGTGCCTGGCCAGATCAGTGCTGAGCAAGGCCAAGATCCTGCTGCTGGATGAGCCT TCTGCCCACCTGGATCCTGTGACCTACCAGATCATCAGGAGGACCCTCAAGCAGGCCTTTGCTGACTGCACAGTCATCCTGTGTGAGCACAGGATTGAGGCCATGCTGGAGTGCCA GCAGTTCCTGGTGATTGAGGAGAACAAAGTGAGGCAGTATGACAGCATCCAGAAGCTGCTGAATGAGAGGAGCCTGTTCAGGCAGGCCATCAGCCCCTCTGATAGAGTGAAGCTGT TCCCCCACAGGAACAGCTCCAAGTGCAAGAGCAAGCCCCAGATTGCTGCCCTGAAGGAGGAGACAGAGGAGGAAGTGCAGGACACCAGGCTGTGAGGGCCCAATCAACCTCTGGAT TACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATGGCTTTCAT TTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCACTGGTTGGG GCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTCGGCTGTTG GGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCTTCGGCCCT CAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCCGCAAGCTT CGCACTTTTTAAAAGAAAAGGGAGGACTGGATGGGATTTATTACTCCGATAGGACGCTGGCTTGTAACTCAGTCTCTTACTAGGAGACCAGCTTGAGCCTGGGTGTTCGCTGGTTA GCCTAACCTGGTTGGCCACCAGGGGTAAGGACTCCTTGGCTTAGAAAGCTAATAAACTTGCCTGCATTAGAGCTCTTACGCGTCCCGGGCTCGAGATCCGCATCTCAATTAGTCAG CAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCC TCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATC ACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCG GTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAG GAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGAT ACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCA CGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAA CCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACA CTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTT TGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAG ATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTAGAAAAACTCATCGAGCATCA AATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGG TATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAA TGGCAACAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGA GACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCT AATACCTGGAATGCTGTTTTTCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAG TCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGA CATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTG TTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACAATTGGTCGACGGATCC SEQ ID NO: 9 Figure 1B The plasmid defined in (pDNA1 pGM830) GGTACCTCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGT ACATTTATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGC GTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACG TCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATG CCCAGTACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTT GACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTGCGATCGCCCGCCCCGTTG ACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGCTGGCTTGTAACTCAGTCTCTTACTAGGAGACCAGCTTGAGCCTGGGTGTTCGCTGGTTAGCC TAACCTGGTTGGCCACCAGGGGTAAGGACTCCTTGGCTTAGAAAGCTAATAAACTTGCCTGCATTAGAGCTTATCTGAGTCAAGTGTCCTCATTGACGCCTCACTCTCTTGAACGG GAATCTTCCTTACTGGGTTCTCTCTCTGACCCAGGCGAGAGAAACTCCAGCAGTGGCGCCCGAACAGGGACTTGAGTGAGAGTGTAGGCACGTACAGCTGAGAAGGCGTCGGACGC GAAGGAAGCGCGGGGTGCGACGCGACCAAGAAGGAGACTTGGTGAGTAGGCTTCTCGAGTGCCGGGAAAAAGCTCGAGCCTAGTTAGAGGACTAGGAGAGGCCGTAGCCGTAACTA CTCTGGGCAAGTAGGGCAGGCGGTGGGTACGCAATTGGGGGCGGCTACCTCAGCACTAAATAGGAGACAATTAGACCAATTTGAGAAAATACGACTTCGCCCGAACGGAAAGAAAAAGTACCAAATTAAACATTTAATATTGGGCAGGCAAGGAGATTGGAGCGCTTCGGCCTCCATGAGAGGTTGTTGGAGACAGAGGAGGGGTGTAAAAGAATCATAGAAGTCCTCTACC CCCTAGAACCAACAGGATCGGAGGGCTTAAAAAGTCTGTTCAATCTTGTGTGCGTGCTATATTGCTTGCACAAGGAACAGAAAGTGAAAGACACAGAGGAAGCAGTAGCAACAGTA AGACAACACTGCCATCTAGTGGAAAAAGAAAAAAGTGCAACAGAGACATCTAGTGGACAAAAGAAAAATGACAAGGGAATAGCAGCGCCACCTGGTGGCAGTCAGAATTTTCCAGC GCAACAACAAGGAAATTGCCTGGGTACATGTACCCTTGTCACCGCGCACCTTAAATGCGTGGGTAAAAGCAGTAGAGGAGAAAAAATTTGGAGCAGAAATAGTACCCATGTTTCAA GCCCTATCGCCTGCAGGCCGTTTGTGCTAGGGTTCTTAGGCTTCTTGGGGGCTGCTGGAACTGCATTGGGAGCAGCGGCGACAGCCCTGACGGTCCAGTCTCAGCATTTGCTTGCT GGGATACTGCAGCAGCAGAAGAATCTGCTGGCGGCTGTGGAGGCTCAACAGCAGATGTTGAAGCTGACCATTTGGGGTGTTAAAAACCTCAATGCCCGCGTCACAGCCCTTGAGAA GTACCTAGAGGATCAGGCACGACTAAACTCCTGGGGGTGCGCATGGAAACAAGTATGTCATACCACAGTGGAGTGGCCCTGGACAAATCGGACTCCGGATTGGCAAAATAAGACTT GGTTGGAGTGGGAAAGACAAATAGCTGATTTGGAAAGCAACATTACGAGACAATTAGTGAAGGCTAGAGAACAAGAGGAAAAGAATCTAGATGCCTATCAGAAGTTAACTAGTTGG TCAGATTTCTGGTCTTGGTTCGATTTCTCAAAATGGCTTAACATTTTAAAAAAGGGATTTTTAGTAATAGTAGGAATAATAGGGTTAAGATTACTTTACACAGTATATGGATGTAT AGTGAGGGTTAGGCAGGGATATGTTCCTCTATCTCCACAGATCCATATAAAGCGGCAATTTTAAAAGAAAGGGAGGAATAGGGGGACAGACTTCAGCAGAGAGACTAATTAATATA ATAACAACACAATTAGAAATACAACATTTACAAACCAAAATTCAAAAAATTTTAAATTTTAGAGCCGCGGAGATCTGTTACATAACTTATGGTAAATGGCCTGCCTGGCTGACTGC CCAATGACCCCTGCCCAATGATGTCAATAATGATGTATGTTCCCATGTAATGCCAATAGGGACTTTCCATTGATGTCAATGGGTGGAGTATTTATGGTAACTGCCCACTTGGCAGT ACATCAAGTGTATCATATGCCAAGTATGCCCCCTATTGATGTCAATGATGGTAAATGGCCTGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACA TCTATGTATTAGTCATTGCTATTACCATGGGAATTCACTAGTGGAGAAGAGCATGCTTGAGGGCTGAGTGCCCCTCAGTGGGCAGAGAGCACATGGCCCACAGTCCCTGAGAAGTT GGGGGGAGGGGTGGGCAATTGAACTGGTGCCTAGAGAAGGTGGGGCTTGGGTAAACTGGGAAAGTGATGTGGTGTACTGGCTCCACCTTTTTCCCCAGGGTGGGGGAGAACCATAT ATAAGTGCAGTAGTCTCTGTGAACATTCAAGCTTCTGCCTTCTCCCTCCTGTGAGTTTGCTAGCCACCATGCAGAGAAGCCCTCTGGAGAAGGCCTCTGTGGTGAGCAAGCTGTTC TTCAGCTGGACCAGGCCCATCCTGAGGAAGGGCTACAGGCAGAGACTGGAGCTGTCTGACATCTACCAGATCCCCTCTGTGGACTCTGCTGACAACCTGTCTGAGAAGCTGGAGAG GGAGTGGGATAGAGAGCTGGCCAGCAAGAAGAACCCCAAGCTGATCAATGCCCTGAGGAGATGCTTCTTCTGGAGATTCATGTTCTATGGCATCTTCCTGTACCTGGGGGAAGTGA CCAAGGCTGTGCAGCCTCTGCTGCTGGGCAGAATCATTGCCAGCTATGACCCTGACAACAAGGAGGAGAGGAGCATTGCCATCTACCTGGGCATTGGCCTGTGCCTGCTGTTCATT GTGAGGACCCTGCTGCTGCACCCTGCCATCTTTGGCCTGCACCACATTGGCATGCAGATGAGGATTGCCATGTTCAGCCTGATCTACAAGAAAACCCTGAAGCTGTCCAGCAGAGT GCTGGACAAGATCAGCATTGGCCAGCTGGTGAGCCTGCTGAGCAACAACCTGAACAAGTTTGATGAGGGCCTGGCCCTGGCCCACTTTGTGTGGATTGCCCCTCTGCAGGTGGCCC TGCTGATGGGCCTGATTTGGGAGCTGCTGCAGGCCTCTGCCTTTTGTGGCCTGGGCTTCCTGATTGTGCTGGCCCTGTTTCAGGCTGGCCTGGGCAGGATGATGATGAAGTACAGG GACCAGAGGGCAGGCAAGATCAGTGAGAGGCTGGTGATCACCTCTGAGATGATTGAGAACATCCAGTCTGTGAAGGCCTACTGTTGGGAGGAAGCTATGGAGAAGATGATTGAAAA CCTGAGGCAGACAGAGCTGAAGCTGACCAGGAAGGCTGCCTATGTGAGATACTTCAACAGCTCTGCCTTCTTCTTCTCTGGCTTCTTTGTGGTGTTCCTGTCTGTGCTGCCCTATG CCCTGATCAAGGGGATCATCCTGAGAAAGATTTTCACCACCATCAGCTTCTGCATTGTGCTGAGGATGGCTGTGACCAGACAGTTCCCCTGGGCTGTGCAGACCTGGTATGACAGC CTGGGGGCCATCAACAAGATCCAGGACTTCCTGCAGAAGCAGGAGTACAAGACCCTGGAGTACAACCTGACCACCACAGAAGTGGTGATGGAGAATGTGACAGCCTTCTGGGAGGA GGGCTTTGGGGAGCTGTTTGAGAAGGCCAAGCAGAACAACAACAACAGAAAGACCAGCAATGGGGATGACTCCCTGTTCTTCTCCAACTTCTCCCTGCTGGGCACACCTGTGCTGA AGGACATCAACTTCAAGATTGAGAGGGGGCAGCTGCTGGCTGTGGCTGGATCTACAGGGGCTGGCAAGACCAGCCTGCTGATGATGATCATGGGGGAGCTGGAGCCTTCTGAGGGC AAGATCAAGCACTCTGGCAGGATCAGCTTTTGCAGCCAGTTCAGCTGGATCATGCCTGGCACCATCAAGGAGAACATCATCTTTGGAGTGAGCTATGATGAGTACAGATACAGGAG TGTGATCAAGGCCTGCCAGCTGGAGGAGGACATCAGCAAGTTTGCTGAGAAGGACAACATTGTGCTGGGGGAGGGAGGCATTACACTGTCTGGGGGCCAGAGAGCCAGAATCAGCC TGGCCAGGGCTGTGTACAAGGATGCTGACCTGTACCTGCTGGACTCCCCCTTTGGCTACCTGGATGTGCTGACAGAGAAGGAGATTTTTGAGAGCTGTGTGTGCAAGCTGATGGCC AACAAGACCAGAATCCTGGTGACCAGCAAGATGGAGCACCTGAAGAAGGCTGACAAGATCCTGATCCTGCATGAGGGCAGCAGCTACTTCTATGGGACCTTCTCTGAGCTGCAGAA CCTGCAGCCTGACTTCAGCTCTAAGCTGATGGGCTGTGACAGCTTTGACCAGTTCTCTGCTGAGAGGAGGAACAGCATCCTGACAGAGACCCTGCACAGATTCAGCCTGGAGGGAG ATGCCCCTGTGAGCTGGACAGAGACCAAGAAGCAGAGCTTCAAGCAGACAGGGGAGTTTGGGGAGAAGAGGAAGAACTCCATCCTGAACCCCATCAACAGCATCAGGAAGTTCAGC ATTGTGCAGAAAACCCCCCTGCAGATGAATGGCATTGAGGAAGATTCTGATGAGCCCCTGGAGAGGAGACTGAGCCTGGTGCCTGATTCTGAGCAGGGAGAGGCCATCCTGCCTAG GATCTCTGTGATCAGCACAGGCCCTACACTGCAGGCCAGAAGGAGGCAGTCTGTGCTGAACCTGATGACCCACTCTGTGAACCAGGGCCAGAACATCCACAGGAAAACCACAGCCT CCACCAGGAAAGTGAGCCTGGCCCCTCAGGCCAATCTGACAGAGCTGGACATCTACAGCAGGAGGCTGTCTCAGGAGACAGGCCTGGAGATTTCTGAGGAGATCAATGAGGAGGAC CTGAAAGAGTGCTTCTTTGATGACATGGAGAGCATCCCTGCTGTGACCACCTGGAACACCTACCTGAGATACATCACAGTGCACAAGAGCCTGATCTTTGTGCTGATCTGGTGCCT GGTGATCTTCCTGGCTGAAGTGGCTGCCTCTCTGGTGGTGCTGTGGCTGCTGGGAAACACCCCACTGCAGGACAAGGGCAACAGCACCCACAGCAGGAACAACAGCTATGCTGTGA TCATCACCTCCACCTCCAGCTACTATGTGTTCTACATCTATGTGGGAGTGGCTGATACCCTGCTGGCTATGGGCTTCTTTAGAGGCCTGCCCCTGGTGCACACACTGATCACAGTG AGCAAGATCCTCCACCACAAGATGCTGCACTCTGTGCTGCAGGCTCCTATGAGCACCCTGAATACCCTGAAGGCTGGGGGCATCCTGAACAGATTCTCCAAGGATATTGCCATCCT GGATGACCTGCTGCCTCTCACCATCTTTGACTTCATCCAGCTGCTGCTGATTGTGATTGGGGCCATTGCTGTGGTGGCAGTGCTGCAGCCCTACATCTTTGTGGCCACAGTGCCTG TGATTGTGGCCTTCATCATGCTGAGGGCCTACTTTCTGCAGACCTCCCAGCAGCTGAAGCAGCTGGAGTCTGAGGGCAGAAGCCCCATCTTCACCCACCTGGTGACAAGCCTGAAG GGCCTGTGGACCCTGAGAGCCTTTGGCAGGCAGCCCTACTTTGAGACCCTGTTCCACAAGGCCCTGAACCTGCACACAGCCAACTGGTTCCTCTACCTGTCCACCCTGAGATGGTT CCAGATGAGAATTGAGATGATCTTTGTCATCTTCTTCATTGCTGTGACCTTCATCAGCATTCTGACCACAGGAGAGGGAGAGGGCAGAGTGGGCATTATCCTGACCCTGGCCATGA ACATCATGAGCACACTGCAGTGGGCAGTGAACAGCAGCATTGATGTGGACAGCCTGATGAGGAGTGTGAGCAGAGTGTTCAAGTTCATTGATATGCCCACAGAGGGCAAGCCTACC AAGAGCACCAAGCCCTACAAGAATGGCCAGCTGAGCAAAGTGATGATCATTGAGAACAGCCATGTGAAGAAGGATGATATCTGGCCCAGTGGAGGCCAGATGACAGTGAAGGACCT GACAGCCAAGTACACAGAGGGGGGCAATGCTATCCTGGAGAACATCTCCTTCAGCATCTCCCCTGGCCAGAGAGTGGGACTGCTGGGAAGAACAGGCTCTGGCAAGTCTACCCTGC TGTCTGCCTTCCTGAGGCTGCTGAACACAGAGGGAGAGATCCAGATTGATGGAGTGTCCTGGGACAGCATCACACTGCAGCAGTGGAGGAAGGCCTTTGGTGTGATCCCCCAGAAA GTGTTCATCTTCAGTGGCACCTTCAGGAAGAACCTGGACCCCTATGAGCAGTGGTCTGACCAGGAGATTTGGAAAGTGGCTGATGAAGTGGGCCTGAGAAGTGTGATTGAGCAGTT CCCTGGCAAGCTGGACTTTGTCCTGGTGGATGGGGGCTGTGTGCTGAGCCATGGCCACAAGCAGCTGATGTGCCTGGCCAGATCAGTGCTGAGCAAGGCCAAGATCCTGCTGCTGG ATGAGCCTTCTGCCCACCTGGATCCTGTGACCTACCAGATCATCAGGAGGACCCTCAAGCAGGCCTTTGCTGACTGCACAGTCATCCTGTGTGAGCACAGGATTGAGGCCATGCTG GAGTGCCAGCAGTTCCTGGTGATTGAGGAGAACAAAGTGAGGCAGTATGACAGCATCCAGAAGCTGCTGAATGAGAGGAGCCTGTTCAGGCAGGCCATCAGCCCCTCTGATAGAGT GAAGCTGTTCCCCCACAGGAACAGCTCCAAGTGCAAGAGCAAGCCCCAGATTGCTGCCCTGAAGGAGGAGACAGAGGAGGAAGTGCAGGACACCAGGCTGTGAGGGCCCAATCAAC CTCTGGATTACAAAATTTGTGAAAGATTGACTGGTATTCTTAACTATGTTGCTCCTTTTACGCTATGTGGATACGCTGCTTTAATGCCTTTGTATCATGCTATTGCTTCCCGTATG GCTTTCATTTTCTCCTCCTTGTATAAATCCTGGTTGCTGTCTCTTTATGAGGAGTTGTGGCCCGTTGTCAGGCAACGTGGCGTGGTGTGCACTGTGTTTGCTGACGCAACCCCCAC TGGTTGGGGCATTGCCACCACCTGTCAGCTCCTTTCCGGGACTTTCGCTTTCCCCCTCCCTATTGCCACGGCGGAACTCATCGCCGCCTGCCTTGCCCGCTGCTGGACAGGGGCTC GGCTGTTGGGCACTGACAATTCCGTGGTGTTGTCGGGGAAATCATCGTCCTTTCCTTGGCTGCTCGCCTGTGTTGCCACCTGGATTCTGCGCGGGACGTCCTTCTGCTACGTCCCT TCGGCCCTCAATCCAGCGGACCTTCCTTCCCGCGGCCTGCTGCCGGCTCTGCGGCCTCTTCCGCGTCTTCGCCTTCGCCCTCAGACGAGTCGGATCTCCCTTTGGGCCGCCTCCCC GCAAGCTTCGCACTTTTTAAAAGAAAAGGGAGGACTGGATGGGATTTATTACTCCGATAGGACGCTGGCTTGTAACTCAGTCTCTTACTAGGAGACCAGCTTGAGCCTGGGTGTTC GCTGGTTAGCCTAACCTGGTTGGCCACCAGGGGTAAGGACTCCTTGGCTTAGAAAGCTAATAAACTTGCCTGCATTAGAGCTCTTACGCGTCCCGGGCTCGAGATCCGCATCTCAA TTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCG AGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCA ATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTCCGCTTCCTCGCTCACTGACTCGC TGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAA AAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACT ATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTC ATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTT GAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTA CGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTT TTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTG GTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTAGAAAAACTCATC GAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAA GATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCC GGTGAGAATGGCAACAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGC CTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGAT ATTCTTCTAATACCTGGAATGCTGTTTTTCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGA TTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTG TATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAACAATTGGTCGACGGATCC SEQ ID NO: 10 Figure 1D The plasmid defined in (pDNA2a pGM297) ATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCT GGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTA CTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTT ATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGC CAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCG TGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTT TAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAG CGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGT GCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGA GTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCC TCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGG GACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAG GGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCG TGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTGC TCGAGACTAGTGACTTGGTGAGTAGGCTTCGAGCCTAGTTAGAGGACTAGGAGAGGCCGTAGCCGTAACTACTCTGGGCAAGTAGGGCAGGCGGTGGGTACGCAATGGGGGCGGCT ACCTCAGCACTAAATAGGAGACAATTAGACCAATTTGAGAAAATACGACTTCGCCCGAACGGAAAGAAAAAGTACCAAATTAAACATTTAATATGGGCAGGCAAGGAGATGGAGCG CTTCGGCCTCCATGAGAGGTTGTTGGAGACAGAGGAGGGGTGTAAAAGAATCATAGAAGTCCTCTACCCCCTAGAACCAACAGGATCGGAGGGCTTAAAAAGTCTGTTCAATCTTG TGTGCGTACTATATTGCTTGCACAAGGAACAGAAAGTGAAAGACACAGAGGAAGCAGTAGCAACAGTAAGACAACACTGCCATCTAGTGGAAAAAGAAAAAAGTGCAACAGAGACA TCTAGTGGACAAAAGAAAAATGACAAGGGAATAGCAGCGCCACCTGGTGGCAGTCAGAATTTTCCAGCGCAACAACAAGGAAATGCCTGGGTACATGTACCCTTGTCACCGCGCAC CTTAAATGCGTGGGTAAAAGCAGTAGAGGAGAAAAAATTTGGAGCAGAAATAGTACCCATGTTTCAAGCCCTATCAGAAGGCTGCACACCCTATGACATTAATCAGATGCTTAATG TGCTAGGAGATCATCAAGGGGCATTACAAATAGTGAAAGAGATCATTAATGAAGAAGCAGCCCAGTGGGATGTAACACACCCACTACCCGCAGGACCCCTACCAGCAGGACAGCTC AGGGACCCTCGCGGCTCAGATATAGCAGGGACCACCAGCTCAGTACAAGAACAGTTAGAATGGATCTATACTGCTAACCCCCGGGTAGATGTAGGTGCCATCTACCGGAGATGGAT TATTCTAGGACTTCAAAAGTGTGTCAAAATGTACAACCCAGTATCAGTCCTAGACATTAGGCAGGGACCTAAAGAGCCCTTCAAGGATTATGTGGACAGATTTTACAAGGCAATTA GAGCAGAACAAGCCTCAGGGGAAGTGAAACAATGGATGACAGAATCATTACTCATTCAAAATGCTAATCCAGATTGTAAGGTCATCCTGAAGGGCCTAGGAATGCACCCCACCCTT GAAGAAATGTTAACGGCTTGTCAGGGGGTAGGAGGCCCAAGCTACAAAGCAAAAGTAATGGCAGAAATGATGCAGACCATGCAAAATCAAAACATGGTGCAGCAGGGAGGTCCAAA AAGACAAAGACCCCCACTAAGATGTTATAATTGTGGAAAATTTGGCCATATGCAAAGACAATGTCCGGAACCAAGGAAAACAAAATGTCTAAAGTGTGGAAAATTGGGACACCTAG CAAAAGACTGCAGGGGACAGGTGAATTTTTTAGGGTATGGACGGTGGATGGGGGCAAAACCGAGAAATTTTCCCGCCGCTACTCTTGGAGCGGAACCGAGTGCGCCTCCTCCACCG AGCGGCACCACCCCATACGACCCAGCAAAGAAGCTCCTGCAGCAATATGCAGAGAAAGGGAAACAACTGAGGGAGCAAAAGAGGAATCCACCGGCAATGAATCCGGATTGGACCGA GGGATATTCTTTGAACTCCCTCTTTGGAGAAGACCAATAAAGACAGTGTATATAGAAGGGGTCCCCATTAAGGCACTGCTAGACACAGGGGCAGATGACACCATAATTAAAGAAAA TGATTTACAATTATCAGGTCCATGGAGACCCAAAATTATAGGGGGCATAGGAGGAGGCCTTAATGTAAAAGAATATAACGACAGGGAAGTAAAAATAGAAGATAAAATTTTGAGAG GAACAATATTGTTAGGAGCAACTCCCATTAATATAATAGGTAGAAATTTGCTGGCCCCGGCAGGTGCCCGGTTAGTAATGGGACAATTATCAGAAAAAATTCCTGTCACACCTGTC AAATTGAAGGAAGGGGCTCGGGGACCCTGTGTAAGACAATGGCCTCTCTCTAAAGAGAAGATTGAAGCTTTACAGGAAATATGTTCCCAATTAGAGCAGGAAGGAAAAATCAGTAG AGTAGGAGGAGAAAATGCATACAATACCCCAATATTTTGCATAAAGAAGAAGGACAAATCCCAGTGGAGGATGCTAGTAGACTTTAGAGAGTTAAATAAGGCAACCCAAGATTTCT TTGAAGTGCAATTAGGGATACCCCACCCAGCAGGATTAAGAAAGATGAGACAGATAACAGTTTTAGATGTAGGAGACGCCTATTATTCCATACCATTGGATCCAAATTTTAGGAAA TATACTGCTTTTACTATTCCCACAGTGAATAATCAGGGACCCGGGATTAGGTATCAATTCAACTGTCTCCCGCAAGGGTGGAAAGGATCTCCTACAATCTTCCAAAATACAGCAGC ATCCATTTTGGAGGAGATAAAAAGAAACTTGCCAGCACTAACCATTGTACAATACATGGATGATTTATGGGTAGGTTCTCAAGAAAATGAACACACCCATGACAAATTAGTAGAAC AGTTAAGAACAAAATTACAAGCCTGGGGCTTAGAAACCCCAGAAAAGAAGGTGCAAAAAGAACCACCTTATGAGTGGATGGGATACAAACTTTGGCCTCACAAATGGGAACTAAGC AGAATACAACTGGAGGAAAAAGATGAATGGACTGTCAATGACATCCAGAAGTTAGTTGGGAAACTAAATTGGGCAGCACAATTGTATCCAGGTCTTAGGACCAAGAATATATGCAA GTTAATTAGAGGAAAGAAAAATCTGTTAGAGCTAGTGACTTGGACACCTGAGGCAGAAGCTGAATATGCAGAAAATGCAGAGATTCTTAAAACAGAACAGGAAGGAACCTATTACA AACCAGGAATACCTATTAGGGCAGCAGTACAGAAATTGGAAGGAGGACAGTGGAGTTACCAATTCAAACAAGAAGGACAAGTCTTGAAAGTAGGAAAATACACCAAGCAAAAGAAC ACCCATACAAATGAACTTCGCACATTAGCTGGTTTAGTGCAGAAGATTTGCAAAGAAGCTCTAGTTATTTGGGGGATATTACCAGTTCTAGAACTCCCGATAGAAAGAGAGGTATG GGAACAATGGTGGGCGGATTACTGGCAGGTAAGCTGGATTCCCGAATGGGATTTTGTCAGCACCCCACCTTTGCTCAAACTATGGTACACATTAACAAAAGAACCCATACCCAAGG AGGACGTTTACTATGTAGATGGAGCATGCAACAGAAATTCAAAAGAAGGAAAAGCAGGATACATCTCACAATACGGAAAACAGAGAGTAGAAACATTAGAAAACACTACCAATCAG CAAGCAGAATTAACAGCTATAAAAATGGCTTTGGAAGACAGTGGGCCTAATGTGAACATAGTAACAGACTCTCAATATGCAATGGGAATTTTGACAGCACAACCCACACAAAGTGA TTCACCATTAGTAGAGCAAATTATAGCCTTAATGATACAAAAGCAACAAATATATTTGCAGTGGGTACCAGCACATAAAGGAATAGGAGGAAATGAGGAGATAGATAAATTAGTGA GTAAAGGCATTAGAAGAGTTTTATTCTTAGAAAAAATAGAAGAAGCTCAAGAAGAGCATGAAAGATATCATAATAATTGGAAAAACCTAGCAGATACATATGGGCTTCCACAAATA