A hybrid dual aav vector system with splice enhancer elements for expression of large genes

Abstract

The present invention relates to a construction of the hybrid dual AAV vector system by utilizing a novel 90bp splice enhancer intron to enhance the efficiency of splicing and the expression of the dual vector system. The new constructs containing the SEI in the 5' head-vector and 3' tail-vector (pGL10) are compared with the alkaline phosphatase fragment containing OAP vectors with respect to the splicing efficacy and the expression efficacy of the LacZ gene. The results indicated that the new hybrid dual AAV vector system, by utilizing a 90bp splice enhancer intron, improved the splicing efficiency and the expression of transgene as well as provided more transgene packaging space, suggesting the use of this dual vector system for the expression of therapeutic genes such as ABCA4, etc.

Description

TITLE OF THE INVENTIONA hybrid dual AAV vector system with splice enhancer elements for expression of large genesPriority Claim:

[0001] This application claims priority from the provisional application numbered 202341034269 filed with Indian Patent Office, Chennai on 16thJune 2023 entitled “A hybrid dual AAV vector system with splice enhancer elements for expression of large genes '., the entirety of which is expressly incorporated herein by reference.Preamble to the Description

[0002] The following specification describes the invention and the manner in which it is to be performed:DESCRIPTION OF THE INVENTIONTechnical field of the invention

[0003] The present invention relates to a construction of a hybrid dual Adeno- associated Virus (AAV) vector system to improve the transgene expression from the dual vector systems. More particularly, the invention relates to the construction of the hybrid dual AAV vector system by utilizing a novel 90bp splice enhancer intron (SEI) to enhance the efficiency of splicing and the expression of the dual vector system.Background of the invention

[0004] Adeno-associated virus as a vector and is a single-stranded DNA parvovirus, the genome of which comprises the rep gene and the cap gene flanked by two inverted terminal repeats. The AAV vector is then used to deliver normal copies of genes to the target tissues or organs in the body, but now it is alsoconsidered to deliver the therapy that has been engineered into it. Currently, AAV technology has advanced to target a wide range of tissues and cells for the treatment of many genetic diseases. AAV has low toxicity, apparent lack of pathogenicity and provides long-term transgene expression.

[0005] Recombinant adeno-associated viral (rAAV) vectors are a leading platform for gene therapies due to its low immunogenicity and greater efficacy as a gene delivery vehicle. Generally, the clinically approved gene therapies use a single rAAV vector carrying only a smaller therapeutic expression cassette. The transgene packaging capacity of rAAV is 4.7 kb and any larger gene is not packaged. Over the past two decades, several studies have addressed this limitation with developments in dual vector strategies, that have demonstrated superiority in final transgene expression.

[0006] In order to overcome the limitations of the previously described platforms such as the lack of preference for directional tail-to-head concatemerization of the trans- splicing approach and the need for optimization of the CDS overlap for each transgene in the overlapping approach, a third transgene-independent dual AAV approach namely hybrid dual AAV vectors are developed. This approach is a combination of the trans-splicing and overlapping approaches, as it is based on the addition of a highly recombinogenic exogenous sequence to the trans-splicing vectors in order to increase recombination efficiency.

[0007] The hybrid dual vector system involves transgene independent homologous recombination, utilising both ITR mediated and an intronic overlapping element mediated recombination between the two half-vector genomes for full transgene reconstitution, which offers additional flexibility with increased recombination events and higher efficacies in transgene reconstitution and expression.

[0008] Previously, various fragment lengths of a highly recombinogenic alkaline phosphatase (AP) sequence have been tested as overlapping sequences in hybrid dual vector system. This element though being highly recombinogenic and functional at transgene reconstitution, has shown to be partially efficacious in termsof transgene expression levels. Moreover, the shortest version of alkaline phosphatase used is 0.26kb.

[0009] The Patent Application No US 12 / 473,651, entitled, “ Hybrid- AAV vectors to deliver large gene expression cassette” discloses a hybrid adeno-associated virus (AAV) vector systems able to efficiently express therapeutic target genes larger than may be carried in a single AAV vector are provided, wherein a highly recombinogenic foreign DNA sequence is incorporated into two or more ITR- mediated AAV vectors. The novel hybrid AAV vector system is a hybrid dual AAV (hdAAV) vector system. The novel hybrid AAV vector system is a hybrid tri AAV (htAAV) vector system. A method of treating a clinical disease caused at least in part by a defective gene is provided, and comprises (1) providing a hybrid AAV vector system capable of expressing a therapeutic target gene, wherein the therapeutic target gene is capable of replacing, restoring or counteracting the effects of the defective gene; and (2) administering a therapeutic amount of said vector system to a subject wherein said therapeutic target gene is expressed at levels having a therapeutic effect.

