Lipid technology for delivery of gene-editing therapies
Gene-editing technology is moving from the research lab to the clinic — one of the most eagerly anticipated biopharma developments in recent history. One of the biggest hurdles to the development of successful gene-editing therapeutics is effective delivery of the large and sensitive components needed to execute the editing functions. Crodas lipid-based nanoparticles present promising solutions to these challenges.
The potential of therapeutic CRISPR gene-editing
Gene-editing technologies are used to manipulate DNA and RNA sequences by cutting, inserting, or replacing genetic sequences in the genome. Three primary technologies have been developed to date:
zinc finger nuclease (ZNF), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeat (CRISPR). Initially, this technology was primarily used for research purposes to identify genes involved in disease pathways — either assessing the consequences of a gene knockout/knockdown or reversing a phenotype by introducing the gene for a functional protein. Today, however, many therapeutic candidates based on gene-editing technologies are now advancing through the clinic.
The developers of the first version of CRISPR/Cas9 gene-editing technology received the 2020 Nobel Prize in Chemistry for their work. A host of advances have been made since the original CRISPR editing tools were introduced — primarily via genetic engineering — with some of the current candidates leveraging second-generation technology and others relying on third-generation approaches that have addressed some of the earlier issues with the precision and persistence required for therapeutic applications. Further improvements are anticipated as candidates move through preclinical and clinical development and more knowledge is gained. A number of companies that have been exploring this approach are currently seeing rapid acceleration of their programs. For instance, Verve Therapeutics and Intellia Therapeutics have begun clinical trials. Many new companies are entering the space as well, quite a number of them focused on in vivo therapies. As gene-editing systems are optimized and become more precise and efficient, the ability to address new and different diseases will become possible, and there will be continued ovement toward in vivo therapeutics.
Two CRISPR-Cas9 components to deliver
Unlike more traditional gene therapies that involve delivery of a single gene of interest, CRISPR/CRISPR-associated system (Cas9) gene-editing requires co-delivery of the Cas9 endonuclease protein and the single guide RNA (sgRNA) that targets the editor protein to a specific genomic location.
The Cas9 protein and sgRNA can be delivered separately or complexed together as a ribonucleoprotein (RNP), which is the active species that performs the genome editing within the cell. Alternatively, plasmid DNA can be delivered to the cell, where it is transcribed into messenger RNA and then translated into Cas9 protein.2 Another option involves the use of Cas9 mRNA, which must be translated into Cas9 protein once it is inside the cell. DNA is more stable than
mRNA, but the kinetics of plasmid delivery are slower than those for mRNA. Regardless of the approach, delivery is
challenging, owing to the large size of the Cas9 protein and the overall CRISPR/Cas9 system, as the components must reach the cell nucleus. Stability is also an issue. The low intracellular delivery efficiency must be overcome to achieve effective gene-editing therapeutics. There are also concerns about the ability to safely and effectively deliver repeated treatments, which for many large tissues and organs will be necessary using current delivery technologies. The need for repeat administration has the potential to result in immunogenicity on the one hand and the development of immunity on the other.
LNPs are attractive because they have low immunogenicity and provide greater flexibility with respect to the cargos for which they can facilitate delivery and the organs or cells that can be targeted.
Why LNPs are attractive delivery vehicles
Several different approaches have been employed for the delivery of CRISPR gene-editing tools, and they can generally be classified as physical, biological, or chemical. Physical delivery methods include electroporation, microinjection, and sonoporation. Electroporation can be effective at delivering Cas9 RNP into hard-to-transfect
cells, including induced pluripotent stem cells. It is inefficient for in vivo delivery, however, so most uses have been in ex vivo applications. Microinjection involves injection of the CRISPR components into cells under a microscope and thus has limited throughput. In addition, while there is no cargo size limitation, it does require specialized equipment and expertise and is largely focused on the zygote stage, which is not appropriate for human therapeutics. Sonoporation combines ultrasound with injection and has been investigated in a few development products.
Biological delivery largely involves the use of viral vectors and virus-like particles. With viral vector delivery, the CRISPR/ Cas9-encoding sequences are integrated into the viral genome, resulting in release of the RNP complex into the targeted cells. There are safety concerns (e.g., mutations, carcinogenesis, immune response) and packaging limitations with certain viral vectors. Exosomes (membrane-bound vesicles) are another natural alternative for CRISPR/Cas9 delivery that are being explored because of their biocompatibility and low immunogenicity.
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Source: Stephen Burgess, Ph.D., Croda brochure “Lipid technology for delivery of gene-editing therapies“, original link of article here
ABOUT THE AUTHOR
![Lipid technology for delivery of gene-editing therapies_Stephen Burgess, Ph.D.]()
Stephen Burgess, Ph.D.
Head of Lipid Technology and Nucleic Acid Delivery, Croda Pharma
Stephen Burgess, Ph.D.Stephen Burgess, Ph.D., serves as Managing Director of Avanti Polar Lipids, and he is Head of Lipid Technology and Nucleic Acid Delivery for Croda Pharma, supporting the group’s vision to become the global leader in empowering biologics delivery. Over the past 30 years, he has worked with numerous researchers to develop novel lipids and lipid formulations to address specific experimental challenges and improve drug delivery. He has played a key role in a diverse list of projects, including drug delivery development, gene therapy, wound healing, and vaccine development. His background in lipid synthesis, lipid biophysical properties, and membrane dynamics has been instrumental in resolving problems associated with these projects.