Spinal muscular atrophy (SMA) is a severe inherited neuromuscular disorder stemming from SMN1 gene deletion or mutation. SMN1 is crucial for snRNP assembly and various cellular processes. SMA's hallmark is lower motor neuron degeneration, resulting in muscle weakness and atrophy. Affecting 1 in 14,848 newborns, it's the leading genetic cause of infant mortality. Humans possess two SMN genes: SMN1 (primary SMN producer) and SMN2 (producing a truncated, unstable protein). Higher SMN2 copy numbers correlate with less severe SMA. Current treatments involve antisense oligonucleotides (AOs) to increase full-length SMN from SMN2, but repetitive administration is necessary. Gene supplementation therapy using scAAV9 delivers a functional SMN1 copy, showing promise but lacking permanent endogenous SMN1 expression due to the episomal nature of AAV vectors. Therefore, in situ correction of endogenous mutated SMN1 is needed. CRISPR-Cas9 technology is a promising tool, but its inefficiency in non-dividing cells, such as motor neurons, and the challenges in delivering editing tools to the spinal cord have hindered progress. A recently developed HITI strategy, effective in both dividing and non-dividing cells, offers a solution. This study introduces Gene DUET, combining SMN1 cDNA supplementation and genome editing using HITI, aiming for long-term SMA treatment.

Existing SMA therapies focus on increasing functional SMN protein levels. Antisense oligonucleotides (AOs) modify SMN2 splicing to produce more full-length SMN protein, showing effectiveness but requiring repeated administrations. Gene supplementation therapy using self-complementary adeno-associated viral vectors (scAAV9) delivers a functional SMN1 gene, leading to improved motor function and survival. However, this approach is limited by the episomal nature of AAV vectors, meaning the added gene doesn't integrate into the host genome and the therapeutic effect isn't permanent. CRISPR-Cas9 gene editing offers potential for a permanent cure, but challenges remain in effectively targeting non-dividing cells within the spinal cord and delivering the editing tools efficiently. The development of homology-independent targeted integration (HITI), effective in both dividing and non-dividing cells, addresses these challenges and provides the basis for this study’s novel approach.

This study utilized SMA model mice (Smn1-/-; Smn2+/+) lacking SMN1 and possessing two human SMN2 alleles. The researchers compared AAV-PHP.eB and standard AAV vectors for gene delivery to the spinal cord, finding AAV-PHP.eB superior in transducing motor neurons. For HITI-mediated gene editing, they targeted intron sequences in Smn1 exon 1/2 to avoid disrupting endogenous exon sequences. They co-injected AAV vectors expressing Cas9, gRNA targeting the Smn1 locus, and a donor construct containing a rat Smn intron 1 segment, codon-optimized mouse Smn1 cDNA (exons 2-8), and a rat 3'UTR. The HITI approach aimed to precisely integrate the corrected Smn1 sequence into the genome via non-homologous end joining (NHEJ). The Gene DUET strategy combined HITI with SMN1 cDNA supplementation using AAV-PHP.eB vectors expressing either mouse Smn cDNA alone or both mouse Smn cDNA and the HITI components. Mice were systemically injected at P0.5 (postnatal day 0.5). Phenotypic improvements (body weight, survival, motor function) were assessed using various methods, including weight measurements, survival curves, righting reflex tests, and open field tests. Molecular analyses included qPCR, Western blotting, RNA sequencing, and target enrichment sequencing to evaluate SMN expression, gene correction efficiency, and transcriptional changes.

AAV-PHP.eB demonstrated superior transduction efficiency in the spinal cord compared to standard AAV. HITI alone improved SMA phenotypes but only transiently (3 weeks). Gene DUET, combining SMN1 cDNA supplementation and HITI-mediated gene correction, significantly enhanced SMA mice's body weight and survival compared to untreated, cDNA-only, and HITI-only groups. The effect was more pronounced in male mice, potentially due to differences in AAV delivery and developmental factors between sexes. The DUET treatment resulted in a more significant restoration of endogenous SMN1 expression levels. RNA sequencing revealed that Gene DUET treatment reversed the dysregulation of pathways related to inflammation (p53 signaling, cytokine-cytokine receptor interactions) and motor neurons in SMA mice's spinal cords. Long-term analysis (20 weeks) confirmed sustained body weight increase and motor improvement in DUET-treated mice. Deep sequencing demonstrated stable gene correction by Gene DUET.

The Gene DUET strategy, combining SMN1 cDNA supplementation and HITI-mediated genome editing, demonstrated significantly improved therapeutic efficacy for SMA compared to either approach alone. The sustained long-term improvement in body weight, survival, and motor function highlights the potential of this combined approach for a more durable and effective treatment of SMA. The observed sex-specific differences in response highlight the importance of considering sex-specific factors in SMA treatment strategies. The reversal of molecular dysregulation observed in RNA sequencing confirms the effects of Gene DUET at a molecular level. The stability of gene correction further supports the use of this approach over current gene supplementation therapies. Although both therapies showed potential benefits, the DUET strategy provided a more sustained and pronounced effect, providing an important advancement in SMA treatment.

This study demonstrates the efficacy of a novel Gene DUET strategy for SMA treatment, combining SMN1 cDNA supplementation with HITI-mediated genome editing. This combined approach yielded superior long-term benefits compared to either approach alone. The stable gene correction achieved by Gene DUET offers a potential for a more durable and effective treatment of SMA, representing a significant advancement in the field. Future research should focus on optimizing the delivery method and dosage of AAV vectors, exploring the sex-specific differences in treatment response further, and assessing the long-term safety and efficacy in larger animal models before clinical trials in humans.

The study primarily utilized a mouse model, and the results may not fully translate to human SMA. The sample size, particularly for the long-term analyses, could be increased to enhance statistical power. The observation of some adverse effects (necrosis) in treated mice necessitates further investigations into the optimal dosage and delivery method to minimize potential side effects. The current study focused solely on SMA and future studies could explore the applicability of the Gene DUET approach to other monogenic diseases.