Vector

Abstract

A vector system for expressing a transgene in a cell, comprising a first vector and a second vector, (a) the first vector comprising, in a 5' to 3' direction, a promoter, an intron, a 5'-end portion of a transgene coding sequence (CDS), a splice donor sequence, and a first recombination induction region, and (b) the second vector comprising, in a 5' to 3' direction, a second recombination induction region, a splice acceptor sequence, and a 3'-end portion of the transgene CDS, wherein the 5'-end portion and the 3'-end portion together constitute the transgene CDS, and the intron is not capable of homologous recombination with the splice donor sequence to excise the 5'-end portion of the transgene CDS.

Description

[Technical field]

[0001] The present invention relates to vectors and vector systems, in particular to vectors and vector systems that allow the delivery of large transgenes to target cells. The present invention also relates to the use of vectors and vector systems in gene therapy. [Background technology]

[0002] Gene therapy, for example using adeno-associated virus (AAV) vectors, is a promising approach for the treatment of many inherited retinal degenerations (IRDs). Indeed, years of preclinical studies and clinical trials on various IRDs have shown that AAV can efficiently deliver therapeutic genes to affected retinal layers (e.g., photoreceptors (PR) and retinal pigment epithelium (RPE)), highlighting its excellent safety and efficacy profile in humans. However, one of the main obstacles in utilizing AAV gene therapy vectors is their capacity to package transgenes, which may be limited to a maximum of about 5 kb. This may be a limiting factor for the development of gene replacement therapy for diseases such as IRDs caused by mutations in genes with coding sequences (CDS) larger than 5 kb.

[0003] There has been great interest in identifying strategies to increase the capacity of AAV. For example, dual AAV vectors, based on the ability of AAV genome to concatemerize through intermolecular recombination, have been successfully exploited to address this issue. Dual AAVs can be generated by splitting a large transgene CDS into separate parts and packaging each into a single normal-sized (NS; less than 5 kb) AAV vector. Reconstitution of the full-length transgene CDS can be achieved upon coinfection of the same cell with both dual AAV vectors followed by either i) inverted terminal repeat (ITR)-mediated tail-to-head concatemerization of the two vector genomes followed by splicing (dual AAV trans-splicing, TS) (Duan et al. (2001) Molecular Therapy: the journal of the American Society of Gene Therapy 4:383-391), ii) homologous recombination between the overlapping regions contained in the two vector genomes (double AAV overlap, OV) (Duan et al. (2001) Molecular Therapy: the journal of the American Society of Gene Therapy 4:383-391), or iii) a combination of the two (double AAV hybrid) (Ghosh et al. (2008). Molecular Therapy: the journal of the American Society of Gene Therapy 16:124-130).

[0004] The most used recombinogenic region in the context of double AAV hybrid vectors is derived from an 872 bp sequence in the central third of the human alkaline phosphatase cDNA that has been shown to confer high levels of double AAV hybrid vector rearrangement. In addition, a 77 bp sequence from the F1 phage genome (AK) has been found to be highly recombinogenic in in vitro and in vivo experiments.

[0005] Although studies have highlighted the potential of dual vector systems, such as AAV vector systems, for delivery and reconstitution of large transgenes in tissues of interest, their translation into clinical use remains a major unmet need. Summary of the Invention

[0006] During the development of a dual vector system for the delivery of large transgenes (e.g., myosin 7A, MYO7A), the inventors unexpectedly found consistent contamination in their preparations of vectors containing the 5'-end portion of the transgene CDS. The inventors analyzed the preparations using Southern blots and identified a band corresponding to the expected vector, and surprisingly also found a smaller sized band of approximately 1.3 kb corresponding to the contaminant.

[0007] Subsequently, the inventors further studied the vector to identify regions of homology between the chimeric promoter intron and the splice donor (SD) site used in the vector. Further sequencing analysis of purified viral DNA confirmed that the presence of these regions of homology within the construct led to a homologous recombination event, which resulted in the deletion of the remaining part of the intron, the 5'-end part of the transgene CDS and the SD site while retaining the AAV inverted terminal repeats (ITR), thus supporting vector production.

[0008] In one aspect, the present invention provides a vector system for expressing a transgene in a cell, the vector system comprising a first vector and a second vector, wherein (a) a first vector comprising, in a 5' to 3' direction, an intron, a 5' terminal portion of a transgene coding sequence (CDS), and a splice donor sequence; (b) a second vector comprising, in the 5' to 3' direction, a splice acceptor sequence and a 3' end portion of a transgene CDS; Here, the 5'-end portion and the 3'-end portion together constitute a transgene CDS, and wherein the intron is not capable of homologous recombination with the splice donor sequence to excise the 5'-end portion of the transgene CDS.

[0009] In another aspect, the present invention provides a vector system for expressing a transgene in a cell, the vector system comprising a first vector and a second vector, wherein: (a) a first vector comprising, in a 5' to 3' direction, a promoter, an intron, a 5' terminal portion of a transgene coding sequence (CDS), and a splice donor sequence; (b) a second vector comprising, in the 5' to 3' direction, a splice acceptor sequence and a 3' end portion of a transgene CDS; Here, the 5'-end portion and the 3'-end portion together constitute a transgene CDS, and wherein the intron is not capable of homologous recombination with the splice donor sequence to excise the 5'-end portion of the transgene CDS.

[0010] In another aspect, the present invention provides a vector system for expressing a transgene in a cell, the vector system comprising a first vector and a second vector, wherein: (a) a first vector includes, in a 5' to 3' direction, a promoter, an intron, a 5' end portion of a transgene coding sequence (CDS), a splice donor sequence, and a first recombinogenic region; (b) a second vector comprising, in the 5' to 3' direction, a second recombinogenic region, a splice acceptor sequence, and a 3'-terminal portion of the transgene CDS; Here, the 5'-end portion and the 3'-end portion together constitute a transgene CDS, and wherein the intron is not capable of homologous recombination with the splice donor sequence to excise the 5'-end portion of the transgene CDS.

[0011] In a preferred embodiment, the intron is a simian virus 40 (SV40) intron. The SV40 intron may be a modified SV40 intron.

[0012] In some embodiments, the intron is a minute virus of mouse (MVM) intron.

[0013] In some embodiments, the intron comprises a nucleotide sequence having at least 95% sequence identity (e.g., at least 96%, 97%, 98% or 99% sequence identity, or 100% sequence identity) to SEQ ID NO: 3 or 4. In preferred embodiments, the intron comprises a nucleotide sequence having at least 95% sequence identity (e.g., at least 96%, 97%, 98% or 99% sequence identity, or 100% sequence identity) to SEQ ID NO: 3.

[0014] In some embodiments, the splice donor sequence comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:5.

[0015] In a preferred embodiment, the first recombinogenic region and the second recombinogenic region are the same.

[0016] In some embodiments, the first recombinogenic region and the second recombinogenic region are both F1 phage recombinogenic regions or fragments thereof.

[0017] In some embodiments, the first recombinogenic region and the second recombinogenic region both comprise a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:7, or a fragment thereof.

[0018] In some embodiments, the first vector and the second vector are viral vectors. The viral vectors can be adeno-associated virus (AAV) vectors, adenovirus vectors, retrovirus vectors, lentivirus vectors, herpes simplex virus vectors, picornavirus vectors or alphavirus vectors. In some embodiments, the first vector and the second vector are plasmids. The first and / or second plasmids can be used, for example, to produce the first and / or second viral vector particles (separately or together in a composition).

[0019] In a preferred embodiment, the first vector and the second vector are AAV vectors.

[0020] In some embodiments, the AAV vectors have the same serotype (e.g., comprise capsids of the same serotype). In some embodiments, the AAV vectors have different serotypes (e.g., comprise capsids of different serotypes).

[0021] In some embodiments, the first vector and the second vector are selected from the group consisting of AAV2, AAV8, AAV5, AAV7, AAV9, AAV-PhP.B and AAV-PhP.eB. In some embodiments, the first vector and the second vector are selected from the group consisting of hu68 (see, e.g., WO 2018 / 160582), Anc library (see, e.g., WO 2015 / 054653 and WO 2017 / 019994) and AAV2-TT (see, e.g., WO 2015 / 121501).

[0022] In some embodiments, the first vector and the second vector are AAV2 vectors. In some embodiments, the first vector and the second vector are AAV8 vectors.

[0023] In some embodiments, the first vector and the second vector comprise a capsid selected from the group consisting of AAV2, AAV8, AAV5, AAV7, AAV9, AAV-PhP.B, and AAV-PhP.eB. In some embodiments, the first vector and the second vector comprise a capsid selected from the group consisting of hu68 (see, e.g., WO 2018 / 160582), Anc library (see, e.g., WO 2015 / 054653 and WO 2017 / 019994), and AAV2-TT (see, e.g., WO 2015 / 121501).

[0024] In some embodiments, the first vector and the second vector are AAV2 capsids. In some embodiments, the first vector and the second vector are AAV8 capsids.

[0025] In some embodiments, the first vector further comprises a 5'ITR and a 3'ITR. In some embodiments, the second vector further comprises a 5'ITR and a 3'ITR. In preferred embodiments, the first vector further comprises a 5'ITR and a 3'ITR and the second vector further comprises a 5'ITR and a 3'ITR.

[0026] In preferred embodiments, the ITRs are AAV ITRs, preferably AAV2 ITRs. In some embodiments, the ITRs are AAV8 ITRs.

[0027] In a preferred embodiment, the first vector and the second vector are AAV2 / 8 vectors.

[0028] In some embodiments, the ITRs are from the same AAV serotype. In some embodiments, the ITRs are from different AAV serotypes.

[0029] In a preferred embodiment, the 3' ITR of the first vector and the 5' ITR of the second vector are derived from the same AAV serotype.

[0030] In a preferred embodiment, the 5'ITR of the first vector and the 5'ITR of the second vector are from the same AAV serotype. In a preferred embodiment, the 3'ITR of the first vector and the 3'ITR of the second vector are from the same AAV serotype.

[0031] In a preferred embodiment, the 5'ITR of the first vector and the 5'ITR of the second vector are AAV2 5'ITR and the 3'ITR of the first vector and the 3'ITR of the second vector are AAV2 3'ITR.

[0032] In some embodiments, the 5'ITR of the first vector and the 5'ITR of the second vector are AAV8 5'ITR, and the 3'ITR of the first vector and the 3'ITR of the second vector are AAV8 3'ITR.

[0033] In some embodiments, the 5'ITR of the first vector and the 5'ITR of the second vector are from different AAV serotypes. In some embodiments, the 3'ITR of the first vector and the 3'ITR of the second vector are from different AAV serotypes. In some embodiments, the 5'ITR of the first vector and the 5'ITR of the second vector are from different AAV serotypes, and the 3'ITR of the first vector and the 3'ITR of the second vector are from different AAV serotypes.

[0034] In some embodiments, the 5' ITR of the first vector and the 3' ITR of the second vector are from different AAV serotypes.

[0035] In some embodiments, the first vector and the second vector are viral vector particles.

[0036] In some embodiments, the promoter is a CBA promoter or a fragment thereof.

[0037] In some embodiments, the first vector further comprises an enhancer sequence. In a preferred embodiment, the enhancer is a CMV enhancer.

[0038] In some embodiments, the second vector further comprises a polyadenylation sequence downstream of the 3' end portion of the transgene CDS. In a preferred embodiment, the polyadenylation sequence is the bovine growth hormone (bGH) polyadenylation sequence.

[0039] In some embodiments, the transgene is selected from the group consisting of myosin 7A (MYO7A), ABCA4, CEP290, CDH23, EYS, USH2a, GPR98 and ALMS1.

[0040] In a preferred embodiment, the transgene is a myosin 7A (MYO7A) transgene. In some embodiments, the transgene is an ABCA4 transgene. In some embodiments, the transgene CDS is a wild-type sequence. In some embodiments, the transgene CDS is codon-optimized (e.g., codon-optimized for expression in humans).

[0041] In a preferred embodiment, (a) the first vector comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 14; and / or (b) the second vector comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:15.

[0042] In a particularly preferred embodiment, (a) the first vector comprises the nucleotide sequence of SEQ ID NO: 14, and / or (b) the second vector comprises the nucleotide sequence of SEQ ID NO:15.

[0043] In some embodiments, the first vector and the second vector are present in a 1:1 genome copy ratio.

[0044] In another aspect, the invention provides a method of expressing a transgene in a cell, comprising transducing or transfecting the cell with a first vector and a second vector as disclosed herein, such that the transgene is expressed in the cell.

[0045] In another aspect, the present invention provides a cell comprising a first vector and a second vector as disclosed herein.

[0046] In another aspect, the invention provides a cell transduced or transfected with a first vector and a second vector as disclosed herein.

[0047] In some embodiments, the cell is a mammalian cell, a human cell, a retinal cell, or a non-embryonic stem cell.

[0048] In another aspect, the present invention provides a vector, wherein the vector is a first vector as disclosed herein.

[0049] In another aspect, the invention provides a vector, wherein the vector is a second vector as disclosed herein.

[0050] In another aspect, the invention provides a vector comprising, in a 5' to 3' direction, an intron, a 5' terminal portion of a transgene coding sequence (CDS), and a splice donor sequence, wherein the intron is not capable of homologous recombination with the splice donor sequence to excise the 5' terminal portion of the transgene CDS.

[0051] In another aspect, the present invention provides a vector comprising, in a 5' to 3' direction, a promoter, an intron, a 5' terminal portion of a transgene coding sequence (CDS), and a splice donor sequence, wherein the intron is not capable of homologous recombination with the splice donor sequence to excise the 5' terminal portion of the transgene CDS.

[0052] In another aspect, the present invention provides a vector comprising, in a 5' to 3' direction, a promoter, an intron, a 5' terminal portion of a transgene coding sequence (CDS), a splice donor sequence, and a recombinogenic region, wherein the intron is not capable of homologous recombination with the splice donor sequence to excise the 5' terminal portion of the transgene CDS.

[0053] In some embodiments, the vector further comprises a 5'ITR and a 3'ITR. In a preferred embodiment, the ITRs are AAV ITRs, preferably AAV2 ITRs.

[0054] In some embodiments, the ITRs are from the same AAV serotype. In some embodiments, the ITRs are from different AAV serotypes.

[0055] In another aspect, the invention provides a vector comprising, in a 5' to 3' direction, a splice acceptor sequence and a 3' terminal portion of a transgene coding sequence (CDS).

