Gene Augmentation of CHM Using Non-Viral Episomal Vectors in Models of Choroideremia

Abstract

Choroideremia (CHM) is an X-linked chorioretinal dystrophy leading to progressive retinal degeneration that results in blindness by late adulthood. It is caused by mutations in the CHM gene encoding the Rab Escort Protein 1 (REP1), which plays a crucial role in the prenylation of Rab proteins ensuring correct intracellular trafficking. Gene augmentation is a promising therapeutic strategy, and there are several completed and ongoing clinical trials for treating CHM using adeno-associated virus (AAV) vectors. However, late-phase trials have failed to show significant functional improvements and have raised safety concerns about inflammatory events potentially caused by the use of viruses. Therefore, alternative non-viral therapies are desirable. Episomal scaffold/matrix attachment region (S/MAR)-based plasmid vectors were generated containing the human CHM coding sequence, a GFP reporter gene, and ubiquitous promoters (pS/MAR-CHM). The vectors were assessed in two choroideremia disease model systems: (1) CHM patient-derived fibroblasts and (2) chm^ru848 zebrafish, using Western blotting to detect REP1 protein expression and in vitro prenylation assays to assess the rescue of prenylation function. Retinal immunohistochemistry was used to investigate vector expression and photoreceptor morphology in injected zebrafish retinas. The pS/MAR-CHM vectors generated persistent REP1 expression in CHM patient fibroblasts and showed a significant rescue of prenylation function by 75%, indicating correction of the underlying biochemical defect associated with CHM. In addition, GFP and human REP1 expression were detected in zebrafish microinjected with the pS/MAR-CHM at the one-cell stage. Injected chm^ru848 zebrafish showed increased survival, prenylation function, and improved retinal photoreceptor morphology. Non-viral S/MAR vectors show promise as a potential gene-augmentation strategy without the use of immunogenic viral components, which could be applicable to many inherited retinal disease genes.

Keywords: S/MAR; choroideremia; inherited retinal disease; non-viral gene therapy.

Figure 1

Generation of pS/MAR-CHM vectors. (A) pS/MAR-CHM vectors were generated by inserting the human CHM coding sequence (CDS) into the S/MAR plasmid backbone with 1 of 5 promoters: CMV (V1), CAG (V2), hPGK (V3), EF1α (V4), or CHMp (V5). The plasmids also contained a GFP sequence. (B) All 5 vectors produced GFP expression in transfected HEK-293 cells, which was observed at varying levels at 48 h post-transfection. (C) REP1 protein expression was examined by Western blot in the non-transfected (NT) and transfected HEK-293 cells, with the CMV (V1) and CAG (V2) promoter versions driving the highest expression levels.

Figure 2

Rescue of CHM patient-derived fibroblasts. (A) Human dermal fibroblasts (HDF) from a CHM patient with the c.126C>G (p.Y42*) mutation were transfected with pS/MAR-CAG-CHM (V2). GFP expression was detected at 48 h post-transfection and was maintained at 120 h post-FACS. (B) Using Western blot, REP1 protein was detected in the transfected CHM patient fibroblasts at 11 days and 35 days post-FACS. REP1 was detected in wild-type control fibroblasts but was almost completely absent in non-transfected CHM fibroblasts. (C) As a measure of prenylation function, an in vitro prenylation assay was performed to detect the size of the unprenylated Rab protein pool in transfected versus non-transfected CHM fibroblasts at 7 days post-transfection. A significant decrease in the presence of unprenylated Rabs was observed in the transfected fibroblasts. * p < 0.05. (ns = non significant).

Figure 3

Micro-injection of chm^ru848 zebrafish with pS/MAR-CHM. Wholemount color and fluorescent images of (A) wild-type, (B) chm^ru848, (C) pS/MAR-CMV-CHM-injected wild-type, and (D) pS/MAR-CMV-CHM-injected chm^ru848 zebrafish at 5 days post-fertilization. (E) Comparison of survival in injected and un-injected chm^ru848 zebrafish larvae. (F) Western blot for human-specific REP1 protein with the 2F1 antibody in injected and un-injected chm^ru848 zebrafish larvae. Human REP1 was detected in the injected zebrafish only. (G) Prenylation assay to detect levels of un-prenylated Rabs in wild-type, injected, and un-injected chm^ru848 zebrafish larvae (* = p < 0.05; *** = p < 0.001).

Figure 4

Retinal expression of pS/MAR-CHM in chm^ru848 zebrafish. Retinal sections from 5 days post-fertilization wild-type, chm^ru848, and pS/MAR-CMV-CHM-injected chm^ru848 zebrafish were stained with PNA lectin and anti-rhodopsin to detect cone and rod cell outer segments, respectively. GFP was also detected in the photoreceptors of the injected zebrafish. Scale bar = 10 µm.