[0010] The Patent Application No. CA3008956A1 entitled ''Improved hybrid dual recombinant AAV vector systems for gene therapy” discloses constructs, vectors, relative host cells and pharmaceutical compositions which allow an effective gene therapy, in particular of genes larger than 5Kb by using an improved hybrid dual recombinant AAV vector system.

[0011] The Patent Application No. US 16 / 952,016 entitled “Improved hybrid dual recombinant AAV vector systems for gene therapy” discloses AAV-based, dualvector systems that facilitate the expression of full-length proteins whose coding sequences exceed that of the polynucleotide packaging capacity of an individual AAV vector. The vector systems are provided that include i) a first AAV vector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide; and ii) a second AAVvector polynucleotide that includes an inverted terminal repeat at each end of the polynucleotide and a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. In another embodiment, the vector system includes i) a first AAV vector polynucleotide comprising an inverted terminal repeat at each end, a suitable promoter followed by a partial coding sequence that encodes an N-terminal portion of a full-length polypeptide followed by a splice donor site and intron and ii) a second AAV vector polynucleotide comprising an inverted terminal repeat at each end, followed by an intron and a splice-acceptor site for the intron, followed by a partial coding sequence that encodes a C-terminal portion of a full-length polypeptide, optionally followed by a polyadenylation (pA) signal sequence. The coding sequence or the intron sequence in the first and second AAV vectors preferably includes a sequence region that overlaps.

[0012] It is observed that the existing recombinant AAV vector systems are targeted for expression of genes of higher size, but the existing vector systems are silent with respect to enhanced efficacy by using splice enhancer.Summary of the invention

[0013] The present invention overcomes the drawbacks of the existing systems in packaging capacity of existing vectors, by providing a dual vector system, wherein a hybrid dual Adeno-associated Virus (AAV) vector system aims to improve the expression of the dual vector systems by enhancing the efficient splicing utilizing a splice enhancer intron (SEI) element.

[0014] The hybrid dual Adeno-associated Virus (AAV) vector system comprises a novel synthetic splice enhancer intron (SEI) element, facilitating increase in the efficiency of splicing and the gene expression. The synthetic splice enhancer element displays improved expression in comparison to existing hybrid dual vector (with AP derived overlapping sequences) systems and increase in the transgene packaging size due to the smaller overlapping sequence size.

[0015] The hybrid dual Adeno-associated Virus (AAV) vector system discloses a novel, smaller 90 base pair splice enhancer intron (SEI) to achieve dual vector reconstitution and efficient splicing. The splice enhancer intron (SEI) vector system includes a synthetic splice enhancer intron (SEI) arranged in tandem with high recombination potential, wherein the smaller size of the overlapping fragment in the splice enhancer intron (SEI) dual vectors, improve the packaging limit of the hybrid Adeno-associated Virus (AAV) dual vectors compared to the OAP vectors. The synthetic splice enhancer intron (SEI) is cloned in a dual LacZ head-tail expression cassette expressed under the control of a cytomegalovirus (CMV) promoter as depicted in SEQ ID: 1 and SEQ ID: 2.

[0016] The recombinant Adeno-associated Virus (AAV) vectors are constructed using the cis-plasmid containing splice enhancer intron (SEI) as depicted in SEQ ID: 3 under the control of the LacZ promoter (SEQ ID: 5), wherein the said vector is constructed (pcis.CMV.5'LacZ-SEI-3'LacZ as depicted in SEQ ID: 7 and SEQ ID: 8) using the 5' Kpnl site and 3' Nrul site for addition within the previously reported parental plasmid. The new dual vectors containing the splice enhancer intron (SEI) are cloned using previously published proviral plasmids pAG54 (CMV.LacZ5'HD vector) and pAG55 (LacZ3'HD with SV40pA (SEQ ID: 10)) replacing the 297bp 5' 1 / 3 AP overlapping sequence. The 5' Sall (SEQ ID: 4a) and 3' BamHI sites (SEQ ID: 4b) within the pAG54 and the 5' Sall and 3' Nrul sites within the pAG55 are used to insert the 90 bp synthetic SEI sequence to generate pGL9, head-vector as depicted in SEQ ID: 1 and pGLIO, tail vector as depicted in SEQ ID: 2 respectively.

