Genome-editing systems have remarkable potential to treat genetic diseases. However, one of the major challenges facing their implementation is the safe and efficient intracellular delivery of genome-editing biomacromolecules, including nucleases and nucleic acids.
Nanoparticles encapsulating genome-editing biomacromolecules must be endocytosed by the cell of interest and reach the nucleus or cytoplasm to function; additional extracellular barriers are present for in vivo delivery.
Physical methods for in vitro and ex vivo delivery of genome-editing tools include electroporation, membrane deformation and microinjection.
Viral vectors are widely used to deliver DNA for genome editing and are typically integrase-defective lentiviral vectors (IDLVs), adenoviruses and adeno-associated viruses (AAVs); AAVs seem to be the most popular vector for in vivo applications.
Non-viral nanoparticles, most often made from synthetic and cationic lipid or polymer delivery materials, can be used to deliver genome-editing tools in vitro, ex vivo and in vivo, sometimes in combination with viral vectors.
At the time of writing, the genome-editing industry is in its infancy and includes ongoing and completed phase I and phase II clinical trials.
In addition to selecting an effective delivery material, scientists must consider safety (that is, off-target effects, immunogenicity and mutagenesis), the required amount of genomic modification for therapeutic benefit, and the duration of effects.
With the recent development of CRISPR technology, it is becoming increasingly easy to engineer the genome. Genome-editing systems based on CRISPR, as well as transcription activator-like effector nucleases (TALENs) and zinc-finger nucleases (ZFNs), are becoming valuable tools for biomedical research, drug discovery and development, and even gene therapy. However, for each of these systems to effectively enter cells of interest and perform their function, efficient and safe delivery technologies are needed. This Review discusses the principles of biomacromolecule delivery and gene editing, examines recent advances and challenges in non-viral and viral delivery methods, and highlights the status of related clinical trials.
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H.Y., K.J.K. and D.G.A. acknowledge funding from the Koch Institute Marble Center for Cancer Nanomedicine and the Cancer Center Support (core) Grant P30-CA14051. H.Y. is supported by Skoltech Center. The authors apologize to those authors whose work was not cited directly owing to space limitations.
D.G.A. and H.Y. have applied for patents relating to delivery technologies for genome editing. D.G.A. is a scientific co-founder of CRISPR Therapeutics.
- RNA interference
(RNAi). Process by which one strand of double-stranded RNA binds to complementary mRNA and degrades or regulates the mRNA via an enzymatic process, usually resulting in a decrease in the expression of a desired protein.
- Antisense oligonucleotides
(ASOs). Short single-stranded DNA or RNA sequences that bind to complementary mRNAs, inhibit translation and/or degrade the targeted mRNA, resulting in a decrease in the expression of a desired protein.
DNA that functions without integrating into the genome: for example, a delivered DNA plasmid.
- Protospacer adjacent motif
A short, typically 2–6 nucleotide-long region of DNA recognized by the Cas9 protein, located immediately next to the target region for the Cas9 nuclease.
The ability of a virus to specifically target particular cells or tissues.
From the same species but not genetically compatible; that is, will induce an immune response.
(RNP). Any biomacromolecule consisting of an RNA in complex with a protein.
- Hydrodynamic injection
A rapid, high-volume intravenous infusion.
Under the control of a single gene.
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Yin, H., Kauffman, K. & Anderson, D. Delivery technologies for genome editing. Nat Rev Drug Discov 16, 387–399 (2017). https://doi.org/10.1038/nrd.2016.280
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