Recombinant adeno-associated viruses for the treatment of cystic fibrosis

Abstract

Provided herein are polynucleotides, rAAV vectors, pharmaceutical compositions, and methods of making and using them, e.g., for the treatment of cystic fibrosis (CF). For example, the present disclosure provides recombinant adeno-associated viruses (rAAVs), which in one embodiment include a polynucleotide comprising an AV.TL65 capsid protein and an F5 enhancer and tg83 promoter operably linked to a CFTRΔR minigene, pharmaceutical compositions thereof, and methods of use thereof, e.g., for the treatment of CF.

Description

[Background technology]

[0001] Gene therapy using adeno-associated viruses (AAVs) is an emerging therapeutic approach, including the treatment of single-gene defects. Cystic fibrosis (CF) is a fatal autosomal recessive disease affecting at least 30,000 people in the United States alone and at least 70,000 worldwide. The average life expectancy of CF patients is approximately 40 years. CF is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR), a channel that conducts chloride and bicarbonate ions across epithelial cell membranes. Impaired CFTR function leads to airway inflammation and progressive bronchiectasis. Because CF has a monogenic etiology and various CFTR mutations exist across patient populations, gene therapy potentially offers a universal cure for CF.

[0002] Adeno-associated virus (AAV), a member of the human parvovirus family, is a nonpathogenic virus that depends on a helper virus for replication. For this reason, recombinant AAV (rAAV) vectors are most frequently used in preclinical and clinical studies of gene therapy. Indeed, clinical trials of CF lung disease using rAAV2 demonstrated both a favorable safety profile and long-term persistence of the viral genome in airway tissue (assessed by biopsy) compared with other gene transfer agents (e.g., recombinant adenovirus). Nevertheless, gene transfer failed to improve lung function in CF patients because rAAV vector-derived CFTR mRNA transcription was not detected.

[0003] Thus, there remains a need in the art for improved compositions and methods for the treatment of CF. Summary of the Invention

[0004] The present disclosure provides, inter alia, rAAVs, pharmaceutical compositions, isolated polynucleotides, and methods of making and using them, e.g., for the treatment of CF. In one aspect, the disclosure features a recombinant adeno-associated virus (rAAV) including: (i) an AV.TL65 capsid protein; and (ii) a polynucleotide including an F5 enhancer and a tg83 promoter operably linked to a CFTRΔR minigene.

[0005] In some embodiments, the AV.TL65 capsid protein comprises the amino acid sequence of SEQ ID NO:13, or a variant thereof having at least 80% amino acid sequence identity to SEQ ID NO:13.

[0006] In some embodiments, the F5 enhancer comprises the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 14, or a variant thereof having at least 80% nucleic acid sequence identity to SEQ ID NO: 1 or SEQ ID NO: 14. In some embodiments, F5 comprises the polynucleotide sequence of SEQ ID NO: 1. In other embodiments, the F5 enhancer comprises the polynucleotide sequence of SEQ ID NO: 14.

[0007] In some embodiments, the tg83 promoter comprises the polynucleotide sequence of SEQ ID NO:2. In some embodiments, the CFTRΔR minigene is a human CFTRΔR minigene.

[0008] In some embodiments, the human CFTRΔR minigene is encoded by a polynucleotide comprising the sequence of SEQ ID NO:4. In some embodiments, the polynucleotide comprises, in the 5' to 3' direction, an F5 enhancer, a tg83 promoter, and a CFTRΔR minigene.

[0009] In another aspect, the disclosure features a pharmaceutical composition including any one of the rAAVs described herein and a pharmaceutically acceptable carrier. In another aspect, the disclosure features a method of treating cystic fibrosis, the method including administering to a subject in need thereof a therapeutically effective amount of any one of the rAAVs described herein or any one of the pharmaceutical compositions described herein. In some embodiments, the method further includes administering one or more additional therapeutic agents to the subject.

[0010] In another embodiment, the mammal is a human. In one embodiment, the human is a newborn. In one embodiment, the human is a juvenile. In another aspect, the disclosure features an rAAV for use in treating cystic fibrosis in a subject in need thereof, the rAAV comprising: (i) an AV.TL65 capsid protein; and (ii) a polynucleotide comprising an F5 enhancer and a tg83 promoter operably linked to a CFTRΔR minigene. In some embodiments, the rAAV is for use in combination with one or more additional therapeutic agents.

[0011] In some embodiments, the one or more additional therapeutic agents are potentiators (e.g., proteasome modulating agents such as anthracyclines (e.g., doxorubicin, idarubicin, aclarubicin, daunorubicin, epirubicin, valproicin, L In some embodiments, the augmenting agent is doxorubicin. In other embodiments, the augmenting agent is idarubicin. In some embodiments, the one or more additional therapeutic agents include an immunosuppressant (e.g., a corticosteroid (e.g., an inhaled corticosteroid)).

[0012] In some embodiments, administration is by inhalation, nebulization, aerosolization, intranasal, intratracheal, intrabronchial, oral, intravenous, subcutaneous, or intramuscular. In some embodiments, administration is by inhalation, nebulization, aerosolization, intranasal, intratracheal, and / or intrabronchial.

[0013] In another aspect, the disclosure features an isolated polynucleotide comprising the sequence of SEQ ID NO:7. In some embodiments, the polynucleotide further comprises, in the 3' direction, a 3' untranslated region (3'-UTR) comprising the sequence of SEQ ID NO:5.

[0014] In some embodiments, the polynucleotide further comprises in the 3' direction a synthetic polyadenylation site comprising the sequence of SEQ ID NO:6. In some embodiments, the polynucleotide further comprises a 5' adeno-associated virus (AAV) inverted terminal repeat (ITR) at the 5' end of the polynucleotide and a 3' AAV ITR at the 3' end of the polynucleotide. In some embodiments, the 5' AAV ITR comprises the sequence of SEQ ID NO: 15, or a variant thereof having at least 80% nucleic acid sequence identity to SEQ ID NO: 15. In some embodiments, the 3' AAV ITR comprises the sequence of SEQ ID NO: 16, or a variant thereof having at least 80% nucleic acid sequence identity to SEQ ID NO: 16.

[0015] In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 11 or SEQ ID NO: 17, or a variant thereof having at least 80% nucleic acid sequence identity to SEQ ID NO: 11 or SEQ ID NO: 17. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 11. In other embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 17.

[0016] In another aspect, the disclosure features an isolated polynucleotide comprising the sequence of SEQ ID NO: 18. In another aspect, the disclosure features a recombinant adeno-associated virus (rAAV) comprising any one of the polynucleotides described herein (e.g., a polynucleotide comprising the sequence of SEQ ID NO: 7, SEQ ID NO: 11, or SEQ ID NO: 17).

[0017] In some embodiments, the rAAV has a tropism for airway cells. In some embodiments, the rAAV has a tropism for airway epithelial cells. In some embodiments, the rAAV has tropism for lung epithelial cells.

[0018] In some embodiments, the rAAV comprises an AV.TL65 capsid protein, an AAV1 capsid protein, an AAV2 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, or an AAV9 capsid protein.

[0019] In some embodiments, the rAAV comprises the AV.TL65 capsid protein. [Brief explanation of the drawings]

[0020] [Figure 1]Figures 1A-1C demonstrate functional complementation of CFTR-mediated chloride transport in polarized human CF airway epithelia. Figure 1A shows that the rAAV2 viral genome AV.TL65-SP183-hCFTRΔR was packaged into three capsid serotypes (AV.TL65, AV.1, and AV.2) and used to apically infect polarized human CF ALI cultures from either the apical (AV.TL65 and AV.1) or basolateral (AV.2) surfaces. Because infection from the basolateral surface is efficient, basolateral infection with AAV2 was used as a positive control. 2.5 μM doxorubucin and 20 μM LLnL were added to the viral inoculum, and ALI cultures were infected for 16 h. Virus was then removed, and cultures were refed in the absence of proteasome inhibitors. Figure 1B shows Isc traces of two cultures for each condition. Arrows indicate the addition of IBMX / forskolin (I&F) and a CFTR inhibitor (GlyH101). Figure 1C shows the mean + / - SEM ΔIsc at 12 days post-infection. [Figure 2] Figures 2A-2D are a series of graphs showing the transfection efficiency of AV.TL65-SP183-hCFTRΔR into the trachea and lungs of ferrets. Figures 2A and 2B show the copy numbers of hCFTR and fCFTR mRNA per 500 ng of RNA in the trachea (Figure 2A) and lungs (Figure 2B). Copy numbers were determined using a standard curve generated from serial dilutions of various plasmid CFTR cDNAs. Figures 2C and 2D show the ratio of transgene-derived hCFTR to endogenous fCFTR mRNA in the trachea (Figure 2C) and lungs (Figure 2D). C1-C3 represent mock-infected animals, and A1-A3 represent AAV-infected animals. The average of three AAV-infected animals is also shown. The dashed line represents the endogenous level of CFTR (ratio = 1). Data are shown as the mean + / - SEM of N = 3 animals in each group. [Figure 3]Figures 3A-3D are a series of graphs showing that AV.TL65 effectively transduces the airways of adult ferrets. Figure 3A shows TaqMan™ RNA-specific PCR (RS-PCR) results for human CFTR mRNA and endogenous ferret GAPDH mRNA in vector- and mock-treated animals. The results show the ratio of hCFTR / fGAPDH mRNA. Figure 3B shows TaqMan™ RS-PCR results for endogenous ferret CFTR mRNA and endogenous ferret GAPDH mRNA in vector- and mock-treated animals. The results show the ratio of fCFTR / fGAPDH mRNA. Figure 3C shows TaqMan™ Q-PCR results for the number of vector genomes per 100 ng of DNA in each sample. Figure 3D shows the ratio of hCFTR / fCFTR mRNA copies in each sample. 1 equals the endogenous level of CFTR (dashed red line). Lung samples contained an average of 3.0 + / - 0.5 copies of transgene-derived hCFTR mRNA per copy of fCFTR mRNA. Transduction of tracheal and nasal tissue was more variable, but averaged one copy of transgene-derived hCFTR / fCFTR mRNA. Results represent the mean + / - SEM of vector-treated animals. [Figure 4] FIG. 4 is a graph showing a representative CF trace of the experiment described in Example 5. [Figure 5] 5 is a series of graphs showing ΔIsc (μA / cm2) under the indicated conditions for CF or non-CF donors from the experiment described in Example 5. Error bars indicate the standard error of the mean (SEM). [Figure 6] FIG. 6 is a series of graphs showing representative Ieq traces (37° C.) from individual wells of a 24-well transwell filter plate from the experiment described in Example 6. [Figure 7] 7 is a series of graphs showing the mean CFTR-mediated chloride secretion after forskolin / IBMX stimulation for each condition (n=4) from the experiment described in Example 6. Error bars indicate SEM. [Figure 8]Figures 8A-8D. In vitro and in vivo comparison of rAAV vector performance. (A) CF(F508del / F508del) human polarized ALI airway cultures were apically infected with AV1-SP183-hCFTRΔR or AV.TL65-SP183-hCFTRΔR (MOI = 100,000 DRP / cell) in the presence of enhancers. Short-circuit current (Isc) measurements were then performed in Ussing chambers 12 days post-infection. ΔIsc responses to forskolin / IBMX and GlyH101 (CFTR inhibitors) are also shown. Data represent the mean ± SD of n = 4 transwells from two donors. Uninfected ALI cultures served as baseline controls (n = 4 from two donors). (B) After Isc measurement, two transwell inserts from each group were pooled and lysed, and vector-derived hCFTRΔR mRNA copies were quantified by reverse transcriptase quantitative PCR (RT-qPCR) and normalized to human GAPDH mRNA copies. Values ​​were then expressed as the ratio of hCFTRΔR / GAPDH. Data represent the mean ± range for n=2. (C) Polarized human and ferret tracheobronchial epithelia at ALI were infected apically with AV.TL65-SP183gLuc at a multiplicity of infection (MOI) of 100,000 DNase-resistant particles (DRP) / cell in the presence of enhancer. Gaussia luciferase activity was measured as relative luminescence units (RLU) 5 days postinfection. Data represent the mean ± SD for n=6 transwells from two individual donors per species. (D) Three-day-old or one-month-old ferrets were intratracheally infected with AV.TL65-SP183-hCFTRΔR (4 × 10 DRP per gram body weight) mixed with enhancer. Mock-infected animals were inoculated with PBS containing enhancer. Tracheas and lungs were then harvested 11 days postinfection, and vector-derived hCFTRΔR and endogenous fCFTR mRNA copies were quantified by RT-qPCR, normalized to GAPDH mRNA copy number. Data represent the ratio of hCFTRΔR to fCFTRΔR mRNA copies (hCFTRΔR / fCFTR). Data represent the mean ± SD of n = 3 animals per group, with ns indicating no significant difference. [Figure 9]Figures 9A-9C. Repeated administration of AV.TL65 to neonatal ferrets. (A) Study design included three groups of neonatal ferrets receiving 0, 1, or 2 doses of virus at 1 x 10 DRP / kg via intratracheal administration. Ferrets receiving one dose received the reporter vector AV.TL65-SP183-gLuc at 4 weeks of age, while ferrets receiving two doses received AV.TL65-SP183-fCFTRΔR at 1 week of age and AV.TL65-SP183-gLuc at 4 weeks of age. Plasma and BALF samples were collected at the indicated ages. (B) Gaussia luciferase activity in plasma at the indicated time points after delivery of AV.TL65-SP183-gLuc. (C) Gaussia luciferase activity in BALF 14 days after delivery of AV.TL65-SP183-gLuc. Results show the mean ± SD of n = 6 animals per group. Statistical significance was analyzed by one-way ANOVA followed by Tukey's post-hoc test. ns, not significant. RLU, relative luminescence units. [Figure 10] Figures 10A-10C. Repeated administration of AV.TL65 to young ferrets. (A) Study design included three groups of young ferrets receiving 0, 1, or 2 doses of virus at 1 x 10 DRP / kg via intratracheal administration. Ferrets receiving one dose received the reporter vector AV.TL65-SP183-gLuc at 8 weeks of age, while ferrets receiving two doses received AV.TL65-SP183-fCFTRΔR at 4 weeks of age and AV.TL65-SP183-gLuc at 8 weeks of age. Plasma and BALF samples were collected at the indicated ages. (B) Gaussia luciferase activity in plasma at the indicated time points after delivery of AV.TL65-SP183-gLuc. (C) Gaussia luciferase activity in BALF 14 days after delivery of AV.TL65-SP183-gLuc. Results show the mean ± SD of n = 9–10 animals per group. Statistical significance was analyzed by one-way ANOVA followed by Tukey's post-hoc test: **P<0.01, ****P<0.0001. RLU, relative luminescence units. [Figure 11]Figures 11A-11D. AV.TL65 neutralizing antibody titers in BALF and plasma of infected ferrets. (A, B) Neonatal ferret samples collected in Figure 9A were evaluated for NAb in (A) BALF and (B) plasma using the transduction inhibition assay. Serial dilutions of BALF or plasma were incubated with AV.TL65-fLuc before infection of A549 cells. NAb titers were calculated as the concentration (dilution ratio) of BALF or plasma that resulted in 50% inhibition of transduction (IC50), as assessed by firefly luciferase activity. Cells infected with AV.TL65-fLuc alone served as a baseline control, and mock-infected cells served as blanks. (C, D) Young ferret samples collected in Figure 10A were evaluated for NAb in (C) BALF and (D) plasma using the transduction inhibition assay described above. Results show the mean ± SD of n = 6 neonatal animals per group and n = 9–10 juvenile animals per group. Statistical significance was analyzed by one-way ANOVA followed by Tukey's post-hoc test: **P<0.01, ****P<0.0001. ns, not significant. [Figure 12] Figures 12A-12B. Development of an ELISA-based assay to quantify anti-capsid antibody isotypes. Immune plasma was generated from pulmonary ferrets infected with AV-TL65 four times at 1- to 2-month intervals starting at 1 month of age. Naive plasma was derived from ferrets of similar age. ELISA plates were coated with (A) AAV5 or (B) AAV2, and binding of immune and naive ferret plasma was assessed. The secondary detection antibody was directed against IgG. Results show the mean ± range of two technical replicates for each sample. [Figure 13]Figures 13A-13F. Quantification of IgG, IgM, and IgA capsid-binding antibodies in the plasma of ferrets infected with AV.TL65. (A-F) Quantification of capsid-binding antibodies in the plasma of (A-C) neonatal and (D-F) juvenile ferrets for (A, D) IgG, (B, E) IgM, and (C, F) IgA. Results show the mean + / - SD of n = 6 neonatal animals per group and n = 9-10 juvenile animals per group. Statistical significance was analyzed by one-way ANOVA followed by Tukey's post-hoc test: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Label-free comparisons between single- and repeated-dose groups were not significantly different. [Figure 14] Figures 14A-14F. Quantification of IgG, IgM, and IgA capsid-binding antibodies in BALF of ferrets infected with AV.TL65. (A-F) Quantification of capsid-binding antibodies in BALF of (A-C) neonatal and (D-F) young ferrets for (A, D) IgG, (B, E) IgM, and (C, F) IgA. Results show the mean + / - SD of n = 6 neonatal animals per group and n = 9-10 young animals per group. Statistical significance was analyzed by one-way ANOVA followed by Tukey's post-hoc test: *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. Label-free comparisons between single- and repeated-dose groups were not significantly different. DETAILED DESCRIPTION OF THE INVENTION

