WO2020070619A2 - Procédé de génération de vecteurs aav glycomodifiés pour une thérapie génique hépatique et oculaire, et produit associé - Google Patents

Procédé de génération de vecteurs aav glycomodifiés pour une thérapie génique hépatique et oculaire, et produit associé

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WO2020070619A2
WO2020070619A2 PCT/IB2019/058288 IB2019058288W WO2020070619A2 WO 2020070619 A2 WO2020070619 A2 WO 2020070619A2 IB 2019058288 W IB2019058288 W IB 2019058288W WO 2020070619 A2 WO2020070619 A2 WO 2020070619A2
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aav2
primer
vectors
aav
seq
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WO2020070619A3 (fr
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Jayandharan Giridhara Rao
Bertin Mary
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Indian Institute Of Technology Kanpur
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0075—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the delivery route, e.g. oral, subcutaneous
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0381—Animal model for diseases of the hematopoietic system
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention is related to hepatic and ocular gene therapy, more particularly it relates to a process for synthesising glycoengineered adeno- associated virus vectors and the glycoengineered adeno-associated virus vectors for hepatic and ocular gene therapy.
  • AAV2 adeno-associated virus
  • AAV2 capsid-specific T cell response was documented that coincided with a rise in liver transaminases and a drop in FIX transgene expression to baseline levels.
  • This CD8+ T cell- mediated immune response was unexpected, as this had not been observed in any pre-clinical animal models.
  • serotypes including AAV5 and AAV8 have been successfully employed for gene therapy of hemophilia B
  • AAV2 is the widely studied serotype (>6.9% of gene therapy trials with AAV vectors) and it’s unrestricted use in clinical space has been widely beneficial to investigators in gene therapy field.
  • strategies to improve transduction of AAV2 vectors in the liver and to attenuate capsid-or transgene specific immune responses would be beneficial to achieve long-term liver directed gene transfer of FIX.
  • AAV2 mediated retinal gene therapy for the treatment of LCA2 has been promising, with substantial clinical benefit seen in more than 30 patients treated in several human trials.
  • LCA is a group of retinal degenerative diseases, that are caused by mutations in >15 different genes and leads to profound impairment in visual function with delayed degeneration of retinal cells.
  • LCA2 caused by mutations in the retinal pigmental epithelium (RPE)65 gene is an ideal target for AAV2 mediated gene transfer, because this form of the disease accounts for -10% of disease in humans, the molecular genetics and functions of the RPE65 gene is well understood and there are several animal models, in which the novel therapies can be tested.
  • a process for synthesising glycoengineered adeno-associated virus vectors includes generating AAV vectors in presence of a group of glycosylation modulators.
  • the process also includes predicting antigenicity of AAV2 capsid protein of the AAV vectors using B-cell and T-cell linear epitope mapping tools in order to refine epitopes of the AAV capsid protein.
  • the process further includes targeting the refined epitopes for site-directed mutagenesis, while using a set of primers, by introducing or abolishing N- and O- glycosylation motifs in the AAV capsid to obtain the plurality of glycoengineered adeno-associated virus (AAV) vectors.
  • AAV glycoengineered adeno-associated virus
  • the site-directed mutagenesis of target amino acids of the AAV capsid comprises N ⁇ Q, Q ⁇ N, S- A, A- T, E/K/Q/R- T and Y/S/T ⁇ N.
  • a glycoengineered adeno-associated virus vectors for hepatic and ocular gene therapy is provided.
  • AAV-2 based gene therapy for diseases such as hemophilia B or Leber congenital amaurosis type 2 (LCA2) has been promising in several clinical trials.
  • AAV Adeno-associated virus
  • LCA2 Leber congenital amaurosis type 2
  • the VP1 region of AAV2 capsid was further analyzed to predict the T- cell and B-cell immunogenic epitopes as well sites of potential glycosylation. Based on this analysis, a total of twenty four glycosylation sites were selected in and around the T-cell and B-cell epitopic regions and further mutagenesis was performed with an aim to create or disrupt a potential glycosylation site within the immunogenic motifs.
  • AAV2-EGFP vectors containing the wild-type (WT) and each one of the 24 glyco-engineered mutants were then evaluated for their transduction efficacy in multiple cell lines (He La, Huh7, ARPE19) in vitro. It was observed a 1.3 to 2.5 fold increase with at least three mutants in all the three cell types.
  • retinal gene transfer of these glycoengineered vectors through intravitreal administration demonstrated an enhanced EGFP expression ⁇ 2 - 14 fold for AAV2- Q259N and AAV2 N705Q vectors and a phenotypic rescue with AAV2-T14N and AAV2- Q259N vectors containing RPE65 gene in a murine mdel of retinal degeneration (rdl2 mice).
  • the present disclosure demonstrates the role of glycosylation during AAV mediated gene transfer and highlights the improved translational potential of these glycoengineered AAV2 vectors for hepatic and ocular gene therapy.
