Modified AAV vectors for enhanced gene therapy in hemophilia b

Abstract

The present invention provides modified adeno-associated virus (AAV) vectors designed for gene therapy for treating hemophilia B The modified AAV vectors are obtained with enhanced transduction efficiency and immune evasion capabilities achieved through precise modifications at post-translational modification (PTM) sites on AAV8 and AAVrh10 capsids. The modified AAV vectors offer advantages including improved therapeutic efficacy at lower vector doses, similar immunogenicity profile as wild type AAV vectors, and versatility for treating hemophilia B and similar liver disorders (e.g., Gaucher's disease). The present invention further encompasses a method for manufacturing the modified AAV vectors, ensuring their precise and reliable production. The modified AAV vectors has potential for long-term gene expression and adaptability for other genetic disorders which makes the present invention a significant advancement in the field of gene therapy.

Description

[0001] MODIFIED AAV VECTORS FOR ENHANCED GENE THERAPY IN HEMOPHILIA B

[0002] EARLIEST PRIORITY DATE:

[0003] This Application claims priority from a Provisional patent application filed in India having Patent Application No. 202311062115, filed on September 15th, 2023, and titled “MODIFIED AAV VECTORS FOR ENHANCED GENE THERAPY IN

[0004] HEMOPHILIA A AND HEMOPHILIA B”

[0005] FIELD OF INVENTION

[0006] The present invention relates to a field of gene therapy for treatment of hereditary bleeding disorders. More specifically, the present invention relates to modification of adeno-associated virus (AAV) vectors to enhance transduction efficiency and immune escape capabilities for treatment of hereditary bleeding disorders, including hemophilia B.

[0007] BACKGROUND

[0008] Hemophilia B is a hereditary bleeding disorder due to a clotting factor IX deficiency (FIX). It is an X-linked genetic disease affecting 1 in every 20000 individuals as disclosed by Saini et. al., 2020. The bleeding disorder manifests itself in different severity depending on the clotting factor activity level Castaman et. al., 2018. Patients with less than 1% clotting factor levels present with severe symptoms such as spontaneous nose bleed, bleeding into joints, and soft tissue including brain hemorrhages leading to life-threatening conditions as disclosed by Kizilocak et. al., 2019. Severe hemophilic patients tend to bleed spontaneously up to 3 times per week. Individuals with clotting factors ranging from 1 to 5% have moderate symptoms such as incessant bleeding upon injury or surgery and fewer spontaneous bleeds (once a month). Mild cases of hemophilia do not generally show any symptoms since the clotting factor level is 5-40% as disclosed by Castaman et. al., 2019, Jayandharan et. al., 2008, Zimmerman et. al., 2013. The standard of care for hemophiliacs includes replacement therapy wherein patients are administered with clotting factor concentrates (CFCs) or pooled plasma as disclosed by Mannucci et. al., 2003. Multiple administrations required for prophylaxis increase the cost of treatment by up to 330000 euros annually as disclosed by Rocha et. al., 2015. The availability of CFCs and pooled plasma in developing countries has been a major concern for the affected people. Due to repeated infusion of clotting factors, patients develop inhibitors against the infused protein leading to the ineffectiveness of the therapy. Replacement therapy has a primary downside of having to provide repeated injections contributing to its increased cost, lack of availability, and reduced patient care as disclosed by Franchini et. al., 2012. To overcome this limitation, gene therapy has evolved to provide a long-term solution by transferring a normal functional copy of a gene into patients’ tissue / cells via a viral or non-viral vector as disclosed by Perrin et. al., 2019. Adeno-associated virus (AAV) is one of the most successful viral-based vectors to be used for gene therapy as disclosed by Naso et. al., 2017. Due to its non-pathogenic nature, wide tissue tropism, and stable gene expression, it has become the vector of choice for various preclinical and clinical studies. AAV is a dependovirus belonging to the parvoviridae family with two open reading frames (ORFs): rep and cap. Recombinant AAV that is used for gene therapy applications contains the therapeutic transgene instead of the rep and cap genes flanked by inverted terminal repeats (ITRs) as disclosed by Colella et. al., 2018.

[0009] Hepatic gene therapy for hemophilia B has been under investigation for over two decades, yet there is only limited success in terms of sustainable gene expression over time as disclosed by George et. al., 2017. Previous clinical study using singlestranded AAV vector carrying FIX-Padua administered at a dose of 5X1011vgs / kg reported 22.9%±9.9% of mean (+SD) FIX activity at 1 year post vector administration. An increase in hepatic transaminases was observed in three out of fifteen patients treated. However, the long-term data for efficacy and safety is not yet reported as disclosed by George et. al., 2017, and George et. al., 2019. Another clinical trial on AAV8 with codon-optimized Padua variant of human FIX was conducted on hemophilia B patients. The study showed a peak FIX activity of 32%- 58.5% in the highest dose cohort administered with a dose of 3X1012vgs / kg. However, the FIX activity level declined in all patients but one after 5 to 11 weeks post vector administration, probably due to immune response against the vector as disclosed by Konkle et. al., 2021. These findings suggest that there is a dire need for an improved vector with better transduction efficacy and immune escape capabilities to achieve a therapeutic level of FIX at a relatively lower dose. This will further reduce the cost of gene therapy and make it accessible to the affected people in distress.

[0010] AAV is known to undergo post-translational modifications during viral synthesis by the host cellular machinery such as glycosylation, SUMOylation, acetylation, ubiquitination, and phosphorylation as disclosed by Mary et. al., 2019. Modifications like phosphorylation were reported to decline the transduction efficiency as a result of degradation via proteasome machinery as disclosed by Zhong et. al., 2008. When tyrosine residues on the AAV2 capsid surface are replaced with phenylalanine, the transduction efficacy of the mutant virus significantly increased compared to the wild-type AAV2 vector as disclosed by Markusic et. al., 2010. The glycosylation status of the AAV2 vector has been studied thoroughly and the abolition of selected glycosylated residues by SDM resulted in increased transduction in the liver and retinal cells as disclosed by Mary et. al., 2019. Another study reported SUMOylation and Neddylation mutant to be superior to wild-type AAV2 vector in terms of phenotypic outcome in hemophilia B and Leber congenital amaurosis 2 as disclosed by Maurya et. al., 2019. These studies confirm the approach to developing novel AAV engineered vectors with improved efficiency and immune escape potential for clinical applications. Liver- directed gene therapy for hemophilia requires AAV vectors with tropism toward hepatocytes such as AAV8 and AAVrhlO as disclosed by Jiang et. al., 2006, and Wang et. al., 2015. One of the most commonly used serotypes for targeting liver tissue is AAV8 which is known to have tropism towards the liver as disclosed by Nathwani et. al., 2014. Multiple studies have shown AAV8-mediated delivery of therapeutic transgene to the liver for the treatment of various diseases such as hemophilia, a-1 antitrypsin deficiency, and phenylketonuria as disclosed by Asokan et. al., 2012. AAV8 has been used in several clinical trials for the delivery of human FIX (hFIX) in hemophilia B patients as disclosed by Konkle et. al., 2021, Nathwani et. al., 2014, and Xue et. al., 2022. AAvrhlO has also been used for hemophilia B gene therapy and phase VII clinical trial data showed a transient increase in FIX expression, which subsequently decreased over time probably due to the immune response generated against the capsid as disclosed by Pipe et. al., 2017. Therefore, vector engineering of these serotypes to generate mutant vectors modified at residues that are known or predicted to have PTMs like glycosylation, SUMOylation, and Neddylation would be useful.