[0021] Detailed Description of the Embodiments of the Present Disclosure Gene therapy is a unique mutation-agnostic approach to treating cystic fibrosis (CF). The present disclosure is based, at least in part, on the discovery that the rAAV vectors described herein (e.g., AV.TL65-SP183-hCFTRΔR) are unexpectedly effective at complementing CFTR-mediated chloride transport in polarized human CF airway epithelia. The rAAV vectors described herein utilize a combination of components to achieve improved functional payload capacity, more effective cellular delivery, and more efficient transgene expression compared to existing CF gene therapy approaches. In particular, the rAAV contains a highly functional CFTR minigene (CFTRΔR), a short but highly active 183-bp synthetic promoter (SP183, including the F5 enhancer and tg83 promoter), and an evolved chimeric rAAV vector, AV.TL65, that is highly effective in the human airway. In one embodiment, the vector is administered to a human. In one aspect, the human is a newborn. In one aspect, the human is a juvenile.

[0022] definition The term "AAV" refers to adeno-associated virus and may be used to refer to the naturally occurring wild-type virus itself or its derivatives. This term encompasses all subtypes, serotypes, pseudotypes, and both native and recombinant forms, unless otherwise indicated. The AAV genome is composed of single-stranded DNA and contains inverted terminal repeats (ITRs) at both ends of the DNA strand and two open reading frames, rep and cap, which encode replication and capsid proteins, respectively. A foreign polynucleotide can replace the native rep and cap genes. AAV can be produced with a variety of different serotype capsids with different transduction profiles, or, as used herein, "tropism" for different tissue types. As used herein, the term "serotype" refers to an AAV identified and distinguished from other AAVs based on capsid protein reactivity with defined antisera, e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAVrhlO. For example, serotype AAV2 is used to refer to an AAV containing a capsid protein encoded by the AAV2 cap gene and a genome containing the 5' and 3' ITR sequences of the same AAV2 serotype. Pseudotyped AAV refers to an AAV containing a viral genome containing the capsid protein of one serotype and the 5'-3' ITR of a second serotype. Pseudotyped rAAV is expected to have the cell surface binding properties of the capsid serotype and genetic properties consistent with the ITR serotype. Pseudotyped rAAV is generated using standard techniques described in the art.

[0023] The term "about" is used herein to mean a value of ±10% of the stated value. As used herein, "administering" refers to a method of providing a subject with a dosage of a composition described herein (e.g., rAAV or a pharmaceutical composition thereof). The compositions utilized in the methods described herein can be administered by any suitable route, including, for example, inhalation, nebulization, aerosolization, intranasal, intratracheal, intrabronchial, oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular), oral, nasal, rectal, topical, or buccal. In some embodiments, the compositions described herein are administered in aerosolized particles intratracheally and / or intrabronchially using an atomizer spray (e.g., using a MADgic™ laryngotracheal mucosal spray device). The compositions utilized in the methods described herein can also be administered locally or systemically. The method of administration can vary depending on various factors (e.g., the components of the composition being administered and the severity of the condition being treated).

[0024] The term "AV.TL65" refers to an evolved chimeric AAV capsid protein that is highly influential on the human respiratory tract. AV.TL65 is described in Excoffon et al. Proc. Natl. Acad. Sci. USA 106(10):3865-3870, 2009, the entire contents of which are incorporated herein by reference, and is also known in the art as AAV2.5T. AV.TL65 is a chimera between AAV2 (aa 1-128) and AAV5 (aa 129-725), with a single point mutation (A581T) substitution. The amino acid sequence of the AV.TL65 capsid is shown below: MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLD KGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQ AKKRVLEPFGLVEEGAKTAPTGKRIDDHFPKRKKARTEEDSKPSTSSDAEAGPSGSQQLQ IPAQPASSLGADTMSAGGGGPLGDNNQGADGVGNASGDWHCDSTWMGDRVVTKSTRTWVL PSYNNHQYREIKSGSVDGSNANAYFGYSTPWGYFDFNRFHSHWSPRDWQRLINNYWGFRP RSLRVKIFNIQVKEVTVQDSTTTIANNLTSTVQVFTDDDYQLPYVVGNGTEGCLPAFPPQ VFTLPQYGYATLNRDNTENPTERSSFFCLEYFPSKMLRTGNNFEFTYNFEEVPFHSSFAP SQNLFKLANPLVDQYLYRFVSTNNTGGVQFNKNLAGRYANTYKNWFPGPMGRTQGWNLGS GVNRASVSAFATTNRMELEGASYQVPPQPNGMTNNLQGSNTYALENTMIFNSQPANPGTT ATYLEGNMLITSESETQPVNRVAYNVGGQMATNNQSSTTAPTTGTYNLQEIVPGSVWMER DVYLQGPIWAKIPETGAHFHPSPAMGGFGLKHPPPMMLIKNTPVPGNITSFSDVPVSSFI TQYSTGQVTVEMEWELKKENSKRWNPEIQYTNNYNDPQFVDFAPDSTGEYRTTRPIGTRY LTRPL (SEQ ID NO: 13).

[0025] A "control element" or "control sequence" is a nucleotide sequence involved in molecular interactions that contribute to the functional regulation of a polynucleotide, including its replication, duplication, transcription, splicing, translation, or degradation. Such regulation can affect the frequency, rate, or specificity of the process and can be activating or repressive in nature. Control elements known in the art include, for example, transcriptional regulatory sequences such as promoters and enhancers. A promoter is a DNA region capable of binding RNA polymerase under specific conditions and initiating transcription of a coding region typically located downstream (3') of the promoter. Promoters include AAV promoters, such as P5, P19, P40, and AAV ITR promoters, as well as heterologous promoters.

[0026] An "expression vector" is a vector containing a region encoding a polypeptide of interest and is used to effect expression of the protein in an intended target cell. Expression vectors also contain regulatory elements operably linked to the coding region to facilitate expression of the protein in the target. The combination of regulatory elements and one or more genes to which they are operably linked for expression is sometimes referred to as an "expression cassette," many of which are known and available in the art or can be readily constructed from components available in the art.

[0027] A "gene" refers to a polynucleotide containing at least one open reading frame that is capable of encoding a particular protein after being transcribed and translated. The term "gene delivery" refers to the introduction of an exogenous polynucleotide into a cell for gene transfer and can include targeting, binding, uptake, transport, localization, replicon integration and expression.

[0028] The term "gene transfer" refers to the introduction of an exogenous polynucleotide into a cell and may include targeting, binding, uptake, transport, localization, and integration of a replicon, but is distinct from and does not imply the subsequent expression of a gene.

[0029] The term "gene expression" or "expression" refers to the processes of transcription, translation, and post-translational modification of a gene. AAV "helper virus" refers to a virus that enables AAV (e.g., wild-type AAV) to be replicated and packaged by mammalian cells. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpesviruses, and poxviruses such as vaccinia. Adenoviruses include several different subgroups, with adenovirus type 5 of subgroup C being the most commonly used. Numerous adenoviruses of human, nonhuman mammalian, and avian origin are known and available from depositories such as the American College of Pathogens (ATCC). Viruses of the herpes family, such as herpes simplex virus (HSV) and Epstein-Barr virus (EBV), as well as cytomegalovirus (CMV) and pseudorabies virus (PRV), are also available from depositories such as the American College of Pathogens (ATCC).

[0030] A "detectable marker gene" is a gene that allows cells carrying that gene to be specifically detected (e.g., distinguished from cells that do not carry the marker gene). A wide variety of such marker genes are known in the art.

[0031] A "selection marker gene" is a gene that allows for specific selection of cells that carry the gene or that do not carry the gene in the presence of a corresponding selection agent. By way of example, an antibiotic resistance gene can be used as a positive selection marker gene that allows for selection of positive host cells in the presence of the corresponding antibiotic. A variety of positive and negative selection markers are known in the art, some of which are described below.

[0032] "Heterologous" means derived from a genotypically distinct entity from that of the other entity to which it is being compared. For example, a polynucleotide introduced into a different cell type by genetic engineering techniques is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide).

[0033] "Host cell," "cell line," "cell culture," "packaging cell line," and other such terms refer to eukaryotic cells useful in this disclosure, e.g., mammalian cells such as human cells. These cells can be used as recipients of recombinant vectors, viruses, or other transfer polynucleotides, and include the progeny of the original transduced cell. It is understood that the progeny of a single cell may not necessarily be completely identical (in morphology or genomic complement) to the original parent cell.

[0034] An "isolated" plasmid, virus, or other substance refers to a preparation of the substance that lacks at least some of the other components that may be present with which the substance or similar substances naturally occur or when the substance was originally prepared. Thus, for example, an isolated substance can be prepared by concentrating it from a source mixture using purification techniques. The degree of enrichment can be measured in absolute terms, such as weight per volume of solution, or in relation to second, potentially interfering substances present in the source mixture. There are various possible degrees of enrichment for embodiments of the present disclosure. Thus, for example, 2-fold enrichment is one possibility, 10-fold enrichment is a further possibility, 100-fold enrichment is a further possibility, and 1000-fold enrichment is a further possibility.

[0035] As used herein, the terms "operably linked" or "operably linked" refer to a physical or functional juxtaposition of the described components that permits them to function in their intended manner. More specifically, for example, two DNA sequences that are operably linked mean that the two DNA sequences are positioned in a relationship (either in cis or trans) that allows at least one DNA sequence to have a physiological effect on the other sequence. For example, an enhancer and / or promoter can be operably linked to a transgene (e.g., a therapeutic transgene such as a CFTRΔR minigene).

[0036] As used herein, "packaging" refers to a series of intracellular events that result in the assembly and encapsidation of viral vectors, particularly AAV vectors. Thus, when a suitable vector is introduced into a packaging cell line under appropriate conditions, it can be assembled into viral particles. The functions associated with packaging viral vectors, particularly AAV vectors, have been described herein and in the art.

[0037] The term "polynucleotide" refers to a polymeric form of nucleotides of any length, comprising deoxyribonucleotides or ribonucleotides, or their analogs. A polynucleotide may also contain modified nucleotides and nucleotide analogs, such as methylated or capped nucleotides, and may be interrupted by non-nucleotide components. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. As used herein, the term polynucleotide refers interchangeably to double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the disclosure described herein that is a polynucleotide encompasses the double-stranded form and each of the two complementary single-stranded forms known or predicted to comprise the double-stranded form.

[0038] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. The terms also include modified amino acid polymers, e.g., those that undergo disulfide bond formation, glycosylation, acetylation, phosphorylation, lipidation, or conjugation with labeling moieties. A polypeptide, such as "CFTR," when discussed in the context of gene therapy and compositions therefor, refers to the respective intact polypeptide or any fragment or genetically engineered derivative thereof that retains the desired biochemical function of the intact protein. Similarly, reference to CFTR and other such genes for use in gene therapy (commonly referred to as "transgenes" that are delivered to recipient cells) includes polynucleotides encoding the intact polypeptide or any fragment or genetically engineered derivative that has the desired biochemical function.

[0039] A "pharmaceutical composition" refers to any composition containing a therapeutic or biologically active agent (e.g., a polynucleotide comprising a transgene (e.g., a CFTRΔR minigene; see, e.g., Ostedgaard et al. Proc. Natl. Acad. Sci. USA 108(7):2921-6, 2011)) that is incorporated into a viral vector (e.g., an rAAV vector) or independent of a viral vector (e.g., incorporated into a liposome, microparticle, or nanoparticle), suitable for administration to a subject. Any of these formulations can be prepared by well-known and accepted methods in the art. See, for example, Remington: The Science and Practice of Pharmacy (21st ed.), A.R. Gennaro (ed.), Lippincott Williams & Wlkins, 2005, and Encyclopedia of Pharmaceutical Technology, J. Swarbrick (ed.), Informa Healthcare, 2006, each of which is incorporated herein by reference.