  • FIG. 1 is an assay depicting deglycosylation of fetuin (positive control for glycosylated protein) and lysozyme (negative control for glycosylated protein) after glycosidase enzymatic treatment in the context of the present invention
  • FIG. 2 denotes the representative gating procedure to count CD3+ lymphocytes in the context of the present invention
  • FIG. 3A depicts MALDI-TOF spectra for N and O-linked glycan profiling in AAV2 capsid protein: Spectra obtained for AAV2 capsid after PNGase F digestion for N-linked glycans in the context of the present invention;
  • FIG. 3B depicts MALDI-TOF spectra for N and O-linked glycan profiling in AAV2 capsid protein: Spectra obtained for AAV2 capsid after permethylation, in the context of the present invention
  • FIG. 4 depicts Enzymatic characterization of glycosylation in AAV2 capsid: 4A) Western blot of AAV2 vectors with Bl antibody displays bands corresponding to untreated (lst lane), reaction buffer control (2nd lane) and AAV2 treated with deglycosidase. A decrease in band intensity is noticed in deglycosylated AAV2 sample [bold arrow]; 4B) Western blot of AAV2 vectors with Bl antibody displays bands corresponding to untreated (I st lane), deglycosylated AAV2 (2nd lane) and reaction buffer control (3rd lane). 4B & 4D) Quantification data from 4A and 4C, respectively. Band intensities for test conditions were compared to that of the untreated AAV2. Error bars indicate SD from two independent measurement by two different users from two biological replicates, * p ⁇ 0.05 vs untreated control;
  • FIG. 6 depicts effect of cellular glycosylation modulators on AAV2 packaging.
  • FIG. 6A scAAV2 vectors were packaged in AAV293 cells pre-treated with (and during transfection) or without glycosylation modulators. Seventy-two hours post transfection of the packaging plasmids, viral particles were purified and titrated by a quantitative PCR. The data depicted are mean of two independent titration assays with polyA specific primers. The viral titers obtained after packaging are ploted as vector genomes/ml and are shown in log scale; FIG. 6B A similar assay performed in AAV293 cells which were treated with the drugs 6 hours post triple plasmid transfection. * p ⁇ 0.05 vs. titers obtained under the absence of glycosylation modulators;
  • FIG. 7 depicts potency of AAV2 vectors packaged in the presence of glycosylation modulator.
  • FIG 7A Transgene expression from scAAV2 vectors packaged in AAV293 cells and pre-treated with cellular glycosylation modulators at a dose of 5000 MOI in HeLa cells was assessed. EGFP expression was quantified by flow cytometry and shown as percentage of GFP positive cells.
  • FIG. 8 is a Schematic representation of AAV2 VP1 capsid with sites of N-linked glycosylation motifs (Blue font) and O-linked glycosylation motifs (Green font);
  • FIG. 9 depicts the sequence of the AAV2 capsid protein.
  • Targets for glycosylation site modification within the putative immune epitopes of AAV2 capsid Sequence of the AAV2 capsid protein VP1 region with the predicted B- cell and T-cell immune epitope regions (red font), loop regions (highlighted in yellow) and endogenous N-glycosylation motif (blue font- N-X-S/T residues) and O-glycosylation motif (green font— S/T residues) is shown.
  • FIG. 10 depicts transduction efficiency of AAV2 glycoengineered mutants in human cells in vitro.
  • HeLa cells were either mock-infected or infected with 5x103 vgs/cell of AAV2-WT or AAV2-glycoengineered mutants
  • FIG. 10A and cells analyzed for EGFP expression 48hrs later by flow cytometry. The percentage EGFP positive cells post-transduction are shown.
  • FIG. 11 is graph plot for neutralisation antibody assay.
  • the transduction potential of AAV2-WT vector expressing EGFP was assessed in the presence of different concentrations of intravenous immunoglobulin (IVIG).
  • IVIG intravenous immunoglobulin
  • the NAb titer is the highest IVIG dilution that inhibited AAV transduction of HeLa cells by 50% or more compared with that of the cells infected with AAV2 alone;
  • FIG. 12 depicts in-vitro neutralization assay with glycoengineered AAV2 mutants in the presence or absence of intravenous immunoglobulin (IVIG).
  • FIG 12A scAAV2-WT or mutant vectors were pre-incubated with IVIG at a dilution of 1:256 and assessed for their transduction in HeLa cells, 48 hours later. The data depicted is the representation of one independent experiment with three replicates.
  • FIG. 15 depicts Glyco-engineered AAV2 vectors demonstrate improved hepatic gene transfer of FIX in a murine model of hemophilia B.
  • Plasma levels of human FIX was measured at 4 weeks, 10 weeks and 12 weeks after hepatic gene therapy. *p ⁇ 0.05 between AAV2-WT Vs.
  • AAV2-T14N injected mice
  • FIG. 16F Number of spots generated by 1x106 splenocyte cells stimulated by AAV2 capsid specific peptide in ELISPOT assay
  • FIG. 16G Representative images of the treatment groups in ELISPOT plate. Data are shown as mean ⁇ SD of the number of spots obtained from splenocytes seeded in duplicate wells for each of the five mice per group. P values were not significant (p>0.05) Vs. WT-AAV2 injected hemophilia B mice;
  • the fold difference in mean GFP intensity is provided in comparison to scAAV2-WT at 2 and 8 weeks. *p ⁇ 0.05 Vs. WT-AAV2 injected mice;
  • FIG. 18 depicts Immunohistochemical analysis of retina from C57BL/6 mice injected with AAV2 vectors. Retinal sections were probed by immunohistochemistry at 16 weeks after intravitreal administration of either PBS or scAAV2-WT or scAAV2-Tl4N mutant vector containing EGFP.
