Rescue of Photoreceptor Degeneration by Curcumin in Transgenic Rats with P23H Rhodopsin Mutation

The P23H mutation in the rhodopsin gene causes rhodopsin misfolding, altered trafficking and formation of insoluble aggregates leading to photoreceptor degeneration and autosomal dominant retinitis pigmentosa (RP). There are no effective therapies to treat this condition. Compounds that enhance dissociation of protein aggregates may be of value in developing new treatments for such diseases. Anti-protein aggregating activity of curcumin has been reported earlier. In this study we present that treatment of COS-7 cells expressing mutant rhodopsin with curcumin results in dissociation of mutant protein aggregates and decreases endoplasmic reticulum stress. Furthermore we demonstrate that administration of curcumin to P23H-rhodopsin transgenic rats improves retinal morphology, physiology, gene expression and localization of rhodopsin. Our findings indicate that supplementation of curcumin improves retinal structure and function in P23H-rhodopsin transgenic rats. This data also suggest that curcumin may serve as a potential therapeutic agent in treating RP due to the P23H rhodopsin mutation and perhaps other degenerative diseases caused by protein trafficking defects

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 β-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. Keywords: Choroideremia, Human iPSCs, Retinal pigmented epithelium, Gene therapy, Adeno-associated virus, Phagocytosis, Prenylation, REP

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

Induced pluripotent stem cells as a model to test gene augmentation therapy for choroideremia (Conference Abstract)

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Induced pluripotent stem cells as a model To test gene augmentation therapy for Choroideremia

Choroideremia (CHM) is an X-linked, blinding, inherited retinal degeneration that is symptomatic in childhood and leads to total blindness in midlife. The disease is characterized by the degeneration of photoreceptors, retinal pigment epithelium (RPE) and choriocapillaris. The causative factor for CHM was identifi ed as a defect in the Rab Escort Protein 1 (REP1) which is involved in prenylation of Rab proteins. The nature and onset of the disease and the size of the gene makes CHM an excellent target for recombinant adeno-associated virus (rAAV) mediated gene therapy. However, the animal model of this disease is not an exact replica of the condition and is not readily available. Therefore, we generated and evaluated induced pluripotent stems cells (iPSCs) from CHM subjects as in vitro models to explore the potential of gene augmentation therapy. A recombinant adeno-associated virus (AAV2) was generated that delivers the wild-type human CHM cDNA driven by a constitutive promoter. Infection of iPSCs with AAV2-CHM resulted in the expression of the CHM protein and rescue of enzymatic function in the defective cells. The gene transfer is effi cient and appears to be safe in the short-term, as shown by studies in affected cells and in normalsighted animal models. These studies not only aid in deciphering the pathogenetic mechanisms of the disease, but also pave the way for a human gene therapy clinical trial for CHM

Bioavailability of curcumin.

<p>Amount of curcumin detected by LC-MS/MS analysis in the eye and brain tissues of curcumin administered SD rats. (A) Representative LC-MS/MS chromatogram of curcumin (standard) and naringenin (internal standard). (B) Delivery of curcumin to ocular tissues and brain of SD rats following oral administration. Data are expressed as mean ± SD for n = 4.</p