Genome Editing with mRNA Encoding TALEN
Transcription activator-like effector nuclease (TALEN)-based gene editing is a commonly used tool for precise and efficient gene editing in live cells. To date, this gene-editing technology has been widely used in a wide range of host systems, including mammals, plants, insects, and bacteria. The effectiveness of TALENs can be improved when delivered to cells in the form of mRNA, as this reduces the off-target effects of the nucleases. The Creative Biogene team is experienced in custom mRNA synthesis, including zinc finger nuclease (ZFN)-mRNA, TALEN-mRNA, but not limited to them. Moreover, we are able to provide the most suitable delivery strategy of TALEN-mRNA according to the specific applications.
TALEN-based gene editing
Similar to ZFNs (the first widely applicable site-specific genome editing tools), TALENs include an artificially engineered nuclease, but TALENs show better specificity and efficiency than ZFNs. TALENs comprise a nonspecific DNA cleavage domain fused to a sequence-specific DNA-binding domain, which acts together to generate nuclease-induced double-stranded breaks (DSBs). The DNA-binding domain contains a highly conserved repeat sequence from transcription activator-like effector (TALE), a protein that was originally discovered in the phytopathogenic Xanthomonas bacteria. There are three domains in TALE protein, including an amino-terminal domain having a transport signal, a DNA-binding domain containing an array of repeating 33- to 35-amino-acid sequence motifs, and a carboxyl-terminal domain possessing a transcription activation domain and a nuclear localization signal. When doing genome editing, a pair of TALEN is used the same way as ZFNs. TALENs have two important advantages over ZFNs, including simpler design and lower number of off-target breaks, mainly due to each domain of TALEN recognizes only one nucleotide (while DNA triplets recognized by ZEF), and the design of TALEN is usually more obvious than ZNF. In addition to successful applications in plant genome engineering, TALEN-based gene editing has been clinically used for cancer therapy by developing universal allogeneic T cells, including relapsed/refractory B acute lymphoblastic leukemia (PALL), follicular lymphoma, acute myeloid leukemia, and multiple myeloma.
Fig1. Transcription activator-like effector nucleases (TALENs) consist of bacterial TALE proteins fused to endonucleases such as FokI. (Ashmore-Harris, C., et al, 2020)
Clinical application of TALEN-mRNA
TALEN-mRNA is utilized in allogenic CAR T cell therapy to generate UCART19. The product is based on allogenic T cells artificially engineered to express CARs against the leukemia antigen CD19. In addition, CAR19 T cells were electroporated with TALEN-mRNA to knockout the CD52 gene and T cell receptors (TCRs). Among them, the knockout of the CD52 confers transplanted allogenic T cells the resistance toward alemtuzumab (a lymphodepleting agent). And the aim of TCRs knockout is to reduce the risk of graft-versus-host disease (GVHD) induced by donor T cells. In addition, TALEN-mRNA has also been applied against HIV infection by knockout the HIV-coreceptor molecule CCR5.
Fig 2. Generation of UCART19 with TALEN-mRNA technology.
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
- Ashmore-Harris, C., & Fruhwirth, G. O. (2020). "The clinical potential of gene editing as a tool to engineer cell-based therapeutics." Clinical and translational medicine, 9(1), 15.
- 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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