  • GCL ganglion cell layer
  • ONL outer nuclear layer
  • OS outer segment
  • RPE retinal pigmented epithelium.
  • FIGs. 19A-D show the retinal permeation characteristics of AAV2-T14N vector packaged in the presence of Tunicamycin, in glycosylation inhibited condition (2x10 8 vgs/eye two weeks post gene delivery) or after treatment with glycosidase enzyme (3x10 8 vgs/eye four weeks post gene delivery) in an aspect of the invention.
  • FIGs. 20A-D depict visual function rescue in rdl2 mice by AAV2 T14N mutant in an aspect of the invention.
  • FIGs. 21 A & B depicts visual function in rdl2 mice after administration of AAV2 Q259N vectors in an aspect of the invention.
  • AAV glycoengineered adeno-associated virus
  • the AAV used herein is AAV serotype 2 (AAV2).
  • the AAV used herein is selected from a group consisting AAV serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 and other AAV variants thereof.
  • the process of for synthesising a plurality of glycoengineered adeno- associated virus (AAV) vectors includes generating AAV vectors in presence of a group of glycosylation modulators.
  • the process also includes predicting antigenicity of AAV2 capsid protein of the AAV vectors using B-cell and T-cell linear epitope mapping tools in order to refine epitopes of the AAV capsid protein.
  • the process further includes targeting the refined epitopes for site- directed mutagenesis, while using a set of primers, by introducing or abolishing N- and O- glycosylation motifs in the AAV capsid to obtain the plurality of glycoengineered adeno-associated virus (AAV) vectors.
  • the site-directed mutagenesis of target amino acids of the AAV capsid comprises N AQ. QAN. SAA, A AT. E/K/Q/RAT and Y/S/TAN.
  • the generation of AAV vectors includes detecting a N-glycan combinations and O-glycans in AAV capsid protein; performing one or more enzymatic deglycosylation assays for a plurality of AAV vectors using PNGase F to analyze N-glycans and a deglycosylation enzyme mix in order to characterize extent of glycosylation on the capsid; and assessing effect of pharmacological modulation of the glycosylation on the capsid on AAV transduction.
  • the present invention provides for an AAV2 vector that has high transduction in ocular and hepatic cells during gene therapy.
  • generation of glycosylation site modified AAV2 capsids is done to improve outcome of gene therapy.
  • the present disclosure provides for designing so as to optimize some of the features that are required from the currently available AAV2 based vectors, for potential gene therapy of hemophilia B and LCA2.
  • the AAV2 capsid protein which constitutes 74% of the vector is the source of foreign antigen during gene therapy but is an essential component for host cell receptor recognition, intracellular trafficking, genome release and T-and B-cell recognition.
  • AAV2 mutant vectors (tyrosine or serine, threonine or lysine) which can bypass intracellular phosphorylation and subsequent proteosomal capsid degradation had shown significantly higher transgene expression after hepatic or ocular gene transfer.
  • tyrosine mutants of AAV2 demonstrated 30-fold increase in FIX expression in murine haemophilia B model.
  • Mutant AAV2/2(7m8) generated by directed evolution strategy exhibited strong transduction potential for photoreceptors upon intra-vitreal administration in the murine rdl model, macaque and human retinal explants.
  • the present invention addresses the paucity of data on other post translational modification (PTM) modifications in AAV capsids.
  • PTM post translational modification
  • AAV1 and AAV6 utilize sialic acid (SA)
  • AAV2 utilize heparan sulfate proteoglycan (HSPG)
  • AAV9 use N-linked glycan as primary receptors for attachment on the cell surface. It is also plausible that the virus which is 20 nm in size and measures 3.9 MegaDaltons in molecular weight, is glycosylated at extremely low levels in a cell specific manner, which is difficult to be detected by standard protein-detection based techniques.
  • the present invention focuses on study of the role of cellular glycosylation in AAV2 life cycle, by use of an expanded panel of glycosylation modulators and assessed their impact on transduction and production efficiency. Perturbation of putative T-cell and B-cell recognition epitopes and imparting or deleting glycosylation targets within these regions in AAV2 capsid may lead to the generation of efficient AAV2 vectors.
  • HeLa Human cervical carcinoma cell line
  • Huh7 hepatic carcinoma cell line
  • AAV293 AAV packaging cell line
  • ATCC American Type Culture Collection
  • ARPE 19 cell line was acquired from Dr.
  • AAV-293 cells grown in forty numbers of l50mm 2 dishes were transfected with three plasmids namely, AAV2 rep/cap, pHelper (AAV helper gene) and either one of the transgene containing plasmid (pdsAAV2-CBA-EGFP or pdsAAV-LPl-hFIX) in the presence of 0.1% PEI (Polysciences Inc., Taipei, Taiwan) at a 1 : 1 ratio.
  • PEI Polysciences Inc., Taipei, Taiwan
  • Cells were harvested after 72 hrs, lysed and treated with benzonase (25 units/ml; Sigma-Aldrich).
