Project Details

Restoring motor neuron function in pediatric spinal muscular atrophy through base editing

Kwangjun Lee, PhD

Summary

BACKGROUND: Spinal muscular atrophy (SMA) is a severe motor neuron disease caused by the loss of the SMN1 gene. Current therapies are either transient (Spinraza and Evrysdi) or may wane (Zolgensma), and do not restore endogenous SMN protein levels. GAP: We previously developed a base editing strategy that enables the efficient conversion of SMN2 to SMN1 genes, which can restore SMN proteins levels to normal and rescues motor phenotypes and lifespan in a severe SMA mouse model. However, mitigating off-target risks and reducing the potential immunogenicity of our base editing for therapeutic use in patients is required. HYPOTHESIS: Engineering high-fidelity and compact base editor (BE)s and adeno associated virus (AAV) architectures will improve in vivo gene editing correction of SMN genes in the target tissue by using a lower AAV-BE dosage with a higher therapeutic index, resulting in improved functional SMN protein restoration in a one-time treatment. METHODS We will design enhanced TadA deaminase variants and miniature Cas9 constructs using AlphaFold and validate their efficacy against our lead BE strategy in SMA mouse embryonic stem cells (mESC) derived from the severe Δ7 SMA mouse model. We will assess editing efficiency and specificity by high throughput sequencing (HTS), and measure SMN protein restoration by western blot. RESULTS: We engineered a bespoke adenine base editor (ABE) to facilitate efficient and precise single nucleotide conversion of SMN2 exon 7 C6T (94±3.4% editing efficiency, 82±1.9% single nucleotide editing purity. With this genome editing approach, we effectively permanently restored wild-type SMN1 gene functionality at endogenous SMN2 alleles in SMA cells. IMPACT: This project aims to develop a safer, more effective, and potentially curative one-time gene therapy for SMA children.