Methods and compositions for inhibiting innate immune responses associated with AAV transduction

Abstract

Disclosed herein are methods and compositions for inhibiting the innate immune response associated with AAV transduction.

Description

[0001] Priority declaration

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 619,468, filed on January 19, 2018, under 35 USC § 119(e), the entire contents of which are incorporated herein by reference.

[0003] Government support statement

[0004] This invention was made with government support under Grant Nos. AI117408, HL125749, AI072176, and AR064369 awarded by the National Institutes of Health. The government has certain rights in this invention.

[0005] Statement Regarding Electronic Submission of Sequence Listings

[0006] In lieu of a paper copy, a sequence listing in ASCII text format is provided, which is submitted pursuant to 37 CFR § 1.821, is named 5470-819WO_ST25.txt, is 7,205 bytes in size, was generated on January 18, 2019, and is submitted via EFS-Web. This sequence listing is hereby incorporated into this specification by reference with respect to its disclosure. Field of the Invention

[0007] The present invention relates to methods and compositions for inhibiting the innate immune response associated with AAV transduction. Background of the Invention

[0009] Adeno-associated virus (AAV) vectors have been successfully applied to patients with hemophilia and blindness disorders in clinical trials. In some patients with hemophilia B, after delivering AAV vectors encoding factor IX (FIX), transgene expression decreased with elevated liver enzymes at weeks 6 to 10. Administration of prednisone prevents FIX from decreasing and increases FIX to previous levels in the blood. This phenomenon has never been observed in rodents and large animals in preclinical trials. Capsid-specific cytotoxic T lymphocytes (CTLs) are detected in these patients; therefore, it has been proposed that treatment failure is due to the removal of AAV transduced hepatocytes mediated by capsid-specific CTLs. The present invention does not fully support this hypothesis.

[0010] First, kinetic studies of AAV capsid antigen presentation showed that effective antigen presentation occurs immediately after AAV administration and gradually decreases to undetectable levels at later time points after AAV transduction. This suggests that capsid-specific CTLs should kill most AAV-transduced cells at early time points, but may not affect transgene expression at later times. Second, if there is CTL-mediated elimination of AAV-transduced target cells, administration of prednisone will not restore transgene expression to previous levels. Third, although capsid-specific CTL responses were observed in some patients, no FIX expression was inhibited. Therefore, after AAV gene delivery, other mechanisms may play a role in FIX reduction. It has been confirmed that innate immune responses are activated immediately after AAV administration through TLR9 and TLR2 recognition; however, there is no study of innate immune response induction at later time points after AAV administration or its role in transgene expression.

[0011] AAV is a single-stranded DNA virus. Its genome contains rep and cap sequences flanked by two inverted terminal repeats (ITRs). It is replaced with a therapeutic cassette (comprising a promoter, one or more therapeutic transgenes, and a poly(A) ("pA") tail) rep and cap Gene production AAV vector constructs. It has been shown that AAV ITRs have promoter function, suggesting that positive-strand RNA transcribed from the 5' ITR and negative-strand RNA transcribed from the 3' ITR can be produced in AAV-transduced cells. This hypothesis is supported by the findings described herein, in which transgene expression increased when a plasmid with a 3'-ITR was deleted as determined by transfection ( Fig. 9 ). The negative strand of RNA transcribed by the 3'-ITR promoter may be used as antisense RNA to knock down transgene expression. The positive strand RNA and negative strand RNA produced from the AAV ITR promoter at both ends can anneal in the cytoplasm of AAV transduced cells and form dsRNA. In addition, it has been shown that some promoters used for gene delivery have bidirectional transcription functions to produce negative strand RNA, which may also form dsRNA. The third possibility for the formation of dsRNA from gene delivery is the formation of mRNA from the secondary structure of the transgene cassette due to modification of the transgene cDNA sequence. This dsRNA formation may activate the innate immune response.

[0012] MDA5 and RIG-I are cytoplasmic viral RNA sensors that can activate type I interferon signal transduction pathways after viral infection, so they play a key role in antiviral innate immunity. MDA5 and RIG-I have a high degree of sequence similarity and a common signal transduction adapter, mitochondrial antiviral signal transduction (MAVS), but they play non-repetitive functions in antiviral immunity by identifying different viruses or viral RNAs. RIG-I recognizes 5′-triphosphorylated (PPP) flat-ended double-stranded RNA (dsRNA) or single-stranded RNA hairpins, which are generally present in various positive and negative strand viruses. MDA5 recognizes relatively long dsRNA in the dsRNA replication intermediate of dsRNA viral genome or positive strand virus, such as encephalomyocarditis virus (EMCV) and poliovirus.

[0013] The present invention overcomes previous shortcomings in the art by providing compositions and methods of use thereof for inhibiting an innate immune response associated with AAV transduction in a subject. SUMMARY OF THE INVENTION

[0015] This overview lists several embodiments of the subject matter of the present disclosure, and in many cases lists variations and permutations of these embodiments. This overview is merely an example of many and varied embodiments. The mention of one or more representative features of a given embodiment is also exemplary. Such embodiments may generally exist with or without the features mentioned; similarly, those features may be applied to other embodiments of the subject matter of the present disclosure, whether listed in this overview or not. In order to avoid excessive repetition, this overview does not list or propose all possible combinations of such features.

[0016] In one embodiment, the present invention provides a recombinant nucleic acid molecule comprising an adeno-associated virus (AAV) 5' inverted terminal repeat (ITR), a nucleotide sequence of interest (NOI) operably associated with a promoter, and an AAV 3' ITR, wherein the recombinant nucleic acid molecule further comprises: a) a poly(A) (pA) sequence oriented 3' to 5' downstream of the 5' ITR and upstream of the promoter and a pA sequence oriented 3' to 5' upstream of the 3' ITR and downstream of the NOI; b) a pA sequence oriented 3' to 5' upstream of the 3' ITR and downstream of the NOI; c) a first pA sequence oriented 3' to 5' upstream of the 3' ITR and downstream of the NOI and a second pA sequence oriented 5' to 3' downstream of the first pA sequence and upstream of the 3' ITR; d) a first pA sequence oriented 3' to 5' upstream of the 3' ITR and downstream of the NOI and a second pA sequence oriented 5' to 3' downstream of the first pA sequence and upstream of the 3' ITR; e) f) the first pA sequence oriented 3' to 5', upstream of the 5' ITR and downstream of the NOI, and the second pA sequence oriented 5' to 3', downstream of the 5' ITR and upstream of the promoter; g) the first pA sequence oriented 5' to 3', downstream of the 5' ITR and upstream of the promoter, the second pA sequence oriented 5' to 3', downstream of the NOI and upstream of the third pA sequence, and the third pA sequence oriented 3' to 5', downstream of the second pA sequence and upstream of the 3' ITR; h) the 5' ITR and upstream of the promoter, the second pA sequence oriented 3' to 5', downstream of the NOI and upstream of the third pA sequence, and the third pA sequence oriented 5' to 3', downstream of the second pA sequence and upstream of the 3' ITR; h) the 5' ITR and upstream of the promoter, the second pA sequence oriented 3' to 5', downstream of the NOI and upstream of the third pA sequence, and the third pA sequence oriented 5' to 3', downstream of the second pA sequence and upstream of the 3' ITR; g) the first pA sequence oriented 5' to 3', downstream of the 5' ITR and upstream of the promoter, the second pA sequence oriented 3' to 5', downstream of the NOI and upstream of the third pA sequence, and the third pA sequence oriented 5' to 3', downstream of the second pA sequence and upstream of the 3' ITR; The invention further comprises a first pA sequence oriented in a 5' to 3' direction downstream of the ITR and upstream of the promoter, a second pA sequence oriented in a 5' to 3' direction downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented in a 3' to 5' direction downstream of the second pA sequence and upstream of the 3' ITR; i) the first pA sequence oriented in a 5' to 3' direction downstream of the 5' ITR and upstream of the promoter, the second pA sequence oriented in a 3' to 5' direction downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented in a 5' to 3' direction downstream of the second pA sequence and upstream of the 3' ITR; j) the first pA sequence oriented in a 3' to 5' direction downstream of the 5' ITR and upstream of the second pA sequence, the second pA sequence oriented in a 5' to 3' direction downstream of the first pA sequence and upstream of the promoter, the third pA sequence oriented in a 5' to 3' direction downstream of the NOI and upstream of the fourth pA sequence, and the third pA sequence oriented in a 5' to 3' direction downstream of the third pA sequence and upstream of the 3' ITR. a fourth pA sequence oriented 3' to 5' upstream of the ITR;k) a first pA sequence oriented 3' to 5', downstream of the 5' ITR and upstream of the second pA sequence, a second pA sequence oriented 5' to 3', downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented 3' to 5', downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented 5' to 3', downstream of the third pA sequence and upstream of the 3' ITR; l) the first pA sequence oriented 5' to 3', downstream of the 5' ITR and upstream of the second pA sequence, a second pA sequence oriented 3' to 5', downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented 5' to 3', downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented 3' to 5', downstream of the third pA sequence and upstream of the 3' ITR; and / or m) 5' A first pA sequence oriented 5' to 3' downstream of the ITR and upstream of the second pA sequence, a second pA sequence oriented 3' to 5' downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented 3' to 5' downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented 5' to 3' downstream of the third pA sequence and upstream of the 3' ITR.;

[0017] In another embodiment, the present invention provides a recombinant nucleic acid molecule comprising an adeno-associated virus (AAV) vector cassette of a first AAV serotype, the vector cassette comprising an AAV 5' inverted terminal repeat (ITR), a nucleotide sequence of interest (NOI) operably associated with a promoter, and an AAV 3' ITR, wherein the recombinant nucleic acid molecule comprises an AAV 5' ITR and / or AAV 3' ITR from a second AAV serotype different from the first AAV serotype and replacing the 5' ITR and / or 3' ITR of the first AAV serotype, and in a specific embodiment, wherein the ITR of the second AAV serotype has no promoter function or has reduced promoter function compared to the promoter function of the ITR of the first AAV serotype. In this embodiment, the first AAV serotype can be any AAV serotype currently known or later identified, and the second AAV serotype different from the first AAV serotype can be any AAV serotype currently known or later identified. In some embodiments, the first AAV serotype is AAV2 and the ITR of the second AAV serotype is AAV5. For example, a recombinant nucleic acid molecule may comprise an AAV vector cassette of AAV2 comprising the 5' and / or 3' ITRs of AAV5.

[0018] In a further embodiment, the present invention provides a recombinant nucleic acid molecule comprising an adeno-associated virus (AAV) 5' inverted terminal repeat (ITR), a nucleotide sequence of interest (NOI) operably associated with a promoter, and an AAV 3' ITR, wherein the 5' ITR and / or the 3' ITR are modified (e.g., by substitution, insertion and / or deletion) to reduce or eliminate promoter activity from the 5' ITR and / or the 3' ITR.

[0019] In another embodiment, the invention provides a recombinant nucleic acid molecule comprising an AAV 5' ITR, a NOI operably associated with a promoter, a pA sequence oriented in a 3' to 5' direction, and an AAV 3' ITR, wherein the NOI sequence is fused to (e.g., in frame with; upstream and / or downstream of) one or more nucleotide sequences encoding an interfering RNA sequence that targets one or more cytoplasmic dsRNA sensors.

[0020] In some embodiments, the present invention provides A) a recombinant nucleic acid molecule comprising an AAV 5' ITR, a NOI operably associated with a first promoter, a first pA sequence oriented in 3' to 5', a nucleotide sequence encoding an interfering RNA sequence targeting a cytoplasmic dsRNA sensor operably associated with a second promoter, a second pA sequence, and an AAV 3' ITR; B) a recombinant nucleic acid molecule comprising an AAV 5' ITR, a NOI operably associated with a first promoter, a pA sequence oriented in 3' to 5', a short hairpin RNA (shRNA) sequence targeting a cytoplasmic dsRNA sensor operably associated with a second promoter, and an AAV 3' ITR; C) a recombinant nucleic acid molecule comprising an AAV 5' ITR, an shRNA targeting a cytoplasmic dsRNA sensor operably associated with a first promoter, a NOI operably associated with a second promoter, a pA sequence oriented in 3' to 5', and an AAV 3' ITR; D) A recombinant nucleic acid molecule comprising, in the following order: an AAV 5' ITR, a NOI and a microRNA (miRNA) sequence targeting a cytoplasmic dsRNA sensor, both operably associated with a promoter, a pA sequence oriented 3' to 5', and an AAV 3' ITR; E) a recombinant nucleic acid molecule comprising, in the following order: an AAV 5' ITR, a miRNA targeting a cytoplasmic dsRNA sensor and the NOI, both operably associated with a promoter, a pA sequence oriented 3' to 5', and an AAV 3' ITR; and / or E) a recombinant nucleic acid molecule comprising, in the following order: an AAV 5' ITR, a NOI, a pA sequence oriented 3' to 5', and an AAV 3' ITR, the NOI comprising a miRNA intron sequence within the NOI, the NOI being operably associated with a promoter.

[0021] Another aspect of the invention relates to a rAAV vector genome comprising the above-mentioned recombinant nucleic acid molecule. Another aspect of the invention relates to an AAV particle comprising a rAAV genome, wherein the rAAV genome comprises the above-mentioned nucleic acid molecule. Another aspect of the invention relates to a composition comprising rAAV particles.

[0022] Further provided herein are compositions comprising a first recombinant nucleic acid molecule comprising an AAV 5' ITR, a NOI operably associated with a promoter, a pA sequence oriented in a 3' to 5' direction, and an AAV 3' ITR, and a second recombinant nucleic acid molecule comprising an interfering RNA sequence that targets a cytoplasmic dsRNA sensor.

[0023] Non-limiting examples of cytoplasmic dsRNAs of the invention include MDA5, MAVS, RIG-1, TRAF6, TRAF5, RIP1, FADD, IRF, TRAF3, NAP1, TBK1, IKK, IκB, TANK, and any other molecules involved in MAVS downstream signal transduction in any combination and order in the recombinant nucleic acid molecules of the invention.

[0024] Non-limiting examples of interfering RNA (RNAi) of the present invention include small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), long double-stranded RNA (long dsRNA), antisense RNA, ribozymes, etc., as known in the art, as well as any other interfering RNA or inhibitory RNA now known or later identified.

[0025] The present invention further provides a recombinant nucleic acid molecule comprising an AAV 5' ITR, a NOI and an inhibitor of MAVS signaling, both operably associated with a promoter, a pA sequence oriented in 3' to 5' orientation, and an AAV 3' ITR.

[0026] Also provided herein is a recombinant nucleic acid molecule comprising an AAV 5' ITR, a NOI operably associated with a first promoter, a first pA sequence oriented 3' to 5', an inhibitor of MAVS signaling operably associated with a second promoter, a second pA sequence oriented 3' to 5', and an AAV 3' ITR.

[0027] In another embodiment, the present invention provides a composition comprising a first recombinant nucleic acid molecule comprising an AAV 5' ITR, a NOI operably associated with a promoter, a pA sequence oriented 3' to 5', and an AAV 3' ITR; and a second recombinant nucleic acid molecule comprising an inhibitor of MAVS signaling and a pA sequence oriented 3' to 5'.

[0028] Non-limiting examples of inhibitors of MAVS signaling include the serine protease NS3-4A from hepatitis C virus, proteases from hepatitis A virus and GB virus B, and hepatitis B virus (HBV) X protein, poly(rC)-binding protein 2, 20S proteasome subunit PSMA7, and mitochondrial fusion protein 2, as well as any other inhibitor of MAVS signaling currently known or later identified.

[0029] Also provided herein are methods of enhancing transduction of an AAV vector in cells of a subject, comprising administering to the subject an AAV vector and an agent that interferes with a dsRNA activation pathway in the cells of the subject.

[0030] Non-limiting examples of agents that interfere with a dsRNA activation pathway include 2-aminopurine, steroids (eg, hydrocortisone, as shown in Figures 28 and 29), and any other agent currently known or later identified that interferes with a dsRNA activation pathway in a cell.

[0031] In some embodiments, the AAV vectors and agents of the invention can be administered to a subject simultaneously and / or sequentially, in any order and at any time intervals (eg, hours, days, weeks, etc.). BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1A and 1B IFN-β was shown to inhibit AAV transgene expression in HeLa cell lines. HeLa cells were treated with 5x10 3 AAV2 / luciferase particles were transduced. (1A) After 24 h, recombinant human IFN-β was added to the culture medium at different doses. Transgene expression was detected by luciferase assay on days 1, 2, 4, and 6 after IFN-β supplementation. (1B) Recombinant human IFN-β was added to the culture medium at 0.5 ng / mL every day. Transgene expression was detected by luciferase assay on days 1, 2, 4, and 6. When compared with no IFN-β treatment ，***p<0.001 .

[0034] Figure 2 Poly(I:C) was shown to inhibit AAV transgene expression in cell lines. HeLa or Huh7 cells were treated with 5x10 3AAV2 / luciferase particles were transduced. 2 μg / mL poly(I:C) was added at different time points: 18 h before AAV transduction, day 0, or day 3. Luciferase expression was measured 3 days after poly(I:C) transfection.

[0035] Figures 3A-3E This shows that the dsRNA immune response is activated at later time points after AAV transduction. HeLa cells were treated with 5x10 3 The expression of MDA5 (3A), RIG-I (3B), and IFN-β (3C) in HeLa cells was detected by Q-PCR at different time points after transduction. When compared with the PBS group, *p<0.05, **p<0.01 . Data represent mean and standard deviation from 3 experiments. For each experiment, PBS or AAV infection groups contained 2 or 3 wells of cells. For Q-PCR data analysis, one sample from the PBS group was normalized to 1 at each time point in each experiment. MDA5 expression in HeLa cells in each group was detected by Western blotting 8 days after dsAAV2 / GFP transduction (3D). The relative level of MDA5 expression was calculated based on the intensity of β-actin (3E). When compared with the PBS group, *** p<0.001 .

[0036] Figure 4A and 4B The dsRNA response profiles in different cell lines are shown. (4A) Huh7, HEK293, and HepG2 cells were treated with 5x10 3 AAV2 / GFP particles were transduced. On day 7, the expression of MDA5, RIG-I, and IFN-β was detected by Q-PCR. When compared with the PBS group, *p<0.05, **p<0.01 (4B) 5x10 3 AAV2 with different transgenes was added to HeLa cells. On the 7th day after AAV transduction, the expression of MDA5, RIG-I and IFN-β was detected by Q-PCR. For Q-PCR data analysis, samples from the PBS group were standardized to 1 in each experiment.

[0037] Figure 5A and 5B Figure 2 shows the innate immune response to dsRNA in human primary hepatocytes after dsAAV2 / GFP transduction. Fresh human primary hepatocytes from 12 individuals were cultured at 5x10 3Each particle is transduced by AAV2 / GFP. At different time points after AAV transduction, the expression of MDA5, RIG-I and IFN-β is detected by Q-PCR. For relative gene expression calculation, the gene expression of the PBS group at each time point is standardized to 1, which is not shown in the figure.

[0038] Fig. 6A and 6B Figure 2 shows the innate immune response to dsRNA in human primary hepatocytes after dsAAV2 / hFIX-opt transduction. Fresh human primary hepatocytes from 10 individuals were cultured at 5x10 3 The particles were transduced by dsAAV8 / hFIX-opt. At different time points after AAV transduction, the expression of MDA5, RIG-I and IFN-β was detected by Q-PCR. For relative gene expression calculation, the gene expression of the PBS group at each time point was normalized to 1, which is not shown in the figure.

[0039] Figures 7A-7C Figure 7 shows the dsRNA response in human hepatocytes from xenograft mice after dsAAV8 / hFIX-opt transduction. (7A) Two human hepatocytes from xenograft mice were transduced with 3x10 11 AAV8 / hFIX-opt particles were injected. At 8 weeks after AAV transduction, the expression of MDA5, RIG-I, and IFN-β in human hepatocytes in mice was detected by Q-PCR. MDA5 protein in mouse liver was detected by Western blot after 8 weeks, and the band intensity was measured to show the relative MDA5 expression based on β-actin, where the data are from 3 independent experiments. When compared with the control group, **p<0.01 (7B) Two mice xenografted with human hepatocytes from another donor were injected with a certain dose of dsAAV8 / hFIX-opt. The expression of MDA5, RIG-I, and IFN-β in human hepatocytes in mice was detected by Q-PCR 4 and 8 weeks after AAV transduction. (7C) MDA5 protein in mouse liver was detected by Western blot after 4 or 8 weeks, and the relative expression level of MDA5 was calculated based on β-actin intensity when compared with the control group. *p<0.05 .

[0040] Figures 8A-8E Knockdown of dsRNA activation pathways increases AAV transgene expression. (8A) HeLa cells were transfected with siControl, siMDA5, or siMAVS. Knockdown efficiency was measured by Western blotting and Q-PCR. (8B) On day 0, HeLa cells were transfected with 5x10 3AAV2 / luciferase particles were transduced. siRNA was transfected into HeLa cells on day 4, and luciferase expression was measured 48 h or 72 h later. As a control, 2 μg / mL poly(I:C) was added on day 3 and siRNA was transfected into HeLa cells on day 4. When compared with the PBS group, *p<0.05, **p<0.01, **p<0.001 (8C) Four days after AAV transduction, siRNA was transfected into HeLa cells, and IFN-β expression was detected by Q-PCR 48 h after siRNA transfection. When compared with the PBS group, **p<0.01 (8D) Four days after AAV transduction, siRNA and IFN-β promoter reporter plasmid were co-transfected into HeLa cells, and luciferase activity was measured after 72 h. (8E) MDA5 expression was detected by Q-PCR 48 h after siRNA transfection.

[0041] Fig. 9 To show the effect of 3'-ITR on transgene expression. 5 293 cells / well were plated in 24-well plates. After 24 hours, 0.5 up of human alpha-1 antitrypsin (AAT) expression plasmid flanked by two AAV ITRs (2TR) or with a 3'ITR deletion (up / TR) or with poly(A) in reverse orientation between the transgene and 3'-ITR (2TR / down-poly AR) was transfected into 293 cells using lipofectamine 2000. AAT levels in the supernatant were measured using ELISA 48 hr after transfection. *p<0.05, **p<0.01 when compared to 2TR plasmid.