  • Vectors were further purified by iodixanol gradient based ultracentrifugation (OptiPrep, Sigma- Aldrich) and by column chromatography (HiTrap SP column; GE Healthcare Life Sciences, Pittsburgh, PA). Purified vectors were concentrated using Amicon Ultra 10K centrifugal filters (Millipore, Bedford, MA) to a final volume of 0.5ml. The purified vectors were quantifed by qPCR based assay using polyA region specific primer after DNase treatment (Sigma-Aldrich) as described elsewhere. The vector titers are expressed as vector genomes (vg)/ ml.
  • FIG. 1 depicts the assay where Feutin and lysozyme were used in this assay as reagent optimization controls. Fetuin and lysozyme (30 pg each) were used as positive and negative controls for deglycosylation assay.
  • control glycosylated protein fetuin showed altered band size 1 hour and 3 hours after digestion with PNGase F and deglycosylation mix.
  • the non- glycosylated protein lysozyme did not show a band shift or change in band intensity, 1 hour and 3 hours after digestion with PNGase F and deglycosylation mix
  • Table 1 List of glycosylation modulators used in cell lines during vector transduction and packaging
  • AAV2 vectors were generated in the packaging cell line, AAV293 as described above but in 20 numbers of l5cm 2 dishes in the presence or absence of pharmacological modulators of glycosylation.
  • Two sets of packaging experiments were performed to understand the efficiency and dynamics of glycosylation modulation during the packaging process. In the first set of packaging experiments, the AAV293 cells were pre-treated with glycosylation modulators prior to triple transfection with plasmid vectors.
  • scAAV2-EGFP vectors were packaged in mock treated cells or in AAV 293 cells pre-treated with tunicamycin (0.125 pg/ml) or all-trans- retinoic-acid (1 mM) for 24 hours and 48 hours respectively. Seventy- two hours post-transfection, cells were harvested, purified and the titers were estimated. In the next set of studies, the AAV293 cells were treated with glycosylation modulators 6 hours after intial step of transfection of plasmids.
  • transfected AAV-293 cells were treated with Alloxane for 12 hours to create cellular O-glycosylation inhibited condition and another set of twenty transfected plates were treated with N-linked glycosylation inhibitor (Tunicamycin) for 24 hours. Twenty transfected plates were maintained as a mock control without any glycosylation modulator treatment. Cell were harvested after 72 hours and viral purification and estimation by qPCR was performed.
  • Antigenicity of the AAV2 capsid protein was predicted using B-cell and T-cell linear epitope mapping tools, IEDB resources server (http://tools.iedb.org/bcell/. http://tools.iedb.org/main/tcell/).
  • the invention further refined the prediction results, with experimentally validated epitopes published previously, to define targets for glycosylation modifications using NetNGlyc (http://www.cbs.dtu.dk/services/NetNGlyc ) and MotifScan (htps://mvhits.isb-sib.ch/cgi-bin/motif scan) tools.
  • Table 2 List of primers used for glycoengineered AAV2 mutant generation. The nucleotides which are mutated in reference to wildtype AAV2 DNA sequence [NC_00l40l.2] are in bold face.
  • an infectivity assay was performed in three different cell lines namely: HeLa, Huh7 and ARPE19. Approximately, 3xl0 4 cells in culture were infected with 5xl0 3 vgs/cell of scAAV2-EGFP (WT) or AAV2 glycoengineered mutant vectors. Forty-eight hours post-infection, GFP expression was quantified by flow cytometry (BD Accuri C6 Plus). Two independent experiments were performed including three intra-assay replicates in each of the experiment. Mean of percentage cells expressing EGFP from six replicate samples were used for comparison between AAV2 WT and AAV2 glycomutants.
  • Huh7 cells were grown overnight in a 24 well plate and infected with either AAV2-WT or glycoengineered mutant vectors expressing human coagulation (h)FIX at a MOI of 5xl0 4 vgs. Forty-eight hours later, total RNA was extracted from the cells using TRIzol reagent (Ambion, Fife technologies, Carlsbad, USA). About lpg of RNA was reverse transcribed using Verso cDNA synthesis kit (Thermo Scientific, Vilnius, Fithuania). Quantitative PCR for hFIX was done in a CFX96 real-time system (Biorad, Hercules, California, USA) using the primers detailed in Table 3. Normalization of data was perfbmed with b-actin as an endogenous control.
  • mice All animal studies were performed after approval by Institute Animal Ethics Committee at Indian Institute of Technology, Kanpur, India. Hepatic gene transfer studies involved the use of Hemophilia B (B6. 129P2-/ "" //Jl 7J) mice and ocular gene transfer was perfomed in C57BL/6 mice. Hemophilia B and C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbour, ME, USA). Studies were performed on mice housed at 22°C in ventilated cages with free access to food and water.
  • Hemophilia B mice (1 to 5 months) were injected intravenously with either PBS (mock) or scAAV2-LPl-hFIX or scAAV2-Tl4N -LPl-hFIX vectors at a dose of 5xl0 10 vgs per animal. Five animals were used in each of these groups. Blood samples in 3.8% citrate buffer were collected from retro-orbital plexus at 4, 10 and 12 weeks after gene transfer to measure plasma FIX antigen levels by enzyme linked immunosorbent assay (EFISA) as per manufacturer's protocol (Asserachrom IX: Ag kit, Diagnostica Stago, France).
