RELATED APPLICATIONS

This application is related to Indian Provisional Application 202021025215 filed 16 ^th Jun, 2020 and is incorporated herein in its entirety.

FIELD OF THE INVENTION

The present invention pertains to novel adeno associated virus (AAV) vector comprising gene encoding anti-VEGF Fab, composition thereof and method of using the same.

BACKGROUND OF THE INVENTION

Age-related macular degeneration (AMD) is a progressive degenerative macular disease attacking the region of highest visual acuity (VA) and the macula. The neovascular "wet" form of the disease (nAMD or wet AMD) is characterized by choroidal neovascularization which is marked by proliferation of blood vessels and cells including those of the retinal pigment epithelium (RPE). Ultimately, photoreceptor death and scar formation result in a severe loss of central vision and the inability to read, write, and recognize faces or drive. Many patients can no longer maintain gainful employment, carry out daily activities and consequently report a diminished quality of life. Preventative therapies have demonstrated little effect and therapeutic strategies have focused primarily on treating the neovascular lesion. Age-related macular degeneration (AMD) is a highly prevalent neurodegenerative disease in which death of photoreceptors and retinal pigmented epithelial (RPE) cells results in gradual loss of central vision. A subgroup of 10% — 15% of patients with AMD develop subretinal neovascularization (NV) resulting in relatively rapid reduction in visual acuity due to leakage of plasma from incompetent new vessels and collection of fluid within and under the retina, which compromises retinal function. This subgroup is said to have neovascular AMD (nAMD).

Glioma or Glioblastoma is an aggressive type of tumor-associated angiogenesis, a cancer that occurs in the brain or spinal cord as a result of intense and aberrant blood vessel development. Glioblastoma forms from cells called astrocytes that support nerve cells derived from neuroglial progenitor cells. This is due to the fact that these cells express high levels of vascular endothelial growth factor (VEGF-A), which plays a key role in angiogenesis and in increasing tumor-vasculature. The level of VEGF expression is correlated with grade of Glioma. As the tumor vasculature supplies nutrients for the tumor growth, the glioma cells inhibit any drug or immune cells from reaching the tumor. Anti-angiogenic therapy is the standard of care for recurrent Glioblastoma. Bevacizumab is the most common monoclonal antibody which binds to vascular endothelial growth factor (VEGF) and most often used in the treatment of Glioblastoma.

Available treatments for nAMD include laser photocoagulation, photodynamic therapy with verteporfin, and intravitreal ("IVT") injections with agents aimed at binding to and neutralizing vascular endothelial growth factor ("VEGF") - a cytokine implicated in stimulating angiogenesis and targeted for intervention. Such anti- VEGF agents used include, e.g., Bevacizumab (a humanized monoclonal antibody (mAh) against VEGF produced in CHO cells), Ranibizumab (the Fab portion of an affinity- improved variant of Bevacizumab made in prokaryotic E. coli), Aflibercept (a recombinant fusion protein consisting of VEGF -binding regions of the extracellular domains of the human VEGF- receptor fused to the Fc portion of human IgGl), or Pegaptanib (a pegylated aptamer (a single- stranded nucleic acid molecule) that binds to VEGF). Each of these therapies has some effect on best- corrected visual acuity; however, their effects appear limited in restoring visual acuity and in duration. Further, these therapeutic approaches have several drawbacks such as the need for repeat dosing, eye infections due to procedural issues, detached retinas, cataracts, prolonged inflammation and increased intraocular pressure. The cost of the treatment, the risk of developing anti -therapeutic factor associated immune responses, and the risk of blinding due to repeated dosing. The need for repeat treatments can incur additional risk to patients and is inconvenient for both patients and treating physicians.

These limitations have prompted the development of gene -based anti-VEGF therapies for wet AMD, Glioblastoma and other eye diseases.

OBJECTS OF THE INVENTION

The Principal object of the present invention is to provide adeno associated virus (AAV) vector comprising nucleotide sequence encoding anti-VEGF single chain antigen binding fragment (scFab).

Another object of the present invention is to provide adeno associated virus (AAV) vector comprising nucleotide sequence of SEQ ID NO: 02 encoding anti-VEGF single chain antigen binding fragment (scFab).

Another object of the present invention is to provide viral genome of recombinant AAV particle which comprises: (a) One or more inverted terminal repeat (ITR) sequences that flank the 5' or 3' terminus of the heterologous polynucleotide sequence;

(b) An expression control element that drives transcription of scFab e.g. a promoter and/or enhancer whereas the promoter is CB7 promoter or (consist of CMV immediate early enhancer and Chicken Beta actin Promoter);

(c) An inducible/switchable/regulatable control element;

(d) An anti-VEGF scFab; and

(e) SV40 PolyA transcription terminator.

