@article {1193,
	title = {Molecular Engineering of Adeno-Associated Virus Capsid Improves Its Therapeutic Gene Transfer in Murine Models of Hemophilia and Retinal Degeneration [Mass Spectrometry - Proteomics Facility]},
	journal = {Mol Pharm},
	year = {2019},
	month = {2019 Oct 22},
	abstract = {<p>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.</p>
},
	issn = {1543-8392},
	doi = {10.1021/acs.molpharmaceut.9b00959},
	author = {Mary, Bertin and Maurya, Shubham and Kumar, Mohit and Bammidi, Sridhar and Kumar, Vikas and Jayandharan, Giridhara R}
}
@article {1139,
	title = {Post-translational modifications in capsid proteins of recombinant adeno-associated virus (AAV) 1-rh10 serotypes [Mass Spectrometry Facility - Proteomics \& Glycomics].},
	journal = {FEBS J},
	year = {2019},
	month = {2019 Jul 22},
	abstract = {<p>Post-translational modifications in viral capsids are known to fine-tune and regulate several aspects of the infective life cycle of several viruses in the host. Recombinant viruses that are generated in a specific producer cell line are likely to inherit unique post-translational modifications during intra-cellular maturation of its capsid proteins. Data on such post-translational modifications in the capsid of recombinant adeno-associated virus serotypes (AAV1-rh10) is limited. We have employed liquid chromatography and mass spectrometry analysis to characterize post-translational modifications in AAV1-rh10 capsid protein. Our analysis revealed a total of 52 post-translational modifications in AAV2-AAVrh10 capsids, including ubiquitination (17\%), glycosylation (36\%), phosphorylation (21\%), SUMOylation (13\%) and acetylation (11\%). While AAV1 had no detectable post-translational modification, at least four AAV serotypes had \&gt;7 post-translational modifications in their capsid protein. About 82\% of these post-translational modifications are novel. A limited validation of AAV2 capsids by MALDI-TOF and western blot analysis demonstrated minimal glycosylation and ubiquitination of AAV2 capsids. To further validate this, we disrupted a glycosylation site identified in AAV2 capsid (AAV2-N253Q), which severely compromised its packaging efficiency (~100-fold vs AAV2 wildtype vectors). In order to confirm other post-translational modifications detected such as SUMOylation, mutagenesis of a SUMOylation site(K258Q) in AAV2 was performed. This mutant vector demonstrated reduced levels of SUMO-1/2/3 proteins and negligible transduction, two weeks after ocular gene transfer. Our study underscores the heterogeneity of post-translational modifications in AAV vectors. The data presented here, should facilitate further studies to understand the biological relevance of post-translational modifications in AAV life cycle and the development of novel bioengineered AAV vectors for gene therapy applications. This article is protected by copyright. All rights reserved.</p>
},
	issn = {1742-4658},
	doi = {10.1111/febs.15013},
	author = {Mary, Bertin and Maurya, Shubham and Arumugam, Sathyathithan and Kumar, Vikas and Jayandharan, Giridhara R}
}