  • EFISA enzyme linked immunosorbent assay
  • AAV2-FIX gene delivery protocol assessed the T cell, B cell and Treg population in animals that had hepatic gene therapy.
  • Peripheral blood was collected from mock or treated hemophilia B mice 12 weeks after gene transfer. Red blood cells were lysed with lysis buffer (l55mM NH 4 Cl, l2mM NaHC0 3 & O. lmM EDTA), pelleted at 250g for 5 mins, washed resuspended in 1XPBS. The samples were then were incubated with FITC labeled anti-CD3, PE-labelled anti-CD8, PerCP labeled anti-CD4, and APC labeled anti-CD 19 antibodies for 30 mins at RT.
  • lysis buffer l55mM NH 4 Cl, l2mM NaHC0 3 & O. lmM EDTA
  • CD3+, CD4+, CD8+ and CD 19+ cells were assessed by flow cytometry (BD Accuri C6 Plus). This data was used to enumerate B-cells (CD 19+) and the the double positive markers among the CD3+ population including, CD4 helper cells (CD3+ CD4+), CD8 cytotoxic cells (CD3+CD8+) in each of the AAV2 vector or PBS administered mice.
  • the representative gating procedure to count CD3+ lymphocytes are denoted in FIG. 2.
  • the Fymphocytes population were dated from PBMCs and CD3+ cells were counted, further CD3+ gate was applied to count CD3+ CD4+ (helper T-cells) CD3+ and CD8+ (Cytotoxic cells) in a FACS analyser [BD Accuri Plus, BD Biosciences]
  • Treg population in mouse splenocytes lxlO 6 cells were stained with PerCP labeled anti-CD4 and APC labeled anti- CD25 antibodies for 30 mins at RT. Stained cells were washed, fixed and permeabilized using mouse Foxp3 buffer set (BD Pharminogen), and further stained with PE-conjugated Foxp3 antibody for 30 mins at RT. Flow cytometry was done to assess the Treg (CD4+ CD25+ Foxp3+) cell population count.
  • mice splenocytes were isolated from the spleen of treated and control animals and subjected to RBC lysis. The viability of the cells were checked by Trypan blue assay. About lxlO 6 cells in IMDM were seeded per well in 96 well IFN-g antibody pre-coated ELISPOT plate (MabTech, Ohio, USA). Subsequently, cells were stimulated with 2 pg/mL of AAV2 serotype T-cell epitope specific peptide (SNYNKSVNV) (JPT Peptide Technologies, GmbH, Germany) overnight and the ELISPOT assay was performed as per the manufacturer' s protocol.
  • SNYNKSVNV AAV2 serotype T-cell epitope specific peptide
  • Concanavalin A (2 pg/mL) was used as positive control for the assay. Plates were incubated for 24hrs and developed using BCIP/NBT. Spot forming units and the images of the wells were captured in an ELISPOT reader (AID reader, GmbH, Germany).
  • Fluorescence imaging was performed after 2 weeks and 8 weeks of vector administration in a Micron IV imaging system as per manufacturer’s instructions (Phoenix Research Lab, Pleasanton, USA). Intensity was set at maximum and gain was set at 15 db , the frame rate was set at 6 fps for imaging of all the groups.
  • EGFP transgene
  • AAV2-T14N vectors expressing EGFP at dose of 3xl0 8 vgs was treated with 250U of a glycosidase enzyme (PNGase F, New England Biolabs, Ipswich, MA, USA) overnight at 37°C.
  • murine eyes were eunucleated and retinal sections prepared. Confocal imaging was performed to assess the transduction and permeation of the vectors after treatment with the glycosidase. Animals administered with only AAV2-T14N vectors were included as experimental controls.
  • lxlO 8 vgs or 7xl0 8 vgs/eye of wild-type or glycosylation site modified AAV2 vector (AAV2-Q259N or AAV2-T14N, respectively) expressing hRPE65 were administered via the subretinal route into groups of 8 weeks old rdl2 mice. Subretinal injections were carried out by the following procedure. The comeal-scleral junction at the limbus was pricked with a beveled 31G needle, releasing the pressure. Care was taken not to injure the cornea and lens. A 33G blunt needle attached to a Hamilton micro syringe was introduced through the aperture at the cornea-scleral junction and it was taken across the vitreous to the retina.
  • the retina/RPE junction was reached by analysing the back pressure and the volume injected in a jet into the space without exerting pressure on the needle or the RPE.
  • a fundus shot was taken post-surgery to check if there was any detachment.
  • a detached bleb signified successful subretinal administration.
  • Scotopic electroretinography was further carried out 6, 10 or 32 weeks later to study the gain in visual physiology. Mice were dark adapted overnight to completely flush any residual photo-transduction activity. Animals were anaesthetized and pupil were dilated with tropicamide/phenylephrine solution. Further, they were placed on a heating pad, to prevent hypothermia, and electrodes viz. comeal contact (objective lens) tail (ground) and head (reference) of the Ganzfeld ERG system (Phoenix Research Labs, Pleasanton, CA, USA), were placed respectively. The cornea was moistened with 2% Hypromellose topical solution and the positive electrode was placed on cornea.