Another object of the present invention is to provide viral genome of recombinant AAV particle comprising nucleotide sequence of SEQ ID NO. 02 encoding:

(a) One or more inverted terminal repeat (ITR) sequences that flank the 5' or 3' terminus of the heterologous polynucleotide sequence;

(b) An expression control element that drives transcription of scFab e.g. a promoter and/or enhancer whereas the promoter is CB7 promoter or (consist of CMV immediate early enhancer and Chicken Beta actin Promoter);

(c) An inducible/switchable/regulatable control element;

(d) An anti-VEGF scFab; and

(e) SV40 PolyA transcription terminator.

Another object of the present invention is to provide recombinant Adeno-associated virus (rAAV) having an AAV, wherein the rAAV comprises a vector genome packaged within the capsid, said vector genome comprising: a) Feft ITR2; b) CB7 Promoter; c) Intron; d) Signal peptide; e) Anti-VEGF scFab; f) SV40 Polyadenylation sequence; and g) Right ITR2.

Another object of the present invention is to provide recombinant Adeno-associated virus (rAAV) having an AAV capsid, wherein the rAAV comprises a vector genome packaged within the capsid, said vector genome comprising nucleotide sequence of SEQ ID NO: 02 encoding: a) Left ITR2; b) CB7 Promoter; c) Intron; d) Signal peptide; e) Anti-VEGF scFab; f) SV40 Polyadenylation sequence; and g) Right ITR2.

Another object of the present invention relates to recombinant AAV particle generated using AAV003 expressing anti-VEGF scFab of ~50 KD where light and heavy chain were expressed along with SC60 linker. The scFab should also bind and neutralize VEGF.

Another object of the present invention is to provide AAV003 for persistent expression of Anti-VEGF to treat ocular angiogenesis and increased vascular permeability e.g. wet age-related macular degeneration (wet AMD), diabetic retinopathy (DR), diabetic macular edema (DME), myopic choroidal neovascularization (mCNV), macular edema following retinal vein occlusion (RVO) and Glioblastoma, using AAV gene therapy based administration of nucleotide sequence encoding anti-VEGF scFab. Another object of the present invention is to provide AAV-003 for method of treatment of wet age related macular degeneration (Wet AMD) using AAV vector genome comprising nucleotide sequence encoding anti-VEGF single-chain antigen binding fragment (scFab).

Another object of the present invention is to provide AAV003 for method of treatment of Glioblastoma using AAV vector genome comprising nucleotide sequence encoding anti-VEGF single-chain antigen binding fragment (scFab).

Another object of the present invention is to provide pharmaceutical formulation comprising:

(a) AAV vector genome comprising nucleotide sequence encoding anti-VEGF single-chain antigen binding fragment (scFab); and

(b) a pharmaceutically acceptable carrier, diluents, excipients, or buffer.

SUMMARY OF THE INVENTION

The principal aspect of the present invention is to provide adeno associated virus (AAV) vector comprising nucleotide sequence encoding anti-VEGF single-chain antigen binding fragment variable (scFab).

Another aspect of the present invention is to provide adeno associated virus (AAV) vector comprising nucleotide sequence of SEQ ID NO: 02 encoding anti-VEGF single-chain antigen binding fragment (scFab).

Another aspect of the present invention is to provide viral genome of recombinant AAV particle which comprises: (a) One or more inverted terminal repeat (ITR) sequences that flank the 5' or 3' terminus of the heterologous polynucleotide sequence;

(b) An expression control element that drives transcription of scFab e.g. a promoter and/or enhancer whereas the promoter is CB7 promoter or (consist of CMV immediate early enhancer Chicken Beta actin Promoter);

(c) An inducible/switchable/regulatable control element;

(d) An anti-VEGF scFab; and

(e) SV40 PolyA transcription terminator.

Another aspect of the present invention is to provide viral genome of recombinant AAV particle comprising nucleotide sequence of SEQ ID NO. 02 encoding:

(a) One or more inverted terminal repeat (ITR) sequences that flank the 5' or 3' terminus of the heterologous polynucleotide sequence;

(b) An expression control element that drives transcription of scFab e.g. a promoter and/or enhancer whereas the promoter is CB7 promoter or (consist of CMV immediate early enhancer Chicken Beta actin Promoter);

(c) An inducible/switchable/regulatable control element;

(d) An anti-VEGF scFab; and

(e) SV40 PolyA transcription terminator.