[0042] Figures 10A-10B Schematic representation of cassettes with (10A) a single poly(A) block and (10B) multiple poly(A) blocks are shown.

[0043] Fig.11 Schematic representation of the ITRs from AAV2 and AAV5 is shown.

[0044] Fig.12 GFP expression from AAV ITR promoter is shown. 5ug of pTR / GFP was co-transfected with 1ug of pCMV / lacZ into 293 cells in a 6-well plate. After 2 days, 293 cells were visualized under fluorescence microscopy and stained for LacZ expression.

[0045] Fig.13 AAT expression from AAV ITR promoter is shown. 2ug of pTR / AAT was transfected into different cells in 12-well plates. After 2 days, the supernatant was harvested for AAT detection using ELISA.

[0046] Fig.14 AAT expression from AAV / ITR-AAT vector is shown. 1x10e9 AAV / ITR / AAT vector particles were added to 1x10e5 293 cells in a 48-well plate. After 2 days, the supernatant was harvested for AAT expression.

[0047] Fig.15 Shown is the administration of 1x10e11 AAV / ITR / AAT vector particles by intramuscular injection in C57BL mice. After 4 weeks, blood was harvested and AAT expression was detected by ELISA.

[0048] Fig.16 Diagrams AF showing the location of shRNA or miRNA.

[0049] Fig.17 Schematic diagrams AC show cassettes for inhibitor expression.

[0050] Fig.18 The effect of hydrocortisone on AAV transduction at later time points is shown. Hela cells were transduced with AAV2 / luc, and 10 ug of hydrocortisone was added to the culture on day 5 after AAV transduction. Luciferase activity from cell lysates was measured 24 hr or 48 hr after the addition of hydrocortisone.

[0051] Fig.19 Shown in the back time point hydrocortisone to the influence of the innate immune response from AAV transduction.Hela cells AAV2 / luc transduction and after AAV transduction, 10ug hydrocortisone was added to the culture on the 5th day.Add hydrocortisone after 24 hr, harvested cells are used for expressing MDA5 (upper figure) and IFN-β (lower figure) by quantitative RT-PCR analysis on the transcriptional level.

[0052] Figures 20A-20B Strand transcript production in AAV-transduced cells is shown. (20A) Overview of gene-specific reverse transcription to detect positive or negative strand transcripts. HeLa cells were harvested 8 days after AAV2 / luciferase transduction. RNA was extracted and treated with DNase. Specific primers for positive or negative strand luciferase were used to synthesize cDNAs of different orientations. PCR was performed using primer pair 1 (F1 and R1) and primer pair 2 (F2 and R2) to detect transcripts in cDNAs of different orientations. (20B) PCR products are shown. PBS was used as a negative control without AAV virus. pTR / luciferase plasmid was used as a positive control for PCR. RNA was used as a template to eliminate the possibility of AAV genomic DNA contamination in the extracted RNA. To measure the yield of transcripts, cDNAs of different orientations were diluted to 20, 200, or 2,000 times as PCR templates.

[0053] Fig.21 Showing high AAV transduction in human hepatocytes with MAVS deficiency. Human hepatocyte cell lines PH5CH8 and PH5CH8 with MAVS knockdown were transduced with different doses / cell of AAV2 / luc vector. Top: 200 vg / cell dose; middle: 5000 vg / cell dose; bottom: 5000 vg / cell dose. Transgene expression was analyzed at the indicated time points.

[0054] Figures 22A-22B shRNA used and Western blot for MAVS shRNA knockdown efficiency are shown. Five different MAVS shRNAs were transfected into Hela cells, and 48 hours later, cells were collected for preparation of cell lysates. Cell lysates were loaded onto SDS-PAGE gels, then transferred to nitrocellulose membranes and stained with MAVS antibodies and GAPDH antibodies. Signals were detected using ECL Western Blot Detection Reagent (GE). (22A) Sequences of MAVS shRNAs. MAVS shRNA #29: SEQ ID NO:36; MAVS shRNA #30: SEQ ID NO:37; MAVS shRNA #31: SEQ ID NO:38; MAVS shRNA #32: SEQ ID NO:39; MAVS shRNA #68: SEQ ID NO:40. (22B) Western blot of MAVS and GAPDH.

[0055] Fig.23 Knockdown of MAVS with shRNA increases AAV transduction. Hela cells were transfected with MAVS shRNA #31 on day -1, and then AAV2 / luc vector was added at a dose of 5000 / cell on day 0. Transgene expression was analyzed on days 1 and 4 after AAV infection. DETAILED DESCRIPTION OF THE INVENTION

[0057] The present invention will now be described more fully hereinafter with reference to the accompanying drawings and specification, in which preferred embodiments of the invention are shown. The present invention may, however, be embodied in different forms and should not be construed as limited to the embodiments set forth herein.

[0058] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. The terms used in the description of the present invention are herein for the purpose of describing specific embodiments only, and are not intended to limit the present invention.

[0059] All publications, patent applications, patents, and other references and accession numbers cited herein are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is provided.

[0060] As used herein, "a", "an" or "the" can refer to one or more than one. For example, "a" cell can refer to a single cell or a plurality of cells.

[0061] Also as used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted in an alternative manner ("or").

[0062] As used herein, the term "about" when referring to a measurable value, such as an amount of a dosage (e.g., an amount of a non-viral vector), etc., is meant to include variations of ±20%, ±10%, ±5%, ±1%, ±0.5% or even ±0.1% of the specified amount.

[0063] As used herein, the transition phrase "consisting essentially of" means that the scope of a claim should be interpreted to include the specified materials or steps described in the claim, as well as those that do not materially affect the basic and novel characteristics of the claimed invention. In re Herz , 537 F.2d 549, 551-52, 190 USPQ 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP § 2111.03. Therefore, the term "consisting essentially of" when used in the claims of the present invention is not intended to be interpreted as equivalent to "comprising."

[0064] Aspects of the present invention relate to the following findings: AAV administration induces an innate immune response caused by long-term AAV transduction in a subject. This innate immune response is late in the infection stage. Without being bound by theory, it is believed that the innate immune response is at least partially triggered by the presence of double-stranded RNA, which is produced by viral infection and / or replication, triggering the cytoplasmic dsRNA recognition pathway. Therefore, when a large amount of negative-strand RNA is synthesized by AAV (e.g., in the late stage of AAV transduction), the innate immune response is activated. This can be at its peak at about the 6th week of transduction. This innate immune response includes, at least in part, an increase in the production of type I IFN-β in recipient cells or subjects and / or an increase in dsRNA sensors (e.g., MDA5 and MAVS). Inhibition of the innate immune response in the late stage after AAV transduction, for example, by inhibiting the expression and / or activity of dsRNA sensors, increases AAV transgene expression in cells or subjects.

[0065] One aspect of the invention relates to nucleic acid molecule cassettes designed to reduce dsRNA production during AAV transduction, thereby reducing the stimulation of an innate immune response, and / or inhibiting an innate immune response that may be produced (e.g., by expressing RNAi, such as siRNA, which specifically targets a response mediator, such as MDA5 and / or MAVS). Various forms of these cassettes are described herein (e.g., shown in Fig. 10A and / or Fig. 10B and / or Fig.16 and / or Fig.17 Another aspect of the invention relates to rAAV vector genomes comprising a nucleic acid molecule cassette as described herein (e.g., as shown in Fig. 10A and / or Fig. 10B and / or Fig.16 and / or Fig.17 ). The AAV genome containing the nucleic acid molecule cassette can be further packaged into a viral capsid to form rAAV particles. Another aspect of the present invention relates to a pharmaceutical preparation comprising a rAAV vector genome or an AAV particle, wherein the rAAV vector genome or the AAV particle comprises the nucleic acid molecule cassette described herein.

[0066] In one embodiment, compared with the rAAV virus particles lacking box elements as described herein, the infection of the rAAV virus particles comprising nucleic acid molecule boxes causes the significant reduction of innate immune response in recipient cells or subjects in the later stage of viral transduction. In one embodiment, compared with the control rAAV virus particles lacking box elements as described herein, the infection of the rAAV virus particles comprising nucleic acid molecule boxes causes the significant increase of transgenic expression in recipient cells or subjects in the later stage of viral transduction. Significant increase is any reproducible statistically significant increase, such as by the method used in the examples chapters herein (for example, compared with the control, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50%, 60%, 75%, 90%, 100%, 2X, 3X, 4X, 5X, 10X or more increase).

[0067] In one embodiment, the present invention provides a recombinant nucleic acid molecule comprising an adeno-associated virus (AAV) 5' inverted terminal repeat (ITR), a nucleotide sequence of interest (NOI) operably associated with a promoter, and an AAV 3' ITR, wherein the recombinant nucleic acid molecule further comprises: a) a poly(A) (pA) sequence oriented 3' to 5' downstream of the 5' ITR and upstream of the promoter and a pA sequence oriented 3' to 5' upstream of the 3' ITR and downstream of the NOI; b) a pA sequence oriented 3' to 5' upstream of the 3' ITR and downstream of the NOI; c) a first pA sequence oriented 3' to 5' upstream of the 3' ITR and downstream of the NOI and a second pA sequence oriented 5' to 3' downstream of the first pA sequence and upstream of the 3' ITR; d) a 3' e) a first pA sequence oriented 3' to 5', upstream of the ITR and downstream of the NOI, and a second pA sequence oriented 5' to 3', downstream of the NOI and upstream of the first pA; f) a first pA sequence oriented 3' to 5', downstream of the 5' ITR and upstream of the promoter, the second pA sequence oriented 5' to 3', downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented 3' to 5', downstream of the second pA sequence and upstream of the 3' ITR; g) a first pA sequence oriented 5' to 3', downstream of the 5' ITR and upstream of the promoter, the second pA sequence oriented 3' to 5', downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented 3' to 5', downstream of the second pA sequence and upstream of the 3' ITR; h) a first pA sequence oriented 5' to 3' downstream of the 5' ITR and upstream of the promoter, a second pA sequence oriented 5' to 3' downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented 3' to 5' downstream of the second pA sequence and upstream of the 3' ITR; i) a first pA sequence oriented 5' to 3' downstream of the 5' ITR and upstream of the promoter, a second pA sequence oriented 3' to 5' downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented 5' to 3' downstream of the second pA sequence and upstream of the 3' ITR; j) a 5' a first pA sequence oriented in 3' to 5' downstream of the ITR and upstream of the second pA sequence, a second pA sequence oriented in 5' to 3' downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented in 5' to 3' downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented in 3' to 5' downstream of the third pA sequence and upstream of the 3' ITR;k) a first pA sequence oriented 3' to 5', downstream of the 5' ITR and upstream of the second pA sequence, a second pA sequence oriented 5' to 3', downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented 3' to 5', downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented 5' to 3', downstream of the third pA sequence and upstream of the 3' ITR; l) the first pA sequence oriented 5' to 3', downstream of the 5' ITR and upstream of the second pA sequence, a second pA sequence oriented 3' to 5', downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented 5' to 3', downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented 3' to 5', downstream of the third pA sequence and upstream of the 3' ITR; and / or m) 5' A first pA sequence oriented 5' to 3' downstream of the ITR and upstream of the second pA sequence, a second pA sequence oriented 3' to 5' downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented 3' to 5' downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented 5' to 3' downstream of the third pA sequence and upstream of the 3' ITR.;

[0068] In one embodiment, a recombinant nucleic acid molecule comprising an adeno-associated virus (AAV) 5' inverted terminal repeat (ITR), a nucleotide sequence of interest (NOI) operably associated with a promoter, and an AAV 3' ITR further comprises: a) a poly A (pA) sequence oriented 3' to 5', downstream of the 5' ITR and upstream of the promoter, and a pA sequence oriented 3' to 5', upstream of the 3' ITR and downstream of the NOI; b) a pA sequence oriented 3' to 5', upstream of the 3' ITR and downstream of the NOI; c) a first pA sequence oriented 3' to 5', upstream of the 3' ITR and downstream of the NOI, and a second pA sequence oriented 5' to 3', downstream of the first pA sequence and upstream of the 3' ITR; d) a first pA sequence oriented 3' to 5', upstream of the 3' ITR and downstream of the NOI, and a second pA sequence oriented 5' to 3', downstream of the NOI and upstream of the first pA; e) a 3' f) the first pA sequence oriented 3' to 5' downstream of the 5' ITR and upstream of the promoter, the second pA sequence oriented 5' to 3' downstream of the NOI and upstream of the third pA sequence, and the third pA sequence oriented 3' to 5' downstream of the second pA sequence and upstream of the 3' ITR; g) the first pA sequence oriented 5' to 3' downstream of the 5' ITR and upstream of the promoter, the second pA sequence oriented 3' to 5' downstream of the NOI and upstream of the third pA sequence, and the third pA sequence oriented 5' to 3' downstream of the second pA sequence and upstream of the 3' ITR; h) the 5' ITR and upstream of the promoter, the second pA sequence oriented 3' to 5' downstream of the NOI and upstream of the third pA sequence, and the third pA sequence oriented 5' to 3' downstream of the second pA sequence and upstream of the 3' ITR; The invention further comprises a first pA sequence oriented in a 5' to 3' direction downstream of the ITR and upstream of the promoter, a second pA sequence oriented in a 5' to 3' direction downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented in a 3' to 5' direction downstream of the second pA sequence and upstream of the 3' ITR; i) the first pA sequence oriented in a 5' to 3' direction downstream of the 5' ITR and upstream of the promoter, the second pA sequence oriented in a 3' to 5' direction downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented in a 5' to 3' direction downstream of the second pA sequence and upstream of the 3' ITR; j) the first pA sequence oriented in a 3' to 5' direction downstream of the 5' ITR and upstream of the second pA sequence, the second pA sequence oriented in a 5' to 3' direction downstream of the first pA sequence and upstream of the promoter, the third pA sequence oriented in a 5' to 3' direction downstream of the NOI and upstream of the fourth pA sequence, and the third pA sequence oriented in a 5' to 3' direction downstream of the third pA sequence and upstream of the 3' ITR. a fourth pA sequence oriented 3' to 5' upstream of the ITR;k) a first pA sequence oriented 3' to 5', downstream of the 5' ITR and upstream of the second pA sequence, a second pA sequence oriented 5' to 3', downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented 3' to 5', downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented 5' to 3', downstream of the third pA sequence and upstream of the 3' ITR; l) a first pA sequence oriented 5' to 3', downstream of the 5' ITR and upstream of the second pA sequence, a second pA sequence oriented 3' to 5', downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented 5' to 3', downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented 3' to 5', downstream of the third pA sequence and upstream of the 3' ITR; or m) A first pA sequence oriented 5' to 3' downstream of the 5' ITR and upstream of the second pA sequence, a second pA sequence oriented 3' to 5' downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented 3' to 5' downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented 5' to 3' downstream of the third pA sequence and upstream of the 3' ITR.;

[0069] In an alternative embodiment, the present invention provides a recombinant nucleic acid molecule comprising an adeno-associated virus (AAV) 5' inverted terminal repeat (ITR), a nucleotide sequence of interest (NOI) operably associated with a promoter, and an AAV 3' ITR, wherein the recombinant nucleic acid molecule further comprises one or more of the following: a) a poly A (pA) sequence oriented 3' to 5' downstream of the 5' ITR and upstream of the promoter and a pA sequence oriented 3' to 5' upstream of the 3' ITR and downstream of the NOI; b) a pA sequence oriented 3' to 5' upstream of the 3' ITR and downstream of the NOI; c) a first pA sequence oriented 3' to 5' upstream of the 3' ITR and downstream of the NOI and a second pA sequence oriented 5' to 3' downstream of the first pA sequence and upstream of the 3' ITR; d) a 3' e) a first pA sequence oriented 3' to 5', upstream of the ITR and downstream of the NOI, and a second pA sequence oriented 5' to 3', downstream of the NOI and upstream of the first pA; f) a first pA sequence oriented 3' to 5', downstream of the 5' ITR and upstream of the promoter, a second pA sequence oriented 5' to 3', downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented 3' to 5', downstream of the second pA sequence and upstream of the 3' ITR; g) a first pA sequence oriented 5' to 3', downstream of the 5' ITR and upstream of the promoter, a second pA sequence oriented 3' to 5', downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented 3' to 5', downstream of the second pA sequence and upstream of the 3' ITR; h) a first pA sequence oriented 5' to 3', downstream of the 5' ITR and upstream of the promoter, a second pA sequence oriented 5' to 3', downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented 3' to 5', downstream of the second pA sequence and upstream of the 3' ITR; i) a first pA sequence oriented 5' to 3', downstream of the 5' ITR and upstream of the promoter, a second pA sequence oriented 3' to 5', downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented 5' to 3', downstream of the second pA sequence and upstream of the 3' ITR; j) a 5' a first pA sequence oriented in 3' to 5' downstream of the ITR and upstream of the second pA sequence, a second pA sequence oriented in 5' to 3' downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented in 5' to 3' downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented in 3' to 5' downstream of the third pA sequence and upstream of the 3' ITR;k) a first pA sequence oriented 3' to 5', downstream of the 5' ITR and upstream of the second pA sequence, a second pA sequence oriented 5' to 3', downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented 3' to 5', downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented 5' to 3', downstream of the third pA sequence and upstream of the 3' ITR; l) a first pA sequence oriented 5' to 3', downstream of the 5' ITR and upstream of the second pA sequence, a second pA sequence oriented 3' to 5', downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented 5' to 3', downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented 3' to 5', downstream of the third pA sequence and upstream of the 3' ITR; or m) a 5' A first pA sequence oriented 5' to 3' downstream of the ITR and upstream of the second pA sequence, a second pA sequence oriented 3' to 5' downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented 3' to 5' downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented 5' to 3' downstream of the third pA sequence and upstream of the 3' ITR.;

[0070] In an alternative embodiment, the present invention provides a recombinant nucleic acid molecule comprising an adeno-associated virus (AAV) 5' inverted terminal repeat (ITR), a nucleotide sequence of interest (NOI) operably associated with a promoter, and an AAV 3' ITR, wherein the recombinant nucleic acid molecule further comprises at least one of the following: a) a poly A (pA) sequence oriented 3' to 5' downstream of the 5' ITR and upstream of the promoter and a pA sequence oriented 3' to 5' upstream of the 3' ITR and downstream of the NOI; b) a pA sequence oriented 3' to 5' upstream of the 3' ITR and downstream of the NOI; c) a first pA sequence oriented 3' to 5' upstream of the 3' ITR and downstream of the NOI and a second pA sequence oriented 5' to 3' downstream of the first pA sequence and upstream of the 3' ITR; d) a 3' e) a first pA sequence oriented 3' to 5', upstream of the ITR and downstream of the NOI, and a second pA sequence oriented 5' to 3', downstream of the NOI and upstream of the first pA; f) a first pA sequence oriented 3' to 5', downstream of the 5' ITR and upstream of the promoter, a second pA sequence oriented 5' to 3', downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented 3' to 5', downstream of the second pA sequence and upstream of the 3' ITR; g) a first pA sequence oriented 5' to 3', downstream of the 5' ITR and upstream of the promoter, a second pA sequence oriented 3' to 5', downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented 3' to 5', downstream of the second pA sequence and upstream of the 3' ITR; h) a first pA sequence oriented 5' to 3', downstream of the 5' ITR and upstream of the promoter, a second pA sequence oriented 5' to 3', downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented 3' to 5', downstream of the second pA sequence and upstream of the 3' ITR; i) a first pA sequence oriented 5' to 3', downstream of the 5' ITR and upstream of the promoter, a second pA sequence oriented 3' to 5', downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented 5' to 3', downstream of the second pA sequence and upstream of the 3' ITR; j) a 5' a first pA sequence oriented in 3' to 5' downstream of the ITR and upstream of the second pA sequence, a second pA sequence oriented in 5' to 3' downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented in 5' to 3' downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented in 3' to 5' downstream of the third pA sequence and upstream of the 3' ITR;k) a first pA sequence oriented 3' to 5', downstream of the 5' ITR and upstream of the second pA sequence, a second pA sequence oriented 5' to 3', downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented 3' to 5', downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented 5' to 3', downstream of the third pA sequence and upstream of the 3' ITR; l) a first pA sequence oriented 5' to 3', downstream of the 5' ITR and upstream of the second pA sequence, a second pA sequence oriented 3' to 5', downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented 5' to 3', downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented 3' to 5', downstream of the third pA sequence and upstream of the 3' ITR; or m) a 5' A first pA sequence oriented 5' to 3' downstream of the ITR and upstream of the second pA sequence, a second pA sequence oriented 3' to 5' downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented 3' to 5' downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented 5' to 3' downstream of the third pA sequence and upstream of the 3' ITR.;

[0071] Non-limiting examples of embodiments of the present invention include Fig. 10A , 10B , 16 and 17, and any cassettes having any combination of elements (e.g., poly(A) sequences) and / or any directional combination as shown in the corresponding cassettes. Poly(A) sequences that can be used in the present invention are known in the art and can be determined by a skilled person. These cassettes and recombinant nucleic acid molecules can be present in a composition or population alone or in any combination and / or in any ratio. The composition or population of the present invention can also comprise, consist essentially of, or consist of a single cassette or recombinant nucleic acid molecule of the present invention.

[0072] In another embodiment, the invention provides a recombinant nucleic acid molecule comprising an adeno-associated virus (AAV) vector cassette of a first AAV serotype, comprising an AAV 5' inverted terminal repeat (ITR), a nucleotide sequence of interest (NOI) operably associated with a promoter, and an AAV 3' ITR, wherein the AAV 5' ITR and / or the AAV 3' ITR are from a second AAV serotype that is different from the first AAV serotype. For example, the 5' ITR and / or the 3' ITR of the first AAV serotype can be replaced by the 5' ITR and / or the 3' ITR from the second AAV serotype.