  • Eye was illuminated with flashes of light stimulus (-1.7 to 3.1 log cd sec/m 2 ) and electrical responses were recorded and analyzed using Labscribe software (Labscribe, iWorx systems, Dover, NH, USA). Representative ERG wave forms were acquired for qualitative analysis and the mean of the amplitudes was plotted against a-wave and b-wave.
  • Glycan profiling detects distinctive signatures on AAV2 capsid
  • Enzymatic deglycosylation reveals the presence of glycans on AAV2 capsid
  • a second set of independent disgestion with a commercial deglycosylation mix containing O-glycosidase, PNGase F, Neuraminidase, b 1-4 Galactosidase, b-N- Acetylglucosaminidase to detect the presence of N and O-linked glycans also showed a similar decrease in the band intensity [-33%, FIGs. 4C & 4D] These data provide additional evidence for the presence of the minimal amount of glycan moities on AAV2 capsid and corroborates the MALDI-TOF data.
  • AAV2 transduction may be due to the activity of inhibitors on glycosylation specific cellular proteins which are known to play a role in intracellular trafficking as reported in many viruses such as Hepatitis C, Hantaan Virus.
  • ATRA All-trans-retinoic-acid
  • AAV293 cells plated in sixty numbers of 150mm dishes were mock-treated or pre-treated with either a N-linked glycosylation inhibitor (Tunicamycin, 0.125 pg/ml) or N-linked glycosylation activator (ATRA, 1 mM) followed by triple transfection with AAV2 packaging plasmids.
  • the recombinant vectors thus generated were further quantified by a qPCR protocol.
  • the potential N-linked and O-linked glycosylation motifs were identified using NetNGlyc, NetOGlyc and MotifScan tools (FIG. 8).
  • the immune epitope regions of the VP1 capsid protein were predicted by the IEDB server and merged with experimentally validated targets in the literature. Putative residues which are located within or near the immune epitope region and are part of predicted glycosylation sites were considered for glycosylation site abolition (O / N mutant). Residues which were part of the predicted epitope and capsid- loop region and that were amenable to host glycosylation motifs were considered for glycosylation-site introduction (0+/N+ mutant).
  • residues thus shortlisted for targeted mutagenesis have been mapped onto the VP1 capsid protein sequence of AAV2 and presented in FIG. 9. Selected residues were further analyzed by NetNGlyc and NetOGlyc tool to predict their glycosylation potential. The results of these predictions after mutagenesis at select residues are given in Table 5.
  • Table 5 List of AAV2 glycoengineered mutants predicted by in-silico prediction analysis. Glycoengineered vectors were generated by introducing mutation at residues to create or abolish glycosylation motif at the selected sites. Glycosylation potential of the modified sites are presented after in-silico analysis
  • Glycoengineered mutants demonstrate improved transduction in ocular and hepatic cell lines in vitro
  • Table 6 List of AAV2 glycoengineered mutants generated and their titer values.
  • AAV2 mutants where O-glycosylation sites were abolished (O ) such as S149A demonstrated an average 11% increase in comparison to cells infected with AAV2-WT vectors alone.
  • the N mutant, N705Q had a partial (-30%) immune escape function as measured by the increased transduction efficiency in the presence of IVIG (47 ⁇ 15% vs l7 ⁇ 3% in scAAV2-WT, FIGs. 12A & 12B).
  • Further screening of AAV2 N705Q vector with a AAV2-specific monoclonal antibody (A20) revealed a similar Nab escape phenomenon.
  • the N705Q vector demonstrated a 15 to 20% increase in EGFP expression in comparison to WT-AAV2 vectors at two different concentrations of A20 antibody (3.9 ng and 2ng) (FIG. 13).
  • a glycoengineered mutant AAV2 T14N demonstrates significantly higher coagulation FIX expression in vitro and in vivo
  • AAV2 T14N, Q259N and N705Q vectors has enhanced reporter gene (GFP) expression in the hepatic cell line, HUH7, further examined, if these vectors could deliver a therapeutic gene such as FIX into hepatic cells in vitro.
  • Huh7 cells were infected with 5x104 vgs of AAV2 expressing hFIX. Forty-eight hours later, measured hFIX transcript levels by qPCR.
  • AAV2 T14N and N705Q-LP1 hFIX vectors exhibited a -2.4 and 2.7-fold higher FIX mRNA expression.
  • the mean hFIX level in AAV2-FIX injected animals at 4, 10, 12 weeks were 20 ⁇ 8%, 30 ⁇ 18% and l4 ⁇ 5% while hFIX levels in AAV2-T14N-LP1 hFIX injected animals were 36 ⁇ 22%, 48 ⁇ 36% and 32 ⁇ 20% (Table 7). This shows an average 1-8-2.3 fold increase for T14N mutants over the WT-AAV2 vectors.
  • the data confirms the superior therapeutic efficiency of AAV2-T14N glycoengineered mutant vectors for hepatic gene transfer of diseases such as hemophilia B.
  • T14N-LP1 hFIX vectors after hepatic gene transfer at 4, 10 and 12 weeks.
  • T-cell and B-cell response to AAV2-T14N mutant is unaltered after its hepatic gene transfer
  • the cytotoxic T lymphocyte population (CD3+CD8+) which are reported to be involved in AAV capsid mediated immune response was 6 ⁇ 4% in PBS-injected mice, 5 ⁇ 3% in AAV2-FIX treated mice and l3 ⁇ l l% in AAV2-T14N-FIX treated group.