[0056] In another aspect, the invention provides a vector comprising, in a 5' to 3' direction, a recombinogenic region, a splice acceptor sequence, and a 3' terminal portion of a transgene coding sequence (CDS).

[0057] In some embodiments, the vector comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:14.

[0058] In a preferred embodiment, the vector comprises the nucleotide sequence of SEQ ID NO:14.

[0059] In another aspect, the present invention provides a kit comprising a first vector as disclosed herein and a second vector as disclosed herein.

[0060] In another aspect, the invention provides a composition comprising a first vector as disclosed herein and a second vector as disclosed herein.

[0061] In some embodiments, the first vector and the second vector are present in a 1:1 genome copy ratio.

[0062] In a preferred embodiment, the composition is a pharmaceutical composition comprising a pharma- ceutically acceptable carrier, diluent or excipient.

[0063] In another aspect, the present invention provides a vector system, vector, kit or composition of the invention for use in therapy.

[0064] In another aspect, the present invention provides a vector system, vector, kit or composition of the invention for use in the treatment of a retinal degeneration. Preferably, the retinal degeneration is an inherited retinal degeneration.

[0065] In another aspect, the present invention provides a first vector as disclosed herein for use in therapy, wherein the first vector is administered in combination, simultaneously, sequentially or separately, with a second vector as disclosed herein.

[0066] In another aspect, the present invention provides a first vector as disclosed herein for use in treating retinal degeneration, wherein the first vector is administered simultaneously, sequentially or separately in combination with a second vector as disclosed herein. Preferably, the retinal degeneration is an inherited retinal degeneration.

[0067] In another aspect, the invention provides a second vector as disclosed herein for use in therapy, wherein the second vector is administered in combination, simultaneously, sequentially or separately, with a first vector as disclosed herein.

[0068] In another aspect, the present invention provides a second vector as disclosed herein for use in treating retinal degeneration, wherein the second vector is administered in combination, simultaneously, sequentially or separately, with a first vector as disclosed herein. Preferably, the retinal degeneration is an inherited retinal degeneration.

[0069] In some embodiments, the use is in the treatment or prevention of Usher syndrome, retinitis pigmentosa, Leber congenital amaurosis (LCA), Stargardt disease, Alström syndrome, or an ABCA4-related disorder.

[0070] In another aspect, the present invention provides a vector system, vector, kit or composition of the invention for use in the treatment of Usher Syndrome.

[0071] In another aspect, the present invention provides a method for treating or preventing retinal degeneration, comprising administering to a subject in need thereof an effective amount of the vector system, vector, kit or composition of the present invention. Preferably, the retinal degeneration is an inherited retinal degeneration.

[0072] In another aspect, the present invention provides a method for treating or preventing Usher Syndrome, comprising administering to a subject in need thereof an effective amount of a vector system, vector, kit or composition of the present invention. [Brief description of the drawings]

[0073] [Figure 1-1] Identification of contaminant vectors. (A) Southern blot image showing genomes corresponding to AAV-5'hMYO7A (indicated by upper arrow) and contaminant vector (indicated by lower arrow). DNAse: treatment with DNAse for degradation of contaminating external DNA, Plasmid: plasmid DNA containing DNA sequence to generate AAV8-CBA-chimeric intron-5'hMYO7A, genomic DNA extracted from 5'AAV genome-CI: AAV8-CBA promoter-chimeric intron-5'hMYO7A, molecular weight markers expressed in kilobases, bp = base pairs. CI: chimeric intron. (B) Representation of AAV-5'hMYO7A genome showing sequences recognized by Southern blot probe. (C) Pairing mechanism between intron of chimeric promoter and SD signal (indicated by dotted line). (D) Representation of contaminant vector genome showing sequences recognized by Southern blot probe. [Figure 1-2]Continuing from FIG. 1-1, (E) Southern blot analysis of AAV preparations containing the following expression cassettes: 1.5' CMV ABCA4 AK (double hybrid), 2.5' CMV ABCA4 TS (double trans-spliced), 3.5' CMV NO INTR ABCA4 OV (double overlap), 4.5' CMV NO INTR ABCA4 AK (double hybrid), 5.5' VMD2 ABCA4 AK (double hybrid), 6.5' RHO ABCA4 AK (double hybrid), 7.5' RHO ABCA4 TS (double trans-spliced). Chimeric introns are present in vectors 1 and 2 and absent in vectors 3-7. Dashed boxes indicate full-length genomes of expected size, solid boxes indicate shorter truncated genomes. [Diagram 2] In vitro comparison of EGFP fluorescence with chimeric introns, SV40 introns, MVM introns and no introns. (A) Representation of plasmids encoding EGFP with chimeric introns, SV40 introns, MVM introns or no introns. (B) Representative fluorescence micrographs of transfected HEK293 cells (10x magnification, scale bar 100 μm). CI: chimeric introns, SV40: simian virus 40, MVM: minute virus of mice. [Figure 3-1]In vitro comparison by Western blot analysis of chimeric introns, SV40 introns, MVM introns and no introns. A) Western blot analysis of HEK293 cells 72 hours after infection with double AAV2-chimeric intron-hMYO7A, double AAV2-SV40 intron-hMYO7A, double AAV2-MVM intron-hMYO7A, double AAV2-intronless-hMYO7A or no vector. Arrows indicate full-length protein, 60 μg of protein was loaded in each lane and for each Western blot, molecular markers are reported on the left. Experiment numbers are reported under each sample set. Negative control: cells that did not receive double AAV2-hMYO7A, @MYO7A: Western blot with anti-myosin 7A (MYO7A) antibody, @filamin: Western blot with anti-filamin antibody, used as loading control. SV40 intron: modified simian virus 40 intron, MVM intron: minute virus of mouse intron. [Figure 3-2] Continuing from FIG. 3-1, B) Quantification of hMYO7A levels expressed upon infection with double AAV2-chimeric intron-hMYO7A, double AAV2-SV40 intron-hMYO7A, double AAV2-MVM intron-hMYO7A or double AAV2-intronless-hMYO7A in HEK293. The levels of hMYO7A are relative to hMYO7A expressed by double AAV2-chimeric intron-hMYO7A. Each filled square represents the value quantified for each sample in the corresponding group. Quantification was performed by Western blot analysis using anti-MYO7A antibody and measurements of human MYO7A band intensity were normalized to filamin. Mean values ​​are reported inside the histograms for each group. SV40 intron: modified simian virus 40 intron, MVM intron: minute virus of mice intron. [Figure 4]Comparison of the chimeric intron, SV40 intron, and MVM intron. (A) Depiction of the expression cassette carried by AAV8-5'hMYO7A. Top: AAV8-5'hMYO7A chimeric intron; middle: AAV8-5'hMYO7A SV40 intron; bottom: AAV8-5'hMYO7A MVM intron. (B) Southern blot of viral genomes derived from the AAV8-5'hMYO7A chimeric intron, AAV8-5'hMYO7A SV40 intron, and AAV8-5'hMYO7A MVM intron. All samples were treated with DNAse to degrade contaminating external DNA before viral genomic DNA was extracted. 5'AAV genome-CI: viral genomic DNA extracted from AAV8-CBA promoter-chimeric intron-5'hMYO7A, 5'AAV genome-SV40: viral genomic DNA extracted from AAV8-CBA promoter-SV40 intron-5'hMYO7A, 5'AAV genome-MVM: viral genomic DNA extracted from AAV8-CBA promoter-SV40 intron-5'hMYO7A, molecular weight markers expressed in kilobases, bp = base pairs. CI: chimeric intron, SV40: simian virus 40 intron, MVM: minute virus of mice intron. (C) Representative Western blot analysis of C57BL / 6 eye cups 2 weeks after subretinal injection of AAV8-5'hMYO7A chimeric intron, AAV8-5'hMYO7A SV40 intron or AAV8-5'hMYO7A MVM intron in combination with AAV8-3'hMYO7A-3XFLAG, or vehicle. Arrows indicate full-length protein, and 150 μg of protein was loaded in each lane. Negative control: vehicle-injected eyes, @Flag: Western blot with anti-Flag to recognize full-length myosin 7A-3XFlag, @Dysferlin: Western blot with anti-dysferlin antibody, used as loading control. (D) Quantification of hMYO7A levels expressed from the AAV8-5'hMYO7A chimeric intron, AAV8-5'hMYO7A SV40 intron, or AAV8-5'hMYO7A MVM intron in combination with AAV8-3'hMYO7A-3XFLAG in the eyecup of subretinally injected C57BL / 6 mice.Levels of hMYO7A-3XFLAG are relative to hMYO7A-3XFLAG expressed by AAV8-5'hMYO7A chimeric intron in combination with AAV8-3'hMYO7A-3XFLAG. The number of eyes positive for hMYO7A-3XFLAG (n) is shown below each bar. Quantification was performed by Western blot analysis using an anti-Flag antibody (Panel C) and measurements of hMYO7A-3XFLAG band intensity were normalized to dysferlin. Mean values ​​are represented above the corresponding bars. Values ​​are expressed as mean ± standard error of the mean (sem). [Figure 5-1] Dose-dependent improvement of apical melanosome localization and hMYO7A protein reconstitution in Shaker mice. (A) Semi-thin retinal sections stained with toluidine blue representative of sh1- / - receiving subretinal injections of either vehicle as a negative control or dual AAV8.hMYO7A (doses of 1.37E+10, 4.4E+9 or 1.37E+9 total GC / eye) and sh1+ / - receiving subretinal injections of vehicle as a positive control. Scale bars (white bars) are 10 μm. Black arrows point to correctly localized melanosomes. (B) Quantification of melanosome localization in the RPE villi of whole retinal sections of sh1 mice 3 months after subretinal delivery of dual AAV8.hMYO7A. The number of apical melanosomes / 100 μm of RPE is reported. Data are presented as single measurements for each eye (dots) and as mean ± sem (cylinders). Statistical analysis was performed using one-way ANOVA followed by Tukey's post-hoc test. p values ​​versus sh1- / - receiving vehicle are **p<0.01, ****p<0.0001. [Figure 5-2]Continuing from FIG. 5-1, (C) Representative Western blot analysis of sh1- / - eyecups 5 weeks after subretinal delivery of dual AAV8.hMYO7A at doses of 1.37E+10, 4.4E+9 or 1.37E+9 total GC / eye. As positive and negative controls, sh1+ / - and sh1- / - received subretinal injections of vehicle (same volume as dual AAV), respectively. α-MYO7A: Western blot with anti-myosin 7A antibody, α-dysferlin: Western blot with anti-dysferlin antibody, used as loading control. (D) Quantification of human MYO7A levels expressed in sh1- / - eyecups 5 weeks after subretinal injection of dual AAV8 vectors as a percentage (%) of endogenous Myo7a expressed in vehicle-injected littermates sh1+ / - eyes. Quantification was performed by Western blot analysis using anti-MYO7A antibody, and measurements of MYO7A and Myo7a band intensity were normalized to dysferlin. Data are presented as mean ± sem (mean values ​​are presented above the corresponding bars). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0074] As used herein, the terms "comprising," "comprises," and "comprised of" are synonymous with "including" or "includes," or "containing" or "contains," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or steps. The terms "comprising," "comprises," and "comprised of" also include the term "consisting of."

[0075] Vector In one aspect, the present invention provides a vector system for expressing a transgene in a cell, the vector system comprising a first vector and a second vector, wherein (a) a first vector comprising, in a 5' to 3' direction, an intron, a 5' terminal portion of a transgene coding sequence (CDS), and a splice donor sequence; (b) a second vector comprising, in the 5' to 3' direction, a splice acceptor sequence and a 3' end portion of a transgene CDS; Here, the 5'-end portion and the 3'-end portion together constitute a transgene CDS, and wherein the intron is not capable of homologous recombination with the splice donor sequence to excise the 5'-end portion of the transgene CDS.

[0076] In another aspect, the present invention provides a vector system for expressing a transgene in a cell, the vector system comprising a first vector and a second vector, wherein: (a) a first vector comprising, in a 5' to 3' direction, a promoter, an intron, a 5' terminal portion of a transgene coding sequence (CDS); and a splice donor sequence; (b) a second vector comprising, in the 5' to 3' direction, a splice acceptor sequence and a 3' end portion of a transgene CDS; Here, the 5'-end portion and the 3'-end portion together constitute a transgene CDS, and wherein the intron is not capable of homologous recombination with the splice donor sequence to excise the 5'-end portion of the transgene CDS.

[0077] In another aspect, the present invention provides a vector system for expressing a transgene in a cell, the vector system comprising a first vector and a second vector, wherein: (a) a first vector includes, in a 5' to 3' direction, a promoter, an intron, a 5' end portion of a transgene coding sequence (CDS), a splice donor sequence, and a first recombinogenic region; (b) a second vector comprising, in the 5' to 3' direction, a second recombinogenic region, a splice acceptor sequence, and a 3'-terminal portion of the transgene CDS; Here, the 5'-end portion and the 3'-end portion together constitute a transgene CDS, and wherein the intron is not capable of homologous recombination with the splice donor sequence to excise the 5'-end portion of the transgene CDS.

[0078] In another aspect, the present invention provides a vector system for expressing a transgene in a cell, the vector system comprising a first vector and a second vector, wherein: (a) a first vector comprising, in a 5' to 3' direction, a 5' end portion of a transgene coding sequence (CDS) and a splice donor sequence; (b) a second vector comprising, in the 5' to 3' direction, a splice acceptor sequence and a 3' end portion of a transgene CDS; Here, the 5'-end portion and the 3'-end portion together constitute the transgene CDS.

[0079] In another aspect, the present invention provides a vector system for expressing a transgene in a cell, the vector system comprising a first vector and a second vector, wherein: (a) a first vector comprising, in a 5' to 3' direction, a promoter, a 5' terminal portion of a transgene coding sequence (CDS), and a splice donor sequence; (b) a second vector comprising, in the 5' to 3' direction, a splice acceptor sequence and a 3' end portion of a transgene CDS; Here, the 5'-end portion and the 3'-end portion together constitute the transgene CDS.

[0080] In another aspect, the present invention provides a vector system for expressing a transgene in a cell, the vector system comprising a first vector and a second vector, wherein: (a) a first vector comprising, in a 5' to 3' direction, a promoter, a 5' end portion of a transgene coding sequence (CDS), a splice donor sequence, and a first recombinogenic region; (b) a second vector comprising, in the 5' to 3' direction, a second recombinogenic region, a splice acceptor sequence, and a 3'-terminal portion of the transgene CDS; Here, the 5'-end portion and the 3'-end portion together constitute the transgene CDS.

