scott.pritchett

Reviewing CRISPR/Cas9 technology

Renowned for its robust editing capabilities and versatile applications, the CRISPR/Cas9 system is composed of two major components: a Cas9 protein and a single-stranded guide RNA (gRNA). To enable modification of the gene target, these two components form a complex called an ribonucleoprotein (RNP). The gRNA brings the complex to the target site in the genome and the Cas9 protein generates a double-stranded DNA break (or a single-stranded break, if using a Cas9 nickase) based on the protospacer adjacent motif (PAM). This can result in either a gene knockout (KO) through mutations introduced by non-homologous end joining (NHEJ) DNA repair systems, or lead to gene knock-in (KI) at a target locus if homology-directed repair (HDR) templates are added. CRISPR-based gene-editing systems provide vast opportunities to tackle a wide range of devastating disorders caused by malignant mutations, and therapies based on the CRISPR/Cas systems are booming. In 2023, the first CRISPR/Cas9-based gene therapy, Casgevy, was approved by the FDA, officially ushering in a new era of gene therapy.

Delivery options for the CRISPR editing system

Delivery of the CRISPR/Cas9 system is a critical step in gene editing, particularly in therapeutic applications. It can be delivered either in vitro or in vivo using one of multiple approaches.

One commonly used method is to deliver the editing system in plasmid DNA form. Plasmids encoding the Cas9 protein and gRNA can achieve intracellular delivery using viruses such as adeno-associated viruses (AAV). The plasmid design and preparation require only standard laboratory setups and are cost-effective. However, plasmids need to enter the nucleus and undergo transcription and translation to express the Cas9 and gRNA, slowing the onset of editing. The longer expression of the system driven by episomal DNA may be beneficial if sustained expression is needed for editing, but this also increases the risk of off-target events. This approach can also cause spontaneous plasmid integration in the genome, posing the risk of insertional mutagenesis.

Another common route is to deliver a pre-complexed Cas9 protein and gRNA ribonucleoprotein (RNP). The direct delivery of Cas9 protein allows immediate and more transient editing, producing high editing efficiency while lowering the chance of off-target effects. Yet, entering the cell and the nucleus is challenging for Cas9 due to its large molecular size. The bacterial origin of Cas9 also trigger immune responses in humans. Moreover, the production and purification of Cas9 protein requires more time, higher costs, and significant effort to maintain the stability and biological activity of the Cas9 protein before injecting it into the subject.

An alternative approach is the delivery of Cas9-encoding mRNA and gRNA. Compared to protein or plasmid delivery, when delivering Cas9 in mRNA form, protein can be produced at cytosolic ribosomal sites without complex cell entry mechanisms of the need to enter the nucleus, enabling quicker expression of the editing system. Moreover, since mRNA is unstable and prone to degradation by RNases, this format allows a more transient expression of Cas9 compared to the longer-lasting effects of plasmid or protein delivery, and thus reduces the risk of off-target events. Lastly, unlike plasmids, mRNA does not pose a risk of insertional mutagenesis in chromosomal DNA.

mRNA-LNP delivery system

RNA delivery particles must successfully perform three major processes to be effective: RNA encapsulation, targeted cell uptake, and endosomal escape. Among a wide array of materials that have been developed for RNA delivery, the most clinically advanced results have been achieved with lipid nanoparticles (LNP), which are used in two FDA-approved COVID-19 mRNA vaccines from Pfizer–BioNTech and Moderna.

LNP are composed of four major components: an ionizable lipid, a helper lipid, cholesterol, and a polyethylene glycol (PEG)-conjugated lipid. The ionizable lipid binds the encapsulated RNA and plays a dominant role in achieving endosomal escape. While it remains neutral at physiological pH, reducing the interaction between anionic membranes of blood cells, the lower pH in endosomes protonates ionizable lipids and promotes membrane destabilization, facilitating the endosomal escape of LNPs. The PEG-conjugated lipid stabilizes the LNP against aggregation and helps the LNP bypass macrophage-mediated clearance and renal filtration. The helper lipid and cholesterol help stabilize the LNP structure and affect the membrane fluidity, which also impacts the efficiency of endosomal escape.

These components allow LNPs to overcome multiple extracellular and intracellular barriers to function in vivo. First, LNPs protect the RNA cargo from nuclease degradation in physiological fluids and systematic clearance such as renal filtration. Second, LNPs direct the cargo to the target cells and enter the cells through endocytosis. Finally, they help RNA escape endosomes and reach the cytoplasm, where translation occurs.