GTAGCAAAAGAGATAGTGGCCATGTGTCCAAAATGTCAGATAAAGGGAGAACCAGTGCATGGACAAGTGGATGCCTCACCTGGAACATGGCAGATGGATTGTACTCATCTAGAAGG AAAAGTAGTCATAGTTGCGGTCCATGTAGCCAGTGGATTCATAGAAGCAGAAGTCATACCTAGGGAAACAGGAAAAGAAACGGCAAAGTTTCTATTAAAAATACTGAGTAGATGGC CTATAACACAGTTACACACAGACAATGGGCCTAACTTTACCTCCCAAGAAGTGGCAGCAATATGTTGGTGGGGAAAAATTGAACATACAACAGGTATACCATATAACCCCCAATCT CAAGGATCAATAGAAAGCATGAACAAACAATTAAAAGAGATAATTGGGAAAATAAGAGATGATTGCCAATATACAGAGACAGCAGTACTGATGGCTTGCCATATTCACAATTTTAA AAGAAAGGGAGGAATAGGGGGACAGACTTCAGCAGAGAGACTAATTAATATAATAACAACACAATTAGAAATACAACATTTACAAACCAAAATTCAAAAAATTTTAAATTTTAGAG TCTACTACAGAGAAGGGAGAGACCCTGTGTGGAAAGGACCAGCACAATTAATCTGGAAAGGGGAAGGAGCAGTGGTCCTCAAGGACGGAAGTGACCTAAAGGTTGTACCAAGAAGG AAAGCTAAAATTATTAAGGATTATGAACCCAAACAAAGAGTGGGTAATGAGGGTGACGTGGAAGGTACCAGGGGATCTGATAACTAAATGGCAGGGAATAGTCAGATATTGGATGA GACAAAGAAATTTGAAATGGAACTATTATATGCATCAGCTGGCGGCCGCGAATTCACTAGTGATTCCCGTTTGTGCTAGGGTTCTTAGGCTTCTTGGGGGCTGCTGGAACTGCAAT GGGAGCAGCGGCGACAGCCCTGACGGTCCAGTCTCAGCATTTGCTTGCTGGGATACTGCAGCAGCAGAAGAATCTGCTGGCGGCTGTGGAGGCTCAACAGCAGATGTTGAAGCTGA CCATTTGGGGTGTTAAAAACCTCAATGCCCGCGTCACAGCCCTTGAGAAGTACCTAGAGGATCAGGCACGACTAAACTCCTGGGGGTGCGCATGGAAACAAGTATGTCATACCACA GTGGAGTGGCCCTGGACAAATCGGACTCCGGATTGGCAAAATATGACTTGGTTGGAGTGGGAAAGACAAATAGCTGATTTGGAAAGCAACATTACGAGACAATTAGTGAAGGCTAG AGAACAAGAGGAAAAGAATCTAGATGCCTATCAGAAGTTAACTAGTTGGTCAGATTTCTGGTCTTGGTTCGATTTCTCAAAATGGCTTAACATTTTAAAAATGGGATTTTTAGTAA TAGTAGGAATAATAGGGTTAAGATTACTTTACACAGTATATGGATGTATAGTGAGGGTTAGGCAGGGATATGTTCCTCTATCTCCACAGATCCATATCCAATCGAATTCCCGCGGC CGCAATTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGAC ATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAA TCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCC CTGCTGTCCATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTA CTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGGTCATAGCTGTTTC CTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCA CTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCGGATCCGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCC GCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAA ACTCATCAATGTATCTTATCATGTCTGTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATC CACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACG AGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCG CTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCA CGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCA GAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAA AGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTT TTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAAT CAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGC CGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTC GTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAACAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCT CGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAAC CGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTTCCGGGGATCGCAGTGGTGAGTAACCATGCATCATC AGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAA ACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAAT CGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAAT GTAACATCAGAGATTTTGAGACACAACAATTGGTCGAC SEQ ID NO: 11 Figure 1E The plasmid defined in (pDNA2b pGM299) TCAATATTGGCCATTAGCCATATTATTCATTGGTTATATAGCATAAATCAATATTGGCTATTGGCCATTGCATACGTTGTATCTATATCATAATATGTACATTT ATATTGGCTCATGTCCAATATGACCGCCATGTTGGCATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACA TAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATG GGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGT ACATGACCTTACGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAATGGGCGTGGATAGCGGTTTGACTCA CGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGG TAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCACTAGAAGCTTTATTGCGGTAGTTTATCACAGTTAAATTGCTAACGCAGTCAGTGCTT CTGACACAACAGTCTCGAACTTAAGCTGCAGAAGTTGGTCGTGAGGCACTGGGCAGGTAAGTATCAAGGTTACAAGACAGGTTTAAGGAGACCAATAGAAACTGGGCTTGTCGAGA CAGAGAAGACTCTTGCGTTTCTGATAGGCACCTATTGGTCTTACTGACATCCACTTTGCCTTTCTCTCCACAGGTGTCCACTCCCAGTTCAATTACAGCTCTTAAGGCTAGAGTAC TTAATACGACTCACTATAGGCTAGCCTCGAGAATTCGATTATGCCCCTAGGACCAGAAGAAAGAAGATTGCTTCGCTTGATTTGGCTCCTTTACAGCACCAATCCATATCCACCAA GTGGGGAAGGGACGGCCAGACAACGCCGACGAGCCAGGAGAAGGTGGAGACAACAGCAGGATCAAATTAGAGTCTTGGTAGAAAGACTCCAAGAGCAGGTGTATGCAGTTGACCGC CTGGCTGACGAGGCTCAACACTTGGCTATACAACAGTTGCCTGACCCTCCTCATTCAGCTTAGAATCACTAGTGAATTCACGCGTGGTACCTCTAGAGTCGACCCGGGCGGCCGCT TCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTA TAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATC GATAAGGATCCGTCGACCAATTGTTGTGTCTCAAAATCTCTGATGTTACATTGCACAAGATAAAAATATATCATCATGAACAATAAAACTGTCTGCTTACATAAACAGTAATACAA GGGGTGTTATGAGCCATATTCAACGGGAAACGTCTTGCTCTAGGCCGCGATTAAATTCCAACATGGATGCTGATTTATATGGGTATAAATGGGCTCGCGATAATGTCGGGCAATCA GGTGCGACAATCTATCGATTGTATGGGAAGCCCGATGCGCCAGAGTTGTTTCTGAAACATGGCAAAGGTAGCGTTGCCAATGATGTTACAGATGAGATGGTCAGACTAAACTGGCT GACGGAATTTATGCCTCTTCCGACCATCAAGCATTTTATCCGTACTCCTGATGATGCATGGTTACTCACCACTGCGATCCCCGGAAAAACAGCATTCCAGGTATTAGAAGAATATC CTGATTCAGGTGAAAATATTGTTGATGCGCTGGCAGTGTTCCTGCGCCGGTTGCATTCGATTCCTGTTTGTAATTGTCCTTTTAACAGCGATCGCGTATTTCGTCTCGCTCAGGCG CAATCACGAATGAATAACGGTTTGGTTGATGCGAGTGATTTTGATGACGAGCGTAATGGCTGGCCTGTTGAACAAGTCTGGAAAGAAATGCATAAGCTGTTGCCATTCTCACCGGA TTCAGTCGTCACTCATGGTGATTTCTCACTTGATAACCTTATTTTTGACGAGGGGAAATTAATAGGTTGTATTGATGTTGGACGAGTCGGAATCGCAGACCGATACCAGGATCTTG CCATCCTATGGAACTGCCTCGGTGAGTTTTCTCCTTCATTACAGAAACGGCTTTTTCAAAAATATGGTATTGATAATCCTGATATGAATAAATTGCAGTTTCATTTGATGCTCGAT GAGTTTTTCTAACTGTCAGACCAAGTTTACTCATATATACTTTAGATTGATTTAAAACTTCATTTTTAATTTAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGACCAA AATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAAC CACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAG TTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAG ACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAG AAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGT CCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTG CTGGCCTTTTGCTCACATGGCTCGACAGATCT SEQ ID NO: 12 Figure 1F The plasmid defined in (pDNA3a pGM301) ATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCT GGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTA CTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTT ATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGC CAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCG TGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTT TAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAG CGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGT GCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGA GTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCC TCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGG GACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAG GGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCG TGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCG ATTGCCATGGCAACATATATCCAGAGAGTACAGTGCATCTCAACATCACTACTGGTTGTTCTCACCACATTGGTCTCGTGTCAGATTCCCAGGGATAGGCTCTCTAACATAGGGGT CATAGTCGATGAAGGGAAATCACTGAAGATAGCTGGATCCCACGAATCGAGGTACATAGTACTGAGTCTAGTTCCGGGGGTAGACTTTGAGAATGGGTGCGGAACAGCCCAGGTTATCCAGTACAAGAGCCTACTGAACAGGCTGTTAATCCCATTGAGGGATGCCTTAGATCTTCAGGAGGCTCTGATAACTGTCACCAATGATACGACACAAAATGCCGGTGCTCCCCAG TCGAGATTCTTCGGTGCTGTGATTGGTACTATCGCACTTGGAGTGGCGACATCAGCACAAATCACCGCAGGGATTGCACTAGCCGAAGCGAGGGAGGCCAAAAGAGACATAGCGCT CATCAAAGAATCGATGACAAAAACACACAAGTCTATAGAACTGCTGCAAAACGCTGTGGGGGAACAAATTCTTGCTCTAAAGACACTCCAGGATTTCGTGAATGATGAGATCAAAC CCGCAATAAGCGAATTAGGCTGTGAGACTGCTGCCTTAAGACTGGGTATAAAATTGACACAGCATTACTCCGAGCTGTTAACTGCGTTCGGCTCGAATTTCGGAACCATCGGAGAG AAGAGCCTCACGCTGCAGGCGCTGTCTTCACTTTACTCTGCTAACATTACTGAGATTATGACCACAATCAGGACAGGGCAGTCTAACATCTATGATGTCATTTATACAGAACAGAT CAAAGGAACGGTGATAGATGTGGATCTAGAGAGATACATGGTCACCCTGTCTGTGAAGATCCCTATTCTTTCTGAAGTCCCAGGTGTGCTCATACACAAGGCATCATCTATTTCTT ACAACATAGACGGGGAGGAATGGTATGTGACTGTCCCCAGCCATATACTCAGTCGTGCTTCTTTCTTAGGGGGTGCAGACATAACCGATTGTGTTGAGTCCAGATTGACCTATATA TGCCCCAGGGATCCCGCACAACTGATACCTGACAGCCAGCAAAAGTGTATCCTGGGGGACACAACAAGGTGTCCTGTCACAAAAGTTGTGGACAGCCTTATCCCCAAGTTTGCTTT TGTGAATGGGGGCGTTGTTGCTAACTGCATAGCATCCACATGTACCTGCGGGACAGGCCGAAGACCAATCAGTCAGGATCGCTCTAAAGGTGTAGTATTCCTAACCCATGACAACT GTGGTCTTATAGGTGTCAATGGGGTAGAATTGTATGCTAACCGGAGAGGGCACGATGCCACTTGGGGGGTCCAGAACTTGACAGTCGGTCCTGCAATTGCTATCAGACCCGTTGAT ATTTCTCTCAACCTTGCTGATGCTACGAATTTCTTGCAAGACTCTAAGGCTGAGCTTGAGAAAGCACGGAAAATCCTCTCGGAGGTAGGTAGATGGTACAACTCAAGAGAGACTGT GATTACGATCATAGTAGTTATGGTCGTAATATTGGTGGTCATTATAGTGATCATCATCGTGCTTTATAGACTCAGAAGGTGAAATCACTAGTGAATTCACTCCTCAGGTGCAGGCT GCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGACATCATGAAGCCCCTTGAGCATCTGAC TTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATT TGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCCCTGCTGTCCATTCCTTATTCCATAGA AAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTACTAGCCAGATTTTTCCTCCTCTCCTG ACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACA ATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCT GTCGTGCCAGCGGATCCGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACT AATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTAACTTGTTTATTGC AGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCT GTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAA AGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGT CAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCT CCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCT GCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACA GAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAA ACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGA ACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACT TGGTCTGACAGTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTC ACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGA AATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAACAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACC AAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATC AACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTTCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGG TCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCA TACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCG TTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAATGTAACATCAGAGATTTTGAGACACAA CAATTGGTCGAC SEQ ID NO: 13 Figure 1G The plasmid defined in (pDNA3b pGM303) ATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCT GGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTA CTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTT ATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGC CAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCG TGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTT TAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAG CGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGT GCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGA GTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCC TCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGG GACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAG GGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCGTGTGACCGGCGGCTCTAGA GCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTCCTCGAGCATGTGGTCTGAGT TAAAAATCAGGAGCAACGACGGAGGTGAAGGACCAGAGGACGCCAACGACCCCCGGGGAAAGGGGGTGCAACACATCCATATCCAGCCATCTCTACCTGTTTATGGACAGAGGGTT AGGGATGGTGATAGGGGCAAACGTGACTCGTACTGGTCTACTTCTCCTAGTGGTAGCACCACAAAACCAGCATCAGGTTGGGAGAGGTCAAGTAAAGCCGACACATGGTTGCTGAT TCTCTCATTCACCCAGTGGGCTTTGTCAATTGCCACAGTGATCATCTGTATCATAATTTCTGCTAGACAAGGGTATAGTATGAAAGAGTACTCAATGACTGTAGAGGCATTGAACA TGAGCAGCAGGGAGGTGAAAGAGTCACTTACCAGTCTAATAAGGCAAGAGGTTATAGCAAGGGCTGTCAACATTCAGAGCTCTGTGCAAACCGGAATCCCAGTCTTGTTGAACAAA AACAGCAGGGATGTCATCCAGATGATTGATAAGTCGTGCAGCAGACAAGAGCTCACTCAGCACTGTGAGAGTACGATCGCAGTCCACCATGCCGATGGAATTGCCCCACTTGAGCC ACATAGTTTCTGGAGATGCCCTGTCGGAGAACCGTATCTTAGCTCAGATCCTGAAATCTCATTGCTGCCTGGTCCGAGCTTGTTATCTGGTTCTACAACGATCTCTGGATGTGTTA GGCTCCCTTCACTCTCAATTGGCGAGGCAATCTATGCCTATTCATCAAATCTCATTACACAAGGTTGTGCTGACATAGGGAAATCATATCAGGTCCTGCAGCTAGGGTACATATCA CTCAATTCAGATATGTTCCCTGATCTTAACCCCGTAGTGTCCCACACTTATGACATCAACGACAATCGGAAATCATGCTCTGTGGTGGCAACCGGGACTAGGGGTTATCAGCTTTG CTCCATGCCGACTGTAGACGAAAGAACCGACTACTCTAGTGATGGTATTGAGGATCTGGTCCTTGATGTCCTGGATCTCAAAGGGAGAACTAAGTCTCACCGGTATCGCAACAGCG AGGTAGATCTTGATCACCCGTTCTCTGCACTATACCCCAGTGTAGGCAACGGCATTGCAACAGAAGGCTCATTGATATTTCTTGGGTATGGTGGACTAACCACCCCTCTGCAGGGT GATACAAAATGTAGGACCCAAGGATGCCAACAGGTGTCGCAAGACACATGCAATGAGGCTCTGAAAATTACATGGCTAGGAGGGAAACAGGTGGTCAGCGTGATCATCCAGGTCAA TGACTATCTCTCAGAGAGGCCAAAGATAAGAGTCACAACCATTCCAATCACTCAAAACTATCTCGGGGCGGAAGGTAGATTATTAAAATTGGGTGATCGGGTGTACATCTATACAA GATCATCAGGCTGGCACTCTCAACTGCAGATAGGAGTACTTGATGTCAGCCACCCTTTGACTATCAACTGGACACCTCATGAAGCCTTGTCTAGACCAGGAAATAAAGAGTGCAAT TGGTACAATAAGTGTCCGAAGGAATGCATATCAGGCGTATACACTGATGCTTATCCATTGTCCCCTGATGCAGCTAACGTCGCTACCGTCACGCTATATGCCAATACATCGCGTGT CAACCCAACAATCATGTATTCTAACACTACTAACATTATAAATATGTTAAGGATAAAGGATGTTCAATTAGAGGCTGCATATACCACGACATCGTGTATCACGCATTTTGGTAAAG GCTACTGCTTTCACATCATCGAGATCAATCAGAAGAGCCTGAATACCTTACAGCCGATGCTCTTTAAGACTAGCATCCCTAAATTATGCAAGGCCGAGTCTTAAGCGGCCGCGCAT GCGAATTCACTCCTCAGGTGCAGGCTGCCTATCAGAAGGTGGTGGCTGGTGTGGCCAATGCCCTGGCTCACAAATACCACTGAGATCTTTTTCCCTCTGCCAAAAATTATGGGGAC ATCATGAAGCCCCTTGAGCATCTGACTTCTGGCTAATAAAGGAAATTTATTTTCATTGCAATAGTGTGTTGGAATTTTTTGTGTCTCTCACTCGGAAGGACATATGGGAGGGCAAATCATTTAAAACATCAGAATGAGTATTTGGTTTAGAGTTTGGCAACATATGCCCATATGCTGGCTGCCATGAACAAAGGTTGGCTATAAAGAGGTCATCAGTATATGAAACAGCCCC CTGCTGTCTATTCCTTATTCCATAGAAAAGCCTTGACTTGAGGTTAGATTTTTTTTATATTTTGTTTTGTGTTATTTTTTTCTTTAACATCCCTAAAATTTTCCTTACATGTTTTA CTAGCCAGATTTTTCCTCCTCTCCTGACTACTCCCAGTCATAGCTGTCCCTCTTCTCTTATGGAGATCCCTCGACCTGCAGCCCAAGCTTGGCGTAATCATGGTCATAGCTGTTTC CTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCA CTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCGGATCCGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCC GCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTT TTGCAAAAAGCTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAA ACTCATCAATGTATCTTATCATGTCTGTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATC CACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACG AGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCG CTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCA CGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCA GAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAA AGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTT TTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAAT CAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGC CGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTC GTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAACAGCTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCT CGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAAC CGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTTCCGGGGATCGCAGTGGTGAGTAACCATGCATCATC AGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAA ACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAAT CGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGATGATATATTTTTATCTTGTGCAAT GTAACATCAGAGATTTTGAGACACAACAATTGGTCGAC SEQ ID NO: 14 Exemplary WPRE component (mWPRE) 1 GGGCCCAATC AACCTCTGGA TTACAAAATT TGTGAAAGAT TGACTGGTAT TCTTAACTAT 61 GTTGCTCCTT TTACGCTATG TGGATACGCT GCTTTAATGC CTTTGTATCA TGCTATTGCT 121 TCCCGTATGG CTTTCATTTT CTCCTCCTTG TATAAATCCT GGTTGCTGTC TCTTTATGAG 181 GAGTTGTGGC CCGTTGTCAG GCAACGTGGC GTGGTGTGCA CTGTGTTTGC TGACGCAACC 241 CCCACTGGTT GGGGCATTGC CACCACCTGT CAGCTCCTTT CCGGGACTTT CGCTTTCCCC 301 CTCCCTATTG CCACGGCGGA ACTCATCGCC GCCTGCCTTG CCCGCTGCTG GACAGGGGCT 361 CGGCTGTTGG GCACTGACAA TTCCGTGGTG TTGTCGGGGA AATCATCGTC CTTTCCTTGG 421 CTGCTCGCCT GTGTTGCCAC CTGGATTCTG CGCGGGACGT CCTTCTGCTA CGTCCCTTCG 481 GCCCTCAATC CAGCGGACCT TCCTTCCCGC GGCCTGCTGC CGGCTCTGCG GCCTCTTCCG 541 CGTCTTCGCC TTCGCCCTCA GACGAGTCGG ATCTCCCTTT GGGCCGCCTC CCCGCAAGCT SEQ ID NO: 15 Exemplary CAG promoter ATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCT GGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGC CCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTA CTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTCGAGGTGAGCCCCACGTTCTGCTTCACTCTCCCCATCTCCCCCCCCTCCCCACCCCCAATTTTGTATTTATTT ATTTTTTAATTATTTTGTGCAGCGATGGGGGCGGGGGGGGGGGGGGGGCGCGCGCCAGGCGGGGCGGGGCGGGGCGAGGGGCGGGGCGGGGCGAGGCGGAGAGGTGCGGCGGCAGC CAATCAGAGCGGCGCGCTCCGAAAGTTTCCTTTTATGGCGAGGCGGCGGCGGCGGCGGCCCTATAAAAAGCGAAGCGCGCGGCGGGCGGGAGTCGCTGCGCGCTGCCTTCGCCCCG TGCCCCGCTCCGCCGCCGCCTCGCGCCGCCCGCCCCGGCTCTGACTGACCGCGTTACTCCCACAGGTGAGCGGGCGGGACGGCCCTTCTCCTCCGGGCTGTAATTAGCGCTTGGTT TAATGACGGCTTGTTTCTTTTCTGTGGCTGCGTGAAAGCCTTGAGGGGCTCCGGGAGGGCCCTTTGTGCGGGGGGAGCGGCTCGGGGGGTGCGTGCGTGTGTGTGTGCGTGGGGAG CGCCGCGTGCGGCTCCGCGCTGCCCGGCGGCTGTGAGCGCTGCGGGCGCGGCGCGGGGCTTTGTGCGCTCCGCAGTGTGCGCGAGGGGAGCGCGGCCGGGGGCGGTGCCCCGCGGT GCGGGGGGGGCTGCGAGGGGAACAAAGGCTGCGTGCGGGGTGTGTGCGTGGGGGGGTGAGCAGGGGGTGTGGGCGCGTCGGTCGGGCTGCAACCCCCCCTGCACCCCCCTCCCCGA GTTGCTGAGCACGGCCCGGCTTCGGGTGCGGGGCTCCGTACGGGGCGTGGCGCGGGGCTCGCCGTGCCGGGCGGGGGGTGGCGGCAGGTGGGGGTGCCGGGCGGGGCGGGGCCGCC TCGGGCCGGGGAGGGCTCGGGGGAGGGGCGCGGCGGCCCCCGGAGCGCCGGCGGCTGTCGAGGCGCGGCGAGCCGCAGCCATTGCCTTTTATGGTAATCGTGCGAGAGGGCGCAGG GACTTCCTTTGTCCCAAATCTGTGCGGAGCCGAAATCTGGGAGGCGCCGCCGCACCCCCTCTAGCGGGCGCGGGGCGAAGCGGTGCGGCGCCGGCAGGAAGGAAATGGGCGGGGAG GGCCTTCGTGCGTCGCCGCGCCGCCGTCCCCTTCTCCCTCTCCAGCCTCGGGGCTGTCCGCGGGGGGACGGCTGCCTTCGGGGGGGACGGGGCAGGGCGGGGTTCGGCTTCTGGCG TGTGACCGGCGGCTCTAGAGCCTCTGCTAACCATGTTCATGCCTTCTTCTTTTTCCTACAGCTCCTGGGCAACGTGCTGGTTATTGTGCTGTCTCATCATTTTGGCAAAGAATTGC TCGAGCCACC SEQ ID NO: 16 Modified SIV / CFTR RNA sequence ucucuuacua ggagaccagc uugagccugg guguucgcug guuagccuaa ccugguuggc 60 caccaggggu aaggacuccu uggcuuagaa agcuauaaaa cuugccugca uuagagcuua 120 ucugaguca guguccucau ugacgccuca cucucuugaa cgggaaucuu ccuuacugggg 180 uucucucucu gaccaggcg agagaacuc cagcaguggc gcccgaacag ggacuugagu 240 gagaguguag gcacguacag cugagaaggc gucggacgcg aaggaagcgc ggggugcgac 300 gcgaccaag aggacuug gugaguaggc ucucgagug ccgggaaaaa gcucgagccu 360 aguagagga cuaggagagg ccguagccgu aacuacug ggcaaguagg gcaggcggug 420 gguacgcaau ugggggcggc uaccucagca cuaauagga ggaauuaga ccaauuugag 480 aaaauacgac uucgcccgaa cggaaagaa aaguaccaaa uaaaacauuu auauauugggc 540 agggaggg auggagcgc ucggcucc augagagguu guuggagaca gaggagggg 600 guaaaagaau cauagaaguc cucuaccccc uagaccaac aggaucggag ggcuuaaaaaa 660 gucuguuca ucugugugc gugcuauauu gcuugcacaca ggacagaaa gugaagaca 720 cagaggaagc aguagcaca guagacaac acugccaucu aguggaaaaaaaaaagug 780 caacagagac aucuagugga caaaagaaaa augacaaggg aauagcagcg ccaccuggug 840 cgagucagaa uuuuccagcg caacaacaag gaauugccu ggguacaugu acccuuguca 900 ccgcgcaccu uaaaugcgug gguaaaagca guagaggaga aaaaauuugg agcagaaaua 960 1020 uggggggcugc uggaacugca uugggagcag cggcgacagc ccugacgguc cagucucagc 1080 auuugcuugc uggguaacug cagcagcaga agaaucugcu ggcggcugug gaggcucaac 1140 agcagauguu gaagcugacc auuuggggug uuaaaaaccu caaugcccgc gucacagccc 1200 uugagaaagua ccuagaggau caggcacgac uaaacuccug ggggugcgca uggaaacaag 1260 uaugucauac cacaguggag uggcccugga caaaucggac uccggauugg caaaauaaga 1320 cuugguugga gugggaaaga caaauagcug auuuggaag caacauuacg agacaauuag 1380 ugaaggcuag agaacaagag gaaaagaauc uagaugccua ucagaaguua acuaguuggu 1440 cagauuuucug gucuugguuc gauuuucucaa aauggcuuaa cauuuuaaaa aagggauuuu 1500 uaguaauagu aggaauaaua ggguuaagau uacuuuacac aguauaugga uguauaguga 1560 ggguuaggca gggauauguu ccucuaucuc cacagaucca uauaaagcgg caauuuuaaa 1620 agaaagggag gaauaggggg acagacuuca gcagagagac uaauuaauau aauaacaaca 1680 caauuagaaa uacaacauuu acaaaccaaa auucaaaaaa uuuuaaauuu uagagccgcg 1740 gagaucuguu acauaacuua ugguaaaugg ccugccuggc ugacugccca augaccccug 1800 cccaaugaug ucaauaauga uguauguucc cauguaaugc caauagggac uuuccauuga 1860 ugucaauggg uggaguauuu augguaacug cccacuuggc aguacaucaa guguaucaua 1920 ugccaaguau gcccccuauu gaugucaaug augguaaaug gccugccugg cauuaugccc 1980 aguacaugac cuuaugggac uuuccuacuu ggcaguacau cuauguauua gucauugcua 2040 uuaccauggg aauucacuag uggagaagag caugcuugag ggcugagugc cccucagugg 2100 gcagagagca cauggcccac agucccugag aaguuggggg gagggguggg caauugaacu 2160 ggugccuaga gaaggugggg cuuggguaaa cugggaaagu gauguggugu acuggcucca 2220 ccuuuuuccc cagggugggg gagaaccaua uauaagugca guagucucug ugaacauca 2280 agcuucugcc uucuccucc ugugaguuug quagccacca ugcagagag cccucuggag 2340 aaggccucug gaggagça gcugucuuc agcuggacca ggcccug gaggaagggc 2400 uacaggcaga gacuggagcu gucugacauc uaccagaucc ccucugugga cucugcugac 2460 aaccugucug agaagcugga gagggagugg gauagagagc uggccagca gagaacccc 2520 augcugauca augcccugag gagaugcuuc augcuggau augcuucuc augcuucuc 2580 cuguaccugg gggaagugac caggcugug cagcucugc ugcugggcag aucauugcc 2640 agcuaugacc cugashaacha ggaggagagg agcauugcca cugaccuggg cauggccug 2700 ugccugcugu ucauugugag gacccugcug cugcacccug ccaucuuugg cugcaccac 2760 auuggcaugc agaagaggau ugccauguuc agccugaucu ahaaaaac ccugaagcug 2820 shake shake shake gaucashauu ggshagcugg shake shake shake shake 2880 cugacaagu uugaugagggg cuggcccug gccacuuug uguggauugc cccucugcag 2940 guggcccugc ugaugggccu gauuugggag cugcugcagg cucugccuu uugggccug 3000 ggcuuccuga uugugcuggc ccuguuucag gcuggccugg gcaggaugau gaugaaguac 3060 agggaccaga gggcaggca gaucagugag agggaccaga ucaccucuga gaugauugag 3120 aacauccagu cugugaggc cucuguugg gaggaagcua uggagau gauugaaaac 3180 cugaggcaga cagagcugaa gcugaccagg aaggcugccu augugagaua cucacagc 3240 quagcuucu cucucucugg cucuucug cugugcugcc cucugcugcc cucugcug 3300 aucaagggga ucauccugag aagauuuuc accaccauca gcuucugcau ugugcugagg 3360 auggcuguga ccagacagou ccccuggggcu gugcagaccu gguaugacag ccuggggggcc 3420 aucacaaga uccaggacuu ccugcagag caggaguaca agacccugga guacaccug 3480 accaccacag aaggugau ggagaaugug accacccuucu gggagggggg 3540 cuguuugaga aggccaagca aaxagaac aaagaagg ggaugaugg ggaugacucc 3600 cuguucuuc cuckoocuc cugcugggc acccugugc ugagacau cuckoo 3660 auugagaggg ggcagcugcu ggcuguggcu ggaucuacag gggcuggcaa gaccagccug 3720 3780 aucagcuuuu gcagccaguu cagcuggauc augccuggca ccaucaaagga gaacaucauc 3840 3900 gagacauca ccaaguuuugc ugagaaggac aacauugugc uggggggaggg aggcauuaca 3960 cugucugggg gccagagagc cagaaucagc cuggccaggg cuguguacaa ggaugcugac 4020 cuguaccugc uggacucccc cuuuggcuac cuggaugugc ugacagagaa ggagauuuu 4080 gagagcugug ugugcaagcu gauggccaac aagaccagaa uccuggugac cagcaagaug 4140 4200 4260 gacagcuuug accaguucuc ugcugagagg aggaacagca uccugacaga gaccugcac 4320 agauucagcc uggagggaga ugccccugug agcuggacag agaccaagaa cgagagcuuc 4380 aagcagacag gggaguuugg ggagaagagg aagacucca uccugaaccc caucaacagc 4440 aucaggaagu ucagcauugu gcagaaaacc ccccugcaga ugaauggcau ugagaagagau 4500 ucugaugagc cccuggagag gagacugagc cuggugccug auucugagca gggagaggcc 4560 auccugccua ggaucucugu gaucagcaca ggcccuacac ugcaggccag aaagggcag 4620 ucugugcuga accugaugac ccacucugug aaccagggcc agaacaucca caggaaaacc 4680 acagccucca ccaggaaagu gagccuggcc ccucaggcca aucugacaga ggugcauc 4740 uacagcagga ggcugucuca ggagacaggc cuggagaauuu cugaggagau caaugaggag 4800 gaccugaaag agugcuucuu ugaugacaug gagagcaucc cugcugugac caccuggaac 4860 4920 gugaucuucc uggcugaagu ggcugcccu cugguggugc uguggcugcu gggaaacacc 4980 ccacugcagg acaagggcaa cagcacccac agcaggaaca acagcuaugc ugugaucauc 5040 accuccaccu ccagcuacua ugugucuac aucuaugugg gaguggcuga uacccugcug 5100 gcuaugggcu ucuuuagagg ccugccccug gugcacacac ugaucacagu gagcaagauc 5160 cuccaccaca agaugcugca cucugugcug caggcuccua ugagcacccu gaauacccug 5220 aaggcugggg gcauccugaa cagauucucc aagcauauug ccauccugga ugaccugcug 5280 ccucucacca ucuuugacuu cauccagcug cugcugauug ugauuggggc cauugcugug 5340 guggcagugc ugcagcccua caucuuugug gccacagugc cugugauugu ggccuucauc 5400 augcugaggg ccuacuuucu gcagaccucc cagcagcuga agcagcugga gucugagggc 5460 agaagcccca ucuucaccca ccuggugaca agccugaagg gccuguggac ccugagagcc 5520 uuuggcaggc agcccuacuu ugacaccug uuccacaagg cccugaaccu gcacacagcc 5580 aacugguucc ucuaccuguc cacccugaga ugguuccaga ugagaauuga gaugaucuuu 5640 gucaucuucu ucauugcugu gaccuucauc agcauucuga ccacaggaga gggagagggc 5700 agagugggca uuauccugac ccuggccaug aacaucauga gcacacugca gugggcagug 5760 aacagcagca uugaugugga cagccugaug aggaguguga gcagaguguu caaguucauu 5820 gauaugccca cagagggcaa gccuaccaag agcaccaagc ccuacaagaa uggccagcug 5880 agcaaaguga ugaucauuga gaacagccau gugaagaagg augauaucug gcccagugga 5940 ggccagauga cagugaagga ccugacagcc aaguacacag aggggggcaa ugcuauccug 6000 gagaacaucu ccuucagcau cuccccuggc cagagagugg gacugcuggg aagaacaggc 6060 ucuggcaagu cuacccugcu gucugccuuc cugaggcugc ugaacacaga gggagagauc 6120 cagauugaug gaguguccug ggacagcauc acacugcagc agguggagaa ggccuuuggu 6180 gugauccccc agaaaguguu caucuucagu ggcaccuuca ggaagaaccu ggaccccuau 6240 gagcaguggu cugaccagga gauuuggaaa guggcugaug aagugggccu gagaagugug 6300 auugagcagu ucccuggcaa gcuggacuuu guccuggugg auggggcug uggcugagc 6360 cauggccaca agcagcugau gugccuggcc agaucagugc ugagcaaggc caagauccug 6420 cugcuggaug agccuucugc ccaccuggau ccugugaccu accagaucau caggaggacc 6480 cucaagcagg ccuuugcuga cugcacaguc auccugug agcacaggau ugaggccaug 6540 cuggagugcc agcaguuccu ggugauugag gagacaaag ugaggcagua ugacagcauc 6600 6660 aagcuguucc cccacaggaa cagcuccaag ugcaagagca agccccagau ugcugcccug 6720 aaggagga cagaggagga agugcaggac accaggcugu gagggcccaa ucaaccucug 6780 gauuacaaaa uuugugaaag auugacuggu auucuuacu auguugcucc uuuuacgcua 6840 uggguaacg cugcuuuau gccuuuguau caugcuauug cuucccguau ggcuuuucauu 6900 uucuccuccu uguauaaauc cugguugcug ucucuuuaug aggaguugug gcccguuguc 6960 aggcaacgug gcguggugug cacuguguuu gcugacgcaa cccccacugg uuggggcauu 7020 gccaccaccu gucagcuccu uuccgggacu uucgcuuucc cccuccuau ugccacggcg 7080 gaacucaucg ccgccugccu ugcccgcugc uggacagggg cucggcuguu gggcacugac 7140 aauuccgugg uguugucggg gaaucaucg uccuuuccuu ggcugcucgc cuguuguugcc 7200 accuggauuc ugcgcgggac guccuucugc uacgucccuu cggcccuacaa uccagcggac 7260 cuuccuuccc gcggccugcu gccggcucug cggccucuuc cgcgucuucg ccuucgcccu 7320 cagacgaguc ggaucucccu uugggccgcc uccccgcaag cuucgcacuu uuuaaaagaa 7380 aagggaggac uggaugggau uuauuacucc gauaggacgc uggcuuguaa cucagucucu 7440 uacuaggaga ccagcuugag ccuggguguu cgcugguuag ccuaaccugg uuggccacca 7500 gggguaagga cuccuuggcu uagaaagcua auaaacuugc cugcauuagagcu 7553 SEQ ID NO: 17 Fct4 protein Gln Ile Pro Arg Asp Arg Leu Ser Asn Ile Gly Val Ile Val Asp Glu 1 5 10 15 Gly Lys Ser Leu Lys Ile Ala Gly Ser His Glu Ser Arg Tyr Ile Val 20 25 30 Leu Ser Leu Val Pro Gly Val Asp Phe Glu Asn Gly Cys Gly Thr Ala 35 40 45 Gln Val Ile Gln Tyr Lys Ser Leu Leu Asn Arg Leu Leu Ile Pro Leu 50 55 60 Arg Asp Ala Leu Asp Leu Gln Glu Ala Leu Ile Thr Val Thr Asn Asp 65 70 75 80 Thr Thr Gln Asn Ala Gly Ala Pro Gln Ser Arg Phe Phe Gly Ala Val 85 90 95 In Gly Thr In Ala Leu Gly Val Ala Thr Ser Ala Gln In Thr Ala 100 105 110 Gly Ile Ala Leu Ala Glu Ala Arg Glu Ala Lys Arg Asp Ile Ala Leu 115 120 125 Ile Lys Glu Ser Met Thr Lys Thr His Lys Ser Ile Glu Leu Leu Gln 130 135 140 Asn Ala Val Gly Glu Gln Ile Leu Ala Leu Lys Thr Leu Gln Asp Phe 145 150 155 160 Val Asn Asp Glu Ile Lys Pro Ala Ile Ser Glu Leu Gly Cys Glu Thr 165 170 175 Pathway Leu Arg Leu Gly Ile Lys Leu Thr Gln His Tyr Ser Glu Leu 180 185 190 Leu Thr Ala Phe Gly Ser Asn Phe Gly Thr Ile Gly Glu Lys Ser Leu 195 200 205 Thr Leu Gln Ala Leu Ser Ser Leu Tyr Ser Ala Asn Ile Thr Glu Ile 210 215 220 Met Thr Thr Ile Arg Thr Gly Gln Ser Asn Ile Tyr Asp Val Ile Tyr 225 230 235 240 Thr Glu Gln Ile Lys Gly Thr Val Ile Asp Val Asp Leu Glu Arg Tyr 245 250 255 Met Val Thr Leu Ser Val Lys Ile Pro Ile Leu Ser Glu Val Pro Gly 260 265 270 Val Leu Ile His Lys Ala Ser Ser Ile Ser Tyr Asn Ile Asp Gly Glu 275 280 285 Glu Trp Tyr Val Thr Val Pro Ser His Ile Leu Ser Arg Ala Ser Phe 290 295 300 Leu Gly Gly Ala Asp Ile Thr Asp Cys Val Glu Ser Arg Leu Thr Tyr 305 310 315 320 Ile Cys Pro Arg Asp Pro Ala Gln Leu Ile Pro Asp Ser Gln Gln Lys 325 330 335 Cys Ile Leu Gly Asp Thr Thr Arg Cys Pro Val Thr Lys Val Val Asp 340 345 350 Ser Leu Ile Pro Lys Phe Ala Phe Val Asn Gly Gly Val Val Ala Asn 355 360 365 Cys Ile Ala Ser Thr Cys Thr Cys Gly Thr Gly Arg Arg Pro Ile Ser 370 375 380 Gln Asp Arg Ser Lys Gly Val Val Phe Leu Thr His Asp Asn Cys Gly 385 390 395 400 Leu Ile Gly Val Asn Gly Val Glu Leu Tyr Ala Asn Arg Arg Gly His 405 410 415 Asp Ala Thr Trp Gly Val Gln Asn Leu Thr Val Gly Pro Ala Ile Ala 420 425 430 Ile Arg Pro Val Asp Ile Ser Leu Asn Leu Ala Asp Ala Thr Asn Phe 435 440 445 Leu Gln Asp Ser Lys Ala Glu Leu Glu Lys Ala Arg Lys Ile Leu Ser 450 455 460 Glu Val Gly Arg Trp Tyr Asn Ser Arg Glu Thr Val Ile Thr Ile Ile 465 470 475 480 Val Val Met Val Val Ile Leo Val Val Ile Ile Val Ile Ile Ile Val 485 490 495 Leo Tyr Arg Leo Arg Arg SEQ ID NO: 18 Fct4 protein (including signal sequence) Met Ala Thr Tyr Ile Gln Arg Val Gln Cys Ile Ser Thr Ser Leu Leu 1 5 10 15 Val Val Leu Thr Thr Leu Val Ser Cys Gln Ile Pro Arg Asp Arg Leu 20 25 30 Ser Asn Ile Gly Val Ile Val Asp Glu Gly Lys Ser Leu Lys Ile Ala 35 40 45 Gly Ser His Glu Ser Arg Tyr Ile Val Leu Ser Leu Val Pro Gly Val 50 55 60 Asp Phe Glu Asn Gly Cys Gly Thr Ala Gln Val Ile Gln Tyr Lys Ser 65 70 75 80 Leu Leu Asn Arg Leu Leu Ile Pro Leu Arg Asp Ala Leu Asp Leu Gln 85 90 95 Glu Ala Leu Ile Thr Val Thr Asn Asp Thr Thr Gln Asn Ala Gly Ala 100 105 110 Pro Gln Ser Arg Phe Phe Gly Ala Val Ile Gly Thr Ile Ala Leu Gly 115 120 125 Val Ala Thr Ser Ala Gln Ile Thr Ala Gly Ile Ala Leu Ala Glu Ala 130 135 140 Arg Glu Ala Lys Arg Asp Ile Ala Leu Ile Lys Glu Ser Met Thr Lys 145 150 155 160 Thr His Lys Ser Ile Glu Leu Leu Gln Asn Ala Val Gly Glu Gln Ile 165 170 175 Leu Ala Leu Lys Thr Leu Gln Asp Phe Val Asn Asp Glu Ile Lys Pro 180 185 190 Ala Ile Ser Glu Leu Gly Cys Glu Thr Ala Ala Leu Arg Leu Gly Ile 195 200 205 Lys Leu Thr Gln His Tyr Ser Glu Leu Leu Thr Ala Phe Gly Ser Asn 210 215 220 Phe Gly Thr Ile Gly Glu Lys Ser Leu Thr Leu Gln Ala Leu Ser Ser 225 230 235 240 Leu Tyr Ser Ala Asn Ile Thr Glu Ile Met Thr Thr Ile Arg Thr Gly 245 250 255 Gln Ser Asn Ile Tyr Asp Val Ile Tyr Thr Glu Gln Ile Lys Gly Thr 260 265 270 Val Ile Asp Val Asp Leu Glu Arg Tyr Met Val Thr Leu Ser Val Lys 275 280 285 Ile Pro Ile Leu Ser Glu Val Pro Gly Val Leu Ile His Lys Ala Ser 290 295 300 Ser Ile Ser Tyr Asn Ile Asp Gly Glu Glu Trp Tyr Val Thr Val Pro 305 310 315 320 Ser His Ile Leu Ser Arg Ala Ser Phe Leu Gly Gly Ala Asp Ile Thr 325 330 335 Asp Cys Val Glu Ser Arg Leu Thr Tyr Ile Cys Pro Arg Asp Pro Ala 340 345 350 Gln Leu Ile Pro Asp Ser Gln Gln Lys Cys Ile Leu Gly Asp Thr Thr 355 360 365 Arg Cys Pro Val Thr Lys Val Val Asp Ser Leu Ile Pro Lys Phe Ala 370 375 380 Phe Val Asn Gly Gly Val Val Ala Asn Cys Ile Ala Ser Thr Cys Thr 385 390 395 400 Cys Gly Thr Gly Arg Arg Pro Ile Ser Gln Asp Arg Ser Lys Gly Val 405 410 415 Val Phe Leu Thr His Asp Asn Cys Gly Leu Ile Gly Val Asn Gly Val 420 425 430 Glu Leu Tyr Ala Asn Arg Arg Gly His Asp Ala Thr Trp Gly Val Gln 435 440 445 Asn Leu Thr Val Gly Pro Ala Ile Ala Ile Arg Pro Val Asp Ile Ser 450 455 460 Leu Asn Leu Ala Asp Ala Thr Asn Phe Leu Gln Asp Ser Lys Ala Glu 465 470 475 480 Leu Glu Lys Ala Arg Lys Ile Leu Ser Glu Val Gly Arg Trp Tyr Asn 485 490 495 Ser Arg Glu Thr Val Ile Thr Ile Ile Val Val Met Val Val Ile Leu 500 505 510 Val Val Ile Ile Val Ile Ile Ile Val Leu Tyr Arg Leu Arg Arg 515 520 525 SEQ ID NO: 19 Fct4 protein (fragment1) Phe Phe Gly Ala Val Ile Gly Thr Ile Ala Leu Gly Val Ala Thr Ser 1 5 10 15 Ala Gln Ile Thr Ala Gly Ile Ala Leu Ala Glu Ala Arg Glu Ala Lys 20 25 30 Arg Asp Ile Ala Leu Ile Lys Glu Ser Met Thr Lys Thr His Lys Ser 35 40 45 Ile Glu Leu Leu Gln Asn Ala Val Gly Glu Gln Ile Leu Ala Leu Lys 50 55 60 Thr Leu Gln Asp Phe Val Asn Asp Glu Ile Lys Pro Ala Ile Ser Glu 65 70 75 80 Leu Gly Cys Glu Thr Ala Ala Leu Arg Leu Gly Ile Lys Leu Thr Gln 85 90 95 His Tyr Ser Glu Leu Leu Thr Ala Phe Gly Ser Asn Phe Gly Thr Ile 100 105 110 Gly Glu Lys Ser Leu Thr Leu Gln Ala Leu Ser Ser Leu Tyr Ser Ala 115 120 125 Asn Ile Thr Glu Ile Met Thr Thr Ile Arg Thr Gly Gln Ser Asn Ile 130 135 140 Tyr Asp Val Ile Tyr Thr Glu Gln Ile Lys Gly Thr Val Ile Asp Val 145 150 155 160 Asp Leu Glu Arg Tyr Met Val Thr Leu Ser Val Lys Ile Pro Ile Leu 165 170 175 Ser Glu Val Pro Gly Val Leu Ile His Lys Ala Ser Ser Ile Ser Tyr 180 185 190 Asn Ile Asp Gly Glu Glu Trp Tyr Val Thr Val Pro Ser His Ile Leu 195 200 205 Ser Arg Ala Ser Phe Leu Gly Gly Ala Asp Ile Thr Asp Cys Val Glu 210 215 220 Ser Arg Leu Thr Tyr Ile Cys Pro Arg Asp Pro Ala Gln Leu Ile Pro 225 230 235 240 Asp Ser Gln Gln Lys Cys Ile Leu Gly Asp Thr Thr Arg Cys Pro Val 245 250 255 Thr Lys Val Val Asp Ser