[0073] In further embodiments of the recombinant nucleic acid molecules of the invention, the ITRs of the second AAV serotype have no promoter function or have reduced promoter function compared to the promoter function of the ITRs of the first AAV serotype. In such embodiments, the first AAV serotype may be any AAV serotype currently known or later identified, and the second AAV serotype different from the first AAV serotype may be any AAV serotype currently known or later identified. In some embodiments, the first AAV serotype is AAV2 and the ITRs of the second AAV serotype are AAV5. For example, the recombinant nucleic acid molecule may comprise an AAV vector cassette of AAV2, the cassette of AAV2 comprising the 5' ITR and / or 3' ITR of AAV5.

[0074] In a further embodiment, the present invention provides a recombinant nucleic acid molecule comprising an adeno-associated virus (AAV) 5' inverted terminal repeat (ITR), a nucleotide sequence of interest (NOI) operably associated with a promoter, and an AAV 3' ITR, wherein the 5' ITR and / or the 3' ITR are modified (e.g., by substitution, insertion and / or deletion) to reduce or eliminate promoter activity from the 5' ITR and / or the 3' ITR.

[0075] In another embodiment, the invention provides a recombinant nucleic acid molecule comprising an AAV 5' ITR, a NOI operably associated with a promoter, a pA sequence oriented in a 3' to 5' direction, and an AAV 3' ITR, wherein the NOI sequence is fused to (e.g., fused in frame to; upstream and / or downstream of) one or more nucleotide sequences encoding an interfering RNA sequence that targets one or more cytoplasmic dsRNA sensors.

[0076] In some embodiments, the present invention provides A) a recombinant nucleic acid molecule comprising an AAV 5' ITR, a NOI operably associated with a first promoter, a first pA sequence oriented in 3' to 5', a nucleotide sequence encoding an interfering RNA sequence targeting a cytoplasmic dsRNA sensor operably associated with a second promoter, a second pA sequence, and an AAV 3' ITR; B) a recombinant nucleic acid molecule comprising an AAV 5' ITR, a NOI operably associated with a first promoter, a pA sequence oriented in 3' to 5', a short hairpin RNA (shRNA) sequence targeting a cytoplasmic dsRNA sensor operably associated with a second promoter, and an AAV 3' ITR; C) a recombinant nucleic acid molecule comprising an AAV 5' ITR, an shRNA targeting a cytoplasmic dsRNA sensor operably associated with a first promoter, a NOI operably associated with a second promoter, a pA sequence oriented in 3' to 5', and an AAV 3' ITR; D) A recombinant nucleic acid molecule comprising, in the following order: an AAV 5' ITR, a NOI and a microRNA (miRNA) sequence targeting a cytoplasmic dsRNA sensor, both operably associated with a promoter, a pA sequence oriented 3' to 5', and an AAV 3' ITR; E) a recombinant nucleic acid molecule comprising, in the following order: an AAV 5' ITR, a miRNA targeting a cytoplasmic dsRNA sensor and the NOI, both operably associated with a promoter, a pA sequence oriented 3' to 5', and an AAV 3' ITR; and / or E) a recombinant nucleic acid molecule comprising, in the following order: an AAV 5' ITR, a NOI, a pA sequence oriented 3' to 5', and an AAV 3' ITR, the NOI comprising a miRNA intron sequence within the NOI, the NOI being operably associated with a promoter.

[0077] Further provided herein is a composition comprising a first recombinant nucleic acid molecule comprising an AAV 5' ITR, a NOI operably associated with a promoter, a pA sequence oriented in 3' to 5' direction, and an AAV 3' ITR, and a second recombinant nucleic acid molecule comprising an interfering RNA sequence targeting a cytoplasmic dsRNA sensor.

[0078] Non-limiting examples of cytoplasmic dsRNAs of the invention include MDA5, MAVS, RIG-1, TRAF6, TRAF5, RIP1, FADD, IRF, TRAF3, NAP1, TBK1, IKK, IκB, TANK, and any other molecules involved in MAVS downstream signal transduction in any combination and order in the recombinant nucleic acid molecules of the invention.

[0079] Non-limiting examples of interfering RNA (RNAi) of the present invention include small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), long double-stranded RNA (long dsRNA), antisense RNA, ribozymes, etc., as known in the art, as well as any other interfering RNA or inhibitory RNA currently known or later identified.

[0080] The present invention further provides a recombinant nucleic acid molecule comprising an AAV 5' ITR, a NOI and an inhibitor of MAVS signaling, both operably associated with a promoter, a pA sequence oriented in 3' to 5' orientation, and an AAV 3' ITR.

[0081] Also provided herein is a recombinant nucleic acid molecule comprising an AAV 5' ITR, a NOI operably associated with a first promoter, a first pA sequence oriented 3' to 5', an inhibitor of MAVS signaling operably associated with a second promoter, a second pA sequence oriented 3' to 5', and an AAV 3' ITR.

[0082] In another embodiment, the present invention provides a composition comprising a first recombinant nucleic acid molecule comprising an AAV 5' ITR, a NOI operably associated with a promoter, a pA sequence oriented 3' to 5', and an AAV 3' ITR, and a second recombinant nucleic acid molecule comprising an inhibitor of MAVS signaling and a pA sequence oriented 3' to 5'.

[0083] Non-limiting examples of inhibitors of MAVS signaling include the serine protease NS3-4A from hepatitis C virus, proteases from hepatitis A virus and GB virus B, hepatitis B virus (HBV) X protein, poly(rC)-binding protein 2, 20S proteasome subunit PSMA7, and / or mitochondrial fusion protein 2, as well as any other inhibitor of MAVS signaling currently known or later identified.

[0084] Also provided herein are methods of enhancing transduction of an AAV vector in cells of a subject, comprising administering to the subject an AAV vector and an agent that interferes with a dsRNA activation pathway in the cells of the subject.

[0085] Non-limiting examples of agents that interfere with a dsRNA activation pathway include 2-aminopurine, steroids (eg, hydrocortisone), and any other agent now known or later identified that interferes with a dsRNA activation pathway in a cell.

[0086] In some embodiments, the AAV vectors and agents of the present invention can be administered to a subject simultaneously and / or sequentially in any order and at any time interval (e.g., hours, days, weeks, etc.). In one embodiment, the AAV vector is administered first, followed by the agent. In one embodiment, the agent is administered first, and then the AAV vector is administered. In one embodiment, the agent is administered at one or several intervals. In one embodiment, after administration of the AAV vector, the agent is administered at intervals (e.g., days, e.g., every 1, 2, 3, 4, 5, 6 days, or weeks, e.g., every 1, 2, 3, 4, 5, 6 weeks or longer).

[0087] definition

[0088] It is expressly intended that the various features of the invention described herein can be used in any combination, unless the context indicates otherwise.

[0089] Furthermore, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features set forth herein may be excluded or omitted.

[0090] To further illustrate, for example, if the specification indicates that a particular amino acid can be selected from A, G, I, L and / or V, this wording also indicates that the amino acid can be selected from any subset of one or more of these amino acids, such as A, G, I or L; A, G, I or V; A or G; only L; etc., as if each such subcombination was explicitly stated herein. In addition, this wording also indicates that one or more of the specified amino acids can be abandoned (e.g., by a negative proviso). For example, in a particular embodiment, the amino acid is not A, G or I; is not A; is not G or V; etc., as if each such possible abandonment was explicitly stated herein.

[0091] All amino acid positions in the AAV capsid protein in the AAV vectors and recombinant AAV nucleic acid molecules of the present invention are designated relative to the VP1 capsid subunit numbering (native AAV2 VP1 capsid protein: GenBank Accession No. AAC03780 or YP680426). Those skilled in the art will appreciate that the modifications described herein, if inserted into the AAV cap Genes can result in modifications in VP1, VP2 and / or VP3 capsid subunits. Alternatively, the capsid subunits can be expressed independently to achieve modifications in only one or two capsid subunits (VP1, VP2, VP3, VP1 + VP2, VP1 + VP3 or VP2 + VP3).

[0092] As used herein, the terms "reduce," "reduces," "reduction," "diminish," "inhibit," and similar terms mean a reduction of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97% or more.

[0093] As used herein, the terms "enhance," "enhances," "enhancement" and similar terms mean an increase of at least about 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more.

[0094] As used herein, the term "parvovirus" includes the Parvoviridae family ( Parvoviridae ), including autonomously replicating parvoviruses and dependent viruses. Autonomous parvoviruses include the genus Parvovirus ( Parvovirus ), Erythrovirus ( Erythrovirus ), Densovirus ( Densovirus ), Repetitive virus genus ( Iteravirus ) and Contravirus ( Contravirus ). Exemplary autonomous parvoviruses include, but are not limited to, mouse parvovirus, bovine parvovirus, canine parvovirus, chicken parvovirus, feline panleukopenia virus, feline parvovirus, goose parvovirus, H1 parvovirus, muscovy duck parvovirus, B19 virus, and any other now known or later discovered autonomous parvovirus. Other autonomous parvoviruses are known to those skilled in the art. See, e.g., BERNARD N. FIELDS et al., VIROLOGY, Vol. 2, Chapter 69 (4th ed., Lippincott-Raven Publishers).

[0095] As used herein, the term "adeno-associated virus" (AAV) includes, but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, and any other now known or later discovered AAV. See, e.g., BERNARD N. FIELDS et al., VIROLOGY, Vol. 2, Chapter 69 (4th Edition, Lippincott-Raven Publishers). Many additional AAV serotypes and clades have been identified (see, e.g., Gao et al., (2004)). J. Virology 78:6381-6388; Moris et al., (2004) Virology 33-:375-383; and Table 3).

[0096] The genomic sequences of various AAV serotypes and autonomous parvoviruses, as well as the sequences of the native terminal repeats (TRs), Rep proteins, and capsid subunits are known in the art. Such sequences can be found in the literature or in public databases such as GenBank. See, for example, GenBank Accession Nos. NC_002077, NC_001401, NC_001729, NC_001863, NC_001829, NC_001862, NC_000883, NC_001701, NC_001510, NC_006152, NC_006261, AF063497, U89790, AF043303, AF0287 05, AF028704, J02275, J01901, J02275, X01457, AF288061, AH009962, AY028226, AY028223, NC_001358, NC_001540, AF513851, AF513852, AY530579; the disclosures of which are incorporated herein by reference for teaching parvovirus and AAV nucleic acid and amino acid sequences. See also, e.g., Srivistava et al. (1983) J. Virology 45:555; Chiorini et al. (1998) J. Virology 71:6823; Chiorini et al., (1999) J. Virology 73:1309; Bantel-Schaal et al., (1999) J. Virology 73:939; Xiao et al., (1999) J. Virology73:3994; Muramatsu et al., (1996) Virology 221:208; Shade et al., (1986) J. Virol. 58:921; Gao et al., (2002) Proc. Nat. Acad. Sci. USA 99:11854; Moris et al., (2004) Virology 33-:375-383; International Patent Publications WO 00 / 28061, WO 99 / 61601, WO 98 / 11244; and U.S. Patent No. 6,156,303; the disclosures of which are incorporated herein by reference for teaching parvovirus and AAV nucleic acid and amino acid sequences. See also Table 3.

[0097] The capsid structures of the autonomous parvoviruses and AAV are described in more detail in BERNARD N. FIELDS et al., VIROLOGY, Vol. 2, Chapters 69 & 70 (4th ed., Lippincott-Raven Publishers). See also, AAV2 (Xie et al., (2002) Proc. Nat. Acad. Sci. 99:10405-10), AAV4 (Padron et al., (2005) J. Virol. 79: 5047-58), AAV5 (Walters et al., (2004) J. Virol. 78: 3361-71) and CPV (Xie et al., (1996) J. Mol. Biol. 6:497-520 and Tsao et al., (1991) Science 251: 1456-64).

[0098] As used herein, the term "tropism" refers to the preferential entry of a virus into certain cells or tissues, optionally followed by expression (e.g., transcription and optionally translation) in the cell of one or more sequences carried by the viral genome, e.g., in the case of a recombinant virus, expression of one or more heterologous nucleic acids of interest.

[0099] Unless otherwise indicated, "efficient transduction" or "efficient tropism" or similar terms can be determined by reference to an appropriate control (e.g., at least about 50%, 60%, 70%, 80%, 85%, 90%, 95%, 100%, 125%, 150%, 175%, 200%, 250%, 300%, 350%, 400%, 500% or more of the transduction or tropism, respectively, of the control). In specific embodiments, the viral vector efficiently transduces or has an effective tropism for neuronal cells and cardiomyocytes. Appropriate controls will depend on a variety of factors, including the desired tropism and / or transduction profile.

[0100] Similarly, whether a virus "does not efficiently transduce" or "does not have an effective tropism" or similar terms for a target tissue can be determined by reference to an appropriate control. In specific embodiments, the viral vector does not efficiently transduce (i.e., does not have an effective tropism) for liver, kidney, gonads, and / or germ cells. In specific embodiments, the transduction (e.g., undesired transduction) of one or more tissues (e.g., liver) is 20% or less, 10% or less, 5% or less, 1% or less, 0.1% or less of the level of transduction of one or more desired target tissues (e.g., skeletal muscle, diaphragm muscle, myocardium, and / or cells of the central nervous system).

[0101] As used herein, unless otherwise indicated, the term "polypeptide" includes peptides and proteins.

[0102] A "polynucleotide" is a sequence of nucleotide bases, and may be RNA, DNA, or a DNA-RNA hybrid sequence (including both naturally occurring and non-naturally occurring nucleotides), but in representative embodiments is a single-stranded or double-stranded DNA sequence.

[0103] As used herein, an "isolated" polynucleotide (e.g., "isolated DNA" or "isolated RNA") means a polynucleotide that is at least partially separated from at least some other components of a naturally occurring organism or virus (e.g., cellular or viral structural components or other polypeptides or nucleic acids with which the polynucleotide is normally found in association). In representative embodiments, the "isolated" nucleotide is enriched by at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more compared to the starting material.

[0104] Likewise, an "isolated" polypeptide means a polypeptide that is at least partially separated from at least some other components of a naturally occurring organism or virus (e.g., cellular or viral structural components or other polypeptides or nucleic acids with which the polypeptide is normally found in association). In representative embodiments, an "isolated" polypeptide is enriched at least about 10-fold, 100-fold, 1000-fold, 10,000-fold, or more, compared to the starting material.

[0105] "Isolated cells" refers to cells that are separated from other components with which they are normally associated in their natural state. For example, isolated cells can be cells in culture medium and / or cells in a pharmaceutically acceptable carrier of the invention. Thus, isolated cells can be delivered to and / or introduced into a subject. In some embodiments, isolated cells can be cells that have been removed from a subject and manipulated ex vivo as described herein and then returned to the subject.

[0106] As used herein, by "isolated" or "purified" (or grammatical equivalents) viral vectors or viral particles or viral particle populations is meant that the viral vectors or viral particles or viral particle populations are at least partially separated from at least some other components in the starting material. In representative embodiments, the "isolated" or "purified" viral vectors or viral particles or viral particle populations are enriched at least about 10-fold, 100-fold, 1000-fold, 10,000-fold or more compared to the starting material.

[0107] A "therapeutic polypeptide" is a polypeptide that can alleviate, reduce, prevent, delay and / or stabilize symptoms caused by the absence or defect of a protein in a cell or subject, and / or a polypeptide that otherwise provides benefit to a subject, for example, anti-cancer effects or improvement of transplant survivability or induction of an immune response.

[0108] By the terms "treat," "treating," or "treatment of" (and grammatical variations thereof) is meant that the severity of the subject's condition is reduced, at least partially improved, or stabilized and / or some alleviation, alleviation, reduction, or stabilization of at least one clinical symptom is achieved and / or there is a delay in the progression of the disease or disorder.

[0109] The terms "prevent", "preventing" and "prevention" (and grammatical variations thereof) refer to the prevention and / or delay of the onset of a disease, disorder and / or one or more clinical symptoms in a subject and / or a reduction in the severity of the onset of the disease, disorder and / or one or more clinical symptoms relative to what would occur in the absence of the methods of the present invention. Prevention can be complete, e.g., the complete absence of a disease, disorder and / or one or more clinical symptoms. Prevention can also be partial, such that the occurrence and / or severity of the onset of a disease, disorder and / or one or more clinical symptoms in a subject is significantly less than what would occur in the absence of the present invention.

[0110] As used herein, a "therapeutically effective" amount is an amount sufficient to provide some improvement or benefit to a subject. In other words, a "therapeutically effective" amount is an amount that will provide some relief, alleviation, reduction, or stabilization of at least one clinical symptom in a subject. One skilled in the art will appreciate that the therapeutic effect need not be complete or curative, as long as some benefit is provided to the subject.

[0111] As used herein, a "prophylactically effective" amount is an amount sufficient to prevent and / or delay the onset of a disease, disorder, and / or clinical symptom in a subject and / or reduce and / or delay the severity of the onset of a disease, disorder, and / or clinical symptom in a subject relative to what would occur without the methods of the present invention. Those skilled in the art will appreciate that the level of prevention need not be complete, as long as some preventive benefit is provided to the subject.

[0112] The terms "nucleotide sequence of interest (NOI)", "heterologous nucleotide sequence" and "heterologous nucleic acid molecule" are used interchangeably herein and refer to a nucleic acid sequence that does not naturally occur in a virus. Typically, the NOI, heterologous nucleic acid molecule or heterologous nucleotide sequence comprises an open reading frame that encodes a polypeptide of interest and / or a non-translated RNA (e.g., for delivery to a cell and / or a subject).

[0113] As used herein, the terms "viral vector," "vector," or "gene delivery vector" refer to viral (e.g., AAV) particles that function as nucleic acid delivery vehicles and contain a vector genome (e.g., viral DNA [vDNA]) packaged in a virion. Alternatively, in some cases, the term "vector" may be used to refer only to the vector genome / vDNA.

[0114] A "recombinant nucleotide sequence," "recombinant nucleic acid molecule," "rAAV vector genome," or "rAAV genome" is an AAV genome (ie, vDNA) that comprises one or more heterologous nucleic acid sequences.

[0115] The term "terminal repeat" or "TR" or "inverted terminal repeat (ITR)" includes any viral terminal repeat or synthetic sequence that forms a hairpin structure and functions as an inverted terminal repeat (i.e., mediates a desired function, such as replication, viral packaging, integration, and / or provirus rescue, etc.). The TR may be an AAV TR or a non-AAV TR. For example, non-AAV TR sequences, such as those of other parvoviruses (e.g., canine parvovirus (CPV), parvovirus of mice (MVM), human parvovirus B-19), or any other suitable viral sequence (e.g., an SV40 hairpin that serves as an SV40 origin of replication), may be used as a TR, which may be further modified by truncation, substitution, deletion, insertion, and / or addition. Further, the TR may be partially or completely synthetic, such as the "double-D sequence" described in U.S. Pat. No. 5,478,745 to Samulski et al.

[0116] "AAV terminal repeats" or "AAV TRs" can be from any AAV, including but not limited to serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 or any other now known or later discovered AAV (see, e.g., Table 3). The AAV terminal repeats need not have native terminal repeats (e.g., the native AAV TR sequence can be altered by insertions, deletions, truncations and / or missense mutations), as long as the terminal repeats mediate the desired function, e.g., replication, viral packaging, integration and / or proviral rescue, etc.

[0117] AAV proteins VP1, VP2 and VP3 are capsid proteins that interact together to form the AAV capsid of icosahedral symmetry. VP1.5 is an AAV capsid protein described in U.S. Publication No. 2014 / 0037585.

[0118] For example, International Patent Publication WO 00 / 28004 and Chao et al., (2000) Molecular Therapy 2:619, the viral vectors of the present invention can further be "targeted" viral vectors (e.g., having a targeted tropism) and / or "hybrid" parvoviruses (i.e., wherein the viral TRs and viral capsids are from different parvoviruses).

[0119] As described in International Patent Publication WO 01 / 92551 (the disclosure of which is incorporated herein by reference in its entirety), the viral vector of the present invention may further be a duplex parvoviral particle. Thus, in some embodiments, the double-stranded (duplex) genome may be packaged into the viral capsid of the invention.

[0120] Furthermore, the viral capsid or genomic elements may contain other modifications, including insertions, deletions and / or substitutions.

[0121] As used herein, a "chimeric" capsid protein means an AAV capsid protein that has been modified by substitution in one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence of the capsid protein relative to the wild type, and insertion and / or deletion of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, etc.) amino acid residues in the amino acid sequence relative to the wild type. In some embodiments, a complete or partial domain, functional region, epitope, etc. from one AAV serotype can be substituted in any combination for the corresponding wild-type domain, functional region, epitope, etc. of a different AAV serotype to produce a chimeric capsid protein of the present invention. The production of chimeric capsid proteins can be carried out according to procedures well known in the art, and a large number of chimeric capsid proteins are described in the literature and herein, which can be included in the capsid of the present invention.

[0122] As used herein, the term "amino acid" or "amino acid residue" includes any naturally occurring amino acid, modified forms thereof and synthetic amino acids.

[0123] Naturally occurring L-amino acids are shown in Table 4.

[0124] Alternatively, the amino acid may be a modified amino acid residue (non-limiting examples are shown in Table 6) and / or may be an amino acid modified by a post-translational modification (e.g., acetylation, amidation, formylation, hydroxylation, methylation, phosphorylation or sulfatation).

[0125] Further, non-naturally occurring amino acids may be as described by Wang et al., Annu Rev Biophys Biomol Struct. 35:225-49 (2006). These unnatural amino acids can be advantageously used to chemically link the target molecule to the AAV capsid protein.

[0126] In some embodiments, the AAV vector of the invention can be a synthetic viral vector designed to exhibit a range of desired phenotypes suitable for different in vitro and in vivo applications. Thus, in one embodiment, the invention provides an AAV particle comprising an adeno-associated virus (AAV) capsid, wherein the capsid comprises capsid protein VP1 in any combination (wherein the capsid protein VP1 is from one or more than one first AAV serotype) and capsid protein VP3, wherein the capsid protein VP3 is from one or more than one second AAV serotype, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes.

