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Luk H. Vandenberghe, Eric Zinn, Livia S Carvalho; Retinal applications for novel, in silico designed adeno-associated viral gene therapy vector platform. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):251.
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© ARVO (1962-2015); The Authors (2016-present)
Gene therapy is a promising approach in the treatment of inherited and common complex disorders of the retina. Preclinical and clinical studies have validated the use of adeno-associated viral vectors (AAV) as a safe and efficient gene delivery vehicle for subretinal gene transfer. RPE and rod, and to a lesser extent cone, photoreceptors can be efficienty targeted with AAV. Other retinal cell types are more challenging targets. Intravitreal gene delivery is inefficient and poses higher inflammatory risk to vector or transgene antigens. Studies here aim to build on AAV as a validated platform while exploring new and optimized vector reagents to overcome current limitations for clinical use. We hypothesized that an ancestral AAV species would be less susceptible to pre-existing AAV immunity (PEI) in human populations and confer an improved clinical safety profile.
We used phylogenetic and bioinformatic methods to infer the ancestral state of AAV at various stages using maximum-likelihood heuristics; most likely AAV capsid sequences libraries were synthesized and members were evaluated for viral assembly and DNA packaging; lead candidates progressed to immunological, in vitro, and in vitro transduction assays for characterizing (a) the ability of sera of various sources to neutralize new AAV virions in comparison to existing serotypes and (b) in vitro/vivo transduction by reporter gene expression from fundus imaging, molecular analyses, and histologically following subretinal injection of C57Bl/6 mice with AAVs for retinal cell targeting.
The most common ancestor of AAV1-3, 6-9 was reconstructed in silico, cloned using synthetic biology methods, and produced as vectors using standard AAV production methods. Particles, named Anc80, were found to be biochemically similar to existing AAV, stable, and infectious in vitro. In vivo retinal transduction in mice demonstrated a high level of RPE and photoreceptor tropism that is quantitatively higher than AAV8. In addition, onset of expression of Anc80 was visible as early as day 1 post injection, and accellerated compared to AAV8. Several variants of Anc80 were evaluated for inner retinal tropism with evidence for improved transduction of Müller glia, RGC, and other inner INL neurons.
Ancestral AAV have significantly increased resistance to PEI in humans and demonstrate enhanced retinal tropism in mouse.
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