Leu Ile Pro Lys Phe Ala Phe Val Asn Gly 260 265 270 Gly Val Val Ala Asn Cys Ile Ala Ser Thr Cys Thr Cys Gly Thr Gly 275 280 285 Arg Arg Pro Ile Ser Gln Asp Arg Ser Lys Gly Val Val Phe Leu Thr 290 295 300 His Asp Asn Cys Gly Leu Ile Gly Val Asn Gly Val Glu Leu Tyr Ala 305 310 315 320 Asn Arg Arg Gly His Asp Ala Thr Trp Gly Val Gln Asn Leu Thr Val 325 330 335 Gly Pro Ala Ile Ala Ile Arg Pro Val Asp Ile Ser Leu Asn Leu Ala 340 345 350 Asp Ala Thr Asn Phe Leu Gln Asp Ser Lys Ala Glu Leu Glu Lys Ala 355 360 365 Arg Lys Ile Leu Ser Glu Val Gly Arg Trp Tyr Asn Ser Arg Glu Thr 370 375 380 Val Ile Thr Ile Ile Val Val Met Val Val Ile Leo Val Val Ile Ile 385 390 395 400 Val Ile Ile Ile Val Leu Tyr Arg Leu Arg Arg 405 410 SEQ ID NO: 20 Fct4 protein (fragment2) Gln Ile Pro Arg Asp Arg Leu Ser Asn Ile Gly Val Ile Val Asp Glu 1 5 10 15 Gly Lys Ser Leu Lys Ile Ala Gly Ser His Glu Ser Arg Tyr Ile Val 20 25 30 Leu Ser Leu Val Pro Gly Val Asp Phe Glu Asn Gly Cys Gly Thr Ala 35 40 45 Gln Val Ile Gln Tyr Lys Ser Leu Leu Asn Arg Leu Leu Ile Pro Leu 50 55 60 Arg Asp Ala Leu Asp Leu Gln Glu Ala Leu Ile Thr Val Thr Asn Asp<0OO2360>65 70 75 80 Thr Thr Gln Asn Ala Gly Ala Pro Gln Ser Arg 85 90 SEQ ID NO: 21 Fct4 protein signal sequence MATYIQRVQC ISTSLLVVLT TLVSC 25 SEQ ID NO: 22 p17 protein sequence Gly Ala Ala Thr Ser Ala Leu Asn Arg Arg Gln Leu Asp Gln Phe Glu 1 5 10 15 Lys Ile Arg Leu Arg Pro Asn Gly Lys Lys Lys Tyr Gln Ile Lys His 20 25 30 Leu Ile Trp Ala Gly Lys Glu Met Glu Arg Phe Gly Leu His Glu Arg 35 40 45 Leu Leu Glu Thr Glu Glu Gly Cys Lys Arg Ile Ile Glu Val Leu Tyr 50 55 60 Pro Leu Glu Pro Thr Gly Ser Glu Gly Leu Lys Ser Leu Phe Asn Leu 65 70 75 80 Val Cys Val Leu Tyr Cys Leu His Lys Glu Gln Lys Val Lys Asp Thr 85 90 95 Glu Glu Ala Val Ala Thr Val Arg Gln His Cys His Leu Val Glu Lys 100 105 110 Glu Lys Ser Ala Thr Glu Thr Ser Ser Gly Gln Lys Lys Asn Asp Lys 115 120 125 Gly Ile Ala Ala Pro Pro Gly Gly Ser Gln Asn Phe 130 135 140 SEQ ID NO: 23 p24 protein sequence Pro Ala Gln Gln Gln Gly Asn Ala Trp Val His Val Pro Leu Ser Pro 1 5 10 15 Arg Thr Leu Asn Ala Trp Val Lys Ala Val Glu Glu Lys Lys Phe Gly 20 25 30 Ala Glu Ile Val Pro Met Phe Gln Ala Leu Ser Glu Gly Cys Thr Pro 35 40 45 Tyr Asp Ile Asn Gln Met Leu Asn Val Leu Gly Asp His Gln Gly Ala 50 55 60 Leu Gln Ile Val Lys Glu Ile Ile Asn Glu Glu Ala Ala Gln Trp Asp 65 70 75 80 Val Thr His Pro Leu Pro Ala Gly Pro Leu Pro Ala Gly Gln Leu Arg 85 90 95 Asp Pro Arg Gly Ser Asp Ile Ala Gly Thr Thr Ser Ser Val Gln Glu 100 105 110 Gln Leu Glu Trp Ile Tyr Thr Ala Asn Pro Arg Val Asp Val Gly Ala 115 120 125 Ile Tyr Arg Arg Trp Ile Ile Leu Gly Leu Gln Lys Cys Val Lys Met 130 135 140 Tyr Asn Pro Val Ser Val Leu Asp Ile Arg Gln Gly Pro Lys Glu Pro 145 150 155 160 Phe Lys Asp Tyr Val Asp Arg Phe Tyr Lys Ala Ile Arg Ala Glu Gln 165 170 175 Ala Ser Gly Glu Val Lys Gln Trp Met Thr Glu Ser Leu Leu Ile Gln 180 185 190 Asn Ala Asn Pro Asp Cys Lys Val Ile Leu Lys Gly Leu Gly Met His 195 200 205 Pro Thr Leu Glu Glu Met Leu Thr Ala Cys Gln Gly Val Gly Gly Pro 210 215 220 Ser Tyr Lys Ala Lys Val Met 225 230 SEQ ID NO: 24 p8 protein sequence Val Gln Gln Gly Gly Pro Lys Arg Gln Arg Pro Pro Leu Arg Cys Tyr 1 5 10 15 Asn Cys Gly Lys Phe Gly His Met Gln Arg Gln Cys Pro Glu Pro Arg 20 25 30 Lys Thr Lys Cys Leu Lys Cys Gly Lys Leu Gly His Leu Ala Lys Asp 35 40 45 Cys Arg Gly Gln Val Asn 50 SEQ ID NO: 25 protease sequence Phe Glu Leu Pro Leu Trp Arg Arg Pro Ile Lys Thr Val Tyr Ile Glu 1 5 10 15 Gly Val Pro Ile Lys Ala Leu Leu Asp Thr Gly Ala Asp Asp Thr Ile 20 25 30 Ile Lys Glu Asn Asp Leu Gln Leu Ser Gly Pro Trp Arg Pro Lys Ile 35 40 45 Ile Gly Gly Ile Gly Gly Gly Leu Asn Val Lys Glu Tyr Asn Asp Arg 50 55 60 Glu Val Lys Ile Glu Asp Lys Ile Leu Arg Gly Thr Ile Leu Leu Gly 65 70 75 80 Ala Thr Pro Ile Asn Ile Ile Gly Arg Asn Leu Leu Ala Pro Ala Gly 85 90 95 Ala Arg Leu Val Met 100 SEQ ID NO: 26 p51 protein sequence Gly Gln Leu Ser Glu Lys Ile Pro Val Thr Pro Val Lys Leu Lys Glu 1 5 10 15 Gly Ala Arg Gly Pro Cys Val Arg Gln Trp Pro Leu Ser Lys Glu Lys 20 25 30 Ile Glu Ala Leu Gln Glu Ile Cys Ser Gln Leu Glu Gln Glu Gly Lys 35 40 45 Ile Ser Arg Val Gly Gly Glu Asn Ala Tyr Asn Thr Pro Ile Phe Cys 50 55 60 Ile Lys Lys Lys Asp Lys Ser Gln Trp Arg Met Leu Val Asp Phe Arg 65 70 75 80 Glu Leu Asn Lys Ala Thr Gln Asp Phe Phe Glu Val Gln Leu Gly Ile 85 90 95 Pro His Pro Ala Gly Leu Arg Lys Met Arg Gln Ile Thr Val Leu Asp 100 105 110 Val Gly Asp Ala Tyr Tyr Ser Ile Pro Leu Asp Pro Asn Phe Arg Lys 115 120 125 Tyr Thr Ala Phe Thr Ile Pro Thr Val Asn Asn Gln Gly Pro Gly Ile 130 135 140 Arg Tyr Gln Phe Asn Cys Leu Pro Gln Gly Trp Lys Gly Ser Pro Thr 145 150 155 160 Ile Phe Gln Asn Thr Ala Ala Ser Ile Leu Glu Glu Ile Lys Arg Asn 165 170 175 Leu Pro Ala Leu Thr Ile Val Gln Tyr Met Asp Asp Leu Trp Val Gly 180 185 190 Ser Gln Glu Asn Glu His Thr His Asp Lys Leu Val Glu Gln Leu Arg 195 200 205 Thr Lys Leu Gln Ala Trp Gly Leu Glu Thr Pro Glu Lys Lys Val Gln 210 215 220 Lys Glu Pro Pro Tyr Glu Trp Met Gly Tyr Lys Leu Trp Pro His Lys 225 230 235 240 Trp Glu Leu Ser Arg Ile Gln Leu Glu Glu Lys Asp Glu Trp Thr Val 245 250 255 Asn Asp Ile Gln Lys Leu Val Gly Lys Leu Asn Trp Ala Ala Gln Leu 260 265 270 Tyr Pro Gly Leu Arg Thr Lys Asn Ile Cys Lys Leu Ile Arg Gly Lys 275 280 285 Lys Asn Leu Leu Glu Leu Val Thr Trp Thr Pro Glu Ala Glu Ala Glu 290 295 300 Tyr Ala Glu Asn Ala Glu Ile Leu Lys Thr Glu Gln Glu Gly Thr Tyr 305 310 315 320 Tyr Lys Pro Gly Ile Pro Ile Arg Ala Ala Val Gln Lys Leu Glu Gly 325 330 335 甘氨酸-谷氨酰胺-色氨酸-丝氨酸-酪氨酸-谷氨酰胺-苯丙氨酸-赖氨酸-谷氨酰胺-谷氨酸-甘氨酸-谷氨酰胺-缬氨酸-亮氨酸-赖氨酸-缬氨酸 340 345 350 甘氨酸-赖氨酸-酪氨酸-苏氨酸-赖氨酸-谷氨酰胺-赖氨酸-天冬酰胺-苏氨酸-组氨酸-苏氨酸-天冬酰胺-谷氨酸-亮氨酸-精氨酸-苏氨酸 355 360 365 亮氨酸-丙氨酸-甘氨酸-亮氨酸-缬氨酸-谷氨酰胺-赖氨酸-异亮氨酸-半胱氨酸-赖氨酸-谷氨酸-丙氨酸-亮氨酸-缬氨酸-异亮氨酸-色氨酸 370 375 380 甘氨酸-异亮氨酸-亮氨酸-脯氨酸-缬氨酸-亮氨酸-谷氨酸-亮氨酸-脯氨酸-异亮氨酸-谷氨酸-精氨酸-谷氨酸-缬氨酸-色氨酸-谷氨酸 385 390 395 400 谷氨酰胺-色氨酸-色氨酸-丙氨酸-天冬氨酸-酪氨酸-色氨酸-谷氨酰胺-缬氨酸-丝氨酸-色氨酸-异亮氨酸-脯氨酸-谷氨酸-色氨酸-天冬氨酸 405 410 415 苯丙氨酸-缬氨酸-丝氨酸-苏氨酸-脯氨酸-脯氨酸-亮氨酸-亮氨酸-赖氨酸-亮氨酸-色氨酸-酪氨酸-苏氨酸-亮氨酸-苏氨酸-赖氨酸 420 425 430 谷氨酸-脯氨酸-异亮氨酸-脯氨酸-赖氨酸-谷氨酸-天冬氨酸-缬氨酸-酪氨酸 435 440 SEQ ID NO: 27 p15 protein sequence 酪氨酸-缬氨酸-天冬氨酸-甘氨酸-丙氨酸-半胱氨酸-天冬酰胺-精氨酸-天冬酰胺-丝氨酸-赖氨酸-谷氨酸-甘氨酸-赖氨酸-丙氨酸-甘氨酸 1 5 10 15 酪氨酸-异亮氨酸-丝氨酸-谷氨酰胺-酪氨酸-甘氨酸-赖氨酸-谷氨酰胺-精氨酸-缬氨酸-谷氨酸-苏氨酸-亮氨酸-谷氨酸-天冬酰胺-苏氨酸 20 25 30 天冬酰胺-苏氨酸-谷氨酰胺-谷氨酰胺-丙氨酸-谷氨酸-亮氨酸-苏氨酸-丙氨酸-异亮氨酸-赖氨酸-甲硫氨酸-丙氨酸-亮氨酸-谷氨酸-天冬氨酸 35 40 45 Ser Gly Pro Asn Val Asn Ile Val Thr Asp Ser Gln Tyr Ala Met Gly 50 55 60 Ile Leu Thr Ala Gln Pro Thr Gln Ser Asp Ser Pro Leu Val Glu Gln 65 70 75 80 Ile Ile Ala Leu Met Ile Gln Lys Gln Gln Ile Tyr Leu Gln Trp Val 85 90 95 Pro Ala His Lys Gly Ile Gly Gly Asn Glu Glu Ile Asp Lys Leu Val 100 105 110 Ser Lys Gly Ile Arg Arg Val Leu 115 120 SEQ ID NO: 28 p31 protein sequence Phe Leu Glu Lys Ile Glu Glu Ala Gln Glu Glu His Glu Arg Tyr His 1 5 10 15 Asn Asn Trp Lys Asn Leu Ala Asp Thr Tyr Gly Leu Pro Gln Ile Val 20 25 30 Ala Lys Glu Ile Val Ala Met Cys Pro Lys Cys Gln Ile Lys Gly Glu 35 40 45 Pro Val His Gly Gln Val Asp Ala Ser Pro Gly Thr Trp Gln Met Asp 50 55 60 Cys Thr His Leu Glu Gly Lys Val Val Ile Val Ala Val His Val Ala 65 70 �? 80 Ser Gly Phe Ile Glu Ala Glu Val Ile Pro Arg Glu Thr Gly Lys Glu 85 90 95 Thr Ala Lys Phe Leu Leu Lys Ile Leu Ser Arg Trp Pro Ile Thr Gln 100 105 110 Leu His Thr Asp Asn Gly Pro Asn Phe Thr Ser Gln Glu Val Ala Ala 115 120 125 Ile Cys Trp Trp Gly Lys Ile Glu His Thr Thr Gly Ile Pro Tyr Asn 130 135 140 Pro Gln Ser Gln Gly Ser Ile Glu Ser Met Asn Lys Gln Leu Lys Glu 145 150 155 160 Ile Ile Gly Lys Ile Arg Asp Asp Cys Gln Tyr Thr Glu Thr Ala Val 165 170 175 Leu Met Ala Cys His Ile His Asn Phe Lys Arg Lys Gly Gly Ile Gly 180 185 190 Gly Gln Thr Ser Ala Glu Arg Leu Ile Asn Ile Ile Thr Thr Gln Leu 195 200 205 Glu Ile Gln His Leu Gln Thr Lys Ile Gln Lys Ile Leu Asn Phe Arg 210 215 220 Val Tyr Tyr Arg Glu Gly Arg Asp Pro Val Trp Lys Gly Pro Ala Gln 225 230 235 240 Leu Ile Trp Lys Gly Glu Gly Ala Val Val Leu Lys Asp Gly Ser Asp 245 250 255 Leu Lys Val Val Pro Arg Arg Lys Ala Lys Ile Ile Lys Asp Tyr Glu 260 265 270 Pro Lys Gln Arg Val Gly Asn Glu Gly Asp Val Glu Gly Thr Arg Gly 275 280 285 Ser Asp Asn 290 SEQ ID NO: 29 Gag protein Met Gly Ala Ala Thr Ser Ala Leu Asn Arg Arg Gln Leu Asp Gln Phe 1 5 10 15 Glu Lys Ile Arg Leu Arg Pro Asn Gly Lys Lys Lys Tyr Gln Ile Lys 20 25 30 His Leu Ile Trp Ala Gly Lys Glu Met Glu Arg Phe Gly Leu His Glu 35 40 45 Arg Leu Leu Glu Thr Glu Glu Gly Cys Lys Arg Ile Ile Glu Val Leu 50 55 60 Tyr Pro Leu Glu Pro Thr Gly Ser Glu Gly Leu Lys Ser Leu Phe Asn 65 70 75 80 Leu Val Cys Val Leu Tyr Cys Leu His Lys Glu Gln Lys Val Lys Asp 85 90 95 Thr Glu Glu Ala Val Ala Thr Val Arg Gln His Cys His Leu Val Glu 100 105 110 Lys Glu Lys Ser Ala Thr Glu Thr Ser Ser Gly Gln Lys Lys Asn Asp 115 120 125 Lys Gly Ile Ala Ala Pro Pro Gly Gly Ser Gln Asn Phe Pro Ala Gln 130 135 140 Gln Gln Gly Asn Ala Trp Val His Val Pro Leu Ser Pro Arg Thr Leu 145 150 155 160 Asn Ala Trp Val Lys Ala Val Glu Glu Lys Lys Phe Gly Ala Glu Ile 165 170 175 Val Pro Met Phe Gln Ala Leu Ser Glu Gly Cys Thr Pro Tyr Asp Ile 180 185 190 Asn Gln Met Leu Asn Val Leu Gly Asp His Gln Gly Ala Leu Gln Ile 195 200 205 Val Lys Glu Ile Ile Asn Glu Glu Ala Ala Gln Trp Asp Val Thr His 210 215 220 Pro Leu Pro Ala Gly Pro Leu Pro Ala Gly Gln Leu Arg Asp Pro Arg 225 230 235 240 Gly Ser Asp Ile Ala Gly Thr Thr Ser Ser Val Gln Glu Gln Leu Glu 245 250 255 Trp Ile Tyr Thr Ala Asn Pro Arg Val Asp Val Gly Ala Ile Tyr Arg 260 265 270 Arg Trp Ile Ile Leu Gly Leu Gln Lys Cys Val Lys Met Tyr Asn Pro 275 280 285 Val Ser Val Leu Asp Ile Arg Gln Gly Pro Lys Glu Pro Phe Lys Asp 290 295 300 Tyr Val Asp Arg Phe Tyr Lys Ala Ile Arg Ala Glu Gln Ala Ser Gly 305 310 315 320 Glu Val Lys Gln Trp Met Thr Glu Ser Leu Leu Ile Gln Asn Ala Asn 325 330 335 Pro Asp Cys Lys Val Ile Leu Lys Gly Leu Gly Met His Pro Thr Leu 340 345 350 Glu Glu Met Leu Thr Ala Cys Gln Gly Val Gly Gly Pro Ser Tyr Lys 355 360 365 Ala Lys Val Met Ala Glu Met Met Gln Thr Met Gln Asn Gln Asn Met 370 375 380 Val Gln Gln Gly Gly Pro Lys Arg Gln Arg Pro Pro Leu Arg Cys Tyr 385 390 395 400 Asn Cys Gly Lys Phe Gly His Met Gln Arg Gln Cys Pro Glu Pro Arg 405 410 415 Lys Thr Lys Cys Leu Lys Cys Gly Lys Leu Gly His Leu Ala Lys Asp 420 425 430 Cys Arg Gly Gln Val Asn Phe Leu Gly Tyr Gly Arg Trp Met Gly Ala 435 440 445 Lys Pro Arg Asn Phe Pro Ala Ala Thr Leu Gly Ala Glu Pro Ser Ala 450 455 460 Pro Pro Pro Pro Ser Gly Thr Thr Pro Tyr Asp Pro Ala Lys Lys Leu 465 470 475 480 Leu Gln Gln Tyr Ala Glu Lys Gly Lys Gln Leu Arg Glu Gln Lys Arg 485 490 495 Asn Pro Pro Ala Met Asn Pro Asp Trp Thr Glu Gly Tyr Ser Leu Asn 500 505 510 Ser Leu Phe Gly Glu Asp Gln 515 SEQ ID NO: 30 Pol protein Met Ser Lys Val Trp Lys Ile Gly Thr Pro Ser Lys Arg Leu Gln Gly 1 5 10 15 Thr Gly Glu Phe Phe Arg Val Trp Thr Val Asp Gly Gly Lys Thr Glu 20 25 30 Lys Phe Ser Arg Arg Tyr Ser Trp Ser Gly Thr Glu Cys Ala Ser Ser 35 40 45 Thr Glu Arg His His Pro Ile Arg Pro Ser Lys Glu Ala Pro Ala Ala 50 55 60 Ile Cys Arg Glu Arg Glu Thr Thr Glu Gly Ala Lys Glu Glu Ser Thr 65 70 75 80 Gly Asn Glu Ser Gly Leu Asp Arg Gly Ile Phe Phe Glu Leu Pro Leu 85 90 95 Trp Arg Arg Pro Ile Lys Thr Val Tyr Ile Glu Gly Val Pro Ile Lys 100 105 110 Ala Leu Leu Asp Thr Gly Ala Asp Asp Thr Ile Ile Lys Glu Asn Asp 115 120 125 Leu Gln Leu Ser Gly Pro Trp Arg Pro Lys Ile Ile Gly Gly Ile Gly 130 135 140 Gly Gly Leu Asn Val Lys Glu Tyr Asn Asp Arg Glu Val Lys Ile Glu 145 150 155 160 Asp Lys Ile Leu Arg Gly Thr Ile Leu Leu Gly Ala Thr Pro Ile Asn 165 170 175 Ile Ile Gly Arg Asn Leu Leu Ala Pro Ala Gly Ala Arg Leu Val Met 180 185 190 Gly Gln Leu Ser Glu Lys Ile Pro Val Thr Pro Val Lys Leu Lys Glu 195 200 205 Gly Ala Arg Gly Pro Cys Val Arg Gln Trp Pro Leu Ser Lys Glu Lys 210 215 220 Ile Glu Ala Leu Gln Glu Ile Cys Ser Gln Leu Glu Gln Glu Gly Lys 225 230 235 240 Ile Ser Arg Val Gly Gly Glu Asn Ala Tyr Asn Thr Pro Ile Phe Cys 245 250 255 Ile Lys Lys Lys Asp Lys Ser Gln Trp Arg Met Leu Val Asp Phe Arg 260 265 270 Glu Leu Asn Lys Ala Thr Gln Asp Phe Phe Glu Val Gln Leu Gly Ile 275 280 285 Pro His Pro Ala Gly Leu Arg Lys Met Arg Gln Ile Thr Val Leu Asp 290 295 300 Val Gly Asp Ala Tyr Tyr Ser Ile Pro Leu Asp Pro Asn Phe Arg Lys 305 310 315 320 Tyr Thr Ala Phe Thr Ile Pro Thr Val Asn Asn Gln Gly Pro Gly Ile 325 330 335 Arg Tyr Gln Phe Asn Cys Leu Pro Gln Gly Trp Lys Gly Ser Pro Thr 340 345 350 Ile Phe Gln Asn Thr Ala Ala Ser Ile Leu Glu Glu Ile Lys Arg Asn 355 360 365 Leu Pro Ala Leu Thr Ile Val Gln Tyr Met Asp Asp Leu Trp Val Gly 370 375 380 Ser Gln Glu Asn Glu His Thr His Asp Lys Leu Val Glu Gln Leu Arg 385 390 395 400 Thr Lys Leu Gln Ala Trp Gly Leu Glu Thr Pro Glu Lys Lys Val Gln 405 410 415 Lys Glu Pro Pro Tyr Glu Trp Met Gly Tyr Lys Leu Trp Pro His Lys 420 425 430 Trp Glu Leu Ser Arg Ile Gln Leu Glu Glu Lys Asp Glu Trp Thr Val 435 440 445 Asn Asp Ile Gln Lys Leu Val Gly Lys Leu Asn Trp Ala Ala Gln Leu 450 455 460 Tyr Pro Gly Leu Arg Thr Lys Asn Ile Cys Lys Leu Ile Arg Gly Lys 465 470 475 480 Lys Asn Leu Leu Glu Leu Val Thr Trp Thr Pro Glu Ala Glu Ala Glu 485 490 495 Tyr Ala Glu Asn Ala Glu Ile Leu Lys Thr Glu Gln Glu Gly Thr Tyr 500 505 510 Tyr Lys Pro Gly Ile Pro Ile Arg Ala Ala Val Gln Lys Leu Glu Gly 515 520 525 Gly Gln Trp Ser Tyr Gln Phe Lys Gln Glu Gly Gln Val Leu Lys Val 530 535 540 Gly Lys Tyr Thr Lys Gln Lys Asn Thr His Thr Asn Glu Leu Arg Thr 545 550 555 560 Leu Ala Gly Leu Val Gln Lys Ile Cys Lys Glu Ala Leu Val Ile Trp 565 570 575 Gly Ile Leu Pro Val Leu Glu Leu Pro Ile Glu Arg Glu Val Trp Glu 580 585 590 Gln Trp Trp Ala Asp Tyr Trp Gln Val Ser Trp Ile Pro Glu Trp Asp 595 600 605 Phe Val Ser Thr Pro Pro Leu Leu Lys Leu Trp Tyr Thr Leu Thr Lys 610 615 620 Glu Pro Ile Pro Lys Glu Asp Val Tyr Tyr Val Asp Gly Ala Cys Asn 625 630 635 640 Arg Asn Ser Lys Glu Gly Lys Ala Gly Tyr Ile Ser Gln Tyr Gly Lys 645 650 655 Gln Arg Val Glu Thr Leu Glu Asn Thr Thr Asn Gln Gln Ala Glu Leu 660 665 670 Thr Ala Ile Lys Met Ala Leu Glu Asp Ser Gly Pro Asn Val Asn Ile 675 680 685 Val Thr Asp Ser Gln Tyr Ala Met Gly Ile Leu Thr Ala Gln Pro Thr 690 695 700 Gln Ser Asp Ser Pro Leu Val Glu Gln Ile Ile Ala Leu Met Ile Gln 705 710 715 720 Lys Gln Gln Ile Tyr Leu Gln Trp Val Pro Ala His Lys Gly Ile Gly 725 730 735 Gly Asn Glu Glu Ile Asp Lys Leu Val Ser Lys Gly Ile Arg Arg Val 740 745 750 Leu Phe Leu Glu Lys Ile Glu Glu Ala Gln Glu Glu His Glu Arg Tyr 755 760 765 His Asn Asn Trp Lys Asn Leu Ala Asp Thr Tyr Gly Leu Pro Gln Ile 770 775 780 Val Ala Lys Glu Ile Val Ala Met Cys Pro Lys Cys Gln Ile Lys Gly 785 790 795 800 Glu Pro Val His Gly Gln Val Asp Ala Ser Pro Gly Thr Trp Gln Met 805 810 815 Asp Cys Thr His Leu Glu Gly Lys Val Val Ile Val Ala Val His Val 820 825 830 Ala Ser Gly Phe Ile Glu Ala Glu Val Ile Pro Arg Glu Thr Gly Lys 835 840 845 Glu Thr Ala Lys Phe Leu Leu Lys Ile Leu Ser Arg Trp Pro Ile Thr 850 855 860 Gln Leu His Thr Asp Asn Gly Pro Asn Phe Thr Ser Gln Glu Val Ala 865 870 875 880 Ala Ile Cys Trp Trp Gly Lys Ile Glu His Thr Thr Gly Ile Pro Tyr 885 890 895 Asn Pro Gln Ser Gln Gly Ser Ile Glu Ser Met Asn Lys Gln Leu Lys 900 905 910 Glu Ile Ile Gly Lys Ile Arg Asp Asp Cys Gln Tyr Thr Glu Thr Ala 915 920 925 Val Leu Met Ala Cys His Ile His Asn Phe Lys Arg Lys Gly Gly Ile 930 935 940 Gly Gly Gln Thr Ser Ala Glu Arg Leu Ile Asn Ile Ile Thr Thr Gln 945 950 955 960 Leu Glu Ile Gln His Leu Gln Thr Lys Ile Gln Lys Ile Leu Asn Phe 965 970 975 Arg Val Tyr Tyr Arg Glu Gly Arg Asp Pro Val Trp Lys Gly Pro Ala 980 985 990 Gln Leu Ile Trp Lys Gly Glu Gly Ala Val Val Leu Lys Asp Gly Ser 995 1000 1005 Asp Leu Lys Val Val Pro Arg Arg Lys Ala Lys Ile Ile Lys Asp 1010 1015 1020 Tyr Glu Pro Lys Gln Arg Val Gly Asn Glu Gly Asp Val Glu Gly 1025 1030 1035 Thr Arg Gly Ser Asp Asn 1040 Examples The present invention will now be described with reference to the following examples. These examples do not limit the scope of the present invention, and those skilled in the art will appreciate that appropriate equivalents can be used within the scope of the present invention. Therefore, the examples are to be considered as an integral part of the present invention, and the individual aspects described therein may be considered as disclosed independently or in any combination.