[0011] The expression of the therapeutic transgene could be further improved by using regulatory elements that are known to enhance gene expression either at the transcriptional or translational level. One such feature is the Kozak sequence which is a nucleic acid motif serving as the protein translation initiation site in most eukaryotic mRNA transcripts. This sequence plays an important role in protein regulation and cellular fitness, influencing human disease manifestation as disclosed by Kozak et. al., 2002, and Kozak et. al., 1989. A study conducted in a family of atrial septal defects reported that a mutation in the Kozak sequence of GATA4 changes the translation status of the protein leading to a disease condition as disclosed by Mohan et. al., 2014. A transgene modification involving the insertion of the Kozak sequence upstream of the transgene increases the transgene expression as disclosed by Gessler et. al., 2017. In this study, we developed Kozak sequence inserted human coagulation factor IX vectors to assess the phenotypic rescue in hemophilia B mice models.

[0012] Therefore, there is a need to develop AAV vectors with modified PTM sites and transgene with regulatory elements for increased and sustained expression of hFIX at a low vector dose.

[0013] SUMMARY The present invention relates to modified adeno-associated virus (AAV) vectors with enhanced transduction efficiency and immune evasion capabilities to achieve therapeutic levels of coagulation factors at lower doses. Specifically, the AAVrhlO and AAV8 capsids are modified at post-translational modification (PTM) sites, including HexNAcylation and neddylation sites, to improve their performance in gene therapy.

[0014] In an embodiment of the present invention, a modified AAV vector for gene therapy is provided. The modified AAV vector comprises an AAV8 or AAVrhlO capsid modified at specific post-translational modification (PTM) sites. The modifications include substitutions of lysine (K) or asparagine (N) residues with glutamine (Q) and substitution of serine (S) residue with alanine (A), resulting in improved transduction efficiency and therapeutic efficacy in treating hemophilia B.

[0015] In another embodiment of the present invention, a method for manufacturing a modified AAV vector for gene therapy is provided. The method begins with identifying specific PTM sites on AAV8 or AAVrhlO capsids using liquid chromatography -mass spectroscopy and in silica tools. PTM sites with high conservation and maximum scores are selected across AAV serotypes. Primers for introducing glutamine (Q) and alanine (A) substitutions are designed at the selected PTM sites. Site-directed mutagenesis plasmids for AAV8 or AAVrhlO capsids are generated with the desired mutations. Cells are transfected with modified plasmids and a therapeutic transgene. The modified AAV vector is produced with improved transduction efficiency and immune escape capabilities.

[0016] The present invention provides modified AAV vectors for gene therapy in the treatment of hemophilia B. The modified AAV vectors exhibit improved transduction efficiency, leading to higher and more sustained expression of the therapeutic gene. Due to enhanced transduction capabilities, the modified AAV vectors enable therapeutic levels of coagulation factors (FVIII or FIX) to be achieved at relatively lower vector doses. This reduces the potential for adverse effects associated with high vector doses. To further clarify the advantages and features of the present invention, a more particular description of the invention will follow by reference to specific embodiments thereof, which are illustrated in the appended figures. It is to be appreciated that these figures depict only typical embodiments of the invention and are therefore not to be considered limiting in scope. The invention will be described and explained with additional specificity and detail with the appended figures.

[0017] BRIEF DESCRIPTION OF THE DRAWINGS

[0018] The disclosure will be described and explained with additional specificity and detail with the accompanying figures in which:

[0019] FIG. 1 depicts an exemplary flowchart depicting a method for preparing a modified AAV vector for gene therapy, in accordance with an embodiment of the present disclosure;

[0020] FIG. 2 is a schematic representation of a plasmid map for pAAVrhlOWT, in accordance with an embodiment of the present disclosure;

[0021] FIG. 3 is a schematic representation of the plasmid map for pAAVrhlOS157A, in accordance with an embodiment of the present disclosure;

[0022] FIG. 4 is a schematic representation of the plasmid map for pAAVrhlOK169Q, in accordance with an embodiment of the present disclosure;

[0023] FIG. 5 is a schematic representation of the plasmid map for pAAVrhl0N306Q, in accordance with an embodiment of the present disclosure;

[0024] FIG. 6 is a schematic representation of the plasmid map for pAAVrhl0K709Q, in accordance with an embodiment of the present disclosure;

[0025] FIG. 7 is a schematic representation of the plasmid map for pAAV8WT, in accordance with an embodiment of the present disclosure;

[0026] FIG. 8 is a schematic representation of the plasmid map for pAAV8K31Q / K61Q, in accordance with an embodiment of the present disclosure; FIG. 9 is a schematic representation of the plasmid map for pAAV LP1 Kozak hFIX, in accordance with an embodiment of the present disclosure;

[0027] FIG. 10 depicts an immunocytochemistry for hFIX protein expression in vitro, in accordance with an embodiment of the present disclosure;

[0028] FIG. 11 depicts hFIX production in hemophilia B mice administered with AAVrhlO WT and mutant vectors, in accordance with an embodiment of the present disclosure;

[0029] FIG. 12 depicts functional rescue in hemophilia B mice administered with AAV8 WT and mutant vectors, in accordance with an embodiment of the present disclosure;

[0030] FIG. 13 depicts functional rescue in hemophilia B mice administered with AAVrhlO WT and mutant vectors, in accordance with an embodiment of the present disclosure;

[0031] FIG. 14 depicts an immune response in hemophilia B mice administered with AAV8 WT and mutant vectors, in accordance with an embodiment of the present disclosure; and

[0032] FIG. 15 depicts the immune response in hemophilia B mice administered with AAVrhlO WT and mutant vectors, in accordance with an embodiment of the present disclosure.

