GENE DELIVERY ADVANCE

Researchers have reported that a self-complimentary adeno-associated virus (AAV) vector with a specific capsid alteration can increase the efficiency of a gene therapy for Leber congenital amaurosis.

The research, led by Dr Cristy Ku and Prof Visvanathan Ramamurthy at the RC Byrd Health Sciences Centre of the University of West Virginia, US, represented the first-ever demonstration in a rapid retinal degeneration model using a self complimentary AAV with mutations of surface-exposed tyrosine residues on the viral capsid.

If successful in larger studies, the technology may have significant implications for broadening the spectrum of patients treatable with ocular gene therapy tools.

Leber congenital amaurosis (LCA) is well characterised as an early onset severe form of inherited retinopathy, representing about five per cent of inherited retinal degenerations. The disease may be caused by mutations in any one of up to 15 different genes, mostly expressed in photoreceptors or the retinal pigment epithelium (RPE).

Recently, researchers have demonstrated proof-of-concept using a gene replacement strategy to treat one form of LCA. Clinical trials involving the replacement of RPE65, delivered with viral vectors, have shown visual improvements in a number of patients. However, with 15 other genes potentially causing the disease, many patients will require delivery of a different gene or some other modification that addresses the particular genetic lesion.

The current studies, which appear in Human Molecular Genetics, [2011, Vol. 20, No. 23, pp 4569-4581] focused on a severe form of LCA caused by mutations in a gene called aryl hydrocarbon receptor interacting protein like 1, or “Aipl1†for short.

Patients with Aipl1 gene mutations account for about five to seven per cent of LCA; the mutation may also arise in juvenile retinitis pigmentosa and cone-rod dystrophy. Aipl1 patients possess a significantly reduced visual acuity by the age of 10 years, often ranging from 20/200 to no light perception. In such instances, time is of the essence, and therapeutic success in part may become a function of delivering the therapy before all recipient photoreceptors have degenerated.

Delivering the right genes to the right cells at the right time are challenges that must be overcome in any gene therapy strategy; the key tool in addressing these challenges is the delivery system. While there are limited types of delivery system to choose from, viral vectors, and in particular adeno-associated virus in retinal medicine, has become the tool of choice. In recent years, there have been significant advances made in the development of viral vector technology and, two advances in particular, have provided considerable optimism in the application of these tools to clinical medicine. Firstly, the development of self-complimentary vectors has provided double-stranded gene vectors that are more stable and eliminate the step of second strand synthesis.

As a consequence, transduction efficiencies of self-complementary vectors have increased and an earlier onset of expression of the delivered gene has been achieved. Secondly, experimentation with the surface exposed tyrosine residues on the viral capsid have led to more accurate targeting of specific cell types and have reduced the ubiquitination of viral particles due to decreased capsid tyrosine phosphorylation.

Combining both these developments – self-complimentary vectors and tyrosine residue alterations – the West Virginia research group used a murine model with no Aipl1 into which a single sub-retinal injection at post-natal day two or post-natal day 10 was performed. The injection contained the self-complementary “AAV-Y733F-RKp-hAipl1†– a viral vector with capsid modifications housing a replacement Ailp1 gene driven by a rhodopsin kinase promoter.

Analysis of the results demonstrated that vision had been restored, including improvements in the ERG a-wave and an increase of up to 45 per cent in the number of photoreceptor cell nuclei. Most importantly, behavioural analysis provided convincing data when single stranded AAV (ssAAV, unmodified) was compared to self-complimentary AAV (scAAV, with tyrosine modifications).

An adapted maze test was used to time reaction speeds under a variety of lighting conditions. Normal subjects with functional Aipl1 had a reaction time of 4.38±0.45 seconds while subjects with no functioning Aipl1 scored a reaction speed of 25.20±6.52 seconds. Following injections at post-natal day 10, subjects without any Aipl1 that received an ssAAV had a reaction time of 24.00±3.51 seconds (similar to no treatment at all) while similar subjects injected with scAAV had a reaction time of 6.69±1.30 seconds – a dramatic difference. The definitive result clearly indicated that, 10 days after birth, use of the scAAV carrying Aipl1 restored certain visual abilities to levels generally found in normal wild-type subjects.

According to the authors of the study, their results represent the first demonstration of the use of a self-complimentary AAV vector with tyrosine modifications in a rapid retinal degeneration.

Combining these advances with increasingly efficient gene packaging may significantly alter the range of retinal degenerations capable of treatment. While RPE65 delivery represented the first clinical success, technologies such as those developed by the US-based researchers may now expand the list of retinopathies that may benefit, most especially to the benefit of rapidly degenerating disorders where time is of the essence and delivery of the gene at the critical time can potentially make an enormous difference to the medical outcome.

“There is an impetus to broaden gene therapy clinical trials to treat a wider severity spectrum of inherited retinal dystrophies,†Prof Ramamurthy commented. “Recent clinical studies report cases of adolescent and adult patients with genetic defects associated with rapid retinal degeneration, who show varying extents of ONL (outer nuclear layer) preservation and inner and outer segment integrity as observed with optical coherence tomography (OCT). These studies show that patients with genetic defects associated with rapid retinal degeneration may potentially benefit from AAV-mediated gene replacement therapyâ€.