[0040] "Pharmaceutically acceptable diluent, excipient, carrier, or adjuvant" means a diluent, excipient, carrier, or adjuvant that is physiologically accepted by a subject while retaining the therapeutic properties of the pharmaceutical composition to which it is administered.

[0041] "Recombinant" as applied to a polynucleotide means that the polynucleotide is the product of various combinations of cloning, restriction, and / or ligation steps and other procedures that result in a construct that differs from that polynucleotide found in nature. A recombinant virus is a viral particle that includes a recombinant polynucleotide. These terms include replicating the original polynucleotide construct and progeny constructs of the original viral construct, respectively.

[0042] "Recombinant adeno-associated virus (AAV)" or "rAAV vector" refers to a recombinantly produced AAV or AAV particle containing a polynucleotide sequence not of AAV origin (e.g., a polynucleotide comprising a transgene that may be operably linked to one or more enhancers and / or promoters) that is delivered to a cell either in vivo, ex vivo, or in vitro. rAAV may use naturally occurring capsid proteins from any AAV serotype. In some embodiments, non-naturally occurring (e.g., chimeric) capsids may be used in the rAAVs described herein, e.g., AV.TL65.

[0043] "Reference" refers to any sample, standard, or level used for comparison purposes. A "normal reference sample" or "wild-type reference sample" can be, for example, a sample from a subject without a disorder (e.g., cystic fibrosis). A "positive reference" sample, standard, or value is a sample, standard, value, or number derived from a subject known to have a disorder (e.g., cystic fibrosis) and can be matched to the subject's sample by at least one of the following criteria: age, weight, stage of disease, and overall health.

[0044] The terms "subject" and "patient" are used interchangeably herein and refer to any mammal (e.g., a human, primate, cat, dog, ferret, cow, horse, pig, goat, rat, or mouse). For example, the subject is a human.

[0045] "Terminator" refers to a polynucleotide sequence that tends to reduce or prevent read-through transcription (i.e., reduce or prevent transcription occurring on one side of the terminator from continuing to the other side of the terminator). The degree to which transcription is disrupted typically depends on the base sequence and / or length of the terminator sequence. In particular, as is well known in many molecular biological systems, certain DNA sequences commonly referred to as "transcription termination sequences" are specific sequences that tend to disrupt read-through transcription by RNA polymerase, presumably by causing the RNA polymerase molecule to terminate and / or disengage from the DNA being transcribed. Typical examples of such sequence-specific terminators include polyadenylation ("polyA") sequences, e.g., SV40 polyA. Inserting a relatively long DNA sequence between the promoter and the coding region in addition to or instead of such a sequence-specific terminator also tends to disrupt transcription of the coding region, roughly in proportion to the length of the intervening sequence. This effect is likely due to the fact that there is always some tendency for an RNA polymerase molecule to cleave off the DNA it is transcribing, and the longer the length of the sequence traversed before reaching the coding region, the greater the likelihood that it will cleave off before transcription of the coding region is complete, and in some cases even before transcription begins. Thus, terminators can prevent transcription from only one direction ("unidirectional" terminators) or both directions ("bidirectional" terminators), and can consist of sequence-specific termination sequences or sequence-nonspecific terminators, or both. A variety of such terminator sequences are known in the art. Exemplary uses of such sequences within the context of the present disclosure are provided below.

[0046] "Therapeutic gene," "prophylactic gene," "target polynucleotide," "transgene," "gene of interest," and the like, collectively refer to one or more genes that are introduced using a vector. Typically, in the context of the present disclosure, such genes are located within an rAAV vector (which is flanked by inverted terminal repeat (ITR) regions and thus can be replicated and encapsidated into rAAV particles). Target polynucleotides can be used in the present disclosure to generate rAAV vectors for many different uses. Such polynucleotides include, but are not limited to: (i) polynucleotides encoding proteins useful in other forms of gene therapy to alleviate defects caused by missing, defective, or suboptimal levels of structural proteins or enzymes; (ii) polynucleotides transcribed into antisense molecules; (iii) polynucleotides transcribed into decoys that bind to transcription or translation factors; (iv) polynucleotides encoding cellular modulators such as cytokines; (v) polynucleotides capable of sensitizing recipient cells to specific drugs, such as the thymidine kinase gene of herpesviruses; (vi) polynucleotides for cancer therapy, such as the E1A tumor suppressor gene or the p53 tumor suppressor gene for the treatment of various cancers; and (vii) polynucleotides for gene editing (e.g., CRISPR). To effect expression of the transgene in recipient host cells, in one embodiment, the transgene is operably linked to a promoter, either its own or a heterologous promoter. Many suitable promoters are known in the art, and their selection depends on the desired expression level of the target polynucleotide; whether constitutive expression, inducible expression, cell-specific expression, or tissue-specific expression is desired, etc. The rAAV vector may also contain a selectable marker.Exemplary transgenes include, but are not limited to, the cystic fibrosis transmembrane conductance regulator (CFTR) or derivatives thereof (e.g., CFTRΔR minigene; see, e.g., Ostedgaard et al., Proc. Natl. Acad. Sci. USA, 2004, 103(1):111-114, which is incorporated herein by reference in its entirety). 108(7):2921-6, 2011), alpha-antitrypsin, beta-globin, gamma-globin, tyrosine hydroxylase, glucocerebrosidase, arylsulfatase A, factor VIII, dystrophin, erythropoietin, alpha 1-antitrypsin, surfactant proteins SP-D, SP-A or SP-C, erythropoietin, or a cytokine, e.g., IFN-alpha, IFN-gamma, TNF, IL-1, IL-17, or IL-6, or an antigen such as a viral, bacterial, tumor or fungal antigen, or a neutralizing antibody or fragment thereof targeting an epitope of an antigen from a human respiratory virus, e.g., influenza virus or RSV, including, but not limited to, an HBoV protein, an influenza virus protein, an RSV protein, or a SARS protein.

[0047] A "therapeutically effective amount" refers to the amount of a composition administered to improve, inhibit, or ameliorate a subject's condition or symptoms of a disorder or disease, e.g., cystic fibrosis, in a clinically relevant manner. Any improvement in the subject is considered sufficient to achieve treatment. In one embodiment, an amount sufficient to treat is an amount that reduces, inhibits, or prevents the onset of, or one or more symptoms of, cystic fibrosis, or reduces the severity of, or the length of time a subject suffers from, one or more symptoms of cystic fibrosis (e.g., by at least about 10%, about 20%, or about 30%, or at least about 50%, about 60%, or about 70%, or at least about 80%, about 90%, about 95%, about 99%, or more compared to a control subject not treated with a composition described herein). Effective amounts of pharmaceutical compositions used to practice the methods described herein (e.g., treating cystic fibrosis) vary depending on the method of administration and the age, weight, and overall health of the subject being treated. A physician or researcher can determine the appropriate amount and dosing regimen.

[0048] As used herein, "transduction" refers to the process of introducing an exogenous polynucleotide, e.g., a transgene within an rAAV, into a host cell, resulting in expression of the polynucleotide, e.g., the transgene, within the cell. This process generally involves 1) endocytosis of the AAV after binding to a cell surface receptor, 2) escape from an endosome or other intracellular compartment within the cytosol of the cell, 3) transport of viral particles or viral genomes to the nucleus, and 4) uncoating of viral particles and generation of expressible double-stranded AAV genome forms, including circular intermediates. The expressible double-stranded forms of the rAAV may persist as nuclear episomes or, optionally, may be integrated into the host genome. Changes in any or a combination of endocytosis of AAV after binding to cell surface receptors, escape from endosomes or other intracellular compartments into the cytosol of the cell, transport of viral particles or viral genomes to the nucleus, or altered uncoating of viral particles and generation of expressible double-stranded AAV genome forms, including circular intermediates, can result in changes in expression levels or persistence of expression, or nuclear transport, or changes in the type or relative number of host cells or cell populations expressing the introduced polynucleotide. Altered expression or persistence of polynucleotides introduced via rAAV can be determined by methods well known in the art, including, but not limited to, protein expression, e.g., ELISA, flow cytometry, and Western blot, measurement of DNA and RNA production by hybridization assays, e.g., Northern blot, Southern blot, and gel shift mobility assay, or quantitative or non-quantitative reverse transcription polymerase chain reaction (PCR), or digital droplet PCR assays.

[0049] "Treatment" of an individual or cell is any type of intervention in an attempt to alter the natural course of the individual or cell at the time treatment is initiated, e.g., to elicit a preventative, curative, or other beneficial effect in the individual. For example, treatment of an individual may be performed to alleviate or limit the symptoms caused by any pathological condition, including, but not limited to, inherited or induced genetic defects (e.g., cystic fibrosis), viral, bacterial, or parasitic infections, neoplastic or aplastic conditions, or immune system dysfunction, such as autoimmunity or immunosuppression. Treatment includes, but is not limited to, the administration of a composition, such as a pharmaceutical composition, and the administration of compatible cells treated with the composition. Treatment can be performed prophylactically or therapeutically, i.e., either before or after the initiation of a pathological event or contact with a pathogenic agent. Treatment may alleviate one or more symptoms of a pathological condition. For example, symptoms of cystic fibrosis are known in the art and include, for example, persistent cough, wheezing, shortness of breath, exercise intolerance, recurring lung infections, inflamed or stuffy nasal passages, foul-smelling or oily stools, poor weight gain and growth, intestinal blockage, constipation, elevated salt levels in sweat, pancreatitis, and pneumonia. Detecting an improvement or absence of one or more symptoms of a disorder (such as cystic fibrosis) indicates successful treatment.

[0050] "Variant" refers to a polynucleotide or polypeptide that is substantially homologous to a native or reference polynucleotide or polypeptide. For example, a variant polynucleotide can be substantially homologous to a native or reference polynucleotide but has a polynucleotide sequence that differs from the polynucleotide sequence of the native or reference polynucleotide due to one or more deletions, insertions, and / or substitutions. In another example, a variant polypeptide can be substantially homologous to a native or reference polypeptide but has an amino acid sequence that differs from the amino acid sequence of the native or reference polypeptide due to one or more deletions, insertions, and / or substitutions. Polynucleotide sequences encoding variant polypeptides include sequences that encode variant proteins or fragments thereof that contain one or more additions, deletions, or substitutions of nucleotides when compared to a native or reference polynucleotide sequence but retain activity. A wide variety of mutagenesis approaches are known in the art and can be applied by those skilled in the art.

[0051] A variant polynucleotide or polypeptide sequence may be at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or more identical to a native or reference sequence. The degree of homology (percent identity) between a native and a variant sequence can be determined, for example, by comparing the two sequences using computer programs commonly used for this purpose, freely available on the World Wide Web (e.g., BLASTp or BLASTn with default settings).

[0052] As used herein, "vector" refers to a polymer or association of polymers that contains or associates with a polynucleotide and can be used to mediate delivery of the polynucleotide to a cell, either in vitro or in vivo. Exemplary vectors include, for example, plasmids, viral vectors, liposomes, and other gene delivery vehicles. The polynucleotide to be delivered, sometimes referred to as a transgene, may contain a coding sequence of interest in gene therapy (such as a gene encoding a therapeutic or protein of interest), a coding sequence of interest in vaccine development (such as a polynucleotide that expresses a protein, polynucleotide, or peptide suitable for eliciting an immune response in a mammal), and / or a selectable or detectable marker.

[0053] Polynucleotides The present disclosure provides polynucleotides that can be incorporated into rAAV vectors or used in the preparation of rAAV vectors. The polynucleotides can include any suitable elements or components, including one or more elements selected from the 5' AAV ITR (e.g., AAV2 5' ITR), F5 enhancer, tg83 promoter, 5' untranslated region (UTR), CFTRΔR minigene, '3 UTR, polyadenylation site, and / or 3' AAV ITR (e.g., AAV2 3' ITR). While polynucleotides are generally incorporated into rAAV vectors, it should be understood that they can be delivered or administered in the context of other types of vectors known in the art.

[0054] In one aspect, the disclosure provides an isolated polynucleotide comprising the sequence of SEQ ID NO: 7 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 7. In some embodiments, the polynucleotide comprises an F5 enhancer comprising the sequence of SEQ ID NO: 1, a tg83 promoter comprising the sequence of SEQ ID NO: 2, and / or an hCFTRΔR minigene comprising the sequence of SEQ ID NO: 4. In other embodiments, the polynucleotide comprises an F5 enhancer comprising the sequence of SEQ ID NO: 14, a tg83 promoter comprising the sequence of SEQ ID NO: 2, and / or an hCFTRΔR minigene comprising the sequence of SEQ ID NO: 4.

[0055] In some embodiments, the polynucleotide further comprises a 3' untranslated region (3'-UTR) in the 3' direction comprising the sequence of SEQ ID NO:5 or a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the polynucleotide sequence of SEQ ID NO:5.

[0056] In some embodiments, the polynucleotide further comprises a synthetic polyadenylation site in the 3' direction (eg, 3' to the 3'-UTR) comprising the sequence of SEQ ID NO:6. In some embodiments, the polynucleotide further comprises a 5' adeno-associated virus (AAV) inverted terminal repeat (ITR) at the 5' end of the polynucleotide and / or a 3' AAV ITR at the 3' end of the polynucleotide. In some embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 11, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 11. In some embodiments, the polynucleotide comprises an F5 enhancer comprising the sequence of SEQ ID NO: 1, a tg83 promoter comprising the sequence of SEQ ID NO: 2, and / or an hCFTRΔR minigene comprising the sequence of SEQ ID NO: 4. In other embodiments, the polynucleotide comprises an F5 enhancer comprising the sequence of SEQ ID NO: 14, a tg83 promoter comprising the sequence of SEQ ID NO: 2, and / or an hCFTRΔR minigene comprising the sequence of SEQ ID NO: 4.

[0057] In other embodiments, the polynucleotide comprises the sequence of SEQ ID NO: 17, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 17. In some embodiments, the polynucleotide comprises an F5 enhancer comprising the sequence of SEQ ID NO: 1, a tg83 promoter comprising the sequence of SEQ ID NO: 2, and / or an hCFTRΔR minigene comprising the sequence of SEQ ID NO: 4. In other embodiments, the polynucleotide comprises an F5 enhancer comprising the sequence of SEQ ID NO: 14, a tg83 promoter comprising the sequence of SEQ ID NO: 2, and / or an hCFTRΔR minigene comprising the sequence of SEQ ID NO: 4.