[0033] Further, those skilled in the art will appreciate that elements in the figures are illustrated for simplicity and may not have necessarily been drawn to scale. Furthermore, in terms of the method steps, chemical compounds, and parameters used herein may have been represented in the figures by conventional symbols, and the figures may show only those specific details that are pertinent to understanding the embodiments of the present disclosure so as not to obscure the figures with details that will be readily apparent to those skilled in the art having the benefit of the description herein. DETAILED DESCRIPTION

[0034] For the purpose of promoting an understanding of the principles of the disclosure, reference will now be made to the embodiment illustrated in the figures and specific language will be used to describe them. It will nevertheless be understood that no limitation of the scope of the disclosure is thereby intended. Such alterations and further modifications in the illustrated system, and such further applications of the principles of the disclosure as would normally occur to those skilled in the art are to be construed as being within the scope of the present disclosure.

[0035] The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such a process or method. Similarly, one or more components, compounds, and ingredients preceded by "comprises... a" does not, without more constraints, preclude the existence of other components or compounds or ingredients or additional components. Appearances of the phrase "in an embodiment", "in another embodiment" and similar language throughout this specification may, but not necessarily do, all refer to the same embodiment.

[0036] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. The system, methods, and examples provided herein are only illustrative and not intended to be limiting.

[0037] In the following specification and the claims, reference will be made to a number of terms, which shall be defined to have the following meanings. The singular forms “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise.

[0038] FIG. 1 depicts an exemplary flowchart depicting a method for preparing a modified AAV vector for gene therapy, in accordance with an embodiment of the present disclosure. Embodiments of the present invention relate to modified adeno-associated virus (AAV) vectors with enhanced transduction efficiency and immune evasion capabilities to achieve therapeutic levels of coagulation factors at lower doses. Specifically, the AAVrhlO and AAV8 capsids are modified at post-translational modification (PTM) sites, including HexNAcylation and neddylation sites, to improve their performance in gene therapy.

[0039] In an embodiment of the present invention, a modified adeno-associated virus (AAV) vector for gene therapy is provided. The modified adeno-associated virus (AAV) vector comprises an AAV capsid selected from a group comprising one of AAV8 capsid and AAVrhlO capsid, and a therapeutic transgene comprising pAAV- LPl-hFIX combined with regulatory elements configured to increase protein expression at translational level. The AAV capsid is modified at post-translational modification (PTM) sites using primers. The primers are designed with substitutions from a group comprising of lysine (K) and asparagine (N) residues with glutamine (Q), and serine (S) residues with alanine (A), resulting in improved transduction efficiency and therapeutic efficacy in treating hemophilia B.

[0040] The AAV8 capsid is modified at specific PTM sites including neddylation site, thereby enhancing immune escape capabilities and facilitating sustained expression of a therapeutic gene at a lower vector dose. The AAVrhlO capsid is modified at specific PTM sites including HexNAcylation and neddylation sites, leading to increased transduction efficiency and phenotypic rescue in preclinical models of hemophilia B.

[0041] The therapeutic transgene comprises modified FIX transgene construct (pAAV- EPl-Kozak-hFIX). The regulatory elements are selected from a group comprising of a combination of liver specific promoter (EPl) and Kozak sequence. The PTM modifications comprises neddylation and HexNAcylation sites configured to increase transduction efficiency and minimize any adverse immune response.

[0042] In another embodiment of the present invention, a method for manufacturing a modified AAV vector for gene therapy is provided. The method begins with identifying post-translational modification (PTM) sites on AAV capsid selected from a group comprising one of AAV8 capsid and AAVrhlO capsid using liquid chromatography -mass spectroscopy and in silica tools, at step 102. Post- translational modification (PTM) sites are selected with high conservation and maximum scores across AAV serotypes, at step 104. Primers for introducing glutamine (Q) and alanine (A) substitutions are designed at the selected PTM sites, at step 106. Primers are selected with substitutions from a group comprising of lysine (K) and asparagine (N) residues with glutamine (Q), and serine (S) residues with alanine (A).

[0043] Designing primers to introduce glutamine (Q) and alanine (A) substitutions at specific post-translational modification (PTM) sites involves a few key steps:

[0044] • Identifying the Target Site: Determining the exact amino acid residue for introducing one of Q and A substitution.

[0045] • Designing Primers: o Forward Primer: Typically, 25-45 bases long, complementary to the template DNA strand just upstream of the target site. o Reverse Primer: Complementary to the template DNA strand downstream of the target site. o The desired change in nucleotide should be flanked by 10-15 nucleotides on either side. o The GC content of the primers needs to be at least 40%. o The primers need to end with a G or C.

[0046] • Incorporate Mutations: o Site-directed mutagenesis techniques are used to introduce the desired Q or A substitutions. o The mutations are ensured to be positioned precisely at the PTM site without disrupting essential regions of the protein.

[0047] • Consideration for PCR: o Primers have similar annealing temperatures to facilitate PCR amplification. o The primers are ensured to be specific enough to bind only to the intended DNA sequence.

[0048] • Validate Primers: o Primer design software is utilized to check for potential secondary structures or primer dimers. o The primers are validated by in silica and ideally through experimental verification such as PCR amplification and sequencing.

[0049] • Experimental Verification: o After primer design and mutation introduction, the resulting mutated sequence is validated using techniques like Sanger sequencing to confirm the presence of the desired glutamine or alanine substitutions.

[0050] By following these steps, primers can be designed effectively to introduce glutamine (Q) and alanine (A) substitutions at specific PTM sites.

[0051] Site-directed mutagenesis plasmids for one of AAV8 and AAVrhlO capsid are generated with the desired mutations, at step 108. Host cells are transfected with modified plasmids and a therapeutic transgene to obtain the modified AAV vector, at step 110. The modified AAV vector is produced with improved transduction efficiency and immune escape capabilities.

[0052] The AAV capsid is selected from a group consisting of AAV8 and AAVrhlO capsids. The PTM sites on the AAV capsid are modified to improve transduction efficiency. The PTM sites on the AAV capsid are modified to enhance immune evasion capabilities. The therapeutic transgene is selected to treat a specific genetic disorder or medical condition. The host cells are cultured under controlled conditions to optimize the production of the modified AAV vector. The modified AAV vector is purified and concentrated after production to obtain the desired modified AAV vector, at step 112. The modified AAV vector is used for gene therapy to treat genetic disorders, including hemophilia B. The post-translational modification (PTM) sites are selected from a group comprising of neddylation and HexNAcylation sites. The AAV capsid is modified at post-translational modification (PTM) sites using primers. The primers are selected with substitutions from a group comprising of lysine (K) and asparagine (N) residues with glutamine (Q), and serine (S) residues with alanine (A). The host cells are cultured under controlled conditions to optimize production of the modified AAV vector. The host cell (AAV293) is cultured in Iscove’s modified Dulbecco’s medium containing 10% fetal bovine serum, lOpg / ml of piperacillin and ciprofloxacin with 5% CO2 at 37 °C. The therapeutic transgene comprises modified FIX transgene construct (pAAV-LPl-Kozak-hFIX). The modified FIX transgene construct (pAAV-LPl- Kozak-hFIX) is obtained by addition of Kozak sequence (GCCGCCACC) to a plasmid comprising human factor IX (hFIX) transgene in AAV backbone (pAAV- LPl-hFIX).