Advantages of using mRNA-LNP for CRISPR gene editing

The mRNA-LNP system offers multiple advantages over other delivery approaches such as viral delivery, particularly when delivering CRISPR/Cas systems in vivo:

• 1. Biosafety

  Uncontrolled integration of foreign sequences into the genome can be dangerous. Although the AAV genome predominantly exists in the nucleus in episomal form, experimental evidence suggests that small portions of genome integration still occur. Since plasmid DNA itself also poses the risk of insertional mutations, delivering the editing agents as the RNA form can help maintain the host genome integrity. In addition, unlike the transient expression of the editing system driven from mRNA-LNP, the prolonged expression from episomal or integrated virus genome leads to increased off-target events. It is also worth noting that the persistent expression of editing agents could trigger immune recognition of edited cells, which may compromise editing outcomes. Lastly, the immunogenicity of synthetic LNPs is much lower than that of viruses.

• 2. Flexibility

  Cargo size is a critical consideration when using AAV as the delivery approach, as it has a packaging capacity of around 4.7 kb. This limits the potential of using AAV to package CRISPR/Cas systems, especially larger systems such as prime editing. The payload size for LNPs is much more flexible in comparison. Also, when it comes to designing therapeutic treatments, the low immunogenicity of LNP allow for repeat administrations, whereas a second injection of AAV has shown to be neutralized by acquired immunity.

• 3. Precise delivery and tissue-targeting

  Currently, LNPs have shown to target hepatic cells efficiently. Other non-liver targeting LNPs are being investigated and developed. For example, by adding charged lipid components, Siegwart’s team proposed and developed selective organ targeting (SORT) LNPs that can direct LNPs to either the lung or the spleen without targeting the liver [5]. Epstein’s team, on the other hand, designed an anti-CD5 antibody-conjugated LNP to target T cells [6]. These advances in tissue-specific LNP delivery broaden the therapeutic applications of gene editing.

• 4. Scalability

  LNPs are composed of synthetic materials, and large-scale production of LNPs has been proven feasible. The use of mRNA also circumvents the production and purification process required when dealing with proteins, allowing for a simpler and more cost-effective manufacturing process.

Success cases: Adopting the LNP-mRNA delivery system to perform CRISPR-based therapies

With these advantages, the LNP-mRNA delivery system has become a promising candidate for delivering CRISPR/Cas systems to achieve optimal therapeutic outcomes. Several CRISPR-based therapies developed with the LNP-mRNA format have shown promising results in both pre-clinical and clinical trials.

1. NTLA-2001 (Intellia Therapeutics)

NTLA-2001 is the first-in-human in vivo CRISPR/Cas9-based gene therapy, developed by Intellia Therapeutics, as a single-dose treatment for transthyretin amyloidosis (also known as ATTR amyloidosis). NTLA-2001 consist of the mRNA-LNP delivery system that carries the gRNA and optimized Cas9 mRNA. It targets and generates insertions and deletions in the transthyretin (TTR) gene, subsequently decreasing the level of both wild-type and mutant TTR, which causes the abnormal accumulation of amyloid fibrils in ATTR amyloidosis patients. Since more than 99% of the circulating TTR is produced in the liver, NTLA-2001’s LNP delivery system with liver tropism maximizes CRISPR editing efficacy while minimizing systemic toxic effects. In preclinical studies, a single administration of TTR-LNP knocked down plasma TTR by 90% and 80% in a transgenic mouse model and a cynomolgus monkey, respectively. In the Phase 1 clinical trial, patients showed a reduction in serum TTR by 47-52% and 80-96% in the low-dose and high-dose groups, respectively, with no serious adverse events observed [7].

2. VERVE-101 and VERVE-102 (Verve Therapeutics)

In addition to delivering a Cas9 nuclease mRNA and gRNA, scientists have shown that LNP is capable of encapsulating and delivering other CRISPR-based gene editing systems such as base editors and prime editors. One successful example is VERVE-101 from Verve Therapeutics. VERVE-101 is a CRISPR-based medicine composed of mRNA encoding an adenine base editor and a guide RNA targeting the PCSK9 gene, encapsulated in LNP, to treat familial hypercholesterolemia. By introducing a premature stop codon, VERVE-101 can permanently turn off the PCSK9 gene, leading to a decrease in low-density lipoprotein cholesterol (LDL-C). In non-human primates, VERVE-101 is able to achieve 70% editing after 14 days of administration, based on the liver biopsy, and maintain 80% and 60% reduction in PCSK9 and LDL-C, respectively, for more than one year. The Phase 1 clinical trial also showed promising efficacy of VERVE-101 with reduction in blood LDL-C from 39 to 55% following VERVE-101 administration as long as 180 days, with follow-up ongoing [8,9].

Both cases demonstrate the broad application of the mRNA-LNP system to achieve significant in vivo genome editing and led to effective and durable therapeutic outcomes.