[0058] Any polynucleotide may include a 5' AAV ITR. Any suitable 5' AAV ITR can be used, including a 5' AAV ITR from any AAV serotype (e.g., AAV2). In some embodiments, the 5' AAV ITR comprises the sequence of SEQ ID NO: 9, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 9. In another example, in some embodiments, the polynucleotide comprises a 5' AAV ITR comprising the sequence of SEQ ID NO: 15, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 15. Any polynucleotide may include a 3' AAV ITR. Any suitable 3' AAV ITR can be used, including 3' AAV ITRs from any AAV serotype (e.g., AAV2). In some embodiments, the 3' AAV ITR comprises the sequence of SEQ ID NO: 10, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 10. In another example, in some embodiments, the polynucleotide comprises a 3' AAV ITR comprising the sequence of SEQ ID NO: 16, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 16. The ITR sequences may be palindromic, for example, as in SEQ ID NO: 15 and SEQ ID NO: 16, where the 5'-end ITR sequence is located on the reverse strand and the 3'-end ITR sequence is located on the forward strand.

[0059] Any polynucleotide can comprise an F5 enhancer. See, e.g., U.S. Patent Application No. 16 / 082,767, incorporated herein by reference in its entirety. In some embodiments, the F5 enhancer comprises the sequence of SEQ ID NO:1 or SEQ ID NO:14, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO:1 or SEQ ID NO:14. In some embodiments, F5 comprises the polynucleotide sequence of SEQ ID NO:1. In other embodiments, the F5 enhancer comprises the polynucleotide sequence of SEQ ID NO:14.

[0060] Any polynucleotide can comprise the tg83 promoter. See, e.g., U.S. Patent Application No. 16 / 082,767. In some embodiments, the tg83 promoter comprises the sequence of SEQ ID NO:2, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO:2.

[0061] Any polynucleotide may comprise a 5'-UTR. Any suitable 5'-UTR can be used. In some embodiments, the 5'-UTR comprises the sequence of SEQ ID NO: 3, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 3.

[0062] Any polynucleotide can comprise a sequence encoding a CFTR minigene. Any suitable CFTR minigene can be used, including human CFTR (hCFTR) or ferret CFTR. In some embodiments, the sequence encoding the hCFTR minigene comprises the sequence of SEQ ID NO:4, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO:4.

[0063] Any polynucleotide may comprise a 3'-UTR. Any suitable 3'-UTR can be used. In some embodiments, the 3'-UTR comprises a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 3, or a variant thereof, such as SEQ ID NO: 5.

[0064] Any polynucleotide may include a polyadenylation site. Any suitable polyadenylation site may be used. In some embodiments, the polyadenylation site comprises the sequence of SEQ ID NO:6, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO:6.

[0065] In one aspect, the disclosure provides an isolated polynucleotide comprising the sequence of SEQ ID NO: 8, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 8. In some embodiments, the polynucleotide comprises an F5 enhancer comprising the sequence of SEQ ID NO: 1, a tg83 promoter comprising the sequence of SEQ ID NO: 2, and / or an hCFTRΔR minigene comprising the sequence of SEQ ID NO: 4. In other embodiments, the polynucleotide comprises an F5 enhancer comprising the sequence of SEQ ID NO: 14, a tg83 promoter comprising the sequence of SEQ ID NO: 2, and / or an hCFTRΔR minigene comprising the sequence of SEQ ID NO: 4.

[0066] In one aspect, the disclosure provides an isolated polynucleotide comprising the sequence of SEQ ID NO: 11, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 11. In some embodiments, the polynucleotide comprises an F5 enhancer comprising the sequence of SEQ ID NO: 1, a tg83 promoter comprising the sequence of SEQ ID NO: 2, and / or an hCFTRΔR minigene comprising the sequence of SEQ ID NO: 4. In other embodiments, the polynucleotide comprises an F5 enhancer comprising the sequence of SEQ ID NO: 14, a tg83 promoter comprising the sequence of SEQ ID NO: 2, and / or an hCFTRΔR minigene comprising the sequence of SEQ ID NO: 4.

[0067] In one aspect, the disclosure provides an isolated polynucleotide comprising the sequence of SEQ ID NO: 12, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 12. In some embodiments, the polynucleotide comprises an F5 enhancer comprising the sequence of SEQ ID NO: 1, a tg83 promoter comprising the sequence of SEQ ID NO: 2, and / or an hCFTRΔR minigene comprising the sequence of SEQ ID NO: 4. In other embodiments, the polynucleotide comprises an F5 enhancer comprising the sequence of SEQ ID NO: 14, a tg83 promoter comprising the sequence of SEQ ID NO: 2, and / or an hCFTRΔR minigene comprising the sequence of SEQ ID NO: 4.

[0068] In another aspect, the disclosure provides an isolated polynucleotide comprising the sequence of SEQ ID NO: 18, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 18. In some embodiments, the polynucleotide comprises an F5 enhancer comprising the sequence of SEQ ID NO: 1, a tg83 promoter comprising the sequence of SEQ ID NO: 2, and / or an hCFTRΔR minigene comprising the sequence of SEQ ID NO: 4. In other embodiments, the polynucleotide comprises an F5 enhancer comprising the sequence of SEQ ID NO: 14, a tg83 promoter comprising the sequence of SEQ ID NO: 2, and / or an hCFTRΔR minigene comprising the sequence of SEQ ID NO: 4.

[0069] The polynucleotide may also contain one or more detectable markers. A variety of such markers are known, including the bacterial beta-galactosidase (lacZ) gene, the human placental alkaline phosphatase (AP) gene, and genes encoding various cell surface markers used as reporter molecules both in vitro and in vivo. The polynucleotide may also contain one or more selectable markers.

[0070] Recombinant AAV vectors Recombinant AAV vectors are potentially powerful tools for human gene therapy, particularly for diseases such as cystic fibrosis. A major advantage of rAAV vectors over other approaches to gene therapy is that they often do not require continuous replication in the target cell, either to reside episomally or to be stably integrated into the host cell. In general, the present disclosure provides an rAAV comprising the AV.TL65 capsid protein and a polynucleotide comprising the F5 enhancer and tg83 promoter operably linked to a transgene.

[0071] For example, in one aspect, the disclosure provides an rAAV comprising: (i) an AV.TL65 capsid protein; and (ii) a polynucleotide comprising an F5 enhancer and a tg83 promoter operably linked to a CFTRΔR minigene.

[0072] In another aspect, the disclosure provides an rAAV for use in treating cystic fibrosis in a subject in need thereof, the rAAV comprising: (i) an AV.TL65 capsid protein; and (ii) a polynucleotide comprising an F5 enhancer and a tg83 promoter operably linked to a CFTRΔR minigene.

[0073] In some embodiments, the AV.TL65 capsid protein comprises the amino acid sequence of SEQ ID NO: 13, or a variant thereof, e.g., an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 13.

[0074] In some embodiments, the F5 enhancer comprises the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 14, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 14. In some embodiments, F5 comprises the polynucleotide sequence of SEQ ID NO: 1. In other embodiments, the F5 enhancer comprises the polynucleotide sequence of SEQ ID NO: 14.

[0075] In some embodiments, the tg83 promoter comprises the polynucleotide sequence of SEQ ID NO:2, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO:2.

[0076] Any suitable CFTR minigene or derivative thereof can be used. In some embodiments, the CFTR minigene is a human CFTR minigene. In other embodiments, the CFTR minigene is a ferret CFTR minigene. In some embodiments, the human CFTR minigene is encoded by a polynucleotide comprising the sequence of SEQ ID NO:4, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO:4.

[0077] In some embodiments, the polynucleotide comprises, from 5' to 3', an F5 enhancer, a tg83 promoter, and a CFTRΔR minigene. In some specific embodiments, the polynucleotide comprises, from 5' to 3', a 5' AAV ITR (e.g., an AAV2 5' ITR), an F5 enhancer, a tg83 promoter, a 5' untranslated region (UTR), a CFTRΔR minigene, a 3' UTR, a polyadenylation site, and a 3' AAV ITR (e.g., an AAV2 3' ITR).

[0078] In another aspect, the present disclosure provides an rAAV comprising a polynucleotide comprising any of the polynucleotides described herein, e.g., the sequence of SEQ ID NO:7, SEQ ID NO:11, or SEQ ID NO:17, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO:7, SEQ ID NO:11, or SEQ ID NO:17. For example, the present disclosure provides an rAAV comprising a polynucleotide comprising the sequence of SEQ ID NO:17, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO:17. In some embodiments, the rAAV has tropism for airway epithelial cells (e.g., lung epithelial cells). In some embodiments, the rAAV comprises an AV.TL65 capsid protein, an AAV1 capsid protein, an AAV2 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, or an AAV9 capsid protein. In some embodiments, the rAAV comprises an AV.TL65 capsid protein. In some embodiments, the polynucleotide comprises an F5 enhancer comprising the sequence of SEQ ID NO:1, a tg83 promoter comprising the sequence of SEQ ID NO:2, and / or an hCFTRΔR minigene comprising the sequence of SEQ ID NO:4. In other embodiments, the polynucleotide comprises an F5 enhancer comprising the sequence of SEQ ID NO:14, a tg83 promoter comprising the sequence of SEQ ID NO:2, and / or an hCFTRΔR minigene comprising the sequence of SEQ ID NO:4.

[0079] The heterologous polynucleotide is recombinantly integrated into or in place of the coding region of the AAV genome (i.e., in place of the AAV rep and cap genes), usually flanked on both sides by AAV inverted terminal repeat (ITR) regions. This means that the ITRs appear directly juxtaposed both upstream and downstream of the coding sequence, without intervening sequences of AAV origin, e.g., to reduce the possibility of recombination that could (but does not necessarily) regenerate a replication-competent AAV genome. However, in some cases, a single ITR is sufficient to perform the functions normally associated with constructs containing two ITRs (see, e.g., WO 94 / 13788), and therefore, vector constructs having only one ITR can be used in conjunction with the packaging and production methods of the present disclosure.

[0080] The native promoter of rep is autoregulatory and can limit the amount of AAV particles produced. The rep gene can also be operably linked to a heterologous promoter, regardless of whether rep is provided as part of the vector construct or separately. A heterologous promoter that is not strongly downregulated by rep gene expression is suitable, but constitutive expression of the rep gene can have adverse effects on host cells, so an inducible promoter is a candidate. A wide variety of inducible promoters are known in the art, including heavy metal ion-inducible promoters (such as the metallothionein promoter); steroid hormone-inducible promoters (such as the MMTV promoter or growth hormone promoter); and promoters such as those derived from T7 phage, which are active in the presence of T7 RNA polymerase. One subclass of inducible promoters is one that is induced by a helper virus used to complement rAAV vector replication and packaging. A number of helper virus-inducible promoters have also been described, including the adenovirus early gene promoter, which is induced by the adenovirus E1A protein; the adenovirus major late promoter; herpesvirus promoters, which are induced by herpesvirus proteins such as VP16 or 1CP4; and vaccinia or poxvirus-inducible promoters.

[0081] Given the relative encapsidation size limitations of various AAV genomes, the insertion of large heterologous polynucleotides into the genome requires the removal of portions of the AAV sequences. Removal of one or more AAV genes is always desirable to reduce the likelihood of generating replication-competent AAV ("RCA"). Thus, in one embodiment, the coding or promoter sequences for rep, cap, or both are deleted, since the functions provided by these genes can be provided in trans.

[0082] The resulting vector is said to be "defective" in these functions. To replicate and package the vector, the missing functions are complemented by one or more packaging genes, which together encode the functions required for the various missing rep and / or cap gene products. In one embodiment, the packaging genes or gene cassettes are not flanked by AAV ITRs and, in one embodiment, do not share substantial homology with the rAAV genome. Therefore, to minimize homologous recombination during replication between the vector sequence and the separately provided packaging genes, it is desirable to avoid overlap of the two polynucleotide sequences. The level of homology and the corresponding frequency of recombination increase with the length of the homologous sequences and the level of identity they share. As is known in the art, the level of homology that poses a concern in a given system can be theoretically determined and experimentally confirmed. However, typically, recombination can be substantially reduced or eliminated if the overlapping sequences are less than about 25 nucleotides and at least 80% identical over their entire length, or less than about 50 nucleotides and at least 70% identical over their entire length. Of course, lower levels of homology further reduce the likelihood of recombination. Even without overlapping homology, some frequency of RCA generation appears to remain. As described by Allen et al. in WO 98 / 27204, "splitting" the replication and encapsidation functions of AAV can further reduce the frequency of RCA generation (e.g., by non-homologous recombination).

[0083] The rAAV vector constructs and complementary packaging gene constructs in this disclosure can be embodied in several different forms: viral particles, plasmids, and stably transformed host cells can all be used to introduce such constructs into packaging cells, either transiently or stably.

[0084] In certain embodiments of the present disclosure, the AAV vector and, if present, complementary packaging genes are provided in the form of a bacterial plasmid, an AAV particle, or any combination thereof. In other embodiments, either the AAV vector sequences, the packaging genes, or both are provided in the form of a genetically modified eukaryotic cell. The development of host cells genetically modified to express the AAV vector sequences, the AAV packaging genes, or both, provides an established source of material that is expressed at reliable levels.

[0085] Thus, a variety of different genetically modified cells can be used in accordance with the present disclosure. Illustratively, mammalian host cells can be used with at least one intact copy of a stably integrated rAAV vector. Replication functions can be provided using an AAV packaging plasmid containing at least the AAVrep gene operably linked to a promoter (as described in U.S. Pat. No. 5,658,776). Alternatively, replication functions can be provided using stable mammalian cell lines with the AAVrep gene operably linked to a promoter (e.g., Trempe et al., (WO 95 / 13392); Burstein et al., (WO 98 / 23018); and Johnson et al., (U.S. Pat. No. 5,656,785)). The AAVcap gene, which provides the encapsidation proteins as described above, can be provided together with or separately from the AAVrep gene (see, e.g., the above-mentioned patent applications and patent documents, as well as Allen et al., (WO 98 / 27204)). Other combinations are possible and are within the scope of the present disclosure.