[0053] In another embodiment of the present invention, a composition for gene therapy is provided. The composition comprises 100%_%_ of a modified adeno-associated virus (AAV) vector, wherein the AAV vector comprises an AAV capsid selected from a group comprising one of AAV8 mutant capsid and AAVrhlO mutant capsid, and a therapeutic transgene combined with regulatory elements configured to increase protein expression at translational level. The AAV capsid is modified at post-translational modification (PTM) sites using primers. The primers are selected with substitutions from a group comprising of lysine (K) and asparagine (N) residues with glutamine (Q), and serine (S) residues with alanine (A). The AAV8 capsid is modified at neddylation sites. The AAVrhlO capsid is modified at both HexNAcylation and neddylation sites. The therapeutic transgene comprises modified FIX transgene construct (pAAV-LPl-Kozak-hFIX). The regulatory elements are selected from a group comprising of a combination of liver specific promoter (LP1) and Kozak sequence. The therapeutic transgene is selected to treat genetic disorders from a group comprising one of hemophilia, diabetes, obesity, and Gaucher's disease.

[0054] FIG. 2 is a schematic representation of a plasmid map for pAAVrhlOWT, in accordance with an embodiment of the present disclosure.

[0055] FIG. 3 is a schematic representation of the plasmid map for pAAVrhlOS157A, in accordance with an embodiment of the present disclosure.

[0056] FIG. 4 is a schematic representation of the plasmid map for pAAVrhlOK169Q, in accordance with an embodiment of the present disclosure.

[0057] FIG. 5 is a schematic representation of the plasmid map for pAAVrhl0N306Q, in accordance with an embodiment of the present disclosure.

[0058] FIG. 6 is a schematic representation of the plasmid map for pAAVrhl0K709Q, in accordance with an embodiment of the present disclosure.

[0059] FIG. 7 is a schematic representation of the plasmid map for pAAV8WT, in accordance with an embodiment of the present disclosure.

[0060] FIG. 8 is a schematic representation of the plasmid map for pAAV8K31Q / K61Q, in accordance with an embodiment of the present disclosure.

[0061] FIG. 9 is a schematic representation of the plasmid map for pAAV LP1 Kozak hFIX, in accordance with an embodiment of the present disclosure. The mutated sites on AAVrhlO capsid sequences 2, 3, 4, 5, and the mutated site on AAV8 capsid sequence 7 are capitalized. LP1 promoter ranges from nucleotide 184-631, Kozak sequence ranges from nucleotide 742-750 and human FIX cDNA ranges from nucleotide 751-2136 in the sequence 8 for pAAV LP1 Kozak hFIX.

[0062] The present invention is explained further in the following specific examples, which are only by way of illustration and are not to be construed as limiting the scope of the invention.

[0063] Examples

[0064] Materials and Methods

[0065] Cell lines:

[0066] AAV293 (Stratagene, San Diego, California, United States), and human hepatocellular carcinoma (Huh7) cell line, a kind gift from Dr. Saumitra Das (IISc, Bangalore), were cultured in Iscoves’s Modified Dulbecco's Medium (Gibco, Life Technologies, Carlsbad, USA) supplemented with FBS (10% vol / vol) (Gibco, Life Technologies, Carlsbad, USA), lOug / ml piperacillin (MP Biomedicals, LLC, USA) and lOug / ml ciprofloxacin (HiMedia Laboratories, Mumbai, India) at 37°C with 5% CO2.

[0067] Selection of Glycosylation, SUMOylation, and Neddylation mutants:

[0068] Liquid chromatography-mass spectroscopy data of post-translational modifications on the surface of AAV8 and AAVrhlO virus including glycosylation and SUMOylation were reviewed and the sites that were not reported previously were considered further. The conserved residue site for glycosylation and SUMOylation across all the AAV serotypes (1 through 10) was listed. Post-translational modifications (PTMs) on the capsid surface of AAV8 and AArhlO were predicted by using in silica tools such as netogly 4.0 for glycosylation, SUMOplot for SUMOylation, and Neddy Preddy for Neddylation. For each serotype, one site for every modification was selected. The residues that were common in LC-MS data and prediction data for SUMOylation and glycosylation were first selected. Next, the predicted sites with the maximum conservation across all the serotypes and the highest score were selected for the generation of mutants. All the lysine (K) or asparagine (N) residues were mutated to glutamine (Q) and serine (S) residue were mutated to alanine (A). The PTM sites chosen for the AAV vectors to be modified are listed in Table 1. Primers were designed to have the required amino acid changes at the desired site. The primer sequence details are mentioned in Table 2.

[0069] Table 1. AAV mutant vectors developed for study

[0070] Table 2. Primer sequence details of SDM mutants for AAV8 and AAVrhlO. The nucleotides which are mutated in reference to wild type sequence are capitalized.

[0071] Generation of site-directed mutagenesis plasmid for AAV8 and AAVrhlO plasmids and Cloning:

[0072] The plasmid constructs containing the desired mutation were generated by PCR. AAV rep-cap plasmid of the desired serotype was used as a template in the PCR mixture containing dNTP (New England Biolabs, Ipswich, Massachusetts, United States), forward and reverse primers for the specific mutation (Eurofins Scientific, Nantes, France), magnesium chloride (New England Biolabs), dimethyl sulfoxide (Sigma Aldrich, St.Louis, MI, USA), Phusion buffer and Phusion Taq Polymerase (New England Biolabs) in nuclease-free water (Promega, Madison, Wisconsin, USA). The PCR reaction mixture was prepared as mentioned in Table 3 and SDM PCR was performed in a hot-start PCR (Bio-Rad, Hercules, California, United States) with the conditions detailed in Table 4. The PCR product was then treated with Dpnl (New England Biolabs) for 1 hour at 37°C. Transformation of PCR product was performed in freshly prepared PEGylated competent DH5a cells. For each mutant, four colonies were inoculated and the plasmid was isolated for further verification by restriction digestion and Sanger sequencing.

