Therapeutic Margins in a Novel Preclinical Model of Retinitis Pigmentosa

Abstract

The third-most common cause of autosomal recessive retinitis pigmentosa (RP) is due to defective cGMP phosphodiesterase-6 (PDE6). Previous work using viral gene therapy on PDE6-mutant mouse models demonstrated photoreceptors can be rescued if administered before degeneration. However, whether visual function can be rescued after degeneration onset has not been addressed. This is a clinically important question, as newly diagnosed patients exhibit considerable loss of rods and cones in their peripheral retinas. We have generated and characterized a tamoxifen inducible Cre-loxP rescue allele, Pde6bStop , which allows us to temporally correct PDE6-deficiency. Whereas untreated mutants exhibit degeneration, activation of Cre-loxP recombination in early embryogenesis produced stable longterm rescue. Reversal at later time-points showed partial long-term or short-lived rescue. Our results suggest stable restoration of retinal function by gene therapy can be achieved if a sufficient number of rods are treated. Because patients are generally diagnosed after extensive loss of rods, the success of clinical trials may depend on identifying patients as early as possible to maximize the number of treatable rods.

Genetic rescue models refute nonautonomous rod cell death in retinitis pigmentosa

“…For both models, we used our genetically engineered RP mouse model Pde6b STOP /Pde6b H620Q , in which one allele of rod-specific Pde6b contains a point mutation and the second allele a floxed STOP cassette. In these mice, PDE6b is dramatically reduced, leading to rod death and secondary degeneration of cones (4). When Pde6b STOP /Pde6b H620Q mice are crossed with a Cre transgenic line, the STOP cassette is removed and PDE6b is expressed in cells where Cre is expressed (5).…”

Halting progressive neurodegeneration in advanced retinitis pigmentosa

“…PCR was performed as previously described (3). To target the Pde6b DNA sequence, we used 3 primers: forward primer, 5′-TGCTCTGTGGTGTTGCTCTGC-3′; reverse primer, 5′-TGG-CGATGCAGAGTGTCCTGA-3′; and internal primer, 5′-GTCCT-GCACGACGCGAGCTG-3′.…”

“…KV1 embryonic stem (ES) cells were resuspended in saline at a density of 4 × 10 7 cells/ml and infected with a linearized targeting vector by electroporation (BioRad electroporator, single pulse, 220 V and 960 μF) (3,49,50). Infected cells were immediately plated on three 10-cm dishes containing mitomycin-c-treated neomycin-resistant primary mouse embryonic fibroblasts, and the medium was replaced 24 hours later.…”

“…In the early stage of the disease, peripheral rod photoreceptor degeneration is underway, and patients experience night blindness; in addition, some cones exhibit structural pathologies (3). Rod degeneration continues in mid and late stages, causing tunnel vision.…”

Gene therapies for inherited retinal disorders

“…In terms of academic and commercial investment in the sector, the infl uence of the LCA preclinical and clinical studies has been monumental. Additional preclinical studies in rodent and dog models of RPE65 -linked LCA (Jacobson et al, 2006 ;Bennicelli et al, 2008 ;Annear et al, 2011 ), employed AAV for gene delivery but utilized different promoter sequences to drive Histology, ERG, OCT, fundus autofl uorescence Tan et al, 2009 ;Sun et al, 2010 ;Ku et al, 2011 ;Testa et al, 2011 Leber congenital amaurosis (LCA), autosomal recessive Acland et al, 2001Acland et al, , 2005Narfstrom et al, 2003Narfstrom et al, , 2005Narfstrom et al, , 2008Jacobson et al, 2006 ;Le Meur et al, 2007 ;Bennicelli et al, 2008 LCA, autosomal recessive Davis et al, 2008 ;Davis et al 2013;Souied et al, 2008 ;Pang et al, 2011 ;Tosi et al, 2011 Usher syndrome (USH; arRP and hearing loss), autosomal recessive Zeng et al, 2004 ;Kjellstrom et al, 2007 ;…”