Seamless Genetic Conversion of SMN2 to SMN1 via CRISPR/Cpf1 and Single-Stranded Oligodeoxynucleotides in Spinal Muscular Atrophy Patient-Specific Induced Pluripotent Stem Cells

Abstract

Spinal muscular atrophy (SMA) is a kind of neuromuscular disease characterized by progressive motor neuron loss in the spinal cord. It is caused by mutations in the survival motor neuron 1 (SMN1) gene. SMN1 has a paralogous gene, survival motor neuron 2 (SMN2), in humans that is present in almost all SMA patients. The generation and genetic correction of SMA patient-specific induced pluripotent stem cells (iPSCs) is a viable, autologous therapeutic strategy for the disease. Here, c-Myc-free and non-integrating iPSCs were generated from the urine cells of an SMA patient using an episomal iPSC reprogramming vector, and a unique crRNA was designed that does not have similar sequences (≤3 mismatches) anywhere in the human reference genome. In situ gene conversion of the SMN2 gene to an SMN1-like gene in SMA-iPSCs was achieved using CRISPR/Cpf1 and single-stranded oligodeoxynucleotide with a high efficiency of 4/36. Seamlessly gene-converted iPSC lines contained no exogenous sequences and retained a normal karyotype. Significantly, the SMN expression and gems localization were rescued in the gene-converted iPSCs and their derived motor neurons. This is the first report of an efficient gene conversion mediated by Cpf1 homology-directed repair in human cells and may provide a universal gene therapeutic approach for most SMA patients.

Advances in Pluripotent Stem Cells: History, Mechanisms, Technologies, and Applications

“…In addition, urine cells from a patient with spinocerebellar ataxia type 3 (autosomal dominant inherited neurodegenerative disease) were transformed into iPSCs with a SeV delivery system, providing a robust platform for further study of this disease's pathogenesis and its susceptibility to pharmacotherapy as well as gene therapy [52]. Recently, iPSCs generated from urine-derived cells from a patient with spinal muscular atrophy with an Epi reprogramming vector (c-Myc-free and non-integrating) combined with CRISPR technology were used to correct the disease-causing mutation at the iPSC level, and these cells were then were developed into motor neurons [53]. Such a protocol may eventually lead to gene therapy for spinal muscular atrophy.…”

Increasing the precision of gene editing in vitro, ex vivo, and in vivo

“…This increases the ratio of precise to imprecise mutations, and reduces the risk of undesired NHEJ products 46 . Most recently this has been shown in spinal muscular atrophy patient iPSCs 47 , suggesting untapped potential for precision gene correction. While Cas12a has obvious benefits for precise gene editing, it has recently been suggested to possess ssDNA cleavage activity, even in the absence of a PAM site 48 ; thus, it may be better suited for dsDNA templates used for large transgenes.…”

Druggable Transcriptional Networks in the Human Neurogenic Epigenome

“…A proximal nucleic acid target currently in clinical trials is the SMN1 locus, in which gene therapy using an artificial regulatory gene construct is delivered via an adenovirus-associated virus vector (AAV) (Zhou et al, 2018). This therapy is intended to ameliorate spinal muscle atrophy, a devastating neuromuscular disease that is the leading cause of death in infants, affecting 1 in 10,000 live births.…”