CRISPR is transforming the treatment of disease, from monogenic rare diseases to immuno-oncology applications. However, the development of safe and efficient approaches to deliver CRISPR components for therapeutic purposes remains a challenge. Multiple factors, including absorption, distribution, metabolism, excretion, and disease characteristics, need to be carefully studied to ensure effective and safe editing in target cells and organs.1 Existing approaches for nucleic acid delivery have been adapted to deliver CRISPR components. These approaches could be broadly classified into two categories: viral and nonviral.
For in vivo applications, where CRISPR components are injected into patients directly to work as drugs for the treatment of disease, the viral approach has been commonly used. The inherent ability of viruses to introduce exogenous genetic material into cells ensures high transduction efficiency. Among current gene therapy studies, adeno-associated viruses (AAVs) are the most commonly used viral vectors, due to their high titer, low immunogenicity, and low genomic integration rate.
This year, we have witnessed the first human adult in the United States to receive a CRISPR-based therapy administered directly in vivo. The clinical trial was sponsored by Editas Medicine and Allergan to evaluate the efficacy and safety of the candidate therapy EDIT-101 for treating Leber congenital amaurosis 10 (LCA10). Additionally, Excision BioTherapeutics is applying CRISPR to cure viral infectious diseases. Excision’s lead candidate, EBT-101, using an all-in-one AAV vector to deliver multiplex sgRNAs and the SaCas9, will enter human clinical trials in 2020.2
Although long-term efficacy has been achieved using AAVs in several clinical trials, the concerns of insertional mutagenesis, carcinogenesis, and immunogenicity associated with viral delivery still linger. A few studies have reported AAV integration in CRISPR cut sites in cultured murine neurons and in mouse brain, muscle, and cochlea.3,4 In addition, a 10-year follow-up study on AAV-treated dogs with hemophilia has shown that an 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.5
Due to the limitations of viral-based CRISPR delivery, nonviral delivery methods are on the rise, especially in immuno-oncology therapeutics. Nonviral approaches utilize CRISPR Cas9 mRNA/protein and synthetic sgRNA ribonucleoproteins (RNPs) to directly deliver CRISPR components, rather than requiring transcription and translation steps as needed for viral CRISPR delivery. Thus, nonviral approaches offer greater control over how long the components linger in cells, and they reduce off-target effects and toxicity. Nonviral delivery approaches can be further classified into physical or chemical carrier–mediated approaches.
Physical methods such as microinjection, electroporation, magnetofection, and hydrodynamic injection employ physical force to facilitate intracellular transport of CRISPR components. Among them, electroporation is the most commonly used method. For example, in the clinical trial of CTX001 (CRISPR Therapeutics and Vertex Pharmaceuticals), it was used to deliver Cas9 RNP to isolated patient cells for the treatment of sickle-cell disease.
In February 2020, a group of scientists led by the University of Pennsylvania’s Edward A. Stadtmauer, MD, and Carl H. June, MD, reported a first-in-human Phase I trial in the United States to test the safety and feasibility of multiplex CRISPR-Cas9 editing to engineer T cells in three patients with refractory cancer. In this study, CRISPR-Cas9 RNP complexes loaded with three sgRNAs were electroporated into the isolated patient T cells to knock out genes encoding endogenous T-cell receptors (TCRs) and the checkpoint PD1.6
However, subsequent delivery of the TCR transgene (NY-ESO-1) in this study was performed using lentiviral transduction. Lentiviral insertion of transgenes has been the dominant method in engineering T cells due to the ability of lentiviral vectors to stably integrate a transgene into a host genome effectively. However, lentiviral vectors have the tendency to insert their transgenes into active transcription units, leading to insertional mutagenesis.
To minimize the chance of random genome insertion, CRISPR offers a more precise way of inserting a transgene into a targeted genome locus. For example, researchers can now knockout multiple immune regulators, as well as precisely insert a new chimeric antigen receptor (CAR) or TCR at an endogenous TCR alpha constant (TRAC) locus in healthy donor- or patient-derived primary T cells using CRISPR agents and an ssDNA donor template to develop safer and more effective CAR T-cell or TCR T-cell therapies.7
Despite its extensive use in T-cell engineering, the electroporation approach is still limited to ex vivo editing applications. Delivery of the highly charged RNA component and large Cas9 protein in vivo remains challenging. Some delivery methods based on chemical carriers have proved highly efficient in cells.
A research team led by the KAUST’s Niveen M. Khashab, PhD, encapsulated CRISPR RNPs with positively charged nanoscale zeolitic imidazole frameworks (ZIFs) with a loading efficiency of 17%. Then the team verified gene editing potential by knocking down the expression of EGFP by 37% in CHO cells over four days.8
In another study, one led by the University of Wisconsin-Madison’s Krishanu Saha, PhD, scientists assembled RNPs with biotinylated oligonucleotides via an RNA aptamer and achieved increased gene editing efficacy in human cells.9 These versatile and preassembled reagents could greatly improve the development of dosing regimens for gene editing in vivo.
