A Systematic Review and Meta-Analyses of Interventional Clinical Trial Studies for Gene Therapies for the Inherited Retinal Degenerations (IRDs).

IRDs are one of the leading causes of visual loss in children and young adults. Mutations in over 271 genes lead to retinal dysfunction, degeneration and sight loss. Though no cure exists, gene augmentation therapy has brought hope to the field. This systematic review sought to assess the efficacy of available gene therapy treatments for IRDs. Databases and public resources were searched for randomised controlled trials (RCTs) and non-randomised studies of interventions (NRSIs). Standard methodological procedures were used, including a risk-of-bias assessment. One RCT and five NRSIs were assessed, all for adeno-associated virus two (AAV2)-mediated treatment of RPE-specific 65 kDa (RPE65)-associated LCA (Leber congenital amaurosis). Five outcomes were reported for meta-analyses. Modest improvements in visual acuity, ambulatory navigation/mobility testing or central retinal thickness was observed. There was significant improvement in red and blue light full-field stimulus testing (FST) (red light risk ratio of 1.89, treated v control, p = 0.04; and blue light risk ratio of 2.01, treated v control, p = 0.001). Study design assessment using a ROBIN-I tool (Cochrane Library) showed risk-of-bias judgement to be "low/moderate", whilst there were "some concerns" for the RCT using a RoB-2 tool (Cochrane Library). Although comparison by meta-analysis is compromised by, amongst other issues, a variable amount of vector delivered in each trial, FST improvements demonstrate a proof-of-principle for treating IRDs with gene therapy.

Improved retinal function in a mouse model of dominant retinitis pigmentosa following AAV-delivered gene therapy.

Mutational heterogeneity represents one of the greatest barriers impeding the progress toward the clinic of gene therapies for many dominantly inherited disorders. A general strategy of gene suppression in conjunction with replacement has been proposed to overcome this mutational heterogeneity. In the current study, various aspects of this strategy are explored for a dominant form of the retinal degeneration, retinitis pigmentosa (RP), caused by mutations in the rhodopsin gene (RHO-adRP). While > 200 mutations have been identified in rhodopsin (RHO), in principle, suppression and replacement may be employed to provide a single mutation-independent therapeutic for this form of the disorder. In the study we demonstrate in a transgenic mouse simulating human RHO-adRP that RNA interference-based suppression, together with gene replacement utilizing the endogenous mouse gene as the replacement, provides significant benefit as evaluated by electroretinography (ERG). Moreover, this is mirrored histologically by preservation of photoreceptors. AAV-based vectors were utilized for in vivo delivery of the therapy to the target cell type, the photoreceptors. The results demonstrate that RNAi-based mutation-independent suppression and replacement can provide benefit for RHO-adRP and promote the therapeutic approach as potentially beneficial for other autosomal dominantly inherited disorders.

RNAi of COL1A1 in mesenchymal progenitor cells.

Given that mutant COL1A1 is known to cause Osteogenesis Imperfecta (OI), tools to modulate COL1A1 expression are likely to be of significant therapeutic value. In this context, we have evaluated RNA interference (RNAi) as a means to downregulate COL1A1 expression in Cos-7 cells and in human mesenchymal progenitor stem cells (MPCs), the latter cells giving rise to bone and therefore representing a target cell type for collagen-related disorders. In addition, allele-specificity, a key factor to the success of RNAi-based suppression, was explored with a view to developing a mutation-independent RNAi-based therapeutic for OI by targeting an intragenic SNP within transcripts derived from the COL1A1 gene. Preferential suppression of individual polymorphic alleles that differed by a single nucleotide was observed.

Sensitivity of photoreceptor-derived cell line (661W) to baculoviral p35, Z-VAD.FMK, and Fas-associated death domain.

PURPOSE: Rod, cone, cone-rod, and macular dystrophies eventually bring about the death of cone photoreceptor cells. The present study explores means of inhibiting apoptosis in addition to inducing a specific apoptotic pathway within a photoreceptor-derived cell line. METHODS: Retinal cell culture of murine 661W photoreceptor-derived cells was used to assess the effect of both a synthetic peptide inhibitor of caspases (benzyloxycarbonyl-Val-Ala-DL-Asp-[Ome] fluoromethylketone [Z-VAD.FMK]) and a natural inhibitor, baculoviral p35. In addition, the effect of transfection of Fas-associated death domain (FADD), a cellular protein implicated in receptor-induced apoptosis, was assessed. Assays were performed by transient transfection of cell cultures, and results were recorded by cell counting, Western blot, and spectrophotometry. RESULTS: Western blot analysis and chromogenic caspase substrate cleavage analysis confirmed the activation of caspases within 661W cells. At a concentration of 80 micro M, Z-VAD.FMK, 72.36% +/- 0.93% of 661W cells survived cytotoxic insult compared with 6.99% +/- 1.35% of control cells. Transient transfection of 1200 ng baculoviral p35 conferred a protection of 75.30% +/- 4.23%, compared with 19.61% +/-1.84% of control cells, and it was additionally observed that as little as 50 ng transfection of FADD was capable of inducing the death of 53.21% +/- 1.33% of cells in 661W cultures. CONCLUSIONS: Apoptotic cell death in 661W cells is caspase dependent and may be inhibited with both a synthetic and natural inhibitor of caspase function. Furthermore, 661W cells are highly sensitive to the FADD protein, which may suggest a number of novel therapeutic approaches to halt photoreceptor cell apoptosis.

Validation in mesenchymal progenitor cells of a mutation-independent ex vivo approach to gene therapy for osteogenesis imperfecta.

Over 100 dominant-negative mutations within the COL1A1 gene have been identified in osteogenesis imperfecta (OI). In terms of human therapeutics, targeting each of these mutations independently is unlikely to be feasible. Here we show that the hammerhead ribozyme Rzpol1a1, targeting a common polymorphism within transcripts from the COL1A1 gene, downregulates COL1A1 transcript in human mesenchymal progenitor cells at a ribozyme to transcript ratio of only 1:1. Downregulation was confirmed at the protein level. Transducing stem cells with Rzpol1A1 ex vivo followed by autologous transplantation could provide a gene therapy for a large proportion of OI patients with gain-of-function mutations using a single therapeutic.