[0251] Example 1 - Transduction Efficiency in Human Bronchial Epithelial Cells (HBEC, F508del / F508del, Type II) Grown in Air-Liquid Interface (ALI) Culture To analyze the transduction efficiency of HBEC (basal cells) subsequently grown in ALI culture, cells were transduced with rSIV.F / HN expressing GFP (vFM107, rSIV.F / HN-GFP) at different multiplicities of infection (MOI) of 3, 10, 30, and 90 in submerged culture and then airlifted 2 days after transduction ( Figure 2 3-4 weeks after the gas lift, the cells were analyzed in a fully differentiated state. On day 21 after the gas lift, the percentage of GFP-positive cells was measured by flow cytometry. Cells transduced at MOIs of 3, 10, 30, and 90 resulted in a significant (p < 0.001, p < 0.0001) dose-dependent increase in transduction efficiency: 7.8 ± 1.1%, 17.6 ± 1.0%, 25.8 ± 1.8%, and 28.3 ± 1.8%, respectively. Figure 2 B).

[0252] Next, on day 28 after transduction, immunofluorescence staining for various epithelial cell markers was performed to examine the cellular profile of ALI derived from transduced basal cells. Colocalization of GFP with ACTUB (ciliated cells), KRT5 (basal cells), SCGB1A1 (club cells), and MUC5AC (goblet cells) was detected, confirming that rSIV.F / HN is expressed in multiple cell types following basal cell transduction. Figure 2 C). These findings were further confirmed by flow cytometry measurement of KRT5 + ACTUB + and SCGB1A1 + The percentage of GFP-positive cells was confirmed. At MOI 10, approximately 20% of cells in each cell population were transduced with the virus, while at MOI 30, this increased to 30%-40% ( Figure 2 D).