[0017] The new constructs containing the SEI in the 5' head- vector (-pGL9) and 3' tail-vector (pGLIO) are compared with the alkaline phosphatase fragment containing OAP vectors (pcis.CMV.5'LacZ-OAP-3'LacZ). The proviral plasmid pcis.RSV.LacZ is used as an intact LacZ positive control.

[0018] The hybrid dual AAV vector system containing SEI (90bp) overlap sequence is compared with the OAP (297bp) i.e., 5’ 1 / 3 of AP; 297 bp overlapsequence within the expressed vector with respect to the splicing efficacy and the protein expression efficacy of the LacZ gene (SEQ ID: 9).Brief Description of the drawings

[0019] FIG la, lb and 1c illustrate the expression of LacZ in dual SEI and OAP vectors in HEK293T cells.

[0020] FIG 2a, 2b and 2c illustrate the splicing efficiency of hybrid dual AAV vector system containing SEI (90bp) and the OAP (297bp) sequences within the introns of hybrid dual AAV vector in HEK293T cells.

[0021] FIG 2d and 2e illustrates the splicing efficiency (in the presence of 10 pM of isoginkgetin) of hybrid dual AAV vector system containing synthetic splice enhancer intron (SEI) and the OAP sequences within the introns of hybrid dual AAV vector in HEK293T cells.

[0022] FIG 3a, 3b, 3c, 3d and 3e illustrates the expression from hybrid dual AAV vectors from cross-sections of the tibialis anterior muscle and eye in the mouse model.

[0023] FIG 4a illustrates the efficiency of hybrid dual AAV vector system containing synthetic splice enhancer intron (SEI) (90bp) in Stargardt disease model of mouse.

[0024] FIG 4b illustrates the function of the retina in Stargardt disease model of mouse transduced with the hybrid dual AAV vector system.

[0025] FIG 4c illustrates the histological staining of the retina in Stargardt disease model of mouse.

[0026] FIG 4d shows the ABCA4 expression from AAV hybrid splice enhancer intron (SEI) dual vectors.Detailed description of the invention

[0027] In order to more clearly and concisely describe and point out the subject matter of the claimed invention, the following definitions are provided for specific terms, which are used in the following written description.

[0028] The term “Adeno- Associated Virus” refers to small viruses that infect humans and some other primate species.

[0029] The term “Splicing” refers to a process by which introns, the noncoding regions of genes, are excised out of the primary messenger RNA transcript, and the exons (i.e., coding regions) are joined together to generate mature messenger RNA.

[0030] The present invention discloses the development of a hybrid dual Adeno- associated Virus (AAV) vector system to improve the expression of the dual vector systems by enhancing the efficient splicing utilizing a novel 90 base pair splice enhancer intron (SEI) thus enhancing the expression of the dual vector system.

[0031] The present invention discloses a hybrid dual AAV vector system with a novel synthetic splice enhancer element to increase the efficiency of splicing and the gene expression. The presence of synthetic splice enhancer element improves the expression in comparison to current hybrid dual vector (with AP derived overlapping sequences) systems and also increases the transgene packaging size due to the smaller overlapping sequence size.

[0032] According to some embodiments of the invention, the development of a hybrid dual AAV vector system using a novel, smaller 90bp splice enhancer intron (SEI) is disclosed, that facilitates achievement of dual vector reconstitution and efficient splicing. The SEI vector system comprises a synthetic splice enhancer intron (SEI) arranged in tandem with high recombination potential. The smaller size of the overlapping fragment in splice enhancer intron (SEI) dual vectors improves the packaging limit of hybrid AAV dual vectors compared to the previous OAP vectors.

[0033] The hybrid dual AAV vector system of the present invention comprises a synthetic splice enhancer intron (SEI) cloned in a dual LacZ head-tail expression cassette expressed under the control of a cytomegalovirus (CMV) promoter as depicted in SEQ ID: 1 and SEQ ID: 2.