Another aspect of the present invention is to provide recombinant Adeno-associated virus (rAAV) having an AAV capsid, wherein the rAAV comprises a vector genome packaged within the capsid, said vector genome comprising: a) Feft ITR2; b) CB7 Promoter; c) Intron; d) Signal peptide; e) Anti-VEGF scFab; f) SV40 Polyadenylation sequence; and g) Right ITR2.

Another aspect of the present invention is to provide recombinant Adeno-associated virus (rAAV) having an AAV capsid, wherein the rAAV comprises a vector genome packaged within the capsid, said vector genome comprising nucleotide sequence of SEQ ID NO: 02 encoding: a) Left ITR2; b) CB7 Promoter; c) Intron; d) Signal peptide; e) Anti-VEGF scFab; f) SV40 Polyadenylation sequence; and g) Right ITR2.

Another aspect of the present invention relates to recombinant AAV particle generated using AAV003 expressing anti-VEGF scFab of ~50 KD where light and heavy chain were expressed along with SC60 linker. The scFab should also bind and neutralize VEGF.

Another aspect of the present invention is to provide AAV003 for persistent expression of Anti-VEGF to treat ocular angiogenesis and increased vascular permeability e.g. wet age-related macular degeneration (wet AMD), diabetic retinopathy (DR), diabetic macular edema (DME), myopic choroidal neovascularization (mCNV), macular edema following retinal vein occlusion (RVO) and Glioblastoma, using AAV gene therapy based administration of nucleotide sequence encoding anti-VEGF scFab. Another aspect of the present invention is to provide AAV003 for method of treatment of wet age related macular degeneration (Wet AMD) using AAV vector genome comprising nucleotide sequence encoding anti-VEGF single-chain antigen binding fragment (scFab).

Another aspect of the present invention is to provide AAV003 for method of treatment of Glioblastoma using AAV vector genome comprising nucleotide sequence encoding anti-VEGF single-chain antigen binding fragment (scFab).

Another aspect of the present invention is to provide pharmaceutical formulation comprising:

(a) AAV vector genome comprising nucleotide sequence encoding anti-VEGF single-chain antigen binding fragment (scFab); and

(b) a pharmaceutically acceptable carrier, diluents, excipients, or buffer.

BRIEF DESCRIPTION OF DRAWINGS

In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figure together with s detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure wherein:

Figure 1: Schematic Representation AAV Vector Map (pGT-AAV003) with Transgene and Regulatory Elements

Figure 2: Schematic Representation of AAV Gene expression Cassette (pGT- AAV003) Figure 3: Schematic Representation of Vector Map pGT scFv/scFab with switch (Representing the inducible/switchable/regulatable control element)

Figure 4: Schematic Representation of Packaging Plasmid Vector Map (plntas- ACG-R2C8) Figure 5: Schematic Representation of pIntas-ACG-R2C8 Packaging DNA Construct

Figure 6: Schematic Representation of Packaging Plasmid Vector Map (plntas- ACG-R2C6)

Figure 7: Schematic Representation of pIntas-ACG-R2C6 packaging DNA Construct

Figure 8: Schematic Representation of Helper Plasmid Vector Map (plntas -Helper) Figure 9: Schematic Representation of plntas-Helper Cassette Figure 10A: Binding Curve Sensogram with Protein F Figure 10B: Binding Curve Sensogram with VEGF Figure 11: Octet Sensogram for binding of pGT003 Culture Supernatant with VEGF

DETAILED DESCRIPTION OF THE INVENTION The rAAV vectors are designed to express an anti-VEGF antibody single chain antigen binding fragment (scFab) in mammalian, and more particularly, human cells. These anti-VEGF scFvs are particularly well suited for treatment of age-related macular degeneration (AMD) and Glioblastoma. As described herein, novel AAV anti-VEGF constructs have been developed which have demonstrated high yield, expression levels, and/or activity.

Adeno-associated virus based viral vector system containing an expression cassette of VEGF neutralizing/ binding antibody, scFab fragment or scFv that is packaged in the optimized viral capsids for specific tissue tropism (i.e. different serotypes of AAV). Such vector can be used for the treatment of wet age-related macular degeneration (wet AMD), diabetic retinopathy (DR), diabetic macular edema (DME), myopic choroidal neovascularization (mCNV), macular edema following retinal vein occlusion (RVO) and Glioblastoma.

Unless otherwise defined, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs.

It is to be noted that the term "a" or "an" refers to one or more. As such, the terms "a" (or "an"), "one or more," and "at least one" are used interchangeably herein.