[0086] Approaches for generating rAAV, e.g., rAAV containing the AV.TL65 capsid protein, are known in the art (see, e.g., Excoffon et al. Proc. Natl. Acad. Sci. USA 106(10):3865-3870, 2009 and U.S. Patent No. 10,046,016, each of which is incorporated herein by reference).

[0087] Enhancer The rAAV described herein can be used in combination with an enhancer of AAV transduction to achieve significantly increased transduction and / or expression of the transgene. Any suitable enhancer can be used. For example, U.S. Patent No. 7,749,491, incorporated herein by reference in its entirety, describes suitable enhancers. The enhancer can be a proteasome modulator. The proteasome modulator can be an anthracycline (e.g., doxorubicin, idarubicin, aclarubicin, daunorubicin, epirubicin, valrubicin, or mitoxantrone), a proteasome inhibitor (e.g., bortezomib, carfilzomib, and ixazomib), a tripeptidyl aldehyde (e.g., N-acetyl-l-leucyl-l-leucyl-l-norleucine (LLnL)), or a combination thereof. In some embodiments, the enhancer is doxorubicin. In other embodiments, the enhancer is idarubicin.

[0088] The rAAV and the enhancing agent can be contacted with a cell or administered to a subject in the same or different compositions (e.g., pharmaceutical compositions). The contact or administration of the rAAV and the enhancing agent can be sequential (e.g., the rAAV followed by the enhancing agent, or vice versa) or simultaneous.

[0089] Pharmaceutical Composition The present disclosure provides pharmaceutical compositions, including pharmaceutical compositions comprising any of the rAAVs described herein. Pharmaceutical carriers can include one or more pharmaceutically acceptable carriers, excipients, diluents, buffers, etc.

[0090] For example, in one aspect, the disclosure provides a pharmaceutical composition comprising an rAAV, the rAAV comprising: (i) an AV.TL65 capsid protein; and (ii) a polynucleotide comprising an F5 enhancer and a tg83 promoter operably linked to a CFTRΔR minigene.

[0091] In another aspect, the disclosure provides a pharmaceutical composition comprising an rAAV for use in treating cystic fibrosis in a subject in need thereof, the rAAV comprising: (i) an AV.TL65 capsid protein; and (ii) a polynucleotide comprising an F5 enhancer and a tg83 promoter operably linked to a CFTRΔR minigene.

[0092] In some embodiments, the AV.TL65 capsid protein comprises the amino acid sequence of SEQ ID NO: 13, or a variant thereof, e.g., an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the amino acid sequence of SEQ ID NO: 13.

[0093] In some embodiments, the F5 enhancer comprises the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 14, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 14. In some embodiments, F5 comprises the polynucleotide sequence of SEQ ID NO: 1. In other embodiments, the F5 enhancer comprises the polynucleotide sequence of SEQ ID NO: 14.

[0094] In some embodiments, the tg83 promoter comprises the polynucleotide sequence of SEQ ID NO:2, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO:2.

[0095] Any suitable CFTR minigene or derivative thereof can be used. In some embodiments, the CFTR minigene is a human CFTR minigene. In other embodiments, the CFTR minigene is a ferret CFTR minigene. In some embodiments, the human CFTR minigene is encoded by a polynucleotide comprising the sequence of SEQ ID NO:4, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO:4.

[0096] In some embodiments, the polynucleotide comprises, from 5' to 3', an F5 enhancer, a tg83 promoter, and a CFTRΔR minigene. In some specific embodiments, the polynucleotide comprises, from 5' to 3', a 5' AAV ITR (e.g., an AAV2 5' ITR), an F5 enhancer, a tg83 promoter, a 5' untranslated region (UTR), a CFTRΔR minigene, a 3' UTR, a polyadenylation site, and a 3' AAV ITR (e.g., an AAV2 3' ITR).

[0097] In another aspect, the present disclosure provides a pharmaceutical composition comprising an rAAV, wherein the rAAV comprises a polynucleotide comprising any of the polynucleotides described herein, e.g., the sequence of SEQ ID NO: 7, 11, or 17, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 7, 11, or 17. For example, the present disclosure provides a pharmaceutical composition comprising an rAAV, wherein the rAAV comprises a polynucleotide comprising the sequence of SEQ ID NO: 17, or a variant thereof, e.g., a sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the polynucleotide sequence of SEQ ID NO: 17. In some embodiments, the rAAV has tropism for airway epithelial cells (e.g., lung epithelial cells). In some embodiments, the rAAV comprises an AV.TL65 capsid protein, an AAV1 capsid protein, an AAV2 capsid protein, an AAV5 capsid protein, an AAV6 capsid protein, or an AAV9 capsid protein. In some embodiments, the rAAV comprises an AV.TL65 capsid protein. In some embodiments, the polynucleotide comprises an F5 enhancer comprising the sequence of SEQ ID NO:1, a tg83 promoter comprising the sequence of SEQ ID NO:2, and / or an hCFTRΔR minigene comprising the sequence of SEQ ID NO:4. In other embodiments, the polynucleotide comprises an F5 enhancer comprising the sequence of SEQ ID NO:14, a tg83 promoter comprising the sequence of SEQ ID NO:2, and / or an hCFTRΔR minigene comprising the sequence of SEQ ID NO:4.

[0098] Pharmaceutical compositions described herein can include rAAV alone or in combination with one or more additional therapeutic agents. Exemplary additional therapeutic agents include, but are not limited to, potentiators (e.g., any potentiator described herein, e.g., doxorubicin or idarubicin), antibiotics (e.g., azithromycin (ZITHROMAX™), amoxicillin and clavulanic acid (AUGMENTIN™), cloxacillin and diclocillin, ticarcillin and clavulanic acid (TIMENTIN™), cephalexin, cefdinir, cefprozil, cefaclor; sulfamethoxazole and trimethoprim (BACTRIM™), erythromycin / sulfisoxazole, erythromycin, clarithromycin, tetracycline, doxycycline, minocycline, tigecycline, vancomycin, imipenem, meripenem, colistimethate ... antihistamines / anticoagulants include fluticasone / COLISTIN™, linezolid, ciprofloxacin, levofloxacin, or a combination thereof), mucus thinning agents (e.g., hypertonic saline or dornase alfa (PULMOZYME™)), ​​CFTR modulators (e.g., ivacaftor (KALYDECO™), lumacaftor, lumacaftor / ivacaftor (ORKAMBI™), tezacaftor / ivacaftor (SYMDEKO™), or TRIKAFTA™ (elexacaftor / ivacaftor / tezacaftor)), mucolytics (e.g., acetylcysteine, ambroxol, bromhexine, carbocysteine, erdosteine, mecysteine, and dornase alfa), immunosuppressants, saline, hypertonic saline, or a combination thereof.

[0099] For example, the pharmaceutical compositions described herein may include one or more immunosuppressants. Any suitable immunosuppressant may be used. For example, non-limiting examples of immunosuppressants include corticosteroids (e.g., inhaled corticosteroids (e.g., beclomethasone (QVAR™), budesonide (PULMICORT™), budesonide / formoterol (SYMBICORT™), ciclesonide (ALVESCO™), fluticasone (FLOVENT HFA™), fluticasone propionate (FLOVENT DISKUS™), fluticasone furoate (ARNUITY ELLIPTA™), fluticasone propionate / salmeterol (ADVAIR™), fluticasone furoate / umeclidinium / vilanterol (TRELEGY™), and the like. ELLIPTA™), mometasone furoate (ASMANEX™), or mometasone / formoterol (DULERA™), predisone, or methylprednisone), polyclonal antilymphocyte antibodies (e.g., antilymphocyte globulin (ALG) and antithymocyte globulin (ATG) antibodies, e.g., from horses or rabbits), monoclonal antilymphocyte antibodies (e.g., anti-CD3 antibodies (e.g., murumomab and alemtuzumab) or anti-CD20 antibodies (e.g., rituximab)), interleukin-2 (IL-2) receptor agonists These include: vasopressin antagonists (e.g., daclizumab and basiliximab), calcineurin inhibitors (e.g., cyclosporine A and tacrolimus), cell cycle inhibitors (e.g., azathioprine, mycophenolate mofetil (MMF), and mycophenolic acid (MPA)), mammalian target of rapamycin (mTOR) inhibitors (e.g., sirolimus (rapamycin) and everolimus), methotrexate, cyclophosphamide, anthracyclines (e.g., doxorubicin, idarubicin, aclarubicin, daunorubicin, epirubicin, valproicin, L bicine, mitoxantrone, or a combination thereof), taxanes (e.g., TAXOL™ (paclitaxel)), and combinations thereof (e.g., a combination of a calcineurin inhibitor, a cell cycle inhibitor, and a corticosteroid).

[0100] In certain embodiments, the pharmaceutical compositions described herein contain one or more corticosteroids (e.g., inhaled corticosteroids (e.g., beclomethasone (QVAR™), budesonide (PULMICORT™), budesonide / formoterol (SYMBICORT™), ciclesonide (ALVESCO™), fluticasone (FLOVENT HFA™), fluticasone propionate (FLOVENT DISKUS™), fluticasone furoate (ARNUTY ELLIPTA™), fluticasone propionate / salmeterol (ADVAIR™), fluticasone furoate / umeclidinium / vilanterol (TRELEGY™)). In some embodiments, the corticosteroid is an inhaled corticosteroid.

[0101] The immunosuppressant (eg, any immunosuppressant described herein) can be administered by inhalation or systemically (eg, intravenously or subcutaneously). Typically, the viral vector is in a pharmaceutically suitable pyrogen-free buffer, such as Ringer's balanced salt solution (pH 7.4). Although not required, the pharmaceutical composition can optionally be provided in a unit dosage form suitable for administration of a precise amount. The pharmaceutical composition is generally sterile.

[0102] Treatment for CF The present disclosure provides methods for treating and / or preventing CF. For example, in one aspect, the disclosure provides a method of treating CF, the method comprising administering to a subject in need thereof a therapeutically effective amount of an rAAV comprising: (i) an AV.TL65 capsid protein; and (ii) a polynucleotide comprising an F5 enhancer and a tg83 promoter operably linked to a CFTRΔR minigene. The rAAV may comprise any of the polynucleotides described herein.

[0103] In another aspect, the disclosure features an rAAV for use in treating cystic fibrosis in a subject in need thereof, the rAAV comprising: (i) an AV.TL65 capsid protein; and (ii) a polynucleotide comprising an F5 enhancer and a tg83 promoter operably linked to a CFTRΔR minigene. In some embodiments, the rAAV is for use in combination with one or more additional therapeutic agents (e.g., any of the enhancing agents described herein). The rAAV may comprise any of the polynucleotides described herein.

[0104] The compositions (e.g., rAAV or pharmaceutical compositions) described herein can be used in vivo and ex vivo. In vivo gene therapy involves administering the vectors of the present disclosure directly to a subject. Pharmaceutical compositions can be supplied as liquid solutions or suspensions, as emulsions, or as solid forms suitable for dissolving or suspending in liquid prior to use. For administration to the respiratory tract, one exemplary form of administration is by aerosol, using a composition that provides either a solid or liquid aerosol when used with an appropriate aerosolization device. Another method of administration to the respiratory tract is to inject the vector using a flexible fiberoptic bronchoscope.

[0105] Compositions (e.g., rAAV or pharmaceutical compositions) described herein can be administered by any suitable route, for example, by inhalation, nebulization, aerosolization, intranasal, intratracheal, intrabronchial, oral, parenteral (e.g., intravenous, subcutaneous, or intramuscular), oral, nasal, rectal, topical, or buccal route. They can also be administered locally or systemically. In some embodiments, compositions described herein are administered in aerosolized particles intratracheally and / or intrabronchially using an atomizer sprayer (e.g., using a MADgic™ laryngotracheal mucosal spray device). In some embodiments, the compositions are administered parenterally. In other embodiments, the compositions are administered systemically. Vectors can also be introduced through bioprostheses, including, by way of example, vascular grafts (PTFE and Dacron), heart valves, intravascular stents, intravascular paving, and other non-vascular prostheses. General techniques regarding the delivery, frequency, composition, and dosage ranges of vector solutions are within the purview of those skilled in the art.

[0106] For administration to the upper (nasal) or lower respiratory tract by inhalation, the compositions described herein (e.g., rAAV or pharmaceutical compositions) are conveniently delivered from an insufflator, nebulizer, or pressurized pack, or other convenient means of delivering an aerosol spray. Pressurized packs may contain a suitable propellant, such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount.

[0107] Alternatively, for administration by inhalation or insufflation, the compositions may take the form of a dry powder, for example a powder mix of the agent and a suitable powder base such as lactose or starch. The powder composition may be presented in unit dosage form in, for example, capsules or cartridges or, for example, gelatin or blister packs from which the powder may be administered with the aid of an inhaler, insufflator or metered-dose inhaler.

[0108] For intranasal administration, the drug can be administered via a liquid spray such as nose drops, a plastic bottle atomizer, or a metered dose inhaler. Typical of atomizers are the Mistometer (Wintrop) and the Mezihaler (Riker).

[0109] Administration of the compositions (e.g., rAAV or pharmaceutical compositions) described herein can be continuous or intermittent, depending, for example, on the physiological condition of the recipient, whether the purpose of administration is therapeutic or prophylactic, and other factors known to those of skill in the art. The compositions described herein can be administered one or more times (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, or more times) at the same or different sites. Administration of the agents of the present disclosure can be essentially continuous over a preselected period of time or can be a series of spaced doses.

[0110] The compositions (e.g., rAAV or pharmaceutical compositions) described herein can be administered as monotherapy. The compositions (e.g., rAAV or pharmaceutical compositions) described herein can also be administered in combination with one or more additional therapeutic agents. Any suitable additional therapeutic agent can be used, including standard of care treatments for CF. In some embodiments, the one or more additional therapeutic agents are antibiotics (e.g., azithromycin (ZITHROMAX™), amoxicillin and clavulanic acid (AUGMENTIN™), cloxacillin and diclocillin, ticarcillin and clavulanic acid (TIMENTIN™), cephalexin, cefdinir, cefprozil, cefaclor; sulfamethoxazole and trimethoprim (BACTRIM™), erythromycin / sulfisoxazole, erythromycin, clarithromycin, tetracycline, doxycycline, minocycline, tigecycline, vancomycin, imipenem, meripenem, colistimethate / COLISTIN™, linezolid). , ciprofloxacin, levofloxacin, or combinations thereof), mucus thinning agents (e.g., hypertonic saline or dornase alfa (PULMOZYME™)), ​​CFTR modulators (e.g., ivacaftor (KALYDECO™), lumacaftor, lumacaftor / ivacaftor (ORKAMBI™), tezacaftor / ivacaftor (SYMDEKO™), or TRIKAFTA™ (elexacaftor / ivacaftor / tezacaftor)), mucolytics (e.g., acetylcysteine, ambroxol, bromhexine, carbocysteine, erdosteine, mecysteine, and dornase alfa), immunosuppressants, saline, hypertonic saline, or combinations thereof.