Current challenges for mRNA-LNP delivery in CRISPR-based gene therapy

Despite the huge potential of the mRNA-LNP system in CRISPR-based therapeutics, one major challenge the needs to be overcome is targeting non-liver cells. Most intravenously administered nanoparticles accumulate in the liver. While these nanoparticles typically fail to reach hepatocytes due to the size restrictions, LNPs are an exception. Through the binding of apolipoproteinE (ApoE) to LNPs, they can reach and enter hepatocytes via low-density lipoprotein receptor (LDLR)-mediated endocytosis. Although the ApoE-LDLR pathway allows LNPs to effectively target liver cells, LNPs need to overcome this pathway dependency to target non-liver cells. Because of this limitation, the application of LNPs is still largely restricted to liver targeting.

Future prospects of mRNA-LNP delivery in CRISPR-based gene therapy

The combined capabilities of CRISPR/Cas systems and the mRNA-LNP system enables safe and effective in vivo editing, largely expanding the therapeutic potential of gene editing. Several CRISPR-based therapies, mostly focusing on editing hepatic cells, are currently in clinical trials and have shown positive results. While the liver tropism of LNPs allows efficient targeting of liver cells, it significantly limits the accessibility of LNPs to other organs. Thus, developing LNPs that enable efficient extrahepatic delivery remains a critical milestone for CRISPR-based therapeutics. Two major directions, adjusting LNP components and conjugating targeting groups, are being investigated. Increased targeting and subsequent editing in non-liver organs, including the lungs, spleen, lymphatic system, and skeleton muscle, have been reported by various groups [5, 10, 11]. This data highlights the vast opportunities of in vivo gene editing therapies as they move toward clinical application. Future advances and refinement of both editing systems and delivery methods will unlock a wide range of in vivo gene editing therapeutic options to tackle a variety of devastating diseases.

To help researchers achieve optimal therapeutic outcomes for their CRISPR-based therapies, GenScript has developed a groundbreaking platform for producing customized, high-quality all-in-one mRNA-LNPs. In this platform, we have optimized both the LNP formulation and the editing system to best meet the needs of therapy developers.

Find out more about our ReadyEdit mRNA-LNP solution for gene editing here.

References

1. Yip, B. H. (2020). Recent advances in CRISPR/Cas9 delivery strategies. Biomolecules, 10(6), 839.

2. Behr, M., Zhou, J., Xu, B., & Zhang, H. (2021). In vivo delivery of CRISPR-Cas9 therapeutics: Progress and challenges. Acta Pharmaceutica Sinica B, 11(8), 2150-2171.

3. Madigan, V., Zhang, F., & Dahlman, J. E. (2023). Drug delivery systems for CRISPR-based genome editors. Nature Reviews Drug Discovery, 22(11), 875-894.

4. Witten, J., Hu, Y., Langer, R., & Anderson, D. G. (2024). Recent advances in nanoparticulate RNA delivery systems. Proceedings of the National Academy of Sciences, 121(11), e2307798120.

5. Cheng, Q., Wei, T., Farbiak, L., Johnson, L. T., Dilliard, S. A., & Siegwart, D. J. (2020). Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR–Cas gene editing. Nature nanotechnology, 15(4), 313-320.

6. Raguram, A., Banskota, S., & Liu, D. R. (2022). Therapeutic in vivo delivery of gene editing agents. Cell, 185(15), 2806-2827.

7. Gillmore, Julian D., et al. "CRISPR-Cas9 in vivo gene editing for transthyretin amyloidosis." New England Journal of Medicine 385.6 (2021): 493-502.

8. Lee, R. G., Mazzola, A. M., Braun, M. C., Platt, C., Vafai, S. B., Kathiresan, S., ... & Khera, A. V. (2023). Efficacy and safety of an investigational single-course CRISPR base-editing therapy targeting PCSK9 in nonhuman primate and mouse models. Circulation, 147(3), 242-253.

9. Vafai, S. B., Gladding, P. A., Scott, R., Kerr, J., Taubel, J., Cegla, J., ... & Bellinger, A. M. (2023). Safety and pharmacodynamic effects of VERVE-101. Presented at the American Heart Association Scientific Sessions, 2023, 12.

10. Kenjo, E., Hozumi, H., Makita, Y., Iwabuchi, K. A., Fujimoto, N., Matsumoto, S., ... & Hotta, A. (2021). Low immunogenicity of LNP allows repeated administrations of CRISPR-Cas9 mRNA into skeletal muscle in mice. Nature communications, 12(1), 7101.

11. Kim, J., Eygeris, Y., Ryals, R. C., Jozić, A., & Sahay, G. (2024). Strategies for non-viral vectors targeting organs beyond the liver. Nature nanotechnology, 19(4), 428-447.