The words "comprise", "comprises", and "comprising" are to be interpreted inclusively rather than exclusively. The words "consist", "consisting", and its variants, are to be interpreted exclusively, rather than inclusively. While various embodiments in the specification are presented using “comprising” language, under other circumstances, a related embodiment is also intended to be interpreted and described using “consisting of or “consisting essentially of’ language.

There are several naturally occurring (“wild-type”) serotypes and over 100 known variants of AAV, each of which differs in amino acid sequence, particularly within the hypervariable regions of the capsid proteins, and thus in their gene delivery properties. No AAV has been associated with any human disease, making recombinant AAV attractive for clinical applications.

For the purposes of the disclosure herein, the terminology “AAV” is an abbreviation for Adeno-associated virus, including, without limitation, the virus itself and derivatives thereof. Except where otherwise indicated, the terminology refers to all subtypes or serotypes and both replication-competent and recombinant forms. The term “AAV” includes, without limitation, AAV type 1 (AAV-1 or AAV1), AAV type 2 (AAV-2 or AAV2), AAV type 3 A (AAV-3A or AAV3A), AAV type 3B (AAV- 3B or AAV3B), AAV type 4 (AAV-4 or AAV4), AAV type 5 (AAV- 5 or AAV5), AAV type 6 (AAV-6 or AAV6), AAV type 7 (AAV-7 or AAV7), AAV type 8 (AAV- 8 or AAV8), AAV type 9 (AAV-9 or AAV 9), AAV type 10 (AAV- 10 or AAV10 or AAVrhlO), avian AAV, bovine AAV, Porcine AAV, canine AAV, caprine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. "Primate AAV" refers to AAV that infect primates, "non-primate AAV" refers to AAV that infect non-primate mammals, "bovine AAV" refers to AAV that infect bovine mammals, etc.

In one embodiment Adeno associated virus is AAV8 or AAV6 selected as preferred choice for the current invention.

The term "packaging" refers to a series of intracellular events that result in the assembly and encapsidation of an AAV particle. AAV "rep" and "cap" genes refer to polynucleotide sequences encoding replication and encapsidation proteins of Adeno- associated virus. AAV rep and cap are referred to herein as AAV "packaging genes."

In one embodiment packaging genes are AAV rep2cap8 (R2C8) and AAV rep2cap6 (R2C6) selected as preferred choice for the current invention.

The terminology "helper virus" for AAV refers to a virus that allows AAV (e.g. wild-type AAV) to be replicated and packaged by a mammalian cell. A variety of such helper viruses for AAV are known in the art, including adenoviruses, herpes viruses and poxviruses such as vaccinia. The adenoviruses encompass a number of different subgroups, although Adenovirus type 5 of subgroup C is most commonly used. Numerous adenoviruses of human, non-human mammalian and avian origin are known and available from depositories such as the ATCC. Viruses of the herpes family include, for example, herpes simplex viruses (HSV) and Epstein-Barr viruses (EBV), as well as cytomegaloviruses (CMV) and pseudorabies viruses (PRV); which are also available from depositories such as ATCC.

The term "gene" refers to a polynucleotide that performs a function of some kind in the cell. For example, a gene can contain an open reading frame that is capable of encoding a gene product. One example of a gene product is a protein, which is transcribed and translated from the gene. Another example of a gene product is an RNA, e.g. a functional RNA product, e.g., an aptamer, an interfering RNA, a ribosomal RNA (rRNA), a transfer RNA (tRNA), a non-coding RNA (ncRNA), a guide RNA for nucleases, etc., which is transcribed but not translated.

The terminology "operatively linked" or "operably linked" refers to a juxtaposition of genetic elements, wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a promoter is operatively linked to a coding region if the promoter helps initiate transcription of the coding sequence. There may be intervening residues between the promoter and coding region so long as this functional relationship is maintained.

The term "pharmaceutically acceptable" refers to compounds and compositions which are suitable for administration to humans and/or animals without undue adverse side effects such as toxicity, irritation and/or allergic response commensurate with a reasonable benefit/risk ratio. The main embodiment of the present invention is to provide adeno associated virus (AAV) vector comprising nucleotide sequence encoding anti-VEGF single chain antigen binding fragment (scFab).

Another embodiment of the present invention is to provide Adeno associated virus (AAV) vector comprising nucleotide sequence of SEQ ID NO: 02 encoding anti- VEGF single chain antigen binding fragment (scFab).

