Abstract
Purpose :
Retinitis pigmentosa (RP), a group of inherited retinal dystrophies affecting 1 in 3000 – 7000 people, is characterized by disruption of rod photoreceptors, leading to rod-cone degeneration. Mutations in the proto-oncogene tyrosine-protein kinase MER (MERTK) gene cause ~3% of RP cases. MERTK is involved in the recycling of photoreceptor outer segments. A previous gene therapy clinical trial for MERTK-associated RP using recombinant adeno-associated virus serotype 2 (rAAV2)-mediated delivery of VMD2-driven hMERTK showed acceptable ocular and systemic safety profiles; however, long term improvement in visual acuity was limited, possibly due to insufficient transgene expression. Therefore, to determine if MERTK expression can be enhanced with the use of a stronger promoter or codon optimization, we transfected four variations of the rAAV2-hMERTK construct into HEK293T and ARPE-19 cell lines.
Methods :
Four rAAV2-hMERTK plasmids were designed to express wildtype (wt) or codon optimized (codop) MERTK cDNA, driven by either the ubiquitous hybrid cytomegalovirus chicken β-actin (CBA) or the RPE-specific vitelliform macular dystrophy-2 (VMD2) promoter. HEK293T and ARPE-19 cells were transfected with VMD2-hMERTKwt, VMD2-hMERTKcodop, CBA-hMERTKwt, or CBA-hMERTKcodop plasmids. For all conditions, MERTK expression was quantified by digital PCR and Western Blot.
Results :
MERTK expression in ARPE-19 cells transfected with CBA-hMERTKwt, VMD2-hMERTKwt, or VMD2-hMERTKcodop was upregulated (700-, 250-, and 3-fold, respectively) from baseline (~21 transcripts/ng), as compared to non-transfected cells in which no change was detected. Protein expression was also upregulated in ARPE-19 cells transfected with VMD2-hMERTKcodop, CBA-hMERTKwt, or CBA-hMERTKcodop. HEK293T cells exhibited a 100-fold upregulation in MERTK expression following transfection with CBA-hMERTKwt, whereas no change in expression was observed following transfection with the RPE-specific VMD2-hMERTKwt negative control.
Conclusions :
rAAV2-hMERTK plasmid constructs efficiently upregulate MERTK expression in HEK293T and ARPE-19 cell lines. These data highlight potential advantages of testing native and codon optimized versions of the transgene under the control of global versus cell-specific promoters to evaluate expression efficiency in cell culture models. Future studies in a Mertk knockout mouse model will test the efficacy of rAAV2-mediated MERTK gene therapy.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.