Gene therapy for congenital muscular dystrophies

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Using gene therapy to treat multiple dystrophies

Congenital Muscular Dystrophy Type 1A (MDC1A) is caused by mutations in the Lama2 gene that cause muscle wasting and destruction of the protective myelin coating around the peripheral nerves. CRISPR has already shown positive preclinical results in Duchenne muscular dystrophy, mainly by targeting mutations in the gene that produces dystrophin, a protein essential for muscle support. Ronald Cohn and his colleagues at Toronto's Hospital for Sick Children (SickKids) used earlier CRISPR-cas9 to eliminate dysfunctional dystrophin exons (inclusion-exon strategy), leaving a shortened but still functional gene. The therapy has been effective for more than a year in mice. In another study researchers applied CRISPR to target exon 51 and successfully restored dystrophin levels to 92% of normal in models of Duchenne muscular dystrophy in dogs.

The problem of the heterogeneity of muscular dystrophies

There are more than 350 nonsense pathogen, missense, splice site and deletion mutations in LAMA2 reported to date.One of the problems in developing a therapy for the treatment of MDC1A is that the heterogeneity of the mutations often leads to varying severity and progression of the disease. Therefore, it is urgent to develop a universal, mutations-independent strategy that offers a therapeutic approach to all patients with MDC1A.

How to extend the application range of CRISPR-Cas9?

Given the number of genomic alterations causing MDC1A, the Crispr-Cas9-mediated correction would require the design and in-depth analysis of several single-guide RNAs (sgRNAs) specific to each mutation, which means that as many therapies of this type should be designed and evaluated thoroughly for each mutation. In addition, safety concerns regarding the potential mutagenic nature of the CRISPR-Cas9 system and the presence of non-targeted effects after gene editing remain, which together may be difficult from the point of view of safety and security. regulation.

A recent study has described the use of the CRISPR transcription activation system to induce expression of target genes in skeletal, renal and hepatic tissues, resulting in a phenotypic increase such as increased muscle mass and improvement. substantial pathophysiology of the disease. However, it relied almost exclusively on a transgenic mouse model expressing Cas9 or on local intramuscular treatments and so it is difficult to extrapolate the efficacy of this strategy to models relevant to the disease.

On the other hand, attenuation of disease pathogenicity by targeted modulation of disease-modifying gene expression would be a potentially safer and more beneficial alternative for all individuals with MDC1A.

An indirect strategy to address all congenital muscular dystrophies

Neuromuscular diseases have provided excellent examples for demonstrating the role of disease modifiers. One of the most potent disease modifiers reported for MDC1A is laminin 1, which is structurally similar to laminin 2. However, laminin 1 is not expressed in skeletal muscle or Schwann cells.

Previous studies have demonstrated that overexpression of transgenic Lama1 healed from myopathy and peripheral neuropathy in mouse models.

Although these studies established a compensation function for laminin 1 in MDC1A, the use of this modifier as postnatal genomic therapy is hampered by the size of the Lama1 cDNA, which exceeds the packaging capacity of AAV vectors. CRISPR-Cas9 technologies have offered opportunities for regulating gene expression and creating epigenetic alterations without introducing double-strand breaks in DNA, known as the CRISPR transcription activation system.

The strategy uses nuclease-deficient Cas9 (dCas9), which is unable to cut DNA due to mutations in the nuclease domains and retains the ability to specifically bind to DNA when guided by sgRNA.

CRISPR activation uses modified versions of dCas9, a mutation of Cas9 without endonuclease activity, with added transcriptional activators on dCas9 or the guide RNAs (gRNAs). Like a standard CRISPR-Cas9 system, dCas9 activation systems rely on similar components such as Cas9 variants for modulation or modification of genes, gRNAs to guide Cas9 to intended targets, and vectors for introduction into cells. However, while a standard CRISPR-Cas9 system relies on creating breaks in DNA through the endonuclease activity of Cas9 and then manipulating DNA Repair mechanisms for gene editing, dCas9 activation systems are modified and employ transcriptional activators to increase expression of genes of interest.

Using previously described Streptococcus pyogenes (Sp dCas9) fused to multiple copies of the VP16 transcription enhancer, Canadian and other researchers have demonstrated the use of the CRISPR-dCas9 system to positively regulate the expression of the modifying genes. vitro.

When the load is too big for viral vectors

A major challenge for in vivo applications is the large size of Sp dCas9 and its derivatives, which exceed the packaging capacity of the AAV genome. Targeting Lama1 presents a particular challenge: the gene is too large to be contained in the viral vectors traditionally used to administer gene therapy. To account for this limitation, the researchers adapted the transcriptional regulatory system and used a considerably smaller Cas9 protein, derived from mStaphylococcus aureus (Sa 9), to positively regulate Lama1.

What results were obtained?

By increasing the expression of Lama1, the treatment not only prevented paralysis in pre-symptomatic mice, but also reversed the progression of the disease in previously symptomatic animals. The treatment resulted in a reduction of fibrosis and an increase in the size of the muscle fiber, thus preventing the appearance of symptoms. More importantly, in mice already suffering from paralysis, the treatment also allowed the animals to get up and move. The researchers also observed a significant increase in nerve conduction velocity, showing a restoration of the myelin sheath and an improvement in neuromuscular function.

Application to Duchenne muscular dystrophy

The Cohn team believes that its strategy of using the CRISPR provided by AAV to upregulate Lama1 could also be applied to Duchenne muscular dystrophy. To achieve greater efficiency, the system could be used in combination with another technology that corrects the mutation. Its application as a combinatorial therapeutic approach, involving simultaneous up-regulation of protective disease-modifying genes and downregulation of harmful genes would represent a new paradigm for reducing disease phenotypes.

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