[0034] The recombinant AAV vectors are constructed according to an embodiment of the invention. The cis-plasmid containing SEI as depicted in SEQ ID: 3 under the control of LacZ promoter (SEQ ID: 5) is constructed (pcis.CMV.5'LacZ-SEI- 3 'LacZ as depicted in SEQ ID: 7 and SEQ ID: 8) using the 5' Kpnl site and 3' Nrul site for addition within the previously reported parental plasmid. The new dual vectors containing the splice enhancer intron (SEI) are cloned using previously published proviral plasmids pAG54 (CMV.LacZ5'HD vector) and pAG55 (LacZ3'HD with SV40pA (SEQ ID: 10)) replacing the 297bp 5' 1 / 3 AP overlapping sequence. The 5' Sall (SEQ ID: 4a) and 3' BamHI sites (SEQ ID: 4b) within the pAG54 and the 5' Sall and 3' Nrul sites within the pAG55 are used to insert the 90 bp synthetic splice enhancer intron (SEI) sequence to generate pGL9, head- vector as depicted in SEQ ID: 1 and pGLIO, tail vector as depicted in SEQ ID: 2 respectively.

[0035] The new constructs containing the splice enhancer intron (SEI) in the 5' head- vector (-pGL9) and 3' tail-vector (pGLIO) are compared with the alkaline phosphatase fragment containing GAP vectors (pcis.CMV.5'LacZ-OAP-3'LacZ). The proviral plasmid pcis.RSV.LacZ is used as an intact LacZ positive control.

[0036] The hybrid dual AAV vector system containing splice enhancer intron (SEI) (90bp) overlap sequence is compared with the OAP (297bp) i.e., 5’ 1 / 3 of AP; 297 bp overlap sequence within the expressed vector with respect to the splicing efficacy and the protein expression efficacy of the LacZ gene (SEQ ID: 9).

[0037] The following examples are offered to illustrate various aspects of the invention. However, the examples are not intended to limit or define the scope of the invention in any manner.Example 1: Evaluation of the expression of LacZ in SEI and OAP containing cis plasmids and AAV9 dual vectors in HEK293T cells

[0038] The expression of LacZ (SEQ ID: 6) is analyzed and compared between splice enhancer intron (SEI) and OAP containing cis plasmids and AAV9 dual vectors in HEK293T cells. The HEK293T cells are transfected with 0.5 pg of cis plasmid using Lipofectamine™ LTX reagent with PLUS™ reagent (Thermo Fisher Scientific, Waltham, MA) or transduced at multiplicity of infection (MOI) of 50,000 in serum-free medium. After three hours of AAV inoculation, fetal bovine serum is brought to 10% and maintained at 37°C, 5% carbon dioxide with 1% Penicillin- Streptomycin- Amphotericin B solution. The cells are harvested after 48 hours of infection and the P-galactosidase activity of the cell lysate is quantified.

[0039] FIG la illustrates the expression of LacZ in dual splice enhancer intron (SEI) and OAP vectors in HEK293T cells. The efficiency of the plasmids and the AAV9 dual vectors of the present invention is analyzed with respect to the gene expression in HEK293T cells that are infected with the dual splice enhancer intron (SEI) and OAP vectors at a multiplicity of infection (MOI) of 50,000 vector genome (vg) per cell for each vector. The enzyme activity levels from the splice enhancer intron (SEI) vectors (0.0082 ± 0.0009) are close to the levels from the intact vector (0.0073 ± 0.0005) and significantly higher (p < 0.0001) when compared to levels from the OAP vectors (0.0034 ± 0.0006). Similarly, dual vectors infected HEK293T cells were subjected to cytological LacZ staining as well. The intact LacZ vectors yielded 12.24 ± 0.91 percent stained LacZ positive cells. The results indicated the higher percentage of LacZ positive cells is observed in the SEI (13.51 ± 3.28) infected HEK293T cells in contrast to OAP (7.292 ± 0.56) infected HEK293T cells as depicted in FIG lb and FIG 1c.Example 2: Evaluation of the splicing efficiency in splice enhancer intron (SEI) and OAP containing cis plasmids and AAV9 dual vectors in HEK293T cells

[0040] The splice enhancer intron (SEI) element has been strategically designed to increase the efficiency of splicing and removal of intronic / ITR region followingrecombination events to form concatemers. The efficacy of the hybrid dual AAV vector system containing splice enhancer introns (SEI) (90bp) is compared with the OAP (297bp) vector with respect to the splicing efficacy in HEK293T cells.

