DESC:RELATED APPLICATIONS

This application is related to Indian Provisional Application 202021025216 filed 16th 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 scFv, 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 (mAb) 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 fragment variable (scFv).

Another object of the present invention is to provide adeno associated virus (AAV) vector comprising nucleotide sequence of SEQ ID NO: 01 encoding anti-VEGF single-chain fragment variable (scFv).

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 scFv 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 scFv; 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. 01 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 scFv 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 scFv; and
(e) SV40 PolyA transcription terminator.

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:
a) Left ITR2;
b) CB7 Promoter;
c) Intron;
d) Signal peptide;
e) Anti-VEGF scFv;
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: 01 encoding:
a) Left ITR2;
b) CB7 Promoter;
c) Intron;
d) Signal peptide;
e) Anti-VEGF scFv;
f) SV40 Polyadenylation sequence; and
g) Right ITR2.

Another object of the present invention relates to recombinant AAV particle generated using AAV-001 expressing anti VEGF scFv of ~26 KD which has a higher affinity to VEGF.

Another object of the present invention is to provide AAV-001 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 scFv.

Another object of the present invention is to provide AAV-001 for method of treatment of wet age related macular degeneration (Wet AMD) using AAV vector genome comprising nucleotide sequence encoding anti-VEGF single-chain fragment variable (scFv).

Another object of the present invention is to provide AAV001 for method of treatment of Glioblastoma using AAV vector genome comprising nucleotide sequence encoding anti-VEGF single-chain fragment variable (scFv).

Another object of the present invention is to provide pharmaceutical formulation comprising:
(a) AAV vector genome comprising nucleotide sequence encoding anti-VEGF single-chain fragment variable (scFv); 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 fragment variable (scFv).

Another aspect of the present invention is to provide adeno associated virus (AAV) vector comprising nucleotide sequence of SEQ ID NO: 01 encoding anti-VEGF single-chain fragment variable (scFv).

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 scFv 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 scFv; 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. 01 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 scFv 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 scFv; 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) Left ITR2;
b) CB7 Promoter;
c) Intron;
d) Signal peptide;
e) Anti-VEGF scFv;
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: 01 encoding:
a) Left ITR2;
b) CB7 Promoter;
c) Intron;
d) Signal peptide;
e) Anti-VEGF scFv;
f) SV40 Polyadenylation sequence; and
g) Right ITR2.

Another aspect of the present invention relates to recombinant AAV particle generated using AAV001 expressing anti VEGF scFv of ~26 KD which has a higher affinity to VEGF.

Another aspect of the present invention is to provide AAV001 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 scFv.

Another aspect of the present invention is to provide AAV001 for method of treatment of wet age related macular degeneration (Wet AMD) using AAV vector genome comprising nucleotide sequence encoding anti-VEGF single-chain fragment variable (scFv).
Another aspect of the present invention is to provide AAV001 for method of treatment of Glioblastoma using AAV vector genome comprising nucleotide sequence encoding anti-VEGF single-chain fragment variable (scFv).

Another aspect of the present invention is to provide pharmaceutical formulation comprising:
(a) AAV vector genome comprising nucleotide sequence encoding anti-VEGF single-chain fragment variable (scFv); 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-AAV001) with Transgene and Regulatory Elements
Figure 2: Schematic Representation of AAV Gene expression Cassette (pGT-AAV001)
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 (pIntas-ACG-R2C8)
Figure 5: Schematic Representation of pIntas-ACG-R2C8 Packaging DNA Construct
Figure 6: Schematic Representation of Packaging Plasmid Vector Map (pIntas-ACG-R2C6)
Figure 7: Schematic Representation of pIntas-ACG-R2C6 packaging DNA Construct
Figure 8: Schematic Representation of Helper Plasmid Vector Map (pIntas-Helper)
Figure 9: Schematic Representation of pIntas-Helper Cassette
Figure 10A: Binding Curve Sensogram with Protein L
Figure 10B: Binding Curve Sensogram with VEGF
Figure 11: Octet Sensogram for binding of pGT001 Culture Supernatant with VEGF
Figure 12: Octet Sensogram Representing Brolucizumab Standard binding to VEGF at different concentration.
Figure 13: Fit Curve for Brolucizumab Standard determined from the Sensogram.