Another embodiment the present invention is to provide viral genome of recombinant AAV particle which comprises:

(a) One or more inverted terminal repeat (ITR) sequences that flank the 5' or 3' terminus of the heterologous polynucleotide sequence;

(b) An expression control element that drives transcription of scFab e.g. a promoter and/or enhancer whereas the promoter is CB7 promoter or (consist of CMV immediate early enhancer Chicken Beta actin Promoter);

(c) An inducible/switchable/regulatable control element;

(d) An anti-VEGF scFab; and

(e) SV40 PolyA transcription terminator.

Another embodiment of the present invention is to provide viral genome of recombinant AAV particle comprising nucleotide sequence of SEQ ID NO. 02 encoding:

(a) One or more inverted terminal repeat (ITR) sequences that flank the 5' or 3' terminus of the heterologous polynucleotide sequence;

(b) An expression control element that drives transcription of scFab e.g. A promoter and/or enhancer whereas the promoter is CB7 promoter or (consist of CMV immediate early enhancer Chicken Beta actin Promoter);

(c) An inducible/switchable/regulatable control element; (d) An anti-VEGF scFab; and

(e) SV40 PolyA transcription terminator.

Another embodiment of the present invention is to provide recombinant Adeno- associated virus (rAAV) having an AAV capsid, wherein the rAAV comprises a vector genome packaged within the capsid, said vector genome comprising: a) Feft ITR2; b) CB7 Promoter; c) Intron; d) Signal peptide; e) Anti-VEGF scFab; f) SV40 Polyadenylation sequence; and g) Right ITR2.

Another embodiment of the present invention is to provide recombinant Adeno- associated virus (rAAV) having an AAV capsid, wherein the rAAV comprises a vector genome packaged within the capsid, said vector genome comprising nucleotide sequence of SEQ ID NO: 02 encoding: a) Feft ITR2; b) CB7 Promoter; c) Intron; d) Signal peptide; e) Anti-VEGF scFab; f) SV40 Polyadenylation sequence; and g) Right ITR2. In certain aspects of the methods described herein, the antigen -binding fragment transgene encodes a leader peptide. A leader peptide may also be referred to as a signal peptide or leader sequence herein.

In certain embodiments, the processing of anti-VEGF scFab is directed by leader peptides that are derived from human IF2 protein. In one embodiment the leader sequence is an interleukin (IF) IF-2 leader sequence, which may be the wild-type human IF2 or a mutated human IF2. Such leader sequences are known in the art. For example, UniProtKB/Swiss-Prot Accession Number: P60568.

In certain embodiments, the viral vectors provided herein comprise one or more untranslated regions (UTRs), e.g., 3 ' and/or 5' UTRs. In certain embodiments, the UTRs are optimized for the desired level of protein expression. In certain embodiments, the UTRs are optimized for the mRNA half-life of the transgene. In certain embodiments, the UTRs are optimized for the stability of the mRNA of the transgene. In certain embodiments, the UTRs are optimized for the secondary structure of the mRNA of the transgene.

Another embodiment of the present invention relates to recombinant AAV particle generated using AAV003 expressing anti-VEGF scFab of ~50 KD where light and heavy chain were expressed along with SC60 linker. The scFab should also bind and neutralize VEGF.

Another embodiment of the present invention is to provide AAV003 for persistent expression of Anti-VEGF to treat ocular angiogenesis and increased vascular permeability e.g. wet age-related macular degeneration (wet AMD), diabetic retinopathy (DR), diabetic macular edema (DME), myopic choroidal neovascularization (mCNV), macular edema following retinal vein occlusion (RVO) and Glioblastoma, using AAV gene therapy based administration of nucleotide sequence encoding anti-VEGF scFab.

Another embodiment of the present invention is to provide AAV003 for method of treatment of wet age related macular degeneration (Wet AMD) using AAV vector genome comprising nucleotide sequence encoding anti-VEGF single-chain antigen binding fragment (scFab).

Another embodiment of the present invention is to provide AAV003 for method of treatment of Glioblastoma using AAV vector genome comprising nucleotide sequence encoding anti-VEGF single-chain antigen binding fragment (scFab).

Another embodiment of the present invention is to provide pharmaceutical formulation comprising:

(a) AAV vector genome comprising nucleotide sequence encoding anti-VEGF single-chain antigen binding fragment (scFab); and

(b) a pharmaceutically acceptable carrier, diluents, excipients, or buffer.

As used herein, the amino acid sequence of antiVEGF-scFab was taken from product monograph of Ranibizumab (Fucentis ATC Code: S01FA04).

