Abstract
Purpose :
Monogenic retinal dystrophies can be treated with gene replacement therapy (GRT) due to easy delivery into retina. Adeno-associated virus vectors (AAVs) mediated gene therapy has become available for clinical use for LCA2. However, they are limited by a 4.7 kb loading capacity in therapies that require delivery of large genes such as Stardardt disease (STGD). Previously, we demonstrated the efficacy of a pH-sensitive lipid ECO nanoparticle-based gene therapy for STGD. Here, we optimized the ABCA4 therapeutic plasmid with an SV40 enhancer to achieve enhanced gene expression and to improve the gene expression efficiency and the therapeutic efficacy of ECO/pABCA4 therapy.
Methods :
The SV40 enhancer was cloned into the ABCA4 plasmid (pCMV-ABCA4). The in vitro gene expressions of ECO/pCMV-ABCA4 and ECO/pCMV-ABCA4-SV40 nanoparticles were evaluated in ARPE-19 cells at different N/P ratios and different pDNA doses (N/P=6, 8, 10; pDNA concentration 1 µg/ml and 2 µg/ml). qRT-PCR was used to evaluate ABCA4 expression 48 h after transfection.
Results :
Increased ABCA4 mRNA expression was observed with the increase of N/P ratios and pDNA doses. Most importantly, after incorporating in the SV40 enhancer, significantly enhanced ABCA4 mRNA expression was observed for pCMV-ABCA4-SV40 across all N/P ratios and doses, demonstrating enhanced ABCA4 expression due to the modification with the SV40 enhancer.
Conclusions :
In conclusion, enhanced ABCA4 mRNA expression was achieved by therapeutic plasmid optimization with the SV40 enhancer. The ECO/pCMV-ABCA4-SV40 nanoparticle-based GRT can be a promising GRT to achieve enhanced ABCA4 expression, thereby improve the therapeutic efficacy for treating Stargardt disease.
This is a 2020 ARVO Annual Meeting abstract.