We present an overview of clinical trials involving gene editing using clustered interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9), transcription activator-like effector nucleases (TALENs), or zinc finger nucleases (ZFNs) and discuss the underlying mechanisms. splicing of the CEP920 gene in Lebers congenital amaurosis. Close concern of safety aspects and education of stakeholders will be essential for a successful implementation of gene editing technology in the clinic. Graphical Abstract Open in a separate window Main Text Conventional Gene Therapy Traditionally, gene therapy relies on viral-based delivery of a protein-coding gene that either semi-randomly integrates Mmp17 into the genome (for retroviruses and lentiviruses) or remains as extrachromosomal DNA copy (for adeno-associated computer virus [AAV]).1, 2, 3 These forms of gene therapy usually use overexpression of a protein that is missing or mutated in human disease. Lentiviral gene therapy has the advantage of being highly efficient and causing long-term efficacy. A drawback of lentiviral gene therapy is the lack of control of the location at which the computer virus integrates into the host genome, with the risk of insertional mutagenesis. By optimizing the lentiviral backbone and by controlling the number of viral copies, it has been exhibited in multiple clinical trials that lentiviral gene therapy is usually safe provided that it is Synephrine (Oxedrine) used with the proper precautions.2,4 AAV-mediated gene therapy Synephrine (Oxedrine) does not rely on integration into the host genome but instead involves delivery of a DNA episome to the nucleus. It is therefore considered to have a Synephrine (Oxedrine) lower risk of genotoxicity compared to lentiviral gene therapy. However, episomal copies of AAV DNA are lost upon cell division, resulting in loss of efficacy. This restricts AAV gene therapy to nondividing cells. In addition, pre-existing immunity to AAV capsid proteins occurs in a significant percentage of the human population and precludes eligibility for the treatment.5 Acquired immunity after a single AAV-mediated gene therapy treatment occurs invariably in patients and precludes eligibility for a second treatment. In both forms of gene therapy, cDNA overexpression can only be used when dosage effects of the transgene product do not apply. Although the desired average number of gene Synephrine (Oxedrine) copies can be approached via the viral titer, it is not possible to precisely control this using viral-based overexpression. Basics of Gene Editing Developments in recent years have enabled the seamless engineering of the human genome using a variety of tools collectively termed gene editing. Precision gene editing strategies allow alteration of the genome of cells at specific loci to generate targeted genomic changes, which are being exploited for multiple applications in medicine. We first introduce the basics of gene editing and then summarize the major challenges for their clinical implementation. Gene editing tools that are currently under investigation in clinical trials include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered interspaced short palindromic repeats (CRISPR) in combination with CRISPR-associated protein (Cas). For a detailed comparison between these tools, we refer to previously published reviews.6,7 In short, target site recognition occurs by sequence-specific DNA-binding proteins (in the case of ZFNs and TALENs) or by a short stretch of RNA termed single guideline RNA (sgRNA; in the case of CRISPR-Cas). Current clinical applications of gene editing rely on the introduction of double-strand DNA breaks (DSBs), mediated by Fok-1 (in the case of ZFNs or TALENs) or by Cas nucleases (in the case of CRISPR-Cas) and the introduction of desired genomic alterations through the cells endogenous DNA repair mechanisms. Two major DNA repair pathways are being exploited to conduct targeted genomic changes in clinical trials: (1) gene editing through homology-directed repair (HDR) used to replace a pathogenic variant or insert foreign DNA elements to restore the wild-type (WT) expression Synephrine (Oxedrine) of a missing (or truncated) gene; and (2) non-homologous end joining (NHEJ) used.