Salk Scientists Tweak CRISPR for Epigenetic Therapies in Diabetes, Kidney Disease and Muscular Dystrophy

Salk Scientists Tweak CRISPR For Epigenetic Therapies In Diabetes, Kidney Disease And Muscular Dystrophy Gene editing systems work by creating DNA double-strand breaks (DSBs). However, these DSBs can have unwanted side effects which are a concern for clinical applications. CRISPR/Cas9 system has evolved to target genes without creating DSBs. This has been difficult in vivo so far. These epigenetic therapies come with a measure of risk as they can affect off-target genes. There is a clear need for targeted epigenetic therapies that work on specific genes without affecting other genes.

A major advance in translational science is CRISPR/Cas9, a bacterial immune system which has led to the development of efficient editing tools to target specific genes. CRISPR/Cas9 can be modified to regulate gene expression and create epigenetic alterations without undesirable effects of DSBs. This technology has tremendous potential to activate/regulate target genes to develop therapeutic interventions for genetic disorders and for epigenetic programming in regenerative medicine.

The first generation of dCas9-VP64 system was unsuccessful in Targeted Gene Activation (TGA) using a single guide RNA (sgRNA). While the second generation CRISPR /Cas9 TGA systems were useful for in vitro genetic studies using sgRNAs, it was still a challenge in vivo. Usually dCas9 is combined with molecular switches to turn on targeted genes, but the protein attached to the activator switches are too bulky to fit into the delivery vehicles which are adeno-associated viruses (AAVs). There is a gap in the delivery tools for such gene therapeutics.


Researcher Izpisua Belmonte’s team used Cas9 and dCas9 (dead form), packaging it into an AAV (adeno-associated viral vector) and used another package for activator switches and guide RNAs. The guide RNAs were optimized so that both packages correctly target the required gene.

The method was tested in mouse models of acute kidney disease, type 1 diabetes and the mdx model of Duchenne muscular dystrophy to enhance the expression of genes which could reverse the symptoms. The mouse models used in acute kidney injury targeted the genes klotho and interleukin 10 (il10). Klotho protects against renal damage and the gene expression is lowering during aging and acute kidney injury. Interleukin 10 is an anti-inflammatory cytokine which protects against renal damage especially after chemotherapy. The researchers found that the CRISPR/Cas9 TGA system worked to improve gene expression of Klotho and Interleukin 10 which provided a prophylactic system to protect against kidney injury. In mouse models with type 1 diabetes, the researchers targeted the pancreatic and duodenal homeobox gene 1 (Pdx1) in liver cells. The researchers found that sufficient expression of Pdx1 resulted in upregulation of insulin 1 (ins1), insulin 2 (ins2) and proprotein convertase subtilisin/kexin type 1 (Pcsk1) in liver cells. In mdx mouse models of Duchenne muscular dystrophy, the researchers used the CRISPR/Cas9 TGA system to upregulate utrophin. Utrophin and dystrophin are similar genes as it encodes similar proteins. The researchers activated the utrophin gene to compensate the loss of dystrophin protein. After two months, they observed that the treated mice greatly improved in muscle strength and tone.

The CRISPR/Cas9 TGA system can be used to develop treatments and cures for a wide range of human diseases which do not have a cure using traditional strategies. The researchers concluded that the in vivo TGA system can be used to transcriptionally activate even large genes to compensate disease-causing mutations.

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Reference:

1. Liao, Hsin-Kai, Fumiyuki Hatanaka, Toshikazu Araoka, Pradeep Reddy, Min-Zu Wu, Yinghui Sui, Takayoshi Yamauchi et al. "In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-epigenetic Modulation." Cell (2017).

Source-Medindia