Treatment Potential for LCA5-Associated Leber Congenital Amaurosis.

Purpose: To determine the therapeutic window for gene augmentation for Leber congenital amaurosis (LCA) associated with mutations in LCA5. Methods: Five patients (ages 6-31) with LCA and biallelic LCA5 mutations underwent an ophthalmic examination including optical coherence tomography (SD-OCT), full-field stimulus testing (FST), and pupillometry. The time course of photoreceptor degeneration in the Lca5gt/gt mouse model and the efficacy of subretinal gene augmentation therapy with AAV8-hLCA5 delivered at postnatal day 5 (P5) (early, n = 11 eyes), P15 (mid, n = 14), and P30 (late, n = 13) were assessed using SD-OCT, histologic study, electroretinography (ERG), and pupillometry. Comparisons were made with the human disease. Results: Patients with LCA5-LCA showed a maculopathy with detectable outer nuclear layer (ONL) in the pericentral retina and at least 4 log units of dark-adapted sensitivity loss. The Lca5gt/gt mouse has a similarly severe and rapid photoreceptor degeneration. The ONL became progressively thinner and was undetectable by P60. Rod- and cone-mediated ERGs were severely reduced in amplitudes at P30 and became nondetectable by P60. Subretinal AAV8-hLCA5 administered to Lca5gt/gt mice at P5 and P15, but not at P30, resulted in structural and functional rescue. Conclusions: LCA5-LCA is a particularly severe form of LCA that was recapitulated in the Lca5gt/gt mouse. Gene augmentation resulted in structural and functional rescue in the Lca5gt/gt mouse if delivered before P30. Retained photoreceptors were visible within the central retina in all patients with LCA5-LCA, at a level equivalent to that observed in rescued Lca5gt/gt mice, suggesting a window of opportunity for the treatment of patients with LCA5-LCA.

Comparative AAV-eGFP Transgene Expression Using Vector Serotypes 1-9, 7m8, and 8b in Human Pluripotent Stem Cells, RPEs, and Human and Rat Cortical Neurons.

Recombinant adeno-associated virus (rAAV), produced from a nonpathogenic parvovirus, has become an increasing popular vector for gene therapy applications in human clinical trials. However, transduction and transgene expression of rAAVs can differ across in vitro and ex vivo cellular transduction strategies. This study compared 11 rAAV serotypes, carrying one reporter transgene cassette containing a cytomegalovirus immediate-early enhancer (eCMV) and chicken beta actin (CBA) promoter driving the expression of an enhanced green-fluorescent protein (eGFP) gene, which was transduced into four different cell types: human iPSC, iPSC-derived RPE, iPSC-derived cortical, and dissociated embryonic day 18 rat cortical neurons. Each cell type was exposed to three multiplicity of infections (MOI: 1E4, 1E5, and 1E6 vg/cell). After 24, 48, 72, and 96 h posttransduction, GFP-expressing cells were examined and compared across dosage, time, and cell type. Retinal pigmented epithelium showed highest AAV-eGFP expression and iPSC cortical the lowest. At an MOI of 1E6 vg/cell, all serotypes show measurable levels of AAV-eGFP expression; moreover, AAV7m8 and AAV6 perform best across MOI and cell type. We conclude that serotype tropism is not only capsid dependent but also cell type plays a significant role in transgene expression dynamics.

Amelioration of Neurosensory Structure and Function in Animal and Cellular Models of a Congenital Blindness.

Most genetically distinct inherited retinal degenerations are primary photoreceptor degenerations. We selected a severe early onset form of Leber congenital amaurosis (LCA), caused by mutations in the gene LCA5, in order to test the efficacy of gene augmentation therapy for a ciliopathy. The LCA5-encoded protein, Lebercilin, is essential for the trafficking of proteins and vesicles to the photoreceptor outer segment. Using the AAV serotype AAV7m8 to deliver a human LCA5 cDNA into an Lca5 null mouse model of LCA5, we show partial rescue of retinal structure and visual function. Specifically, we observed restoration of rod-and-cone-driven electroretinograms in about 25% of injected eyes, restoration of pupillary light responses in the majority of treated eyes, an ∼20-fold decrease in target luminance necessary for visually guided behavior, and improved retinal architecture following gene transfer. Using LCA5 patient-derived iPSC-RPEs, we show that delivery of the LCA5 cDNA restores lebercilin protein and rescues cilia quantity. The results presented in this study support a path forward aiming to develop safety and efficacy trials for gene augmentation therapy in human subjects with LCA5 mutations. They also provide the framework for measuring the effects of intervention in ciliopathies and other severe, early-onset blinding conditions.

