Protocol for Delivery of CRISPR/dCas9 Systems for Epigenetic Editing into Solid Tumors Using Lipid Nanoparticles
Eleanor A Woodward1,2, Edina Wang1,2, Christopher Wallis1,2
1Cancer Epigenetics Group, Harry Perkins Institute of Medical Research, Perth, WA, Australia.
Methods in Molecular Biology (Clifton, N.J.)
|July 16, 2024
Summary
CRISPR epigenetic editing offers cancer therapy potential. Researchers developed mRNA and lipid nanoparticle delivery for in vivo cancer treatment, overcoming previous delivery barriers.
Area of Science:
- Biotechnology
- Molecular Biology
- Cancer Research
Background:
- CRISPR epigenetic editing systems, utilizing deactivated Cas9 (dCas9) fused to effector domains, show promise for targeting cancer transcriptional abnormalities.
- Current limitations in CRISPR-based cancer therapy include inefficient in vivo delivery, large molecule size, and potential immunogenicity.
- Viral delivery methods for CRISPR face challenges like limited packaging capacity, genomic integration risks, and immunogenicity.
Purpose of the Study:
- To describe the design of optimized messenger RNA (mRNA) and guide RNA (gRNA) for CRISPR epigenetic editing.
- To outline lipid nanoparticle (LNP) formulations for efficient delivery of dCas9-effector mRNA.
- To provide protocols for co-encapsulating mRNA and gRNA into LNPs for in vitro and in vivo delivery.
Main Methods:
- Design and optimization of dCas9-effector mRNAs and gRNAs for targeted gene activation or repression.
- Formulation of lipid nanoparticles (LNPs) for encapsulating mRNA and gRNA.
- Co-encapsulation of dCas9-effector mRNA and gRNA into LNPs.
- In vitro delivery to cell lines and in vivo delivery to mouse models of breast cancer.
Main Results:
- Successful design of optimized dCas9-effector mRNAs and gRNAs for epigenetic editing.
- Development of suitable LNP formulations for dCas9 mRNA delivery.
- Establishment of protocols for co-encapsulating mRNA and gRNA into LNPs.
- Demonstration of in vitro and in vivo delivery of CRISPR components using LNPs in breast cancer models.
Conclusions:
- mRNA and LNP-based delivery systems offer a viable, non-viral strategy for CRISPR epigenetic editing in cancer therapy.
- This approach overcomes key delivery challenges associated with dCas9 fusion proteins and multimodal delivery.
- The developed protocols facilitate the translation of CRISPR epigenetic editing into potential clinical applications for cancer treatment.
