Modified gene-editing technique reverses disease without cutting DNA

Californian researchers have developed a modified version of CRISPR-Cas9 genome-editing technology that alters the activity, rather than the underlying sequence, of disease-associated genes, thus allowing them to activate genes without creating breaks in the DNA.

Writing in the journal Cell, the researchers revealed that their technique potentially circumvents a major hurdle to using gene-editing technologies to treat human diseases, having already been used to treat several different diseases in mice. Their findings are said to be the first to provide evidence that one can alter the phenotype of an animal with an epigenetic editing technology, preserving DNA integrity.

Much of the enthusiasm around CRISPR-Cas9 technology centres on the ability to insert or remove genes or to repair disease-causing mutations. However, most CRISPR-Cas9 systems work by creating ‘double-strand breaks’ (DSBs), or cuts, in regions of the genome targeted for editing or for deletion, and researchers are opposed to creating such cuts in the DNA of living humans. They are concerned about how cells respond to these cuts and how they are repaired; with frequency, they say, the technique leaves new mutations in its wake — and uncertain side effects.

“Cutting DNA opens the door to introducing new mutations,” said Juan Carlos Izpisua Belmonte of the Salk Institute for Biological Studies, whose laboratory developed the new technique. “That is something that is going to stay with us with CRISPR or any other tool we develop that cuts DNA. It is a major bottleneck in the field of genetics — the possibility that the cell, after the DNA is cut, may introduce harmful mistakes.”

This fact was very much in the mind of the Izpisua Belmonte researchers as they developed a new technique using a modified CRISPR-Cas9 system that does not cut the DNA. The principal idea behind the technique is the use of two adeno-associated viruses (AAVs) as the machinery to introduce their genetic manipulation machinery to cells in post-natal mice.

In the original CRISPR/Cas9 system, the enzyme Cas9 is coupled with guide RNAs that target it to the right spot in the genome to create DSBs. Recently, researchers have started using a ‘dead’ form of Cas9 (dCas9), which can still target specific places in the genome but no longer cuts DNA. Instead, dCas9 has been coupled with transcriptional activation domains — molecular switches — that turn on targeted genes. Unfortunately, the resulting protein — dCas9 attached to the activator switches — is too large and bulky to fit into AAVs, making it difficult to use in clinical applications.

Izpisua Belmonte’s team combined Cas9/dCas9 with a range of different activator switches to uncover a combination that worked even when the proteins were not fused to one another. After inserting the gene for the Cas9 enzyme into one AAV virus, they used another AAV virus to introduce a short single guide RNA (sgRNA), which specifies the precise location in the mouse genome where Cas9 will bind, and a transcriptional activator. The shorter sgRNA is only 14 or 15 nucleotides compared with the standard 20 nucleotides used in most CRISPR-Cas9 techniques, and this prevents Cas9 from cutting the DNA.

In other words, Cas9 or dCas9 was packaged into one AAV, and the switches and guide RNAs were packaged into another. The researchers also optimised the guide RNAs to make sure all the pieces ended up at the desired place in the genome, and that the targeted gene was strongly activated.

“Basically, we used the modified guide RNA to bring a transcriptional activator to work together with the Cas9 and delivered that complex to the region of the genome we were interested in,” said Hsin-Kai Liao, co-first author on the study.

The complex sits in the region of DNA of interest and promotes expression of a gene of interest. Similar techniques could be used to activate virtually any gene or genetic pathway without the risk of introducing potentially harmful mutations.

“The components all work together in the organism to influence endogenous genes,” said Liao. In this way, the technology operates epigenetically, meaning it influences gene activity without changing the DNA sequence.

The team has already used the technique to demonstrate disease reversal in several disease models in mice, in each case engineering their CRISPR-Cas9 system to boost the expression of an endogenous gene that could potentially reverse disease symptoms. In the case of kidney disease, they activated two genes known to be involved in kidney function, and observed not only increased levels of the proteins associated with those genes but also improved kidney function following an acute injury. For type 1 diabetes, they aimed to boost the activity of genes that could generate insulin-producing cells. Once again the treatment worked, lowering blood glucose levels in a mouse model of diabetes.

The lab’s advanced in vivo Cas9-based epigenetic gene activation system enhances skeletal muscle mass (top) and fibre size growth (bottom) in a treated mouse (right) compared with an independent control (left). The fluorescent microscopy images at bottom show purple staining of the laminin glycoprotein in tibialis anterior muscle fibres. Image credit: Salk Institute.

Preliminary data suggest that the technique is safe and does not produce unwanted genetic mutations. The researchers are currently pursuing further studies to ensure safety, practicality and efficiency before considering bringing it to a clinical environment.

As for the future, the team is working to improve the specificity of the system and to apply it to more cell types and organs to treat a wider range of human diseases, with Izpisua Belmonte eyeing neurological disorders such as Alzheimer’s and Parkinson’s diseases. He additionally hopes it can be used to rejuvenate specific organs and to reverse the ageing process and age-related conditions, such as hearing loss and macular degeneration.