[0081] In another aspect, the present invention provides a vector combination for expressing a transgene in a cell, the combination comprising a first vector and a second vector, wherein (a) a first vector comprising, in a 5' to 3' direction, an intron, a 5' terminal portion of a transgene coding sequence (CDS), and a splice donor sequence; (b) a second vector comprising, in the 5' to 3' direction, a splice acceptor sequence and a 3' end portion of a transgene CDS; Here, the 5'-end portion and the 3'-end portion together constitute a transgene CDS, and wherein the intron is not capable of homologous recombination with the splice donor sequence to excise the 5'-end portion of the transgene CDS.

[0082] In another aspect, the present invention provides a vector combination for expressing a transgene in a cell, the combination comprising a first vector and a second vector, wherein (a) a first vector comprising, in a 5' to 3' direction, a promoter, an intron, a 5' terminal portion of a transgene coding sequence (CDS), and a splice donor sequence; (b) a second vector comprising, in the 5' to 3' direction, a splice acceptor sequence and a 3' end portion of a transgene CDS; Here, the 5'-end portion and the 3'-end portion together constitute a transgene CDS, and wherein the intron is not capable of homologous recombination with the splice donor sequence to excise the 5'-end portion of the transgene CDS.

[0083] In another aspect, the present invention provides a vector combination for expressing a transgene in a cell, the combination comprising a first vector and a second vector, wherein (a) a first vector includes, in a 5' to 3' direction, a promoter, an intron, a 5' end portion of a transgene coding sequence (CDS), a splice donor sequence, and a first recombinogenic region; (b) a second vector comprising, in the 5' to 3' direction, a second recombinogenic region, a splice acceptor sequence, and a 3'-terminal portion of the transgene CDS; Here, the 5'-end portion and the 3'-end portion together constitute a transgene CDS, and wherein the intron is not capable of homologous recombination with the splice donor sequence to excise the 5'-end portion of the transgene CDS.

[0084] In another aspect, the present invention provides a vector combination for expressing a transgene in a cell, the vector system comprising a first vector and a second vector, wherein: (a) a first vector comprising, in a 5' to 3' direction, a 5' end portion of a transgene coding sequence (CDS) and a splice donor sequence; (b) a second vector comprising, in the 5' to 3' direction, a splice acceptor sequence and a 3' end portion of a transgene CDS; Here, the 5'-end portion and the 3'-end portion together constitute the transgene CDS.

[0085] In another aspect, the present invention provides a vector combination for expressing a transgene in a cell, the vector system comprising a first vector and a second vector, wherein: (a) a first vector comprising, in a 5' to 3' direction, a promoter, a 5' terminal portion of a transgene coding sequence (CDS), and a splice donor sequence; (b) a second vector comprising, in the 5' to 3' direction, a splice acceptor sequence and a 3' end portion of a transgene CDS; Here, the 5'-end portion and the 3'-end portion together constitute the transgene CDS.

[0086] In another aspect, the present invention provides a vector combination for expressing a transgene in a cell, the vector system comprising a first vector and a second vector, wherein: (a) a first vector comprising, in a 5' to 3' direction, a promoter, a 5' end portion of a transgene coding sequence (CDS), a splice donor sequence, and a first recombinogenic region; (b) a second vector comprising, in the 5' to 3' direction, a second recombinogenic region, a splice acceptor sequence, and a 3'-terminal portion of the transgene CDS; Here, the 5'-end portion and the 3'-end portion together constitute the transgene CDS.

[0087] The vector system or vector combination of the present invention may be used to deliver a transgene to a cell when the transgene cannot be packaged by a single vector, for example due to size constraints of the vector, for example, AAV vectors may have the capacity to package transgenes limited to a maximum of about 5 kb.

[0088] When the first vector and the second vector are introduced into a cell, the transgene CDS can be reconstituted from the 5' and 3' end portions. The reconstituted transgene can be expressed in the cell.

[0089] For example, reconstitution of the full-length transgene CDS can be achieved upon introduction of both the first and second vector into the same cell by i) inverted terminal repeat (ITR)-mediated tail-to-head concatenation of the two vector genomes followed by splicing (double vector trans-splicing, TS), ii) homologous recombination between overlapping regions contained in the two vector genomes (double AAV overlap, OV), or iii) a combination of the two (double vector hybrid).

[0090] In some embodiments, the portion of the transgene CDS (e.g., the 5' and / or 3' end portions) is less than or equal to 10 kb, for example, less than or equal to 9.5 kb, 9 kb, 8.5 kb, 8 kb, 7.5 kb, 7 kb, 6.5 kb, 6 kb, 5.5 kb, 5 kb, or 4.5 kb. In preferred embodiments, the portion of the transgene CDS (e.g., the 5' and / or 3' end portions) is less than or equal to 5 kb.

[0091] In some embodiments, the 5' terminal portion and the 3' terminal portion do not contain overlapping sequences.

[0092] In some embodiments, the transgene CDS is split into a 5' end portion and a 3' end portion at a natural exon-exon junction.

[0093] As used herein, the term "not capable of homologous recombination" may mean that when the vector is prepared under standard conditions (e.g., as disclosed in the Examples herein, for example, transfection of HEK293 cells with a plasmid encoding (a) the vector genome, (b) Rep and Cap proteins, and (c) the adenovirus helper genes (e.g., E2, E4 and / or VARNA) required for AAV production, followed by purification), homologous recombination is not detectable or is substantially not detectable (e.g., as disclosed in the Examples herein, for example, using Southern blot analysis). If the intron is not capable of homologous recombination with the splice donor sequence, excision of the 5'-end portion of the transgene CDS may be minimized or prevented, for example, thereby increasing the amount of transgene CDS reconstituted from the 5'- and 3'-end portions when the first and second vectors are introduced into a cell.

[0094] In some embodiments, the intron does not comprise a region of at least 20, 30, 40, 50, 60, 70, 80, 90 or 100 contiguous nucleotides having at least 95%, 96%, 97%, 98%, 99% or 100% (preferably 100%) sequence identity to a region of the splice donor sequence. Because the intron does not share homology with the splice donor sequence, the intron is not capable of homologous recombination with that sequence.

[0095] In another aspect, the present invention provides a vector system for expressing a transgene in a cell, the vector system comprising a first vector and a second vector, wherein: (a) a first vector comprising, in a 5' to 3' direction, an intron, a 5' terminal portion of a transgene coding sequence (CDS), and a splice donor sequence; (b) a second vector comprising, in the 5' to 3' direction, a splice acceptor sequence and a 3' end portion of a transgene CDS; wherein the 5'-end portion and the 3'-end portion together constitute the transgene CDS, and wherein the intron does not include a region of at least 20, 30, 40, 50, 60, 70, 80, 90 or 100 consecutive nucleotides having at least 95%, 96%, 97%, 98%, 99% or 100% (preferably 100%) sequence identity to a region of the splice donor sequence.

[0096] In another aspect, the present invention provides a vector system for expressing a transgene in a cell, the vector system comprising a first vector and a second vector, wherein: (a) a first vector comprising, in a 5' to 3' direction, a promoter, an intron, a 5' terminal portion of a transgene coding sequence (CDS), and a splice donor sequence; (b) a second vector comprising, in the 5' to 3' direction, a splice acceptor sequence and a 3' end portion of a transgene CDS; wherein the 5'-end portion and the 3'-end portion together constitute the transgene CDS, and wherein the intron does not include a region of at least 20, 30, 40, 50, 60, 70, 80, 90 or 100 consecutive nucleotides having at least 95%, 96%, 97%, 98%, 99% or 100% (preferably 100%) sequence identity to a region of the splice donor sequence.

[0097] In another aspect, the present invention provides a vector system for expressing a transgene in a cell, the vector system comprising a first vector and a second vector, wherein: (a) a first vector includes, in a 5' to 3' direction, a promoter, an intron, a 5' end portion of a transgene coding sequence (CDS), a splice donor sequence, and a first recombinogenic region; (b) a second vector comprising, in the 5' to 3' direction, a second recombinogenic region, a splice acceptor sequence, and a 3'-terminal portion of the transgene CDS; wherein the 5'-end portion and the 3'-end portion together constitute the transgene CDS, and wherein the intron does not include a region of at least 20, 30, 40, 50, 60, 70, 80, 90 or 100 consecutive nucleotides having at least 95%, 96%, 97%, 98%, 99% or 100% (preferably 100%) sequence identity to a region of the splice donor sequence.

[0098] In another aspect, the present invention provides a vector combination for expressing a transgene in a cell, the combination comprising a first vector and a second vector, wherein: (a) a first vector comprising, in a 5' to 3' direction, an intron, a 5' terminal portion of a transgene coding sequence (CDS), and a splice donor sequence; (b) a second vector comprising, in the 5' to 3' direction, a splice acceptor sequence and a 3' end portion of a transgene CDS; wherein the 5'-end portion and the 3'-end portion together constitute the transgene CDS, and wherein the intron does not include a region of at least 20, 30, 40, 50, 60, 70, 80, 90 or 100 consecutive nucleotides having at least 95%, 96%, 97%, 98%, 99% or 100% (preferably 100%) sequence identity to a region of the splice donor sequence.

[0099] In another aspect, the present invention provides a vector combination for expressing a transgene in a cell, the combination comprising a first vector and a second vector, wherein (a) a first vector comprising, in a 5' to 3' direction, a promoter, an intron, a 5' terminal portion of a transgene coding sequence (CDS), and a splice donor sequence; (b) a second vector comprising, in the 5' to 3' direction, a splice acceptor sequence and a 3' end portion of a transgene CDS; wherein the 5'-end portion and the 3'-end portion together constitute the transgene CDS, and wherein the intron does not include a region of at least 20, 30, 40, 50, 60, 70, 80, 90 or 100 consecutive nucleotides having at least 95%, 96%, 97%, 98%, 99% or 100% (preferably 100%) sequence identity to a region of the splice donor sequence.

[0100] In another aspect, the present invention provides a vector combination for expressing a transgene in a cell, the combination comprising a first vector and a second vector, wherein (a) a first vector includes, in a 5' to 3' direction, a promoter, an intron, a 5' end portion of a transgene coding sequence (CDS), a splice donor sequence, and a first recombinogenic region; (b) a second vector comprising, in the 5' to 3' direction, a second recombinogenic region, a splice acceptor sequence, and a 3'-terminal portion of the transgene CDS; wherein the 5'-end portion and the 3'-end portion together constitute the transgene CDS, and wherein the intron does not include a region of at least 20, 30, 40, 50, 60, 70, 80, 90 or 100 consecutive nucleotides having at least 95%, 96%, 97%, 98%, 99% or 100% (preferably 100%) sequence identity to a region of the splice donor sequence.

[0101] Promoters and Enhancers The vector of the present invention may include a promoter. Suitably, the 5'-end portion of the transgene CDS is operably linked to the promoter. The term "operably linked" as used herein means that the portions (e.g., transgene and promoter) are linked together in such a manner that they can both perform their functions substantially unimpeded.

[0102] Any suitable promoter may be used, the selection of which can be easily made by those skilled in the art. The promoter sequence may be constitutively active (i.e., operable in any host cell background) or may be active only in a specific host cell environment, thus allowing targeted expression of the transgene in a specific cell type (e.g., tissue-specific promoters). The promoter may exhibit inducible expression in response to the presence of another factor, e.g., a factor present in the host cell. When the vector is administered for therapy, it is preferred that the promoter is functional in the target cell (e.g., retinal cell).

[0103] In some embodiments, the promoter is selected from the group consisting of a cytomegalovirus promoter, a rhodopsin promoter, a rhodopsin kinase promoter, an interphotoreceptor retinoid binding protein promoter, and a vitelliform macular degeneration 2 promoter, or a fragment thereof.

[0104] In a preferred embodiment, the promoter is the chicken beta-actin (CBA) promoter or a fragment thereof.

[0105] Exemplary CBA promoters include: TIFF2024517957000001.tif15160TIFF2024517957000002.tif15160(Sequence Number 1) TIFF2024517957000003.tif35160 (SEQ ID NO: 28) Includes.

[0106] In some embodiments, the promoter comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:1, or a fragment thereof, wherein preferably the promoter substantially retains the native function of the promoter of SEQ ID NO:1.

[0107] In some embodiments, the promoter comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:28, or a fragment thereof, wherein preferably the promoter substantially retains the native function of the promoter of SEQ ID NO:28.

[0108] In a preferred embodiment, the promoter comprises or consists of the nucleic acid sequence of SEQ ID NO: 1 or a fragment thereof.

[0109] In a preferred embodiment, the first vector comprises a promoter comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:1 or a fragment thereof.

[0110] An exemplary rhodopsin (Rho) promoter sequence is: TIFF2024517957000004.tif66160TIFF2024517957000005.tif28160(SEQ ID NO:29) It is.

[0111] In some embodiments, the promoter comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:29, or a fragment thereof, wherein preferably the promoter substantially retains the native function of the promoter of SEQ ID NO:29.

[0112] An exemplary vitelloid macular degeneration 2 (VMD2) promoter sequence is TIFF2024517957000006.tif79160 (SEQ ID NO: 30) It is.

[0113] In some embodiments, the promoter comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide sequence identity to SEQ ID NO:30, or a fragment thereof, wherein preferably the promoter substantially retains the native function of the promoter of SEQ ID NO:30.

[0114] The vector of the present invention may comprise an enhancer. Suitably, the 5' end portion of the transgene CDS is operably linked to the enhancer.

[0115] In some embodiments, the enhancer is upstream of the promoter (ie, toward the 5' end of the vector).

[0116] An "enhancer" is a region of DNA that can increase the likelihood that transcription of a particular gene will occur upon binding of a protein (activator). Enhancers are cis-acting. Enhancers can be located up to 1 Mbp (1,000,000 bp) away from the gene, either upstream or downstream of the start site.

[0117] In a preferred embodiment, the enhancer is a CMV enhancer.

[0118] An exemplary CMV enhancer sequence is TIFF2024517957000007.tif50160 (Sequence Number 2) It is.

[0119] In some embodiments, the enhancer comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:2, or a fragment thereof, wherein preferably the enhancer substantially retains the native function of the enhancer of SEQ ID NO:2.

