Abstract
Purpose :
Adeno-associated virus (AAV)-mediated gene therapy using sub-retinal (SR) injections is promising for some ocular diseases. Intravitreal (IVT) administration of existing AAV serotypes is far less efficient, notably due to the inner limiting membrane (ILM), which is a profound barrier in the primate retina. 2.5T and ShH10 can transduce photoreceptor in pig explants or in SR-injected mice. However, they are unable to efficiently cross the ILM and penetrate the entire non-human primate (NHP) retina when delivered IVT perhaps due to their inability to sufficiently bind heparan sulfate proteoglycan (HSPG) receptors that are abundant on the ILM. We are developing AAV vectors that can overcome this limitation and effectively transduce target cells following IVT injection for diseases where SR delivery is not a preferred method.
Methods :
We attempted to create hybrid vectors for enhanced IVT delivery by inserting a HSPG-binding domain or a peptide loop. We engineered 2.5T-HSPG swap variants by either substituting two positively charged resides, a short region comprising of 6 binding residues, or a longer region consisting of the binding domain and flanking residues in the receptor-binding region of 2.5T, from AAV2. We generated ShH10/7m8 by inserting the 10 amino acid 7m8-loop in the surface exposed, receptor-binding region of ShH10. We performed a binding assay using the HiTrap Heparin HP column and we analyzed the collected fractions by a dot blot using the α-AAV B1 antibody. We evaluated transgene expression and distribution in pig retinal explants, and in IVT-delivered gerbil and NHP retinas.
Results :
A majority of the 2.5T-HSPG swap variants packaged successfully and demonstrated varying levels of HSPG-binding compared to the parent capsid. An interesting observation in pig explants was altered tropism compared to the parent capsid. Also, several 2.5T-HSPG variants showed significantly improved transgene expression in gerbils after IVT delivery. Although the ShH10/7m8 hybrid had an enhanced expression profile in pig explants, it was not significantly improved over ShH10 in IVT-transduced gerbil retinas. Efficacy of candidate variants is currently being tested in NHPs.
Conclusions :
The resulting 2.5T-HSPG chimeric variants did indeed acquire the desired HSPG-binding ability, which resulted in improved tropism in gerbil retina.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.