  • the B-cells were relatively unaltered among the mock (60 ⁇ 13%) and the treated groups (57 ⁇ 27 % in AAV2 treated and 73 ⁇ 5% in AAV2-T14N).
  • Further quantitation of immune modulatory T regulatory cells (Treg- CD4+CD25&FoxP3+) in splenocyte population denoted decrease in the Treg population similarly as AAV2 WT.
  • AAV2 T14N vector has better transduction efficiency after ocular gene transfer in vivo
  • AAV2-T14N vectors that were packaged with tunicamycin under glycosylation inhibited condition or treated with glycosidase (PNGase F) were administered intravitreally at a dose of 2xl0 8 vgs/eye or 3xl0 8 vgs/eye, respectively. These vectors, two or four weeks later, demonstrated reduced transduction/permeation in the murine retina at different doses (FIGs. 19A-D). This further suggested that the efficiency of the AAV2-T14N mutant vectors is likely due to the introduction of a capsid glycosylation site.
  • PNGase F glycosidase
  • FIGs. 19A-D show the retinal permeation characteristics of AAV2-T14N vector packaged in the presence of Tunicamycin, in glycosylation inhibited condition (2xl0 8 vgs/eye two weeks post gene delivery) or after treatment with glycosidase enzyme (3x10 8 vgs/eye four weeks post gene delivery).
  • GCL ganglion cell layer
  • ONL outer nuclear layer
  • INL inner nuclear layer
  • OS outer segment
  • RPE retinal pigmented epithelium.
  • the magnification-400X was used herein. Phenotypic correction of visual function in rdl2 mice
  • FIG 20A-D which depict visual function rescue in rdl2 mice by AAV2 T14N mutant:
  • FIG 20A is representative image of the rescue in ERG wave forms in AAV2-T14N injected eyes when compared to wild type vector injected and mock controls in rdl2 mice at 6 weeks and FIG 20B 10 weeks.
  • FIG. 20C is dot plot for a wave and b wave plotted against the mean amplitude obtained at 3.1 log cd sec/m 2 shows significant rescue in a wave form (left graph) and b wave (right graph) in the mutant vector injected group when compared to wild type vector injected and mock controls at 6 weeks and FIG. 20D 10 weeks post gene transfer.
  • n 4-6 eyes. Values represented ⁇ SEM, where ** p ⁇ 0.0l, ***p ⁇ 0.00l.
  • FIGs. 21A and B depict visual function in rdl2 mice after administration of AAV2 Q259N vectors.
  • FIG 21A The data shows the representative images of the ERG wave forms in AAV2-Q259N injected eyes in comparison to wild type vector or mock injected rdl2 mice at 32 weeks after ocular gene transfer. A rescue in physiological vision is represented by the regain in qualitative wave form.
  • Viruses are dependent on various host cellular factors to establish an infective life cycle.
  • the typical events following a viral infection includes the binding of the virus to the cellular receptors, its internalization, cytoplasmic trafficking towards nucleus and nuclear entry all of which require its interaction with a number of cellular factors.
  • AAV2 was reported to interact with membrane associated heparan sulfate proteoglycan and utilize it as the primary cellular receptor. Removal of heparan sulfate proteoglycan from cellular surface revealed that infectivity of AAV2 is compromised. Subsequently, several studies have reported various cellular factors including post translational modifications (PTM) such as phosphorylation, ubiquitination that modulate the AAV transduction, but very little data exists on other PTMs.
  • PTM post translational modifications
  • glycosylation inhibitors target different cellular proteins/pathway involved in glycosylation.
  • tunicamycin is known to target GlcNAc transferase while swainsonine is known to target a-mannosidase II proteins.
  • the data generated here suggests that cellular glycosylation status is an important determinant of AAV transduction.
  • the present results are consistent with similar reports on other viruses, where inhibition of glycosylation is known to enhance entry of HIV 2 into CD4 cells.
  • an activator of glycosylation such as ATRA is known to modulate replication of HIV or herpes simplex virus.
  • ATRA an activator of glycosylation
  • the reduced AAV vector yield in the presence of glycosylation modifiers noted here could be due to altered functionality of cellular proteins necessary for virus packaging or alteration of viral Rep and helper proteins that participate in AAV DNA replication.
  • Nash et al. have identified 188 cellular proteins from 16 different functional categories which interact with AAV rep protein, known to be involved in in genome replication, control of viral and cellular transcription and protein translation.
  • Several of these proteins associated with AAV2 rep are known to be heavily glycosylated.
  • N-linked or O-linked oligosaccharides have an important role in protein folding and in the genesis of complex viral protein structures, required for the optimal functionality of viral capsid in Adenovirus, Rotavirus and Influenza virus.
  • MALDI-TOF screening of AAV2 capsid which showed the presence of various N-linked glycans on AAV2 capsids including fucosylated moieties ( Figure 1A & B). Fucosylated capsids/proteins are known to induce evasion of immune response in other viruses.
  • fucose-containing glycans can reduce the binding affinity of immunoglobulins and also have a significant role in maintaining viral protein conformation in viruses like HIV. Further screening by deglycosidase enzyme combinations, confirmed the presence of these glycan attachments but also demonstrated that these modifications are less abundant (FIGs. 2A &B).