[0120] In a preferred embodiment, the enhancer comprises or consists of the nucleic acid sequence of SEQ ID NO: 2, or a fragment thereof.

[0121] In a preferred embodiment, the first vector comprises an enhancer comprising or alternatively consisting of the nucleic acid sequence of SEQ ID NO:2, or a fragment thereof.

[0122] Introns Introns can be included in vectors to increase transgene expression. Any suitable intron can be used, and its selection can be easily made by those skilled in the art, provided that the intron of the first vector is not capable of homologous recombination with the splice donor sequence to excise the 5'-end portion of the transgene CDS.

[0123] An example intron sequence is TIFF2024517957000008.tif13160 (SEQ ID NO:31, wild type small T antigen intron) TIFF2024517957000009.tif40160 (SEQ ID NO:32; wild-type large T antigen intron) TIFF2024517957000010.tif18160 (SEQ ID NO:33, SV40 intron, e.g., upstream sequences are from the large T antigen intron and downstream sequences are from the SV40 cds), Includes.

[0124] In some embodiments, the intron comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO: 31, 32 or 33, and preferably the intron substantially retains the native function of the intron of SEQ ID NO: 31, 32 or 33, respectively.

[0125] In a preferred embodiment, the intron is a simian virus 40 (SV40) intron. The SV40 intron may be a modified SV40 intron (see, e.g., Nathwani et al. (2006) Blood 107:2653-2661).

[0126] In some embodiments, the intron is a minute virus of mice (MVM) intron.

[0127] An example SV40 intron sequence is TIFF2024517957000011.tif18160 (SEQ ID NO:3, modified SV40 intron) It is.

[0128] In preferred embodiments, the intron comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:3, wherein preferably the intron substantially retains the native function of the intron of SEQ ID NO:3.

[0129] In some embodiments, the intron comprises or consists of the nucleic acid sequence of SEQ ID NO:3, or a variant thereof having 4, 3, 2 or 1 nucleotide substitutions, additions or deletions, wherein preferably the intron substantially retains the native function of the intron of SEQ ID NO:3.

[0130] In a preferred embodiment, the intron comprises or alternatively consists of the nucleic acid sequence of SEQ ID NO:3.

[0131] In a preferred embodiment, the first vector comprises an intron comprising or alternatively consisting of the nucleic acid sequence of SEQ ID NO:3.

[0132] An example MVM intron sequence is TIFF2024517957000012.tif18160 (Sequence Number 4) It is.

[0133] In some embodiments, the intron comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:4, wherein preferably the intron substantially retains the native function of the intron of SEQ ID NO:4.

[0134] In some embodiments, the intron comprises or consists of the nucleic acid sequence of SEQ ID NO:4, or a variant thereof having 4, 3, 2 or 1 nucleotide substitutions, additions or deletions, wherein preferably the intron substantially retains the native function of the intron of SEQ ID NO:4.

[0135] In a preferred embodiment, the intron comprises or alternatively consists of the nucleic acid sequence of SEQ ID NO:4.

[0136] In a preferred embodiment, the first vector comprises an intron comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:4.

[0137] In another aspect, the present invention provides a vector system for expressing a transgene in a cell, the vector system comprising a first vector and a second vector, wherein: (a) a first vector comprising, in a 5' to 3' direction, an intron, a 5' terminal portion of a transgene coding sequence (CDS), and a splice donor sequence; (b) a second vector comprising, in the 5' to 3' direction, a splice acceptor sequence and a 3' end portion of a transgene CDS; wherein the 5'-end portion and the 3'-end portion together constitute a transgene CDS, wherein the intron comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO: 3 or 4, and wherein preferably the intron substantially retains the native function of the intron of SEQ ID NO: 3 or 4, respectively.

[0138] In another aspect, the present invention provides a vector system for expressing a transgene in a cell, the vector system comprising a first vector and a second vector, wherein: (a) a first vector comprising, in a 5' to 3' direction, a promoter, an intron, a 5' terminal portion of a transgene coding sequence (CDS), and a splice donor sequence; (b) a second vector comprising, in the 5' to 3' direction, a splice acceptor sequence and a 3' end portion of a transgene CDS; wherein the 5'-end portion and the 3'-end portion together constitute a transgene CDS, wherein the intron comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO: 3 or 4, and wherein preferably the intron substantially retains the native function of the intron of SEQ ID NO: 3 or 4, respectively.

[0139] In another aspect, the present invention provides a vector system for expressing a transgene in a cell, the vector system comprising a first vector and a second vector, wherein: (a) a first vector includes, in a 5' to 3' direction, a promoter, an intron, a 5' end portion of a transgene coding sequence (CDS), a splice donor sequence, and a first recombinogenic region; (b) a second vector comprising, in the 5' to 3' direction, a second recombinogenic region, a splice acceptor sequence, and a 3'-terminal portion of the transgene CDS; wherein the 5'-end portion and the 3'-end portion together constitute a transgene CDS, wherein the intron comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO: 3 or 4, and wherein preferably the intron substantially retains the native function of the intron of SEQ ID NO: 3 or 4, respectively.

[0140] Splice donor and acceptor 1 RNA splicing is a form of RNA processing in which newly made precursor messenger RNA (pre-mRNA) transcripts are transformed into mature messenger RNA (mRNA). During splicing, introns (non-coding regions) are removed and exons (coding sequences) are joined together.

[0141] Within an intron, a donor site (at the 5' end of the intron), a branch site (near the 3' end of the intron) and an acceptor site (at the 3' end of the intron) are required for splicing. The splice donor site contains the nearly invariant sequence GU at the 5' end of the intron within a larger, less highly conserved region. The splice acceptor site at the 3' end of the intron terminates the intron with a nearly invariant AG sequence. Upstream (5') from the AG is a region rich in pyrimidines (C and U), or a polypyrimidine tract. Additionally, upstream from the polypyrimidine tract, there is a branch point.

[0142] A "splice donor sequence" is a nucleotide sequence that can function as a donor site at the 5' end of an intron. Consensus sequences and frequencies of human splice site regions are described in Ma et al. (2015) PLoS One 10(6):p.e0130729.

[0143] A "splice acceptor sequence" is a nucleotide sequence that can function as an acceptor site at the 3' end of an intron. Consensus sequences and frequencies of human splice site regions are described in Ma et al. (2015) PLoS One 10(6):p.e0130729.

[0144] Exemplary splice donor sequences include TIFF2024517957000013.tif18160 (sequence number 5).

[0145] In some embodiments, the splice donor sequence comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:5, wherein preferably the splice donor sequence substantially retains the native function of the splice donor sequence of SEQ ID NO:5.

[0146] In a preferred embodiment, the splice donor sequence comprises or alternatively consists of the nucleic acid sequence of SEQ ID NO:5.

[0147] In a preferred embodiment, the first vector comprises a splice donor sequence comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:5.

[0148] Exemplary splice acceptor sequences include TIFF2024517957000014.tif8160 (sequence number 6).

[0149] In some embodiments, the splice acceptor sequence comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:6, wherein preferably the splice acceptor sequence substantially retains the native function of the splice acceptor sequence of SEQ ID NO:6.

[0150] In a preferred embodiment, the splice acceptor sequence comprises or alternatively consists of the nucleic acid sequence of SEQ ID NO:6.

[0151] In a preferred embodiment, the second vector comprises a splice acceptor sequence that comprises, or alternatively consists of, the nucleic acid sequence of SEQ ID NO:6.

[0152] Recombinogenic region Recombinogenic regions can be added to the dual vectors to increase recombination. Preferably, a first recombinogenic region is located downstream of the splice donor sequence in the first vector, and a second recombinogenic region is located upstream of the splice acceptor sequence in the second vector.

[0153] In a preferred embodiment, the first recombinogenic region and the second recombinogenic region are the same.

[0154] In some embodiments, the first recombinogenic region and the second recombinogenic region are both F1 phage recombinogenic regions or fragments thereof. In a preferred embodiment, the first recombinogenic region and the second recombinogenic region are both AK recombinogenic regions or fragments thereof.

[0155] An exemplary recombinogenic region sequence (AK) is: TIFF2024517957000015.tif15160 (SEQ ID NO: 7) TIFF2024517957000016.tif15160 (SEQ ID NO: 34) Includes.

[0156] In some embodiments, the recombinogenic region (e.g., the first recombinogenic region and the second recombinogenic region) comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:7, or a fragment thereof, wherein preferably the recombinogenic region substantially retains the native function of the recombinogenic region of SEQ ID NO:7.

[0157] In some embodiments, the recombinogenic region (e.g., the first recombinogenic region and the second recombinogenic region) comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:34, or a fragment thereof, wherein preferably the recombinogenic region substantially retains the native function of the recombinogenic region of SEQ ID NO:34.

[0158] In a preferred embodiment, the recombinogenic region (eg, the first recombinogenic region and the second recombinogenic region) comprises or consists of the nucleic acid sequence of SEQ ID NO: 7, or a fragment thereof.

[0159] In a preferred embodiment, the first vector comprises a recombinogenic region comprising or alternatively consisting of the nucleic acid sequence of SEQ ID NO: 7 or a fragment thereof.

[0160] In a preferred embodiment, the second vector comprises a recombinogenic region comprising or alternatively consisting of the nucleic acid sequence of SEQ ID NO: 7 or a fragment thereof.

[0161] In some embodiments, the first recombinogenic region and the second recombinogenic region are both derived from an alkaline phosphatase gene, such as AP (NM 001632, bp 823-1100, SEQ ID NO: 35), AP1 (XM 005246439.2, bp 1802-1516, SEQ ID NO: 36), or AP2 (XM_005246439.2, bp 1225-938, SEQ ID NO: 37).

[0162] Exemplary AP recombinogenic region sequences include: TIFF2024517957000017.tif36160 (SEQ ID NO: 35; AP) TIFF2024517957000018.tif36160 (SEQ ID NO: 36; AP1) TIFF2024517957000019.tif36160 (SEQ ID NO: 37; AP2) Includes.

[0163] In some embodiments, the recombinogenic region (e.g., the first recombinogenic region and the second recombinogenic region) comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:35, or a fragment thereof, wherein preferably the recombinogenic region substantially retains the native function of the recombinogenic region of SEQ ID NO:35.

[0164] In some embodiments, the recombinogenic region (e.g., the first recombinogenic region and the second recombinogenic region) comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:36, or a fragment thereof, wherein preferably the recombinogenic region substantially retains the native function of the recombinogenic region of SEQ ID NO:36.

[0165] In some embodiments, the recombinogenic region (e.g., the first recombinogenic region and the second recombinogenic region) comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:37, or a fragment thereof, wherein preferably the recombinogenic region substantially retains the native function of the recombinogenic region of SEQ ID NO:37.

[0166] Polyadenylation sequence The vector of the present invention may comprise a polyadenylation sequence. Suitably, the transgene is operably linked to the polyadenylation sequence. The polyadenylation sequence may be inserted downstream of the transgene to improve transgene expression.

[0167] Polyadenylation sequences usually comprise a polyadenylation signal, a polyadenylation site and downstream elements: the polyadenylation signal contains a sequence motif recognized by the RNA cleavage complex, the polyadenylation site is the cleavage site where the polyA tail is added to the mRNA, and the downstream element is usually a GT-rich region located immediately downstream of the polyadenylation site, which is important for efficient processing.

[0168] In some embodiments, the second vector further comprises a polyadenylation sequence downstream of the 3' end portion of the transgene CDS.

[0169] In some embodiments, the polyadenylation sequence is the bovine growth hormone (bGH) polyadenylation sequence or the SV40 polyadenylation sequence.

[0170] In a preferred embodiment, the polyadenylation sequence is the bovine growth hormone (bGH) polyadenylation sequence.

[0171] Exemplary polyadenylation sequences include: TIFF2024517957000020.tif29160 (SEQ ID NO:8) TIFF2024517957000021.tif34160 (SEQ ID NO: 38) Includes.

[0172] In some embodiments, the polyadenylation sequence comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:8, wherein preferably the polyadenylation sequence substantially retains the native function of the polyadenylation sequence of SEQ ID NO:8.

[0173] In some embodiments, the polyadenylation sequence comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:38, wherein preferably the polyadenylation sequence substantially retains the native function of the polyadenylation sequence of SEQ ID NO:38.

[0174] In a preferred embodiment, the polyadenylation sequence comprises or alternatively consists of the nucleic acid sequence of SEQ ID NO:8.

[0175] In a preferred embodiment, the second vector comprises a polyadenylation sequence comprising, or alternatively consisting of, the nucleic acid sequence of SEQ ID NO:8.

[0176] vector A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. According to the present invention and by way of example, some vectors used in recombinant nucleic acid technology allow an entity, such as a segment of nucleic acid (e.g., a heterologous DNA segment, e.g., a heterologous cDNA segment), to be transferred to a target cell. A vector serves the purpose of maintaining the heterologous nucleic acid (DNA or RNA) in a cell, may facilitate the replication of a vector containing a segment of nucleic acid, or may facilitate the expression of a protein encoded by a segment of nucleic acid. A vector can be non-viral or viral. Examples of vectors used in recombinant nucleic acid technology include, but are not limited to, plasmids, mRNA molecules (e.g., in vitro transcribed mRNA), chromosomes, artificial chromosomes, and viruses. A vector can also be, for example, a naked nucleic acid (e.g., DNA). In its simplest form, the vector itself can be the nucleotide of interest.

[0177] Vectors can be introduced into cells using a variety of techniques known in the art, such as transfection, transformation and transduction. Several such techniques are known in the art, such as infection with recombinant viral vectors, such as retroviruses, lentiviruses (e.g., integration-defective lentiviruses), adenoviruses, adeno-associated viruses, baculoviruses and herpes simplex virus vectors, direct injection of nucleic acids and biolistic transformation.

[0178] Non-viral delivery system includes, but is not limited to, DNA transfection method. Here, transfection includes the process of using non-viral vector to deliver gene to target cell. Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compact DNA-mediated transfection, liposome, immunoliposome, lipofectin, cationic drug-mediated transfection, cationic facial amphiphile (CFA) (Nat.Biotechnol.(1996)14:556) and combinations thereof.

[0179] Viral Vectors In a preferred embodiment, the vector is a viral vector, e.g., comprises a viral (preferably AAV) vector genome. The viral vector may exist in the form of a viral vector particle.