[0073] Table 3. PCR reaction mixture for generation of site-directed mutants

[0074] Table 4. PCR conditions for generation of site-directed mutants

[0075] Development of modified hFIX transgene for improved transgene expression

[0076] A Kozak sequence (GCCGCCACC) was added to the plasmid containing human factor IX (hFIX) transgene in AAV backbone (pAAV-LPl-hFIX) (GenScript, USA) to generate the modified FIX transgene construct (pAAV-LPl-Kozak-hFIX). The insertion of the kozak sequence before the promoter was confirmed by Sanger sequencing.

[0077] Immunocytochemistry for FIX gene expression in vitro.

[0078] Huh7 cells were seeded at a density of 105cells on poly-L-lysine coated coverslip and transfected with 2 pg of pAAV-LPl-hFIX or pAAV-LPl-Kozak-hFIX by using 0.1% polyethyleneimine (PEI). After 3 hours post-transfection, the media was changed with a supplement of vitamin KI at a concentration of 6 pg / ml. After 48 hours of transfection, immuno staining was performed to assess FIX expression driven by different constructs. The cells were probed with hFIX specific antibody (1: 100, GAFFIX-AP, Affinity Biologicals, Ontario, Canada) for 1 hr at room temperature (RT). Further, secondary antibody donkey anti-goat Cy3 (1:500, 705- 165-003, Jackson Immunoreasearch, Pennsylvania, USA) was added and incubated for 40 minutes followed by counterstaining with 4',6-diamidino-2-phenylindole (DAPI). Mounting was done using Fluorsave (Merck Millipore) before imaging in fluorescence microscope (Dmi8, Leica Microsystems, Germany). The fluorescence images were acquired by using Leica Dmi8 Fluorescence microscope (Leica Microsystems, Wetzlar, Germany).

[0079] Plasmid preparation for vector packaging:

[0080] Plasmids required for vector packaging including plasmid containing transgene, a rep-cap plasmid for WT and mutants, and helper plasmid were generated in large quantities (in mgs). Briefly, plasmids were transformed into CaCh-treated competent DH5alpha cells expanded in 4 liters of LB broth, and incubated at 37°C for 12-14 hrs. Cells were collected by centrifuging at 6000 rpm for 20 minutes. The cells were resuspended in lysozyme buffer containing lysozyme (12mg / ml) (Bio Basic, Markham ON, Canada,) for the breakdown of the cell wall. Cells were gently lysed in lysis buffer containing 0.2N sodium hydroxide (Sigma Aldrich) and 1% sodium dodecyl sulfate (Sigma Aldrich) which also caused denaturation of genomic and plasmid DNA due to high pH. This was followed by neutralization of pH by adding 3M sodium acetate (Rankem, Gurugram, India) resulting in the renaturation of nucleic acids that led to precipitation of genomic DNA owing to its large size. The supernatant containing the plasmid is separated by centrifugation to remove protein and genomic DNA contamination. The nucleic acid is again purified by precipitation using cold polyethylene glycol (Sigma Aldrich). The pellet is then dissolved in water and RNA contamination is removed by the addition of 5.5M lithium chloride (Bio Basic) which precipitates RNA. The supernatant is collected, and plasmid DNA is purified by isopropanol precipitation. The plasmid DNA pellet is dissolved in Tris-EDTA buffer which is further purified by cesium chloride (Bio Basic) density gradient ultracentrifugation (Beckman Coulter, Brea, California, United States). The purest form of plasmid DNA in supercoiled form is collected and verified by restriction digestion before use.

[0081] Viral packaging and titration:

[0082] AAV vectors were produced in AAV293 cells by the triple transfection method. Plasmids carrying the transgene, helper genes, and rep-cap genes were transfected into 40 15cm2dishes with AAV293 cells at 80% confluency using 0.1% PEI as a transfection agent, and media was changed after 6 hours of transfection. Cells were harvested 72 hours post-transfection, lysed, and treated with benzonase nuclease (25 units / ml; Sigma-Aldrich). The virus was purified by iodixanol gradient centrifugation (Sigma Aldrich) followed by column purification (GE Healthcare Life Sciences, Pittsburgh, United States). The virus was further concentrated using a 15ml concentrator (Merck Millipore, Massachusetts, USA). Viruses were treated with DNase I as per manufacturer protocol (Sigma Aldrich) and titration of the virus was performed by qPCR (Bio-Rad) using ITR-specific primers.

[0083] Experimental animals and AAV vector administration:

[0084] Hemophilia B mice were procured from Jackson Laboratory (Maine, USA) and housed in the animal facility in proper housing condition and provided with food and water ad libitum. All animal procedures and experiments were approved by IAEC at IIT, Kanpur. AAV vectors were administered via tail vein injection. Hemophilia B mice were administered with AAV8WT and mutant vectors at two different doses of 5X1O10vgs / mice and 1X1O10with at least n=4 in each group. For AAVrhlOWT and mutant vectors, hemophilia B mice were injected at a dose of 1X1O10vgs / mice in n=4-6 mice per group for preclinical evaluation of bioengineered vectors. hFIX expression measurement by ELISA:

[0085] Blood was collected in 3.8% sodium citrate by retro-orbital bleeding at multiple time points after vector administration. Plasma was isolated by centrifugation at 2000 rpm for 15 minutes. Human FIX expression in plasma was measured using Asserachrom FIX: Ag ELISA kit (Diagnostica Stage, asnieres sur seine, He de france, France) as per the manufacturer’s instructions. Briefly, mouse plasma was diluted 1:51 fold as recommended for the assay. 50pl of standards and test samples were added to the coated wells and incubated for 1 hr at room temperature (RT). After washing with the washing buffer provided for 5 times, an alkaline phosphatase-conjugated antibody against hFIX was added and incubated for 1 hr at RT. The substrate was added after washing and incubated for exactly 5 min. Sulphuric acid (IM) was added to stop the reaction and incubated for 15 minutes before reading absorbance at 450nm. hFIX level measurement using clotting assay:

[0086] Plasma samples were diluted using Owren Koller buffer and clotting assay was performed using STA-Deficient FIX plasma to measure the FIX level in the plasma samples being tested as per manufacturer’s protocol.

