Department of Ophthalmology and Visual Sciences

University of Iowa, Institute for Vision Research Center

Iowa City, Iowa

BASIC RESEARCH PROJECT

Gene therapy vectors to treat retinal degenerations associated with ABCA4, USH1D, and USH1F

Research Interests

Mutations in the CEP290 gene are a leading cause of Leber congenital amaurosis (LCA), a hereditary retinal dystrophy that causes severe vision loss in early childhood. Although gene therapy has shown promise for treating inherited retinal degenerations, CEP290LCA remains as a challenging target because of the gene’s large size and diverse mutations found throughout the gene. Our interest is aimed at overcoming the limitation of current gene therapy approaches and developing a novel strategy to treat CEP290-LCA by developing a mutation-independent, generic gene therapy vector for the gene.  Successful completion of this study will not only move us forward to the cure of CEP290-LCA but also provide a framework for the development of gene therapy vectors targeting other large gene associated genetic diseases.

The overarching goal of Dr. Seo’s research program is to develop highly efficient, adenoassociated virus (AAV)-based large gene delivery systems for retinal gene therapy. Although AAV has been proven to be a safe and effective gene delivery vehicle for retinal gene therapies, its main drawback is the limited packaging capacity. With the ultimate goal of developing versatile and potent gene delivery methods for large therapeutic genes, in the proposed study, we will focus on evaluating gene therapy vectors designed for the treatment of retinal degenerations associated with ABCA4, PCDH15 (USH1F), and CDH23 (USH1D).

Plans for 2024

In 2024, the Seo lab will continue their study on dual AAV-ABCA4 gene therapy vectors in our ABCA4 rat model. They will evaluate the long-term therapeutic efficacies of the dual AAVABCA4 vectors, using the two phenotypes identified in Abca4 knockout rats as key indicators for assessment. In addition, they will subretinally deliver dual AAV-PCDH15 and triple AAV-CDH23 vectors to mouse retinas, determine optimal doses for each vector set, and assess the production and localization of full-length PCDH15 and CDH23 proteins.

Specific Aims:

Aim 1) Evaluate the long-term therapeutic efficacies of dual AAV-ABCA4 vectors in Abca4 knockout rats;

Aim 2) Assess the restoration of CDH23 (USH1D) and PCDH15 (USH1F) expressions in mouse retinas.

Progress in 2023

Novel dual AAV approaches to treat ABCA4-associated retinal degeneration

Dr. Seo developed three strategies for the delivery of large genes using AAV: i) the gp41 split-intein-mediated protein trans-splicing, ii) the SpyTag/SpyCatcher pair-mediated protein ligation, and iii) the CRE/lox-mediated unidirectional DNA reconstitution systems.

Following his preliminary study in Abca4 knockout rats, Dr. Seo opted for the proteinlevel reconstitution methods for ABCA4. His lab also identified two distinct phenotypes in Abca4 knockout rats, which serve as valuable indicators for evaluating the therapeutic efficacies of their gene therapy vectors. Furthermore, the team created gene therapy vectors for USH1D and USH1F utilizing these three approaches. After assessing their reconstitution efficiencies in 293T cells, Dr. Seo determined that the CRE/lox-mediated DNA reconstitution system is the most efficient choice for these genes.

In 2023, Dr. Seo proposed to focus his efforts on determining optimal compositions of the three sets of dual AAV-ABCA4 gene therapy vectors in his ABCA4 animal model. The Seo lab will determine the ratios and doses of the dual AAV vectors to maximize the production of full-length ABCA4 proteins and therapeutic effects, while minimizing the production of truncated proteins and potential cellular toxicities, and compare therapeutic efficacies of the three approaches in Abca4-null rats. The outcome of this study will provide crucial pre-clinical data before clinical studies in human patients.

Specific Aims:

Aim 1. Determine the optimal ratios of the N- and the C-terminal vectors of the split intein and the SpyTag/SpyCatcher sets.

Aim 2. Determine the optimal doses of the AAV-ABCA4 vector sets.

Aim 3. Assess the ability of AAV-ABCA4 vectors to restore ABCA4 functions in-vivo and determine the most effective approach for ABCA4 gene therapy.

Progress in 2022

Novel dual AAV approaches for efficient delivery of large genes

The overarching goal of the 2022 research program was to develop highly effective, AAV-based large gene delivery systems for retinal gene therapy. Although adeno-associated virus (AAV) is a proven safe gene delivery vehicle for retinal gene therapy, its main drawback is the limited packaging capacity. The proposed study was to develop generic and effective gene therapy strategies for large therapeutic genes.

Dr. Seo has established three approaches to deliver large genes using AAV. One approach utilizes gp41 split intein-mediated protein trans-splicing to re-join protein fragments. The second approach utilizes a pair of high-affinity polypeptides (SpyTag and SpyCatcher) that form a covalent bond upon binding. The third uses the CRE/lox-mediated site-specific DNA recombination system to facilitate the assembly of AAV genomes in a pre-designed configuration. These approaches were used to produce ABCA4 and CEP290 gene therapy vectors and compared their reconstitution efficiencies in cultured mammalian cells. Additionally, the lab began to test the therapeutic efficacies of ABCA4 gene therapy vectors in an animal model.

Specific Aims: Aim 1: Validate and optimize novel dual AAV vector strategies in a human cell line. Aim 2: Evaluate the safety and efficacy of the new gene therapy strategies using Abca4null mice in-vivo.

Progress in 2021

Development of mutation-independent gene therapy approaches for CEP290-LCA

Dr. Seo generated an array of split CEP290 constructs with high-affinity peptide pairs to facilitate the re-joining of N- and C-terminal halves of CEP290. His research team is testing their functionality in CEP290 mutant cells. They also generated a set of short promoters that drive transgene expression at various levels in mouse retinas. These promoters will be used in dual AAV vectors for moderate- to low-level transgene expression. In addition, the Seo lab developed new strategies to improve the reconstitution efficiencies of the split constructs either at the DNA or protein levels.