[0041] The HEK293T cells are transfected with 0.5 pg of cis plasmid using Lipofectamine™ LTX reagent with PLUS™ reagent (Thermo Fisher Scientific, Waltham, MA) or transduced at multiplicity of infection (MOI) of 50,000 in serum- free medium. The cells are harvested after 48 hours of transduction, and the RNA is isolated using RNeasy Qiagen kit and cDNA is synthesized using iScriptTM cDNA synthesis kit, according to an embodiment of the invention. The forward and reverse primer pairs are used to identify spliced (Ml) and unspliced products (M2 for head half and M3 for tail half). The forward primer Ml and M2 isGAGTGTGATCATCTGGTCGC, the reverse primer Ml isCGCGTACATCGGGCAAATA, the reverse primer M2 isGCCCAGTTTCTATTGGTCTCC, the forward primer M3 isGTCTTACTGACATCCACTTTGC and the reverse primer M3 isTTGATGGACCATTTCGGCA. The ratio and percentage of spliced versus unspliced product are analyzed using quantitative real time PCR and qualitativePCR. In order to determine the splicing efficiency in the presence a pre-mRNA splice inhibitor, transfection experiments with cis-plasmids containing the splice enhancer intron (SEI) and OAP within the intron was used at a concentration of 0.5 pg of pDNA and cells were harvested at 24 hours post transfection.

[0042] FIG 2a, 2b and 2c illustrate the splicing efficiency of hybrid dual AAV vector system containing splice enhancer intron (SEI) (90bp) and the OAP (297bp) vector in HEK293T cells. The results indicated that the unspliced products are not identified in intact vectors as the M2, M3 primers are designed specifically to sequence spanning 5' half LacZ split region and splice donor, splice acceptor and 3' half LacZ split region as depicted in FIG 2a. The hybrid dual AAV vector system containing splice enhancer intron (SEI) (90bp) demonstrated slightly greater percentages of spliced / unspliced product ratio (6.83%) compared to dual OAP vectors (2.83%) as depicted in FIG 2b. Similar results are also observed on agarosegel as depicted in FIG 2c. It is interpreted that the splicing efficiency mediated by splice enhancer intron (SEI) element is superior to OAP element.

[0043] The splicing efficiency was further validated in the presence of 10 pM isoginkgetin (IGG), a pre-mRNA splicing inhibitor, when HEK293T cells were pretreated with the IGG drug and transfection was performed. The hybrid dual AAV vector system containing splice enhancer intron (SEI) (90bp) demonstrated greater spliced / unspliced product ratio (7.245 ± 1.892) compared to dual OAP vectors (3.898 ± 0.9235) as depicted in FIG 2d. Further, similar results are also observed on agarose gel as depicted in FIG 2e.Example 3: Evaluation of the expression of the transgene in muscle and retina of mouse model.

[0044] The efficiency of the hybrid dual AAV vector system containing splice enhancer intron (SEI) (90bp) is compared with the OAP (297bp) vector with respect to the transgene expression in the mouse model.

[0045] The hybrid dual AAV vector system containing splice enhancer intron (SEI) (90bp) is compared with the OAP (297bp) vector are tested for stable gene expression in vivo in mouse tibialis anterior (TA) muscles and retina. The mice are injected with AVV9.RSV.intact LacZ with a dosage of 1 x 10A10 vg particles and the dual AAV9 splice enhancer intron (SEI) and OAP vectors (l x 10A10 vg particles of each head and tail vector particles per muscle) into the tibialis anterior muscles. Further, subretinal injections are administered to the eyes of the same animal at vector dose of 2.5 xlOA9 for the intact vector and each of the vectors of the dual AAV system. The expression of LacZ is analyzed 6 weeks later.