[0087] Immune profiling of AAV8-hFIX and AAVrhlO-hFIX hepatic gene therapy:

[0088] We assessed the T-cell, B-cell, and regulatory T-cell (Treg) population in experimental animals 32 weeks after gene transfer to assess the immunogenicity of the AAV8-hFIX gene therapy. For AAVrhlO vector-administered mice, we evaluated the T-cell and B-cell population 50 weeks post-vector administration. This assessment aimed to determine the potential for sustained immunogenicity of AAVrhlO-hFIX. Following red blood cell lysis, pelleted cells were incubated for 30 minutes at room temperature with FITC-labeled anti-CD3, PE-labeled anti-CD8, PerCP-labeled anti-CD4, and APC-labeled anti-CD19 antibodies. Flow cytometry was then used to determine the percentage of CD3+, CD4+, CD8+, and CD 19+ cells (BD Accuri C6 Plus). These data were used to count B-cells (CD19+) and double positive markers among the CD3+ population, such as CD4 helper cells (CD3+CD4+) and CD8 cytotoxic cells (CD3+CD8+) in each of the vector or PBS - treated mice. To calculate the percentage of Tregs in mouse splenocytes, 1X106cells were stained for 30 minutes with FITC-labeled CD3, PerCP-labeled anti-CD4 and APC-labeled anti-CD25 antibodies. Cells were then washed, fixed, and permeabilized with the mouse Foxp3 buffer set (BD Pharminogen) followed by staining for 30 minutes with the PE-conjugated Foxp3 antibody. The Treg (CD3+CD4+ CD25+ Foxp3+) cells were counted using flow cytometry.

[0089] Data analysis

[0090] The gene expression analysis by qPCR was analysed using CFX manager 3.0. An unpaired student’s t-test and one-way Anova were used for statistical analysis. P- value < 0.05 was considered to be statistically significant. All the graphs were rendered using GraphPad Prism 8.0.2.

[0091] Results

[0092] Viral Packaging and titration of AAV8 vectors:

[0093] Viral titres of the AAV8 vectors containing human FIX are detailed in Table 5. Both the WT and mutant vector titres were similar and within expected range.

[0094] Table 5. Viral titres of AAV8 carrying hFIX as transgene

[0095] FIG. 10 depicts an immunocytochemistry for hFIX protein expression in vitro, in accordance with an embodiment of the present disclosure. hFIX expression was detected by probing the cells with anti-hFIX antibody post-transfection. Fluorescence images were captured using Leica Dmi8 fluorescence microscope. Representative images are shown. Scale bar -75pm. Densitometric analysis was performed by Image J analysis. Data are expressed as mean ± SD, n = 3, ns-not significant.

[0096] Immunofluorescence assay FIX gene expression:

[0097] Immuno- staining of FIX protein in Huh7 cells showed a similar expression pattern in pAAV-LPl-hFIX and pAAV-LPl-Kozak-hFIX transfected group (FIG. 10). Upon densitometric analysis, no significant difference in the FIX gene expression was observed amongst transfected groups.

[0098] Viral Packaging and titration of AAVrhlO vectors:

[0099] Viral titers of the AAVrhlO vectors containing Kozak-hFIX are detailed in Table 6. One of the AAVrhlO mutant vectors (AAVrhl0N306Q) showed lower titers than wild type vectors.

[0100] Table 6. Viral titers of AAVrhlO carrying Kozak-hFIX as a transgene.

[0101] FIG. 11 depicts hFIX production in hemophilia B mice administered with AAVrhlO WT and mutant vectors, in accordance with an embodiment of the present disclosure. AAVrhlOWT (n=6), AAVrhlOS157A (n=6), AAVrhlOK169Q (n=6), AAVrhl0N306Q (n=6), and AAVrhl0K709Q (n=4) vectors containing Kozak-human FIX transgene were administered in hemophilia B mice via tail vein injection at a dose of IxlO10vgs / mice. Blood was collected from retro-orbital sinus at 5 weeks and 51 weeks post- vector administration. Plasma levels of hFIX were measured using Asserachrom FIX: Antigen ELISA kit. The data shown here is mean+SD. * versus AAVrhlOWT; *p-value<O.O5, **p-value<0.01, ***p- value<O.OOl.

[0102] Human factor IX expression in hemophilia B mice by ELISA:

[0103] ELISA-based hFIX specific assay demonstrated that in mice administered with AAVrhlOWT and mutant vectors, the average hFIX circulating levels in animals that received the mutant vectors S 157A and K169Q after 5 weeks of vector injection were 30.44% ± 3.69%, and 59.32% ± 10.85%, respectively whereas in animals that received the wild-type AAVrhlO and K709Q vectors, were having comparable FIX level of 8.17% ± 3.30% and 8.96% ± 1.57%, respectively. Long-term follow-up at 51 weeks post vector administration showed persistent gene expression (-20.24%) in AAVrhlOK169Q group (FIG. 11).

[0104] FIG. 12 depicts functional rescue in hemophilia B mice administered with AAV8 WT and mutant vectors, in accordance with an embodiment of the present disclosure. AAV8WT (n=5), AAV8K31Q / K61Q (n=5) vectors containing human FIX transgene were administered in hemophilia B mice via tail vein injection at a dose of 5xlO10vgs / mice. Blood was collected from retro-orbital sinus at 4 weeks post-vector administration. Plasma levels of hFIX were measured by clotting assay using FIX STA-Deficient IX. The data shown here is mean+SD. * versus AAV8WT; *p-value<0.05.

[0105] FIG. 13 depicts functional rescue in hemophilia B mice administered with AAVrhlO WT and mutant vectors, in accordance with an embodiment of the present disclosure. AAVrhlOWT (n=6), AAVrhlOS157A (n=6), AAVrhlOK169Q (n=6), and AAVrhl0K709Q (n=4) vectors containing Kozak-human FIX transgene were administered in hemophilia B mice via tail vein injection at a dose of IxlO10vgs / mice. Blood was collected from retro-orbital sinus at 5 weeks, 10 weeks, 28 weeks and 51 weeks post-vector administration. Plasma levels of hFIX was measured by clotting assay using FIX STA-Deficient IX. Data shown here is mean+SD. * versus AAVrhlOWT; **p-value<0.01, ***p-value<0.001.

[0106] Human FIX levels in hemophilia B mice by clotting assay: FIX-specific clotting assay revealed the FIX level in the mice administered with AAV8K31Q / K61Q vector to be significantly higher (352%) than those injected with AAV8WT vector (204%) after 4 weeks of gene transfer (FIG. 12). In the case of AAVrhlO serotype, the average FIX activity after 5 weeks of gene transfer in mice that received the mutant vectors S157A and K169Q was 38.59% ± 5.61%, and 83.87% ± 15.93%, respectively, whereas mice that received WT and K709Q vectors had comparable FIX activity of 10.58% ± 3.5% and 10.85% ± 1.97%, respectively. hFIX expression was stable 10 weeks post vector administration and the FIX activity in animals injected with mutants S157A, K169Q and K709Q was 32.34% ± 6.13%, 76.19% ± 14.40%, and 10.18%±3.65, respectively. Long-term follow-up at 28- and 51 -weeks post vector administration showed persistent and significantly higher FIX expression of -54% and -67%, respectively in AAVrhlOK169Q group when compared to the wild-type AAVrhlO administered animals (FIG. 13).