  • N-glycosylation site mutant capsids developed, four of them (N+ mutants: T14N, Q259N and S412N and N- mutant: N705Q) had enhanced transduction in all the cell lines in vitro.
  • N+ mutants T14N, Q259N and S412N and N- mutant: N705Q
  • these studies highlight that in general, modification of N- linked sites had relatively higher transgene expression from mutant AAV2 vectors.
  • the nature and type of glycosylation site modification did not yield a distinctive phenotype of gene expression from AAV vectors.
  • N+ mutants E499N, G504N, R513T, Q677N
  • a N- mutant, N382Q were non-infectious. This shows a considerable heterogeneity in the phenotypic expression of the mutants generated. Similar phenomenon has been seen earlier with tyrosine or serine/threonine or lysine mutants [32, 43] Thus, it stands to reason that the efficacy of any AAV capsid mutant generated is sequence, context and cell-type specific. Interestingly, among all the mutants generated, only one N-glycosylation site abolished mutant demonstrated partial immune escape to Nab (IVIG) or to an AAV2 monoclonal antibody, A20. ( Figure 9 &10).
  • N705Q virus capsid Abolition of N-glycosylation site in case of N705Q virus capsid, has shown to decrease antibody recognition. It was speculated that the Nab escape phenotype of N705Q mutant is due to the fact that this residue is within a B-cell epitope recognition sequence and whose involvement as an immunogenic epitope is experimentally verified in C57BL/6 mice model. Further detailed studies are necessary to understand the mechanistic basis of the diverse phenotypes observed here with the present glyco-engineered vectors.
  • AAV2 serotype is the most utilized vector for human gene therapy applications and whose safety is established in multiple trials for diverse therapeutic genes.
  • One of the advantages of this AAV2 glyco- engineered capsids is that this approach is applicable to all single-stranded or self complementary AAV transgene cassettes, and further could be used for any of the clinical trials that use AAV2 vectors for diseases such as AAT deficiency, hemophilia A or hemophilia B and LCA2.
  • adaptation of a glycoengineered mutant capsid to test the expression of the therapeutic genes with or without cell-specific promoters for the desired tissue tropism, will provide further insights into its therapeutic potential.
  • the present invention utilized the most consistent glyco-engineered mutant in the present in vitro studies, AAV2- T14N to package hFIX under the control of a liver specific promoter, LP1.
  • the use of T14N mutant capsid improved the circulating levels of hFIX consistently by 2-fold in hemophilia B mice at 4, 10 and 12 weeks after hepatic gene transfer (Fig. 16).
  • Present data also demonstrated no significant difference in T and B cell activation between WT-AAV2 treated and T14N treated hemophilia B mice (Fig. 16).
  • glycol- engineered mutant capsids are excellent candidates to test the efficacy of the therapeutic gene, RPE65 in a suitable transgenic model of FCA2.
  • a differential transgene enhancement from the EGFP and hFIX vector-mediated by T14N vectors was observed, (1.49 vs 2-fold), this could be due to multiple differences including the host cell type (retinal vs hepatic cells), dose (3x10 vgs vs. 5xl0 10 vgs/animal) and also due to the nature of protein expressed (intracellular vs. circulating protein).
  • AAV2-T14N vector showed reduction in the permeation of the retina and a reduced gene expression when treated with PNGase F or packaged under glycosylation inhibited condition, demonstrating the presence of glycan modification in T14N site (FIG 19).
  • These data suggest that such glycosylation site modified mutant capsids are excellent candidates to test the efficacy of the therapeutic gene, hRPE65 in a suitable transgenic model of LCA2.
  • subretinal administration of these vectors such as AAV2-T14N and AAV2- Q259N substantially rescued the visual parameters in rdl2 mice.
  • the dose at which this phenotypic rescue was observed is significantly lower (lxl0 8 vgs- 7xl0 8 vgs/eye) than reported in other studies.
  • present invention has uncovered the potential of glycosylation modulation to improve AAV2 infection. It provides for the novel glycoengineered AAV2 vectors that demonstrate significant therapeutic potential for ocular and hepatic gene therapy applications in humans. [87] 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.

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Abstract

La présente invention porte sur un procédé de synthèse de vecteurs à virus adéno-associé glycomodifiés. Le procédé consiste à : générer des vecteurs AAV en présence d'un groupe de modulateurs de glycosylation; prédire l'antigénicité de la protéine capsidique AAV2 des vecteurs AAV à l'aide d'outils de cartographie d'épitopes linéaires de lymphocytes B et de lymphocytes T afin d'affiner des épitopes de la protéine capsidique d'AAV; cibler les épitopes affinés pour une mutagenèse dirigée sur site, au moyen d'un ensemble d'amorces, en introduisant ou en supprimant des motifs de glycosylation N- et O- dans la capside d'AAV afin d'obtenir la pluralité des vecteurs à virus adéno-associés glycomodifiés (AAV). La mutagenèse dirigée sur site d'acides aminés cibles de la capside d'AAV comprend NQ, QN, SA, AT, E/K/Q/RT et Y/S/TN. Les vecteurs à virus adéno-associé glycomodifiés présentent une expression génique supérieure, par rapport au type sauvage, après le transfert d'un gène hépatique et oculaire.
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