[0127] In some embodiments, the AAV particle may comprise a capsid comprising capsid protein VP2 in any combination, wherein the capsid protein VP2 is from one or more than one third AAV serotype, wherein at least one of the one or more than one third AAV serotypes is different from the first AAV serotype and / or the second AAV serotype. In some embodiments, the AAV capsid described herein may comprise capsid protein VP1.5. VP1.5 is described in U.S. Patent Publication No. 20140037585, and the amino acid sequence of VP1.5 is provided herein.

[0128] In some embodiments, the AAV particles of the present invention may comprise a capsid comprising capsid protein VP1.5 in any combination, wherein the capsid protein VP1.5 is from one or more than one fourth AAV serotype, wherein at least one of the one or more than one fourth AAV serotype is different from the first AAV serotype and / or the second AAV serotype. In some embodiments, the AAV capsid protein described herein may comprise capsid protein VP2.

[0129] The present invention also provides an AAV vector of the present invention, which comprises an AAV capsid, wherein the capsid comprises a capsid protein VP1 (wherein the capsid protein VP1 is from one or more than one first AAV serotype) and a capsid protein VP2 in any combination, wherein the capsid protein VP2 is from one or more than one second AAV serotype, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes.

[0130] In some embodiments, the AAV vector of the present invention may comprise a capsid comprising capsid protein VP3 in any combination, wherein the capsid protein VP3 is from one or more than one third AAV serotype, wherein at least one of the one or more than one third AAV serotype is different from the first AAV serotype and / or the second AAV serotype. In some embodiments, the AAV capsid described herein may comprise capsid protein VP1.5.

[0131] The present invention further provides an AAV vector comprising an adeno-associated virus (AAV) capsid, wherein the capsid comprises capsid protein VP1 (wherein the capsid protein VP1 is from one or more than one first AAV serotype) and capsid protein VP1.5 in any combination, wherein the capsid protein VP1.5 is from one or more than one second AAV serotype, and wherein at least one of the first AAV serotypes is different from at least one of the second AAV serotypes.

[0132] In some embodiments, the AAV vector of the present invention may comprise a capsid comprising capsid protein VP3 in any combination, wherein the capsid protein VP3 is from one or more than one third AAV serotype, wherein at least one of the one or more than one third AAV serotypes is different from the first AAV serotype and / or the second AAV serotype. In some embodiments, the AAV capsid protein described herein may comprise capsid protein VP2.

[0133] In some embodiments of the capsid of the AAV vector described herein, the one or more than one first AAV serotype, the one or more than one second AAV serotype, the one or more than one third AAV serotype, and the one or more than one fourth AAV serotype are selected from the AAV serotypes listed in Table 3 in any combination.

[0134] In some embodiments of the AAV vectors of the invention, the AAV capsid described herein lacks the capsid protein VP2.

[0135] In some embodiments of the AAV vector of the present invention, the capsid may comprise a chimeric capsid VP1 protein, a chimeric capsid VP2 protein, a chimeric capsid VP3 protein and / or a chimeric capsid VP1.5 protein.

[0136] The present invention further provides a composition, which may be a pharmaceutical preparation comprising the viral vector or AAV particle of the present invention and a pharmaceutically acceptable carrier.

[0137] In embodiments of the invention, cell transduction by the AAV particles of the invention is at least about 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold or more the level of transduction by the AAV particles described herein that induce a dsRNA-mediated immune response.

[0138] Heterologous molecules (e.g., nucleic acids, proteins, peptides, etc.) are defined as those not naturally found in AAV infection, e.g., those not encoded by the wild-type AAV genome. Further, therapeutically useful molecules can be associated with transgenes for transfer of the molecules into host target cells. Such associated molecules can include DNA and / or RNA.

[0139] The modified capsid protein and capsid may further comprise any other modification currently known or identified later. It will be appreciated by those skilled in the art that for some AAV capsid proteins, the corresponding modification will be insertion and / or substitution, depending on whether the corresponding amino acid position is partially or completely present in the virus, or is completely absent. Similarly, when modifying an AAV other than AAV2, one or more specific amino acid positions may be different from the position in AAV2 (see, e.g., Table 5). As discussed elsewhere herein, one or more corresponding amino acid positions will be apparent to those skilled in the art using well-known techniques. Non-limiting examples of corresponding positions in many other AAVs are shown in Table 5 (Position 2).

[0140] In a representative embodiment, the viral vector of the present invention is a recombinant viral vector comprising a heterologous nucleic acid encoding a polypeptide of interest and / or a functional RNA. The recombinant viral vector is described in more detail below.

[0141] Those skilled in the art will appreciate that in certain embodiments, the capsid proteins, viral capsids, viral vectors and AAV particles of the invention exclude those capsid proteins, capsids, viral vectors and AAV particles as they would exist or be found in their natural state.

[0142] Methods for producing viral vectors

[0143] The present invention further provides methods for producing the AAV particles and vectors of the present invention. Therefore, the present invention provides methods for preparing AAV particles, which include: a) transfecting host cells with one or more plasmids, which together provide all the functions and genes required for assembling AAV particles; b) introducing one or more nucleic acid constructs into a packaging cell line or a production cell line to together provide all the functions and genes required for assembling AAV particles; c) introducing one or more recombinant baculovirus vectors into host cells, which together provide all the functions and genes required for assembling AAV particles; and / or d) introducing one or more recombinant herpes virus vectors into host cells, which together provide all the functions and genes required for assembling AAV particles. Non-limiting examples of various methods for preparing viral vectors of the present invention are described in Clement and Greiger ("Manufacturing of recombinant adeno-associated viral vectors for clinical trials" Mol. Ther. Methods Clin Dev.3:16002 (2016)) and Greiger et al. (“Production of recombinant adeno-associated virus vectors using suspensionHEK293 cells and continuous harvest of vector from the culture media for GMPFIX and FLT1 clinical vector” Mol Ther 24(2):287-297 (2016)), the entire contents of which are incorporated herein by reference.

[0144] In an exemplary embodiment, the invention provides a method for producing AAV particles, the method comprising providing to a cell: (a) a nucleic acid template comprising at least one TR sequence (e.g., an AAV TR sequence), and (b) an AAV sequence sufficient for replication and encapsidation of the nucleic acid template into an AAV capsid (e.g., an AAV rep AAV sequences and AAV encoding the AAV capsid of the invention cap Sequence). Optionally, the nucleic acid template further comprises at least one heterologous nucleic acid sequence. In a specific embodiment, the nucleic acid template comprises two AAV ITR sequences, which are located 5' and 3' to the heterologous nucleic acid sequence (if present), although they need not be directly adjacent thereto.

[0145] Providing the nucleic acid template and the AAV under conditions such that a viral vector comprising the nucleic acid template packaged within an AAV capsid is produced in the cell rep and cap Sequence. The method may further include the step of collecting the viral vector from the cells. The viral vector may be collected from the culture medium and / or by lysing the cells.

[0146] The cell may be a cell that is permissive for AAV virus replication. Any suitable cell known in the art may be used. In a specific embodiment, the cell is a mammalian cell. Alternatively, the cell may be a trans-complementing packaging cell line that provides the function missing from the replication-defective helper virus, for example, a 293 cell or other E1a trans-complementing cell.

[0147] AAV replication and capsid sequences can be provided by any method known in the art. Current protocols generally express AAV on a single plasmid. rep / cap Genes. AAV replication and packaging sequences need not be provided together, although it may be convenient to do so. rep and / or cap The sequences can be provided by any viral or non-viral vector. For example, rep / cap Sequences can be provided by hybrid adenoviral or herpesvirus vectors (e.g., indels in the E1a or E3 regions of an adenoviral vector). Epstein-Barr virus (EBV) vectors can also be used to express AAV cap and rep One advantage of this approach is that the EBV vector is episomal, but will remain in high copy number throughout successive cell divisions (i.e., stably integrated into the cell as an extrachromosomal element, termed an "EBV-based nuclear episome", see Margolski, (1992) Curr. Top. Microbiol. Immun. 158:67).

[0148] As a further alternative, rep / cap The sequence is stably incorporated into the cell.

[0149] Generally, AAV rep / cap The sequences will not be flanked by TRs to prevent rescue and / or packaging of these sequences.

[0150] The nucleic acid template can be provided to the cell using any method known in the art. For example, the template can be provided by a non-viral (e.g., plasmid) or viral vector. In a specific embodiment, the nucleic acid template is provided by a herpes virus or adenovirus vector (e.g., in the E1a or E3 region of an indel adenovirus). As another example, Palombo et al., J. Virology 72:5025 (1998) describes a baculovirus vector carrying a reporter gene flanked by AAV TRs. EBV vectors can also be used to deliver templates, as described above with respect to rep / cap Described by genes.

[0151] In another representative embodiment, the nucleic acid template is provided by a replicating rAAV virus. In yet other embodiments, the AAV provirus comprising the nucleic acid template is stably integrated into the chromosome of the cell.

[0152] To increase viral titers, helper virus functions (e.g., adenovirus or herpes virus) that promote productive AAV infection can be provided to the cells. Helper virus sequences necessary for AAV replication are known in the art. Typically, these sequences will be provided by a helper adenovirus or herpes virus vector. Alternatively, the adenovirus or herpes virus sequences can be provided by another non-viral or viral vector, for example, as a non-infectious adenovirus miniplasmid that carries all the helper genes that promote efficient AAV production, as described by Ferrari et al., (1997) Nature Med. 3:1295, and as described in U.S. Patent Nos. 6,040,183 and 6,093,570.

[0153] Further, helper virus functions can be provided by packaging cells that have helper sequences embedded in the chromosome or maintained as stable extrachromosomal elements. Typically, helper virus sequences cannot be packaged into AAV virions, for example, are not flanked by TRs.

[0154] Those skilled in the art will appreciate that it may be advantageous to provide AAV replication and capsid sequences and helper virus sequences (e.g., adenovirus sequences) on a single helper construct. The helper construct may be a non-viral or viral construct. As a non-limiting illustration, the helper construct may be a construct comprising AAV rep / cap Genetic hybrid adenovirus or hybrid herpes virus.

[0155] In one embodiment, AAV rep / cap The sequences and adenoviral helper sequences are supplied by a single adenoviral helper vector. The vector may further comprise a nucleic acid template. rep / cap The sequences and / or rAAV template can be inserted into a deleted region of the adenovirus (eg, the E1a or E3 region).

[0156] In a further embodiment, the AAV rep / cap The sequences and adenoviral helper sequences are supplied by a single adenoviral helper vector. According to the described embodiment, the rAAV template may be provided as a plasmid template.

[0157] In another illustrative embodiment, AAV rep / cap The sequences and adenoviral helper sequences are provided by a single adenoviral helper vector, and the rAAV template is integrated into the cell as a provirus. Alternatively, the rAAV template is provided by an EBV vector that is maintained in the cell as an extrachromosomal element (e.g., as an EBV-based nuclear episome).

[0158] In a further exemplary embodiment, AAV rep / cap The sequences and adenoviral helper sequences are provided by a single adenoviral helper virus. The rAAV template can be provided as a separate replicative viral vector. For example, the rAAV template can be provided by rAAV particles or a second recombinant adenoviral particle.

[0159] According to the aforementioned methods, the hybrid adenoviral vector generally contains adenoviral 5' and 3' cis sequences (i.e., adenoviral terminal repeats and PAC sequences) sufficient for adenoviral replication and packaging. rep / cap The sequences and, if present, the rAAV template are embedded in the adenoviral backbone and flanked by 5' and 3' cis sequences so that these sequences can be packaged into the adenoviral capsid. As described above, the adenoviral helper sequences and AAV rep / capThe sequences are generally not flanked by TRs so that these sequences are not packaged into AAV virions.

[0160] Zhang et al. (2001) Gene Ther. 18:704-12) describes adenovirus and AAV rep and cap A chimeric helper virus containing both genes.

[0161] Herpes viruses can also be used as helper viruses in AAV packaging methods. Hybrid herpes viruses encoding one or more AAV Rep proteins can advantageously facilitate scalable AAV vector production protocols. rep and cap Hybrid herpes simplex virus type 1 (HSV-1) vectors expressing the gene have been described (Conway et al. (1999) Gene Therapy 6:986 and WO 00 / 17377.

[0162] As a further alternative, baculovirus vectors can be used to deliver rep / cap Genes and rAAV templates are used to produce the viral vectors of the invention in insect cells, as described, for example, by Urabe et al. (2002) Human Gene Therapy 13:1935-43.

[0163] AAV vector stocks free of contaminating helper viruses can be obtained by any method known in the art. For example, AAV and helper viruses can be easily distinguished based on size. AAV can also be separated from helper viruses based on affinity for a heparin substrate (Zolotukhin et al. (1999) Gene Therapy 6:973). A deleted, replication-defective helper virus can be used so that any contaminating helper virus is not replication-competent. As a further alternative, an adenoviral helper virus lacking late gene expression can be used, since only adenoviral early gene expression is required to mediate AAV virus packaging. Adenoviral mutants defective for late gene expression are known in the art (e.g., ts100K and ts149 adenoviral mutants).

[0164] Recombinant viral vector

[0165] The invention provides methods for administering a nucleic acid molecule to a cell, the method comprising contacting the cell with a viral vector, AAV particle, composition and / or pharmaceutical preparation of the invention.

[0166] The invention further provides a method of delivering a nucleic acid to a subject, the method comprising administering to the subject a viral vector, AAV particle, composition and / or pharmaceutical formulation of the invention.

[0167] The subject of the present invention can be any animal, and in some embodiments, the subject is a mammal, and in some embodiments, the subject is a human. In some embodiments, the subject suffers from or is at risk of a disorder that can be treated by an immunotherapy and / or gene therapy procedure. Non-limiting examples of such disorders include muscular dystrophy, including Duchenne or Becker muscular dystrophy, hemophilia A, hemophilia B, multiple sclerosis, diabetes, Gaucher's disease, Fabry's disease, Pompe disease, cancer, arthritis, muscle wasting, heart disease, including congestive heart failure or peripheral arterial disease, intimal hyperplasia, neurological disorders, including epilepsy, Huntington's disease, Parkinson's disease or Alzheimer's disease, autoimmune diseases, cystic fibrosis, thalassemia, Hurler's syndrome, Seeley syndrome, Shea's syndrome, Hurler-Scheie syndrome, Hunter's syndrome, Sanfilep syndrome A, B, C, D, Morquio syndrome, Marshall-Lan syndrome, Krabbe's disease, phenylketonuria, Batten's disease, spinocerebral ataxia, LDL receptor deficiency, hyperammonemia, anemia, arthritis, retinal degenerative diseases, including macular degeneration, adenosine deaminase deficiency, metabolic disorders, and cancer, including tumor-forming cancer.

[0168] In the methods described herein, the viral vectors, AAV particles and / or compositions or pharmaceutical preparations of the invention can be administered / delivered to the subject of the invention by systemic routes (e.g., intravenous, intraarterial, intraperitoneal, etc.) and / or local direct injection (e.g., intramuscular injection, direct brain injection, injection into CSF, injection into the eye, etc.). In some embodiments, the viral vectors and / or compositions can be administered to the subject by intraventricular, intracisternal, intraparenchymal, intracranial and / or intrathecal routes.

[0169] The viral vectors of the present invention are used to deliver nucleic acid molecules to cells in vitro, ex vivo and in vivo. In particular, viral vectors can be advantageously used to deliver or transfer nucleic acid molecules to animal cells, including mammalian cells.

[0170] Any one or more heterologous nucleic acid sequences of interest can be delivered in the viral vectors of the present invention. Target nucleic acid molecules include nucleic acid molecules encoding polypeptides, including therapeutic (e.g., for medical or veterinary use) and / or immunogenic (e.g., for vaccines) polypeptides.

[0171] Therapeutic polypeptides include, but are not limited to, cystic fibrosis transmembrane regulator (CFTR), dystrophin (including mini- and micro-dystrophin, see, e.g., Vincent et al. (1993) Nature Genetics 5:130; U.S. Patent Publication No. 2003 / 017131; International Publication WO / 2008 / 088895; Wang et al. Proc. Natl. Acad. Sci. USA 97:13714-13719 (2000); and Gregorevic et al. Mol. Ther. 16:657-64 (2008)), myostatin propeptide, follistatin, soluble type II activator protein receptor, IGF-1, anti-inflammatory peptides such as dominant mutant Ikappa B, sarcospan, utrophin (Tinsley et al. (1996) Nature 384:349), mini-utrophin, coagulation factors (e.g., coagulation factor VIII, coagulation factor IX, coagulation factor X, etc.), erythropoietin, angiostatin, endostatin, catalase, tyrosine hydroxylase, superoxide dismutase, leptin, LDL receptor, lipoprotein lipase, ornithine transcarbamylase, β-globin, α-globin, spectrin, α1-antitrypsin, adenosine deaminase, hypoxanthine guanine phosphoribosyltransferase, β-glucocerebrosidase, sphingomyelinase, lysosomal hexosaminidase A, branched-chain ketoacid dehydrogenase, RP65 protein, cytokines (e.g., interferon α, interferon β, interferon-γ, interleukin-2, interleukin-4, granulocyte macrophage colony-stimulating factor, lymphotoxin, etc.), peptide growth factors, neurotrophins and hormones (e.g., somatotropin, insulin, insulin-like growth factor 1 and 2, platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, neurotrophin-3 and -4, brain-derived neurotrophic factor, bone morphogenetic proteins [including RANKL and VEGF], glial-derived growth factor, transforming growth factor-α and -β, etc.), lysosomal acid α-glucosidase, α-galactosidase A, receptors (e.g., tumor necrosis growth factor α soluble receptor), S100A1, parvalbumin, adenylate cyclase type 6, molecules that regulate calcium handling (e.g., SERCA 2A, inhibitors of PP1 1 and fragments thereof [e.g., WO 2006 / 029319 and WO 2007 / 100465]), molecules that affect knockdown of G protein-coupled receptor kinase type 2, such as truncated constitutively active bARKct, anti-inflammatory factors such as IRAP, anti-myostatin proteins, aspartate acyltransferase (aspartoacylase), monoclonal antibodies (including single-chain monoclonal antibodies; an exemplary Mab is Herceptin ® Mab), neuropeptides and fragments thereof (e.g., galanin, neuropeptide Y (see, US 7,071,172), angiogenesis inhibitors such as Vasohibins and other VEGF inhibitors (e.g., Vasohibins 2 [see WO JP 2006 / 073052]). Other illustrative heterologous nucleic acid sequences encode "suicide" gene products (e.g., thymidine kinase, cytosine deaminase, diphtheria toxin and tumor necrosis factor), proteins that confer resistance to drugs used in cancer treatment, tumor suppressor gene products (e.g., p53, Rb, Wt-1), TRAIL, FAS-ligand and any other polypeptide that has a therapeutic effect in a subject in need thereof. AAV vectors can also be used to deliver monoclonal antibodies and antibody fragments, for example, antibodies or antibody fragments against myostatin (see, e.g., Fang et al. Nature Biotechnology 23:584-590(2005)).

[0172] Heterologous nucleic acid sequences encoding polypeptides include those encoding reporter polypeptides (e.g., enzymes). Reporter polypeptides are known in the art and include, but are not limited to, green fluorescent protein (GFP), luciferase, β-galactosidase, alkaline phosphatase, luciferase, and chloramphenicol acetyltransferase genes.

[0173] Optionally, the heterologous nucleic acid molecule encodes a secreted polypeptide (eg, a polypeptide that is secreted in its native state or has been artificially engineered to be secreted, eg, by operably associating with a secretory signal sequence as known in the art).

[0174] Alternatively, in certain embodiments of the invention, the heterologous nucleic acid molecule may encode an antisense nucleic acid molecule, a ribozyme (e.g., as described in U.S. Pat. No. 5,877,022), RNAs that affect spliceosome-mediated trans-splicing (see, Puttaraju et al. (1999) Nature Biotech. 17:246; U.S. Pat. No. 6,013,487; U.S. Pat. No. 6,083,702), interfering RNAs (RNAi), including siRNA, shRNA or miRNA that mediate gene silencing (see, Sharp et al. (2000) Science287:2431) and other non-translated RNAs, such as "guide" RNAs (Gorman et al. (1998) Proc. Nat. Acad. Sci. USA 95:4929; U.S. Pat. No. 5,869,248 to Yuan et al.), etc. Exemplary non-translated RNAs include RNAi against multidrug resistance (MDR) gene products (e.g., to treat and / or prevent tumors and / or to be administered to the heart to prevent damage from chemotherapy), RNAi against myostatin (e.g., for Duchenne muscular dystrophy), RNAi against VEGF (e.g., to treat and / or prevent tumors), RNAi against phospholamban (e.g., to treat cardiovascular disease, see, e.g., Andino et al. J. Gene Med. 10:132-142 (2008) and Li et al. Acta Pharmacol Sin. 26:51-55 (2005)); phospholamban inhibitory or dominant negative molecules, such as phospholamban S16E (e.g., to treat cardiovascular disease, see, e.g., Hoshijima et al. Nat. Med. 8:864-871 (2002)), RNAi to adenosine kinase (e.g., for epilepsy), and RNAi against pathogenic organisms and viruses (e.g., hepatitis B and / or C virus, human immunodeficiency virus, CMV, herpes simplex virus, human papillomavirus, etc.).

[0175] Further, nucleic acid sequences that direct alternative splicing can be delivered. To illustrate, antisense sequences (or other inhibitory sequences) complementary to the 5' and / or 3' splice sites of dystrophin exon 51 can be delivered with the U1 or U7 nuclear small (sn) RNA promoter to induce skipping of this exon. For example, a DNA sequence comprising a U1 or U7 snRNA promoter located 5' to one or more antisense / inhibitory sequences can be packaged and delivered in a modified capsid of the invention.