[0111] For example, any one of the compositions (e.g., rAAV or pharmaceutical compositions) described herein can be administered in combination with one or more immunosuppressive agents. Any suitable immunosuppressive agent can be used. For example, non-limiting examples of immunosuppressive agents include corticosteroids (e.g., inhaled corticosteroids (e.g., beclomethasone (QVAR™), budesonide (PULMICORT™), budesonide / formoterol (SYMBICORT™), ciclesonide (ALVESCO™), fluticasone (FLOVENT HFA™), fluticasone propionate (FLOVENT DISKUS™), fluticasone furoate (ARNUITY ELLIPTA™), fluticasone propionate / salmeterol (ADVAIR™), fluticasone furoate / umeclidinium / vilanterol (TRELEGY ... ELLIPTA™), mometasone furoate (ASMANEX™), or mometasone / formoterol (DULERA™), predisone, or methylprednisone), polyclonal antilymphocyte antibodies (e.g., antilymphocyte globulin (ALG) and antithymocyte globulin (ATG) antibodies, e.g., from horses or rabbits), monoclonal antilymphocyte antibodies (e.g., anti-CD3 antibodies (e.g., murumomab and alemtuzumab) or anti-CD20 antibodies (e.g., rituximab)), interleukin-2 (IL-2) receptor agonists These include: vasopressin antagonists (e.g., daclizumab and basiliximab), calcineurin inhibitors (e.g., cyclosporine A and tacrolimus), cell cycle inhibitors (e.g., azathioprine, mycophenolate mofetil (MMF), and mycophenolic acid (MPA)), mammalian target of rapamycin (mTOR) inhibitors (e.g., sirolimus (rapamycin) and everolimus), methotrexate, cyclophosphamide, anthracyclines (e.g., doxorubicin, idarubicin, aclarubicin, daunorubicin, epirubicin, valproicin, L bicine, mitoxantrone, or a combination thereof), taxanes (e.g., TAXOL™ (paclitaxel)), and combinations thereof (e.g., a combination of a calcineurin inhibitor, a cell cycle inhibitor, and a corticosteroid).

[0112] In certain embodiments, any one of the compositions (e.g., rAAV, pharmaceutical composition, and / or potentiator) described herein is administered in combination with one or more corticosteroids (e.g., inhaled corticosteroids (e.g., beclomethasone (QVAR™), budesonide (PULMICORT™), budesonide / formoterol (SYMBICORT™), ciclesonide (ALVESCO™), fluticasone (FLOVENT HFA™), fluticasone propionate (FLOVENT DISKUS™), fluticasone furoate (ARNUTY ELLIPTA™), fluticasone propionate / salmeterol (ADVAIR™), fluticasone furoate / umeclidinium / vilanterol (TRELEGY™), fluticasone furoate / umeclidinium / vilanterol (TRELEGY™)). In some embodiments, the corticosteroid is an inhaled corticosteroid.

[0113] The immunosuppressant (eg, any immunosuppressant described herein) can be administered by inhalation or systemically (eg, intravenously or subcutaneously). The compositions (e.g., rAAV or pharmaceutical compositions) described herein can be administered to a mammal alone or in combination with a pharmaceutically acceptable carrier. As noted above, the relative proportions of active ingredient and carrier will be determined by the solubility and chemical properties of the compounds, the chosen route of administration, and standard pharmaceutical practice.

[0114] The dosage of the composition will vary depending on the mode of administration, the particular compound selected, and the physiological characteristics of the particular patient being treated. It is desirable to utilize the lowest effective concentration of the virus to reduce the risk of undesirable effects such as toxicity.

[0115] Example The present invention will be more fully understood by reference to the following examples, which, however, should not be construed as limiting the scope of the present invention. It is understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes will be suggested to those skilled in the art in light thereof and are within the spirit and scope of this application and the appended claims.

[0116] Example 1: Development of AV.TL65-SP183-CFTRΔR and functional complementation of CFTR-mediated chloride transport in polarized human CF airway epithelia The limited packaging capacity of rAAV vectors has hindered the development of this virus for gene therapy of cystic fibrosis (CF). For example, viral genomes with a total size greater than 4.9 kb result in small deletions at the ends of the genome. In the case of CFTR vectors, where the transgene cassette is directly linked to the ITRs, this can lead to reduced CFTR function.

[0117] This study describes the development of an rAAV vector, designated AV.TL65-SP183-hCFTRΔR, which utilizes an evolved chimeric AAV capsid protein, AV.TL65, highly effective in the human airway; a short but highly active 183-base pair synthetic enhancer and promoter (SP183, containing the F5 enhancer and tg83 promoter); and a highly functional CFTR minigene (called hCFTRΔR). This vector represents a promising combination of elements that may overcome many of the obstacles that have hindered CF lung gene therapy efforts. The examples described herein used an rAAV vector containing a polynucleotide including a 5' AAV ITR containing the sequence of SEQ ID NO:15, an F5 enhancer containing the sequence of SEQ ID NO:14 (which may contain a 5' EcoRI site and a 3' XhoI site, as in SEQ ID NO:1), a tg83 promoter containing the sequence of SEQ ID NO:2, a 5' UTR containing the sequence of SEQ ID NO:3, an hCFTRΔR minigene containing the sequence of SEQ ID NO:4, a 3' UTR containing the sequence of SEQ ID NO:5, an s-pA containing the sequence of SEQ ID NO:6, and a 3' AAV ITR containing the sequence of SEQ ID NO:16. For example, the packaged polynucleotide may contain the sequence of SEQ ID NO:17. AV.TL65-SP183-hCFTRΔR can be used alone or in combination with one or more enhancers of rAAV transduction (e.g., small molecule enhancers). Interestingly, as described herein, we confirmed that AV.TL65 can also infect the respiratory tract of ferrets, enabling the use of a ferret CF model. A ferret version of the CFTRΔR minigene (fCFTRΔR) can also be used in such models.

[0118] Strikingly, AV.TL65-SP183-hCFTRΔR outperformed AV.1-SP183-hCFTRΔR in a direct comparison in CF air-liquid interface (ALI) cultures assessing CFTR-mediated chloride transport (Figures 1A-1C). In these experiments, the rAAV2 viral genome AV.TL65-SP183-hCFTRΔR was packaged into three capsid serotypes (AV.TL65, AV.1, and AV.2) and used to infect polarized human CF ALI cultures apically (AV.TL65 and AV.1) or basolaterally (AV.2). Because infection is efficient via the basolateral surface, basolateral infection with AAV2 was used as a positive control. 2.5 μM doxorubucin and 20 μM LLnL were added to the viral inoculum, and ALI cultures were infected for 16 h. The virus was then removed, and the cultures were refed in the absence of proteasome inhibitors. Prior to the development of AV.TL65, rAAV serotype 1 (AV.1) was the best-performing vector tested in human ALI cultures and chimpanzee lungs. AV.TL65-SP183-hCFTRΔR outperformed AV.1-SP183-hCFTRΔR by approximately twofold (Figures 1B and 1C). Thus, these data demonstrate functional complementation of CFTR-mediated chloride transport in polarized human CF airway epithelia.

[0119] Example 2: In vivo expression of AV.TL65-SP183-hCFTRΔR This study describes testing of the clinical candidate vector AV.TL65-SP183-hCFTRΔR for hCFTR expression in the airways of neonatal and adult ferrets. The endpoint of these analyses was the ratio of transgene-derived human CFTR (hCFTR) to endogenous ferret CFTR (fCFTR) mRNA. Three-day-old neonatal ferrets were inoculated with 6 x 10 IgG in a 100 μl volume of 500 μM doxorubicin. 11DRP mice were infected with AV.TL65-SP183-hCFTRΔR. Uninfected animals received the same volume of vehicle containing doxorubicin. At 10 days postinfection, lungs and whole tracheas were harvested and snap-frozen in liquid nitrogen. Tissues were pulverized to generate mRNA and cDNA for Q-PCR of human and ferret CFTR. As shown in Figures 2A-2D, AV.TL65-SP183-CFTRΔR increased human CFTR expression by 240% compared to endogenous (ferret) CFTR after gene delivery to the lung. Unexpectedly, treated ferrets also showed a ∼90-fold increase in endogenous CFTR in the lung (but not the trachea) compared to controls. This suggests that receptor binding of AV.TL65-SP183-CFTRΔR and / or the infection process may induce endogenous CFTR expression. Without wishing to be bound by theory, this may provide additional therapeutic benefit to patients taking partial-function CFTR mutants or CFTR modulators.

[0120] Neonatal ferrets are born with immature airways lacking submucosal glands and containing few ciliated cells. By the end of the third postnatal week, ciliary formation and submucosal gland formation are complete throughout the ferret's cartilaginous airways. Given that the phenotype of the ferret airway epithelium and secretions within the airways change during this maturational period, we assessed whether AV.TL65 transduces the mature ferret airways. To this end, we evaluated the ability of AV.TL65 to transduce the lungs of 1-month-old ferrets. The lungs of 1-month-old ferrets (N=3) were transduced with 7.5 x 10 cells containing SP183-hCFTRΔR cDNA in a 500 μl volume of PBS in the presence of 250 μM doxorubicin. 12 Mock-infected control animals (N=1) received 500 μl of PBS without vector in the presence of 250 μM doxorubicin. Vector was delivered to the lungs with a PennCentury microspray via tracheal intubation. Nasal delivery in the same animals was also achieved by liquid instillation with 250 μM doxorubicin and 1.5 × 10 12Mock infections were performed using 100 μl of PBS containing DRP. For intranasal delivery, mice were given PBS containing 250 μM doxorubicin. On day 12 postinfection, lung lobes were harvested separately along with the trachea, carina, and nasal turbinates with surrounding adventitia. Tissues were snap-frozen, and pulverized samples were processed separately for mRNA and DNA. In 1-month-old adult ferrets (Figures 3A-3D), AV.TL65-SP183-hCFTRΔR resulted in three-fold higher expression of human CFTR compared to endogenous (ferret) CFTR after gene delivery to the lung. While AV.TL65 was developed to effectively transduce the apical surface of differentiated human airway epithelia, these findings suggest that the receptors and coreceptors that determine AV.TL65 efficacy are conserved in ferrets. Notably, in these experiments, the induction of endogenous ferret CFTR transcripts observed in 3-day-old ferrets after AV.TL65 infection (Figure 2B) was not observed in the lungs of adult ferrets (Figure 3B), suggesting that this biology may be unique to the neonatal airway. These findings from neonatal and adult ferrets demonstrate that our approach to gene therapy for CF can be reliably translated in vivo.

[0121] Example 3: Large safety margin demonstrates clinical feasibility of inhaled doxorubicin to enhance gene therapy The clinical feasibility of inhaled proteasome inhibitors has been demonstrated with doxorubicin. Doxorubicin was tested in two clinical trials in patients with lung cancer or metastases administered as an inhaled aerosol formulation. The maximum tolerated dose in these studies was 6.0 mg / m 2 once every 3 weeks. 2 and 7.5 mg / m 2 The dose of doxorubicin that achieved efficacy in the lungs of adult ferrets (Figures 3A-3D) was 100 μl of 250 μM doxorubicin, which covers a body surface area of ​​0.043 m². 2 Assuming that the 2 Therefore, the FDA 2Using allometric scaling, we predict an 18- to 22-fold safety margin between the effective ferret dose and the maximum tolerated human dose. This large safety margin with doxorubicin supports the concept of utilizing inhalation enhancers to improve transduction efficiency with rAAV.

[0122] Example 4: CFTR functional complementation by nasal potential difference (PD) measurements and bacterial clearance in juvenile and adult CF ferrets infected with AV.TL65-SP183-fCFTRΔR This example describes a model clinical trial in CF ferrets to demonstrate functional complementation of nasal PD measures and enhanced bacterial clearance following infection with AV.TL65-SP183-fCFTRΔR. These studies utilize a gut-corrected CFTR-KO ferret model in which immune responses to ferret CFTR are prevented. Delivery of AV.TL65-SP183-fCFTRΔR to the nasal epithelium of CF animals is expected to result in CFTR-dependent changes in Vt.

[0123] Experimental Design and Methods Gene therapy into the nasal epithelium of CF ferrets. AV.TL65-SP183-fCFTRΔR was administered to the nasal epithelium of 5-month-old adult CF ferrets at 1 × 10 12DRP is delivered at a dose of 1000 mg / kg alone or with enhancers. Age-matched non-CF controls are also assessed in the absence of vector and / or enhancers to determine baseline values. Nasal transepithelial voltage (Vt) measurements are performed at baseline and at days 10, 20, and 30 post-infection using the previously described protocol. Transepithelial voltage measurements are assessed using the sequential addition of the following drugs / solutions to the epithelial perfusate after baseline measurements: amiloride (100 μM), Cl-free solution, isoproterenol (10 μM), ATP (100 μM), and GlyH-101 (100 μM). A change in transepithelial voltage in the presence of isoproterenol reflects CFTR-mediated Cl permeability, and the addition of GlyH-101 should block this voltage change if it is due to CFTR. In one example, eight CF animals (four males and four females) and eight non-CF controls (four males and four females) are evaluated.

[0124] Pulmonary gene therapy in CF ferrets. AV.TL65-SP183-fCFTRΔR was injected into the lung epithelium of 1-month-old CF ferrets at 1 × 10 doses using a Penn-Century microsprayer. 13 DRP / kg is delivered alone or with enhancer (similar to the experiments described in Figures 3A-3D). Control CF and non-CF ferrets receive a control (e.g., vehicle or enhancer only). At the time of gene transfer, both CF and non-CF animals are removed from antibiotics used during husbandry to prevent bacterial colonization of the lungs. At 12 days post-infection or after control delivery of enhancer only, animals are challenged with ampicillin-resistant Pseudomonas aeruginosa (PA01) (1 x 10) using a Penn-Century microsprayer, using a procedure similar to that previously described in neonatal CF and non-CF ferrets demonstrating a defect in CF bacterial clearance. 6 CFU / 100 grams body weight) and erythromycin-resistant Staphylococcus pseudintermedius (1 × 10 6The animals are challenged with an equal mixture of CFTR CFU / 100 grams body weight. In one example, 16 CF animals are evaluated with and without vector administration (4 males and 4 females in each condition) and 8 non-CF controls (4 males and 4 females). 24 hours after bacterial challenge, whole lung homogenates are generated for quantification of the following endpoints: 1) total bacterial CFU on blood agar, 2) ampicillin-resistant bacterial CFU on blood agar, 3) erythromycin-resistant bacterial CFU on blood agar, 4) transgene and endogenous CFTR mRNA, and 5) vector-derived genome.

