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C6 - Gene Targeting and Gene Correction New Technologies

11: Efficacy and Integration of a Non-Viral ABCA4 Transposon in Treating Stargardt Disease: Evidence from Mice and Primate Studies

Type: Oral Abstract Session

Presentation Details
Session Title: New Technologies for Gene Targeting and Gene Correction

Introduction: Stargardt disease (STGD1) is a genetic disorder causing progressive central blindness. Novel integrative non-viral gene therapy applications overcome the limitations of low immunogenicity and durability. In addition, other advantages specific to STGD1 include the potential to deliver the large (6.8 kb) ATP Binding Cassette Subfamily A Member 4 (ABCA4) gene to correct the numerous ABCA4 mutations in patients. The dysfunctional ABCA4 transporter causes STGD1 by altering the transfer of all-trans-retinal (ATR) and the accumulation of diretinoid-pyridinium-ethanolamine (A2E) and retinal dimer conjugates. In Abca4-/- and Rdh8-/- double knock-out (dKO) mice, A2E accumulation is accelerated compared to Abca4-/- single KO mice because the deficient all-trans-retinol dehydrogenase enzyme (Rdh8) augments the ATR defects. As such, we performed subretinal injections of an optimized lipid nanoparticle (LNP) to deliver an mRNA encoding a novel bioengineered transposase derived from the mammal, Myotis lucifugus (bMLT), and a DNA expressing full-length human ABCA4 (fl-ABCA4) under the control of a constitutive promoter. This transposition process, called Gene Coding technology, was also tested in non-human primates (NHPs) to confirm interspecies translation. Methods: We co-encapsulated bMLT mRNA and fl-ABCA4 cDNA in LNPs to evaluate dosing, tolerability, and subretinal administration (1uL per eye) in three groups of dKO mice at postnatal day 14 to 17: vehicle alone (n=2) and treated groups with low dose (LD) (n=4) and high dose (HD) (n=6). The three dKO groups were compared to wild-type (WT) mice (n=9) by serial fundoscopy, optical coherence tomography (OCT) both within and outside the treated retinal area, and histological sectioning. In 10-week-old mice, terminal A2E measurements were analyzed in the three dKO groups and age-matched WT mice at 50 days after subretinal injection using a reversed phase chromatography (RP-HPLC) method with analyte detection at a wavelength of 430 nanometers. To assess ABCA4 expression and genomic integration, we administered the same DNA/RNA co-encapsulated LNP by subretinal injection in African Green monkeys (n=2). Immunohistochemical (IHC) studies co-staining for ABCA4 and Arrestin-3, digital droplet polymerase chain reaction (ddPCR), and quantitative reverse transcription (RTqPCR) were used to show ABCA4 integration and photoreceptor localization. Results: There were no detectable differences in retinal thickness by OCT outside the treated area in 10-week-old dKO mice compared to WT mice. Within the treated area, LD and HD treated dKO mouse groups showed no differences in OCT retinal layer measurements compared to the untreated controls, implying good tolerability. Untreated dKO mice showed a significant (p<0.0001) increase in A2E levels compared to age-matched WT mice. At 50 days post LNP injection, dKO HD treated mice showed a 40% reduction in A2E compared to untreated controls (p=0.03). NHP retinal IHC staining showed ABCA4 protein co-localization with the cone photoreceptor marker, Arrestin-3, and evidence of integration by ddPCR. Conclusions: Fifty days after treatment, dKO mice exhibited a reduction in A2E levels compared to untreated dKO mice suggesting correction of the Stargardt phenotype. NHP studies confirmed expression of ABCA4 in photoreceptors and the interspecies translatability of Gene Coding technology. This non-viral integrative gene therapy represents a potentially promising, one-time treatment for STGD1.

Plain Language Summary
Stargardt disease, an inherited eye disease, causes central blindness. There is no effective treatment available today. Precision genetic medicines for inherited eye disorders like Stargardt, need to deliver large genes to specific specialized cells in the eyes called photoreceptors. Existing therapies, based on genetic tools found in bacteria, viruses, and other lower life forms, are unable to do this and have significant safety risks. We have developed a new kind of gene therapy, based on an enzyme that naturally occurs in mammals, that is able to insert the Stargardt gene in these photoreceptors in mice and monkeys. We show in mice with Stargardt that the therapy safely corrects the disease process. This next generation of gene therapy is designed to avoid the potential harm caused by older types of therapies and to offer a new tool to stop serious diseases like Stargardt with a single treatment.

Michelle E. LeBlanc, Chuanqi Peng, Shashank Shukla, Mitchell R. Kopacz, Kelsey A. Rush, Jungyeon Hwang, Michael J. Crowley, Subhadeep Dutta, Hugues Bernard, Ria Vashishth, Jonathan T. Lu, Sandeep Nema, Joseph J. Higgins, Joseph J. Senn

SalioGen Therapeutics, Lexington, MA"

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