[0176] The viral vector may also comprise a heterologous nucleic acid molecule that shares homology with a locus on a host cell chromosome and recombines therewith. For example, the method may be used to correct a genetic defect in a host cell.

[0177] The present invention also provides viral vectors expressing immunogenic polypeptides, peptides and / or epitopes, for example, for vaccination. The nucleic acid molecule can encode any desired immunogen known in the art, including, but not limited to, immunogens from human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), influenza virus, HIV or SIV gag protein, tumor antigens, cancer antigens, bacterial antigens, viral antigens, etc.

[0178] The use of parvovirus as a vaccine vector is known in the art (see, e.g., Miyamura et al. (1994) Proc. Nat. Acad. Sci USA 91:8507; U.S. Pat. No. 5,916,563 to Young et al., U.S. Pat. No. 5,905,040 to Mazzara et al., U.S. Pat. No. 5,882,65 to Samulski et al., U.S. Pat. No. 5,863,541 to Samulski et al.). The antigen may be present in the parvoviral capsid. Alternatively, the immunogen or antigen may be expressed by a heterologous nucleic acid molecule introduced into the genome of the recombinant vector. The viral vectors of the present invention may provide any immunogen or antigen of interest as described herein and / or as known in the art.

[0179] The immunogenic polypeptide can be any polypeptide, peptide and / or epitope suitable for eliciting an immune response and / or protecting a subject from infection and / or disease, including, but not limited to, microbial, bacterial, protozoan, parasitic, fungal and / or viral infection and disease. For example, the immunogenic polypeptide can be an orthomyxovirus immunogen (e.g., an influenza virus immunogen, such as an influenza virus hemagglutinin (HA) surface protein or an influenza virus nucleoprotein, or an equine influenza virus immunogen) or a lentivirus immunogen (e.g., an equine infectious anemia virus immunogen, a simian immunodeficiency virus (SIV) immunogen or a human immunodeficiency virus (HIV) immunogen, such as an HIV or SIV envelope GP160 protein, an HIV or SIV matrix / capsid protein, and an HIV or SIV gag , pol and env The immunogenic polypeptide can also be an arenavirus immunogen (e.g., a Lassa fever virus immunogen, such as a Lassa fever virus nucleocapsid protein and a Lassa fever envelope glycoprotein), a poxvirus immunogen (e.g., a vaccinia virus immunogen, such as a vaccinia L1 or L8 gene product), a flavivirus immunogen (e.g., a yellow fever virus immunogen or a Japanese encephalitis virus immunogen), a filovirus immunogen (e.g., an Ebola virus immunogen or a Marburg virus immunogen, such as NP and GP gene products), a bunyavirus immunogen (e.g., an RVFV, CCHF and / or SFS virus immunogen), or a coronavirus immunogen (e.g., an infectious human coronavirus immunogen, such as a human coronavirus envelope glycoprotein, or a porcine transmissible gastroenteritis virus immunogen, or an avian infectious bronchitis virus immunogen). The immunogenic polypeptide can further be a polio immunogen, a herpes immunogen (e.g., CMV, EBV, HSV immunogens), a mumps immunogen, a measles immunogen, a rubella immunogen, a diphtheria toxin or other diphtheria immunogen, a pertussis antigen, a hepatitis (e.g., hepatitis A, hepatitis B, hepatitis C, etc.) immunogen, and / or any other vaccine immunogen now known in the art or later identified as an immunogen.

[0180] Alternatively, the immunogenic polypeptide may be any tumor or cancer cell antigen. Optionally, the tumor or cancer antigen is expressed on the surface of a cancer cell. Exemplary cancer and tumor cell antigens are described in S. A. Rosenberg ( Immunity 10:281 (1991). Other illustrative cancer and tumor antigens include, but are not limited to: BRCA1 gene product, BRCA2 gene product, gp100, tyrosinase, GAGE-1 / 2, BAGE, RAGE, LAGE, NY-ESO-1, CDK-4, β-catenin, MUM-1, caspase-8, KIAA0205, HPVE, SART-1, PRAME, p15, melanoma tumor antigen (Kawakami et al. (1994) Proc. Natl. Acad. Sci. USA 91:3515; Kawakami et al. (1994) J. Exp. Med. , 180:347; Kawakami et al. (1994) Cancer Res. 54:3124), MART-1, gp100 MAGE-1, MAGE-2, MAGE-3, CEA, TRP-1, TRP-2, P-15, tyrosinase (Brichard et al. (1993) J. Exp. Med. 178:489); HER-2 / neu gene product (U.S. Pat. No. 4,968,603), CA 125, LK26, FB5 (endosialoprotein), TAG 72, AFP, CA19-9, NSE, DU-PAN-2, CA50, SPan-1, CA72-4, HCG, STN (sialyl Tn antigen), c-erbB-2 protein, PSA, L-CanAg, estrogen receptor, milk fat globulin, p53 tumor suppressor protein (Levine. (1993) Ann. Rev. Biochem . 62:623); mucin antigens (International Patent Publication No. WO 90 / 05142); telomerase; nuclear matrix protein; prostatic acid phosphatase; papillomavirus antigens; and / or antigens now known or later discovered to be associated with melanoma, adenocarcinoma, thymoma, lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer, and any other cancer or malignant condition now known or later identified (see, e.g., Rosenberg, (1996) Ann. Rev. Med . 47:481-91).

[0181] As a further alternative, the heterologous nucleic acid molecule can encode any polypeptide, peptide and / or epitope that is desired to be produced in cells in vitro, ex vivo or in vivo. For example, a viral vector can be introduced into cultured cells and the expressed gene product can be isolated therefrom.

[0182] Those skilled in the art will appreciate that one or more heterologous nucleic acid molecules of interest may be operably associated with appropriate control sequences. For example, the heterologous nucleic acid molecule may be operably associated with expression control elements, such as transcription / translation control signals, replication origins, polyadenylation signals, internal ribosome entry sites (IRES), promoters and / or enhancers, and the like.

[0183] Furthermore, regulatable expression of one or more heterologous nucleic acid molecules of interest can be achieved at the post-transcriptional level, for example, by regulating alternative splicing of different introns through the presence or absence of oligonucleotides, small molecules and / or other compounds that selectively block splicing activity at specific sites (e.g., as described in WO 2006 / 119137).

[0184] Those skilled in the art will appreciate that a variety of promoter / enhancer elements may be used, depending on the desired level and tissue-specific expression. Depending on the desired expression pattern, the promoter / enhancer may be constitutive or inducible. The promoter / enhancer may be native or exogenous, and may be a native or synthetic sequence. Exogenous is intended to mean that the transcription initiation region is not found in the wild-type host into which the transcription initiation region is introduced.

[0185] In a specific embodiment, the promoter / enhancer element may be native to the target cell or the subject to be treated. In a representative embodiment, the promoter / enhancer element may be native to the heterologous nucleic acid sequence. The promoter / enhancer element is usually selected so that it works in one or more target cells of interest. Further, in a specific embodiment, the promoter / enhancer element is a mammalian promoter / enhancer element. The promoter / enhancer element may be constitutive or inducible.

[0186] Inducible expression control elements are generally advantageous in those applications where it is desired to provide regulation of expression of one or more heterologous nucleic acid sequences. Inducible promoter / enhancer elements for gene delivery can be tissue-specific or preferred promoter / enhancer elements, and include muscle-specific or preferred (including heart, bone and / or smooth muscle-specific or preferred), neural tissue-specific or preferred (including brain-specific or preferred), eye-specific or preferred (including retina-specific and cornea-specific), liver-specific or preferred, bone marrow-specific or preferred, pancreas-specific or preferred, spleen-specific or preferred, and lung-specific or preferred promoter / enhancer elements. Other inducible promoter / enhancer elements include hormone-inducible and metal-inducible elements. Exemplary inducible promoter / enhancer elements include, but are not limited to, Tet on / off elements, RU486-inducible promoters, ecdysone-inducible promoters, rapamycin-inducible promoters, and metallothionein promoters.

[0187] In embodiments where one or more heterologous nucleic acid sequences are transcribed and then translated in the target cell, specific initiation signals are generally included for efficient translation of the inserted protein coding sequence. These exogenous translation control sequences, which may include the ATG initiation codon and adjacent sequences, may have a variety of origins, both natural and synthetic.

[0188] Viral vectors according to the present invention provide means for delivering heterologous nucleic acid molecules to numerous cells, including dividing cells and non-dividing cells. Viral vectors can be used for delivering target nucleic acid molecules to cells in vitro, for example, to produce polypeptides in vitro or for in vitro or in vivo gene therapy. The viral vectors are additionally used for delivering nucleic acids to a method for a subject in need thereof, for example, to express immunogenic or therapeutic polypeptides or functional RNA. In this way, polypeptides or functional RNA can be produced in vivo in a subject. The subject may need the polypeptide, because the subject lacks the polypeptide. Further, the method can be put into practice, because the production of polypeptides or functional RNA in a subject can confer some beneficial effects.

[0189] Viral vectors can also be used to produce a polypeptide or functional RNA of interest in cultured cells or in a subject (e.g., using the subject as a bioreactor to produce the polypeptide or observe the effect of the functional RNA on the subject, e.g., in conjunction with screening methods).

[0190] In general, the viral vectors of the invention can be used to deliver heterologous nucleic acid molecules encoding polypeptides or functional RNAs to treat and / or prevent any condition or disease state for which delivery of a therapeutic polypeptide or functional RNA is beneficial. Exemplary disease states include, but are not limited to: cystic fibrosis (cystic fibrosis transmembrane regulator) and other lung diseases, hemophilia A (coagulation factor VIII), hemophilia B (coagulation factor IX), thalassemia (β-globin), anemia (erythropoietin) and other blood disorders, Alzheimer's disease (GDF; neprilysin), multiple sclerosis (β-interferon), Parkinson's disease (glial cell line-derived neurotrophic factor [GDNF]), Huntington's disease (repeat-removing RNAi), amyotrophic lateral sclerosis, epilepsy (galanin, neurotrophic factors) and other neurological disorders, cancer (endostatin, angiostatin, TRAIL, FAS-ligand, cytokines including interferons; RNAi including RNAi to VEGF or multidrug resistance gene products, mir-26a [e.g., for hepatocellular carcinoma]), diabetes (insulin), muscular dystrophy including Duchenne (dystrophin, mini-dystrophin, insulin-like growth factor I, sarcoglycan [e.g., α, β, γ], RNAi against myostatin, myostatin propeptide, follistatin, type II activin soluble receptor, anti-inflammatory polypeptides such as dominant mutants of Ikappa B, sarcospan, dystrophin, mini-dystrophin, antisense or RNAi against splice junctions in the dystrophin gene to induce exon skipping [see, e.g., WO / 2003 / 095647], against U7 antisense to snRNAs to induce exon skipping [see, e.g., WO / 2006 / 021724], and antibodies or antibody fragments to myostatin or myostatin propeptide) and Becker, Gaucher disease (glucocerebrosidase), Hurler's disease (α-L-iduronidase), adenosine deaminase deficiency (adenosine deaminase), glycogen storage diseases (e.g., Fabry disease [α-galactosidase] and Pompe disease [lysosomal acid α-glucosidase]) and other metabolic disorders, congenital emphysema (α1-antitrypsin), Levy-Nez-Zah syndrome (hypoxanthine guanine phosphoribosyltransferase), Nieto-Pen disease (sphingomyelinase), Tay-Sachs disease (lysosomal hexosaminidase A), maple syrup urine disease (branched-chain ketoacid dehydrogenase), retinal degeneration (and other eye and retinal diseases);For example, PDGF and / or vasohibin or other inhibitors of VEGF or other angiogenesis inhibitors for macular degeneration, to treat / prevent retinal disorders, e.g., in type I diabetes), solid organ diseases such as brain (including Parkinson's disease [GDNF], astrocytoma [endostatin, angiostatin and / or RNAi to VEGF], glioblastoma [endostatin, angiostatin and / or RNAi to VEGF]), liver, kidney, heart, including congestive heart failure or peripheral arterial disease (PAD) (e.g., by delivering protein phosphatase inhibitor I (I-1) and its fragments (e.g., I1C), serca2a, zinc finger proteins that regulate phospholamban genes, Barkct, beta 2-adrenergic receptor, beta2-adrenergic receptor kinase (BARK), phosphoinositide-3 kinase (PI3 kinase), S100A1, parvalbumin, adenylate cyclase type 6, molecules that affect knockdown of G protein-coupled receptor kinase type 2, such as truncated constitutively active bARKct; calsarcin, RNAi against phospholamban; phospholamban inhibitory or dominant negative molecules, such as phospholamban S16E, etc.), arthritis (insulin-like growth factor), joint disorders (insulin-like growth factor 1 and / or 2), intimal hyperplasia (e.g., by delivery of enos, inos), improved survival of cardiac transplants (superoxide dismutase), AIDS (soluble CD4), muscle wasting (insulin-like growth factor I), kidney deficiency (kidney deficiency) (erythropoietin), anemia (erythropoietin), arthritis (anti-inflammatory factors, such as IRAP and TNFα soluble receptors), hepatitis (α-interferon), LDL receptor deficiency (LDL receptor), hyperammonemia (ornithine transcarbamylase), Krabbe's disease (galactocerebrosidase), Batten's disease, spinocerebral ataxia including SCA1, SCA2 and SCA3, phenylketonuria (phenylalanine hydroxylase), autoimmune diseases, etc. The present invention can be further used to increase the success of transplantation and / or reduce the negative side effects of organ transplantation or adjuvant therapy after organ transplantation (e.g., by blocking cytokine production by administering immunosuppressants or inhibitory nucleic acids). As another example, bone morphogenetic proteins (including BNP 2, 7, etc., RANKL and / or VEGF) can be administered with bone allografts, for example, after amputation or surgical removal in cancer patients. ;

[0191] The present invention can also be used to produce induced pluripotent stem cells (iPS). For example, the viral vector of the present invention can be used to deliver one or more stem cell-related nucleic acids to non-pluripotent cells, such as adult fibroblasts, skin cells, hepatocytes, kidney cells, adipocytes, cardiac cells (cardiac cell), neural cells, epithelial cells, endothelial cells, etc. The nucleic acids encoding factors related to stem cells are known in the art. Non-limiting examples of such factors related to stem cells and pluripotency include Oct-3 / 4, SOX family (e.g., SOX1, SOX2, SOX3 and / or SOX15), Klf family (e.g., Klf1, Klf2, Klf4 and / or Klf5), Myc family (e.g., C-myc, L-myc and / or N-myc), NANOG and / or LIN28.

[0192] The invention can also be practiced to treat and / or prevent metabolic disorders, such as diabetes (e.g., insulin), hemophilia (e.g., coagulation factor IX or coagulation factor VIII), lysosomal storage disorders, such as mucopolysaccharidosis (e.g., Seeley syndrome [β-glucuronidase], Hurler syndrome [α-L-iduronidase], Shea syndrome [α-L-iduronidase], Hurler-Scheie syndrome [α-L-iduronidase], Hunter syndrome [iduronic acid sulfatase], Sanfilep syndrome A [heparan sulfamidase], B [N-acetylglucosaminidase], C [acetyl-CoA:α-glucosamine acetyltransferase], D [N-acetylglucosamine 6-sulfatase], Morquio syndrome A [galactose-6-sulfate sulfatase], B [N-acetylglucosaminidase], C [acetyl-CoA:α-glucosamine acetyltransferase], D [N-acetylglucosamine 6-sulfatase], [β-galactosidase], Marran syndrome [N-acetylgalactosamine-4-sulfatase], etc.), Fabry disease (α-galactosidase), Gaucher disease (glucocerebrosidase), or glycogen storage disorders (eg, Pompe disease; lysosomal acid α-glucosidase).

[0193] Gene transfer has considerable potential for understanding disease states and providing treatment for them. There are many genetic diseases in which the defective genes are known and have been cloned. Generally, the above disease states are divided into two categories: deficiency states, which are usually enzymes that are usually inherited in a recessive manner, and imbalance states, which may involve regulatory or structural proteins, and which are generally inherited in a dominant manner. For diseases in deficiency states, gene transfer can be used to bring normal genes into the affected tissues for replacement therapy, and to produce animal models of the diseases using antisense mutations. For unbalanced disease states, gene transfer can be used to produce disease states in model systems, which can then be used in efforts to offset the disease states. Therefore, viral vectors according to the present invention allow treatment and / or prevention of genetic diseases.

[0194] The viral vector according to the present invention can also be used to provide functional RNA to cells in vitro or in vivo. For example, the expression of functional RNA in cells can reduce the expression of cells to specific target proteins. Therefore, functional RNA can be used to reduce the expression of specific proteins in subjects in need thereof. Functional RNA can also be used to regulate gene expression and / or cell physiology in vitro to cells, for example, to optimize cell or tissue culture systems or in screening methods.

[0195] Furthermore, the viral vectors according to the invention are used in diagnostic and screening methods, whereby a nucleic acid of interest is transiently or stably expressed in cell culture systems or transgenic animal models.

[0196] As will be apparent to those skilled in the art, the viral vectors of the invention may also be used for a variety of non-therapeutic purposes, including but not limited to, in procedures for evaluating gene targeting, clearance, transcription, translation, etc. Viral vectors may also be used for the purpose of evaluating safety (transmission, toxicity, immunogenicity, etc.). For example, the United States Food and Drug Administration considers such data as part of the regulatory approval process prior to evaluating clinical efficacy.

[0197] As a further aspect, the viral vector of the present invention can be used to generate an immune response in a subject. According to the embodiment, a viral vector comprising a heterologous nucleic acid sequence encoding an immunogenic polypeptide can be administered to a subject, and an active immune response is initiated by the subject against the immunogenic polypeptide. The immunogenic polypeptide is as described above. In some embodiments, a protective immune response is elicited.

[0198] An "active immune response" or "autoimmunity" is characterized by "the involvement of host tissues and cells following encounter with an immunogen. It involves the differentiation and proliferation of immunocompetent cells in the lymphoreticular tissue, which results in the synthesis of antibodies or the development of cell-mediated reactivity, or both." Herbert B. Herscowitz, Immunophysiology: Cell Function and Cellular Interactions in Antibody Formation, In IMMUNOLOGY: BASIC PROCESSES 117 (Joseph A. Bellanti, ed., 1985). In other words, an active immune response is mounted by the host after exposure to an immunogen through infection or vaccination. Active immunity can be contrasted with passive immunity, which is achieved by "transfer of preformed substances (antibodies, transfer factors, thymic grafts, interleukin-2) from an actively immune host to a non-immune host." Ibid.

[0199] As used herein, a "protective" immune response or "protective" immunity indicates that the immune response confers some benefit to the subject because it prevents or reduces the incidence of disease. Alternatively, a protective immune response or protective immunity can be used to treat and / or prevent disease, particularly cancer or tumors (e.g., by preventing cancer or tumor formation, by causing cancer or tumor regression and / or by preventing metastasis and / or by preventing the growth of metastatic nodules). The protective effect can be complete or partial, as long as the benefits of the treatment outweigh any disadvantages.

[0200] In certain embodiments, viral vectors or cells comprising heterologous nucleic acid molecules can be administered in an immunogenic effective amount, as described below.

[0201] The viral vectors of the present invention can also be administered for cancer immunotherapy by expressing one or more cancer cell antigens (or immunologically similar molecules) or any other immunogens of the viral vectors that produce an immune response to cancer cells. For example, an immune response to cancer cell antigens can be produced in a subject by administering a viral vector comprising a heterologous nucleic acid encoding a cancer cell antigen, for example to treat a patient with cancer and / or to prevent the development of cancer in a subject. As described herein, the viral vectors can be administered to a subject in vivo or by using an ex vivo method. Alternatively, the cancer antigen can be expressed as part of the viral capsid or otherwise associated with the viral capsid (e.g., as described above).

[0202] As another alternative, any other therapeutic nucleic acid (eg, RNAi) or polypeptide (eg, cytokine) known in the art can be administered to treat and / or prevent cancer.

[0203] As used herein, the term "cancer" includes cancers that form tumors. Likewise, the term "cancerous tissue" includes tumors. "Cancer cell antigens" include tumor antigens.

[0204] The term "cancer" has its meaning as understood in the art, e.g., uncontrolled growth of tissue with the potential to spread to distant parts of the body (i.e., metastasis). Exemplary cancers include, but are not limited to, melanoma, adenocarcinoma, thymoma, lymphoma (e.g., non-Hodgkin's lymphoma, Hodgkin's lymphoma), sarcoma, lung cancer, liver cancer, colon cancer, leukemia, uterine cancer, breast cancer, prostate cancer, ovarian cancer, cervical cancer, bladder cancer, kidney cancer, pancreatic cancer, brain cancer, and any other cancer or malignant condition now known or later identified. In representative embodiments, the present invention provides methods for treating and / or preventing cancers that form tumors.

[0205] The term "tumor" is also understood in the art to be an abnormal, undifferentiated mass of cells, such as in a multicellular organism. Tumors can be malignant or benign. In representative embodiments, the methods disclosed herein are used to prevent and treat malignant tumors.

[0206] The term "treating cancer", "treatment of cancer" and equivalent terms are intended to refer to reducing or at least partially eliminating the severity of cancer and / or slowing down and / or controlling the progression of the disease and / or stabilizing the disease. In specific embodiments, these terms indicate preventing or reducing or at least partially eliminating the metastasis of cancer, and / or preventing or reducing or at least partially eliminating the growth of metastatic nodules.

[0207] The term "prevention of cancer" or "preventing cancer" and equivalent terms are intended to mean that the method at least partially eliminates or reduces and / or delays the incidence and / or severity of the onset of cancer. In other words, the onset of cancer in a subject can be reduced and / or delayed in likelihood or probability.