[0253] In addition, the average degree of integration in the genome (vector copy number (VCN)) was analyzed in DNA samples from ALI cultures that were independently transduced with rSIV.F / HN expressing GFP (vGM107) or CFTR (vGM058). Whole-body DNA analysis showed a dose-related increase in VCN for both rSIV.F / HN expressing GFP and rSIV.F / HN expressing CFTR (30.0±5.2 / 35.8±4.0, 60.3±11.9 / 58.6±6.3, 124.2±25.2 / 87.2±10.5 copies / ng DNA in cells transduced with rSIV.F / HN expressing GFP / CFTR at MOIs of 3, 10, and 90). No difference in VCN was observed between rSIV.F / HN expressing GFP and rSIV.F / HN expressing CFTR at any MOI analyzed ( Figure 2 E).

[0254] Vector-derived woodchuck hepatitis posttranscriptional regulatory elements ( WPRE ) mRNA expression and was again demonstrated between cells transduced with rSIV.F / HN expressing GFP (vGM107) or with rSIV.F / HN expressing CFTR (vGM058). WPRE No difference in performance ( Figure 2 A high variability in the number of WPRE copies per cell was observed, indicating that some cells could be identified as high expressors and other cells as medium / low expressors. Figure 2(E, F) show that the transduction efficiency of rSIV.F / HN expressing GFP and rSIV.F / HN expressing CFTR is comparable.

