The emerging CRISPR technology has expanded the scope and applications of gene therapy. Translation of CRISPR-based gene editing to in vivo application however, is still not easy. A vital challenge is to develop safe, efficient, and clinically suitable in vivo delivery approaches of the CRISPR components.
Viral based CRISPR delivery approaches have been extensively studied both in vitro and in vivo, and AAV is the most commonly used viral vector for in vivo gene therapy studies. The inherent ability of virus to introduce exogenous genetic material into the cells ensures the high transduction efficiency of viral delivery. Eric N. Olson’s team used AAV to deliver CRISPR/Cas9 and restored dystrophin expression in a canine model of Duchenne muscular dystrophy (DMD), reaching up to ~80% of WT levels in some muscles after 8 weeks (Leonela Amoasii et al., 2018). Earlier this year, EDIT-101, a AAV5 vector engineered with sequence encoding two gRNAs and cas9 protein, is under clinical investigation for the treatment of Leber congenital amaurosis 10 (LCA10). (Nat Biotechnol 38, 382. 2020). In March 2020, a patient treated with EDIT-101 became the first human patient to receive a CRISPR/Cas9 gene therapy administered directly in vivo.
Although long-term efficacy has been achieved in several clinical trials, the concerns of insertional mutagenesis, carcinogenesis, and immunogenicity associated with viral delivery still linger. For example, Casey Maguire and Bence Gyorgy reported high levels of AAV integration (up to 47%) into Cas9-induced double-strand breaks (DSBs) in therapeutically relevant genes in cultured murine neurons, mouse brain, muscle and cochlea (Killian S. Hanlon et al., 2019). Nelson et al. also reported AAV integration at CRISPR cut sites in a mouse muscular dystrophy model (Christopher E. Nelson et al., 2019). In addition, a 10-year follow up study on AAV treated dogs with hemophilia also shown that AAV vector can readily insert its payload into the host’s DNA near genes that control cell growth, hinting that AAV-based gene therapy may pose cancer risks (Jocelyn Kaiser, Science, 2020).
An alternative non-viral delivery method is to directly deliver Cas9 protein and synthetic gRNA ribonucleoproteins (RNPs), which offers greater control over how long the components linger in the cells, thus reduce the off-target effects and toxicity. Currently, CTX001, the ex vivo gene therapy developed by CRISPR Therapeutics and Vertex Pharmaceuticals is in Phase I clinical study, where CRISPR RNP is delivered using electroporation to isolated patient cells. However, delivering large RNA and protein molecules into target cells in vivo is a relatively new research field and remains challenging.
Several nanoparticle delivery approaches are now under investigation for effectiveness and efficacy, including lipid based and metal based nanoparticles. Nanoparticle is attractive in gene therapeutics because of its specificity, scale-up ability, easy customization, minimized immune response, and minimal exposure to nucleases (Fengqian Chen et al, 2019). For example, Lee et al. developed a delivery vehicle composed of gold nanoparticles (CRISPR-Gold) for CRISPR/Cas9 RNP, which efficiently corrected the DNA mutation that causes DMD with reduced off-target effect in mice (Kunwoo Lee et al., 2017) and corrected mutations in the brains of adult mice (Bumwhee Lee and Kunwoo Lee et al., 2018). Lipid nanoparticles (LNPs) are another clinically advanced approach with high efficiency, low cytotoxicity, and low immunogenicity. In 2018, Intellia Therapeutics reported that a biodegradable LNP-based CRISPR/Cas9 delivery system achieved significant transthyretin (TTR) gene editing, and >97% serum protein level reduction that persisted for 12 months following a single administration in mice and rats (Jonathan D Finn et al., 2018). Recently, Daniel J. Siegwart’s team reported a strategy termed selective organ targeting (SORT), wherein multiple classes of lipid nanoparticles are systematically engineered to exclusively edit extrahepatic tissues, expanding the application of LNPs in specific organs and tissues outside the liver (Qiang Cheng et al., 2020).
Despite the advances, non-viral delivery approaches for CRISPR/Cas9 delivery in vivo still face some hurdles. For instance, the serum stability of polyplexes after systemic administration and the relatively low transfection efficiency compared with viral vectors (Ling Li et al., 2018).
The development of safer and more efficient delivery systems is accelerating the use of CRISPR technology in human disease therapy. Though each of these delivery strategies has advantages and shortcomings and faces unique challenges, we are already on the way to translating CRISPR technology to the clinic. However, the question remains as to which delivery approach is more promising, viral or non-viral?
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Which delivery approach is more promising for CRISPR-based gene therapy?
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