[0125] Example 5: CFTR functional complementation in polarized human CF airway epithelia In this example, short-circuit current was measured to assess the rescue of functional CFTR using AV.TL65-SP183-fCFTRΔR. This assay evaluates cAMP-gated chloride channel activity in the apical membrane of human bronchial epithelium (HBE) in an Ussing chamber. Amiloride inhibits the Na+ channel activity of the epithelium. + used to block channel activity, and the change in short-circuit current (ΔIsc) during subsequent manipulation indicates Cl - The anion transport inhibitor 4,4'-diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) was used to treat low Cl - When applied in a buffer containing Cl - / HCO3 - Blockade of putative anion transporter-1 in the exchanger was achieved at approximately 120 mM Cl -This is more effective than physiological Ringer's solution at a concentration of 100 mg / mL. The cAMP agonists forskolin and IBMX activate CFTR via a cyclic adenosine monophosphate (cAMP)-dependent mechanism. GlyH101 is a specific inhibitor of CFTR, allowing the relative contribution of CFTR (versus other anion transport pathways) to be determined using an Ussing chamber system. In this study, CF HBE (dF508 / dF508) cells (N = 6) were compared with non-CF HBE cells (N = 6). Cells were grown in USG medium, and assays were performed 7 days after AAV and PI treatment (AAV, 10K MOI). In this experiment, a ferret CFTRΔR minigene was used; however, human CFTRΔR can be used in other cases.

[0126] Representative CF traces are shown in Figure 4. Forskolin-stimulated CFTR-mediated chloride transport (Isc) in CF HBEs treated with either AV.TL65 + doxorubicin or AV.TL65 + idarubicin was >50% of that in non-CF HBEs (Figure 5). Transepithelial resistance (TEER) measurements before and after the addition of AAV, PI, or both were unchanged, indicating no significant effect (if any, slight increase in TEER). This indicates a lack of significant toxicity or cell death in HBEs in response to AV.TL65 + doxorubicin or AV.TL65 + idarubicin treatment.

[0127] Example 6: AV.TL65-SP183-hCFTRΔR can significantly increase CFTR activity compared to standard of care Complementation of CFTR activity by AV.TL65-SP183-hCFTRΔR was compared with the current standard of care, VX-809 (LUMACAFTOR) and VX-770 (IVACAFTOR)-ORKAMBI™ combinations. The effects of AV.TL65-SP183-hCFTRΔR plus Dox or Ida were compared with VX-770 / VX-809 ("VX") in two separate CF HBE P3 cell lines: dF508 / dF508 and dF508 / R553X. Cells were treated with proteasome inhibitors (PIs) or vehicle and AV.TL65 (MOIs = 10K, 25K, 80K). Cells were grown in BronchiaLife / Vertex air-liquid interface (ALI) medium and assayed 7 days after AAV and PI treatment. A transepithelial cell current clamp amplifier (TECC-24) for 24-well plate assays was performed. CFTR activation by forskolin / VX caused an increase in Cl conductance, resulting in membrane depolarization; addition of DMSO produced only a small deviation.

[0128] Figure 6 shows a representative I staining from an individual well of a 24-well transwell filter plate. eq Traces (37°C) are shown. An MOI-dependent increase in CFTR activity in AV.TL65-SP183-hCFTRΔR-treated cells was observed in the presence of PI. No change in CFTR activity was observed in cells treated with AV.TL65-SP183-hCFTRΔR without PI. VX-809 / VX-770 significantly increased CFTR activity.

[0129] Figure 7 shows area under the curve (AUC) graphs showing the mean CFTR-mediated chloride secretion after forskolin stimulation for each condition, n = 4. Transepithelial resistance measurements before and after addition of AAV and / or PI showed no change and were unaffected.

[0130] These data demonstrate that CFTR activity is significantly increased by AV.TL65-SP183-hCFTRΔR compared to current standard of care, such as VX-770 / VX-809 combination therapy.

[0131] Sequence Listing

[0132] [Table 1-1]

[0133] [Table 1-2]

[0134] [Table 1-3]

[0135] [Table 1-4]

[0136] [Table 1-5]

[0137] [Table 1-6]

[0138] [Table 1-7]

[0139] [Table 1-8]

[0140] [Table 1-9]

[0141] [Table 1-10]

[0142] [Table 1-11]

[0143] [Table 1-12]

[0144] [Table 1-13]

[0145] [Table 1-14]

[0146] [Table 1-15]

[0147] [Table 1-16]

[0148] [Table 1-17]

[0149] Example 7: Repeated administration of AV.TL65 into the lungs of ferrets induces an antibody response that reduces transduction in an age-dependent manner Treating cystic fibrosis (CF) lung disease using gene therapy may require repeated administration of recombinant adeno-associated virus (rAAV). However, little is known about rAAV-mediated immune responses in the lung. Here, we demonstrated that ferrets are a suitable species for preclinical testing of AV.TL65 for pulmonary CFTR delivery and characterization of neutralizing antibody (NAb) responses. AV.TL65-hCFTR efficiently transduced both human and ferret airway epithelial cultures and upregulated CFTR Cl in CF airway cultures. - Current data complemented this study. Delivery of AV.TL65-hCFTRΔR to the lungs of neonatal and young ferrets produced hCFTR mRNA at levels 200–300% higher than endogenous fCFTR. A single dose of AV.TL65 (AV.TL65-gLuc) or multiple doses (AV.TL65-fCFTRΔR followed by AV.TL65-gLuc) were administered to neonatal and young ferrets. Repeated administration significantly reduced transgene expression (11-fold) and increased bronchoalveolar lavage fluid (BALF) NAb levels in young ferrets, but not in neonatal ferrets, despite similar plasma NAb responses in both age groups. Notably, both age groups showed a decrease in BALF anti-capsid-binding IgG, IgM, and IgA antibodies after repeated administration. Unique to young ferrets was the suppression of plasma anti-capsid-binding IgM levels after the second vector administration. Thus, age-dependent immune system maturation and isotype switching may influence the development of high-affinity lung NAbs after repeated administration of AV.TL65, potentially providing a pathway to blunt AAV neutralization responses in the lung.

[0150] The above results were carried out in detail as follows. result The ferret is a suitable preclinical species for evaluating pulmonary AV.TL65 gene therapy.

[0151] To assess whether the AV.TL65 capsid variant can complement CFTR function in the airways, the AV.TL65-SP183-hCFTRΔR virus inhibited CFTR-mediated Cl in human CF ALI cultures after apical infection. -We tested the ability of rAAV1 to modify CFTR-mediated Cl currents. Because rAAV1 has previously been shown to be one of the best serotypes for apical transduction of human ALI cultures, we pseudopackaged the same AV2-F5tg83-hCFTRΔR viral genome into AAV1 capsids and performed a comparative analysis with AV.TL65. This comparison demonstrated that apical infection with AV.TL65-SP183-hCFTRΔR virus resulted in higher levels of CFTR-mediated Cl currents than those following infection with rAAV1 virus containing the same genome (AV1.SP183-hCFTRΔR). - The results showed that the CFTR signal produced electrical current (Fig. 8A) and CFTR mRNA (Fig. 8B).

[0152] To assess whether AV.TL65 could also transduce ferret airway epithelia, we first performed in vitro transduction assays in well-differentiated tracheobronchial ALI cultures from humans and ferrets using the secreted Gaussia luciferase (gLuc) reporter vector AV.TL65-SP183gLuc (Figure 8C). Apical infection of these cultures with AV.TL65-SP183gLuc revealed no significant differences in the level of gLuc transgene expression between the two species. To confirm the tropism of AV.TL65 for ferret lungs in vivo, we assessed the transduction efficiency of AV.TL65-SP183-hCFTRΔR in neonatal and juvenile ferrets after intratracheal delivery. In these studies, expression of transgene-derived hCFTRΔR mRNA was referenced to endogenous fCFTR mRNA as an indicator of transduction efficiency (i.e., the ratio of hCFTRΔR / fCFTR mRNA copies). Using this metric, lung hCFTRΔR mRNA expression was 2-3 times greater than endogenous fCFTR mRNA in both neonatal and juvenile ferrets (Figure 8D). In contrast, tracheal expression of hCFTRΔR mRNA was lower than endogenous fCFTR mRNA in neonatal pups and nearly equivalent in juvenile animals. The low tracheal transduction of AV.TL65 in neonatal pups and highly variable in juvenile animals may be due to the delivery method, which used surgery to inject the virus into the center of the trachea. Overall, these in vitro and in vivo studies demonstrate that ferrets are a suitable species for studying the pulmonary immunological response to AV.TL65 infection.

[0153] Previous exposure of the lungs of young, but not neonatal, ferrets to AV.TL65 impairs transduction following a second dose. We evaluated the feasibility of repeated administration of AV.TL65 into ferret lungs using two rAAV vectors (AV.TL65-SP183-fCFTRΔR and AV.TL65-SP183-gLuc). AV.TL65-SP183-fCFTRΔR was selected for the initial viral infection because this vector should not initiate an immune response against the transgene (i.e., ferret CFTR, or fCFTR). For the second viral infection, we selected the secreted gLuc reporter vector AV.TL65-SP183-gLuc because we needed a robust reporter that would allow temporal and quantitative analysis of transgene expression. Ferrets in the single-dose group were infected with the AV.TL65-SP183-gLuc vector alone, while ferrets in the repeated-dose group were infected first with AV.TL65-SP183-fCFTRΔR and then with AV.TL65-SP183-gLuc. We first evaluated repeated dosing in young animals (Figure 9). These studies began with neonatal ferrets. At 1 week of age, a repeated-dose group was infected with AV.TL65-SP183-fCFTRΔR. Three weeks later, both the repeated-dose and single-dose (naive) groups were infected with AV.TL65-SP183-gLuc virus (Figure 9A). Luciferase activity was monitored in blood samples 14 days after infection with AV.TL65-SP183-gLuc and in BALF samples at the end of the experiment. Findings from this study showed that gLuc activity in plasma peaked by day 5 postinfection and remained stable up to day 14 in both treatment groups (Figure 9B). There was also no significant difference in plasma gLuc activity levels between the two treatment groups. Similarly, gLuc activity in BALF at day 14 postinfection was not significantly different between the two treatment groups (Figure 9C). In both plasma and BALF, gLuc activity was well above background levels in naive (uninfected) controls (FIGS. 9B and 9C).

[0154] This study in neonatal ferrets demonstrated that AV.TL65 rechallenge was possible without significantly reducing lung transduction. However, the possibility remained that the immature immune system of neonatal ferrets might create an immune state tolerant to AAV capsids. For these reasons, we repeated the experiment in young ferrets by initiating the initial infection with AV.TL65-SP183-fCFTRΔR at 1 month of age, roughly equivalent to a 1- to 2-year-old infant, followed 4 weeks later by delivery of a gLuc reporter vector (AV.TL65-SP183-gLuc) to both the single-dose and repeat-dose groups (Figure 10A). Results from this second study showed maximal plasma gLuc activity 5 days postinfection in both groups, but the repeat-dose group had lower plasma gLuc activity (15- to 34-fold lower) at all time points tested. In contrast to the stable plasma gLuc expression in the single- and repeat-treated neonatal groups (Figure 9B), a gradual decline in plasma gLuc activity was observed in both juvenile groups, with a more rapid trend in the repeat-treated animals (Figure 10B). Similarly, BALF gLuc activity was significantly lower (11-fold) in the repeat-treated juvenile group (Figure 10C). Cumulatively, these studies suggested the possibility of an NAb response to AAV capsids in juvenile, but not neonatal, ferrets.

[0155] Repeated administration of AV.TL65 induces higher NAb responses in BALF and plasma Given the reduced efficiency of AV.TL65 transduction in the lungs of young ferrets previously exposed to this virus, we sought to evaluate NAbs in the BALF and plasma of test animals. We assessed the potency of anti-AV.TL65 NAbs by measuring the IC of inhibition of AV.TL65-SP183-fLuc transduction in A594 cells, a human airway cell line. 50The NAb titers were determined as . Consistent with similar levels of transgene expression in single- and repeat-administered neonatal ferrets, BALF NAb titers were not significantly different between the two administration conditions (Figure 11A). In contrast, BALF NAb titers in young ferrets were significantly higher in the repeat-administered group compared with the single-administered group (Figure 11B). Furthermore, absolute NAb titers in older animals, both in the single- and repeat-administered groups, were higher (3- to 5-fold) than in the neonatal test group, suggesting a more fully developed immune response in older ferrets.

[0156] Similar analyses of plasma samples demonstrated the absence of pre-existing NAb in the control naive group (Figures 11C and 11D) and the test group prior to AV.TL65 infection. In both age groups, single- and repeat-dose animals showed a gradual, time-dependent increase in plasma NAb titers after infection, with repeat-dose young ferrets producing slightly higher plasma NAb titers (2- to 2.8-fold) than neonatal ferrets. Young ferrets also produced NAb more rapidly in plasma after single-dose infection, appearing at 5 days postinfection compared with 10 days postinfection in neonatal ferrets. Plasma NAb levels in the repeat-dose group were also significantly higher than those in the single-dose group at both ages, except at 14 days postinfection in young ferrets.

