# Development of novel genetic medicine approaches to treat orphan diseases

> **NIH NIH R44** · KINEA BIO, INC. · 2024 · $303,065

## Abstract

SUMMARY:
Adeno-associated viral (AAV) vectors hold great promise for treating genetic disorders. AAVs efficiently
transduce tissues, including skeletal and cardiac muscles, which is attractive for treating multisystemic muscular
diseases. The primary shortcoming of using AAV vectors is their limited cargo capacity (~5 kb), which excludes
many disorders, such as dystro- or dysferlinopathies, from using these vectors as gene transporters. Due to the
large coding sequences of the defective genes, respectively DMD or DYSF, these muscular dystrophies have
not fully taken advantage of this promising therapeutic modality. To circumvent this limitation, miniaturized forms
(i.e, micro-dystrophin or mini-dysferlin) can be delivered by a single AAV vector. However, therapeutic minigenes
have failed to provide complete rescue in animal models, pointing to the need to express full-length proteins for
maximal benefit.
We and others produced full-length dysferlin or larger dystrophins using simultaneous delivery of two
independent AAV vectors, each one carrying the 5’ or 3’ part of the cDNA flanked by a short region of homology.
This dual vector strategy allows recombination of AAV genomes into an open-reading frame, leading to the
expression of large and highly functional proteins when administrated locally. However, low protein levels occur
after systemic delivery of high AAV doses.
To address this fundamental challenge in delivering large genetic cargo in AAVs, we propose to test a novel
method, SIMPLI-GT (Split Intein-Mediated Protein Ligation for Gene Therapy). This strategy expresses large
stable proteins with high efficiency by use of split inteins to mediate peptide trans-splicing extending the use of
AAV gene transfer to genes exceeding the maximum cargo capacity. In a first in vivo proof-of-concept study, we
showed the feasibility of dual or triple AAV approaches to express mini- or full-length dystrophin. Moreover, using
AAV-Myo, we found that low dose (2x1013 vg/kg) generates large dystrophins in striated muscles with nearly
complete physiological rescue. Further, we used this method to efficiently reconstitute full-length dysferlin in
vitro. Therefore, we propose to apply SIMPLI-GT for the treatment of dysferlinopathies:
Phase 1: Aim1: Demonstrate the feasibility of dual-AAV delivery of split dysferlin by IM injections.
Phase 1, Aim 2: Study the functionality of intein-generated dysferlin in murine and human-derived myoblast.
Phase 2: Aim 1: Evaluate and optimize systemic delivery regimen of AAV-Myo intein/dysferlin in mice.
Phase 2, Aim 2: Validate the effectiveness of full-length intein-generated dysferlin in human iPSC-derived 3D-
engineered muscle tissues.
Phase 2, Aim3: Refine the optimal stoichiometry of dual vector in a dose-finding study in mice.
Phase 2, Aim 4: Evaluate the biodistribution of AAV-Myo split intein/dysferlin following intravenous delivery in
large animal.

## Key facts

- **NIH application ID:** 10922611
- **Project number:** 1R44AR083801-01A1
- **Recipient organization:** KINEA BIO, INC.
- **Principal Investigator:** Martin K Childers
- **Activity code:** R44 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $303,065
- **Award type:** 1
- **Project period:** 2024-09-19 → 2025-03-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10922611

## Citation

> US National Institutes of Health, RePORTER application 10922611, Development of novel genetic medicine approaches to treat orphan diseases (1R44AR083801-01A1). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10922611. Licensed CC0.

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