DETAILED DESCRIPTION OF THE INVENTION

The rAAV vectors are designed to express an anti-VEGF antibody single chain fragment variable (scFv) 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, Fab 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-l 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 fragment variable (scFv).

Another embodiment of the present invention is to provide adeno associated virus (AAV) vector comprising nucleotide sequence of SEQ ID NO: 01 encoding anti-VEGF single-chain fragment variable (scFv).

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 scFv 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 scFv; 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. 01 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 scFv 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 scFv; 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) Left ITR2;
b) CB7 Promoter;
c) Intron;
d) Signal peptide;
e) Anti-VEGF scFv;
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: 01 encoding:
a) Left ITR2;
b) CB7 Promoter;
c) Intron;
d) Signal peptide;
e) Anti-VEGF scFv;
f) SV40 Polyadenylation sequence; and
g) Right ITR2.

In certain embodiments, the processing of anti-VEGF scFv is directed by leader peptides that are derived from human IL2 protein. In one embodiment the leader sequence is an interleukin (IL) IL-2 leader sequence, which may be the wild-type human IL2 or a mutated human IL2. Such leader sequences are known in the art. For example, UniProtKB/Swiss-Prot Accession Number: P60568.

Another embodiment of the present invention relates to recombinant AAV particle generated using AAV001 expressing anti VEGF scFv of ~26 KD which has a higher affinity to VEGF.

Another embodiment of the present invention is to provide AAV001 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 scFv.

Another embodiment of the present invention is to provide AAV001 for method of treatment of wet age related macular degeneration (Wet AMD) using AAV vector genome comprising nucleotide sequence encoding anti-VEGF single-chain fragment variable (scFv).

Another embodiment of the present invention is to provide AAV001 for method of treatment of Glioblastoma using AAV vector genome comprising nucleotide sequence encoding anti-VEGF single-chain fragment variable (scFv).

Another embodiment of the present invention is to provide pharmaceutical formulation comprising:
(a) AAV vector genome comprising nucleotide sequence encoding anti-VEGF single-chain fragment variable (scFv); and
(b) a pharmaceutically acceptable carrier, diluents, excipients, or buffer.

As used herein, the amino acid sequence of antiVEGF-scFv was taken from KEGG DRUG database (Entry: D11083, Name: Brolucizumab) and BLA of Brolucizumab (Application number: 761125) which are already known in the prior art.

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 2001091800. 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 mAb 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-AAV001, 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 ELISA and Octet based systems. Two constructs (i.e. pGT-AAV001 (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 comparative binding curve sensogram with protein L and VEGF respectively. Based on these binding data, Inventors ranked vector candidates in the following order: AAV001>AAV003>AAV002 and AAV001 was selected for the future development.

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

1. Plasmid Vector: pGTAAV 001 (pGT antiVEGF scFv)
The plasmid vector pGT AAV001 as shown in Figure-1 comprises of a vector backbone with a functional origin of replication for E. coli and Kanamycin as selection marker. The whole transcription assembly as shown in 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 corresponding amino acid sequence having and nucleotide sequence of anti-VEGF scFv corresponding to amino acids sequence of was placed along with SV40 PolyA transcription termination sequence. The construct was intended to produce a fully functional and secretory scFv polypeptide sequence.

pGTAAV 001 expresses approximately ~26 kDa protein anti-VEGF scFv, which has a very high affinity to multiple isoform of VEGF-A and neutralization of its binding to its receptor. It is a humanized single chain variable fragment. The amino acid sequence of antiVEGF-scFv was taken from KEGG DRUG database (Entry: D11083, Name: Brolucizumab) which was back translated to DNA sequence and codon optimized for optimal expression in human.

2. Packaging Plasmid Vector: pIntas-ACG-R2C8
The packaging plasmid vector pIntas-ACG-R2C8 as shown in Figure-4 comprises of a vector backbone with a functional origin of replication for E. coli and Ampicillin as selection marker.

The transcription assembly as shown in 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.

