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CRISPR delivery challenges

CRISPR, short for Clustered Regularly Interspaced Short Palindromic Repeats, are the hallmark of a bacterial defense system that forms the basis for CRISPR-Cas9 genome editing technology. Most commonly used CRISPR techniques involve the use of Cas proteins with guide RNAs for site-specific editing. CRISPR gene editing technology has developed rapidly since its discovery in the early 1990s, which has opened up a new path for the treatment of a variety of gene-related diseases. Multiple preclinical studies and clinical results have shown its powerful therapeutic effects. Nonetheless, one of the main obstacles in the field of CRISPR-Cas editing approaches is efficient cellular delivery , which ultimately determines the power of the application.¹ Delivery with viral vectors generate an autoimmune response that impacts delivery effectiveness. The commonly used electroporation method cannot be used in vivo, and lipo transfection is highly cytotoxic. Lipid nanoparticles (LNPs), on the other hand, have become an attractive non-viral delivery platform for CRISPR-mediated genome editing due to their low immunogenicity and application flexibility.

What are LNPs?

Lipid nanoparticles (LNPs) are tiny, spherical vesicles composed of lipids that can encapsulate and deliver various types of therapeutic molecules including nucleic acids like mRNA and CRISPR components such as guide RNA and DNA knock-in templates. Apart from the nucleic acid payload, LNP components generally include, cationic or ionizable lipids, and some accessory lipids- which are usually phospholipids (e.g., DOPE, DSPC), cholesterol, and pegylated lipids (e.g., DMG-PEG2000, DSPE-PEG2000) (Figure 1).² The principle behind nucleic acid loading to LNPs is that cationic or ionizable lipid substances bind to electronegative nucleic acids through electrostatic interactions. In addition, cationic or ionizable lipid can mediate the electrostatic interaction between LNPs and cytoplasmic or endosomal membranes, promoting cellular uptake and endosomal release of nucleic acids.2 Among lipid adjuvants, phospholipids are the main components of natural biofilms. Due to their cylindrical geometry, it is beneficial for nanoparticles to form a bilayer phase, which can improve the stability of LNPs.³ Cholesterol stabilizes LNPs by filling the gaps between phospholipids, and in the presence of serum proteins, cholesterol-containing LNPs are more stable than cholesterol-free LNPs.⁴ In addition, cholesterol is one of the components of natural biofilms, which can promote the fusion of membranes and facilitate the entry of LNPs into cells. The proportion of pegylated lipids determines the particle size of LNPs, and the higher the ratio, the smaller the particle size. Although LNPs with small particle size are more likely to enter tumor cells, excessive pegylated lipids can cause excessive colloidal stability of LNPs, hindering the internalization and release of LNPs into cells. Due to the high Enhanced Permeability and Retention (EPR) effect, pegylated lipids give LNPs higher tumor penetration, tumor accumulation, reduced serum protein clearance, and prolonged circulation time.⁵ The hydrophilia of pegylated lipids also facilitate the passage of LNPs through viscous mediators, such as lung mucus, reducing the loss of LNPs in the humoral circulation.⁶

LNP advantages and applications

Overcoming the challenge of inefficient CRISPR delivery warrants sophisticated packaging and delivery systems. An important advantage of using LNPs as drug carriers is their ability to escape recognition by the innate immune system and have higher circulation time.⁷ These features are especially useful for delivering hydrophobic drugs with short circulation half-lives such as nucleic acids and proteins. Sufficient circulation time enables LNPs (including those containing CRISPR components in either nucleic acid or protein forms) to reach target tissues and induce efficient on-target therapeutic genome editing.

CRISPR components in a variety of formats can be delivered to cells using LNPs. The most common approaches include the encapsulation of: (1) plasmid DNA (pDNA) encoding both Cas9 protein and gRNA or pDNA encoding Cas9 protein in combination with gRNA oligos, (2) Cas9 mRNA and gRNA, or (3) a Cas9 protein/sgRNA RNP complex.⁷ There are advantages and limitations to each of these techniques, so each approach must use a unique set of LNP specific formulation criteria to ensure optimal compatibility without compromising function.

In November 2021, Intellia Therapeutics announced a second clinical trial which aims to prevent angioedema attacks in patients with hereditary angioedema (HAE). The company’s CRISPR/Cas9 platform, NTLA-2002, utilizes LNP delivery to target the KLKB1 gene. The therapy is currently in clinical trials.