[0046] FIG 3a illustrates the cross-sections of the tibialis anterior muscle in the mouse model. The results indicated that the tibialis anterior muscle cross-sections stained for LacZ, showed greater expression in the intact LacZ infected muscles, followed by splice enhancer intron (SEI) vectors and OAP vectors. The OAP vectors showed partially positive area of LacZ stained fibers i.e., 42.63% ±8.94,whereas the percentage of LacZ positive fibers were significantly (P <0.01) higher in SEI infected muscles i.e., 86.09% ±3.41, almost close to the positive fiber numbers seen with the intact vector as depicted in FIG 3b. Beta-galactosidase activity is significantly (P <0.050) higher in the splice enhancer intron (SEI) vector i.e., 0.0083 ± 0.00099 infected muscles compared to OAP i.e., 0.0012 ± 0.00025 as depicted in FIG 3c. In the retina, LacZ expression measured as diffusion area percentage is significantly (P = 0.03) higher with splice enhancer intron (SEI) (45.8 ±0.8) compared to OAP (40.5 ±1.4) as depicted in FIG 3d and 3e. Further, according to embodiment of the invention, the synthetic splice enhancer intron (SEI) is positioned within the 5' portion of the target gene sequence, where the target gene sequence may be derived from LacZ and ABCA4 genes.Example 4: Evaluation of the expression of the transgene in disease mouse model.

[0047] The hybrid dual AAV vector system containing splice enhancer intron (SEI) (90bp) is analyzed for the efficacy of this unique element in a disease mice model. The ABCA4 gene is split at the exon 22 / 23 junction and accommodated in two separate vectors. The AAV9.CMV.ABCA4 head and tail vector containing the novel overlapping SEI element is injected in ABCA4- / - to correct the Stargardt disease phenotype. The ABCA4- / - mice are administered with subretinal injection of the dual vectors at a dosage of lxlOA9 vg of each vector at 6 months of age. Single 5' head vector at the same dosage is injected as untreated control in control mice. The animals are assessed weekly by FFA, AF and PS-OCT.

[0048] FIG 4a illustrates the efficiency of hybrid dual AAV vector system containing splice enhancer intron (SEI) (90bp) in Stargardt disease model of mouse. The results indicated that after eight weeks, the lipofuscin deposits are greatly reduced resulting in low autofluorescence in the eyes treated with hybrid dual AAV vector system containing splice enhancer intron (SEI) (90bp) compared to the high autofluorescence seen in naive ABCA4 KO mice eyes. In addition, the electroretinogram results interpreted the recovery of retinal function in mice treatedwith hybrid dual AAV vector system containing splice enhancer intron (SEI) (90bp), whereas non-injected control eyes did not show functional recovery of the retina as shown in FIG 4b.

[0049] FIG 4c illustrates the histological staining of the retina in Stargardt disease model of mouse. The results indicated that AAV dual vectors are well tolerated in the retina when delivered by sub-retinal injections and did not cause any structural anomalies. FIG 4d shows the ABCA4 expression from AAV hybrid splice enhancer intron (SEI) dual vectors are observed in the retinal photoreceptor layer when delivered by sub-retinal injections. The above results interpreted that the hybrid dual AAV vector system containing splice enhancer intron (SEI) (90bp) is effective in maintaining and regenerating photoreceptors in ABCA4- / - mice and validating the application of this novel overlapping element in ABCA4 dual vectors for the treatment of Stargardt disease by rescue and reversal of retinal pathology.

[0050] The hybrid dual AAV vector system containing splice enhancer intron (SEI) (90bp) of the present invention is constructed and exhibits improved efficiency in splicing and the gene expression in vitro and in vivo.

[0051] According to some embodiments of the invention, the hybrid dual AAV vector system containing splice enhancer intron (SEI) (90bp) of the present invention comprising a unique splice enhancer intron (SEI) is effective in enhancing the splicing and the transgene expression in HEK293T cells and in mouse model. The results of the present invention with the enhanced ability of the expression thus indicating the utility of hybrid dual AAV vector system containing splice enhancer intron (SEI) (90bp) for the expression of therapeutic genes such as ABCA4, etc.

[0052] The hybrid dual AAV vector system has several applications covering atleast one gene, where the length of the transgene exceeds the AAV packaging limit of 4.7kb. The genes include, but not limited to Dystrophin, Dysferlin, Sarcoglycans, Nebulin, gene editing enzymes including Cas9 and others, EYS, Myo7A, ALMS1, RP1, OTOG, NF1, TSC2, LAMA2, ATM, SPG11 / 15, etc, are enabled to be packaged in the hybrid dual AAV vectors using the SEI hybrid dualvector system. Further, the hybrid dual AAV vector system is compatible with different tissue types for reconstitution of split genes.

