Genome Editing with mRNA Encoding ZFN
Based on target-sequence-specific engineered or bacterial nucleases, gene-editing technology is an emerging therapeutic approach for directly manipulating genomic sequences in almost all eukaryotic cells. Genome editing techniques have been applied to build more accurate animal and cellular models of pathological processes and show remarkable potential in a variety of fields, including basic research, applied biotechnology as well as biomedical research. In vitro-transcribed (IVT) mRNA delivery of nucleases holds great potential for therapeutic application due to no risk of genomic integration, a potentially low off-target rate, high editing efficiency, and transient expression with in vivo and in vitro delivery. Creative Biogene is a leading service provider for mRNA-based drug research and development. Here, we focus on delivery systems for nucleases and ZFN-mRNA for gene editing therapy. And zinc finger nuclease (ZFN) is one of three main types of gene editing nucleases.
The delivery system for nucleases
Figure1. Barriers for non-viral delivery.
(Zhang, H. X., et al, 2019)
The key to successfully transforming genome-editing techniques into drugs is to deliver nucleases to the right cells and tissues at the right time. Genome-editing nucleases currently can be delivered in the forms of protein, DNA, and mRNA. Among them, protein-mediated delivery of nucleases is the most straightforward way, which can provide the best control over nuclease dosage without any signal amplification. Besides, protein-mediated delivery prevents the transcriptional and translational processes, offering the most transient genome-editing duration with reduced off-target effects, thus, it is suitable for ex vivo cell therapy. While, there are several limitations of protein-mediated delivery for in vivo therapy, such as inducing the immune response, limited systemic delivery due to charge and large size as well as the challenges of protein production purification. DNA-mediated delivery of nucleases is cost-effective and now commonly used for basic research. In this delivery system, viruses and plasmids are the most popular vectors to deliver nuclease, and they usually offer persistent expressions. However, DNA-mediated delivery has several drawbacks, including the sustained nuclease expression, the low delivery efficiency of the nuclease, and cellular toxicity, which limit its applications. The mRNA-mediated delivery system might be the ideal delivery system that allows transient nuclease activity by considering the potential mutagenicity and immunogenicity of nucleases.
Table 1. Modes of nuclease delivery.
| DNA | mRNA | Protein | |
| Transcription in nuclear | yes | no | no |
| Translation in cytoplasm | yes | yes | no |
| End products function in nuclear | yes | yes | yes |
| Starting time after transfection (h) | >8 | 4-6 | 3 |
| Stability | great | good (modified mRNA) | poor |
| Duration | 1 week | several days | ∼24-48 h |
| Integration risk | yes | no | no |
| Off-target | high | low | low |
* Zhang, H. X., et al, 2019 (Molecular Therapy)
ZFN-mRNA for gene editing therapy
Fig 2. Zinc finger nuclease (ZFN) recognizes its target sites which is composed of two zinc finger monomers that flank a short spacer sequence recognized by the FokI cleavage domain. (Khalil, A. M, 2020)
Zinc finger nucleases (ZFNs) are artificially engineered by restriction enzymes for custom site-specific genome editing. As one of the transcription factors, ZFNs have 3–4 bases on each finger. The action of ZFNs is based on zinc-finger DNA binding domains and the FokI endonuclease domain. As one of the DNA binding motifs found in the DNA-binding domains of many eukaryotic transcription factors, the zinc finger is responsible for DNA identification. When two zinc-finger proteins are near in space, the fused FokI endonuclease form a functional dimer that acts like genomic scissors, cleaving DNA in the target loci. A properly designed pair of ZFNs can be applied to any gene in any organism in principle. At present, ZFN-mRNA has been applied in cell engineering and the generation of genetically modified animal models for research and clinical trials. And the translation to clinic of ZFN-mRNA is currently focused on ex vivo applications. We show recent clinical trials of ZFN-mRNA for gene editing therapy.
Table 2. Clinical trials of ZFN-mRNA for gene editing therapy.
| Disease | Biological Active | Target Protein | Strategy/Delivery System | Administration Route |
| HIV | SB-728mR | CCR5 | Ex vivo/Autologous CD4+ T Cells | Intravenous |
| SB-728mR | CCR5 | Ex vivo/Autologous CD4 CAR+ T cells | Intravenous | |
| SB-728mR-T | CCR5 | Ex vivo/Autologous T cells | Intravenous | |
| SB-728mR-HSPC | CCR5 | Ex vivo/Autologous CD34+ hHSPCs | Intravenous | |
| Sickle Cell Disease | BIVV003 | B-cell lymphoma/leukemia 11A (BCL11A) | Ex vivo/Autologous CD34 + hematopoietic stem cells (HSPC) | Intravenous |
* Gómez-Aguado, I., et al, 2020 (Nanomaterials)
Creative Biogene is offering a wide series of mRNA research services, which can accelerate the progress of mRNA-based genome editing for customers worldwide. We can support our customers with the most affordable, high-quality custom mRNAs according to the desired applications. If you are interested in this area, please feel free to contact us. We'll get back to you in time.
References
- Khalil, A. M. (2020). "The genome editing revolution." Journal of Genetic Engineering and Biotechnology, 18(1), 1-16.
- Zhang, H. X., et al. (2019). "Genome editing with mRNA encoding ZFN, TALEN, and Cas9." Molecular Therapy, 27(4), 735-746.
- Gómez-Aguado, I., et al. (2020). "Nanomedicines to deliver mRNA: state of the art and future perspectives." Nanomaterials, 10(2), 364.
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