[0208] It is known in the art that immune responses can be enhanced by immunomodulatory cytokines (e.g., α-interferon, β-interferon, γ-interferon, ω-interferon, τ-interferon, interleukin-1α, interleukin-1β, interleukin-2, interleukin-3, interleukin-4, interleukin-5, interleukin-6, interleukin-7, interleukin-8, interleukin-9, interleukin-10, interleukin-11, interleukin-12, interleukin-13, interleukin-14, interleukin-18, B cell growth factor, CD40 ligand, tumor necrosis factor-α, tumor necrosis factor-β, monocyte chemoattractant protein-1, granulocyte macrophage colony stimulating factor and lymphotoxin). Therefore, immunomodulatory cytokines (preferably, CTL-inducing cytokines) can be administered to a subject together with a viral vector.

[0209] The cytokines may be administered by any method known in the art. Exogenous cytokines may be administered to the subject, or, alternatively, nucleic acids encoding the cytokines may be delivered to the subject using a suitable vector and the cytokines produced in vivo.

[0210] Subjects, drug formulations and administration methods

[0211] The viral vectors and AAV particles according to the present invention are used in both veterinary and medical applications. Suitable subjects include birds and mammals. The term "birds" as used herein includes, but is not limited to, chickens, ducks, geese, quail, turkeys, pheasants, parrots, parakeets, etc. The term "mammals" as used herein includes, but is not limited to, humans, non-human primates, bovines, sheep, goats, equines, felines, canines, lagomorphs, etc. Human subjects include neonates, infants, young, adults, and elderly subjects.

[0212] In representative embodiments, a subject is "in need of" the methods of the invention.

[0213] In a specific embodiment, the invention provides a pharmaceutical composition comprising a viral vector and / or capsid and / or AAV particle of the invention in a pharmaceutically acceptable carrier, and optionally, other pharmaceutical agents, pharmaceutical agents, stabilizers, buffers, carriers, adjuvants, diluents, etc. For injection, the carrier will generally be a liquid. For other methods of administration, the carrier may be a solid or a liquid. For inhalation administration, the carrier will be suitable for breathing, and may optionally be in the form of solid or liquid particles. For administration to a subject or for other pharmaceutical uses, the carrier will be sterile and / or physiologically compatible.

[0214] By "pharmaceutically acceptable" is meant a material that is not toxic or otherwise undesirable, ie, the material can be administered to a subject without causing any undesirable biological effects.

[0215] One aspect of the present invention is a method for transferring nucleic acid molecules to cells in vitro. Viral vectors can be introduced into cells at an appropriate multiplicity of infection according to standard transduction methods appropriate for the specific target cells. Depending on the type and number of target cells and the specific viral vector, the titer of viral vector to be administered can vary and can be determined by one skilled in the art without undue experimentation. In a representative embodiment, at least about 10 3 infectious units, optionally at least about 10 5 of infectious units into cells.

[0216] One or more cells into which the viral vector is introduced can be of any type, including but not limited to neural cells (including cells of the peripheral and central nervous systems, particularly brain cells, such as neurons and oligodendrocytes), lung cells, eye cells (including retinal cells, retinal pigment epithelium and corneal cells), epithelial cells (e.g., intestinal and respiratory epithelial cells), muscle cells (e.g., skeletal muscle cells, cardiomyocytes, smooth muscle cells and / or diaphragmatic cells), dendritic cells, pancreatic cells (including islet cells), hepatocytes, cardiomyocytes, osteocytes (e.g., bone marrow stem cells), hematopoietic stem cells, spleen cells, keratinocytes, fibroblasts, endothelial cells, prostate cells, germ cells, etc. In representative embodiments, the cell can be any progenitor cell. As a further possibility, the cell can be a stem cell (e.g., neural stem cell, liver stem cell). As a further alternative, the cell can be a cancer or tumor cell. In addition, as described above, the cell can be from a species of any origin.

[0217] In order to administer the modified cells to the subject, the viral vector can be introduced into the cell in vitro. In a specific embodiment, the cell has been removed from the subject, the viral vector is introduced therein, and the cell is then administered back to the subject. The method of removing cells from the subject for ex vivo manipulation followed by introduction back into the subject is known in the art (see, e.g., U.S. Patent No. 5,399,346). Alternatively, the recombinant viral vector can be introduced into cells from a donor subject, cultured cells, or cells from any other suitable source, and the cell is administered to a subject in need thereof (i.e., a "recipient" subject).

[0218] Suitable cells for ex vivo nucleic acid delivery are described above. The cell dosage administered to a subject will vary depending on the age, condition and species of the subject, the type of cells, the nucleic acid expressed by the cells, the mode of administration, etc. Generally, at least about 10 cells / mL will be administered per dose in a pharmaceutically acceptable carrier. 2 to about 10 8 cells or at least about 10 3 to about 10 6 In certain embodiments, cells transduced with a viral vector are combined with a pharmaceutical carrier and administered to a subject in a therapeutically or prophylactically effective amount.

[0219] In some embodiments, the viral vector is introduced into the cell, and the cell can be administered to the subject to elicit an immunogenic response to the delivered polypeptide (e.g., expressed as a transgene or in a capsid). Generally, a certain amount of cells expressing an immunogenic effective amount of polypeptides in combination with a pharmaceutically acceptable carrier is administered. An "immunogenic effective amount" is an amount of the expressed polypeptide that is sufficient to cause an active immune response to the polypeptide in a subject to which the pharmaceutical formulation is administered. In a specific embodiment, the dosage is sufficient to produce a protective immune response (as defined above). The degree of protection conferred does not need to be complete or permanent, as long as the benefits of administering the immunogenic polypeptide outweigh any disadvantages thereof.

[0220] A further aspect of the present invention is a method of administering a viral vector and / or a viral capsid to a subject. Administration of a viral vector and / or a capsid according to the present invention to a human subject or animal in need thereof can be performed by any means known in the art. Optionally, the viral vector and / or capsid are delivered in a pharmaceutically acceptable carrier in a therapeutically effective or prophylactically effective dose.

[0221] The viral vectors and / or capsids of the present invention may be further administered to elicit an immunogenic response (e.g., as a vaccine). Generally, the immunogenic compositions of the present invention comprise an immunogenic effective amount of viral vectors and / or capsids in combination with a pharmaceutically acceptable carrier. Optionally, the dose is sufficient to produce a protective immune response (as defined above). The degree of protection conferred does not need to be complete or permanent, as long as the benefits of administering the immunogenic polypeptide outweigh any disadvantages thereof. The subject and immunogen are as described above.

[0222] The dosage of the viral vector and / or capsid administered to a subject depends on the mode of administration, the disease or condition to be treated and / or prevented, the condition of the individual subject, the particular viral vector or capsid and the nucleic acid to be delivered, etc., and can be determined in a routine manner. An exemplary dosage for obtaining a therapeutic effect is at least about 10 5 , 10 6 , 10 7 , 10 8 , 10 9 , 10 10 , 10 11 , 10 12 , 10 3 , 10 14 , 10 15 Transduction units, about 10 8 to about 10 13 The titer of transducing units.

[0223] In certain embodiments, more than one administration (e.g., two, three, four, five, six, seven, eight, nine, ten, etc., or more administrations) may be used to achieve desired gene expression levels over a range of time periods at various intervals, e.g., hourly, daily, weekly, monthly, yearly, etc.

[0224] Exemplary modes of administration include oral, rectal, transmucosal, intranasal, inhalation (e.g., by aerosol), buccal (e.g., sublingual), vaginal, intrathecal, intraocular, transdermal, intrauterine (or intraocular), intravenous ( in ovo )), parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular [including administration to skeletal muscle, diaphragm and / or myocardium], intradermal, intrapleural, intracerebral and intraarticular), topical (e.g., to both skin and mucosal surfaces, including respiratory surfaces, and transdermal administration), intralymphatic, etc., and direct tissue or organ injection (e.g., to the liver, skeletal muscle, myocardium, diaphragm or brain). Administration to a tumor (e.g., in or near a tumor or lymph node) is also possible. In any given case, the most appropriate route will depend on the nature and severity of the condition being treated and / or prevented, as well as the nature of the specific carrier being used.

[0225] The viral vector and / or capsid can be administered intravenously, intra-arterially, intraperitoneally, by limb perfusion (optionally, independent limb perfusion of the leg and / or arm; see, e.g., Arruda et al. (2005) Blood 105: 3458-3464) and / or direct intramuscular injection for delivery. In specific embodiments, the viral vectors and / or capsids are administered to a limb (arm and / or leg) of a subject (e.g., a subject with muscular dystrophy such as DMD) by limb perfusion, optionally independent limb perfusion (e.g., by intravenous or intraarticular administration). In embodiments of the invention, the viral vectors and / or capsids of the invention can advantageously be administered without the use of "fluid dynamics" techniques. Tissue delivery of prior art vectors (e.g., to muscle) is often enhanced by fluid dynamics techniques (e.g., large volume intravenous / intravenous administration), which increases pressure in the vasculature and promotes the ability of the vector to cross the endothelial cell barrier. In specific embodiments, the viral vectors and / or capsids of the invention can be administered without fluid dynamics techniques such as large volume infusion and / or elevated intravascular pressure (e.g., greater than normal systolic pressure, e.g., less than or equal to 5%, 10%, 15%, 20%, 25% increase in intravascular pressure compared to normal systolic pressure). This approach may reduce or avoid the adverse effects associated with fluid dynamic techniques, such as edema, nerve damage, and / or compartment syndrome.

[0226] The invention can also be practiced to produce antisense RNA, RNAi or other functional RNA (eg, ribozymes) for systemic delivery.

[0227] Injectables can be prepared in conventional forms, as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. Alternatively, one can administer the viral vectors and / or viral capsids of the invention in a local rather than systemic manner, for example, in a depot or sustained release formulation. Further, the viral vectors and / or viral capsids can be delivered attached to a surgically implantable matrix (e.g., as described in U.S. Patent Publication No. US-2004-0013645-A1).

[0228] In certain embodiments, the delivery vectors of the invention can be administered to treat diseases of the CNS, including genetic disorders, neurodegenerative conditions, psychiatric disorders, and tumors. Illustrative diseases of the CNS include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, Canavan disease, Leigh's disease, Refsum's disease, Tourette's syndrome, primary lateral sclerosis, amyotrophic lateral sclerosis, progressive muscular atrophy, Pick's disease, muscular dystrophy, multiple sclerosis, myasthenia gravis, Binswanger's disease, trauma due to spinal cord or head injury, Tay-Sachs disease, Lesch-Nyan disease, epilepsy, cerebral infarction, psychiatric disorders, including affective disorders (e.g., depression, bipolar disorder, persistent affective disorder, secondary affective disorder), schizophrenia, drug dependence (e.g., alcoholism and other substance dependence), neurotic disorders (e.g., anxiety, obsessional disorder, somatoform disorder, dissociative disorder, grief, postpartum depression), psychosis (e.g., hallucinations and delusions), dementia, paranoia, attention deficit disorder, psychosexual disorders, sleep disorders, pain disorders (e.g., disorders), eating or weight disorders (e.g., obesity, cachexia, anorexia nervosa, and bulemia), and cancers and tumors of the CNS (e.g., pituitary tumors).

[0229] Disorders of the CNS include eye disorders involving the retina, posterior tract, and optic nerve (eg, retinitis pigmentosa, diabetic retinopathy and other retinal degenerative diseases, uveitis, age-related macular degeneration, glaucoma).

[0230] Most, if not all, ocular diseases and disorders are associated with one or more of the following three types of indications: (1) angiogenesis, (2) inflammation, and (3) degeneration. The delivery vehicles of the present invention can be used to deliver anti-angiogenic factors; anti-inflammatory factors; factors that delay cell degeneration, promote cell immunity, or promote cell growth, and combinations thereof.

[0231] For example, diabetic retinopathy is characterized by angiogenesis. Diabetic retinopathy can be treated by delivering one or more anti-angiogenic factors intraocularly (e.g., in the vitreous) or periocularly (e.g., in the sub-Tenon's region). One or more neurotrophic factors can also be co-delivered intraocularly (e.g., in the vitreous) or periocularly.

[0232] Uveitis involves inflammation. One or more anti-inflammatory factors can be administered by intraocular (eg, vitreous or anterior chamber) administration of the delivery vehicle of the invention.

[0233] In contrast, retinitis pigmentosa is characterized by retinal degeneration.In representative embodiments, retinitis pigmentosa can be treated by intraocular (eg, intravitreal administration) delivery of a vector encoding one or more neurotrophic factors.

[0234] Age-related macular degeneration involves both angiogenesis and retinal degeneration. The condition can be treated by intraocular (e.g., vitreous) administration of a delivery vector of the invention encoding one or more neurotrophic factors and / or intraocular or periocular (e.g., in the sub-Tenon's region) administration of one or more anti-angiogenic factors.

[0235] Glaucoma is characterized by elevated ocular pressure and loss of retinal ganglion cells. Treatment of glaucoma involves administering one or more neuroprotective agents that protect cells from excitotoxic injury using the delivery vehicles of the invention. Such agents include N-methyl-D-aspartate (NMDA) antagonists, cytokines, and neurotrophic factors delivered intraocularly, optionally intravitreally.

[0236] In other embodiments, the invention can be used to treat seizures, e.g., to reduce the onset, incidence, or severity of seizures. The efficacy of therapeutic treatment of seizures can be assessed by behavioral (e.g., eye or mouth shaking, twitches) and / or electrographic means (most seizures have characteristic electrographic abnormalities). Thus, the invention can also be used to treat epilepsy, which is marked by multiple seizures over time.

[0237] In a representative embodiment, somatostatin (or its active fragment) is administered to the brain using the delivery vector of the present invention to treat pituitary tumors. According to the embodiment, the delivery vector encoding somatostatin (or its active fragment) is administered by microinfusion into the pituitary gland. Similarly, this treatment can be used to treat acromegaly (abnormal growth hormone secretion from the pituitary gland). The nucleic acid sequence (e.g., GenBank Accession No. J00306) and amino acid (e.g., GenBank Accession No. P01166; containing processed active peptides somatostatin-28 and somatostatin-14) sequences of somatostatin are known in the art.

[0238] In certain embodiments, the vector may comprise a secretion signal, as described in US Pat. No. 7,071,172.

[0239] In representative embodiments of the present invention, viral vectors and / or viral capsids are delivered to the CNS (e.g., to the brain or to the eye) after systemic administration. Viral vectors and / or capsids may be introduced into the spinal cord, brainstem (medulla oblongata, pons), midbrain (hypothalamus, thalamus, epithalamus, pituitary gland, substantia nigra, pineal gland), cerebellum, telencephalon (striatum, cerebrum, including occipital, temporal, parietal and frontal lobes, cortex, basal ganglia, hippocampus and amygdala), limbic system, neocortex, striatum, cerebrum and hypothalamus. After peripheral administration, viral vectors and / or capsids may also be delivered to different areas of the eye, such as the retina, cornea and / or optic nerve.

[0240] Viral vectors and / or capsids can be delivered into the cerebrospinal fluid (e.g., by lumbar puncture) for more dispersed administration of the delivery vector. In cases where the blood-brain barrier has been disturbed (e.g., brain tumors or cerebral infarction), viral vectors and / or capsids can be further administered intravascularly to the CNS.

[0241] The viral vectors and / or capsids can be administered to one or more desired areas of the body by any route known in the art, including, but not limited to, intrathecal, intraocular, intracerebral, intraventricular, intravenous (e.g., in the presence of a sugar such as mannitol), intranasal, intraauricular, intraocular (e.g., intravitreal, subretinal, anterior chamber), and periocular (e.g., sub-Tenon's area) delivery, and intramuscular in the case of retrograde delivery to motor neurons.

[0242] In certain embodiments, the viral vector is administered to a desired area or compartment in the CNS by direct injection (e.g., stereotactic injection) in a liquid formulation. In other embodiments, the viral vector and / or capsid can be provided by topical application to the desired area or by intranasal administration of an aerosol formulation. Administration to the eye can be by topical application of droplets. As a further alternative, the viral vector and / or capsid can be administered as a solid, sustained release formulation (see, e.g., U.S. Pat. No. 7,201,898).

[0243] In yet additional embodiments, viral vectors can be used for retrograde transport to treat and / or prevent diseases and disorders involving motor neurons (e.g., amyotrophic lateral sclerosis (ALS); spinal muscular atrophy (SMA); etc.). For example, a viral vector can be delivered to muscle tissue, from which the viral vector can migrate into neurons.

[0244] The invention may be defined as in any of the following numbered paragraphs:

[0245] 1. A recombinant nucleic acid molecule comprising an adeno-associated virus (AAV) 5' inverted terminal repeat (ITR), a nucleotide sequence of interest (NOI) operably associated with a promoter, and an AAV 3' ITR, wherein the recombinant nucleic acid molecule further comprises:

[0246] a) a poly A (pA) sequence oriented in 3' to 5' direction downstream of the 5' ITR and upstream of the promoter and a pA sequence oriented in 3' to 5' direction upstream of the 3' ITR and downstream of the NOI;

[0247] b) a pA sequence oriented in 3' to 5' direction upstream of the 3' ITR and downstream of the NOI;

[0248] c) a first pA sequence oriented in 3' to 5', upstream of the 3' ITR and downstream of the NOI, and a second pA sequence oriented in 5' to 3', downstream of the first pA sequence and upstream of the 3' ITR;

[0249] d) a first pA sequence oriented in 3' to 5', upstream of the 3' ITR and downstream of the NOI, and a second pA sequence oriented in 5' to 3', downstream of the NOI and upstream of the first pA;

[0250] e) a first pA sequence oriented in 3' to 5' direction, upstream of the 3' ITR and downstream of the NOI, and a second pA sequence oriented in 5' to 3' direction, downstream of the 5' ITR and upstream of the promoter;

[0251] f) a first pA sequence oriented in 3' to 5', downstream of the 5' ITR and upstream of the promoter, a second pA sequence oriented in 5' to 3', downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented in 3' to 5', downstream of the second pA sequence and upstream of the 3' ITR;

[0252] g) a first pA sequence oriented in 5' to 3', downstream of the 5' ITR and upstream of the promoter, a second pA sequence oriented in 3' to 5', downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented in 5' to 3', downstream of the second pA sequence and upstream of the 3' ITR;

[0253] h) a first pA sequence oriented in 5' to 3', downstream of the 5' ITR and upstream of the promoter, a second pA sequence oriented in 5' to 3', downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented in 3' to 5', downstream of the second pA sequence and upstream of the 3' ITR;

[0254] i) a first pA sequence oriented in 5' to 3', downstream of the 5' ITR and upstream of the promoter, a second pA sequence oriented in 3' to 5', downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented in 5' to 3', downstream of the second pA sequence and upstream of the 3' ITR;

[0255] j) a first pA sequence oriented in 3' to 5', downstream of the 5' ITR and upstream of the second pA sequence, a second pA sequence oriented in 5' to 3', downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented in 5' to 3', downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented in 3' to 5', downstream of the third pA sequence and upstream of the 3' ITR;

[0256] k) a first pA sequence oriented in 3' to 5', downstream of the 5' ITR and upstream of the second pA sequence, a second pA sequence oriented in 5' to 3', downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented in 3' to 5', downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented in 5' to 3', downstream of the third pA sequence and upstream of the 3' ITR;

[0257] l) a first pA sequence oriented 5' to 3' downstream of the 5' ITR and upstream of the second pA sequence, a second pA sequence oriented 3' to 5' downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented 5' to 3' downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented 3' to 5' downstream of the third pA sequence and upstream of the 3' ITR; and / or

[0258] m) a first pA sequence oriented in 5' to 3' direction downstream of the 5' ITR and upstream of the second pA sequence, a second pA sequence oriented in 3' to 5' direction downstream of the first pA sequence and upstream of the promoter, a third pA sequence oriented in 3' to 5' direction downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented in 5' to 3' direction downstream of the third pA sequence and upstream of the 3' ITR.

[0259] 2. A recombinant nucleic acid molecule comprising an adeno-associated virus (AAV) vector cassette of a first AAV serotype, the vector cassette comprising an AAV 5' inverted terminal repeat (ITR), a nucleotide sequence of interest (NOI) operably associated with a promoter, and an AAV 3' ITR, wherein the AAV 5' ITR and / or the AAV 3' ITR are from a second AAV serotype different from the first AAV serotype.

[0260] 3. The recombinant nucleic acid molecule of paragraph 2, wherein the first AAV serotype is AAV2 and the second AAV serotype is AAV5.

[0261] 4. A recombinant nucleic acid molecule comprising an adeno-associated virus (AAV) 5' inverted terminal repeat (ITR), a nucleotide sequence of interest (NOI) operably associated with a promoter, and an AAV 3' ITR, wherein the 5' ITR and / or 3' ITR are modified to reduce or eliminate promoter activity from the 5' ITR and / or 3' ITR.

[0262] 5. A recombinant nucleic acid molecule comprising an AAV 5' ITR, a NOI operably associated with a promoter, a pA sequence oriented in 3' to 5' direction, and an AAV 3' ITR, wherein the NOI sequence is fused to one or more than one nucleotide sequence encoding an interfering RNA sequence that targets a cytoplasmic dsRNA sensor.

[0263] 6. A recombinant nucleic acid molecule comprising an AAV 5' ITR, a NOI operably associated with a first promoter, a first pA sequence oriented 3' to 5', a nucleotide sequence encoding an interfering RNA sequence targeting a cytoplasmic dsRNA sensor operably associated with a second promoter, a second pA sequence, and the AAV 3' ITR.

[0264] 7. A recombinant nucleic acid molecule comprising an AAV 5' ITR, a NOI operably associated with a first promoter, a pA sequence oriented 3' to 5', a short hairpin RNA (shRNA) sequence targeting a cytoplasmic dsRNA sensor operably associated with a second promoter, and the AAV 3' ITR.

[0265] 8. A recombinant nucleic acid molecule comprising an AAV 5' ITR, an shRNA targeting a cytoplasmic dsRNA sensor operably associated with a first promoter, a NOI operably associated with a second promoter, a pA sequence oriented in 3' to 5' direction, and an AAV 3' ITR.

[0266] 9. A recombinant nucleic acid molecule comprising, in the following order: AAV 5' ITR, NOI and a microRNA (miRNA) sequence targeting a cytoplasmic dsRNA sensor, both operably associated with a promoter, a pA sequence oriented 3' to 5', and AAV 3' ITR.

