Therapeutic strategy for spinal muscular atrophy by combining gene supplementation and genome editing

Background

Spinal Muscular Atrophy (SMA) is a debilitating genetic disease characterized by muscle weakness and atrophy. This disease is caused by mutations in the SMN1 gene, which encodes the survival motor neuron 1 (SMN1) protein. Currently, the therapeutic strategies for SMA focus on prolonging life span and on improving the quality of the patients’ life. These include small molecule or gene supplementary therapies which aim to deliver a fully functional copy of SMN1. However, these strategies only have a beneficial effect for a limited amount of time and coming up with long-term therapies, such as the correction of the endogenous mutation, remains a significant challenge. In this preprint, the authors describe a novel gene therapy approach for treating spinal muscular atrophy (SMA) in a mouse model. They combined two existing therapies, gene supplementation and gene editing, into a single approach called Gene-DUET.

Homology-independent targeted integration

Genome editing using the CRISPR/Cas9 system exploits either homologous -directed repair (HDR), which is seen only in proliferating cells, or non-homologous end joining (NHEJ), which is seen both in proliferating and non-proliferating cells. To achieve CRISPR/Cas9 mediated knock-out, both HDR or NHEJ mechanisms can be used. However, achieving knock-ins – particularly in non-proliferating or differentiated cells – is more challenging as it requires HDR. In 2016, the lab of the preprint authors developed a CRISPR/Cas9- based genome editing technology which utilises NHEJ, known as homology-independent targeted integration (HITI). With this technique, the authors previously showed successful knock-in in non-proliferating cells¹. The HITI donor plasmid includes the transgene which is flanked by Cas9 cleavage sites, alongside the sgRNA which specifies the target loci and the direction of insertion. The resulting Cas9 cleavage occurs both in the donor plasmid and the genomic target, thereby releasing the transgene from the donor which then gets incorporated at the site of the double strand break (DSB) in the genomic loci ^1,2.

Key findings

AAV vectors for transducing non-dividing cells

Delivering therapeutic genes to the neurons in the spinal cord can be challenging. AAV vectors are often the first choice for safe delivery of therapeutic genes, and in the case of SMA, AAV-PHP.eB and AAV9 have been commonly used. To compare the efficacy of these two AAV vectors, the authors injected the respective AAV-GFP in the neonatal wildtype mouse and observed that AAV-PHP.eB was more efficient in transducing spinal motor neurons than AAV9.

HITI-mediated knock-in in spinal motor neurons

The authors then tested the in vivo efficacy of HITI in the spinal cord by delivering AAV-PHP.eB along with a guide RNA expression cassette into neonatal mice. The results showed GFP expression in the nucleus of motor neurons and liver, suggesting HITI-mediated knock-in is successful in spinal motor neurons.

Taken together, the findings described above show that AAV-PHP.eB is an efficient gene delivering carrier in the spinal cord and that HITI-mediated genome editing is successful in vivo in non-dividing, spinal motor neurons.

Synergistic effect of genome editing and gene supplementation

The authors used the HITI approach to correct the SMA mutation in the SMA mouse model. Although the HITI-treated mice showed improved phenotypes, the effect was insufficient for this treatment to be considered a long-term therapeutic strategy. It was observed that protein expression following gene editing was too late already for an effective rescue. Hence, in their next step, the authors developed the Gene-DUET method as part of which the AAV-PHP.eB vector was redesigned to include mSmn1 CDS for overexpression and the corrected mSmn1/SMN1 gene for genome editing (AAV-PHP.eB-SMN1-DUET). The authors then systematically delivered this vector into P0.5 SMA mice with or without Cas9, expressed by AAV-PHP.eB-Cas9 . Both the cDNA (without Cas9) and the DUET (with Cas9) restored molecular dysfunctions in the spinal cord, thereby improving the phenotype significantly. Furthermore, the authors reported that the Gene-DUET approach was more effective than cDNA supplementation only, as the effects were stable and long-lasting. This suggests it could be a promising approach for treating genetic diseases. 

Why I like this preprint

As someone who has had the chance to engage with SMA affected patients and hearing their therapeutic requirements, this preprint piqued my interest.

The Gene-DUET approach which was shown to be more effective than the traditional gene supplementation therapy might bring promising remedies for a wide range of neurological diseases in the future. Additionally, the finding that the AAV-PHP.eB can efficiently transduce spinal neurons can have important implications for therapies for neurological disease. Overall, these findings provide the basis for further research and development for SMA- and related diseases.

Questions to the authors

1. What safety concerns could the Gene-DUET system pose if it is to be implemented for clinical trials?
2. What are the limitations of the HITI technology in terms of efficiency, specificity and toxicity?
3. What further research is needed to fully explore the potential of the HITI strategy for treating genetic diseases?

References

1. Suzuki, K. et al. In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration. Nature 540, 144–149 (2016).
2. Suzuki, K. & Izpisua Belmonte, J. C. In vivo genome editing via the HITI method as a tool for gene therapy. J. Hum. Genet. 63, 157–164 (2018).

Tags: crispr, gene therapy, gene-duet, neurological disease, spinal muscular atrophy

Posted on: 3 May 2023 , updated on: 12 May 2023

doi: https://doi.org/10.1242/prelights.34510

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