Thanks, John! Hope all is well

Please check out these links:
- free link to our manuscript: rdcu.be/eFAEz
- MGH press release: www.massgeneralbrigham.org/en/about/new...
- coverage in Fierce Biotech: www.fiercebiotech.com/research/cus...
- coverage in Precision Medicine Online: www.precisionmedicineonline.com/regulatory-n...

Together, our results demonstrate the value in using the mutant allele as a substrate in engineering campaigns, resulting in a bespoke enzyme with improved efficacy and safety compared to wild-type SpCas9 base editors.
🔧🧬 🚀

This was a wonderful collaboration and was funded by $$ from the NIH.

In vivo base editing via AAV9 or a smooth muscle cell tropic capsid AAV-PR (@casey-maguire-lab.bsky.social) substantially extended lifespan of treated MSMDS mice (+ key phenotypic changes). Notably, most AAV-ABE treated MSMDS mice died from bowel impaction, which should be avoidable in human.

@markelindsay.bsky.social and Dr. Musolino's labs created an MSMDS mouse model (that recapitulated many human phenotypes!), which we then utilized for in vivo experiments to deliver our optimized enhanced VRQR (eVRQR) ABE via a dual AAV approach. 🐁

We then performed a comprehensive off-target nomination workflow via an updated GUIDE-seq2 pipeline and the base editor-specific CHANGE-seq-BE assay, followed by rhAmpSeq-based validation of OTs (finding evidence of a small number of intergenic OTs).

To maximize on-target editing at this NGA PAM target site, we undertook a mutant allele-specific engineering approach to develop a bespoke enzyme with enhanced efficiency. Stacking activity-potentiating mutations with VRQR improved correction with minimal bystanders! 💯 ⬆️

To avoid this bystander we spatially minimized access to the M178 adenine by shifting the edit window of the ABE by 4 nt. Using either a PAM-relaxed SpG enzyme (@russelltwalton.bsky.social) or our more selective SpCas9-VRQR, this new target site dramatically minimized the M178V bystander! 🍻

By testing the impact of ACTA2 variant overexpression in human smooth muscle cells, we found that the M178V bystander edit resulted in disrupted actin polymerization; not as severely as the MSMDS R179H mutation, but this is a bystander edit that should be avoided. 🫨

When testing latest generation ABEs in an ACTA2 R179H cell line, we observed high levels of R179H correction (🎉!), but unfortunately most corrected alleles also contained a bystander edit that causes an ACTA2 M178V mutation (😩🤔). Does this bystander edit matter?

MSMDS is caused by a point mutation in the ACTA2 gene, which results in an R179H amino acid substitution - a mutation that should be correctable using adenine base editors (ABEs).

Multi-systemic smooth muscle dysfunction syndrome (MSMDS) is a rare a genetic vascular disease affecting children that Dr. Musolino sees in clinic, known to cause stroke, aortic dissection, and death. There are no therapies, motivating us to explore genome editing-based Tx for these patients.

This project has been a 5+ yr collaboration with Patricia Musolino and @markelindsay.bsky.social's groups, involving 30 co-authors! 🤝🙏🤲

Huge thanks to co-first authors Christiano Alves, Sabya Das, and Vijay Krishnan, and members from my group Leillani Ha, Hannah Stutzman, Lauren Fox, and others!

For more about how Rachel created this bespoke Cas9 protein, see here!

bsky.app/profile/bkle...