[0053] According to some embodiments of the invention, the hybrid dual AAV vector system is compatible to be used for any AAV capsid serotype for specific applications encompassing any human or veterinary diseases. Further, the hybrid dual AAV vector system may be used to treat human retinal blindness diseases including, but not limited to Stargardt disease. Additionally, the system helps to achieve efficient treatment of atleast one human neuromuscular diseases including, but not limited to Duchenne Muscular Dystrophy, Limb-girdle muscular dystrophy (LGMD), and atleast one human blood diseases including but not limited to haemophilia, sickle cell disease, etc.

Claims

Claims:I Claim:

1. A hybrid dual Adeno-associated Virus (AAV) vector system with one or more splice enhancer elements for expression of plurality of large genes, the system comprising: a. a synthetic splice enhancer intron (SEI) vector having atleast one base pair, wherein the synthetic splice enhancer intron (SEI) vector comprises a dual LacZ head-tail expression cassette expressed under the control of a cytomegalovirus (CMV) promoter; and b. a portion of the target gene sequence, wherein the synthetic splice enhancer intron (SEI) vector facilitates splicing and reconstitution of the full-length target gene upon co-infection of a host cell; wherein the hybrid dual Adeno-associated Virus (AAV) vector is generated using a cis-plasmid containing the synthetic splice enhancer intron (SEI) under the control of a LacZ promoter, constructed using 5 ' Kpnl and 3 ' Nrul sites.

2. The system as claimed in claim 1, wherein the hybrid dual Adeno-associated Virus (AAV) vector with overlapping sequence with the 90 bp SEI sequence such that pGL9 head-vector vector is generated as depicted in SEQ ID: 1 and pGLIO and a tail vector as depicted in SEQ ID: 2, where the system enhances the packaging limits of the dual AAV hybrid vector by an additional ~400bp beyond the GAP vectors.

3. The system as claimed in claim 1, wherein the synthetic splice enhancer intron (SEI) is positioned within the 5' portion of the target gene sequence, where the target gene sequence may be derived from LacZ and ABCA4 genes.

4. The system as claimed in claim 1, wherein the synthetic splice enhancer intron (SEI) vector includes sequences depicted in SEQ ID: 1 and SEQ ID: 2.

5. The system as claimed in claim 1, wherein the efficiency of gene expression measured by P-galactosidase activity from the reconstituted LacZ gene is efficient due to the usage of the 90bp sequence.

6. The system as claimed in claim 1, wherein the percentage of spliced / unspliced product ratio of the hybrid dual Adeno-associated Virus (AAV) vector system containing splice enhancer intron (SEI) (90bp) is higher with 6.83%, compared to the dual OAP vectors with 2.83%.

7. The system as claimed in claim 1, wherein in the presence of a splicing inhibitor spliced / unspliced product ratio is greater in splice enhancer intron (SEI) (7.245 ± 1.892) compared to OAP cis-plasmids (3.898 ± 0.9235).

8. The system as claimed in claim 1, wherein the administration of the hybrid dual Adeno-associated Virus (AAV) vector through subretinal injection leads to expression of the ABCA4 gene in retinal photoreceptor cells.

9. The system as claimed in claim 1, wherein the hybrid dual Adeno-associated Virus (AAV) vector may include atleast one gene where the length of the transgene exceeds the AAV packaging limit of 4.7kb, where the gene may be Dystrophin, Dysferlin, Sarcoglycans, Nebulin, gene editing enzymes including Cas9, EYS, Myo7A, ALMS1, RP1, OTOG, NF1, TSC2, LAMA2, ATM, SPG11 / 15, etc, whereby the genes are enabled to be packaged in AAV vectors using the SEI hybrid dual vector system.

10. The system as claimed in claim 1, wherein the hybrid dual Adeno-associated Virus (AAV) vector system is adaptable with all tissue types for reconstitution of split genes delivered using AAV vectors.

11. The system as claimed in claim 1, wherein the hybrid dual Adeno-associated Virus (AAV) vector system may be used for any AAV capsid serotype.

12. The system as claimed in claim 1, wherein the hybrid dual Adeno-associated Virus (AAV) vector system may be used to treat at least one human retinalblindness diseases including Stargardt disease, atleast one human neuromuscular diseases including Duchenne Muscular Dystrophy, Limb-girdle muscular dystrophy (LGMD), and atleast one human blood diseases including haemophilia, sickle cell disease, etc.

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