CRISPR-Cas9 Gene Therapy

Genome editing has a wide range of applications in therapeutic medicine and biomedical research. In recently years, CRISPR-Cas9 has become a cost-effective and convenient tool for various genome editing. CRISPR-Cas9 is revolutionizing many areas of medical research and one of the most amazing areas is certainly gene therapy, which can provide novelty to the way infectious diseases and genetic disorders are treated. Just several months after its introduction into mammalian cells, CRISPR-Cas9 demonstrated its potentials in gene therapy by mutating HIV-1 to decrease its expression in human T cells. Since then, much effort has been made to explore the therapeutic potentials.

CRISPR-Cas9 can be applied for therapeutic purposes in several methods. It can correct the causal mutations in monogenic disorders and thus rescue the disease phenotypes, which currently represents the most translatable field in CRISPR-Cas9-mediated gene therapy. CRISPR-Cas9 can also engineer pathogen genome such as HIV for therapeutic purposes, or induce protective or therapeutic mutations in host tissues. Moreover, CRISPR-Cas9 has shown potentials in cancer gene therapy such as inactivation or clearance of oncogenic virus, inducing oncosuppressor expressions, and correcting genetic disorders and so on.

CRISPR-Cas9 Mediated Cancer Gene Therapy

CRISPR-Cas9 plays an important role in cancer gene therapy. For instance, the ability of cancer cells to develop resistance to chemotherapy drugs is a primary cause of failure of chemotherapy. The application of the CRISPR-Cas9 system to inactivate drug resistance genes in a given cancer is a potential therapeutic strategy to increase the efficacy of chemotherapy. Another aspect of CRISPR-Cas9 in cancer gene therapy is to enhance the host cells immune response to cancer. This could be possible through CRISPR-Cas9 mediated modification of T-cells. The reinfusion of genetically modified T-cells into cancer patients has shown promising results in clinical trials and could be a way forward for anti-cancer therapies.

Oncolytic virotherapy is one of the most promising fields in cancer gene therapy. Oncolytic viruses (OVs) can be developed by attenuating and modifying viruses and/or arming the virus with a therapeutic gene. The genetically engineered OVs have antitumor properties. They can infect and replicate in cancer cells, and then kill the cancer cells through virus-mediated cytotoxicity or enhanced anticancer immune response without causing any harm to the normal cells. CRISPR-Cas9 can play a part in oncolytic virotherapy in several ways, such as adding a cancer-specific promoter to genes that are indispensable for viral replication, or inducing mutations in viral genome whose defects can be complemented by cancer specific metabolites.

CRISPR-Cas9 Gene Therapy in Genetic Disorders

Various researches in the past years have confirmed the efficiency of CRISPR-Cas9 as a probable method to overcome genetic diseases in humans via experimentation in animal and human cellular models. Targeted mutation using CRISPR-Cas9 can manipulate genetic material by deleting and replacing causal mutations, host mutations can also be induced that will provide protection to the host. CRISPR-Cas9 technology can be used with ease to treat monogenic diseases; where a correction in the culprit gene could reverse the genetic disease. On the other hand, polygenic diseases are not so straightforward, having multiple mutations in the genome; they possess a far strenuous challenge to treat in comparison to monogenic diseases.

CRISPR-Cas9 components delivered through intramuscular, intraperitoneal or intravenous injection can correct the culprit gene mutation in mouse models of Duchenne muscular dystrophy (DMD) and rescued the disease phenotype. DMD is a monogenic disease caused by mutations in the gene encoding a protein termed dystrophin that is necessary for muscle cell integrity. In mouse models of DMD generated by a nonsense mutation in exon 23 of Dmd gene, CRISPR-Cas9 targeting intron 22 and intron 23 of Dmd gene removed the disease-causing mutation in a proportion of muscle cells in neonatal or adult mice, resulting in restoration of dystrophin expression and muscle function.

Challenges and Solutions of CRISPR-Cas9 Gene Therapy

Although these recent advances represent a significant step forward to the eventual application of CRISPR-Cas9 to the clinic, there are still many challenges to overcome, such as the off-target effects of CRISPR-Cas9, HDR rate, efficacy of homology-directed repair, fitness of edited cells, immunogenicity of therapeutic CRISPR-Cas9 components, as well as efficiency, specificity, and translatability of in vivo delivery methods. Delivery and editing efficiency has long been a crux of gene therapy applications. The solutions of this problem lie in future the development of more efficient delivery vectors, more powerful sgRNA, and more potent Cas9.

Off-Target Effects: Because CRISPR-Cas9 causes permanent genome alterations, its off-target effects must be accurately profiled and controlled when applied in gene therapy. Several methods have been developed to reduce the off-target effects. Firstly, both the structure and composition of the guide RNA can affect the frequency of off-target effects. Secondly, the using of a pair of Cas9 nickases to generate paired nicks on the two strands of target sequence can significantly increase target specificity because off-target single nicks are faithfully repaired. Thirdly, sgRNA truncated by 2-3nt are reported to reduce off-target effects possibly because shorter sgRNA sequence has a decreased mismatch tolerance. The delivery vehicles by which Cas9 and sgRNAs enter into cells can also affect on- to off-target activity ratio. Lastly, combination of CRISPR-Cas9 with other nuclease may also help.

HDR Rate: The DSBs produced by CRISPR-Cas9 are subsequently processed by NHEJ or HDR. HDR-mediated gene correction has broader application spectrum than NHEJ-mediated gene deletion or inactivation because there are far more diseases whose treatments necessitate precise gene correction than those requiring only culprit gene deletion or inactivation. In many cases, HDR results in desired gene modifications while NHEJ gives rise to undesired indels that cause uncontrollable gene disruptions. Therefore, increasing HDR rate can improve the efficiency and reliability of CRISPR-Cas9-mediated gene therapy while decrease genomic toxic effects concurrently. Many methods have been reported to increase HDR rate, such as rational design of single-stranded DNA donors, inhibition of NHEJ pathway, and increasing the extent of similarity between donor templates and the double-strand break sites.

CRISPR-Cas9 Gene Therapy Related Information

CRISPR-Cas9 Gene Therapy Related References

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3. Xiao-Jie L, et al. CRISPR-Cas9: a new and promising player in gene therapy. J Med Genet 2015;52:289–296.
5. Ding et al. Recent Advances in Genome Editing Using CRISPR/Cas9. Frontiers in Plant Science. May 2016, Volume 7, Article 703.