A team led by researchers from the University of California Berkeley has demonstrated the first use of a gold nanoparticle system to effectively deliver CRISPR components, Cas9 ribonucleoprotein and donor DNA, into a wide variety of cell types, as well as in an in vivo mouse model.
Currently, the most advanced method of Cas9 delivery is the use of engineered adeno-associated viruses (AAV). This method has drawbacks, however. A fraction of the human population has pre-existing AAV immunity, making them ineligible for treatment. Furthermore, the physical size of AAV limits what can be packaged, and multiple viruses are needed to deliver all of the CRISPR components, decreasing the editing efficiency.
To overcome this challenge, the team used gold nanoparticles as the core scaffold for delivery. Gold is favorable due to its ability to bind with proteins through noncovalent, electrostatic forces. It also possesses low toxicity and is readily taken up by cells. To set up the vehicle, the gold is first coated with donor DNA, followed by Cas9 bound to the guide RNA (also known as the Cas9 ribonucleoprotein complex). The finished complex is called, CRISPR-Gold.
When tested on human embryonic stem cells, iPSCs, and dendritic cells, the efficiency of DNA repair was significantly greater than those of controls. These first set of findings demonstrate the safety and effectiveness of CRISPR-Gold delivery into a wide range of cells. The authors then tested the treatment on a mouse model of Duchenne muscular dystrophy (DMD). DMD is the most common muscular disorder and 30% of patients have single base mutations or small deletions that could be readily amenable to genetic therapy.
A single injection of CRISPR-Gold into the muscle tissue of DMD mice restored 5.4% of the disease-causing gene, dystrophin, back to its normal wild-type variant. This correction rate was approximately 18 times higher than in mice Cas9 and donor DNA by themselves, which was a 0.3% rate. Phenotypic effects in muscle tissue were also observed. Compared to controls, CRISPR-Gold-treated mice showed a twofold increase in hanging time, a common test for mouse strength and agility.
Additional experiments were conducted to assess for safety. Analysis of mouse plasma 24 hours and two weeks post injection showed no acute upregulation of inflammatory cytokines, showing CRISPR-Gold is non-immunogenic. Deep sequencing analysis to quantify the degree of off-target editing also showed minimal DNA damage.
"CRISPR-Gold and, more broadly, CRISPR-nanoparticles open a new way for safer, accurately controlled delivery of gene-editing tools," says leading author Michael Conboy. "Ultimately, these techniques could be developed into a new medicine for Duchenne muscular dystrophy and a number of other genetic diseases."
A new start-up company, GenEdit, has since been formed by some of the study co-authors to focus on translating the CRISPR-Gold technology into humans. Co-authors Murthy and Conboy are now also working on the next generation of particles for delivering CRISPR into tissues via the bloodstream. The ideal targets are adult stem cells, due to their capacity for self-renewal and differentiation.
The study was published earlier this week in Nature Biomedical Engineering.