Examining advances in AAV vector for retinal gene therapy

Gainesville, FL-Much work is being done with Adeno-associated virus (AAV) vectors for application in retinal gene therapy, with modifications being made to the capsid and genome of the vector to generate novel variants with unique transduction profiles, according to Shannon Boye, PhD.

“The AAV vector toolkit is expanding rapidly,” said Dr. Boye, assistant professor, University of Florida, Gainesville.

Sequence differences in the large variable regions of the AAV capsid account for functional differences between serotypes in receptor biology and immunologic profiles, among others.

Rational design and directed evolution are two methods used to modify the variable regions, she explained. The former involves using knowledge of vector biology to make structure-informed mutations to the capsid sequence to generate novel variants, e.g., the tyrosine-phenylalanine story led by Arun Srivastava, PhD, at the University of Florida.

The latter method can also generate novel variants, Dr. Boye said.

“This method harnesses nature to mine capsid sequences that confer vectors with the ability to best overcome a selective pressure,” she explained.

Two variants thus mined were “7m8”, which showed an increased transduction of photoreceptors after intravitreal injection, and “shh10,” which demonstrated preferential transduction of Müller cells after intravitreal injection.

Dr. Boye said that a unique feature of these novel variants and those rationally designed in her laboratory is their differential affinity for heparan sulfate proteoglycan (HSPG)-abundant in the internal limiting membrane-relative to their unmodified parent serotypes. While HSPG binding is necessary for transducing neural retina via the vitreous, Dr. Boye and her colleagues wanted to determine its necessity for photoreceptor transduction by comparing a variant with very high HSPG affinity, AAV2 (tripleY-F+T-V) to one that lacked canonical HSPG binding residues, AAV2 Max delta HS.

The latter exhibited no retinal transduction after intravitreal injection. When injected subretinally, however, the same variant exhibited robust transduction of photoreceptors and retinal pigment epithelium (similar to that seen with photoreceptor-phillic AAV5 or AAV8 serotypes).

Regarding modifications to the genome, various cellular promoters can be used to restrict transgene expression to specific retinal cell types. Newer promoters have activity specifically in retinal ganglion and ON bipolar cells.

Dr. Boye and her colleagues are exploring an ON bipolar-specific promoter based on regulatory regions of the PCP2 gene in combination with a rationally designed variant for its ability to drive therapeutic nyctalopin expression after intravitreal injection in a mouse model of X-linked congenital stationary night blindness.

Because of the limited packaging capacity of standard AAV vectors (~5kb), dual AAV vectors have been designed to double the payload. This involves packaging the front half of a gene in one vector and the back half of a gene in a second vector. The two vectors can be co-infected and the front and back half portions of the large gene can recombine to form the full-length gene and go on to produce therapeutic protein.

For the first time, the sequence fidelity of mRNA transcript emanating from recombined genomes was 100% accurate, she noted.

“This has implications for regulatory approval of dual-vector platforms and opens the door for treating AAV, retinal diseases associated with mutations in large genes such as MYO7A Usher 1B, ABCA4 Stargardt’s disease, and CEP290 Leber’s congenital amaurosis,” Dr. Boye said.

This article is adapted from Dr. Boye's presentation at the 2014 meeting of the Association for Research in Vision and Ophthalmology.

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