In certain embodiment inverted terminal repeats (ITR) from AAV2 may be selected. Vectors having ITRs from a different source than its capsid are termed "pseudotyped". In certain embodiments, ITRs from a source other than AAV2 may be selected for this construct to generate another pseudotyped AAV. Alternatively, ITRs from the same source as the capsid may be selected. In certain embodiments, ITRs may be selected to generate a self-complementary AAV, such as defined infra. In certain embodiments, the promoter is CB7, a hybrid between a cytomegalovirus (CMV) immediate early enhancer and the chicken beta actin promoter. In other embodiments, the promoter is a ubiquitin C (UbC) promoter. See, e.g., WO 2001/091800. See, e.g., GenBank® accession numbers AF232305 (rat) and D63791 (human), respectively. Still other promoters and/or enhancers may be selected. See, e.g. cytomegalovirus (CMV) immediate early enhancer (260 bp, C4; GenBank # K03104.1). Chicken beta-actin promoter (281 bp; CB; GenBank # X00182.1). In still other embodiments, multiple enhancers and/or promoters may be included.

In certain embodiments, an intron is included. One suitable intron is a chicken beta- actin intron. However, other suitable introns may be selected.

In certain embodiments the vector genomes described herein include a polyadenylation signal (polyA). A variety of suitable polyA are known. In one example, the polyA is SV40 polyA signal. Still other suitable polyA sequences may be selected. Optionally, other suitable vector elements may be selected which may include, e.g., a UTR sequence or a Kozak sequence.

In one embodiment the DNA sequence packaged in-side the virus contains ITR sequences at 3 ’and 5’ end along with CB7 Promoter (which contains CMV Immediate Early enhancer and chicken beta actin promoter) linked with intron and Kozak sequence. Downstream of the Kozak sequence, the nucleotide sequence encoding anti-VEGF scFv (as in vector AAV001) and anti-VEGF scFab (Fab with SC60 linker sequence) was placed along with SV40 Poly A transcription termination sequence (as in vector AAV003). The construct is intended to produce a fully functional and secretory scFv or scFab polypeptide sequence that will provide continuous source of Anti-VEGF that binds with high affinity to than the current Anti-VEGF mAh used for similar therapy. For use in producing an AAV viral vector (e.g., a recombinant (r) AAV), the expression cassettes can be carried on any suitable vector, e.g. a plasmid, which is delivered to a packaging host cell. The plasmids useful may be engineered such that they are suitable for replication and packaging in prokaryotic cells, mammalian cells, or both. Suitable transfection techniques and packaging host cells are known and/or can be readily designed by one of skill in the art.

EXPERIMENTAL DETAILS Vector Designing:

Three different vector constructs (i.e. pGT-AAVOOl, pGT-AAV002 and pGT- AAV003) were designed to identify the nucleotide sequence of potential candidate which has high binding affinity to VEGF and good genome packaging in different serotype of AAV.

• AAV 001 Transgene expresses ~ 26 KD scFv proteins which binds and neutralize VEGF.

• AAV 002 Transgene express ~ 50 KD Fab proteins where light and heavy chain were co-expressed in tandem using T2A linker. The T2A linker gets processed by proteolytic activity of the cell resulting in anti VEGF Fab expression which binds and neutralize VEGF.

• AAV 003 Transgene express ~ 50 KD scFab proteins where light and heavy chain were expressed along with SC60 linker. The scFab should also bind and neutralize VEGF.

The vector was chemically synthesized and propagated in E. coli for bulk DNA preparation. HEK 293 cells were transiently transfected with all three vectors and supernatant were harvested for protein expression. The expression level and binding affinity of the scFv and scFab transgenes were evaluated with EFISA and Octet based systems. Two constructs (i.e. pGT-AAVOOl (SEQ ID NO: 01) and pGT-AAV003 (SEQ ID NO: 02) were finally identified for AAV VLP generation, purification, characterization and in vitro analysis. Figure 10A and 10B shows binding curve sensogram with protein L and VEGF respectively.

The embodiments of the present invention are further described using specific examples herein after. The examples are provided for better understanding of certain embodiments of the invention and not, in any manner, to limit the scope thereof. Possible modifications and equivalents apparent to those skilled in the art using the teachings of the present description and the general art in the field of the invention shall also form the part of this specification and are intended to be included within the scope of it.

