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#apaperaday: CRISPR-Based Gene Therapies: From Preclinical to Clinical Treatments

In today’s #apaperaday, Prof. Aartsma-Rus reads and comments on the paper titled: CRISPR-Based Gene Therapies: From Preclinical to Clinical Treatments

Today’s pick is about CIRSPR-based gene therapies with both a preclinical and clinical focus by Laurent et al in @Cells_MDPI DOI: 10.3390/cells13100800

Gene editing targets the direct cause of genetic diseases. With CRISPR Cas9 system this can now be done straightforwardly in model systems. The system involves an endonuclease (cas9) & singe guide RNAs that direct the cas9 to a location in the DNA to induce a double strand break.

This is then repaired by non homologous end joining (causing small errors), or in dividing cells by homology directed repair, which uses a template for error free repair. Base editing instead uses a single stranded break and correction of a single nucleotide change.

With prime editing small insertions or deletions can be induced. Finally with dCas9 chromatin can be remodeled to increase or decrease expression of a gene. With these tools preclinical research had been done for blood diseases and neuromuscular diseases.

Authors first explain how Casgevy, the first approved gene editing drug, works on hematopoietic stem cells. Here editing happens ex vivo (cells are isolated from a patient, edited in the lab and then transplanted back after quality controls).

Beta-globin is a crucial component of hemoglobin. When there are pathogenic variants in the beta globin gene this leads to e.g. sickle cell disease or beta-thalassemia. During fetal development, fetal hemoglobin is used, so this disease has a post natal onset.

As a therapeutic approach researchers wanted to activate the production of fetal hemoglobin in patients using gene editing, which targets the BCL11a enhancer, which is erythroid specific for beta globin production.

When this enhancer is disrupted, this activates the production of fetal hemoglobin, to compensate for loss of beta globin in patients with sickle cell disease and transfusion dependent beta thalassemia (now approved).

Authors outline other efforts for gene editing in sickle cell disease & beta thalassemia & primary immunodeficiencies that are in preclinical and clinical trials. Note for some immunodeficiencies gene replacement with lentiviral vectors is available as an approved treatment.

Authors then move to Duchenne, which is caused by lack of dystrophin. Partially functional dystrophins give rise to milder Becker muscular dystrophy. Exon skipping aims to allow Duchenne patients to make Becker-type dystrophins.

However this acts on RNA level so repeated dosing is required, with weekly intravenous infusions for currently approved exon skipping treatments. Gene editing would act on DNA so less frequent treatment would be required.

Authors outline how gene editing has been used to generate cell and animal models for Duchenne and preclinical work on how non homologous end joining gene editing was used to delete exons, or disrupt splice sites or reframe exons to allow production of Becker-type dystrophin.

Base & prime editing have been used to correct small mutations or to disrupt splice sites. Finally chromatin targeting has been used to increase expression of utrophin for all mutations, or the Purkinje dystrophin isoform for patients with deletions involving the muscle promotor.

They compare the blood diseases with an approved treatment with Duchenne where all research is preclinical. The difference is that for blood diseases it is possible to edit stem cells ex vivo while for Duchenne you need to treat muscle in vivo.

This involves more risks, as AAV is needed to deliver editing components. This means long term expression and higher likelihood of unwanted edits. Here the risk of unwanted effects per se can be studied in Casgevy treated patients.

It also involves the risks associated with AAV in general, with side effects and inability to treat patients with preexisting immunity. As satellite cells are not targeted durability of effect is unclear.

One thing authors do not mention is the risk of autoimmunity due to expression of a bacterial protein (cas) in muscle and heart. For me this is the biggest concern after hearing the impact of the anti-microdystrophin auto-immune response.

The review is a nice comprehensive overview of past and ongoing work in blood diseases and Duchenne. However, the focus is rather narrow as gene editing is evaluated for many other genetic diseases.

I understand authors wanted to compare the approved Casgevy with Duchenne to outline differences but they could have e.g. expanded to neuromuscular diseases, as for blood diseases they did not only focus on the Casgevy indication.