[0107] FIG. 14 depicts an immune response in hemophilia B mice administered with AAV8 WT and mutant vectors, in accordance with an embodiment of the present disclosure. AAV8WT (n=5) and AAV8K31Q / K61Q (n=5) vectors containing human FIX transgene were administered in hemophilia B mice via tail vein injection at a dose of IxlO10vgs / mice. Blood was collected from retro-orbital sinus at 32 weeks post-vector administration. The levels of helper T cells, cytotoxic T cells, B cells, and regulatory T cells were measured by flow cytometry. The data shown here is mean+SD.

[0108] FIG. 15 depicts the immune response in hemophilia B mice administered with AAVrhlO WT and mutant vectors, in accordance with an embodiment of the present disclosure. AAVrhlOWT (n=6), AAVrhlOS157A (n=6) AAVrhlOK169Q (n=6), and AAVrhl0K709Q (n=5) vectors containing Kozak-human FIX transgene were administered in hemophilia B mice via tail vein injection at a dose of IxlO10vgs / mice. Blood was collected from retro-orbital sinus at 50 weeks post-vector administration. The levels of cytotoxic T cells, helper T cells, and B cells were measured by flow cytometry. Data shown here is mean+SD. ns- non- significant. Evaluation of T-cell and B-cell response to vector after hepatic gene therapy:

[0109] We measured cytotoxic and helper T lymphocytes and B lymphocytes after FIX gene therapy to determine whether the mutant vectors altered the host immunogenic response. Peripheral T lymphocyte (CD3+, CD4+, and CD8+) counts were not significantly different between AAV8WT-hFIX and AAV8K31Q / K61Q-hFIX administered groups, as shown in FIG. 14. Similarly, the B-cell count was similar in the mock (53%) and treated groups (66% in AAV8 and 69% in AAV8K31Q / K61Q). A similar finding was found when immune modulatory T regulatory cells (CD3+CD4+CD25+ and FoxP3+) were quantified in splenocytes. Likewise, the immune response against wild type and mutant AAVrhlO vectors was assessed 50 weeks following hepatic gene transfer in hemophilia B mice. The counts of peripheral T and B lymphocytes (CD3+, CD4+, CD8+, and CD 19+) exhibited no significant difference between AAVrhlOWT and mutant groups, as shown in FIG. 15.

[0110] It is well known that AAV capsid undergoes post-translational modification during packaging as well as infection Mary et al., 2019. PTMs like phosphorylation and ubiquitination on the AAV capsid have been shown to have a significant impact on transduction efficiency in a few studies Yan et al., 2002, and Zhang et al., 2015 Despite the fact that AAV is a reliable vector, host immune and cellular degradation systems remain a significant barrier Manno et al., 2006, and Zhong et al., 2008. A variety of approaches, including pseudo packaging and capsid modification, have been employed to circumvent them Duan et al., 2001, and Gabriel et al., 2013. Each method focuses on the molecular mechanisms that prevent AAV transmission or intracellular processes that break down capsid proteins and interact with immune effectors. Previous research has demonstrated a functional overlap between the ubiquitination process and PTM modifications like SUMOylation and Neddylation. Neddylation has been shown to control host- viral interactions Whitby et al., 1998, and McManus et al., 2017. Through the E3 ligase HDM2, Neddylation of polymerase basic protein 2 (PB2) prevents influenza A virus 9 from replicating. Studies conducted in vitro have demonstrated that altering a PB2 residue that is involved in Neddylation made PB2 more stable and promoted viral replication Zhang et al., 2017. Similar to this, it is well known that SUMOylation contributes to the interaction between hosts and viruses Hblscher et al., 2015. Previous studies on abolishment of PTM site on AAV 2 vectors have been successful in generation of better performing vectors than wild type AAV vectors Mary et al., 2019, Maurya et al., 2019, Gabriel et al., 2013, and Zhong et al., 2008. The present invention aims to generate such improvised vector with increased transduction potential or immune escape capabilities for AAV8 and AAVrhlO serotype for liver directed gene therapy. There are multiple reports on importance of PTMs in protein folding that forms the icosahedral structure of AAV capsid El Andari et al., 2021, and Agbandje-McKenna et al., 2011. AAV8 WT and mutant vectors resulted in physiological level of FIX at relatively low dose of 5xl010vgs / mice. The FIX level measured by clotting assay revealed AAV8K31Q / K61Q having significantly higher FIX level than WT at 4 weeks post vector administration. Immune profiling of AAV8WT and AAV8K31Q / K61Q vector showed that the mutant vector has an immunogenic response similar to AAV8WT vector.

[0111] Several preclinical studies for hepatic gene transfer have utilized AAVrhlO vectors Wang et al., 2015, and Pipe et al., 2017. AAVrhlO vectors have been reported to have continued hepatic transgene expression when compared to other serotypes Hu et al., 2010. In one such study, the AAVrhlO S671A modified mutant partially restored hepatic gene expression when tested in the presence of human IVIG Selot et al., 2017. The positive effect of downregulation of the SUMOylation pathway enzymes SAE1, SAE2, and UBC9 on AAV transduction has been demonstrated in a previous study Holscher et al., 2015. A number of viruses, including HIV and influenza, have altered antigenicity due to mutations at the site of glycosylation without affecting their biological activity Abe et al., 2004. Recent research on Neddylation, SUMOylation, and glycosylation site modification has demonstrated improved phenotypic recovery for hepatic and ocular gene disorders Mary et al., 2019, and Maurya etal., 2019. We carried out a similar study using bioengineered AAVrhlO vectors and a hFIX transgene with the Kozak sequence. In the present invention for preclinical assessment of these AAVrhlO mutant vectors, hemophilia B mice were systemically administered with a low vector dose of 1X1O10vg / mice. The functional activity of FIX after 5 weeks and 10 weeks of gene transfer was consistent among groups with Neddylation mutant (AAVrhlOK169Q) resulting in a physiological level of circulating FIX. Long-term follow-ups of 28 weeks and 51 weeks demonstrated persistent and substantially higher FIX activity with a 5-fold increase in FIX levels in animals that received the AAVrhlOK169Q vectors compared to AAVrhlOWT vector administered animals. Furthermore, the enumeration of immune cells at 50 weeks post-vector administration indicated that the immune profiles of both AAVrhlOWT and mutant vectors closely resemble those of the mock group. Notably, no significant differences were observed in the populations of T-cells and B -cells. In conclusion, our work has generated bioengineered AAV capsids and mutant vectors of some serotypes such as AAVrhlO, which have an improved therapeutic efficiency during hepatic gene therapy in preclinical models of hemophilia B. Additionally, the development of such AAV platform vectors may be advantageous for therapeutic gene transfer in other hepatic diseases such as hemophilia B, diabetes and obesity and, Gaucher’s disease.