[0180] The viral vector can be an adeno-associated viral (AAV) vector, an adenoviral vector, a retroviral vector, a lentiviral vector, a herpes simplex viral vector, a picornavirus vector or an alphavirus vector.

[0181] In a preferred embodiment, the first vector and the second vector are AAV vectors. The AAV vector may be in the form of an AAV vector particle.

[0182] Adeno-associated virus vector AAV vector or AAV vector particle may comprise AAV genome or its fragment or derivative. AAV genome is a polynucleotide sequence that may code for functions required for the production of AAV particles. These functions include functions that operate in the replication and packaging cycle of AAV in host cells, including the encapsidation of AAV genome into AAV particles. Naturally occurring AAV is replication-defective and depends on the provision of helper functions in trans for the completion of replication and packaging cycles. Thus, AAV genome is usually replication-defective.

[0183] The AAV genome may be in single-stranded form, either the positive or negative strand, or alternatively in double-stranded form. The use of the double-stranded form allows for the avoidance of a DNA replication step in the target cell, and therefore accelerates transgene expression.

[0184] Naturally occurring AAVs can be classified according to different biological systems. The AAV genome can be derived from any naturally occurring serotype, isolate, or clade of AAV.

[0185] AAV can be referred to in terms of its serotype. A serotype corresponds to a variant subspecies of AAV, which has a characteristic reactivity that can be used to distinguish it from other variant subspecies due to its profile of capsid surface antigen expression. Usually, AAV vector particles with a specific AAV serotype do not cross-react efficiently with neutralizing antibodies specific for any other AAV serotype. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV-PhP.B and AAV-PhP.eB.

[0186] AAVs may also be referred to in terms of clades or clones, which refers to the phylogenetic relationships of naturally occurring AAVs and, usually, to a lineage of AAVs that can be traced back to a common ancestor, including all of its descendants. Additionally, AAVs may also be referred to in terms of specific isolates, i.e., genetic isolates of a particular AAV found in nature. The term genetic isolate refers to a population of AAVs that has undergone limited genetic mixing with other naturally occurring AAVs, thereby defining a population that is recognizably distinct at the genetic level.

[0187] Typically, the AAV genome of a naturally occurring serotype, isolate or clade of AAV contains at least one inverted terminal repeat (ITR). The ITR sequence acts in cis to provide a functional origin of replication and to allow integration and excision of the vector from the genome of a cell. Suitably, one or more ITR sequences flank the transgene or a portion thereof.

[0188] The AAV genome may also include packaging genes, such as the rep and / or cap genes, which code for packaging functions for AAV particles. A promoter may be operably linked to each of the packaging genes. Specific examples of such promoters include the p5, p19 and p40 promoters. For example, the p5 and p19 promoters are commonly used to express the rep gene, whereas the p40 promoter is commonly used to express the cap gene. The rep gene codes for one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or variants thereof. The cap gene codes for one or more capsid proteins, such as VP1, VP2 and VP3 or variants thereof.

[0189] The AAV genome can be the complete genome of a naturally occurring AAV. For example, a vector containing a complete AAV genome can be used to prepare an AAV vector or vector particle.

[0190] Suitably, the AAV genome is derivatized for administration to a patient. Such derivatization is standard in the art, and the present invention encompasses the use of any known derivative of the AAV genome, and derivatives that can be produced by applying techniques known in the art. The AAV genome may be a derivative of any naturally occurring AAV. Suitably, the AAV genome is a derivative of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11.

[0191] Derivatives of the AAV genome include any truncated or modified AAV genome that allows expression of a transgene from the AAV vector of the present invention in vivo. Usually, the AAV genome can be significantly truncated to include minimal viral sequences but retain the above functions. This can reduce the risk of recombination of the vector with wild-type virus and avoid the induction of a cellular immune response due to the presence of viral gene proteins in target cells.

[0192] Typically, the derivative comprises at least one inverted terminal repeat (ITR), and optionally more than one ITR, for example, two ITRs or more. One or more of the ITRs may be derived from an AAV genome with a different serotype, or may be a chimeric or mutated ITR. A suitable mutated ITR is one that has a deletion of the trs (terminal resolution site). This deletion allows for continuous replication of the genome to generate a single-stranded genome containing both the coding sequence and the complementary sequence, i.e., a self-complementary AAV genome. This allows for the avoidance of DNA replication in the target cell, and therefore allows for accelerated transgene expression.

[0193] An AAV genome can include one or more ITR sequences from any naturally occurring serotype, isolate, or clade of AAV, or variants thereof. An AAV genome can include at least one, e.g., two, of an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 ITR, or variants thereof.

[0194] One or more ITRs may flank the transgene or a portion thereof at both ends. The inclusion of one or more ITRs can aid in the formation of AAV vector concatemers in the nucleus of the host cell, for example after conversion of single-stranded vector DNA to double-stranded DNA by the action of host cell DNA polymerase. The formation of such episomal concatemers protects the AAV vector during the life of the host cell, thereby allowing long-term expression of the transgene in vivo.

[0195] Suitably, the ITR elements are the only sequences retained from the natural AAV genome in the derivative. Suitably, the derivative may not include the rep and / or cap genes of the natural genome and any other sequences of the natural genome. This may reduce the possibility of the vector integrating into the host cell genome. Furthermore, the size of the AAV genome may be reduced to allow greater flexibility in incorporating transgenes or parts thereof as well as other sequence elements (e.g., regulatory elements) into the vector.

[0196] Thus, in the derivatives of the invention, the following may be deleted: one inverted terminal repeat (ITR) sequence, the replication (rep) and the capsid (cap) genes. However, the derivatives may further comprise one or more of the rep and / or cap genes or other viral sequences of the AAV genome. Naturally occurring AAV integrates at a high frequency into a specific site on human chromosome 19 and shows a negligible frequency of random integration, therefore retention of integration capacity in AAV vectors may be acceptable in therapeutic settings.

[0197] The invention further encompasses the provision of sequences of the AAV genome in an order and arrangement that differs from the order and arrangement of the sequences in the native AAV genome. The invention also encompasses the replacement of one or more AAV sequences or genes with sequences from another virus or with chimeric genes composed of sequences from more than one virus. Such chimeric genes may be composed of sequences from two or more related viral proteins from different viral species.

[0198] AAV vector particles may be encapsidated by capsid proteins. Suitably, AAV vector particles may be in a transcapsidated form, in which an AAV genome or derivative with ITRs of one serotype is packaged in a capsid of a different serotype. AAV vector particles also include mosaic forms, in which a mixture of unmodified capsid proteins from two or more different serotypes constitutes the viral capsid. AAV vector particles also include chemically modified forms with ligands adsorbed to the capsid surface. For example, such ligands can include antibodies for targeting specific cell surface receptors.

[0199] Where the derivative comprises capsid proteins, i.e., VP1, VP2 and / or VP3, the derivative may be a chimeric, shuffled or capsid-modified derivative of one or more naturally occurring AAV. In particular, the present invention encompasses the provision of capsid protein sequences from different serotypes, clades, clones or isolates of AAV within the same vector (i.e., pseudotyped vectors). The AAV vector may be in the form of a pseudotyped AAV vector particle.

[0200] Chimeric, shuffled or capsid modified derivatives are usually selected to provide one or more desired functionalities to the AAV vector. Thus, these derivatives may show increased efficiency of gene delivery and / or decreased immunogenicity (humoral or cellular) compared to AAV vectors that contain naturally occurring AAV genomes. For example, increased efficiency of gene delivery may be achieved by improved receptor or co-receptor binding on the cell surface, improved internalization, improved transport into cells and nuclei, improved uncoating of viral particles and improved conversion of single-stranded genome to double-stranded form.

[0201] Chimeric capsid proteins include those produced by recombination between two or more capsid coding sequences of naturally occurring AAV serotypes. This can be done, for example, by a marker rescue approach, in which a non-infectious capsid sequence of one serotype is co-transfected with a capsid sequence of a different serotype, and directed selection is used to select for capsid sequences with desired properties. The capsid sequences of different serotypes can be altered in cells by homologous recombination to produce novel chimeric capsid proteins.

[0202] Chimeric capsid proteins also include those generated by engineering of capsid protein sequences to transfer particular capsid protein domains, surface loops or particular amino acid residues between two or more capsid proteins, e.g., between two or more capsid proteins of different serotypes.

[0203] Shuffled or chimeric capsid proteins may also be generated by DNA shuffling or by error-prone PCR. Hybrid AAV capsid genes can be generated by randomly fragmenting the sequences of related AAV genes, such as AAV genes encoding capsid proteins of several different serotypes, and then subsequently reassembling the fragments in a self-priming polymerase reaction that may also cause crossovers in regions of sequence homology. A library of hybrid AAV genes thus generated by shuffling the capsid genes of several serotypes can be screened to identify viral clones with the desired functionality. Similarly, error-prone PCR can be used to randomly mutate AAV capsid genes to generate a diverse library of variants that can then be selected for desired properties.

[0204] The sequence of the capsid gene may also be genetically engineered to introduce specific deletions, substitutions or insertions relative to the natural wild-type sequence. In particular, the capsid gene may be modified by the insertion of a sequence of an unrelated protein or peptide within the open reading frame of the capsid coding sequence or at the N-terminus and / or C-terminus of the capsid coding sequence. The unrelated protein or peptide may advantageously act as a ligand for a particular cell type, thereby conferring improved binding to the target cell or improving the specificity of targeting of the vector to a particular cell population. The unrelated protein may also be one that aids in the purification of the viral particles as part of the production process, i.e. an epitope or affinity tag. The site of insertion is usually selected so as not to interfere with other functions of the viral particle, such as internalization, transport of the viral particle.

[0205] The capsid protein may be an artificial or mutated capsid protein. As used herein, the term "artificial capsid" means that the capsid particle includes an amino acid sequence that does not occur in nature or that contains an amino acid sequence that has been engineered (e.g., modified) from a naturally occurring capsid amino acid sequence. In other words, the artificial capsid protein includes a mutation or variation in the amino acid sequence compared to the sequence of the parent capsid from which the artificial capsid amino acid sequence is derived, when the artificial capsid amino acid sequence and the parent capsid amino acid sequence are aligned.

[0206] In some embodiments, the first vector and the second vector are selected from the group consisting of hu68 (see, e.g., WO 2018 / 160582), Anc library (see, e.g., WO 2015 / 054653 and WO 2017 / 019994), and AAV2-TT (see, e.g., WO 2015 / 121501).

[0207] An exemplary 5'ITR sequence is: TIFF2024517957000022.tif23160 (SEQ ID NO: 9) It is.

[0208] In some embodiments, the 5' ITR comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:9, wherein preferably the 5' ITR substantially retains the native function of the 5' ITR of SEQ ID NO:9.

[0209] In a preferred embodiment, the 5'ITR comprises or alternatively consists of the nucleic acid sequence of SEQ ID NO:9.

[0210] In a preferred embodiment, the first vector and the second vector comprise a 5' ITR that comprises, or alternatively consists of, the nucleic acid sequence of SEQ ID NO:9.

[0211] An exemplary 3'ITR sequence is: TIFF2024517957000023.tif23160 (SEQ ID NO: 10) It is.

[0212] In some embodiments, the 3' ITR comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:10, wherein preferably the 3' ITR substantially retains the native function of the 3' ITR of SEQ ID NO:10.

[0213] In a preferred embodiment, the 3'ITR comprises or alternatively consists of the nucleic acid sequence of SEQ ID NO:10.

[0214] In a preferred embodiment, the first vector and the second vector comprise a 3' ITR that comprises, or alternatively consists of, the nucleic acid sequence of SEQ ID NO:10.

[0215] Transgene In some embodiments, the transgene is selected from the group consisting of myosin 7A (MYO7A), ABCA4, CEP290, CDH23, EYS, USH2a, GPR98 and ALMS1.

[0216] In a preferred embodiment, the transgene is a myosin 7A (MYO7A) transgene.

[0217] An exemplary MYO7A nucleotide sequence is: TIFF2024517957000024.tif100160TIFF2024517957000025.tif244160TIFF2024517957000026.tif244160TIFF2024517957000027.tif127160 (SEQ ID NO: 11) It is.

[0218] An exemplary 5' end portion of the MYO7A transgene is: TIFF2024517957000028.tif93160TIFF2024517957000029.tif244160(SEQ ID NO:12) It is.

[0219] In some embodiments, the 5'-end portion of the transgene comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:12, wherein preferably the 5'-end portion of the transgene substantially retains the native function of the 5'-end portion of the transgene of SEQ ID NO:12.

[0220] In a preferred embodiment, the 5' terminal portion of the transgene comprises or alternatively consists of the nucleic acid sequence of SEQ ID NO:12.

[0221] In a preferred embodiment, the first vector comprises a 5' terminal portion of a transgene comprising or consisting of the nucleic acid sequence of SEQ ID NO:12.

[0222] An exemplary 3' end portion of the MYO7A transgene is: TIFF2024517957000030.tif153160TIFF2024517957000031.tif230160(SEQ ID NO:13) It is.

[0223] In some embodiments, the 3'-terminal portion of the transgene comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:13, wherein preferably the 3'-terminal portion of the transgene substantially retains the native function of the 3'-terminal portion of the transgene of SEQ ID NO:13.

[0224] In a preferred embodiment, the 3' terminal portion of the transgene comprises or alternatively consists of the nucleic acid sequence of SEQ ID NO:13.

[0225] In a preferred embodiment, the first vector comprises a 3' terminal portion of the transgene comprising or consisting of the nucleic acid sequence of SEQ ID NO:13.

[0226] A further exemplary MYO7A nucleotide sequence is: TIFF2024517957000032.tif165160TIFF2024517957000033.tif245160TIFF2024517957000034.tif245160TIFF2024517957000035.tif63160 (SEQ ID NO:39; NM_000260.3 nucleotides 273-6920) It is.

[0227] A further exemplary 5' end portion of the MYO7A transgene is TIFF2024517957000036.tif159160TIFF2024517957000037.tif179160 (SEQ ID NO: 40; NM_000260.3 nucleotides 273-3380) It is.

[0228] In some embodiments, the 5'-terminal portion of the transgene comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:40, wherein preferably the 5'-terminal portion of the transgene substantially retains the native function of the 5'-terminal portion of the transgene of SEQ ID NO:40.

[0229] In a preferred embodiment, the 5' terminal portion of the transgene comprises or alternatively consists of the nucleic acid sequence of SEQ ID NO:40.