[0255] Based on these data, the transduction rate of GFP-expressing rSIV.F / HN was used as a surrogate readout to assess the transduction level of CFTR-expressing rSIV.F / HN, thereby enabling correlation analysis between transduction level and the extent of functional CFTR restoration.

[0256] Example 2 - Transduction of CF ALI cultures (F508del / F508del, Class II) with rSIV.F / HN-CFTR (vGM058) results in expression of transgenic CFTR, restoration of CFTR chloride currents, and increased ciliary beat frequency To determine whether rSIV.F / HN-CFTR-transduced CF ALI can effectively produce vector-derived codon-optimized 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, whereas no expression was observed in cells transduced with rSIV.F / HN-GFP (vGM107). Figure 3 The expression of endogenous CFTR was also analyzed, and no difference was observed between the untransduced samples, the samples transduced with rSIV.F / HN-GFP, and the samples transduced with rSIV.F / HN-CFTR at MOI 10 ( Figure 3 B). The expression of coCFTR mRNA is 10-fold higher than that of endogenous CFTR ( Figure 3 C), demonstrating that low numbers of transduced high CFTR-expressing cells (17% for an MOI of 10) are sufficient to restore CFTR expression to above endogenous levels.

[0257] To analyze the functionality of channels expressing rSIV.F / HN-CFTR, CFTR-mediated chloride currents were measured in Ussing chambers using ALI cultures transduced with rSIV.F / HN-CFTR (vGM058). First, sodium channels were blocked with amikacin (ENaC), followed by stimulation of CFTR with forskolin, and the change in short-circuit current (ΔIsc) (peak and plateau values) was calculated. In some experiments, the small molecule CFTR enhancer ivacaftor, which increases the probability of channel openness, was added for additional stimulation of CFTR currents. Finally, chloride currents were blocked with the CFTR inhibitor 172 ( Figure 3 D).

[0258] As expected, untransduced cells (MOI 0) or cells transduced with rSIV.F / HN-GFP did not respond to forskolin or CFTR inhibitors, confirming the absence of functional CFTR channels. In contrast, a dose-dependent increase in CFTR chloride currents was observed in ALI cells transduced with rSIV.F / HN-CFTR. At MOI 3, 49 ± 6% (peak, p < 0.0001) and 38 ± 4% (plateau, p < 0.01) of non-CF chloride currents were restored. When rSIV.F / HN-CFTR (MOI 3) was combined with the enhancer ivacaftor, stimulation of chloride currents was significantly increased (61 ± 4% for both peak and plateau). With an MOI of 10, we observed significantly higher recovery of non-CF chloride currents (94 ± 11% peak, 66 ± 6% plateau, p < 0.0001); the combination of MOI 10 and ivacaftor further increased recovery (121 ± 11% for both peak and plateau). Higher MOIs (30 and 90) resulted in further significant increases: 144 ± 27% and 162 ± 49% recovery (peak, p < 0.0001) and 101 ± 37% and 114 ± 36% (plateau, p < 0.0001), respectively.

[0259] Thus, rSIV.F / HN-CFTR is able to fully restore CFTR-associated chloride currents to non-CF values ​​and the combination of rSIV.F / HN-CFTR and ivacaftor can amplify this effect of gene therapy by 1.3-1.8 fold. These data compare favorably with currently approved modulator therapies that have also been evaluated. Thus, treatment with lumacaftor + ivacaftor and tesacaftor + ivacaftor resulted in 34% and 21% recovery of CFTR currents, respectively, while treatment with elexacaftor + tesacaftor + ivacaftor resulted in 81% recovery ( Figure 3 E, F; Table 1).

[0260] Table 1: Ussing chamber results of class II F508del / F508del CF ALI cultures transduced with rSIV.F / HN (vGM058) deal with LV dose <![CDATA[ΔFsk 峰 ]]> <![CDATA[ΔFsk 平稳阶段 ]]> <![CDATA[ΔCFTR-inh172 峰 ]]> <![CDATA[ΔCFTR-inh172 平稳阶段 ]]> <![CDATA[WT (峰) %]]> <![CDATA[WT (平稳阶段) %]]> Non-CF (WT) - - 32.3 ± 3.5 31.8 ± 3.2 -34.3 ± 3.4 -33.6 ± 3.4 100 100 CF - - -0.003 ± 0.1 -0.003 ± 0.1 -0.4 ± 0.1 -0.4 ± 0.1 - - (Same as above) rSIV.F / HN-GFP MOI 90 -0.6 ± 0.07 -0.6 ± 0.07 -0.7 ± 0.1 -0.7 ± 0.1 - - (Same as above) rSIV.F / HN-CFTR MOI 3 14.1 ± 1.9 9.7 ± 1.2 -16.8 ± 2.1 -12.9 ± 1.5 49 38 (Same as above) (Same as above) MOI 10 30.1 ± 3.6 18.1 ± 1.6 -32.4 ± 3.6 -22.5 ± 1.9 94 67 (Same as above) (Same as above) MOI 30 38.2 ± 6.1 23.3 ± 6.7 -49.4 ± 9.3 -34.6 ± 12.8 144 103 (Same as above) (Same as above) MOI 90 47.3 ± 14.6 33.9 ± 11.4 -55.5 ± 16.8 -39.1 ± 12.4 162 116 (Same as above) Iva - -0.1 ± 0.3 -0.1 ± 0.3 -2.1 ± 0.3 -2.1 ± 0.3 - - (Same as above) rSIV.F / HN-CFTR + Iva MOI 3 20.4 ± 1.6 20.4 ± 1.6 -21.0 ± 1.5 -21.0 ± 1.5 61 62 (Same as above) (Same as above) MOI 10 35.3 ± 3.3 35.3 ± 3.3 -41.4 ± 3.8 -41.4 ± 3.8 121 123 (Same as above) Luma + Iva - 12.1 ± 1.1 12.1 ± 1.1 -11.6 ± 1.1 -11.6 ± 1.1 34 34 (Same as above) Teza + Iva - 7.4 ± 0.5 7.4 ± 0.5 -7.1 ± 0.6 -7.1 ± 0.6 21 21 (Same as above) Elexa + Teza + Iva - 33.5 ± 0.5 33.5 ± 0.5 -27.9 ± 3.4 -27.9 ± 3.4 81 83 LV - lentivirus, Fsk - forskolin, CFTR-inh172 - CFTR inhibitor 172, Iva - ivacaftor, Luma - lumacaftor, Teza - tesacaftor, Elexa - elexaacaftor Since the transduction efficiencies of rSIV.F / HN-GFP and rSIV.F / HN-CFTR were similar ( Figure 2 E, F), Correlation analysis between GFP transduction level and CFTR chloride current recovery degree ( Figure 3 G). Approximately 17% of transduced cells was sufficient to restore CFTR chloride current to physiological levels. When rSIV.F / HN-CFTR was combined with ivacaftor, complete recovery was achieved in approximately 14% of transduced cells.

