BioRxiv. 2023 Jan 21
Christiano R. R. Alves, Leillani L. Ha, Rebecca Yaworski, Cicera R. Lazzarotto, Kathleen A. Christie, Aoife Reilly, Ariane Beauvais, Roman M. Doll, Demitri de la Cruz, Casey A. Maguire, Kathryn J. Swoboda, Shengdar Q. Tsai, Rashmi Kothary, and Benjamin P. Kleinstiver
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AAV vector packaging For genome editing experiments, two AAV9 vectors encoding ABE8e-SpRY split into N-term and C-terminal fragments via an Npu intein (as described above and similar to as previously reported59) paired with gRNA A8 were packaged by PackGene Biotech Inc. (Worcester, MA). Request Quote

Research Field: spinal muscular atrophy

Keywords: CRISPR, genome editing, adenine base editors (ABEs), neuromuscular diseases, SMA

AAV Serotype: AAV9

Dose: For tissue transduction tests, intracerebroventricular (ICV) injections were performed in two litters of SMND7 mice at postnatal day 1 (P1). For each mouse, 3 μL ICV injections contained 2 x 10^10 vg AAV9-EGFP or AAV-FEGFP.

Routes of Administration: intracerebroventricular (ICV) injections

Targeted organ: brain

Animal or cell line strain: SMND7 mice (FVB.Cg-Grm7Tg(SMN2)89Ahmb Smn1tm1Msd Tg (SMN2*delta7) 4299Ahmb/J)


Spinal muscular atrophy (SMA) is a devastating neuromuscular disease caused by mutations in the SMN1 gene. Despite the development of various therapies, outcomes can remain suboptimal in SMA infants and the duration of such therapies are uncertain. SMN2 is a paralogous gene that mainly differs from SMN1 by a C•G-to-T•A transition in exon 7, resulting in the skipping of exon 7 in most SMN2 transcripts and production of only low levels of survival motor neuron (SMN) protein. Genome editing technologies targeted to the SMN2 exon 7 mutation could offer a therapeutic strategy to restore SMN protein expression to normal levels irrespective of the patient SMN1 mutation. Here, we optimized a base editing approach to precisely edit SMN2, reverting the exon 7 mutation via an A•T-to-G•C base edit. We tested a range of different adenosine base editors (ABEs) and Cas9 enzymes, resulting in up to 99% intended editing in SMA patient-derived fibroblasts with concomitant increases in SMN2 exon 7 transcript expression and SMN protein levels. We generated and characterized ABEs fused to high-fidelity Cas9 variants which reduced potential off-target editing. Delivery of these optimized ABEs via dual adeno-associated virus (AAV) vectors resulted in precise SMN2 editing in vivo in an SMA mouse model. This base editing approach to correct SMN2 should provide a long-lasting genetic treatment for SMA with advantages compared to current nucleic acid, small molecule, or exogenous gene replacement therapies. More broadly, our work highlights the potential of PAMless SpRY base editors to install edits efficiently and safely.

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