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Fixing Duchenne muscular dystrophy
Scientists at the University of California, Berkeley have engineered a new way to deliver the CRISPR-Cas9 gene-editor. CRISPR-Gold, as the new delivery system is called, demonstrated repair for the mutation causing Duchenne muscular dystrophy (DMD). A severe muscle-wasting disease, a single injection of CRISPR-Gold into DMD mice led to a higher correction rate with a two-fold increase in DMD mouse strength and agility.
Since 2012, when study co-author Jennifer Doudna, a professor of molecular and cell biology and of chemistry at UC Berkeley, and colleague Emmanuelle Charpentier, of the Max Planck Institute for Infection Biology, repurposed the Cas9 protein creating a cheap, precise and easy-to-use gene editor, they hoped therapies based on CRISPR-Cas9 would revolutionize treatment for genetic diseases.
Yet, developing treatment for genetic disease remains a big challenge. Genetic diseases can only be cured if the disease-causing gene mutation can be changed into a normal gene sequence, impossible to do with conventional therapies.
CRISPR/Cas9, however, can correct gene mutations by cutting out the mutated DNA, thereby triggering DNA self repair. However, strategies for safely delivering the necessary components into cells has to be developed before the potential of CRISPR-Cas9-based therapeutics can be realized. A common technique to deliver CRISPR-Cas9 uses adeno-associated viruses (AAVs), but that method has complications. Viruses can’t be used with pre-existing immunities or ones not identified. AAVs can also attach to genes not intended for repair, causing damage.
CRISPR-Gold does not need viral delivery.
In the new study, the laboratories of Berkeley bioengineering professors Niren Murthy and Irina Conboy demonstrated how CRISPR-Gold uses gold nanoparticles to deliver the Cas9 protein to bind to and cut DNA using the Cas9 protein, an RNA guide — and then insert donor DNA, fixing a gene mutations in living organisms.
"CRISPR-Gold is the first example of a delivery vehicle that can deliver all of the CRISPR components needed to correct gene mutations, without the use of viruses."
The study was published Oct. 2 in the journal Nature Biomedical Engineering.
CRISPR-Gold repairs DNA mutations using homology-directed repair (HDR) in order to correct disease-causing gene mutations by returning them to their original wild-type or normal sequence. Scientists struggle to develop HDR therapeutics requiring the almost simultaneous activity of the Cas9 protein attaching to a mutation and then using donor DNA to make the correction.
To overcome this challenge, Berkeley scientists invented a delivery vessel binding all repair components together that then releases them when the delivery vessel is inside a wide variety of cell types. CRISPR-Gold's gold nanoparticles then coat the donor DNA while binding Cas9. When injected into mice, mouse cells recognize a marker within CRISPR-Gold and incoporate the delivery vessel. CRISPR-Gold is then released through a series of cellular mechanisms into the cells' cytoplasm breaking apart and rapidly releasing Cas9 and the donor DNA.
A single injection of CRISPR-Gold into muscle tissue of mice that model DMD restored 5.4 percent of the dystrophin gene to the wild- type, or normal, sequence. This correction rate was approximately 18 times higher than in mice treated with Cas9 and donor DNA by themselves, which experienced only a 0.3 percent correction.
Importantly, the study authors note, CRISPR-Gold faithfully restored the normal sequence of dystrophin, which is a significant improvement over previously published approaches that only removed the faulty part of the gene, making it shorter and converting one disease into another, though milder version of muscle disease.
CRISPR-Gold also reduced tissue fibrosis or formation of excess connective tissue, a hallmark in muscle dysfunction diseases, while enhancing strength and agility in mice with DMD. CRISPR-Gold-treated mice showed a two-fold increase in their "hang-time" which is a common test for mouse strength and agility, when compared to mice injected with a control.
"These experiments suggest that it will be possible to develop non-viral CRISPR therapeutics that can safely correct gene mutations, via the process of homology-directed repair, by simply developing nanoparticles that can simultaneously encapsulate all of the CRISPR components."
Researchers quantified CRISPR-Gold's off-target DNA damage and found them similar to typical DNA sequencing errors in a typical cell not exposed to CRISPR (0.005 - 0.2 percent). To test for possible provocation of an immune response, the blood stream cytokine levels of mice were analyzed 24 hours and two weeks after the CRISPR-Gold injection. CRISPR-Gold did not cause an acute increase in inflammatory cytokines after multiple injections, or weight loss, suggesting that CRISPR-Gold can be used multiple times safely, with a high therapeutic window for gene editing in muscle tissue.
"CRISPR-Gold and, more broadly, CRISPR-nanoparticles open a new way for safer, accurately controlled delivery of gene-editing tools," says Conboy. "Ultimately, these techniques could be developed into a new medicine for Duchenne muscular dystrophy and a number of other genetic diseases."
A human clinical trial will be next to discern the effectiveness of CRISPR-Gold as treatment for genetic disease. The labs of Murthy and Conboy, and the company GenEdit, are working on the next generation of particles to deliver CRISPR into tissues via the blood stream, preferentially to target adult stem cells considered the best targets for gene correction. Stem and progenitor cells are capable of gene editing, self-renewal and differentiation.
"Genetic diseases cause devastating levels of mortality and morbidity, and new strategies for treating them are greatly needed," Murthy adds. "CRISPR-Gold was able to correct disease-causing gene mutations in vivo, via the non-viral delivery of Cas9 protein, guide RNA and donor DNA, and therefore has the potential to develop into a therapeutic for treating genetic diseases."
Clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR associated protein 9 (Cas9)-based therapeutics, especially those that can correct gene mutations via homology-directed repair, have the potential to revolutionize the treatment of genetic diseases. However, it is challenging to develop homology-directed repair-based therapeutics because they require the simultaneous in vivo delivery of Cas9 protein, guide RNA and donor DNA. Here, we demonstrate that a delivery vehicle composed of gold nanoparticles conjugated to DNA and complexed with cationic endosomal disruptive polymers can deliver Cas9 ribonucleoprotein and donor DNA into a wide variety of cell types and efficiently correct the DNA mutation that causes Duchenne muscular dystrophy in mice via local injection, with minimal off-target DNA damage.
Authors: Kunwoo Lee, Michael Conboy, Hyo Min Park, Fuguo Jiang, Hyun Jin Kim, Mark A. Dewitt, Vanessa A. Mackley, Kevin Chang, Anirudh Rao, Colin Skinner, Tamanna Shobha, Melod Mehdipour, Hui Liu, Wen-chin Huang, Freeman Lan, Nicolas L. Bray, Song Li, Jacob E. Corn, Kazunori Kataoka, Jennifer A. Doudna, Irina Conboy & Niren Murthy
The study was funded by the National Institutes of Health, the W.M. Keck Foundation, the Moore Foundation, the Li Ka Shing Foundation, Calico, Packer, Roger's and SENS, and the Center of Innovation (COI) Program of the Japan Science and Technology Agency.
Study co-authors Kunwoo Lee and Hyo Min Park formed GenEdit as a start-up company, Murthy has an ownership stake in GenEdit, focused on translating CRISPR-Gold technology into humans.
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CRISPR–Gold uses gold nanoparticles to encapsulate all of the elements needed for
CRISPR/Cas9 gene editing and deliver them directly to cells.
Image source: University of California, Berkeley