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Mol Pharm. 2019 Nov 4;16(11):4738-4750. doi: 10.1021/acs.molpharmaceut.9b00959. Epub 2019 Oct 22.

Molecular Engineering of Adeno-Associated Virus Capsid Improves Its Therapeutic Gene Transfer in Murine Models of Hemophilia and Retinal Degeneration.

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Department of Biological Sciences and Bioengineering , Indian Institute of Technology Kanpur , Kanpur 208 016 , Uttar Pradesh , India.
Mass Spectrometry and Proteomics Core Facility , University of Nebraska Medical Center , Omaha 68198 , Nebraska , United States.


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.


AAV2; Leber congenital amaurosis; glycosylation; hemophilia B

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