Summary
The vast number of inherited blinding disorders has made ocular gene therapy an active research field in recent
years, culminating in well-publicized clinical trials. However, efficacious therapies are still elusive for two
reasons: gene expression often remains inadequate in duration and levels, and the limited packaging capacity
of standard vectors prohibits the inclusion of disease genes with their cis-regulatory elements. Overcoming
these barriers is critical for the advancement of the field and widespread clinical application. Current gene
delivery strategies use cDNA-based vectors that lack the non-coding and cis-elements found in genomic DNA
that regulate gene expression. These genomic sequences can preserve the stability of the transcript, improve
translation and produce physiologically relevant levels of expression. In this application, we test the hypothesis
that providing the entire corrective gene including its authentic promoter, enhancer, introns and untranslated
regions (UTRs) improves the levels and duration of transgene expression in mouse model of RPE65-associated
Leber Congenital Amaurosis (LCA), a disease that affects the retinal pigment epithelium (RPE). This disease
manifests in early childhood leading to a gradual vision loss often resulting in blindness. Due to the high
prevalence of RPE65-associated LCA, several clinical trials have been conducted using adeno-associated
viruses (AAV) to deliver human RPE65 cDNA. However, almost all attempts failed to halt the ongoing visual
loss. We thus aim to establish an effective therapeutic approach by delivering the whole human RPE65 gene
with its 5’/3’ regulatory elements, exons and introns to treat a mouse model of LCA (RPE65-/-). We aim to achieve
full regulation and long-term of expression in a cell-specific manner to ensure rescue of the LCA disease
phenotype. We have cloned the human RPE65 genomic fragment into an expression vector and will develop
an effective delivery platform utilizing naked DNA or DNA formulated as nanoparticles (NPs) with polylysine
peptides conjugated to polyethylene glycol (CK30PEG). We will test the longevity and levels of gene expression
after RPE65 delivery using these platforms, and evaluate their ability to mediate full phenotypic rescue in the
RPE65-/- mice. In aim1, we will engineer vectors that can achieve therapeutic levels of expression in the RPE
and in aim 2 we will evaluate long-term rescue and transduction efficiency in the RPE of the LCA model before
and after the onset of the disease phenotype. In summary, results from this application will facilitate the
advancement of non-viral gene therapy for RPE-associated diseases.