3. Packaging Plasmid Vector: pIntas-ACG-R2C6
The packaging plasmid vector pIntas-ACG-R2C6 as shown in Figure-6 comprises of a vector backbone with a functional origin of replication for E. coli and Ampicillin as selection marker.
The transcription assembly as shown in 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.

4. Helper Plasmid : pIntas Helper
The sequences of E2A, E4 and VA helper genes belong to Adenovirus Type 5 and were taken from published sequence, GenBank accession: AF369965.1. The expression of the said genes is driven by inherent promoters. The vector map and transcription elements are shown in Figure-8 & Figure-9 respectively.

EXAMPLE 2: TRANSFORMATION, BANKING AND BULK DNA PREPARATION

1. Transformation, Clone Selection and Banking
The plasmid DNA was transformed in a suitable bacterial host strain most preferably E. coli. DH5 alpha, Top10 and/or preferentially in Stbl3 or Stbl4.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 Selection Marker
Vector Name Selection marker in E. coli
pGT-AAV001 Kanamycin
pIntas-ACG-R2C8 Ampicillin
pIntas-ACG-R2C6 Ampicillin
pIntas-Helper Ampicillin
.
2. Bulk DNA Preparation
An ampule or vial of the glycerol stock was removed from -80° C 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 shift was done at 42º 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 001 VECTORS

1. Single Plasmid Transfection
In - Vitro anti-VEGF scFv binding to VEGF has been demonstrated and accepted widely in the prior arts. The vector pGTAAV001 was transiently transfected in HEK 293 cells maintained in serum free media at 37 ºC, 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 on Octet System.

2. 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 antiVEGF scFv present in cell culture supernatant to VEGF. The VEGF immobilized sensor tips and samples were loaded in Octet 365QK. The interaction and run in binding or kinetic mode to measure interaction between the VEGF and antiVEGF ScFv. Analysis software can quickly determine kinetic constants from binding data. Figure-11 shows the resulting dissociation rates of the pGTAAV001 samples in the experiment described above. This demonstrates the difference in binding affinity of Anti-VEGF ScFv sample and Ranibizumab to VEGF using octet. It was observed that Anti-VEGF ScFv has a higher affinity to VEGF as compared to Ranibizumab.

This functional assay measures not only the biological activity and functionality of human cell expressed antiVEGF scFv but also employs the functionality of vector pGTAAV001.

EXAMPLE 4: rAAV8 antiVEGF scFv 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 proteins for AAV serotype 6 or 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% CO2 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 001), packaging Plasmid vector (pIntas-ACG-R2C8) plasmid encoding capsid proteins for AAV serotype 8 and Helper plasmid (pIntas 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 -80º C till the purification.

EXAMPLE 5: rAAV6 antiVEGF scFv PRODUCTION

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% CO2 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 001), packaging Plasmid vector (pIntas-ACG-R2C6) plasmid encoding capsid proteins for AAV serotype 6 and Helper plasmid (pIntas 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

Quanti?cation of AAV genomes was done using gene specific primers and probes on Droplet Digital PCR (ddPCR). ddPCR is a method for performing digital PCR which is based on water-oil emulsion droplet technology. A sample is fractionated into 20,000 droplets, and PCR amplification of the template molecules occurs in each individual droplet. Some of these reactions contain the target molecule (positive) while others do not (negative) which is quantified using droplet reader.

The Vector genome Quantification method comprises two parts i.e. sample processing and droplet generation and reading. Sample processing step was optimized for removal of unwanted plasmid and genomic DNA (non-encapsidated DNA) present in the sample using DNase1 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 ddPCR™ Super mix for probes - no dUTP and Primer probe for antiVEGF.

Reagents and workflows were similar to those divides used for most standard TaqMan probe-based assays. Droplet generator PCR samples into water-in-oil droplets generating approximately 20000 droplets. PCR amplification of the droplet was performed as per standard format keeping the annealing temperature of 61° C. Following PCR, each droplet was read to determine the fraction of PCR-positive droplets in the original sample. These data were then analyzed using poisson statistics to determine the target DNA template concentration in the original sample.