Nanoparticles, including metal-based nanoparticles and lipid nanoparticles (LNPs), are attractive because of their specificity, scale-up ability, easy customization, minimal immunogenicity, and minimal exposure to nucleases. Delivery vehicles composed of gold nanoparticles have been developed by researchers led by Niren Murthy, PhD, a scientist at the University of California, Berkeley, and Hye Young Lee, PhD, a scientist at the University of Texas Health Science Center at San Antonio. The vehicles delivered CRISPR/Cas9 RNP, which efficiently corrected the DNA mutation that causes Duchenne muscular dystrophy, as well as mutations in the brains of mice.10,11
LNPs also represent a clinically advanced approach with high efficiency, low cytotoxicity, and low immunogenicity. In 2018, Intellia Therapeutics published preclinical data in Cell Reports demonstrating effective CRISPR/Cas9 editing using an LNP-based delivery method.12 The company has used its modular LNP system to deliver CRISPR/Cas9 in its lead in vivo program, for treating transthyretin amyloidosis (ATTR), as well as in its potential hereditary angioedema program.
Recently, a team of scientists at the University of Texas Southwestern Medical Center led by Daniel J. Siegwart, PhD, developed a new strategy termed selective organ targeting (SORT). It can expand the application of LNPs in specific organs and tissues outside the liver.13
Overall, viral vectors have shown high efficiency in gene delivery and expression. However, viral vectors also pose immunogenicity and carcinogenicity risks, and they provide limited packaging capacity. In contrast, lower delivery efficiency is a main barrier of current nonviral delivery methods, despite a safer editing profile. In the near future, we believe both methods will continue to improve, overcoming the obstacles that limit their application. More clinical trials and follow-up data will also be available for us to make better delivery choices.
GenScript provides comprehensive CRISPR reagents and services to help accelerate the development of safer gene and cell therapy products, including the RNP system with HPLC-purified synthetic gRNAs with end modifications for the best editing results and minimum cytotoxicity, as well as long single-stranded DNA as a transgene knockin template for precise gene insertion.
1. Li B, Niu Y, Ji W, Dong Y. Strategies for the CRISPR-Based Therapeutics. Trends Pharmacol. Sci. 2020; 41(1): 55–65. DOI: 10.1016/j.tips.2019.11.006.
2. Yin C, Zhang T, Qu X, et al. In Vivo Excision of HIV-1 Provirus by saCas9 and Multiplex Single-Guide RNAs in Animal Models. Mol. Ther. 2017; 25(5): 1168–1186. DOI: 10.1016/j.ymthe.2017.03.012.
3. Hanlon KS, Kleinstiver BP, Garcia SP, et al. High levels of AAV vector integration into CRISPR-induced DNA breaks. Nat. Commun. 2019; 10: 1–11. DOI: 10.1038/s41467-019-12449-2.
4. Nelson CE, Wu Y, Gemberling MP, et al. Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy. Nat. Med. 2019; 25(3): 427–432. DOI: 10.1038/s41591-019-0344-3.
5. Kaiser J. Virus used in gene therapies may pose cancer risk, dog study hints. Science 2020. Published January 6, 2020. DOI: 10.1126/science.aba7696.
6. Stadtmauer EA, Fraietta JA, Davis MM, et al. CRISPR-engineered T cells in patients with refractory cancer. Science 2020; 367(6481): eaba7365. DOI: 10.1126/science.aba7365.
7. Roth TL, Puig-Saus C, Yu R, et al. Reprogramming human T cell function and specificity with non-viral genome targeting. Nature 2018; 559(7714): 405–409. DOI: 10.1038/s41586-018-0326-5.
8. Alsaiari SK, Patil S, Alyami M, et al. Endosomal Escape and Delivery of CRISPR/Cas9 Genome Editing Machinery Enabled by Nanoscale Zeolitic Imidazolate Framework. J. Am. Chem. Soc. 2018; 140(1): 143–146. DOI: 10.1021/jacs.7b11754.
9. Carlson-Stevermer J, Abdeen AA, Kohlenberg L, et al. Assembly of CRISPR ribonucleoproteins with biotinylated oligonucleotides via an RNA aptamer for precise gene editing. Nat. Commun. 2017; 8(1): 1711. DOI: 10.1038/s41467-017-01875-9.
10. Lee K, Conboy M, Park HM, et al. Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair. Nat. Biomed. Eng. 2017; 1: 889–901. DOI: 10.1038/s41551-017-0137-2.
11. Lee B, Lee K, Panda S, et al. Nanoparticle delivery of CRISPR into the brain rescues a mouse model of fragile X syndrome from exaggerated repetitive behaviours. Nat. Biomed. Eng. 2018; 2(7): 497–507. DOI: 10.1038/s41551-018-0252-8.
12. Finn JD, Smith AR, Patel MC, et al. A Single Administration of CRISPR/Cas9 Lipid Nanoparticles Achieves Robust and Persistent In Vivo Genome Editing. Cell Rep. 2018; 22(9): 2227–2235. DOI: 10.1016/j.celrep.2018.02.014.
13. Cheng Q, Wei T, Farbiak L, et al. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR–Cas gene editing. Nat. Nanotechnol. 2020; 15(4): 313–320. DOI: 10.1038/s41565-020-0669-6.