UC Berkeley

Inherited retinal degenerations are genetically heterogeneous conditions affecting roughly 1:3000 people and are characterized by the loss of photoreceptors. Progressive retinal degenerative disease is the leading cause of vision loss in industrialized countries, and is the result of a wide range of mutations, mostly in rod-specific transcripts. Over 140 disease-causing genes have been identified to date. As the genetic mechanisms underlying inherited forms of retinal degeneration are identified, gene therapy is becoming a promising approach for the treatment of many inherited blinding diseases.

Indeed, the recent success of three clinical trials using adeno-associated virus (AAV) to deliver a normal copy of the RPE65 gene to the retinas of Leber congenital amaurosis (LCA) patients illustrates the potential of gene therapy in the retina. AAV has been shown safe and effective especially in a younger cohort of patients. Some important obstacles remain, however, for AAV-mediated gene therapy to become widely applicable across the range of existing retinal degenerative diseases. It will be essential to carefully evaluate the method used to deliver therapeutic genetic material to the retina, as this will determine the success of the treatment.

The serotype of vector used, the promoter chosen to drive expression and the method of injection are important components of the gene delivery system. A wide variety of AAV serotypes exist with different tropisms for cell populations in the retina, potentially allowing treatments to be targeted to specific cell types. The retinal cell types AAV can infect differs, however, depending on whether the vector is delivered into the vitreous cavity or the subretinal space. Subretinal injections, which were used in the LCA trials, result in the creation of a retinal detachment and localized injury to the retina while delivering high concentrations of transgene to only a limited area. An intravitreal approach has the potential to transduce panretinally and is less invasive, and therefore preferable, but naturally occurring serotypes of AAV transduce photoreceptors poorly from the vitreous, as a result of structural barriers that exist on the inner surface of the retina.

Recent advances in the understanding of AAV and the production of viral vectors have shown the flexibility of this virus, indicating that its function can be altered and tailored to the requirements of retinal gene therapy. A directed evolution approach has been used to select, out of a highly diverse library of AAV capsid variants, a novel variant with improved tropism for Müller glia. And in a parallel approach, residues on the capsid surface have been mutated to avoid ubiquitination and altering the nuclear trafficking of the virus.

This dissertation examines the use of engineered viral vectors for gene therapy in the retina. The creation of a novel variant of AAV, called 7m8, which is characterized by increased transduction of photoreceptors from the vitreous, is described below. 7m8 was derived from an AAV2 peptide insertion library and contains a 7mer motif. Injected intravitreally, 7m8 transduces cells throughout the retina, including photoreceptors in the outer retina, significantly more efficiently compared to the parental serotype. Expression was restricted to photoreceptors using a rhodopsin promoter. This virus, as well as the previously described Müller-specific variant ShH10, was used to deliver a wild-type copy of the retinoschisin gene to mice lacking this protein. Retinoschisin is secreted from photoreceptors, and retinas deficient in this protein are severely structurally impaired. Subretinal injections, which are damaging in nature, are therefore suboptimal because they are likely to cause additional injury. We show that 7m8 is able to efficiently target photoreceptors via intravitreal injection in this mouse model, leading to high levels of retinoschisin protein production, as well as structural and functional rescue. This rescue is longer lasting than that seen using ShH10, indicating the importance of targeting photoreceptors in this disease model.

AAV9 has been shown to transduce the murine retina when injected intravenously through the tail vein. We used two surface tyrosine-to-phenylalanine mutations to improve the retinal expression of AAV9, and demonstrated that these mutations lead to higher infectivity of all retinal layers, most dramatically in photoreceptors and the inner nuclear layer, but also including the retinal pigment epithelium and ganglion cells. This novel vector was then used to explore the bifunctionality of the Nxnl1 gene, which encodes two isoforms of the rod-derived cone viability factor (RdCVF). The short form of RdCVF is secreted and has been shown to support cone survival, while the long isoform is retained intracellularly and has been implicated in redox signaling. AAV92YF and 7m8 were used to express the two isoforms of RdCVF in the rd10 mouse model of retinitis pigmentosa. RdCVF rescued cone survival when injected intravenously or intravitreally, but had little effect on rod survival. Early expression of RdCVFL in dark-reared rd10 mice delayed rod, and subsequently cone death.