Viral and non-viral delivery approaches for CRISPR-based gene therapy, which one is more promising?

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? Share your opinion in the comment section. 

Which delivery approach is more promising for CRISPR-based gene therapy?

  • Viral
  • Non-viral
  • See my opinion in the comment section

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The Cas9 protein was subjected to site-directed mutation to form eSpCas9 and SpCas9-HF1. The mutant weakened the non-specific binding ability of the Cas9 protein to DNA, and enhanced the competitiveness of the targeting sequence to bind to the Cas9 protein.

Particle bombardment has a given name called biolistics, which is a funny portmanteau.

The most concerning issue is the off-target effects of viral delivery. Given time the non-viral delivery efforts are more promising. The issues with transfection efficiencies can be overcome.

There's still a long way to go for non-viral approaches for safe and efficient clinical use. The mainstream approach for a long time will still be viral vectors and I believe lots of improvement will be made after so many clinical studies. But if we imagine a distant future, non-viral approaches may give us more surprises.

Given CRISPR's popularity and global use, we will likely see developments in both types of delivery systems. As DNA synthesis becomes cheaper, it will be interesting to see what aspects of viral systems can be modified or redesigned (lower immune response? lower instances of viral integration?). Non-viral systems will no doubt see improvements in the coming years; I believe that these systems will be more flexible than viral systems and allow us to better tailor therapies to different situations. Whether an improved virus platform will be able to compete with these upcoming non-viral systems remains to be seen.

Non-viral delivery methods have difficulties in yielding proper ratio for gene editing and lack transduction efficacy. Therefore, it lacks the potential to deliver CRISPR/Cas9 in clinical settings. On the contrary, viral delivery methods have off-target effects that can be minimized by prolonged research. Their boon is far-reaching as they have made to clinical trials for the treatment of cancer, small gene therapies, and many screening procedures.

Non-viral delivery of CAR with CRISPR technology has multiple advantages: specific insertion, controlable insertion copy number, multiplex purpose for T cell gene editing. Compared viral methed, cost is much lower. With CRISPR technology development, eg lower off target or high KI efficiency, non viral method will be much superior.

Non-viral delivery of Cas9 protein or transient expression of Cas9 reduces the immune response caused by persistent expression of Cas9 and off-target effects in vivo.  sgRNA and donor template can be constructed into a viral vector for persistent expression. Therefore, in my opinion, the combination of viral vector with non-viral vector may be an optimal approach for precise medicine based on CRISPR technology.

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