EXAMPLE 1: VECTOR DESIGNING Plasmid vector: pGTAAV 003 (pGT antiVEGF scFab)

The plasmid vector pGT AAV 003 (Figure- 1) consist of a vector backbone with a functional origin of replication for E. coli and Kanamycin as selection marker. The whole transcription assembly (Figure-2) was synthesized and cloned in this vector backbone by GenScript. The transcription assembly comprises of all the Cis regulatory elements namely ITR sequences at 5 ’and 3 ’end along with CB7 Promoter (which contains CMV, Immediate Early enhancer and chicken beta actin promoter), intron and Kozak sequence. Downstream of the Kozak sequence, the nucleotide sequence encoding human IL2 signal sequence and anti-VEGF scFab was placed along with SV40 Poly A transcription termination sequence. The construct was intended to produce a fully functional and secretory scFab polypeptide sequence. pGT AAV 003 expresses approximately ~55 kDa protein anti-VEGF scFab, which has a high affinity to multiple isoforms of VEGF-A and neutralizes binding of VEGF-A to its receptor. The amino acid sequence of antiVEGF-scFv was taken from product monograph of Ranibizumab (Fucentis ATC Code: S01FA04) which was back translated to DNA sequence and codon optimized for optimal expression in human. Packaging Plasmid vector: pIntas-ACG-R2C8

The packaging plasmid vector pIntas-ACG-R2C8 (Figure-4) comprises of a vector backbone with a functional origin of replication for E. coli and Ampicillin as selection marker. The transcription assembly (Figure-5) was generated by synthesizing 5’UTR (untranslated Region), Rep2 coding sequence, spacer sequence and 3’UTR (taken from AAV2 genome - NCBI Reference Sequence: NC_001401.2) along with AAV8 capsid coding sequence (taken from AAV8 genome - NCBI Reference Sequence: NC_006261.1) placed between Rep2 and 3’UTR. ACG was used as start codon instead of ATG. Packaging Plasmid vector: pIntas-ACG-R2C6

The packaging plasmid vector pIntas-ACG-R2C6 (Figure-6) comprises of a vector backbone with a functional origin of replication for E. coli and Ampicillin as selection marker.

The transcription assembly (Figure-7) was generated by synthesizing 5’UTR (untranslated Region), Rep2 coding sequence, spacer sequence and 3’UTR (taken from AAV2 genome - NCBI Reference Sequence: NC_001401.2) along with AAV6 capsid coding sequence (taken from AAV6 genome - Gen Bank Reference Sequence: AF028704.1) placed between Rep2 and 3’ UTR. Helper Plasmid : plntas Helper The sequences of E2A, E4 and VA helper genes belong to Adenovirus Type 5 and were taken from published sequence, GenBank: AF369965.1. The expression of the said genes is driven by inherent promoters. The vector map and transcription elements are shown in Figure-8 & 9 respectively. EXAMPLE 2: Transformation, Banking and Bulk DNA Preparation Transformation, Clone Selection and Banking The plasmid DNA were transformed in a suitable bacterial host strain most preferably E. coli DH5 alpha, ToplO and/or preferentially in Stbl3. Transformants obtained were scored over LB plates on the basis of antibiotic resistance as described in Table- 1. Plasmid DNA isolated from few such colonies was analyzed for the presence gene of interest using various restriction enzymes. One of the fully characterized clones was chosen to prepare glycerol stock.

Table- 1 List of Vectors with their selection marker. Bulk DNA Preparation

An ampule or vial of the glycerol stock was removed from -80°C freezer and inoculated in LB medium containing the same combination of antibiotic provided and described in Table- 1.

Cultures were allowed to grow overnight at 37 °C with shaking for 16 to 18 hours. Post 16 hours incubation temperature was shifted to 42°C with shaking for 4 to 6 hours (optional) and subsequently it was harvested in a centrifuge at maximum speed for time sufficient to pellet down the bacterial cells.

The plasmid DNA extraction was performed using miniprep or maxiprep or gigaprep according to the protocol of the kit.

The pellets were resuspended in resuspension buffer using a pipette followed by alkaline lysis, neutralization and column purification according procedure mentioned in the kit. EXAMPLE 3: EXPRESSION AND ACTIVITY OF pGT-AAV 003 VECTORS Single Plasmid Transfection In vitro anti-VEGF scFab binding to VEGF has been demonstrated and accepted widely in the prior arts. The vector pGTAAV 003 was transiently transfected in HEK 293 cells maintained in serum free media at 37° C, 8% CO2 and 125 RPM. The media supernatant was collected after 4-7 days and concentrated. Post concentration, supernatant was analyzed for VEGF binding using Octet System. Binding Analysis on Octet

Forte Bio Streptavidin (SA) biosensors (Cat no. 18-5019 Forte Bio) are suitable for immobilization of biotin labeled proteins, hence these biosensors were immobilized with Biotinylated VEGF (Cat no. 11066-H27H-B Sino Biological Inc.) and used for the interaction analysis of anti VEGF scFab present in cell culture supernatant to VEGF. The VEGF immobilized sensor tips and samples were loaded in Octet 365QK. The method was run in binding or kinetic mode to measure interaction between the VEGF and antiVEGF ScFab. Analysis software can quickly determine kinetic constants from binding data. Figure- 11 shows the resulting binding rates of the pGTAAV 003 samples in the experiment described above.