[0267] 10. A recombinant nucleic acid molecule comprising, in the following order: AAV 5' ITR, a miRNA and a NOI targeting a cytoplasmic dsRNA sensor, both operably associated with a promoter, a pA sequence oriented in 3' to 5' direction, and AAV 3' ITR.

[0268] 11. A recombinant nucleic acid molecule comprising, in the following order: an AAV 5' ITR, a NOI, a pA sequence oriented in a 3' to 5' direction, and an AAV 3' ITR, wherein the NOI comprises a miRNA intron sequence within the NOI, wherein the NOI is operably associated with a promoter.

[0269] 12. A composition comprising a first recombinant nucleic acid molecule comprising an AAV 5' ITR, a NOI operably associated with a promoter, a pA sequence oriented 3' to 5', and an AAV 3' ITR, and a second recombinant nucleic acid molecule comprising an interfering RNA sequence targeting a cytoplasmic dsRNA sensor.

[0270] 13. The composition of paragraph 12, wherein the interfering RNA sequence is shRNA.

[0271] 14. A recombinant nucleic acid molecule comprising:

[0272] AAV 5′ ITR;

[0273] an inhibitor of NOI and MAVS signaling, both operably associated with the promoter;

[0274] a pA sequence in a 3' to 5' orientation; and

[0275] AAV 3' ITR.

[0276] 15. The recombinant nucleic acid molecule of paragraph 14, wherein the inhibitor of MAVS signaling is selected from the group consisting of: serine protease NS3-4A from hepatitis C virus, protease from hepatitis A virus, protease from GB virus B, hepatitis B virus (HBV) X protein, poly (rC)-binding protein 2, 20S proteasome subunit PSMA7, mitochondrial fusion protein 2, and any combination thereof.

[0277] 16. A recombinant nucleic acid molecule comprising:

[0278] AAV 5′ ITR;

[0279] a NOI operably associated with a first promoter;

[0280] a first pA sequence oriented in 3' to 5' direction;

[0281] an inhibitor of MAVS signaling operably associated with a second promoter;

[0282] a second pA sequence oriented in a 3' to 5' direction; and

[0283] AAV 3' ITR.

[0284] 17. An rAAV vector genome comprising the recombinant nucleic acid molecule of any one of paragraphs 1-12 and 13-16.

[0285] 18. An rAAV particle comprising the rAAV genome of paragraph 17.

[0286] 19. A composition comprising the rAAV particles of paragraph 18.

[0287] 20. A composition comprising a first recombinant nucleic acid molecule comprising an AAV 5' ITR, a NOI operably associated with a promoter, a pA sequence oriented 3' to 5', and an AAV 3' ITR, and a second recombinant nucleic acid molecule comprising an inhibitor of MAVS signaling and a pA sequence oriented 3' to 5'.

[0288] 21. A method of enhancing transduction of an AAV vector in cells of a subject, comprising administering to the subject an AAV vector and an agent that interferes with a dsRNA activation pathway in the cells of the subject.

[0289] 22. The method of paragraph 21, wherein the agent that interferes with the dsRNA activation pathway in the subject's cells is 2-aminopurine.

[0290] 23. The method of paragraphs 21-22, wherein the AAV vector and the agent are administered to the subject simultaneously.

[0291] 24. The method of paragraphs 21-22, wherein the AAV vector and the agent are administered at separate times.

[0292] The subject matter of the present invention will now be described more fully below with reference to the accompanying examples, in which representative embodiments of the presently disclosed subject matter are shown. However, the presently disclosed subject matter can be embodied in different forms and should not be construed as being limited to the embodiments provided herein. Rather, these embodiments are provided so that the present disclosure will be comprehensive and complete and fully convey the scope of the presently disclosed subject matter to those skilled in the art. Example

[0293] The following examples provide illustrative embodiments. Certain aspects of the following examples are disclosed in accordance with techniques and procedures that the inventors have found or expect to work well in the practice of the embodiments. Based on this disclosure and the general level of skill in the art, the skilled person will appreciate that the following examples are intended to be exemplary only, and that many variations, modifications, and changes may be used without departing from the scope of the presently claimed subject matter.

[0294] Example 1

[0295] cell.HeLa cells, 293 cells, Huh7 cells, and HepG2 cells were grown at 37°C in 5% CO2 in Dulbecco's modified Eagle's medium with 10% FBS and 1% penicillin-streptomycin. Human primary hepatocytes were purchased from Triangle Research Labs. Information about fresh human primary hepatocytes is listed in Table 1. Primary hepatocytes were plated in Williams' E medium (ThermoFisher Scientific) with Hepatocyte Thawing and Plating Supplement Pack and maintained in Williams' E medium (Thermo Fisher Scientific) with Hepatocyte Maintenance Supplement Pack and HepExtend™ Supplement.

[0296] AAV virus production. AAV virus production was described before using triple plasmid transfection. Briefly, HEK-293 cells were transfected with AAV transgenic plasmids (single-stranded (ss) pTR-CBA-luciferase, double-stranded (ds) pTR-CBh-GFP, ss pTR-CMV-GFP, sspTR-CBA-AAT, dspTR-shRNA-mixed and dspTR-TTR-FΙX-opt), Rep and Cap AAV helper plasmids and adenovirus helper plasmid pXX6-80. 48 hours after transfection, cells were harvested. After lysis of HEK-293 cells, AAV virus was purified by cesium chloride (CsCl) gradient density centrifugation. Virus titer was determined by Q-PCR.

[0297] Mouse. Human xenograft mice with 70% human hepatocyte regeneration were purchased from Yecuris Inc. Mice were maintained in a specific pathogen-free facility at the University of North Carolina at Chapel Hill. The University of North Carolina Institutional Animal Care and Use Committee approved all procedures.

[0298] In vitro transduction. HeLa, Huh7, 293 or HepG2 cells were cultured at 5 × 10 3 Transduced cells were harvested at different time points. For long-term AAV transduction studies, 1 × 10 5 HeLa cells were cultured at 5 × 10 3AAV particles were transduced. On day 3 after transduction, cells were split 1:5 and then cultured for up to 5 days with daily medium changes. AAV-transduced cells were harvested at designated time points and cell lysates were used to measure luciferase activity.

[0299] Transduction of primary human hepatocytes. The suspended hepatocytes were plated onto collagen I-coated plates and cultured at a dose of 5 × 10 3 AAV vectors (AAV2 / GFP or AAV2 / FIX-opt) were added per 10000 particles / cell. One day later, the plating medium was replaced with the maintenance medium. Primary hepatocytes were cultured for 10 days, with the medium changed daily. Hepatocytes were harvested at different time points for detection of MDA-5, RIG-1, and IFN-β.

[0300] Mouse experiments. Human hepatocytes from xenograft mice were expressed at 3x10 11 AAV8 / FIX-opt particles were administered by retro-orbital injection. At 4 or 8 weeks after AAV injection, mice were sacrificed and livers were harvested for RNA extraction and protein analysis by Western blot.

[0301] Luciferase assay. Cells transduced with AAV2 / luciferase were treated with passive lysis buffer (Promega) for 20 min. Luciferase activity was measured using luciferase assay reagent (Promega) according to the manufacturer's instructions. Luciferase activity was measured using a Wallac 1420 Victor3 plate reader.

[0302] Transfection assay. For poly(I:C) transfection, cells were transfected with 2 µg poly(I:C) by Lipofectamin 3000 (Thermo Fisher Scientific) in 12-well plates at different time points: 18 h before AAV transduction, simultaneously or 3 days after AAV transduction. For siRNA transfection, HeLa cells were split on day 3 after AAV vector transduction, and 24 h later, cells were transfected with 1 µg siRNA (siMDA5: CUGAAUCUGCUCCUUCACC (SEQ ID NO:1), siMAVS: AUACAACUGACCCUGUGGG (SEQ ID NO:2), siMAVS-2: UAGUUGAUCUCGCGGACGA (SEQ ID NO:3) and CCGUUUGCUGAAGACAAGA (SEQ ID NO:4), siControl: UGUGAUCAAGGACGCUAUG, SEQ ID NO:5). At 48 or 72 h after transfection, cells were harvested for luciferase assay or RNA extraction.

[0303] RNA isolation and real-time PCR. RNA from cultured cells or mouse liver tissue was isolated using TRIzol Reagent (Invitrogen). First-strand cDNA was synthesized from RNA template using RevertAid First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific). Real-time PCR was performed using a LightCycler 480 instrument (Roche). Primers used for real-time PCR are listed in Table 2.

[0304] Western blotting. Cells or tissues were treated with RIPA buffer. 60 μg of protein per lane was loaded into SDS-PAGE gel. After protein transfer to NC membrane, the membrane was stained with rabbit monoclonal MDA5 antibody (Thermo Fisher Scientific) or β-actin antibody (Thermo Fisher Scientific). Signals were detected using ECL Western Blotting Detection Reagent (GE). Data analysis was performed using ImageJ software.

[0305] IFN-β promoter reporter assay. 1 × 10 in a 6-well plate 5 HeLa cells were cultured at 5 × 10 3 The cells were transduced with AAV2 / GFP particles. On day 3 after transduction, the cells were split 1:5. 24 h later, the cells were co-transfected with IFN-β promoter reporter plasmid and siRNA. Luciferase activity was then measured 72 h after transfection.

[0306] Statistical analysis. All statistical calculations were performed using statistical software (GraphPad Prism 7.0 software). Differences between different groups were evaluated by Student's t-test and were considered statistically significant when P value < 0.05.

[0307] IFN-β inhibits transgene expression from AAV-transduced cells. Type I IFN-β expression is a hallmark of innate immune activation. To investigate the effects of innate immune response activation on transgene expression, we investigated the effects of IFN-β on transgene expression after in vitro AAV transduction. HeLa cells were infected with an AAV2 / luc vector encoding a firefly luciferase transgene, and 24 hr later, different doses of IFN-β were added. Luciferase activity was measured at different time points after IFN-β supplementation. Luciferase transgene expression was significantly reduced compared to the PBS (no IFN) group. Inhibition was dose-dependent ( Figure 1A When IFN-β was added daily starting from day 1 after AAV transduction, stronger inhibition of luciferase expression was observed from long-term cultures ( Figure 1B). This result suggests that activation of the innate immune response can inhibit AAV transduction.

[0308] Poly(I:C) inhibits AAV transduction. In clinical trials, in patients with hemophilia, a reduction in transgene expression was observed in the 6th to 10th week after AAV administration. At this time point, most AAV virions have entered the nucleus for effective transgene expression. The possibility of triggering an innate immune response from endosomes in the cytoplasm or pattern recognition receptors (PRPs) for sensors of DNA seems low. We assume that dsRNA can be produced from AAV vector-mediated transgene delivery and can activate innate immune responses. In order to determine whether innate immunity from dsRNA affects transgene expression from AAV transduction, we transduced cells with AAV2 / luc, where at different time points, i.e., 18 hours before AAV2 transduction or simultaneously or on the 3rd day after AAV2 transduction, transfection of polyinosinic acid-polycytidylic acid (poly (I: C)) as a synthetic analog of dsRNA was performed. After 72 hours after poly (I: C) transfection, cell lysates were harvested for luciferase activity analysis. Regardless of the time point, transfection of poly (I: C) in both HeLa cells and Huh7 cells inhibited transgene expression ( Figure 2 ). The data suggest that innate immune responses triggered from dsRNA affect AAV transduction.

[0309] Double-stranded RNA innate immune responses in HeLa cells triggered from late AAV transduction. To investigate whether AAV transduction activates the innate immune response triggered by dsRNA, we examined the expression kinetics of dsRNA sensors at the transcriptional level: MDA5 and RIG1. As shown in Figure 3, upregulation of MDA5 was observed on day 6 after scAAV / GFP vector transduction in HeLa cells. There was no MDA5 activation before day 5 of transduction ( Figure 3A There was no increase in RIG1 expression during AAV transduction in HeLa cells ( Figure 3B There was no increase in IFN-β during days 3-6, however high IFN-β expression was obtained on day 8 ( Figure 3C We also examined the expression of MDA-5 at the translational level. On day 8 after AAV transduction, MDA-5 expression was found to be high in cell lysates based on the intensity of Western blot results ( Figure 3D and 3E ). This study suggests that AAV transduction can activate dsRNA-mediated innate immune responses.

[0310] AAV transduction-mediated activation of dsRNA innate immune responses is cell-specific and transgene-dependent.In clinical trials, in patients with hemophilia B, transgenic FIX is driven by a liver-specific promoter and expressed primarily in hepatocytes. The results from the above studies have confirmed that dsRNA immune responses in HeLa cells (a non-hepatocyte cell line) are triggered at a later time after AAV transduction. Next, we want to know whether dsRNA innate immune responses are also triggered in other human cell lines, including cell lines derived from hepatocytes. After AAV2 / GFP vector infection, dsRNA responses were evaluated at different time points. Only the upregulation of MDA5 and IFN-β was observed in human hepatocyte Huh7 cells and HepG2 cells, but not in 293 cells ( Figure 4A ).

[0311] To investigate the activation of dsRNA-mediated innate immune responses from different transgenes, we transduced HeLa cells with AAV2 encoding different transgenes, including luciferase, shRNA-scrambled, and antitrypsin. MDA5 transcription was detected on day 8 after AAV transduction. Higher MDA-5 expression was observed in HeLa cells transduced with ssAAV / GFP, AAV2 / luc, AAV2 / shRNA-scrambled, but not with AAV2 / AAT transduction compared to the control group ( Figure 4B ). However, IFN-β expression was upregulated in all AAV2 vector-transduced cells, regardless of the transgene.

[0312] dsRNA innate immune responses are induced in AAV / GFP-transduced primary human hepatocytes. It has been shown that AAV transduction triggers dsRNA innate immune responses in the human hepatocyte cell line Huh7 cells, and as mentioned above, we wondered if this finding could be applied to human primary hepatocytes. AAV2 has been shown to effectively transduce primary human hepatocytes in vitro. We used the scAAV2 / GFP vector to transduce primary human hepatocytes from 6 different subjects. At different time points after AAV2 transduction, RNA was harvested from human hepatocytes for transcriptional expression of MDA5, RIG1, and IFN-β. After day 5 after AAV transduction, MDA5 was upregulated in 6 of the 12 subjects ( Figure 5A ), where higher RIG1 expression was observed in only 3 subjects (subjects 1, 5, and 12). The other 6 subjects did not show changes in MDA5 or RIG-I expression ( Figure 5B). However, higher IFN-β expression was detected in all subjects after AAV transduction (Fig. 5). MDA5 expression reached a peak at the 5th day or later time after AAV transduction, and then decreased to baseline. High IFN-β expression has no specific pattern, and in most cases, at the later time point (≥5 days) after AAV transduction, the increased IFN-β expression is accompanied by MDA5 expression. In some subjects, high IFN-β expression (subjects 1, 3, 7 and 9) was detected at the very early time point (within 1 day) after AAV transduction, but there was no change for dsRNA sensors. This result may support the activation of the innate immune response by dsDNA-TLR9 approach, as reported in other studies.

[0313] dsRNA innate immune responses are induced in AAV / FIX-opt-transduced primary human hepatocytes. Next, we tested whether AAV vector delivery of the therapeutic transgenic FIX also triggers a dsRNA innate immune response. We transduced human primary hepatocytes from 10 subjects with AAV vectors to deliver a clinically used FIX cassette-optimized human FIX with a mutation at R338L for enhanced coagulation activity (hFIX-R338L-opt). After transduction with AAV2 / hFIX-R338L-opt, MDA5 and IFN-β were upregulated in 5 of the 10 subjects on or after day 5 of AAV infection ( Fig. 6A ), and the other 5 subjects showed only high IFN-β expression ( Figure 6B ). RIG-I was upregulated in subjects 8 and 12 on day 7 after AAV transduction ( Fig. 6A ). The results indicate that the dsRNA innate immune response is activated in human primary hepatocytes transduced with AAV vectors encoding clinical therapeutic transgenes (Figure 6).

[0314] Activation of dsRNA innate immune responses in AAV-transduced human hepatocytes from humanized mice. All the above results support that the cytosolic dsRNA innate immune response in human cells is activated at a later time after AAV transduction in vitro. Next, we examined the dsRNA innate immune response in human hepatocytes in vivo using a human chimeric mouse model. In these mice, human hepatocytes were transplanted into the mouse liver at 70% regeneration. In the first set of experiments, we injected the clinical vector AAV8 / hFIX-opt in two mice, and at week 8, the mouse livers were harvested for RNA extraction. Then, MDA5 and RIG1 expression were detected at the transcriptional level in human hepatocytes. Fig. 7AAs shown, the expression of both MDA5 and RIG1 increased. In addition, IFN-β expression was higher in AAV8 / hFIX-opt treated mice than in untreated mice. In a second experiment, mouse livers were harvested for analysis of dsRNA immune responses at weeks 4 and 8 after AAV8 / hFIX-opt injection. The mRNA levels for both MDA and RIG-1 increased at week 4 and decreased to control levels at week 8 after AAV8 administration ( Figure 7B ). Higher MDA5 expression was confirmed at the protein level ( Figure 7C ). AAV-treated mice had high IFN-β expression not only at week 4 but also at week 8 ( FIG. 7 ).

[0315] Blockade of the dsRNA activation pathway increased transgene expression and suppressed IFN-β expression from AAV-transduced cells. In the described experiments, induction of the innate immune response and addition of IFN-β reduced AAV transduction. Next, we wondered whether blocking the dsRNA innate immune response would affect transgene expression at later time points after AAV transduction. Because MAD5 is the major dsRNA sensor in HeLa cells with AAV transduction, we used siRNA specific for MDA5 and MAVS (a common adaptor of MDA5 and RIG1) to knock down their expression, and studied transgene expression and IFN-β expression. Transfection of siRNA was able to effectively inhibit the transcriptional expression of MDA5 and MAVS ( Fig. 8A ). Initially, we examined the effect of siRNA on poly(I:C) inhibition of AAV transgene expression. Poly(I:C) was added on day 3 after AAV2 / luc transduction. siRNA was transfected on day 4. Transgene expression was measured 48 or 72 hours after siRNA. When siRNA for MDA5 or MAVS was used, luciferase expression was significantly increased at both 48 and 72 hours. Next, siRNA was transfected on day 4 after AAV transduction, and IFN-β expression was measured 48 and 72 hours later. Similar to the findings from the poly(I:C) application, higher luciferase expression was achieved by administering siRNA. Finally, we investigated the effect of siRNA on IFN-β expression after AAV transduction. Consistent with the above studies, high IFN-β expression was shown by siControl RNA transfection ( Figure 8B As expected, IFN-β expression was almost completely inhibited at both the transcriptional and translational levels when siRNA to MDA5 or MAVS was used ( Figure 8C and Fig.8D ). It is important to note that MDA5 upregulation was rescued when siMAVS was used ( Fig. 8E ). These results suggest that blockade of the dsRNA activation pathway is able to attenuate the innate immune response at later stages after AAV transduction, which results in higher transgene expression.

[0316] The innate immune response system is the first line of defense against pathogens, and its activation in AAV transduction has been studied. Compared with other pathogenic viruses, adeno-associated virus (AAV) infection or its recombinant vector transduction after AAV transduction only induces transient and low innate immunity. In addition, all studies on the innate immune response to viruses focus on the early time point after viral infection. In this study, we first confirmed that the innate immune response is triggered at the later time point after long-term AAV transduction. Late innate immune response activation occurs in different cell lines and human primary hepatocytes. Most importantly, late innate immune response is also detected in human hepatocytes from the liver of human xenotransplanted mice after AAV transduction. Late innate immune response is mediated by dsRNA activation pathways. Blocking of dsRNA sensors or adapters can weaken innate immune response and increase AAV transduction.

[0317] For innate immune response activation, the production of co-stimulatory molecules and inflammatory cytokines is usually upregulated by pattern recognition receptors (PRRs) recognizing pathogen-associated molecular patterns (PAMPs). PRRs have been divided into several families: Toll-like receptors (TLRs), RIG-I-like receptors (RLRs), NOD-like receptors (NLRs), C-type lectin receptors (CLRs), AIM2-like receptors (ALRs), and cytosolic DNA sensors. Previous studies have demonstrated that innate immune responses are induced from AAV infection through the TLR9-MyD88 pathway in plasmacytoid DCs and TLR2 in human non-parenchymal liver cells. Another study has shown that when AAV vectors are administered in mice by systemic administration, increased TLR9 signaling is observed in the liver. According to these studies, innate immune responses are detected within 24 hours.

[0318] In further studies, it was found that strong IFN-α secretion was achieved 18 hours after pDC was infected by AAV transduction. The activation of the innate immune response was triggered in non-parenchymal liver cells (NPC) within 24 hours. It has been confirmed that in mice, systemic administration of AAV vectors leads to rapid induction of inflammatory cytokines, which return to baseline 6 h after AAV injection. This early activation of the innate immune response affects the long-term stable transgene expression after AAV transduction. However, after AAV vector liver targeting, the early activation of the innate immune response in some patients with hemophilia B after the 6th week may not promote the decline of transgene expression.

[0319] In this study, upregulation of IFN-β expression was observed in HeLa cells at day 6 after AAV transduction. In primary human hepatocytes, the pattern of IFN-β expression was inconsistent. In general, high expression of IFN-β was achieved after day 5 in all samples. Finally, we also detected upregulation of IFN-β in human hepatocytes from humanized mice from week 4 to week 8 after AAV administration. These results strongly support the view that activation of the innate immune response is triggered from long-term AAV transduction. This may suppress late transgene expression, as has been shown in clinical trials in some patients with hemophilia.