Use of induced pluripotent stem cell models to probe the pathogenesis of Choroideremia and to develop a potential treatment.

Choroideremia (CHM) is a rare monogenic, X-linked recessive inherited retinal degeneration resulting from mutations in the Rab Escort Protein-1 (REP1) encoding CHM gene. The primary retinal cell type leading to CHM is unknown. In this study, we explored the utility of induced pluripotent stem cell-derived models of retinal pigmented epithelium (iPSC-RPE) to study disease pathogenesis and a potential gene-based intervention in four different genetically distinct forms of CHM. A number of abnormal cell biologic, biochemical, and physiologic functions were identified in the CHM mutant cells. We then identified a recombinant adeno-associated virus (AAV) serotype, AAV7m8, that is optimal for both delivering transgenes to iPSC-RPEs as well as to appropriate target cells (RPE cells and rod photoreceptors) in the primate retina. To establish the proof of concept of AAV7m8 mediated CHM gene therapy, we developed AAV7m8.hCHM, which delivers the human CHM cDNA under control of CMV-enhanced chicken ß-actin promoter (CßA). Delivery of AAV7m8.hCHM to CHM iPSC-RPEs restored protein prenylation, trafficking and phagocytosis. The results confirm that AAV-mediated delivery of the REP1-encoding gene can rescue defects in CHM iPSC-RPE regardless of the type of disease-causing mutation. The results also extend our understanding of mechanisms involved in the pathophysiology of choroideremia.

Mutant spastin proteins promote deficits in axonal transport through an isoform-specific mechanism involving casein kinase 2 activation.

Mutations of various genes cause hereditary spastic paraplegia (HSP), a neurological disease involving dying-back degeneration of upper motor neurons. From these, mutations in the SPAST gene encoding the microtubule-severing protein spastin account for most HSP cases. Cumulative genetic and experimental evidence suggests that alterations in various intracellular trafficking events, including fast axonal transport (FAT), may contribute to HSP pathogenesis. However, the mechanisms linking SPAST mutations to such deficits remain largely unknown. Experiments presented here using isolated squid axoplasm reveal inhibition of FAT as a common toxic effect elicited by spastin proteins with different HSP mutations, independent of microtubule-binding or severing activity. Mutant spastin proteins produce this toxic effect only when presented as the tissue-specific M1 isoform, not when presented as the ubiquitously-expressed shorter M87 isoform. Biochemical and pharmacological experiments further indicate that the toxic effects of mutant M1 spastins on FAT involve casein kinase 2 (CK2) activation. In mammalian cells, expression of mutant M1 spastins, but not their mutant M87 counterparts, promotes abnormalities in the distribution of intracellular organelles that are correctable by pharmacological CK2 inhibition. Collectively, these results demonstrate isoform-specific toxic effects of mutant M1 spastin on FAT, and identify CK2 as a critical mediator of these effects.

Early effects of high glucose in retinal tissue cultures Renin-Angiotensin system-dependent and -independent signaling.

The early effects of the diabetic milieu on retinal tissue and their relation to the Renin-Angiotensin system (RAS) activation are poorly known. Here we investigated RAS signaling in retinas explanted from adult rats exposed for 48 h to high glucose (HG), with or without the Angiotensin Converting Enzyme inhibitor enalaprilat, which blocks RAS. HG was observed to i) initiate a phosphotyrosine-dependent signaling cascade; ii) up-regulate Angiotensin(1) Receptor (AT(1)R); iii) activate src tyrosine kinase and increase phosphorylation of Pyk2, PLCgamma1 and ERK1/2; and iv) activate Akt and the transcription factor CREB. In the presence of enalaprilat, tyrosine phosphorylation signal and AT(1)R upregulation decreased and activation of PLCgamma1 and CREB reverted, showing their relation to RAS signaling. In line with Akt activation, no apoptosis or synapse degeneration was found. Müller glia was activated, but in a RAS-independent manner. Our results suggest that, in early phases of HG exposure, a pro-survival cell program may be induced in the retina.