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Abstract
Recombinant adeno-associated virus (AAV)-based gene therapy has been promising, but several host-related transduction or immune challenges remain. For this mode of therapy to be widely applicable, it is crucial to develop high transduction and permeating vectors that infect the target at significantly low doses. Because glycosylation of capsid proteins is known to be rate limiting in the life cycle of many viruses, we reasoned that perturbation of glycosylation sites in AAV2 capsid will enhance gene delivery. In our first set experiments, pharmacological modulation of the glycosylation status in host cells, modestly decreased (1-fold) AAV2 packaging efficacy while it improved their gene expression (~74%) in vitro. We then generated 24 mutant AAV2 vectors modified to potentially create or disrupt a glycosylation site in its capsid. Three of them demonstrated a 1.3–2.5-fold increase in transgene expression in multiple cell lines (HeLa, Huh7, and ARPE-19). Hepatic gene transfer of these vectors in hemophilia B mice, resulted in a 2-fold increase in human coagulation factor (F)IX levels, while its T/B-cell immunogenic response was unaltered. Subsequently, intravitreal gene transfer of glycosylation site-modified vectors in C57BL6/J mice demonstrated an increase in green fluorescence protein expression (~2- to 4-fold) and enhanced permeation across retina. Subretinal administration of these modified vectors containing RPE65 gene further rescued the photoreceptor response in a murine model of Leber congenital amarousis. Our studies highlight the translational potential of glycosylation site-modified AAV2 vectors for hepatic and ocular gene therapy applications.
Highlights
Recombinant adeno-associated virus (AAV) vectors based on serotype 2 have gained prominence in gene therapy applications because of its excellent safety profile
AAV5 and AAV8 have been successfully employed for gene therapy of hemophilia B or Leber congenital amaurosis type 2 (LCA2),6 AAV2 is the widely studied serotype (>6.9% of gene therapy trials) and its unrestricted use in clinical space has been widely beneficial to investigators in the gene therapy field
To check the effect of glycosylation inhibitors on AAV2 transduction, human cervical carcinoma (HeLa) cells were cultured in the presence of N-linked and O-linked glycosylation inhibitors and further infected with scAAV2EGFP vectors
Summary
Recombinant adeno-associated virus (AAV) vectors based on serotype 2 have gained prominence in gene therapy applications because of its excellent safety profile. To assess the efficacy of the AAV2 glycosylation site-modified vectors, an infectivity assay was performed in three different cell lines, namely, HeLa, Huh[7], and ARPE-19. To assess the transduction ability of the AAV2 mutants during ocular gene transfer, we performed intravitreal administration of scAAV2 vectors T14N, Q259N, and N705Q carrying EGFP into the eyes of C57BL6/J mice. T14N and N705Q vectors, whereas the AAV2-Q259N vector had a significantly higher expression in the inner nuclear layer (INL) of retina, in comparison to AAV2-WT vectoradministered group These data underscore the significant therapeutic potential of glycosylation site-modified AAV2 vectors for ocular gene therapy. Two or four weeks later, these vectors demonstrated reduced transduction/permeation in the murine retina at different doses (Figure S7) This further suggested that the efficiency of the AAV2-T14N mutant vectors is due to the introduction of a capsid glycosylation site. Phenotypic Correction of Visual Function in rd[12]
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