Project Summary/Abstract:
Our goal is to advance our current intravitreal gene therapy platform consisting of DNA nanoparticles (DNA-
NPs)/hyaluronic acid nanospheres (HA-NSs) to deliver large genes in order to develop safe/effective therapies
for visual loss in Usher Syndrome type 2A (USH2A). Currently, there are no treatments for USH2A. Our
complementary expertise in nanoformulation and in molecular biology/physiology of the visual and auditory
systems will facilitate the advancement of our non-viral therapy to rescue vision loss associated with the most
common USH2A mutation in usherin, c.2299delG. Our platform consists of: 1) CK30PEG DNA-NPs that
efficiently transfect photoreceptors (PRs), exert no toxic effects after multiple injections, and are distributed
throughout the subretinal space in both mouse and baboon models. Critically, these DNA-NPs provide
structural/functional rescue in multiple mouse retinal disease models. 2) HA-NS made of hyaluronic acid which
house DNA or DNA-NPs for controlled long-term DNA release. HA-NSs are capable of reaching the PRs after
intravitreal delivery. 3) Sulfotyrosine (ST), which is a non-toxic small molecule that enhances retinal penetration
of intravitreally delivered HA-NS. 4) Engineered DNA vectors with genomic elements to carry and promote
expression of large therapeutic genes. Developing an effective treatment for USH2A has been challenging due
to its large coding sequence (15.8 kb) that has precluded its delivery using standard approaches and the
presence of multiple isoforms with functions that are not fully understood. We have already cloned two usherin
isoforms to be tested with our innovative platform (DNA-NP/HA-NS/ST) to safely advance gene therapy for
USH2A. We will use three previously generated knockin mouse models to learn more about usherin transcripts,
isoforms, and their role in PRs to help develop well-designed therapies for USH2A. The models are: 1) UNYFP,
where YFP with a 3XFlag tag was inserted after the secretory sequence at the N-terminus of Ush2a; 2)
UCmKate, where mKATE2 with a 3XMyc tag was inserted at the C-terminus before the transmembrane domain
of Ush2a; and 3) ush2aG/G, where the mouse-equivalent of human c.2299delG mutation was introduced followed
by the human 20 aa extension followed by 3XFlag tag. In aim 1, we will evaluate Ush2a isoforms in the retina
vs cochlea, the mechanism of auditory and visual defects in the ush2aG/G model, and in vitro testing of the most
suitable Ush2a isoform(s) for gene therapy. In aim 2, we will investigate the transduction efficiency and
therapeutic efficacy of Ush2a-containing intravitreally delivered DNA-NPs/HA-NSs/ST in the ush2aG/G model.
First, we will perform short-term studies to evaluate the ability of intravitreal vs subretinally delivered usherin to
generate gene expression in PRs in the ush2aG/G retina. Second, we will longitudinally assess long-term
therapeutic efficacy of the platform/vectors/isoform identified from Part I to provide lasting phenotypic rescue in
the ush2aG/G model. In summary, this proposal will provide a solid foundation for understanding the function of
each usherin isoform and develop an effective gene therapy platform to treat USH2A associated visual defects.