EXAMPLE 7: CAPSID TITER ANALYSIS

The Capsid titer was determined by using a dilution series of sample in the Progen AAV8 Titration ELISA, against a standard curve prepared from pre-tittered rAAV8 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 scFv AND rAAV6 anti VEGF scFv

Culture harvest from post transfection HEK293 cells (Example 4 and Example 5) was lysed and clarified by centrifugation followed by 0.2µ filter. The AAV preparation were purified by methods using affinity chromatography, anion exchange chromatography, CsCl or Iodixanol gradient purification. The purified product was characterized for Vector genome titer and Capsid titer by ddPCR and ELISA respectively.

EXAMPLE 9: IN-VITRO EVALUATION OF rAAV8 antiVEGF scFv and rAAV6 antiVEGF scFv

ARPE 19 Cell line (ATCC No. CRL-2302) was propagated and maintained in T75 flask in DMEM media containing 10% FBS. At more than 90% confluency of the T75 flask the cells were trypsinised and plated in a 24 well plate. The 24 well plate was incubated till more than 90% confluency in each well. The cells were then transduced with rAAV8 antiVEGF scFv and rAAV6 antiVEGF scFv purified product diluted in DMEM F12 and in the presence of Etoposide. 16 hours post transduction the media was changed with DMEM F12 with 10% FBS (complete media). 72 hours post media change the supernatant was collected and stored at – 20° C. These samples were analyzed using octet for the post transduction expression of scFv in the media supernatant. (Table-2 & 3)

Table-2: Binding Rate of Brolucizumab Standard with VEGF Determined by Octet
Sr. No. Sample ID Type Binding Rate Known Conc. (µg/ml) Calc Conc. (µg/ml) Residual (%) R2
1 Brolucizumab Standard Standard 0.2665 20 20 0.22 0.999
2 Brolucizumab Standard Standard 0.1931 15 14.9 -0.86 0.999
3 Brolucizumab Standard Standard 0.1249 10 10.1 1.17 0.998
4 Brolucizumab Standard Standard 0.0528 5 5.04 0.79 0.998
5 Brolucizumab Standard Standard 0.0155 2.5 2.31 -7.4 1.000
6 Brolucizumab Standard Standard 0.0017 1.25 1.25 0 0.998
7 1XPBS Reference 0 N/A 0 N/A 0.000

Table-3: Post Transduction antiVEGF scFv Expression Determined by Octet.
Sample Detail
MOI* Post Transduction
antiVEGF scFv Expression (µg/ml)
AAV8 antiVEGF scFv 1.20E+05 5.9
6.00E+04 5.65
AAV6 antiVEGF scFv 1.20E+05 9.1
6.00E+04 8.15 ,CLAIMS:We Claim,

1. An adeno associated virus (AAV) vector comprising nucleotide sequence encoding anti-VEGF single-chain fragment variable (scFv).

2. The AAV vector according to claim 1, having nucleotide sequence of SEQ ID NO: 01.

3. The AAV vector according to claim 1, for use in the method of treatment of wet age related macular degeneration (Wet AMD) and glioblastoma.

4. A recombinant AAV particle comprising nucleotide sequence of SEQ ID NO: 01 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 scFv;
(c) An inducible/switchable/regulatable control element;
(d) An anti-VEGF scFv; and
(e) SV40 PolyA transcription terminator.

5. The recombinant AAV particle according to claim 4, wherein expression control element is CB7 promoter.

6. A 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) Left ITR2;
(b) CB7 Promoter;
(c) Intron;
(d) Signal peptide;
(e) Anti-VEGF scFv;
(f) SV40 Polyadenylation sequence; and
(g) Right ITR2.

7. The recombinant adeno-associated virus (rAAV) vector according to claim 6, having nucleotide sequence of SEQ ID NO: 01.

8. The recombinant adeno-associated virus (rAAV) vector according to claim 6, wherein AAV capsid is selected from AAV6 and AAV8.

9. The recombinant adeno-associated virus (rAAV) vector according to claim 6, for use in the method of treatment of wet age related macular degeneration (Wet AMD) and glioblastoma.

10. A pharmaceutical formulation comprising:
(a) AAV vector genome comprising nucleotide sequence encoding anti-VEGF single-chain fragment variable (scFv); and
(b) A pharmaceutically acceptable carrier, diluents, excipients, or buffer thereof.