[0230] In a preferred embodiment, the first vector comprises a 5' end portion of the transgene comprising or alternatively consisting of the nucleic acid sequence of SEQ ID NO:40.

[0231] An exemplary 3' end portion of the MYO7A transgene is: TIFF2024517957000038.tif216160TIFF2024517957000039.tif167160 (SEQ ID NO: 41; NM_000260.3 nucleotides 3381-6920) It is.

[0232] In some embodiments, the 3'-terminal portion of the transgene comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:41, wherein preferably the 3'-terminal portion of the transgene substantially retains the native function of the 3'-terminal portion of the transgene of SEQ ID NO:41.

[0233] In a preferred embodiment, the 3' terminal portion of the transgene comprises or alternatively consists of the nucleic acid sequence of SEQ ID NO:41.

[0234] In a preferred embodiment, the first vector comprises a 3' terminal portion of the transgene comprising or consisting of the nucleic acid sequence of SEQ ID NO:41.

[0235] In some embodiments, the transgene is an ABCA4 transgene.

[0236] An exemplary ABCA4 nucleotide sequence is: TIFF2024517957000040.tif225160TIFF2024517957000041.tif244160TIFF2024517957000042.tif244160TIFF2024517957000043.tif20160 (SEQ ID NO: 42) It is.

[0237] An exemplary 5' end portion of the ABCA4 transgene is: TIFF2024517957000044.tif198160TIFF2024517957000045.tif118160(SEQ ID NO:43) It is.

[0238] In some embodiments, the 5'-terminal portion of the transgene comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:43, wherein preferably the 5'-terminal portion of the transgene substantially retains the native function of the 5'-terminal portion of the transgene of SEQ ID NO:43.

[0239] An exemplary 3' end portion of the ABCA4 transgene is: TIFF2024517957000046.tif67160TIFF2024517957000047.tif246160TIFF2024517957000048.tif106160 (SEQ ID NO: 44) It is.

[0240] In some embodiments, the 3'-terminal portion of the transgene comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:44, wherein preferably the 5'-terminal portion of the transgene substantially retains the native function of the 5'-terminal portion of the transgene of SEQ ID NO:44.

[0241] The polynucleotides used in the present invention are codon-optimized. In some embodiments, the transgene is codon-optimized. Codon optimization has been previously described in WO 1999 / 41397 and WO 2001 / 79518. Different cells differ in their use of certain codons. This codon bias corresponds to a bias in the relative abundance of a particular tRNA in a cell type. Expression can be increased by altering the codons in the sequence so that the codons are adjusted to match the relative abundance of the corresponding tRNA. Similarly, expression can be decreased by deliberately selecting codons whose corresponding tRNAs are known to be rare in a particular cell type. Thus, a further degree of transcriptional control is available. Codon usage tables are known in the art for mammalian cells, as well as for a variety of other organisms.

[0242] Exemplary Vectors An exemplary sequence of the first vector of the invention is: TIFF2024517957000049.tif245160TIFF2024517957000050.tif239160(SEQ ID NO:14) It is.

[0243] JPEG2024517957000051.jpg60155

[0244] In some embodiments, the first vector comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:14, wherein preferably the first vector substantially retains the native function of the first vector of SEQ ID NO:14.

[0245] In a preferred embodiment, the first vector comprises or alternatively consists of the nucleic acid sequence of SEQ ID NO:14.

[0246] An exemplary sequence of the second vector of the invention is: TIFF2024517957000052.tif102160TIFF2024517957000053.tif244160TIFF2024517957000054.tif128160 (SEQ ID NO: 15) It is.

[0247] JPEG2024517957000055.jpg46158

[0248] In some embodiments, the second vector comprises or consists of a nucleic acid sequence having at least 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% nucleotide identity to SEQ ID NO:15, wherein preferably the second vector substantially retains the native function of the second vector of SEQ ID NO:15.

[0249] In a preferred embodiment, the second vector comprises or alternatively consists of the nucleic acid sequence of SEQ ID NO:15.

[0250] In a particularly preferred embodiment, the first vector comprises, or alternatively consists of, the nucleic acid sequence of SEQ ID NO:14 and the second vector comprises, or alternatively consists of, the nucleic acid sequence of SEQ ID NO:15.

[0251] composition The vectors, vector systems and cells of the invention may be formulated for administration to a subject with a pharma- ceutically acceptable carrier, diluent or excipient. Suitable carriers and diluents include isotonic saline, such as phosphate buffered saline, potentially containing human serum albumin.

[0252] Materials used to formulate pharmaceutical compositions should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material can be determined by one skilled in the art depending on the route of administration.

[0253] Pharmaceutical compositions are usually in liquid form. Liquid pharmaceutical compositions generally include liquid carriers such as water, petroleum, animal or vegetable oils, mineral oils or synthetic oils. Physiological saline, magnesium chloride, dextrose or other sugar solutions, or glycols, ethylene glycol, propylene glycol or polyethylene glycol may be included. In some cases, surfactants such as pluronic acid (PF68) 0.001% may be used.

[0254] For injection, the active ingredient may be in the form of a pyrogen-free aqueous solution, with appropriate pH, isotonicity and stability. Those skilled in the art are well able to prepare appropriate solutions using isotonic vehicles such as, for example, Sodium Chloride Injection, Ringer's Injection or Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and / or other additives may be included as required.

[0255] For delayed release, the drug may be included in a pharmaceutical composition which has been formulated for sustained release according to methods known in the art, for example, in microcapsules formed from biocompatible polymers, or in liposome carrier systems.

[0256] Handling of cell therapy products should preferably be carried out in compliance with the FACT-JACIE International Standards for Cell Therapy.

[0257] Treatment method In one aspect, the present invention provides a vector system, vector, kit or composition of the invention for use in therapy.

[0258] In another aspect, the present invention provides a vector system, vector, kit or composition of the invention for use in the treatment of a retinal degeneration. Preferably, the retinal degeneration is an inherited retinal degeneration.

[0259] In some embodiments, the use is in the treatment or prevention of Usher syndrome, retinitis pigmentosa, Leber congenital amaurosis (LCA), Stargardt disease, Alström syndrome, or an ABCA4-related disorder.

[0260] In another aspect, the present invention provides a vector system, vector, kit or composition of the invention for use in the treatment of Usher Syndrome.

[0261] In a preferred embodiment, the Usher syndrome is Usher syndrome type 1B.

[0262] In another aspect, the present invention provides a method for treating or preventing retinal degeneration, comprising administering to a subject in need thereof an effective amount of the vector system, vector, kit or composition of the present invention. Preferably, the retinal degeneration is an inherited retinal degeneration.

[0263] In another aspect, the present invention provides a method for treating or preventing Usher Syndrome, comprising administering to a subject in need thereof an effective amount of a vector system, vector, kit or composition of the present invention.

[0264] In some embodiments, the localization of melanosomes to the retinal pigment epithelium (RPE) apical villi is increased or normalized (e.g., increased to a level that is approximately the same as that of a healthy subject). The increase may be compared to RPE apical villi from an eye that is not treated according to the present invention (e.g., an eye from a subject that is diseased but otherwise under substantially the same conditions). The increase (e.g., in number per 100 μm) may be, for example, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold. The increase may increase the number of melanosomes (e.g., in number per 100 μm) to within, for example, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 99% of the number for a healthy subject. Methods for analyzing melanosomes are well known to those of skill in the art and include, for example, the inventions disclosed herein.

[0265] With a global prevalence of 1 / 2,000, inherited retinal degenerations (IRDs) are the leading cause of blindness worldwide. Among the most frequent and severe IRDs are retinitis pigmentosa (RP), Leber congenital amaurosis (LCA) and Stargardt disease (STGD), which are in most cases inherited as monogenic conditions. The majority of mutations causing IRDs are found in genes expressed in neuronal photoreceptors (PRs), rods and / or cones in the retina.

[0266] AAV vectors are among the most efficient vectors in targeting both PR and retinal pigment epithelium (RPE) for long-term treatment upon single subretinal administration. The present invention allows for the treatment of diseases such as those listed in the table below that are likely difficult to treat with a single AAV vector (which may have a maximum cargo dose of about 5 kb).

[0267] [Table 1]

[0268] Usher syndrome type 1B (USH1B) is the most severe form of RP and deafness caused by mutations in the MYO7A gene (CDS: 6648 bp), which encodes atypical MYO7A, an actin-based motility protein expressed in both PR and RPE in the retina.

[0269] Stargardt disease (STGD) is the most common form of inherited macular degeneration caused by mutations in the ABCA4 gene (CDS: 6822 bp), which encodes an all-trans retinal transporter located in the PR outer segment.

[0270] Cone-rod dystrophy type 3, fundus flaviforme, age-related macular degeneration type 2, early-onset severe retinal dystrophy and retinitis pigmentosa type 19 are also associated with ABCA4 mutations (ABCA4-related disorders).

[0271] All references herein to treatment include curative, palliative and prophylactic treatment. The treatment of mammals, particularly humans, is preferred. Both human and veterinary treatments are within the scope of the present invention.

[0272] Administration In some embodiments, the vector, vector system, or cells are administered locally to the subject.

[0273] In some embodiments, the vector, vector system or cells are administered to the eye of the subject. Administration may be by injection, for example a subretinal injection.

[0274] The first and second vectors may be administered in combination simultaneously, sequentially or separately.

[0275] As used herein, the term "combination" or the terms "in combination," "used in combination with," or "combined preparation" can refer to the simultaneous, sequential or separate combined administration of two or more agents.

[0276] As used herein, the term "concurrently" means that the agents are administered at the same time, i.e., the same time.

[0277] As used herein, the term "sequentially" means that the agents are administered one after the other.

[0278] As used herein, the term "separately" means that the agents are administered independent of each other, but within a time interval that allows the agents to exert a combined effect, preferably a synergistic effect. Thus, administering "separately" may allow one agent to be administered, for example, within 1 minute, 5 minutes, or 10 minutes after the other.

[0279] Dosage Those skilled in the art can easily determine the appropriate dose of the agent of the present invention to be administered to a subject. Usually, a physician will determine the actual dosage that is most suitable for an individual patient, which will depend on a variety of factors, such as the activity of the specific compound used, the metabolic stability and duration of action of the compound, age, body weight, general health, sex, diet, mode and timing of administration, excretion rate, drug combinations, the severity of the specific condition, and the therapy the individual is undergoing. Of course, there may be individual situations where higher or lower dosage ranges are merited, and these are within the scope of the present invention.

[0280] The dose can be sufficient to treat or prevent, for example, retinal degeneration. The dose can be sufficient to treat or prevent, for example, Usher syndrome, retinitis pigmentosa, Leber congenital amaurosis (LCA), Stargardt disease, Alström syndrome, or an ABCA4-related disease.

[0281] In some embodiments, the dose is 1×10 per eye. 9 ~1.5×10 10 In some embodiments, the dose is 4×10 total genome copies per eye. 9 ~1.5×10 10 In some embodiments, the dose is 1 x 10 total genome copies per eye. 9 ~8×109 total genome copies, 2 × 10 per eye 9 ~7×10 9 total genome copies, 3 × 10 per eye 9 ~6×10 9 total genome copies or 4 × 10 per eye 9 ~5×10 9 In some embodiments, the dose is 7×10 total genome copies per eye. 9 ~5×10 10 total genome copies, 8 × 10 per eye 9 ~4×10 10 total genome copies, 9 × 10 per eye 9 ~3×10 10 1 x 10 total genome copies or 1 x 10 per eye 10 ~2×10 10 The total genome copies are

[0282] Equivalent doses optimized for human subjects may be used. In some embodiments, the dose is 1×10 per eye. 9 ~2×10 12 In some embodiments, the dose is 1 x 10 total genome copies per eye. 10 ~2×10 12 In some embodiments, the dose is 1 x 10 total genome copies per eye. 11 ~2×10 12 The total genome copies are

[0283] In some embodiments, the dose is 1×10 per eye. 11 ~1.5×10 12 In some embodiments, the dose is 4×10 total genome copies per eye. 11 ~1.5×10 12 In some embodiments, the dose is 1 x 10 total genome copies per eye. 11 ~8×10 11 total genome copies, 2 × 10 per eye 11 ~7×10 11 total genome copies, 3 × 10 per eye 11 ~6×10 11total genome copies or 4 × 10 per eye 11 ~5×10 11 In some embodiments, the dose is 7×10 total genome copies per eye. 11 ~5×10 12 total genome copies, 8 × 10 per eye 11 ~4×10 12 total genome copies, 9 × 10 per eye 11 ~3×10 12 1 x 10 total genome copies or 1 x 10 per eye 11 ~2×10 12 The total genome copies are

[0284] Equivalent doses that are optimized for different non-human subjects may be used.

[0285] subject As used herein, the term "subject" refers to either a human or a non-human animal.

[0286] Examples of non-human animals include vertebrates, such as mammals, such as non-human primates (particularly higher primates), dogs, rodents (e.g., mice, rats or guinea pigs), pigs and cats. The non-human animals may be pets.

[0287] Preferably, the subject is a human.

[0288] Variants, Derivatives, Analogs, and Fragments In addition to the specific proteins and polynucleotides mentioned herein, the present invention also encompasses variants, derivatives, and fragments thereof.

[0289] In the context of the present invention, a "variant" of any given sequence is a sequence in which a specific sequence of residues (whether amino acid or nucleic acid residues) has been modified such that the polypeptide or polynucleotide retains at least one of its endogenous functions. Variant sequences can be obtained by addition, deletion, substitution, modification, replacement and / or mutation of at least one residue present in a naturally occurring polypeptide or polynucleotide.

[0290] The term "derivative" as used herein in reference to a protein or polypeptide of the invention includes any substitution, mutation, modification, replacement, deletion and / or addition of one (or more) amino acid residues from or to the sequence, provided that the resulting protein or polypeptide retains at least one of its endogenous functions.

[0291] Typically, amino acid substitutions, for example from one, two or three to ten or twenty substitutions, can be made, provided that the altered sequence retains the required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues.

[0292] Proteins used in the present invention may also have deletions, insertions or substitutions of amino acid residues that result in silent changes and result in functionally equivalent proteins. Deliberate amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or amphipathicity of the residues, so long as the intrinsic function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid, positively charged amino acids include lysine and arginine, and amino acids with uncharged polar head groups with similar hydrophilicity values ​​include asparagine, glutamine, serine, threonine and tyrosine.

