Molecular Evolution of Adeno-associated Virus for Enhanced Glial Gene Delivery

Abstract

Due to the natural tropism of most viral vectors, including adeno-associated viral (AAV) vectors, efficient gene delivery within the central nervous system and retina occurs primarily to neurons and epithelia. Despite the clinical relevance of glia for homeostasis in neural tissue, and as causal contributors in genetic disorders such as Alzheimer's and amyotrophic lateral sclerosis, efforts to develop more efficient gene delivery vectors for glia have met with limited success. Recently, viral vector engineering involving high-throughput random diversification and selection has enabled the rapid creation of novel AAV vectors with valuable new gene delivery properties. We have engineered novel AAV variants capable of efficient glia transduction by employing directed evolution with a panel of four distinct AAV libraries, including a new semi-random peptide replacement strategy. Several novel variants transduced both human and rat astrocytes in vitro up to 15-fold higher than their parent serotypes, and injection into the rat striatum led to astrocyte transduction levels up to 16% of the total transduced cell population. Furthermore, one variant exhibited a substantial shift in tropism towards Müller glia within the retina, further highlighting the general utility of these variants for efficient glia transduction within the CNS and retina.

Targeted ablation of Crb2 in photoreceptor cells induces retinitis pigmentosa

“…AAV stocks were generated and purified as described previously (44). Briefly, plasmids containing the transgene flanked by the ITRs were co-transfected with the AAV5 pDP5RS packaging plasmid (Plasmid Factory, Bielefeld, Germany) or AAV-ShH10Y445F packaging plus pHelper plasmids (27) into HEK293 T cells to generate cross-packaged AAV2/5 and AAV2/ShH10Y445F viral vectors (45,46). At the third day after transfection, the medium was changed for lysis buffer (50 mM Tris, 2 mM MgCl 2 , 150 mM NaCl and 0.1% Triton X-100).…”

Enhanced gene delivery to the neonatal retina through systemic administration of tyrosine-mutated AAV9

“…Unfortunately, there are a number of formidable barriers impeding gene delivery to the retina from the vitreous such as diffusion in the vitreous cavity and sequestering of viral particles in the inner limiting membrane. Although much progress has been made in the understanding of barriers to retinal transduction from the vitreous for AAV 7,8 as well as improving the limited transduction of the naturally occurring serotypes by rational 9,10 and directed evolution approaches 11,12 major obstacles remain in reaching therapeutically effective levels of gene expression, especially in larger animals where the inner limiting membrane is significantly thicker than in rodents. 13 An alternative approach for transducing large areas of retina is to administer vectors through the ocular vasculature.…”

Progress and prospects: Immunobiology of gene therapy for neurodegenerative disease: prospects and risks

“…89 In addition, by using directed evolution with a diverse array of novel AAV libraries, a new generation of AAV vectors capable of highly efficient delivery to astrocytes has been engineered. 90 Self-complementary AAV vectors, which bypass the required second-strand DNA synthesis to achieve transcription of the transgene have recently been used for retinal gene therapy without adverse immune responses. Transgene expression lasted for over 3.5 months in rats and 2.35 years in monkeys without any reported adverse effects in these studies.…”