This functional assay measures not only the biological activity and functionality of human cell expressed antiVEGF scFab but also employs the functionality of vector pGTAAV 003.

EXAMPLE 4: rAAV8 antiVEGF scFab PRODUCTION Viruses packed with transgene was generally produced by co-transfection of HEK 293T cells with the AAV transgene plasmid, a helper plasmid encoding capsid protein for AAV serotype 8, and helper plasmid with adenovirus helper functions.

HEK 293 cells were propagated and maintained in in 1000 mL Shake Flask in a suitable mammalian cell culture media. The cells were seeded at 0.8 million/mL in 1000 mL Shake Flask with 300 mL media and maintained at 37° C, 125 rpm and 8% C02 for 48 hours or until the cell density reaches between 2.5 million/mL to 3.5 million/mL.

A triple co-transfection of HEK 293 cells was done with the AAV transgene plasmid (pGTAAV 003), packaging plasmid vector (pIntas-ACG-R2C8) plasmid encoding capsid proteins for AAV serotype 8 and Helper plasmid (plntas Helper) in the ratio of 1:1:2. The transfection of DNA was done using PEI-MAX reagent. Transfected HEK 293 cells were maintained at 37°C 125 RPM and 8% CO2 for 72 to 96 hours in shaker incubator. At the termination time culture was harvested and stored in -80° C till the purification.

EXAMPLE 5: rAAV6 antiVEGF scFab PRODUCTION

Viruses packed with transgene was generally produced by co-transfection of HEK 293T cells with the AAV transgene plasmid, a helper plasmid encoding capsid protein for AAV serotype 6, and helper plasmid with adenovirus helper functions.

HEK 293 cells were propagated and maintained in in 1000 mL Shake Flask in a suitable mammalian cell culture media. The cells were seeded at 0.8 million/mL in 1000 mL Shake Flask with 300 mL media and maintained at 37° C, 125 RPM and 8% C02 for 48 hours or until the cell density reaches between 2.0 million/mL to 3.5 million/mL

A triple co-transfection of HEK 293 cells was done with the AAV transgene plasmid (pGTAAV 003), packaging Plasmid vector (pIntas-ACG-R2C6) plasmid encoding capsid proteins for AAV serotype 6 and Helper plasmid (plntas Helper) in the ratio of 1:1:2. The transfection of DNA was done using PEI-MAX reagent. Transfected HEK 293 cells were maintained at 37° C 125 RPM and 8% CO2 for 72 to 96 hours in shaker incubator. At the termination time culture was harvested and stored in -80° C till the purification.

EXAMPLE 6: VECTOR GENOME ANALYSIS

Quantification of AAV genomes was done using Q-PCR with SV40 primers and probes. The Vector genome Quantification method comprises two parts i.e. sample processing and Q-PCR. Sample processing step was optimized for removal of unwanted plasmid and genomic DNA (non-encapsidated DNA) present in the sample using DNasel followed by Proteinase K treatment for removal of capsid and unbounding of packaged DNA in AAV particle. Post processing the samples were appropriately diluted and mixed with TaqMan® 2X universal PCR master Mix and Primer probe for SV40. Reagents and workflows were similar to those used for most standard TaqMan probe-based assays. PCR was performed as per standard format keeping the annealing temperature at 55°C.

EXAMPLE 7: CAPSID TITER ANALYSIS

The Capsid titer was determined by using a dilution series of sample in the Progen AAV8 Titration ELISA and Progen AAV6 Titration ELISA for rAAV8 antiVEGF scFab and rAAV6 antiVEGF scFab, respectively, against a standard curve prepared from pre-tittered rAAV8/rAAV6 standard preparation provided in the kit. The ELISA procedure was performed as per the instruction manual provided in the kit. The ELISA does not differentiate the empty or filled capsid but reports the total assembled capsid particles.

EXAMPLE 8: PURIFICATION OF rAAV8 antiVEGF scFab AND rAAV6 anti VEGF scFab Culture harvest from post transfection HEK293 cells (Example 4 and Example 5) was lysed and clarified by centrifugation followed by 0.2 pm filter. The AAV preparations were purified by methods using affinity chromatography, anion exchange chromatography, and CsCl or Iodixanol gradient purification. The purified product was characterized for Vector genome titer and Capsid titer by ddPCR and ELISA respectively.