CRISPR/Cas9 Gene Editing: Recent Advancements To Improve Clinical Potential
More than 3000 human genes have been strongly correlated to cancer and genetic disorders. Elucidation of genetic mutations and human disease is becoming increasingly urgent. Perhaps the most functional genome editing method was influenced by an adaptive immune response mechanism, whereby bacteria capture and cleave viral DNA to protect against viral attacks. The mechanism was later adapted by Doudna and Charpentier et al. and named the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated system (Cas) for studying genomic alterations in humans. Today, CRISPR/Cas gene editing is an integral part of gene-based drug discovery to uncover target genes and perform knockouts, which offers insight into the resulting phenotype. From this perspective, this technology is a huge milestone towards correlating patient genome to phenotype and empowering precision medicine.
Like every other genomic modality, CRISPR/Cas gene therapy has to be administered in an efficient and patient-friendly way. However, current gene delivery methods have demonstrated off-target effects and a lack of efficient knockdown.
In this Drug Targets Review podcast, Dr. Pietro De Angeli, the Institute for Ophthalmic Research of the University Hospital Tübingen, and Dr. Maarten, the Princess Maxima Center, discuss the safety profiles of CRISPR/Cas 9 delivery methods as well as current trends involving scalability and the use of artificial intelligence (AI) methods.
Dive into the conversation where we discuss the opportunities CRISPR/Cas technology offers to revolutionize gene drug discovery.
Safety profiles in focus: DNA, mRNA, and RNPs in CRISPR/Cas delivery
For CRISPR/Cas9 gene editing to work seamlessly, the complex must reach the nucleus, where it can locate the target genes.
CRISPR delivery is mediated by plasmid DNA (pDNA), mRNA, or ribonucleoproteins (RNPs), and the former two impose safety challenges. Delivery through plasmid DNA or mRNA can often result in the activation of the innate immune response, and pDNA has the added risk of incorporation into the host genome, leading to long-term adverse effects. Although ex-vivo strategies can help predict and mitigate these risks, CRISPR/Cas9 specificity remains unresolved.
CRISPR/Cas9 Mechanism. The Cas9 enzyme is activated by first binding to a guide RNA, then binding to the matching genomic sequence that immediately precedes 3-nucleotide PAM sequence. The Cas9 enzyme then creates a double-strand break, and either the NHEJ or the HDR pathway is used to repair the DNA, resulting in an edited gene sequence.
To de-risk gene editing, scientists are turning to RNPs, consisting of the Cas9 protein in complex with a targeting gRNA. This delivery method offers improved specificity and safety. To date, Maarten points to the inverse correlation between the time the complex spends in the host and the precision of CRISPR/Cas9 gene editing: “mRNA or for DNA, need up to a week for the transcription and translation of Cas9, while a RNP is active as soon as it enters the nucleus and can do the job in less than 24 hours. The longer Cas9 stays in the nucleus, the more likely it will find and accumulate off-target sites. That’s why the safest gene editing method is giving a very short Cas9 pulse through RNP.” Another advantage of RNPs is the ease of optimization, Pietro adds. “When using RNPs, it’s easier to tune the concentration of your reagents, which impacts target efficiency. Whereas in DNA-based CRISPR delivery, the transfection efficiency and the number of DNA copies are hard to predict and finetune.”
Another challenge posed by DNA or mRNA-based CRISPR/Cas9 is that it could alter the transcriptomic and proteomic profile of the host. This shift mainly occurs because the host re-organizes its energetic resources for the transcription and translation of DNA and mRNA forms. The resulting metabolic load can perturb the host metabolism, which alters the transcriptome and proteasome. In contrast, the delivery of CRISPR/Cas9 as an RNP is a complex ready to function and exert the desired gene editing, so it does not cause a shift in the genome towards expressing Cas9.
Ex-vivo applications: Navigating the scalability and feasibility of CRISPR
The CRISPR/Cas9 system cannot be delivered to the nucleus as a naked complex, so the appropriate CRISPR delivery vehicle plays a significant role in protecting the integrity of the complex. To address current efforts, the scientists share pioneering work from research labs.
Prof. David Liu, vice-chair of the Faculty at the Broad Institute of MIT and Harvard, engineered lipid nanoparticles (LNPs) of viral origin that can encapsulate the complex. LNPs demonstrated efficient delivery in all forms of CRISPR/Cas9, including the preformed RNP. This machinery guided the delivery of the complex to specific organs of interest (Banskota et al., 2022, Cell 185, 250–265). Meanwhile, Jennifer Doudna's lab enhanced the site specificity of Cas9 by adding nucleic localization signals to the C- and the N- terminus of the protein. In vivo experiments confirmed a precise gene editing in the mice brain (Stahl EC, et al., Mol Ther. 2023 Aug 2;31(8):2422-2438).
While the delivery methods continue to improve, the clinical potential of CRISPR/Cas9 gene editing has not been fully realized, mainly because viral delivery, associated with prolonged off-target editing, is still the gold standard. Amongst non-viral delivery methods, electroporation stands out for its safe and robust delivery capabilities compatible with RNPs. This method creates pores in the cell membrane, allowing seamless RNP entry into the cytoplasm and preventing its endocytosis or endosomal escape.