[0293] Conservative substitutions may be made, for example, according to the following table. Amino acids in the same block in the second column and in the same line in the third column may be substituted for each other:

[0294] [Table 2]

[0295] Typically, the variants may have a certain identity with the wild-type amino acid sequence or the wild-type nucleotide sequence.

[0296] In this context, a variant sequence is taken to include an amino acid sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical to the subject sequence, suitably at least 95%, 96% or 97% or 98% or 99% identical. Although variants can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties / functions), it is preferred in the context of the present invention to be expressed in terms of sequence identity.

[0297] In this context, a variant sequence is taken to include a nucleotide sequence which may be at least 50%, 55%, 65%, 75%, 85% or 90% identical to the subject sequence, suitably at least 95%, 96% or 97% or 98% or 99% identical. Variants can also be considered in terms of similarity, although in the context of the present invention they are preferably expressed in terms of sequence identity.

[0298] Suitably, reference to a sequence having a percent identity to any one of the SEQ ID NOs detailed herein refers to a sequence having that stated percent identity over the entire length of the referenced SEQ ID NO.

[0299] Sequence identity comparisons can be performed by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the percent identity between two or more sequences.

[0300] Percent identity may be calculated over a contiguous sequence, i.e., one sequence is aligned with the other and each amino acid or nucleotide in one sequence is directly compared, one residue at a time, to the corresponding amino acid or nucleotide in the other sequence. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively small number of residues.

[0301] While this is a very convenient and consistent method, it does not take into account that, for example, in an otherwise identical sequence pair, a single insertion or deletion in an amino acid or nucleotide sequence may exclude the following residue or codon from the alignment, thus resulting in a large decrease in percent identity when a global alignment is performed. As a result, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall identity score. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local identity.

[0302] However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment, so that a sequence alignment that contains as few gaps as possible, reflecting a higher relatedness between the two compared sequences for the same number of identical amino acids or nucleotides, scores higher than one that contains a large number of gaps. An "affine gap cost" is usually used, which imposes a relatively high cost on the existence of a gap and a smaller penalty on each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will naturally produce an optimized alignment that contains fewer gaps. Most alignment programs allow the gap penalty to be modified. However, it is preferred to use the default values ​​when using such software for sequence comparison. For example, when using the GCG Wisconsin Bestfit package, the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0303] Therefore, the calculation of maximum percent identity first requires the creation of an optimal alignment, taking into account gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Research 12:387). Examples of other software that can carry out sequence comparison include, but are not limited to, the BLAST package (see Ch. 18 of Ausubel et al. (1999) ibid.), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410), EMBOSS Needle (Madeira, F. et al., 2019. Nucleic acids research, 47(W1), pp. W636-W641) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al. (1999) ibid., pages 7-58 to 7-60). However, for some applications it is preferred to use the GCG Bestfit program. Another tool, BLAST 2 Sequences, is also available for comparing protein and nucleotide sequences (FEMS Microbiol. Lett. (1999) 174(2):247-50; FEMS Microbiol. Lett. (1999) 177(1):187-8).

[0304] Although a final percent identity can be measured, the alignment process itself is usually not based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​typically used that assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin programs typically use either the public default values ​​or a custom symbol comparison table if provided (see user manual for further details). For some applications, it is preferred to use the public default values ​​for the GCG package, or in the case of other software, a default matrix such as BLOSUM62.

[0305] Once the software has produced an optimal alignment, it is possible to calculate the percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. The percent sequence identity can be calculated as the number of identical residues as a percentage of the total residues in the referenced SEQ ID NO.

[0306] A "fragment" is also a variant, and this term generally refers to a selected region of a polypeptide or polynucleotide of interest functionally or, for example, in an assay. Thus, a "fragment" refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0307] Such variants, derivatives, and fragments may be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. Where an insert is to be made, synthetic DNA may be made that encodes the insert together with 5' and 3' flanking regions corresponding to the naturally occurring sequence on either side of the insertion site. The flanking regions contain convenient restriction sites corresponding to sites in the naturally occurring sequence, so that the sequence may be cleaved with the appropriate enzyme(s) and the synthetic DNA ligated to the cuts. The DNA is then expressed in accordance with the invention to make the encoded protein. These methods are merely illustrative of the many standard techniques known in the art for the manipulation of DNA sequences, and other known techniques may be used.

[0308] It will be understood by those skilled in the art that it is possible to combine all of the features of the invention disclosed herein without departing from the scope of the invention disclosed.

[0309] Preferred features and embodiments of the present invention will now be described by way of non-limiting example.

[0310] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology which are within the skill of those in the art. Such techniques are explained in the literature. See, e.g., Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F. M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J., and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J. M., and McGee, J. O'D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M. J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D. M., and Dahlberg, J. E. (1992) Methods in Enzymology: DNA Structures Part 1, pp. 111-115, 2002. A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general purpose texts is incorporated herein by reference.

[0311] [Example] [Example 1] Results and Discussion Optimization of dual adeno-associated virus (AAV) vectors During characterization of dual AAV8 vectors for delivery of human myosin 7A (hMYO7A), the inventors discovered a contaminant vector in a preparation of vector containing the 5'-terminal portion of the transgene coding sequence CDS (AAV8-5'hMYO7A).

[0312] Southern blot analysis developed using a probe recognizing the tribebeta-actin (CBA) promoter used in the vector showed a larger band corresponding to the expected AAV8-5'hMYO7A and a smaller band of approximately 1.3 Kb corresponding to a contaminant (Figures 1A and 1B).

[0313] Smaller genomic contaminants were consistently present in vector preparations but not in the plasmids used to generate them, therefore we hypothesized that this problem was related to the viral genome and that, since the original plasmid genome was apparently intact, generation of the smaller products occurs during or after production of the vector particles.

[0314] Next, we identified an 82 base pair region of homology between two sequences: the chimeric promoter intron and the splicing donor (SD) signal (Figure 1C, see sequence below).

[0315] Chimeric introns (Bothwell et al. (1981) Cell 24:625-637): TIFF2024517957000058.tif19160 (SEQ ID NO: 16)

[0316] Splicing donor (SD) sequence: TIFF2024517957000059.tif16160 (SEQ ID NO: 17)

[0317] The underlined sequences are identical: the SD sequence is identical to nucleotides 1-82 of the chimeric intron.

[0318] Using subcloning and Sanger sequencing of purified viral DNA, we confirmed that a homologous recombination event occurred due to the presence of regions of homology within the construct, resulting in deletion of the intron, the 5' hMYO7A sequence, and the remainder of the SD signal, while the new construct still retained the AAV inverted terminal repeats (ITRs), thus supporting vector production (Figure 1D).

[0319] Similar contaminants were observed in other vectors (eg, containing other transgenes and promoters) containing intron and SD sequences (FIG. 1E) and were eliminated by removing the intron sequences.

[0320] We then replaced the chimeric intron with a sequence that is not homologous to the SD sequence. We cloned plasmids encoding enhanced green fluorescent protein (EGFP) with either the chimeric intron, a modified version of the simian virus 40 (SV40) intron (Nathwani et al. (2006) Blood 107:2653-2661), the minute virus of mice (MVM) intron (Wu et al. (2008) Mol. Ther. 16:280-289), or no intron, to compare EGFP expression in HE293 cells by transfection. Fluorescence imaging (Figure 2B) shows that EGFP expression from constructs containing the SV40 intron or the MVM intron is similar to that containing the chimeric intron.

[0321] After cloning the SV40 and MVM introns, we generated respective AAV2 vectors containing the 5'-end portion of the hMYO7A CDS (AAV2-SV40 intron-5'hMYO7A and AAV2-MVM intron-5'hMYO7A) and performed a second in vitro comparison in HEK293 cells by MYO7A expression against AAV2-chimeric intron-5'hMYO7A and AAV2-intronless-5'hMYO7A.

[0322] Western blot analysis demonstrated that both the SV40 and MVM introns induced comparable expression levels of hMYO7A in vitro (Figure 3).

[0323] Finally, we generated AAV8 vectors (AAV8-SV40 intron-5'hMYO7A and AAV8-MVM intron-5'hMYO7A) each containing the 5'-end portion of the hMYO7A CDS. We then performed a third comparison to AAV8-chimeric intron-5'hMYO7A by Southern blot of purified viral DNA, and we also co-injected AAV8-3'hMYO7A-3XFlag subretinally into C57BL / 6 mice to assess hMYO7A expression levels by Western blot analysis.

[0324] We found that both the SV40 and MVM introns achieved similar hMYO7A expression levels in vivo, avoiding the formation of contaminant vectors (Figure 4). We decided to use AAV8-SV40 intron-5'hMYO7A for the production of dual AAV8-5'hMYO7A to be used in preclinical and clinical studies.

[0325] Materials and Methods Generation of AAV vector plasmids The plasmid used for AAV vector production contained the inverted terminal repeats (ITRs) of AAV serotype 2. The two AAV vector plasmids (5' and 3') required to generate the dual AAV vector contained several elements. The 5' plasmid contained the tribeta-actin promoter (CBA) and CMV enhancer linked to a chimeric promoter intron consisting of a 5'-donor site from the first intron of the human β-globin gene and a 3'-acceptor site from an intron between the branch and leader and the body of the immunoglobulin gene heavy chain variable region (Bothwell et al. (1981) Cell 24:625-637), a modified version of the simian virus 40 promoter intron (SV40) (Nathwani et al. (2006) Blood 107:2653-2661) or a minute virus of mouse intron (Wu et al. (2008) Mol. Ther. 16:280-289); the N-terminal portion of the transgene coding sequence (CDS); and a splice donor sequence. The 3' plasmid contained a splice acceptor sequence and the C-terminal portion of the transgene CDS followed by BGH polyA. For some experiments, the 3' portion of hMYO7A with a C-terminal 3XFlag-tag was used.

[0326] The hMYO7A CDS was split between exons 24 and 25 at natural exon-exon junctions (5' half: NM_000260.3, bp 273-3380; 3' half: NM_000260.3, bp 3381-6920).

[0327] The splice donor (SD) and splice acceptor (SA) sequences contained in the dual AAV vector plasmid are as follows:

[0328] SD: TIFF2024517957000060.tif16160 (SEQ ID NO: 18)

[0329] SA: TIFF2024517957000061.tif11160 (SEQ ID NO: 19)

[0330] The recombinogenic sequences contained in the hybrid AK vector plasmid were derived from the phage F1 genome (Gene Bank Accession No: J02448.1; bp 5850-5926). The AK sequence is TIFF2024517957000062.tif11160 (SEQ ID NO: 20) It is.

[0331] AAV vector production and characterization The dual AAV-hMYO7A vector was produced by the TIGEM AAV Vector Core. The vector was produced by triple transfection of HEK293 cells followed by two rounds of CsCl2 purification (Grimm et al. (1998) Hum. Gene Ther. 2760:2745-2760; Liu et al. (2003) Biotechniques 34:184-189; Salvetti et al. (1998) Hum. Gene Ther. 9:695-706; Zolotukhin et al. (1999) Gene Ther. 6:973-985). The physical titer [genome copies (GC) / ml] was determined for each virus preparation by TaqMan quantitative PCR (Applied Biosystems, Carlsbad, CA, USA). Primers and probes were designed to anneal with 5'-hMYO7A for AAV-5'hMYO7a and with BGH pA for AAV-3'hMYO7A. Alkaline Southern blot analysis for AAV-5'hMYO7A was performed as follows: 3E+10 GC viral DNA was extracted from AAV particles. To digest unpackaged genomes, the vector solution was incubated with 1 U / μL DNase I (Roche, Milan, Italy) for 2 h at 37°C in a total volume of 300 μL containing 40 mM TRIS-HCl, 10 mM NaCl, 6 mM MgCl2, 1 mM CaCl2, pH 7.9. DNase I was then inactivated with 50 mM EDTA before incubating with proteinase K and 2.5% N-lauroyl-sarkosyl solution at 50°C for 45 min to dissolve the capsids. DNA was extracted twice with phenol-chloroform and precipitated with two volumes of absolute ethanol and 10% sodium acetate (3 M, pH 7). Purified DNA was run on an alkaline agarose gel and imaged using a digoxigenin non-radioactive method (Roche, Milan, Italy). 10 μL of 1 kb DNA ladder (N3232L; New England Biolabs, Ipswich, MA, USA) was loaded as a molecular weight marker.The Southern blot probe was obtained by enzymatic digestion of 5' AAV plasmid DNA with KpnI-XhoI and the 544 base pair probe was extracted and purified.

[0332] Cell culture and transfection HEK293 cells were maintained in DMEM supplemented with 10% fetal bovine serum (FBS) (Gibco, Thermo Fisher Scientific, Waltham, MA, USA). Cells were plated in 6-well plates (HEK293 1E+6 cells / well) and 24 h later wells were transfected using calcium phosphate + 1.5 μg of the corresponding plasmid. After 4 h, the medium was replaced with 2 mL of fresh pre-warmed medium. 72 h after transfection, cells were harvested and lysed.

[0333] Subretinal injection of AAV vectors in mice This study was carried out in accordance with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research and with the Italian Ministry of Health regulations on animal procedures (authorization n°301 / 2020-PR).

[0334] C57BL / 6 and Shaker mice were housed in TIGEM animal house (Pozzuoli, Italy) and maintained under a 12-h light / dark cycle (10-50 lux exposure during the light phase). Surgery was performed under anesthesia, and every effort was made to minimize discomfort. Adult mice were anesthetized with an intraperitoneal injection of 2 mL / 100 g body weight of ketamine / medetomidine. Equal volumes of vector solution or vehicle were delivered subretinal via a posterior transscleral transchoroidal approach as described by Liang et al. (Liang et al. (2000) Vis. Res. Protoc. 47:125-139).

[0335] Western blot analysis Cells and eye cups (cups + retina) for Western blot (Wb) analysis were lysed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP40, 0.5% Na-deoxycholate, 1 mM EDTA pH 8.0, 0.1% SDS) to extract MYO7A. The lysis buffer was supplemented with 0.5% phenylmethylsulfonyl fluoride (PSMF) (Sigma-Aldrich, St. Louis, MO, USA) and 1% cOmplete EDTA-free protease inhibitor cocktail (Roche, Milan, Italy). Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo-Scientific). After lysis, samples were denatured for 5 min at 99 °C in 4 × Laemmli sample buffer (Bio-rad, Milan, Italy) supplemented with β-mercaptoethanol 1:10. Samples were separated on a 7% acrylamide gel. Antibodies used for immunoblotting were: anti-3XFlag (1:1000, monoclonal, A8592; Sigma) to recognize full-length hMYO7A-3XFlag, anti-dysferlin (1:500, MONX10795; Tebu-bio, Le Perret-en-Yvelines, France). Quantification of Wb bands was performed using ImageJ software, and hMYO7A expression was normalized to that of dysferlin.

