Читать книгу Genome Editing in Drug Discovery - Группа авторов - Страница 15

Genome engineering describes the specific introduction of new genetic elements into target genes to mediate either a disruption of the gene sequence, and consequent loss of gene expression, or a change in the protein sequence of the gene being transcribed. The ability to modify gene sequences at precise locations in the genome arose in the 1990s with the development of meganucleases and Zinc Finger Nuclease (ZFN) technology (Kim et al. 1996; Bibikova et al. 2001). ZFNs are synthetic restriction enzymes created by fusing one or more zinc finger DNA‐binding domains, engineered to target a specific DNA sequence, to a DNA nuclease, typically the restriction enzyme Fok1. ZFNs are able to recognize and cut a specific site in the genome to create a double‐stranded DNA break that can be repaired by non‐homologous end joining (NHEJ) or Homology Directed Repair (HDR) to result in gene inactivation or gene repair following the introduction of a DNA repair template. A boost to the technology came in 2009 with the discovery of transcription activator‐like effector nucleases (TALENs) (Boch et al. 2009; Moscou and Bogdanove 2009). TALENs are synthetic restriction enzymes engineered to cut specific sequences of DNA. These are created by fusing a TAL effector DNA‐binding domain designed to recognize the target DNA sequence with a nuclease to mediate DNA cleavage. TALEN constructs can be introduced into cells to cut the DNA at specific sequences to create a double‐stranded DNA break, that following repair by NHEJ, results in the inactivation of the target gene as a consequence of the introduction of additional DNA sequences as part of the repair process. Furthermore, TALENs can be used to change specific nucleotides within a gene, or to introduce new sequences into the genome following transfection of cells with a TALEN’s construct and a DNA repair template. Following HDR at the DNA cut site, the new sequence, encoded by the DNA repair template, can be introduced into the genome, albeit at a low editing efficiency. ZFNs and TALENs technologies have been used extensively in genome engineering projects in medical research and to modify plant genomes. Furthermore, Zinc Finger technology has been used by Sangamo Therapeutics and others to create genome editing medicines. Treatments for a range of diseases are in development with the most advanced project, a treatment for hemophilia currently in Phase 3 clinical studies. However, the widespread adoption of these technologies has been limited due to the requirement for expertise in protein engineering to create a ZFN or TALEN that precisely targets a specific DNA sequence, the relatively low editing efficiencies observed, and the potential for editing at multiple sites in the genome.