Recombinant AAVs are carriers of choice for retinal gene therapy.
Inherited retinal dystrophies in humans typically progress over a period of decades. Clinical applications are therefore likely to require an efficient therapy, sustained over the long term. Administration of therapeutic genes using rAAV vectors meets this requirement.
The stable transduction of retinal cells by rAAV vectors has allowed efficient and sustained gene therapy. The safety and efficacy of rAAV vectors-mediated retinal gene transfer has been demonstrated in several species, including humans.
The lack of pathogenicity and low immunogenicity are the main characteristics of rAAVs. This feature argues in favour of their use over other potential vectors for human gene therapy.
Recombinant AAVs: How are they built?
rAAV vectors are based on a non-pathogenic, replication deficient member of the parvovirus family. This wild-type virus encodes rep and cap genes, which are required for viral replication and synthesis of capsid proteins, respectively. Via a combination of alternative translation start and splicing sites, the small genome is able to express four rep and three cap gene products. Humans are likely infected through the respiratory tract. Infection of a cell in the absence of a helper virus results in the onset of the latent cycle, in which the virus may integrate with preference to a specific site in human genome. In the presence of a helper virus (such as adenovirus, herpes virus, or others), the AAV genome is excised and replicated through intermediate double-stranded forms. The rep gene products and sequences in the inverted terminal repeats (145 bp ITRs, which flank the genome) are critical in this process.
To create a recombinant AAV vector (rAAV), the wild type, viral coding sequences of the AAV i.e., rep and cap, are removed and replaced with an expression cassette for the therapeutic, healthy copy of the gene of interest. The rAAV vectors are therefore devoid of viral coding sequences. While the ITR has been shown to possess mild promoter activity, for maximum levels of transgene expression, the cassette typically includes a promoter/enhancer combination, a small intron sequence, the cDNA of the therapeutic gene, and a polyadenylation signal. For the production of rAAV vectors, the rep and cap (i.e. cap gene of the chosen capsid serotype) and helper functions are provided apart during the production process (other plasmid(s)).
The therapeutic gene, packaged into the viral capsid, can then be delivered into the target retinal cells.
In a rAAV vector, the capsid serotype (1 to 9 etc.) confers the specificity of targeting. The promoter has a direct impact on the regulation of the expression level of the protein.
A technology adapted to ophthalmology practice
The recombinant vector is injected subretinally between the photoreceptors and the retinal pigment epithelium allowing the healthy gene to access the cells where it was originally missing or mutated.