[0261] 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 was demonstrated in CF ALI (6.9 ± 0.8 Hz compared to 8.2 ± 1 Hz in non-CF ALI, p < 0.05). Transduction with rSIV.F / HN-CFTR was able to restore CBF to non-CF values ​​(9.3 ± 1.5, 9.7 ± 2.6, and 9.5 ± 2.2 Hz for MOIs 3, 10, and 30, respectively, all p > 0.0001). Figure 3 H).

[0262] Example 3 - Generation of CFTR knockout hSABCi cell line as a model for CF translational class I mutations and transduction of CFTR knockout cells with rSIV.F / HN (vGM107, vGM058) Class I CFTR knockout mutations result in a complete absence of full-length CFTR protein and are therefore not amenable to functional correction by current CFTR modulators. Gene therapy offers the opportunity to establish disease-modifying treatments for patients with all mutation types, including those homozygous for these knockout mutations. Primary airway epithelial cells derived from the few patients with class I mutations are difficult to obtain. Therefore, in order to enable functional characterization of rSIV.F / HN-CFTR in a class I mutation background, we used the previously described immortalized human airway small basal cell line hSABCi-NS1.1 (hSABCi) to CFTR A mutant model was generated by CRISPR / Cas9-mediated bi-allele knockout (KO) of the CFTR gene in exon 4. This cell line maintained basal cell characteristics (TP63+, KRT5+) after more than 200 cell division cycles and 70 passages.

[0263] When cultured under air-liquid interface conditions, hSABCi cells consistently form tight junctions and differentiate into ciliated (ARL13B+), rod-shaped (SCGB1A1+), goblet-shaped (MUC5AC+, MUC5B+), neuroendocrine (CHGA+), ionocyte (FOXI1+), and surfactant protein-positive cells (SFTPA+, SFTPB+, SFTPD+). Furthermore, 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 analyze editing efficiency, the edited locus was amplified by PCR and subsequently Sanger sequenced. Editing efficiencies of selected individual strains were determined using established bioinformatics programs to generate inferred CRISPR editing (ICE) metrics (data not shown). The strain (strain 5) with the highest out-of-frame editing / gene knockout efficiency of 99.4% (data not shown) was selected for further experiments.

[0264] To further characterize the CFTR-KO phenotype, CFTR protein levels were analyzed by Western blotting using a monoclonal hCFTR antibody (R&D systems, Minneapolis, MN, US). A weak signal of mature CFTR was observed in the unedited hSABCi cell line, whereas no mature CFTR signal was detectable in the CFTR KO cells ( Figure 4 A), thus confirming CFTR deficiency.

[0265] Next, the efficiency of lentiviral transduction in CFTR KO cells grown in ALI culture was analyzed. CFTR KO cells were transduced and analyzed with respect to primary F508del / F508del cells. Flow cytometry analysis revealed that transduction with rSIV.F / HN-GFP (vGM107) at MOIs of 3, 10, 30, and 90 resulted in an average of 12.2 ± 1.7 (p < 0.0001), 26.9 ± 1.2 (p < 0.0001), 36.3 ± 1.1 (p < 0.01), and 32.6 ± 2.4% GFP+ cells, respectively. Figure 4In addition, DNA VCN analysis of CFTR KO ALI cultures independently transduced with rSIV.F / HN-GFP (vGM107) or rSIV.F / HN-CFTR (vGM058) showed no differences in VCN between rSIV.F / HN-GFP-transduced and rSIV.F / HN-CFTR-transduced cells at all MOIs analyzed, with the exception of an MOI of 30 where GFP-transduced cells showed a slight increase in VCN compared to CFTR-transduced cells (p<0.01, Figure 4 C). These data indicate that rSIV.F / HN-GFP and rSIV.F / HN-CFTR have similar transduction efficiencies. Finally, rSIV.F / HN-GFP-mediated transduction of CFTR KO cells was analyzed at the cellular level 28 days after transduction by immunofluorescence staining for various epithelial cell markers. Colocalization of GFP with ACTUB, KRT5, SCGB1A1, and MUC5AC was observed, indicating that rSIV.F / HN-GFP is capable of transducing ciliary, basal, club, and goblet cells, respectively. Figure 2 In addition, flow cytometric measurement of the percentage of GFP+ cells in KRT5+, ACTUB+, and SCGB1A1+ cells confirmed the previous findings in type II cells (Example 2). At an MOI of 10, an average of 16.5±1.8%, 58±2.1%, and 26.3±0.8% of ciliated, club-shaped, and basal cells were virally transduced ( Figure 4 E).

[0266] Example 4 - Transduction of CFTR KO ALI cultures (Class I) with rSIV.F / HN-CFTR (vGM058) results in expression of coCFTR mRNA and restoration of CFTR currents, whereas ion channel modulators fail to restore CFTR currents Codon-optimized CFTR mRNA expression was analyzed in CFTR KO ALI transduced with rSIV.F / HN-CFTR (vGM058). A dose-dependent increase in coCFTR expression was observed in cells transduced with rSIV.F / HN-CFTR compared to untransduced cells, whereas no coCFTR expression was observed in cells transduced with rSIV.F / HN-GFP at all MOIs, as expected. Figure 5 Functional analysis via Ussing chamber assays demonstrated a dose-related increase in CFTR currents in CFTR KO ALI (class I) transduced with rSIV.F / HN-CFTR (vGM058) ( Figure 5(B, C). Untransduced cells did not show any CFTR current activation, thus supporting the functional effectiveness of genome editing. Cells transduced with rSIV.F / HN-CFTR at an MOI of 3 produced 58 ± 2% CFTR current recovery at the forskolin peak and 54 ± 2% recovery at the plateau (p < 0.0001 for both). The combination of MOI 3 and the enhancer drug ivacaftor increased this effect to 77 ± 4%. MOI 10 produced 112 ± 6% CFTR current recovery at the peak (103 ± 6% at the plateau, both p < 0.0001), and combined with ivacaftor produced 138 ± 10% CFTR current recovery (p < 0.05 for the plateau). MOIs of 30 and 90 produced 142 ± 11% and 153 ± 12% recovery at peak and 135 ± 9% and 149 ± 11% at plateau, respectively (all p < 0.0001); combination with ivacaftor increased these effects to 183 ± 13% (p < 0.01 for peak and p < 0.001 for plateau) and 161 ± 10% ( Figure 5 Thus, the combination of rSIV.F / HN-CFTR and ivacaftor increased the effect of gene therapy by 1.3-1.4 fold. In addition, the analysis of clinically used CFTR modulators did not show signs of CFTR restoration as expected ( Figure 5 B, C; Table 2).

[0267] Since the transduction efficiencies of rSIV.F / HN-GFP and rSIV.F / HN-CFTR were similar ( Figure 4 C), we can infer the correlation between the percentage of transduced cells and the degree of CFTR current recovery in CFTR KO cells ( Figure 5 D). Approximately 23% of cells transduced with rSIV.F / HN-CFTR were sufficient to restore CFTR currents to the levels of the parental hSABCi cell line. When rSIV.F / HN-CFTR was combined with ivacaftor, complete recovery was achieved in approximately 17% of transduced cells.

[0268] Example 5 - Comparison of preclinical candidate vGM058 and clinical candidate vGM244 (BI 3720931) To compare the preclinical candidate virus (vGM058) with the clinical candidate virus (vGM244), class II HBEC were transduced with rSIV.F / HN expressing GFP (vGM107) and rSIV.F / HN expressing CFTR (vGM058 and vGM244) at MOIs of 3 and 10. On day 21 after aeration, cells were harvested and the average degree of integration in the genome (vector copy number (VCN)) was analyzed in DNA samples from ALI cultures transduced at MOI 10.

[0269] No difference in VCN levels was observed betw...

Claims

1. A combination for use in a method of treating cystic fibrosis (CF), which is a combination of: (i) a lentiviral vector pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxovirus, wherein the lentiviral vector comprises a cystic fibrosis transmembrane conductance regulator (CFTR) transgene; and (ii) a CFTR modulator.

2. The combination according to claim 1, characterized in that The lentiviral vector is a SIV vector and the respiratory paramyxovirus is Sendai virus.

3. The combination according to claim 1 or 2, characterized in that: The transgene is operably linked to a promoter selected from the group consisting of a cytomegalovirus (CMV) promoter, an elongation factor 1a (EF1a) promoter, and a hybrid human CMV enhancer / EF1a (hCEF) promoter.

4. A combination according to any one of the preceding claims, characterised in that The lentiviral vector comprises a hybrid human CMV enhancer / EF1a (hCEF) promoter, which either comprises or consists of a nucleotide sequence having at least 90% identity to SEQ ID NO:

2.

5. A combination according to any one of the preceding claims, characterised in that The CFTR transgene is a codon-optimized CFTR transgene, which either comprises a nucleotide sequence having at least 90% identity with SEQ ID NO: 1 or consists of the nucleotide sequence.

6. A combination according to any one of the preceding claims, characterised in that The lentiviral vectors were produced using codon-optimized plasmids.

7. A combination according to any one of the preceding claims, characterised in that The lentiviral vector is produced using (i) pGM691 and / or (ii) pGM830 or pGM326; and preferably also using pGM299, pGM301 and / or pGM303.

8. A combination according to any one of the preceding claims, characterised in that The lentiviral vector is vGM058, vGM195 or vGM244.

9. A combination according to any one of the preceding claims, characterised in that The lentiviral vector is a SIV vector pseudotyped by Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) protein, wherein: the vector comprises a modified retroviral RNA sequence, which comprises the nucleic acid sequence of SEQ ID NO: 16 or consists of the nucleic acid sequence.

10. A combination according to any one of the preceding claims, characterised in that The lentiviral vector comprises an F protein having a first subunit comprising or consisting of an amino acid sequence of SEQ ID NO: 19 and a second subunit comprising or consisting of an amino acid sequence of SEQ ID NO:

20.

11. A combination according to any one of the preceding claims, characterised in that The lentiviral vector further comprises: (a) a p17 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 22; (b) a p24 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 23; (c) a p8 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 24; (d) a protease comprising or consisting of the amino acid sequence of SEQ ID NO: 25; (e) a p51 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 26; (f) a p15 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 27; (g) a p31 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 28; (h) a Gag protein comprising or consisting of the amino acid sequence of SEQ ID NO: 29; and / or (i) a Pol protein comprising or consisting of the amino acid sequence of SEQ ID NO: 30; Optionally, the carrier comprises each of (a) to (g).

12. A combination according to any one of the preceding claims, characterised in that The CFTR modulator is a CFTR enhancer and / or a CFTR corrector, preferably a CFTR enhancer.

13. The combination according to claim 12, characterized in that The CFTR modulator is selected from ivacaftor, tezacaftor, elexacaftor or lumacaftor, or a combination thereof.

14. The combination according to claim 12 or 13, characterized in that The CFTR modulator is ivacaftor.

15. A combination for use in a method of treating cystic fibrosis (CF), which is a combination of: A. SIV vector pseudotyped with Sendai virus hemagglutinin-neuraminidase (HN) and fusion (F) proteins, wherein: (a) the vector comprises a modified retroviral RNA sequence comprising or consisting of the nucleic acid sequence of SEQ ID NO: 16; and (b) the F protein comprises a first subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 19 and a second subunit comprising or consisting of the amino acid sequence of SEQ ID NO: 20; and B. Ivacaftor.

16. The combination according to claim 15, characterized in that The vector further comprises one or more of the following: (a) a p17 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 22; (b) a p24 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 23; (c) a p8 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 24; (d) a protease comprising or consisting of the amino acid sequence of SEQ ID NO: 25; (e) a p51 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 26; (f) a p15 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 27; (g) a p31 protein comprising or consisting of the amino acid sequence of SEQ ID NO: 28; (h) a Gag protein comprising or consisting of the amino acid sequence of SEQ ID NO: 29; and / or (i) a Pol protein comprising or consisting of the amino acid sequence of SEQ ID NO: 30; Optionally, the vector comprises each of (a) to (g).

17. A combination according to any one of the preceding claims, characterised in that The patient to be treated has at least one Class I, Class II, Class III, Class IV, Class V, or Class VI CFTR mutation.

18. The combination according to claim 17, characterized in that The patient to be treated has at least one class I and / or class II CFTR mutation.

19. A combination according to claim 17 or 18, characterized in that: (a) the combination is suitable for use independently of the CFTR mutation in 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. A combination according to any one of the preceding claims, characterised in that The lentiviral vector and the CFTR modulator are administered simultaneously or sequentially.

21. A combination according to any one of the preceding claims, characterised in that: (a) the lentiviral vector is administered by inhalation; and / or (b) The CFTR modulator is administered orally.

22. A combination according to any one of the preceding claims, characterised in that: (a) The lentiviral vector is approximately 8 8 to about 10 14 The dose is between 10 transduction units (TU), preferably about 10 6 to about 10 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 modulators are administered at a concentration used for monotherapy of each modulator or lower.

23. A combination according to any one of the preceding claims, characterised in that Treatment restored CFTR activity to at least 10% of the CFTR activity of healthy controls.

24. The combination according to claim 23, characterized in that Treatment restored CFTR activity to at least 50% of the CFTR activity of healthy controls.

25. A combination according to any one of the preceding claims, characterised in that This treatment increased CFTR activity by at least 1.2-fold compared to treatment with the lentiviral vector alone.

26. The combination according to claim 25, characterized in that 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 to treatment with the lentiviral vector alone.

27. A combination according to any one of the preceding claims, characterised in that The patient to be treated has a class I CFTR mutation and the treatment: (i) restores CFTR activity to at least 10% of the CFTR activity of 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 to treatment with the lentiviral vector alone.

28. A combination according to any one of the preceding claims, characterised in that The patient to be treated has a class II CFTR mutation and the treatment: (i) restores CFTR activity to at least 10% of the CFTR activity of 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 to treatment with the lentiviral vector alone.

29. A combination according to any one of the preceding claims, characterised in that A transduction rate of between about 10% and about 20%, preferably between about 14% and about 17%, is sufficient to achieve a therapeutic effect on CFTR activity as defined in any one of claims 23 to 28.

30. A method of treating CF in an individual in need thereof, characterized in that The invention comprises 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 respiratory paramyxovirus, wherein the lentiviral vector comprises a cystic fibrosis transmembrane conductance regulator (CFTR) transgene; and (ii) a CFTR modulator.

31. Use of a lentiviral vector pseudotyped with hemagglutinin-neuraminidase (HN) and fusion (F) proteins from respiratory paramyxovirus, wherein the lentiviral vector comprises a cystic fibrosis transmembrane conductance regulator (CFTR) transgene, for the manufacture of a medicament for use in a method for treating CF, wherein the method further comprises administering a CFTR modulator.

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