Muscle-Targeted Cell-Penetrating Peptides for Delivery of Cas9-RNPs and Modified mRNA to Dystrophic Muscle
Abstract
This project is based on the exiting findings that we can formulate genetic therapies/biotherapeutics with a new generation of drug delivery vectors based on cell-penetrating peptides (CPPs), short peptides with a remarkable ability to enter cells and deliver large drug cargos. We have shown that by mere mixing of these peptides with Cas9-RNPs, a macromolecular protein complex that can promote editing of genes, or mRNA, used for expression of therapeutic protein, nanoparticles are formed that promote robust gene editing in vitro and mRNA-mediated gene expression in mice. Importantly, such nanoparticles display negligible toxicity and distribute to several different organs, and in part to muscle, when administered systemically through intravenous injections. This makes this platform unique over other existing non-viral delivery vectors that predominantly distribute to liver upon systemic delivery in mice. Hence, it is here our intention to explore this vector system to deliver disease-relevant biotherapeutics in the context of Duchenne muscular dystrophy (DMD). DMD is a fatal disease caused by a progressive degeneration of muscle that finally leads to cardiac and respiratory failure and premature death. Despite extensive efforts to develop new drugs for DMD, most are merely pallative or do not sufficiently correct the disease phenotype. We aim to develop next-generation CPP-based nanoparticles targeted to muscle and loaded with both microdystrophin mRNA (genetic material encoding for a short functional isoform of dystrophin, the protein DMD patients lack) and Cas9-RNPs (an enzyme that can cut DNA to create a functional protein in DMD patients). The mRNA will be used as a short-term treatment until the DNA can be corrected by Cas9-RNPs to be able to save as much muscle as possible from the first day of diagnosis. This is a unique combinatorial approach that can immediately benefit DMD patients. In addition, and importantly, using this approach, virtually all patients are amenable to treatment, which is not the case with many other promising treatment modalities. Although we have a delivery system that can deliver large molecules to cells in animals without any visible toxicity, the delivery to muscle tissue today is still too low for a clinical benefit in DMD patients. Therefore, we propose to further modify the non-toxic CPP to infer muscle-targeting capabilities for the nanoparticles to be able to deliver large molecules to muscle. We will evaluate the efficacy of the delivery in both cell cultures and DMD mice and have a pre-clinical package on efficacy ready by the end of the 2 years. Our team has taken other novel treatments for DMD from the pre-clinical stage to the clinic within 10 years and if successful here, this can thus be fast-tracked to the clinic. In summary, the DMD field has several promising treatment modalities, including microdystrophin expression and CRISPR-Cas9 gene editing. However, all novel treatments rely on large molecules that are hard to deliver to cells. Microdystrophin and CRISPR-Cas9 can be delivered via virus particles, but there are several problems associated with viral-associated delivery. The most crucial of these is the immune response against the virus, which can cause severe side effects. Another problem is the risk of integration of the delivered cargo into the genome of the patient. Our novel nanoparticles will overcome all these drawbacks and can be adapted to deliver both classes of large molecules and hence, transform the treatment options for DMD patients. If the delivery efficacy to muscle can be increased, we can utilize all existing treatment modalities developed already for DMD patients. Therefore, we believe the impact of our research can be tremendous for both DMD patients and patients with other muscle-related diseases, such as traumatic muscle injury. The treatment would benefit Service members’ families suffering from DMD directly; however, since our approach is flex
Document Details
- Document Type
- DoD Grant Award
- Publication Date
- Mar 10, 2021
- Source ID
- W81XWH2010678
Entities
People
- Samir El Andaloussi
Organizations
- Karolinska Institutet
- United States Army