[0320] The high expression of IFN-β on the first day of AAV transduction in some primary human hepatocytes can be attributed to the innate immune response mediated by TLR9, but not to the TLR2 pathway, as suggested from earlier studies. The mechanism of IFN-β upregulation at later time points after AAV transduction has not been studied. It is unlikely that the activation of the innate immune response in the later stages is triggered by the same mechanism as in the early stages after AAV transduction. In early AAV infection, TLR9 recognizes the dsAAV genome or TLR2 recognizes the AAV capsid, respectively, and plays an important role in activating the innate immune response in pDC or non-parenchymal liver cells. TLR only recognizes PRRs located on the cell surface or in endosomes. After long-term AAV transduction, if PRRs from the AAV vector (dsDNA AAV genome or AAV capsid protein) remain in the endosome, TLRs should continue to recognize these PRRs and induce sustained IFN-β expression. This hypothesis is contrary to our observations that IFN-β expression was at baseline levels during days 2-4 after AAV transduction in HeLa cells in this study. Therefore, some other mechanism should be involved in the activation of the innate immune response in the later stages after AAV transduction. In addition to transmembrane TLRs, cytoplasmic PRRs can also detect viral nucleic acids or proteins from viral infections. In general, RIG-I and MDA5 can recognize cytosolic dsRNA from RNA viruses, and several DNA sensors in the cytoplasm have been identified. NLR proteins are also involved in the innate immune response to viral infections.

[0321] It has been demonstrated that AAV ITRs have promoter function and that the 3' ITR can transcribe negative-strand RNA, which serves as antisense to inhibit transgene expression ( Fig. 9). This antisense RNA can bind to the sense RNA to form dsRNA by annealing in the cytoplasm. The dsRNA produced by AAV vector transduction has the potential to trigger dsRNA innate immune responses by regulating RIG-1 and MDA5 expression. MDA-5 and RIG-1 bind to the common adapter MAVS to promote direct or indirect transcriptional induction of many genes by activating several essential transcription factors, including interferon-regulatory factors (IRFs) and NF-κB to produce IFN-β and inflammatory cytokines. In fact, at later time points after AAV transduction, activation of MDA5 was observed in HeLa cells, primary human hepatocytes, and hepatocytes from humanized mice.

[0322] Upregulation of MDA5, but not RIG-1, further supports our hypothesis that dsRNA can be formed from negative-strand RNA from AAV 3'-ITR because MDA5 recognizes long dsRNA. This result shows that dsRNA-mediated activation of the innate immune response is triggered after long-term AAV transduction. Using siRNA to block the dsRNA sensor MDA5 or the adapter MAVS, IFN-β expression was inhibited and transgene expression was increased at the later time point of AAV infection. These results further support that in patients with hemophilia B receiving AAV gene therapy, dsRNA-activated innate immune responses promote therapeutic FIX reduction at later times. Regarding how dsRNA-mediated activation of the innate immune response is only detected in the late stages of AAV transduction, one of the possible mechanisms is that the promoter of AAV ITR is very weak. Therefore, it takes a relatively long time to produce enough antisense RNA from AAV 3'ITR to reach the threshold and form dsRNA. In addition, the biology of AAV vector transduction can play a role in dsRNA formation in the late stages of AAV transduction. Unlike adenoviral vectors, transgene expression reaches its peak at week 6 in preclinical and clinical trials and remains constant for a long time after AAV vector administration. Therefore, large amounts of negative-strand RNA can be synthesized only in the late stages of AAV transduction.

[0323] In short, our study revealed a new mechanism by which long-term AAV transduction activates the innate immune response through the cytoplasmic dsRNA recognition pathway in transduced cells, which leads to the production of type I IFN-β. Transient blockade of the dsRNA pathway in AAV-transduced cells reduced IFN-β expression and increased transgene expression. These results provide valuable information that helps us design effective methods to interfere with the dsRNA pathway to improve AAV transduction.

[0324] Example 2

[0325] Blocking AAV ITR promoter function.The exact mechanism by which dsRNA transduction from AAV induces an innate immune response is unknown. One possibility is the promoter function of the ITR and the potential bidirectional function of the promoter for transgene expression. Negative-strand RNA transcription from the 3'-ITR or promoter, and positive-strand RNA from the promoter or 5'-ITR can form double-stranded RNA, which triggers an innate immune response. To prevent transcription initiated through the ITR, we added poly(A) downstream of the 5'-ITR or upstream of the promoter and 3'-ITR to block long RNA transcripts. Poly(A) can act as a single segment ( Fig. 10A ) or in combination ( Fig. 10B ) are located in different locations.

[0326] Modify AAV ITRs to reduce their promoter function or use promoter-free ITRs from different serotypes ITR is optional. Until recently, 13 AAV serotypes and more than 100 variants have been isolated. These AAV serotypes and mutants use different ITRs for viral replication and packaging. In particular, we studied the promoter function from the AAV5 ITR because there are some differences between the AAV5 ITR and the AAV2 ITR ( Fig.11 and Table 7). AAV5 ITR has 5 repeats for RBE and a longer spacer between RBE and trs ( Fig.11 and Table 7). To compare promoter functions from different ITRs, we first generated cassettes with different ITRs to drive the GFP transgene. After co-transfection of plasmids with CMV / Laz as an internal control into 293 cells, 293 cells were visualized under a fluorescent microscope and stained with LacZ 2 days later ( Fig.12 ).

[0327] When compared to ITR2 / GFP, almost no GFP-positive cells were seen from the transfection of ITR5 / GFP. To further confirm the results from GFP expression, we used the ITRs to generate other cassettes to drive the human alpha-1 antitrypsin transgene (AAT). After transfection into different cells, the AAT levels in the supernatant were significantly lower in the ITR5 group than in the ITR2 group in all cell lines tested ( Fig.13 ). We packaged ITR5 / AAT or ITR2 / AAT into AAV2 or AAV5 capsids. After transduction of 293 cells, consistent with the results from plasmid transfection, lower AAT expression was observed from AAV / ITR5 / AAT transduction regardless of the different capsids ( Fig.14 ). Following intramuscular injection of these vectors, AAT expression in the blood was measured at week 4 after AAV administration. Similar to the in vitro transduction data, ITR5 induced significantly lower AAT expression than AAV2 ( Fig.15). In summary, these results suggest that AAV5 ITRs have weaker promoter function than AAV2 ITRs. It is possible that ITRs from other serotypes or variants may have no promoter function. These ITRs without promoter function will be used to generate AAV cassettes.

[0328] Knockdown of dsRNA sensors. MDA5 and RIG-I and protein kinase (PKR) are cytoplasmic dsRNA sensors. Silencing these molecules can block the innate immune response triggered by cytoplasmic dsRNA. siRNA for specific sensors can be used at different time points after AAV transduction. For transgenic expression of specific sensors, shRNA driven by RNA polymerase III or miRNA driven by the same RNA polymerase II can be used with a separate vector ( Fig.16 , Figure A) or as a single vector linked to a transgene cassette. When a single vector is used, the shRNA or miRNA can be located at different locations.

[0329] 1. For shRNA between poly(A) and 3' AAV ITR ( Fig.16 , Figure B)

[0330] 2. For shRNA between 5' AAV ITR and promoter ( Fig.16 , Figure C)

[0331] 3. For miRNA between the transgene and 3' AAV ITR ( Fig.16 , Figure D)

[0332] 4. For miRNA between promoter and transgene ( Fig.16 , Figure E)

[0333] 5. miRNA is inserted into the transgenic intron ( Fig.16 , Figure F)

[0334] Silencing of molecules involved in pathways that activate the innate immune response to dsRNA.Cytosolic viral RNA is recognized by receptors RIG-I and MDA5, which activate mitochondrial antiviral signaling protein (MAVS) through caspase-recruitment domain (CARD)–CARD interactions. MAVS recruits various signaling molecules to trigger downstream signaling, such as TNF receptor-associated factor 6 (TRAF6) and TRAF5. TRAF6, as well as other intracellular proteins, activate NF-κB signaling through receptor-interacting protein 1 (RIP1) and FAS-associated death domain protein (FADD). NF-κB signaling phosphorylates NF-κB inhibitor-α (IκBα) and initiates proinflammatory cytokine gene expression. MAVS also activates interferon regulatory factor (IRF) signaling. Knockdown of MAVS or molecules involved in MAVS downstream signaling using the same strategy as above will block dsRNA innate immune responses.

[0335] MAVS signaling can also be inhibited by various molecules from viral infection. For example, the serine protease NS3–4A from hepatitis C virus, the protease from hepatitis A virus and GB virus B, and the hepatitis B virus (HBV) X protein. During viral infection, some endogenous proteins, such as poly(rC)-binding protein 2, 20S proteasome subunit PSMA7, and mitochondrial fusion protein 2, can inhibit MAVS signaling. These proteins (inhibitors) can be delivered with different vectors ( Fig.17 , Figure A) or in a single vector fused to a transgene ( Fig.17 , Figure B) or driven by different promoters ( Fig.17 , Figure C) to block the dsRNA immune response during therapeutic transgene expression.

[0336] Blocking dsRNA innate immune response activation pathways. In addition to blocking the genetic pathways of the innate immune response to dsRNA, chemicals can also be used to interfere with the dsRNA activation pathway. PKR is phosphorylated and activated by dsRNA and promotes the induction of type I interferons, such as IFN-β, which can further increase its expression. 2-Aminopurine (2-AP) is a potent inhibitor of double-stranded RNA (dsRNA)-activated protein kinase (PKR). Steroids, such as hydrocortisone ( Fig.18 and 19 ).

[0337] Example 3

[0338] Produced from negative strand RNA of the AAV 3′-ITR following AAV transduction.To investigate whether negative-strand RNA can be generated from the AAV 3′-ITR promoter, HeLa cells were infected with the AAV2 / luciferase vector and harvested 8 days later for RNA extraction. cDNA synthesis was performed using sense or antisense primers for the luciferase transgene (Table 2). Two pairs of luciferase-specific PCR primers were used to detect positive or negative-strand transcripts ( Fig. 20A Both positive and negative strand transcripts were detected after AAV transduction, and there were no PCR products when RNA was used as template ( Fig. 20B ). The negative strand transcript was detected only when the cDNA template was diluted 200-fold. However, we were able to detect the positive strand transcript even at a 2,000-fold dilution of the cDNA template ( Fig. 20B ). This result shows that reverse-oriented transcripts can be generated from AAV transduction, and the efficiency of negative-strand RNA formation is significantly lower than that of positive-strand RNA. It also supports the possibility that positive-strand RNA and negative-strand RNA generated from different orientations can form dsRNA in AAV-transduced cells.

[0339] Increased AAV transduction in cells with MAVS knockdown. Inhibition of MAVS expression with siRNA oligos has been shown to increase AAV transduction. We examined transduction efficiency in cells with MAVS deficiency. Following transduction of AAV2 / luc vectors, higher transgene expression was consistently achieved in a human hepatocyte cell line with MAVS knockdown (PH5CH8-MAVS-KO) than in PH5CH8 cells ( Fig.21 ). The increased transduction was independent of vector dose and transduction duration.

[0340] Efficient knockdown with MAVS shRNA. We designed five shRNAs driven by the U6 promoter that have the potential to silence human MAVS ( Fig.22A After transfecting shRNA plasmids into Hela cells, we examined the expression of MAVS and found that #31 induced the strongest MAVS knockdown ability ( Fig. 22B ). Therefore, MAVS shRNA #31 was selected for subsequent studies.

[0341] Enhanced AAV transduction in cells with shRNA silencing of MAVS. To investigate whether knocking down MAVS with shRNA increases AAV transduction, we first transfected Hela cells with MAVS shRNA #31. The next day, AAV2 / luc vector was added. Transgene expression was measured on days 1 and 4 after AAV transduction. Fig.23 As shown, higher AAV transduction was observed in cells with MAVS silencing.

[0342] In conclusion, increased AAV transduction was achieved when target cells lacked MAVS. This result suggests that integration of MAVS shRNA into the AAV cassette can induce higher AAV transduction by blocking dsRNA-mediated activation of the innate immune response.

[0343] Although specific embodiments of the present invention are shown and described, it should be understood that the present invention is not limited thereto, but may be embodied and implemented in other ways within the scope of the appended claims. Because many modifications and optional embodiments of the present invention are obvious to those skilled in the art, this description should be interpreted as being merely illustrative and for the purpose of teaching those skilled in the art the best way to implement the present invention. Therefore, all suitable modifications and equivalents may be considered to fall within the scope of the appended claims.

[0344] Table 1 : Information about human primary hepatocytes

[0345]

[0346] Table 2 Primers used in this study

[0347]

[0348]

[0349] Table 3 :

[0350]

[0351] Table 4 : Amino acid residues and abbreviations

[0352]

[0353] Table 5 :

[0354]

[0355] Table 6 :

[0356]

[0357] Table 7 : Comparison between TR2 and TR5

[0358]

Claims

1. A recombinant adeno-associated virus (AAV) vector designed to reduce the production of double-stranded RNA in AAV vector transduction to inhibit the innate immune response that may be generated by AAV vector transduction, comprising a nucleotide sequence of interest (NOI) operably associated with a promoter in a 5' to 3' orientation, an adeno-associated virus (AAV) 5' inverted terminal repeat (ITR) located upstream of the NOI, and an AAV 3' ITR located downstream of the NOI, and further comprising one or more poly A (pA) sequences selected from the following: a) a poly A (pA) sequence oriented in 3' to 5' direction downstream of the 5' ITR and upstream of the promoter and a pA sequence oriented in 3' to 5' direction upstream of the 3' ITR and downstream of the NOI, wherein the 3' ITR has promoter activity; b) a pA sequence oriented in 3' to 5' direction upstream of the 3' ITR and downstream of the NOI, wherein the 3' ITR has promoter activity; c) a first pA sequence oriented in 3' to 5' direction, upstream of the 3' ITR and downstream of the NOI, and a second pA sequence oriented in 5' to 3' direction, downstream of the first pA sequence and upstream of the 3' ITR, wherein the 3' ITR has promoter activity; d) a first pA sequence oriented in 3' to 5' direction, upstream of the 3' ITR and downstream of the NOI, and a second pA sequence oriented in 5' to 3' direction, downstream of the NOI and upstream of the first pA, wherein the 3' ITR has promoter activity; e) a first pA sequence oriented in 3' to 5' direction, upstream of the 3' ITR and downstream of the NOI, and a second pA sequence oriented in 5' to 3' direction, downstream of the 5' ITR and upstream of the promoter, wherein the 3' ITR has promoter activity and wherein the 5' ITR has promoter activity; f) a first pA sequence oriented in 3' to 5', downstream of the 5' ITR and upstream of the promoter, a second pA sequence oriented in 5' to 3', downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented in 3' to 5', downstream of the second pA sequence and upstream of the 3' ITR, wherein the 3' ITR has promoter activity; g) a first pA sequence oriented in 5' to 3' direction downstream of the 5' ITR and upstream of the promoter, a second pA sequence oriented in 3' to 5' direction downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented in 5' to 3' direction downstream of the second pA sequence and upstream of the 3' ITR, wherein the 5' ITR has promoter activity and wherein the 3' ITR has promoter activity; h) a first pA sequence oriented in 5' to 3' direction downstream of the 5' ITR and upstream of the promoter, a second pA sequence oriented in 5' to 3' direction downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented in 3' to 5' direction downstream of the second pA sequence and upstream of the 3' ITR, wherein the 5' ITR has promoter activity and wherein the 3' ITR has promoter activity; i) a first pA sequence oriented in 5' to 3' direction downstream of the 5' ITR and upstream of the promoter, a second pA sequence oriented in 3' to 5' direction downstream of the NOI and upstream of the third pA sequence, and a third pA sequence oriented in 5' to 3' direction downstream of the second pA sequence and upstream of the 3' ITR, wherein the 3' ITR has promoter activity; j) a first pA sequence oriented in 3' to 5', downstream of the 5' ITR and upstream of the second pA sequence, a second pA sequence oriented in 5' to 3', downstream of the first pA sequence and upstream of a promoter, a third pA sequence oriented in 5' to 3', downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented in 3' to 5', downstream of the third pA sequence and upstream of the 3' ITR, wherein the 5' ITR has promoter activity and wherein the 3' ITR has promoter activity; k) a first pA sequence oriented in 3' to 5', downstream of the 5' ITR and upstream of the second pA sequence, a second pA sequence oriented in 5' to 3', downstream of the first pA sequence and upstream of a promoter, a third pA sequence oriented in 3' to 5', downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented in 5' to 3', downstream of the third pA sequence and upstream of the 3' ITR, wherein the 5' ITR has promoter activity and wherein the 3' ITR has promoter activity; l) a first pA sequence oriented 5' to 3', downstream of the 5'ITR and upstream of the second pA sequence, a second pA sequence oriented 3' to 5', downstream of the first pA sequence and upstream of a promoter, a third pA sequence oriented 5' to 3', downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented 3' to 5', downstream of the third pA sequence and upstream of the 3'ITR, wherein the 5'ITR has promoter activity and wherein the 3'ITR has promoter activity; and / or m) a first pA sequence oriented 5' to 3', downstream of the 5'ITR and upstream of the second pA sequence, a second pA sequence oriented 3' to 5', downstream of the first pA sequence and upstream of a promoter, a third pA sequence oriented 3' to 5', downstream of the NOI and upstream of the fourth pA sequence, and a fourth pA sequence oriented 5' to 3', downstream of the third pA sequence and upstream of the 3'ITR, wherein the 5'ITR has promoter activity and wherein the 3'ITR has promoter activity; The rAAV vector does not further contain a NOI in a 3' to 5' orientation and / or a promoter in a 3' to 5' orientation.

2. The rAAV vector of claim 1, wherein the one or more pA sequences are b) a pA sequence upstream of the 3' ITR and downstream of the NOI in a 3' to 5' orientation.

3. The rAAV vector of claim 1 or 2, wherein the 5'ITR and / or 3'ITR is modified to reduce or eliminate promoter activity from the 5'ITR and / or 3'ITR.

4. The rAAV vector of claim 3, wherein the 5'ITR and / or the 3'ITR is an AAV5 ITR or an AAV2 ITR.

5. The rAAV vector of any one of claims 1 to 4, wherein the NOI sequence is fused to one or more nucleic acids encoding an interfering RNA (RNAi) or a functional RNA targeting a cytoplasmic dsRNA sensor and / or a nucleic acid encoding an inhibitor of MAVS signal transduction.

6. The rAAV vector of claim 5, wherein the one or more nucleic acids encoding RNAi or functional RNA targeting a cytoplasmic dsRNA sensor are operably associated with a second promoter.

7. The rAAV vector of claim 5 or 6, wherein the RNAi is small interfering RNA (siRNA), short hairpin RNA (shRNA), micro RNA (miRNA), long double-stranded RNA (long dsRNA), antisense RNA or ribozyme.

8. The rAAV vector of claim 7, wherein the RNAi is siRNA or shRNA that inhibits MDA5 or siRNA or shRNA that inhibits MAVS.

9. The rAAV vector of claim 8, wherein the siRNA that inhibits MDA5 is SEQ ID NO: 1 and the siRNA that inhibits MAVS is SEQ ID NO: 2, SEQ ID NO: 3 or SEQ ID NO: 4, and the shRNA that inhibits MAVS is SEQ ID NO:

38.

10. The rAAV vector of claim 5 or 8, wherein the inhibitor of MAVS signaling is selected from: serine protease NS3-4A from hepatitis C virus, protease from hepatitis A virus, protease from GB virus B, hepatitis B virus (HBV) X protein, poly (rC)-binding protein 2, 20S proteasome subunit PSMA7, mitochondrial fusion protein 2, and any combination thereof.

11. The rAAV vector of any one of claims 7-9, wherein the vector comprises an AAV 5' ITR, an siRNA operably associated with a first promoter, a NOI operably associated with a second promoter, a pA sequence oriented in 3' to 5' direction, and an AAV 3' ITR.

12. The rAAV vector of any one of claims 7 to 9, comprising, in the following order, an AAV 5' ITR, a NOI and a siRNA operably associated with a promoter, a pA sequence oriented in 3' to 5' orientation, and an AAV 3' ITR.

13. The rAAV vector of any one of claims 7 to 9, comprising, in the following order, an AAV 5' ITR, an siRNA and a NOI operably associated with a promoter, a pA sequence oriented in 3' to 5' direction, and an AAV 3' ITR.

14. The rAAV vector of any one of claims 7-9, comprising, in the following order, an AAV 5'ITR, a NOI comprising an siRNA intron sequence inside it, the NOI operably associated with a promoter, a pA sequence oriented in 3' to 5' direction, and an AAV 3'ITR.

15. An rAAV vector particle comprising the rAAV vector of any one of claims 1-14.

16. The rAAV particle of claim 15, which is a first AAV serotype, wherein the AAV 5' ITR and / or AAV 3' ITR is from a second AAV serotype different from the first AAV serotype.

17. The rAAV particle of claim 16, wherein the first AAV serotype is AAV2 and the second AAV serotype is AAV5.

18. The rAAV vector particle of any one of claims 15-17, wherein the rAAV vector is the rAAV vector of claim 2.

19. A composition comprising the rAAV vector particle of any one of claims 15-18.

20. The composition of claim 19, further comprising a recombinant nucleic acid molecule encoding an interfering RNA sequence targeting a cytoplasmic dsRNA sensor and / or a recombinant nucleic acid molecule encoding an inhibitor of MAVS signaling.

21. Use of the rAAV vector particle comprising the rAAV vector of claim 15 or the composition of claim 19 or 20 in the preparation of a medicament for reducing the innate immune response associated with AAV transduction.

22. The use of claim 21, further comprising use of an agent that interferes with a dsRNA activation pathway in cells of a subject in the preparation of a medicament for enhancing transduction of an AAV vector in cells of a subject.

23. The use of claim 22, wherein the agent that interferes with the dsRNA activation pathway in the cells of the subject is 2-aminopurine or hydrocortisone.

24. The use of claim 22 or claim 23, wherein the AAV vector and the agent are used simultaneously.

25. The use of claim 22 or claim 23, wherein the AAV vector and the agent are administered at separate times.

26. A plasmid comprising the rAAV vector of any one of claims 1-14.

27. A cell comprising the plasmid of claim 26.

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