Abstract Details

B5 - Neurologic Diseases (excluding Ophthalmic and Auditory Diseases)

36: Developing an AAV-Based Gene Replacement Therapy for Mitochondrial Alanyl-tRNA Synthetase 2 (AARS2) Leukodystrophy

Type: Oral Abstract Session

Presentation Details

Session Title: Neurologic Diseases I






The nuclear AARS2 gene encodes mitochondrial alanyl-tRNA synthetase 2 that is responsible for attaching alanine onto the cognate tRNA in mitochondria. The resulting alanyl-tRNA participates in mitochondrial translation that produces the key proteins constituting the mitochondrial respiratory complexes. AARS2 mutations have been implicated in two recessive diseases: infantile cardiomyopathy and late-onset leukodystrophy. Currently, there is no treatment available for AARS2 related diseases.Animal models that recapitulate human disease are of paramount importance to AAV gene therapy development. However, homozygous Aars2 knockout (KO) mice are embryonically lethal. Therefore, we sought to engineer an AARS2 leukodystrophy patient-derived mutation in the mouse endogenous Aars2, and successfully generated mice homozygous for the M146T mutation. The Aars2^M146T/M146T mice showed 50%-80% reduction in AARS2 protein (but not mRNA) in multiple tissues compared with WT littermates. We are currently characterizing the phenotype of the Aars2^M146T/M146T mice as they age. As another approach to overcome embryonic lethality due to constitutive Aars2 knockout, we generated a mouse line harboring a loxP-flanked human AARS2 transgene, which is expected to rescue endogenous Aars2 KO and enable conditional AARS2 depletion after crossing with a Cre driver line. Resulting mouse strains will allow us to dissect the impact of AARS2 deficiency in specific tissue and cell types, and to test targeted AAV-based gene therapy.To develop a potential gene replacement therapy, we generated recombinant AAV9 vectors expressing human AARS2 driven by a ubiquitous promoter (rAAV9.hAARS2), and injected wild-type (WT) mice to test the safety of AARS2 over-expression after birth. We found that systemic delivery to neonatal mice via facial vein injection caused animal death within a week, whereas intracerebroventricular injection was well tolerated. TUNEL assay revealed apoptotic hepatocytes in the animals receiving facial vein treatment, suggesting liver toxicity. Therefore, we modified the expression cassette to include miR122 binding sites that enable liver-specific, miR122-mediated transgene silencing (rAAV9.hAARS2.miR122BS). Indeed, the modified vector design prevented hepatocyte apoptosis and animal lethality following facial vein delivery. Together, our preliminary results support the safety of brain-directed, liver-detargeted AARS2 gene replacement therapy. Studies on disease pathomechanisms and in vivo gene therapy efficacy involving various mouse models will be presented and discussed. (^#Co-corresponding authors)

Plain Language Summary

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Ruxiao Xing¹, Lingzhi Ren¹, Nan Liu¹, Chen Zhou¹, Jialing Liang¹, Jiaming Wang^1,2, Guangping Gao#^1,2, Dan Wang#<sup>1,3

1</sup>Horae Gene Therapy Center, University of Massachusetts Chan Medical School, Worcester, MA,²Department of Microbiology and Physiological Systems, University of Massachusetts Chan Medical School, Worcester, MA,³RNA Therapeutics Institute, University of Massachusetts Chan Medical School, Worcester, MA"