Another set of issues surface when it comes to scalability. While the Cas9 endonuclease can be produced in large batches without much cost, the guide RNA (gRNA) is specific not only to the disease or therapeutic approach but also to individuals. Pietro believes that the path to gRNA scale-up is paved with risks. "At the moment, what I see as a problem is not the technology itself, but rather the legal framework. Big pharmaceutical companies are focusing on more common disorders because they are looking for commercially viable opportunities. A patient belonging to this group has a higher chance to access gene editing treatments. For the treatment of rare diseases, academic and non-profit research institutions have to spend effort to facilitate patient access." According to Maarten, the regulatory procedure for rare diseases must be different: "For every disease, the safety of the complex must be demonstrated via substantial ex-vivo data. For rare diseases with a limited number of patients around the world, generating this comprehensive report is not possible. Currently, initiatives sponsored by Jennifer Doudna and other CRISPR experts are liaising with the FDA to soften the preclinical data requirements and IND filing for rare diseases." This proposal involves using gene editing ex-vivo to generate edited cells that can be administered back to patients, so that genome editing is regarded as a reagent rather than a drug.
Enhancing CRISPR precision medicine: The synergy of CRISPR/Cas with AI and ML
Machine learning (ML) and AI tools - similar to their impact in many other industries - are considered game changers in gene editing and gene-driven drug discovery. In the case of CRISPR/-Cas9, they can be used to assemble large data sets of site-specific and off-target editing and correlate these to different base editors or 3D RNP structures. Machine learning algorithms can give you the gRNA sequence needed to target a specific gene as well as all the modifications at the protein level necessary to achieve the level of specificity. Thus, scientists can now design the safest and the most efficient gRNA.
AI tools have also been used to predict the efficacy of more evolved variants of CRISPR/Cas9. For improved prime editing, researchers developed nCas9 - Cas9 nickase fused to reverse transcriptase - to streamline various genome editing types, such as point mutations, insertions, and deletions. Although this novel system yields better prime editing efficiency in theory, its design and safety must be assessed thoroughly, which is where the predictive power of AI tools comes in. Maarten emphasizes the value of AI in gene-based drug discovery: "It helps us scan hundreds of Cas9 - gRNA combinations to detect the top 10 best-performing ones."
The future of CRISPR: Transforming therapeutic approaches
Conventional genome sequencing does not suffice to elucidate the genetic context of diseases. While researchers can isolate cells from one patient and compare the genome to healthy ones, this comparison does not necessarily cover the thousands of point mutations contributing to the disease phenotype. CRISPR/Cas9 genome editing helps overcome these barriers and greatly benefits from precise disease modeling and personalized medicine to target specific mutations. To Pietro, treatment is only one side of the coin, "We can use it not only as a treatment tool but also as a means for disease research. 3D cellular models, such as induced pluripotent stem cells (iPSC)-derived organoids, are incredibly valuable for emulating complex human biology. So, by introducing a disease-associated mutation to the iPSC genome, we can generate cell cultures that can differentiate into the disease phenotype." This aspect of CRISPR/Cas9 is worth paying attention to because the genetic background of a disease is paramount to developing powerful therapeutic tools.
Of course, the role of 3D cell modeling cannot be overstated. Through 3D cell culture technologies and CRISPR/Cas9, researchers can generate isogenic cell models, which encompass the entire genomic landscape with cells extracted from only a few patients.
Conclusion - Next steps in CRISPR research and application
The therapeutic opportunities unlocked by CRISPR/Cas9 are expansive, especially in editing point mutations and gene-repairing ex vivo and - in the future - in vivo. This can increase treatment access to patients with rare diseases who carried previously hard-to-target mutations.
It is important to note that CRISPR is not a one-size-fits-all solution. Especially when talking about cancer, it does not invalidate the value of chemotherapeutic approaches. In contrast, it can create a synergistic effect by delving into the genomic roots of the disease, while the drug compound inhibits disease-associated mechanisms on a chemoenzymatic level. Combining CRISPR with FDA-approved drugs can accelerate its entry into mainstream clinical cancer treatment applications.
In the podcast, we explored innovative CRISPR delivery methods, including RNPs, engineered viral particles, and nanoparticle formulations, highlighting their potential to propel CRISPR-Cas9 applications closer to clinical use. The emphasis was on the necessity for precise and efficient gene editing techniques. To support these scientific advancements, Molecular Devices' ImageXpress high-content imaging systems offer critical insights by allowing detailed observation of these delivery mechanisms within cells. Furthermore, the integration of tools like SpectraMax microplate readers and the FLIPR Penta system enhances our understanding of cell growth, maintenance, and functionality, especially when working with complex models such as organoids. While discussing the importance of streamlining the research process, the conversation also touched upon the role of automation in modern laboratories. Solutions such as the CellXpress.ai Automated Cell Culture System support the automation of the cell culture process, contributing to more reliable and reproducible outcomes in scientific research. Automated solutions streamlining the IND filing during clonal screening are available, such as the ClonePix 2 Colony Picker with Monoclonality Assurance. Custom lab automation solutions were mentioned as a way to collaborate with scientists, adapting workflows to meet the specific needs of their CRISPR-Cas9 studies, thereby aligning technological capabilities with the pursuit of groundbreaking scientific discoveries.