Abstract
Purpose :
Therapeutics that deliver proteins directly to cells would offer several advantages over small molecule and viral vector-based approaches including unmatched specificity, low immunogenicity, and rapid transient expression. Protein therapies would be especially beneficial for treating inner retinal cell types like retinal ganglion cells (RGC) and glia, which in humans are difficult to transfect with viral vectors. However, efficient protein delivery to the inner retina has not been previously achieved. We are testing whether combinations of modified cell penetrating peptides (CPPs) and cationic amphiphilic drug (CAD) that enhance protein delivery to retinal cells. Effective protein therapies for the inner retina, would be impactful for drug development for retinal diseases including optic neuropathies like glaucoma. Our platform could be used to deliver therapeutic factors for a wide range of applications including neuroprotection, cellular regeneration, or gene editing.
Methods :
We tested the ability of two modified TAT CPPs (dLLTAT, dWWTAT) to enhance transduction of TAT-cre in adult mouse retina. TAT-cre alone or with 5-80µM of TAT CPPs was intravitreally injected into Ai9 mice, which carry a cre-dependent TdTomato reporter. TAT CPPs+TAT-cre were also combined with acepromazine, a readily available and approved CAD, which was predicted to enhance endosomal release. After 7 days, retinas were collected to measure cre-recombination efficiency and assess toxicity. Retinal whole mounts were immunostained for markers that label RGCs, microglia, astrocytes, and Müller glia markers and imaged by confocal microscopy to determine the cell type-specificity of transduction.
Results :
We have determined that TAT CPPs dramatically improve transduction of TAT-cre in RGCs, astrocytes, and Müller glia. However, some RGC toxicity was observed at higher doses. We are currently performing quantification of marker colocalization to determine transduction efficiency in each of the previous cell populations.
Conclusions :
Our CPP platform demonstrates enhanced nuclear delivery of proteins to inner retinal cell populations. Further testing is required but our results demonstrate that incorporating these methods can be a powerful tool for delivery of therapeutic proteins to RGCs and glia in the inner retina. Optimization of these compounds has the potential to be transformative for the treatment of blinding diseases.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.