Vector engineering for non-viral delivery of large genomic DNA to the RPE

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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.