Gene-editing therapeutics based on the clustered regularly interspaced short palindromic repeats (CRISPR) show great promise for treating genetic diseases. CRISPR consists of a DNA-cutting enzyme called Cas9 and a short RNA that guides the enzyme to a specific region on the genome so that it can cut this region out. Some treatments also contain donor DNA too.

“This technique is known as homology-directed repair (HDR) and corrects disease-causing gene mutations to their wild-type, or normal, sequence, “explain co-team leaders Niren Murthy and Irina Conboy. “HDR could potentially cure the vast majority of genetic diseases thanks to this mechanism of action.”

No viruses needed

At present, researchers mainly rely on viruses to carry the genes for Cas9 and the RNA guide strand. A non-virus technique would be better, however, since most people have pre-existing antibodies to the viruses employed or being tested. Viral-based Cas9 delivery can also significantly damage genes not specifically targeted by the treatment. What is more, because they have a small packing size, multiple viruses are needed to deliver the CRISP components, thus decreasing the overall efficiency of the treatment.

Murthy, Conboy and colleagues have now developed a delivery vehicle comprising gold nanoparticles conjugated to DNA and then complexed with Cas9 protein and gRNA, and finally the cationic endosomal disruptive polymer PAsp(DET). “We inject the CRISPR-Gold, as we call it, into mice and it is internalized into cells via a process called endocytosis,” explains Murthy. “The PAsp(DET) then disrupts the cell endosome and allows the Cas9 protein, gRNA and donor DNA access to the cell cytoplasm and nucleus.”

Minimal off-target DNA damage

“We successfully used our technique to correct the DNA mutation that causes Duchenne muscular dystrophy in mice via local injection, with minimal off-target DNA damage,” say the researchers.

“The technique is efficient and proves that it is possible to correct gene mutations in vivo via HDR using non-viral delivery methods,” says Murthy. “Our work also shows that nanoparticles that can encapsulate Cas9 protein, gRNA and DNA within the same particles have tremendous potential for treating genetic diseases and provide a straightforward design principle for developing the next-generation of such particles.”

Towards clinical trials

The team says that it is now looking to bring the CRISPR-Gold technology into clinical trials. “Kunwoo Lee and Hyo Min Park, two of the joint first authors of this paper, have set up a start-up company called GenEdit that is focused on translating the technology,” says Murthy. “I am also an equity holder in the company and we are interested in developing strategies to improve the gene editing efficiency of CRISPR-Gold.”

One hurdle that needs to be overcome is to ensure that CRISPR-Gold can be delivered from the blood stream into any tissue without the need to rely on multiple injections in the tissue, adds Conboy. “Our second future goal is to target CRISPR-Gold to dividing stem and progenitor cells since CRISPR gene editing only works on cells that are dividing. Most of our skeletal muscle, heart and brain cells, for example, are composed of (already differentiated) non-dividing cells and we will not be able to correct mutations in these cells.

“Rare degenerative (stem) cells in our organs, on the other hand, are the only ones that are responsive to CRISPR-based gene editing, so boosting their proliferation and homing CRISPR-Gold to these cells should enhance the efficiency of gene correction. This will be needed for clinical translation.”

The research is detailed in Nature Biomedical Engineering doi:10.1038/s41551-017-0137-2.