Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo

Abstract

The combination of Cas9, guide RNA and repair template DNA can induce precise gene editing and the correction of genetic diseases in adult mammals. However, clinical implementation of this technology requires safe and effective delivery of all of these components into the nuclei of the target tissue. Here, we combine lipid nanoparticle–mediated delivery of Cas9 mRNA with adeno-associated viruses encoding a sgRNA and a repair template to induce repair of a disease gene in adult animals. We applied our delivery strategy to a mouse model of human hereditary tyrosinemia and show that the treatment generated fumarylacetoacetate hydrolase (Fah)-positive hepatocytes by correcting the causative Fah-splicing mutation. Treatment rescued disease symptoms such as weight loss and liver damage. The efficiency of correction was >6% of hepatocytes after a single application, suggesting potential utility of Cas9-based therapeutic genome editing for a range of diseases.

Exosome–Liposome Hybrid Nanoparticles Deliver CRISPR/Cas9 System in MSCs

“…[[qv: 5b,6]] However, the possible cytotoxicity, immunogenic response, long‐term expression, and off‐target effects of viral vectors, continue to be the clinical application concern 7. Also, the nonviral delivery methods, such as electroporation,[[qv: 7a]] microinjection,8 lipid and lipid‐like nanoparticles delivery,9 and ribonucleoprotein delivery10 are reported, which are also limited by their stability, accessibility, safety, or efficiency.…”

Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents

“…Cas9-based genome editing agents can mediate targeted gene disruption or repair 10–13 . For applications that seek a one-time, permanent modification of genomic DNA, we and others have shown that the delivery of non-replicable, transient Cas9:single guide RNA (sgRNA) ribonucleotide protein (RNPs) complexes in vivo offer improved DNA specificity and potentially greater safety and applicability 14,15 compared with methods that introduce DNA expressing these agents.…”

In vivo gene therapy potentials of CRISPR-Cas9

“…In conclusion, these recent advances [10][11][12]14,15 represent significant steps forward to the final application of CRISPR-Cas9 to human gene therapy. However, there are still many challenges lying ahead, such as delivery efficiency, HDR efficiency, off-target effects and the fitness of edited cells.…”

“…In these two studies, the authors used either a dual viral vector systems 14 or a combination of viral vector and lipid nanoparticles 15 to deliver the three therapeutic components of CRISPR-Cas9 (sgRNA, Cas9 and donor template), and gained a HDR efficiency level sufficient to rescue the disease phenotypes in mouse models of human hereditary liver diseases whose treatments necessitate HDR-mediated gene replacement.…”

“…In 2014, Yin et al 8 reported in vivo gene correction in adult mice with hereditary tyrosinemia by hydrodynamic injection of therapeutic CRISPR-Cas9 components through tail-vein, but this approach remains inapplicable to humans due to its potential damages to liver and cardiovascular functions. 16 On the contrary, these five recent studies [10][11][12]14,15 demonstrated the effectiveness of CRISPR-Cas9-mediated gene therapy through in vivo delivery approaches that are translatable to humans (intra-muscular, intra-peritoneal or intravenous injection). In mouse models of DMD, 10-12 the ratio of dystrophin-positive myofibers was observed to increase with time, 12 indicating that the corrected gene was persistently expressed, and may impose a growth advantage.…”