CRISPR-Cas9: a new prospective for Duchenne muscular dystrophy treatment

da | Giu 15, 2019 | Biologia Molecolare

Duchenne muscular dystrophy (DMD) is a genetic hereditary myopathy associated to mutations in the dystrophin gene which is located on the X chromosome. The absence of dystrophin causes the degeneration of skeletal and cardiac muscles inducing muscular atrophy and weakness with fast progression which ends with death caused by heart or respiratory failure. Therapies are actually based on the use of drugs for treatment of DMD, however these allow the restoration of less than 1% of the normal level of dystrophin [1].

A new approach to restore the DMD is explored by Yi-Li Min and his collaborators; it consists of a genome editing strategy applying the CRISPR-Cas9 technology to restore the correct expression of dystrophin by reframing the relative gene [2]. Endonucleases Cas9 derived from Staphylococcus pyogenes is able to cut a double strand break in a specific DNA region thanks to its association with a sgRNA which works as a guide, targeting the cut in a complementary region. In the presence of a double strand break, cells apply two possible strategies to repair the damaged DNA: homologous directed repair (HDR) which consists in repairing the cut by using a DNA template with high level of homology for the sequence surrounding the cut to copy the correct sequence or non-homologous end joining (NHEJ). In this latter strategy the two ends of DNA are joined together by insertions or deletions of a small number of nucleotides in the cutting site [3].

The article presented by Yi-Li Min and his research group concerns the application of this genome editing strategy to correct one of the most common dystrophin gene mutations in human that cause the deletion of exon 44. In fact, The lack of exon 44 results in an incomplete and dysfunctional dystrophin due to premature termination by wrong reading frame. The authors of this work decided to target the dystrophin genes producing a deletion in the region adjacent of the deletion (exon 43 and 45) with the aim to restore the correct open reading frame. This can be obtained in two cases: skipping the entire exon 43 or 45 implementing the splicing respectively between exons 42 and 45 or 43 and 46; or reframing the two exons exploiting the insertion or deletion of small number of nucleotides caused by the NHEJ repairing system.

The research has been conducted in three consequential steps: preliminary in vitro study using human cells derived by patients affected by DMD ΔEx44; then generating a mouse model with the same mutation of dystrophin gene; and finally correct the DMD exon 44 deletion in vivo by the CRISPR-Cas9 technology. To study the efficiency of gene therapy, they produced an induced pluripotential stem cell line (iPSCs) using cells deriving from peripheral blood mononuclear cells (PBMCs) taken from patients with DMD deletion of exon 44. As healthy control, they use an iPSCs line deriving from the patient’s brother with a normal dystrophin gene.

Figure: schematic representation of CRISPR-Cas9 technology applied to restore the correct ORF in dystrophin gene.

These iPSCs lines were infected with two adeno-associated viruses of serotype 9 (AAV9) encoding the CRISP-Cas9 gene and the singles guide RNAs (sgRNAs). Subsequently these transfected cell lines are differentiated in cardiomyocytes. They tested eight sgRNAs to target either the exons 43 or 45. The efficiency of the different sgRNAs was verified with a T7 Endonucleases assay (T7E1). Three sgRNAs with the highest efficiency were chosen to be inserted in the AA9-sgRNA vector to be used for the genome editing.  Western blot and immunostaining analysis confirmed the recovery of dystrophin expression in both cases of editing exon 43 or 45.

For the following study in mice, Yi-Li Min and his group chose an sgRNA which directed the cut in a 100% conserved region between human and miceon the exon 45. For the in vivo study they generated a mouse model with DMD deletion of exon 44 (DMD ΔEx44). On this mice model Yi-Li Min and collaborators have chosen to deliver SpCas9 and sgRNA through an intramuscular injection and further through an intraperitoneal injection. To validate the efficacy of the strategies they performed Western Blot and immunostaining and tests to analyze muscle function. cDNA amplicon sequencing shows the predominance of reframing events compared to exon skipping events. The most common event is a single A insertion at the cutting site, probably caused by the single T insertion mediated by Cas9. Multiple ratio of AAV9-G6 to AAV9-Cas9 were tested to determine how the systemic editing efficiency can change. Increasing the dosage of AAV9-G6 the researchers observed different results between skeletal muscles and heart.

The restoration of dystrophin in skeletal muscles was correlated with the dosage of AAV-G6, it increased at higher levels of sgRNA (1:10 ratio of AAV-Cas9: AAV-G6). In contrast, in the heart, high levels of dystrophin were present at low dosage of sgRNA. On the basis of this results the authors improved CRISPR/Cas9 technology just changing ratios of the genomic editing system components. The difference shown is probably correlated with the number of nuclei in each cell. Both skeletal muscle and heart have multinucleated cells, but a single cardiomyocyte contains one to four nuclei on average, one myofiber may contain hundreds. Generating dystrophin-expressing myocytes by editing nuclei in one cardiomyocyte is more efficient than in one myofiber. Is also known that AAV9 has better tropism for heart then skeletal muscle [4].

All these results highlight the effectiveness of single-cut CRISPR gene editing for efficient restoration of dystrophin in vivo. This approach proved to be better than the double cut approach, used in previous studies [5, 6]. In other studies, no immune response was observed after short period [7], but remains to be investigated a possible immunological response to Cas9 or the modified dystrophin over long term. Of course, it also remains to be determined if the marked effects observed in mice can be scaled up to humans with much larger muscles over a longer time frame and if there are off target sites on the human genome. One of the most limitation of this study is to establish whether the effects may fade over time, especially in the skeletal muscles where the turnover can replace the dystrophin-expressing generated myocytes. Considering that cardiomyocytes don’t turn over, we expect that the benefits of dystrophin restoration in the heart will be lifelong. For a long-term maintenance of dystrophin expression, a future prospect is to infect satellite cells in vivo, but AAV9 delivery to these cells is hard to obtain.

References

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Loris Curatolo

Master Industrial Biotechnology student

Giacomo Antonicelli

Master Industrial Biotechnology student