The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas system has significantly simplified the gene editing workflow. Genome modifications, thought impossible a decade ago, are currently executed with an astonishing ease, efficiency, and specificity. Although two main CRISPR/Cas systems, class 1 and class 2, have been identified and classified based on their effector protein composition, the majority of the current gene editing approaches rely on the CRISPR/Cas9 system, which belongs to the second class. Particularly, the Streptococcus pyogenes Cas9 (SpCas9) nuclease is broadly used.
Significant advances have been made to expand the versatility of the CRISPR/Cas9 toolbox. However, several challenges remain related to off-target mutation frequency, PAM associated constraints on targeting, efficiency of editing, incidence of HDR, and immune reactivity to Cas9 (Moon et al. 2019). Particularly, CRISPR/Cas9 delivery approaches represent a significant bottleneck for in vivo and potential therapeutic applications.
Viral and non-viral approaches may be leveraged for the delivery of the CRISPR/Cas9 complexes. One universal challenge for CRISPR/Cas9 delivery is the large molecular size of the SpCas9 nuclease (~160kDa). Whether delivering the CRISPR/Cas9 complex through viral or non-viral approaches, the large size of the nuclease often calls for creative ways to achieve efficient delivery of the full editing cargo (Rosenblum et al. 2020).
| Delivery Strategy | mRNA | DNA | Ribonucleoprotein (RNP) |
|---|---|---|---|
| Viral |
Lentivirus (LV), Adenovirus (AV), Adeno-associated virus (AAV) |
||
| Non-Viral |
Electroporation, Lipid-based nanoparticle, Microinjection |
Electroporation, Hydrodynamic delivery, Lipid-based nanoparticle, Microinjection |
Cell penetrating peptide, DNA nanoclews, Electroporation, Extracellular vesicle (EVs), Gold nanoparticle, Hydrodynamic delivery , iTOP, Lipid-based nanoparticle, Microinjection |
(Lino et al. 2018, Yip, 2020)
Viral vectors such as AAV offer several advantages for in vivo applications and potential therapeutic use including their low immunogenicity, inability to replicate, and lack of insertional mutagenesis risk. In contrast, LVs and AVs are not feasible CRISPR/Cas9 delivery systems for therapeutic applications due to their potential for random integration and high immunogenicity, respectively.
Non-viral approaches rely on physical and chemical methods for delivering the CRISPR/Cas9 complex as mRNA, plasmid DNA (e.g., DNA plasmid encoded Cas9 and gRNA), or ribonucleoprotein complex (RNP) (Lino et al. 2018, Yip, 2020). Among these options, the use and delivery of CRISPR/Cas9 as RNP complexes presents several advantages including fast gene editing, high editing efficiency, as well as low off-target effects and immunogenicity.
However, some disadvantages still need to be addressed, primarily in relation to the lack of effective strategies for the delivery of the large RNP complexes to desired target tissues while remaining functional. Recently, modified lipid nanoparticles containing supplemental permanently cationic lipids have been developed and used to deliver RNP complexes to the liver and lungs in mice (Wei et al. 2020).
In order to improve systemic delivery of RNPs to targeted organs, modified lipid nanoparticles (LNPs) are being developed, most recently by a team at the University of Texas Southwestern Medical Center led by Daniel Siegwart, PhD. The newly modified LNPs contain a supplemental permanently-cationic lipid (DOTAP), which helps stabilize the RNPs during the process of LNP encapsulation under neutral buffer conditions. Cellular uptake of these modified lipid nanoparticles, 5A2-DOT-10, was shown to occur via lipid-raft mediated endocytosis and led to efficient knockout in vitro.
Most relevant to the potential use of this approach for therapeutic applications, by modulating the percentage of DOTAP within the LNPs from 5 to 60%, investigators were able to target specific tissues in mice following LNP IV administration. For example, LNPs with low concentration of DOTAP resulted in RNP targeting and efficient gene editing in the liver, while the highest DOTAP concentration primarily led to gene editing in the lungs.
Highlighting the strength of this delivery approach for animal model based studies, investigators were able to target specifically the liver and lungs for editing of multiple genes leading to the formation of organ-specific tumor lesions. Finally, in an animal model of Duchenne Muscular Dystrophy, intramuscular injection of LNPs loaded with RNPs targeting the dystrophin gene, successfully restored dystrophin expression in tibialis anterior muscles.