Currently, different LNP delivery strategies have been developed for CRISPR. By altering the structure of ionizable liposomes, specific organ targeting can be achieved. Different classes of lipid materials, including diketopiperazine lipids with degradable ester linkages, diethylamino lipids with adamantane tails, and piperazine lipids with either hydrazine or ethanolamine linkers, can deliver nucleic acids to various cell types in the spleen. Furthermore, doping LNPs with a lipid containing a quaternary ammonium headgroup, a component termed a selective organ targeting (SORT) molecule, enables the lung-selective delivery of either mRNA or Cas9–single guide RNA (sgRNA) ribonucleoproteins for gene editing in endothelial, epithelial, and immune cells.^8-12

GenScript solutions for LNP CRISPR

First, GenScript is at the forefront of nucleic acid production. At present, GenScript provides sgRNA plus ssDNA, closed-end dsDNA, and circular dsDNA HDR templates from RUO to GMP level, which can efficiently satisfy the needs of customers at different stages of therapy development. We can accommodate order quantities ranging from µg up to 100 grams in scale.

Our LNP formulation service provides an effective delivery option for our nucleic acid production, regardless of your application. We offer four ionizable lipid formulations that are not active targeting, and will launch specific tissue-target formulations later this year. We are also actively developing an all-in-one CRISPR-LNP solution. At present, this solution has achieved knockout efficiencies of more than 80% in experimental in vitro data, and knock-in efficiencies of 40%. Stay tuned for an official launch announcement for this service later this year.

We have also developed a pre-complexed Cas9/sgRNA RNP service to further serve our customers' CRISPR needs, which will be launching soon.

Future Outlook

It is important to note that many therapeutic applications of the CRISPR/Cas9 system involve the use of the HDR pathway for gene correction, instead of NHEJ. This means an additional donor DNA template containing the desired edits must also be present. The success rate of HDR is largely dependent upon the availability of the donor template at close proximity to the Cas9 cut site, which complicates the delivery scheme by requiring encapsulation of an additional DNA component. Current research on LNP formulations mostly focuses on creating indels with the NHEJ pathway or base editing, where the delivery of only Cas9 and gRNA are sufficient. Efficient delivery of DNA, sgRNA, and mRNA together is a pain point that requires further work before LNP delivery can sufficiently meet the needs of researchers working on this application.

Interested in learning more about our current CRISPR services? Click here.

References

[1] Xue. W., Chen. S., Yin. H. CRISPR-mediated direct mutation of cancer genes in the mouse liver. Nature. 2014, 514, 380–384.

[2] X. Cheng., R.J. Lee. The role of helper lipids in lipid nanoparticles(LNPs) designed for oligonucleotide delivery. Adv. Drug Deliver. Rev. 2016, 99, 129-137.

[3] J. L. Thewalt., M. Bloom. Phosphatidylcholine: cholesterol phase diagrams. Biophys. J. 1992, 63, 1176-1181.

[4] F. Sakurai., T. Nishioka., F. Yamashita., Y. Takakura., M. Hashida. Effects of erythrocytes and serum proteins on lung accumulation of lipoplexes containing cholesterol or DOPE as a helper lipid in the single-pass rat lung perfusion system. Eur. J. Pharm. Biopharm. 2001, 52, 165-172.

[5] Y. Bao., Y. Jin., P. Chivukula., J. Zhang., Y. Liu., J. Liu et al., Effect of PEGylation on Biodistribution and Gene Silencing of siRNA/Lipid Nanoparticle Complexes. Pharm. Res. 2013, 30, 342-351.

[6] Q. Xu., L.M. Ensign., N.J. Boylan., A. Schoen., X. Gong., J.-C. Yang et al., Impact of surface polyethylene glycol(PEG) density on biodegradable nanoparticle transport in mucus ex vivo and distribution in vivo. ACS Nano. 2015, 9, 9217-9227.

[7] Puri., A et al. Lipid-Based Nanoparticles as Pharmaceutical Drug Carriers: From Concepts to Clinic. Crit. Rev. Ther. Drug Carrier Syst. 2009, 26, 523−580.

[8] Fenton., O. S. et al. Customizable lipid nanoparticle materials for the delivery of siRNAs and mRNAs. Angew. Chem. Int. Ed. 2018, 57, 13582–13586.

[9] Lokugamage, M. P., Sago, C. D., Gan, Z., Krupczak, B. R. & Dahlman, J. E. Constrained nanoparticles deliver siRNA and sgRNA to T cells in vivo without targeting ligands. Adv. Mater. 2019, 31, e1902251.

[10] Ramishetti, S. et al. A combinatorial library of lipid nanoparticles for RNA delivery to leukocytes. Adv. Mater. 2020, 32, e1906128.

[11] Cheng, Q. et al. Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR–Cas gene editing. Nat. Nanotechnol. 2020, 15, 313–320.

[12] Wei, T., Cheng, Q., Min, Y. L., Olson, E. N. & Siegwart, D. J. Systemic nanoparticle delivery of CRISPR–Cas9 ribonucleoproteins for effective tissue specific genome editing. Nat. Commun. 2020, 11, 3232.