[0157] Development of an ELISA-based assay to quantify anti-AV.TL65 capsid antibody isotypes The VP2 and most abundant VP3 capsid proteins of AV.TL65, evolved from an AAV2 / AAV5 capsid shuffling library, are derived from AAV5 with a single A581T mutation in VP1. The VP1 of AV.TL65 is a hybrid of AAV2 and AAV5 capsids, containing the N-terminal unique sequence (VP1u) from aa 1 to 131 of AAV2 VP1 followed by aa 128 to 724 of the AAV5 capsid containing the A581T mutation. The AAV VP1u contains the phospholipase A2 (PLA2) catalytic domain, which is thought to be important for virion escape from endosomes. To assess AV.TL65 capsid-specific immunoglobulins (IgG, IgM, and IgA) in the plasma and BALF of ferrets infected with AV.TL65, we developed an ELISA assay using AAV virions as coating antigens. To validate this method, we used plasma collected from 1-month-old ferrets in which AV.TL65 virus was delivered into the lungs four times at 1- to 2-month intervals. When AAV5 particles were used as the coating antigen, differences in IgG binding between naive and AV.TL65-immune plasma were observed starting at a dilution of 1:50. Naive plasma binding disappeared by a dilution of 1:1250, whereas antibody binding remained high in AV.TL65-immune plasma (Figure 12A). In contrast, when AAV2 was used as the coating antigen, there was no difference in plasma IgG binding between immune and naive plasma at any dilution, and the IgG detection sensitivity was much lower than that of AAV5 (Figure 12B). These findings suggest that the surface antigen epitope of AV.TL65 presents immunogenicity similar to that of the AAV5 capsid. For these reasons, we decided to use AAV5 as the coating antigen for characterizing anti-capsid antibody isotypes in the BALF and plasma of test animals.

[0158] Next, we used this ELISA method to classify anti-capsid antibody isotypes (IgG, IgM, and IgA) in the BALF and plasma of test animals (Figures 12 and 13). Overall, newborn and young ferrets elicited similar AAV5-reactive IgG responses in the plasma of both single- and repeated-dose groups, but titers were higher after repeated infection (Figures 13A and 13D). In contrast, plasma AAV5-reactive IgM (Figures 13B and 13E) and IgA (Figures 13C and 13F) responses showed differences from the IgG responses depending on the animal's age and dosing regimen. For example, capsid-bound plasma IgM levels were suppressed only in young animals in the repeated-dose group (Figures 13B and 13E), whereas capsid-bound plasma IgA levels were suppressed in both age groups after repeated dosing. Furthermore, neonatal animals initially mounted large anti-capsid IgA responses following the second virus challenge that declined over time, whereas juvenile animals lacked this response (Figures 13C and 13F). These findings suggest that age-dependent differences in antibody isotype switching may be influenced by prior exposure to AV.TL65. Contrary to expectations, AAV5-reactive IgG, IgM, and IgA in BALF were significantly higher in the single-dose group compared with the repeat-dose group in both neonatal and juvenile animals (Figure 14). Furthermore, the absolute levels of capsid-bound IgG, IgM, and IgA were similar between both age groups and treatment conditions, despite higher levels of NAbs in the BALF of juvenile animals exposed twice to the virus (Figures 11A and 11B).

[0159] Materials and Methods Generation of recombinant AV.TL65 viral vector pAV.TL65repcap (Excoffon et al., 2009, supra) is the AAV helper plasmid used to generate AV.TL65 capsids for production of AV1-SP183-hCFTRΔR, AV.TL65-SP183-hCFTRΔR, AV.TL65-SP183-fCFTRΔR, AV.TL65-SP183-fLuc, and AV.TL65-SP183-gLuc. The rAAV proviral plasmids used for packaging were pAV2.F5tg83-hCFTRΔR and pAV2.F5tg83-fCFTRΔR, as well as pAV2-F5tg83fLuc (firefly luciferase reporter) and pAV2-F5tg83gLuc (Gaussia luciferase reporter). The AV.TL65 vector was produced at the Vector Core at Children's Hospital of Philadelphia (CHOP) using a triple-plasmid transfection method. Briefly, the AAV helper pAV.TL65repcap and adenovirus helper pAd were transfected into HEK293 cells along with one of the AAV proviral vectors. rAAV vectors generated from transfected HEK293 cells were purified on a CsCl density gradient. Titers were determined by quantitative real-time polymerase chain reaction (qPCR) using transgene-specific primers and probes, and the purity of the vector stock was assessed by SDS-PAGE after silver staining.

[0160] In vitro evaluation of the AV.TL65 vector in human and ferret airway epithelia To assess whether ferrets are a suitable species for analyzing AV.TL65, we first performed in vitro transduction experiments in well-differentiated tracheobronchial ALI cultures derived from humans and ferrets. The reporter vector AV.TL65-SP183gLuc was inoculated apically into human (n = 6 transwells from two donors) and ferret (n = 6 transwells from two donors) airway epithelial ALI cultures at an MOI (multiplicity of infection) of 10,000 DRPs (DNase-resistant particles) / cell. Doxorubicin was added to the culture medium at a final concentration of 4 μM during the infection period, and the relative luminescence units (RLU) of Gaussia luciferase activity were measured 5 days postinfection using a Renilla Luciferase Activity Assay Kit (Promega) designed to measure Gaussia luciferase and Renilla luciferase activity, according to the manufacturer's instructions. Two uninfected transwells served as controls.

[0161] In vitro comparison of CFTR-mediated currents after infection of human CF airway epithelia with AV1-SP183-hCFTR.DELTA.R and AV.TL65-SP183-hCFTR.DELTA.R viruses The efficacy of AV.TL65-SP183-hCFTRΔR and AV1-SP183-hCFTRΔR on hCFTRΔR expression and complementation of CFTR function was evaluated in polarized human ALI cultures (F508del / F508del) derived from the proximal airways of CF patients. Each vector was applied to the apex of ALI cultures (n = 4 transwells from two donors) at an MOI of 100,000 DRP / cell in the presence of doxorubicin (2.5 μM) and LLnL (20 μM). These two proteasome-modulating agents have been shown to enhance transduction by several AAV serotypes. At 12 days postinfection, CFTR-mediated Cl -Currents were measured in Ussing chambers as described previously, and changes in short-circuit current (ΔIsc) following cAMP stimulation (IBMX / forskolin) and CFTR inhibition (GlyH101) were measured. Uninfected ALI cultures (n = 4 transwells from two donors) were used as baseline controls. After measuring ΔIsc, two inserts from each virus-infected group were pooled, and total RNA was lysed using the RNeasy™ Plus Mini Kit (Qiagene). After converting the mRNA to cDNA, vector-derived hCFTRΔR mRNA was quantified by TaqMan™ PCR and normalized to human GAPDH mRNA.

[0162] Analysis of AV.TL65 transduction in the lungs of neonatal and young ferrets Three-day-old neonatal ferrets (n = 3) or one-month-old infant ferrets (n = 3) were administered 4 × 10 per gram of body weight mixed with doxorubicin (final concentration 250 μM). 10 DRP AV.TL65-SP183-hCFTRΔR virus was administered intratracheally. Mock-infected ferrets (n = 3) were inoculated with Dox (250 μM) in PBS alone. Eleven days after infection, animals were euthanized, and tracheal and lung tissues were collected separately, snap-frozen, and pulverized for total RNA extraction. Vector-derived mRNA for the transgene hCFTRΔR and endogenous fCFTR were quantified using TaqMan™. hCFTRΔR and fCFTRΔR copy numbers were normalized to GAPDH and expressed as the ratio of hCFTRΔR / fCFTR.

[0163] Administration of AV.TL65-SP183-fCFTRΔR and / or AV.TL65-SP183-gLuc to Ferrets for Humoral Response Studies The following experimental design was used to evaluate repeated administration of AV.TL65 vectors to neonatal and young ferrets. Neonatal ferrets: The AV.TL65-SP183-gLuc reporter vector was administered intratracheally to 4-week-old ferrets that were either naive to the AV.TL65 capsid or previously infected with AV.TL65-SP183-fCFTAR at 1 week of age. Young ferrets: The AV.TL65-SP183-gLuc reporter vector was administered intratracheally to 8-week-old ferrets that were either naive to the AV.TL65 capsid or previously infected with AV.TL65-SP183-fCFTRΔR at 4 weeks of age. For each dose, animals received either the AV.TL65-SP183gLuc or AV.TL65-SP183-fCFTRΔR vector (1 × 10 13 One-week-old neonatal ferrets received an inoculum containing 150 μl of the kit-administered inoculum via surgical intratracheal instillation under anesthesia with a mixture of isoflurane and oxygen. At other ages, virus was administered intratracheally using a MicroSprayer™ aerosolizer under anesthesia with a subcutaneous injection of a mixture of ketamine and xylazine. The volume of vector / doxorubicin inoculum for aerosolization was normalized to the ferret's body weight (5 ml / kg).

[0164] Blood collection and bronchoalveolar lavage fluid collection for measurement of Gaussia luciferase activity Plasma was collected from anesthetized ferrets into heparinized tubes on days 0, 5, 10, and 14 after delivery of the AV.TL65-SP183-gLuc report vector. Animals were euthanized with EUTHASOL™ (Virbac AH Inc.), and bronchoalveolar lavage fluid (BALF) was collected from the tracheal / lung cassette by instillation of 5 ml of PBS per 300 grams of body weight. gLuc activity in plasma and BALF was measured immediately after sample collection.

[0165] Antibody neutralization assay using plasma and BALF Microneutralization assays were performed using a modified version of a previously reported method (Wu et al. Front Immunol. 8:1649, 2017). NAb titers in plasma and BALF were quantified as the reduction in reporter gene expression after infection of A549 cells with AV.TL65-SP183-fLuc virus incubated with serially diluted plasma or BALF before infection. Briefly, all plasma samples from ferrets were heat-inactivated (56°C, 30 min). Five-fold serial dilutions of plasma (starting at 1:50 and ending at 1:156,250) were incubated with AV.TL65-SP183-fLuc in a total volume of 100 μl. For BALF, the same conditions were applied, except that the serial dilutions started at 1:5 and ended at 1:3125. These mixtures were incubated at 37°C for 1 h to promote antibody binding and neutralization, and then cultured in a 48-well plate (1 x 10 5 Each dilution was applied in duplicate to a monolayer of A549 cells at a concentration of 1000 DRP / well (MOI = 5000 DRP / cell). After incubating the cells with the virus mixture for 1 hour at 37°C / 5% CO2, DMEM containing 2% fetal bovine serum was added to the wells and incubated for an additional 24 hours. Firefly luciferase activity in the cell lysates was then measured using a firefly luciferase assay kit (Promega) according to the manufacturer's instructions. Each time the assay was performed, A549 cells infected with AV.TL65-SP183-fLuc alone served as a 100% transduction reference control. The neutralization titer of each plasma or BALF sample was calculated as half the maximal inhibitory concentration (IC50).

[0166] ELISA measurement of capsid-bound IgG, IgM, and IgA in plasma and BALF An ELISA procedure was used to capture and quantitate total capsid-bound IgG, IgM, and IgA in plasma and BALF. Briefly, rAAV5 in carbonate buffer was bound to a 96-well ELISA plate overnight at 4°C (1 × 10 9The plasma samples (diluted 1:2000 for IgG and IgM, and 1:20 for IgA) and undiluted BALF samples were applied to each well and incubated for 1 hour at room temperature. After washing three times with PBS-T (0.05% Tween™-20), diluted HRP-conjugated secondary antibodies were added and incubated for 1 hour at room temperature. The HRP-conjugated secondary antibodies included chicken anti-ferret IgG (Gallus Immunotech or Abeam) and goat anti-ferret IgM or IgA (Life-Bio Inc). The HRP reaction product was then quantified by absorbance on a plate reader.

[0167] statistical analysis Experimental data are expressed as mean ± SD. Prism7 (GraphPad Software, Inc., San Diego, CA, USA) was used for data analysis. Statistical significance was analyzed by one-way analysis of variance (ANOVA) followed by Tukey's test (*P<0.05; **P<0.01; ***P<0.001, ****P<0.0001).

[0168] Ethics statement for animal care All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee at the University of Iowa.

[0169] All publications, patents, and patent applications are incorporated herein by reference. In the foregoing detailed description, the invention has been described in connection with certain preferred embodiments thereof, and numerous details have been set forth for purposes of illustration, but it will be apparent to those skilled in the art that the invention is capable of additional embodiments and that the specific details herein may be varied considerably without departing from the underlying principles of the invention.

Claims

1. 1. An rAAV comprising: (i) an AV.TL65 capsid protein comprising the amino acid sequence of SEQ ID NO:13; and (ii) an isolated polynucleotide comprising the sequence of SEQ ID NO:

7.

2. The rAAV of claim 1, further comprising a 3' untranslated region (3'-UTR) in the 3' direction comprising the sequence of SEQ ID NO:

5.

3. 3. The rAAV of claim 1 or 2, further comprising a synthetic polyadenylation site in the 3' direction comprising the sequence of SEQ ID NO:

6.

4. The rAAV of any one of claims 1 to 3, further comprising a 5' adeno-associated virus (AAV) inverted terminal repeat sequence at the 5' end of the polynucleotide and a 3' AAV ITR at the 3' end of the polynucleotide.

5. The rAAV of claim 4, wherein the polynucleotide comprises the sequence of SEQ ID NO:

17.

6. A pharmaceutical composition comprising the rAAV of any one of claims 1 to 5 and a pharmaceutically acceptable carrier.

7. A pharmaceutical for treating cystic fibrosis, comprising the rAAV of any one of claims 1 to 5.

8. A medicament for treating cystic fibrosis, comprising the pharmaceutical composition of claim 6.

9. 9. The pharmaceutical of claim 7 or 8, formulated to be administered together with one or more additional therapeutic agents.

10. 10. The method of claim 9, wherein the one or more additional therapeutic agents comprise an antibiotic, a mucus thinner, a CFTR modulator, a mucolytic agent, saline, hypertonic saline, an immunosuppressant, or a combination thereof.

11. The pharmaceutical of claim 9 , wherein the one or more additional therapeutic agents include a potentiator.

12. The pharmaceutical composition of claim 11 , wherein the enhancer comprises a proteasome modulator, a proteasome inhibitor, a tripeptidyl aldehyde, or a combination thereof.

13. The pharmaceutical composition of claim 11, wherein the enhancer comprises doxorubicin, idarubicin, aclarubicin, daunorubicin, epirubicin, valrubicin, mitoxantrone, bortezomib, carfilzomib, ixazomib, N-acetyl-l-leucyl-l-leucyl-l-norleucine (LLnL), or a combination thereof.

14. The pharmaceutical composition of claim 11 , wherein the enhancer comprises doxorubicin.

15. 15. The medicament of any one of claims 7 to 14, formulated for administration by inhalation, nebulization, or aerosolization, and / or for intranasal, intratracheal, intrabronchial, oral, intravenous, subcutaneous, or intramuscular administration.

16. 16. The medicament of claim 15, formulated for administration by inhalation, nebulization, or aerosolization, and / or for intranasal, intratracheal, or intrabronchial administration.

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