[0336] Vector sequence The sequences of the MYO7A-encoding vectors used in the experiments are disclosed herein as SEQ ID NOs:14 and 15.

[0337] The sequences of further vectors used in the experiments are as follows: 5'CMV-ABCA4-AK TIFF2024517957000063.tif101160TIFF2024517957000064.tif245160TIFF2024517957000065.tif144160 (SEQ ID NO: 21)

[0338] JPEG2024517957000066.jpg53168

[0339] 5'CMV-ABCA4-TS TIFF2024517957000067.tif244160TIFF2024517957000068.tif230160(SEQ ID NO:22)

[0340] JPEG2024517957000069.jpg46156

[0341] 5'CMV NO INTRON ABCA4 OV TIFF2024517957000070.tif172160TIFF2024517957000071.tif246160TIFF2024517957000072.tif75160 (SEQ ID NO: 23)

[0342] JPEG2024517957000073.jpg32157

[0343] 5'CMV NO INTRON ABCA4-AK TIFF2024517957000074.tif108160TIFF2024517957000075.tif244160TIFF2024517957000076.tif99160 (SEQ ID NO: 24)

[0344] JPEG2024517957000077.jpg47156

[0345] 5'VMD2 ABCA4-AK TIFF2024517957000078.tif61160TIFF2024517957000079.tif245160TIFF2024517957000080.tif160160 (SEQ ID NO: 25)

[0346] JPEG2024517957000081.jpg53156

[0347] 5'RHOABCA4-AK TIFF2024517957000082.tif238160TIFF2024517957000083.tif238160(SEQ ID NO:26)

[0348] JPEG2024517957000084.jpg53156

[0349] 5'RHO ABCA4-TS TIFF2024517957000085.tif160160TIFF2024517957000086.tif245160TIFF2024517957000087.tif61160 (SEQ ID NO: 27)

[0350] JPEG2024517957000088.jpg47156

[0351] [Example 2] Dual AAV8.hMYO7A response study The therapeutic efficacy of the dual AAV8.hMYO7A containing AAV8.5'MYO7A with an SV40 intron was evaluated in the Shaker mouse model of Usher-1b. - / - (sh1 - / - To select the dose to be used in Usher syndrome type 1B (USH1B) subjects, we performed a dose-response study using a duplex AAV8.hMYO7A (i.e., tox lot) produced under good manufacturing-like practices. A mouse model of USH1B injected subretinally, sh1 - / -Mice were analyzed for rescue of retinal defects and protein hMYO7A levels. We selected three different doses: 1,37E+9 (low dose or LD), 4,4E+9 (medium dose or MD), and 1,37E+10 (high dose or HD) total GCs / eye. Unaffected heterozygous mice and affected mice injected with AAV vehicle only (phosphate buffered saline supplemented with NaCl 35 mM and 0.001% Poloxamer 188) were used as positive and negative controls, respectively. As melanosomes are rarely located in the retinal pigment epithelium (RPE) apical villi, Sh1 - / - Mice exhibit retinal ultrastructural defects. Three months after injection, we confirmed a dose-dependent effect by measuring the number of melanosomes precisely localized to the RPE apical villi (Figures 5A-5B). HD and MD injections of dual AAV8.hMYO7A significantly improved the survival of sh1 mice receiving vehicle alone. - / - The retinal defect was significantly rescued compared to the non-affected and affected eyes treated with HD, and no statistical difference was observed between the affected and unaffected eyes treated with HD (Figure 5B, pANOVA values: affected sh1 - / - Vs. unaffected sh1 injected with formulation buffer + / - <0,0001 vs. high dose treated sh1 - / - <0,0001, compared to medium dose treated sh1 - / - <0.01 or vs. low dose treated sh1 - / - = 0, 313; sh1 treated with high dose - / - Vs. unaffected sh1 injected with formulation buffer + / - = 0,105, compared to sh1 treated at a medium dose - / - = 0,113 or sh1 treated with low doses - / - <0,01; sh1 treated with medium dose - / - Vs. unaffected sh1 injected with formulation buffer + / - <0,001 or low dose treated sh1 - / - = 0, 442; unaffected sh1 injected with formulation buffer + / - vs. sh1 treated with low dose - / -<0,0001). LD-treated Sh1 - / - The unaffected sh1 eyes also showed correction of the retinal phenotype compared to the negative controls. + / - There was some within-group variability, and therefore we did not find any significant differences in the results for unaffected sh1 + / - The ANOVA analysis was repeated without and reached statistical significance for LD as well (Figure 5B, pANOVA values: diseased sh1 - / - vs. high dose treated sh1 - / - <0,0001, compared to medium dose treated sh1 - / - <0,0001 or vs. low dose treated sh1 - / - <0,01; sh1 treated with high dose - / - vs. sh1 treated at medium doses - / - <0,001 or vs. low dose treated sh1 - / - <0,0001; sh1 treated with medium dose - / - vs. sh1 treated with low dose - / - <0.05). Five weeks after retinal injection, sh1 - / - Western blot analysis of lysed eye cups (RPE+neural retina) from mice shows expression of full-length hMYO7A for all selected doses of dual AAV8.hMYO7A (Figure 5C-5D). A greater number of eyes were positive for hMYO7A expression using HD and MD compared to LD (Figure 5D). Considering that the human retina is 100 times larger than the mouse retina (Panda-Jonas et al. (1994) Ophthalmology 101:519-523; Remtulla et al. (1985) Vision Res. 25:21-31), we can infer that the equivalent therapeutic dose in humans could be in the range of 1.37E+11 to 1.37E+12 total GCs / eye of dual AAV8.hMYO7A.

[0352] Materials and Methods Western blot analysis Eye cups (cups + retina) for Western blot (WB) analysis were lysed in RIPA buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1% NP40, 0.5% Na-deoxycholate, 1 mM EDTA pH 8.0, 0.1% SDS). Lysis buffer was supplemented with 0.5% phenylmethylsulfonyl fluoride (PSMF) (Sigma-Aldrich, St. Louis, MO) and 1% complete EDTA-free protease inhibitor cocktail (Roche, Milan, Italy). Protein concentration was determined using the Pierce BCA Protein Assay Kit (Thermo-Scientific, Waltham, MA). After lysis, samples were denatured for 5 min at 99 °C in 4 × Laemmli sample buffer (Bio-rad, Milan, Italy) supplemented with β-mercaptoethanol (Sigma-Aldrich) diluted 1:10. Samples for MYO7A analysis on 4-20% gradient precast TGX gels (Bio-rad). The following antibodies were used for immunoblotting: custom made anti-hMYO7A (1:200, polyclonal; Primm Srl, Milan, Italy), which recognizes a peptide corresponding to amino acids 941-1070 of the hMYO7A protein (DMVDKMFGFLGTSGGLPGQEGQAPSGFEDLERGRREMVEEDLDAALPLPDEDEEDLSEYKFAKFAATYFQGTTTHSYTRRPLKQPLLYHDDEGDQLAALAVWITILRFMGDLPEPKYHTAMSDGSEKIPV; underlined amino acids differ (1.6%) from mouse Myo7A), anti-dysferlin (1:500, MONX10795; Tebu-bio, Le Perret-en-Yvelines, France). Quantification of WB bands was performed using ImageJ software. hMYO7A expression was normalized to dysferlin expression.

[0353] Melanosome localization analysis Eyes from pigmented sh1 mice (+ / - or - / -) were enucleated 3 months after AAV injection and cauterized on the temporal side of the cornea. Fixation was performed overnight using 2% glutaraldehyde-2% paraformaldehyde in 0.1M PBS, rinsed in 0.1M PBS, and examined under a light microscope. The temporal part of the eyecup was embedded in Araldite 502 / EMbed 812 (Araldite 502 / EMbed 812 KIT, Cat. #13940; Electron Microscopy Sciences, Hatfield, PA, USA). Semi-thin (0.5 μm) sections were cut in cross-section on a Leica Ultramicrotome RM2235 (Leica Microsystems, Bannockburn, IL, USA), mounted on glass slides, and stained with toluidine blue and borace stain. Melanosomes were counted by a masked operator in montages of whole retinal sections obtained through the acquisition of overlapping fields using a Zeiss Apotome (Carl Zeiss, Oberkochen, Germany) at 100x magnification, and whole retinal sections were subsequently reconstructed with Photoshop software (Adobe, San Jose, CA). Melanosome counts and retinal pigment epithelium (RPE) measurements were performed using ImageJ software. Melanosome numbers were normalized by the length of the RPE divided by 100 μm.

[0354] statistical analysis One-way analysis of variance (ANOVA) followed by Tukey's post-hoc test was used to perform multiple pairwise comparisons between groups in Figure 5. Figure 5: Dose-dependent effects on melanosomes precisely localized to the retinal pigment epithelium: ANOVA p-values ​​are: - / - Vs. unaffected sh1 injected with formulation buffer + / - (pANOVA<0,0001), vs. high dose treated sh1 - / - (pANOVA<0,0001), vs. sh1 treated at medium dose - / - (pANOVA<0,01) or vs. sh1 treated with low dose - / -(pANOVA=0,313); sh1 treated with high dose - / - Vs. unaffected sh1 injected with formulation buffer + / - (pANOVA=0,105), vs. sh1 treated at the medium dose - / - (pANOVA=0,113) or sh1 treated with low dose - / - (pANOVA<0,01); sh1 treated with medium dose - / - Vs. unaffected sh1 injected with formulation buffer + / - (pANOVA<0,001) or vs. low dose treated sh1 - / - (pANOVA 0,442); unaffected sh1 injected with formulation buffer + / - vs. sh1 treated with low dose - / - (pANOVA<0,0001). The effect of injection of formulation buffer on the ANOVA analysis was not significant. + / - Due to variability in the ANOVA p-values, the comparisons were analyzed again without the non-affected controls: affected sh1 injected with formulation buffer - / - vs. high dose treated sh1 - / - (pANOVA<0,0001), vs. sh1 treated at medium dose - / - (pANOVA<0,0001) or vs. low dose treated sh1 - / - (pANOVA<0,01); sh1 treated with high dose - / - vs. sh1 treated at medium dose (pANOVA<0,001) or vs. sh1 treated at low dose - / - (pANOVA<0,0001); sh1 treated with medium dose - / - vs. sh1 treated with low dose - / - (pANOVA<0.05). Data are presented as mean values ​​[±standard error of the mean (sem)] calculated using independent in vitro experiments or numbers of eyes (not repeated measurements of the same sample). A statistical p-value ≦0.05 was considered significant.

[0355] All publications mentioned in the above specification are incorporated herein by reference. Various modifications and alterations of the disclosed vectors, systems, methods or uses of the present invention will be apparent to those skilled in the art without departing from the scope and spirit of the present invention. Although the present invention has been disclosed in connection with certain preferred embodiments, it should be understood that the invention described in the claims should not be unduly limited to such specific embodiments. Indeed, various modifications of the disclosed modes for carrying out the invention will be apparent to those skilled in the art and are intended to be within the scope of the appended claims.

Claims

1. A vector system for expressing a transgene in a cell, comprising a first vector and a second vector, (a) a first vector comprising, in a 5' to 3' direction, a promoter, an intron, a 5' end portion of a transgene coding sequence (CDS), a splice donor sequence, and a first recombinogenic region; (b) a second vector comprising, in the 5' to 3' direction, a second recombinogenic region, a splice acceptor sequence, and a 3' end portion of the transgene CDS; The 5'-end portion and the 3'-end portion together constitute a transgene CDS, and the intron is not capable of homologous recombination with the splice donor sequence to excise the 5'-end portion of the transgene CDS. The vector system.

2. 2. The vector system of claim 1, wherein the intron does not include a region of at least 20, 30, 40, 50, 60, 70, 80, 90 or 100 contiguous nucleotides having at least 95%, 96%, 97%, 98%, 99% or 100% (preferably 100%) sequence identity to a region of the splice donor sequence.

3. The intron is (a) a simian virus 40 (SV40) intron or a minute virus of mice (MVM) intron; and / or (b) comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 3 or 4; The vector system of claim 1.

4. The vector system of claim 1 , wherein the splice donor sequence comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:

5.

5. The first recombinogenic region and the second recombinogenic region (a) both are F1 phage recombinogenic regions or fragments thereof; and / or (b) both contain a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:7 or a fragment thereof, The vector system of claim 1.

6. The vector system of claim 1 , wherein the first vector and the second vector are viral vectors.

7. 2. The vector system of claim 1, wherein the first vector and the second vector are AAV vectors, and optionally the first vector further comprises a 5'ITR and a 3'ITR, and the second vector further comprises a 5'ITR and a 3'ITR.

8. The vector system of claim 1 , wherein the promoter is a CBA promoter or a fragment thereof.

9. The vector system of claim 1 , wherein the second vector further comprises a polyadenylation sequence downstream of the 3'-end portion of the transgene CDS.

10. 2. The vector system of claim 1, wherein the transgene is a myosin 7A (MYO7A) transgene.

11. (a) the first vector comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO: 14; and / or (b) the second vector comprises a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:15; The vector system of claim 1.

12. 13. A method for expressing an introduced gene in a cell, the method comprising transducing or transfecting the cell with a first vector and a second vector as defined in claim 1 such that the introduced gene is expressed in the cell.

13. A vector comprising, in the 5' to 3' direction, a promoter, an intron, a 5' end portion of a transgene coding sequence (CDS), a splice donor sequence, and a recombination induction region, wherein the intron is not capable of homologous recombination with the splice donor sequence to excise the 5' end portion of the transgene CDS.

14. The vector of claim 13, comprising a nucleotide sequence having at least 95% sequence identity to SEQ ID NO:

14.

15. 10. The vector system of claim 1 for use in therapy.

16. 2. The vector system of claim 1 for use in the treatment of Usher syndrome, optionally Usher syndrome type 1B.

17. 13. A method for treating or preventing Usher syndrome, comprising administering an effective amount of the vector system of claim 1 to a subject in need of treatment or prevention, optionally wherein the Usher syndrome is Usher syndrome type 1B.

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