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.

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).

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).

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.