[0112] Therefore, the present invention provides modified AAV vectors for gene therapy in the treatment of hemophilia B. The modified AAV vectors exhibit improved transduction efficiency, leading to higher and more sustained expression of the therapeutic gene. Due to enhanced transduction capabilities, the modified AAV vectors enable therapeutic levels of coagulation factors (FVIII or FIX) to be achieved at relatively lower vector doses. This reduces the potential for adverse effects associated with high vector doses.

[0113] The modification of AAV capsids at post-translational modification (PTM) sites enhances immune escape capabilities. The present invention can be applied to AAV8 and AAVrhlO capsids, making it versatile for the treatment of hemophilia B, which are distinct genetic disorders. The method for manufacturing the modified AAV vectors involves a systematic approach, including the identification of specific PTM sites, design of primers for site-directed mutagenesis, and in vitro verification. This precision ensures the reliable production of vectors with improved characteristics. While primarily designed for hemophilia treatment, the modified AAV vectors and manufacturing method can be adapted for other genetic disorders and liver-directed gene therapy applications, such as diabetes, obesity, Gaucher's disease, and more. Therefore, the present invention represents a significant advancement in the field of gene therapy, offering improved treatment options for individuals with hemophilia B and the potential for broader applications in other genetic disorders.

[0114] While specific language has been used to describe the invention, any limitations arising on account of the same are not intended. As would be apparent to a person skilled in the art, various working modifications may be made to the method in order to implement the inventive concept as taught herein.

[0115] The figures and the foregoing description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single functional element. Alternatively, certain elements may be split into multiple functional elements. Elements from one embodiment may be added to another embodiment. For example, order of processes described herein may be changed and are not limited to the manner described herein. Moreover, the actions of any flow diagram need not be implemented in the order shown; nor do all of the acts need to be necessarily performed. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples.

Claims

WE CLAIM:

1. A modified adeno-associated virus (AAV) vector for gene therapy, comprising: an AAV capsid selected from a group comprising one of AAV8 capsid and AAVrhlO capsid, wherein the AAV capsid is modified at post-translational modification (PTM) sites using primers; wherein the primers are selected with substitutions from a group comprising of lysine (K) and asparagine (N) residues with glutamine (Q), and serine (S) residues with alanine (A), wherein the AAV8 capsid is modified at neddylation sites, and wherein the AAVrhlO capsid is modified at both HexNAcylation and neddylation sites; and a therapeutic transgene combined with regulatory elements configured to increase gene and protein expression at translational level.

2. The modified AAV vector as claimed in claim 1, wherein the therapeutic transgene comprises modified FIX transgene construct (pAAV-LPl-Kozak-hFIX).

3. The modified AAV vector as claimed in claim 1, wherein the regulatory elements are selected from a group comprising of a combination of liver specific promoter (LP1) and Kozak sequence.

4. The modified AAV vector as claimed in claim 1, wherein the PTM modification contains neddylation and HexNAcylation sites configured to increase transduction efficiency and minimize adverse immune response.

5. A method for preparing a modified AAV vector, comprising the steps of:identifying post-translational modification (PTM) sites on AAV capsid selected from a group comprising one of AAV8 capsid and AAVrhlO capsid using liquid chromatography -mass spectroscopy and in silica tools; selecting post-translational modification (PTM) sites with high conservation and maximum scores across AAV serotypes; designing primers for introducing glutamine (Q) and alanine (A) substitutions at the selected PTM sites, wherein primers are selected with substitutions from a group comprising of lysine (K) and asparagine (N) residues with glutamine (Q), and serine (S) residues with alanine (A); generating site-directed mutagenesis plasmids for one of AAV8 capsid and AAVrhlO capsid with the desired mutations to obtain modified plasmids; transfecting host cells with the modified plasmids and a therapeutic transgene to obtain the modified AAV vector with increased transduction efficiency and immune escape capabilities; and purifying followed by concentrating the modified AAV vector.

6. The method as claimed in claim 5, wherein the post-translational modification (PTM) sites are selected from a group comprising of neddylation and HexNAcylation sites.

7. The method as claimed in claim 5, wherein the AAV capsid is modified at post-translational modification (PTM) sites using primers.

8. The method as claimed in claim 7, wherein the primers are selected with substitutions from a group comprising of lysine (K) and asparagine (N) residues with glutamine (Q), and serine (S) residues with alanine (A).

9. The method as claimed in claim 5, wherein the host cells are cultured under controlled conditions to optimize production of the modified AAV vector.

10. The method as claimed in claim 9, wherein the controlled conditions comprise culturing host cell (AAV293) in Iscove’s modified Dulbecco’s medium comprising 10% fetal bovine serum, lOpg / ml of piperacillin and ciprofloxacin with 5% carbon dioxide (CO2) at 37 °C.

11. The method as claimed in claim 5, wherein the therapeutic transgene comprises modified FIX transgene construct (pAAV-LPl-Kozak-hFIX).

12. The method as claimed in claim 11, wherein the modified FIX transgene construct (pAAV-LPl-Kozak-hFIX) is obtained by addition of Kozak sequence (GCCGCCACC) to a plasmid comprising human factor IX (hFIX) transgene in AAV backbone (pAAV-LPl-hFIX).

13. A composition for gene therapy, comprising:100% of a modified adeno-associated virus (AAV) vector, wherein the AAV vector comprises an AAV capsid selected from a group comprising one of AAV8 capsid and AAVrhlO capsid, wherein the AAV capsid is modified at post-translational modification (PTM) sites using primers, wherein the primers are selected with substitutions from a group comprising of lysine (K) and asparagine (N) residues with glutamine (Q), and serine (S) residues with alanine (A), wherein the AAV8 capsid is modified at neddylation sites, and wherein the AAVrhlO capsid is modified at both HexNAcylation and neddylation sites; and a therapeutic transgene combined with regulatory elements configured to increase gene and protein expression at translational level.

14. The composition as claimed in claim 13, wherein the therapeutic transgene comprises modified FIX transgene construct (pAAV-LPl-Kozak-hFIX).

15. The composition as claimed in claim 13, wherein the regulatory elements are selected from a group comprising of a combination of liver specific promoter (LP1) and Kozak sequence.

16. The composition as claimed in claim 13, wherein the therapeutic transgene is selected to treat genetic disorders from a group comprising one of hemophilia, diabetes, obesity, and Gaucher's disease.

Images