Translat Regulat Sci. 2(3): 100–106, 2020; doi: 10.33611/trs.2020-007 REVIEW Genome/Epigenome Application of genome editing technology in human gene therapy Kohnosuke MITANI1* 1 Division of Gene Therapy and Genome Editing, Research Center for Genomic Medicine, Saitama Medical University, 1397-1 Yamane, Hidaka, Saitama 350-1241, Japan Abstract. In recent years, gene therapy drugs have finally been approved in Europe, the U.S., and Japan. In parallel with this, advances in genome editing technologies have enabled therapeutic strategies by gene knockout and gene repair, which were difficult with conventional so-called gene addition therapy. Worldwide, over 30 clinical trials of genome editing therapy have already been conducted, and some protocols have shown not only safety but also therapeutic efficacy. In the clinical application of genome editing, in addition to the technical hurdles of conventional gene therapy, there are problems specific to genome editing technology, such as the risk of introducing DNA mutations due to off-target activity of enzymes and the immune response to the artificial nucleases. It is necessary to consider the objective risk and benefit in comparison to existing therapeutic protocols. It is also essential to further develop technologies for therapeutic application in a wider range of diseases. Key words: gene knockout, gene repair, gene therapy, genome editing Highlights In recent years, genome editing has quickly become a popular biological tool. The applications range from basic science to therapeutic drugs, in medicine and other biological fields. The therapeutic application of genome editing can be considered a form of gene therapy, which has also been drawing increased attention. However, genome editing, like conventional gene addition therapy, has many technical challenges to overcome, from off-target activities to immunogenicity, before its potential can be fully realized. This minireview summarizes recent progress in genome editing therapy and discusses these challenges. Introduction recent preclinical and clinical studies of genome editing and discusses the problems that may arise in the clinical setting. From Glybera (adeno-associated viral vector (AAV) for There will also be issues on intellectual property related to the treatment of familial lipoprotein lipase deficiency) in artificial nucleases and vectors and problems related to vec- 2012 to Zynteglo (a lentiviral vector for the treatment of tor production. However, I will leave these to other articles. β-thalassemia) in 2019, gene therapy drugs are being ap- Among gene editing strategies, gene knockout by inac- proved one after another in Europe, the United States, and curate non-homologous end-joining (NHEJ) has opened up Japan. The recent success is based on the steady progress new strategies for gene therapy. In addition, if homology- in basic research on gene therapy-related area over 20–30 directed repair (HDR) using genome editing is used, the years. In addition to conventional gene therapy using the treatment of dominant genetic diseases and a safe and stable overexpression of therapeutic genes, the development of gene expression—due to site-specific chromosomal integra- therapeutic strategies utilizing genome editing technology tion of the therapeutic gene—can be obtained (Fig. 1). As is also rapidly advancing [1]. Gene therapy utilizing gene points to be considered in the clinical application of ge- knockout or gene knockin is currently being evaluated nome editing, there are potential problems unique to the in clinical trials, as described below. New genome edit- genome editing technology, in addition to the problems of ing tools, such as a base editor that enables single base conventional gene therapy. Progress in the efficiency and substitution without introducing double-strand breaks safety of gene delivery methods (vectors) and advances in into chromosomal DNA and a prime editor that enables understanding on host immune responses to vectors and the deletion and insertion of tens of bases [2], will also be therapeutic genes are the keys to the recent success of gene used clinically in the near future. This paper introduces the therapy. In particular, it has been critical that the improve- ment of vector technology has made it possible to introduce *Correspondence to: Mitani, K.: [email protected] genes into various target tissues with efficiencies close to Received: Apr. 30, 2020; Accepted: Jun. 16, 2020; 100%. Using this vector technology, artificial nucleases can Advanced Epub in J-STAGE: Aug. 5, 2020 ©2020 Catalyst Unit This is an open-access article distributed under the terms of the Creative Commons Attribution Non-Commercial No Derivatives (by-nc-nd) License. 100 (CC-BY-NC-ND 4.0: https://creativecommons.org/licenses/by-nc-nd/4.0/) Application of genome editing technology in human gene therapy Fig. 1. Comparison of conventional gene addition therapy and genome editing therapy for inherited diseases. Among the gene therapy strategies, genome editing technology added new options, such as efficient gene knockout and precise and seamless repair of DNA mutations, in gene therapy strategies. also be introduced and expressed in target cells with high hematopoietic stem cells. Currently, gene knockout with efficiency. However, successful genomic editing requires >90% efficiency and 10–20% HDR is possible in this type of subsequent steps of efficient chromosome breaks and DNA cells. Because X-linked severe combined immunodeficiency repair (Fig. 1). Thus, in order for genome editing to be ap- can be potentially cured when 10% of the stem cells in the plied to a wider range of diseases, it is necessary to increase patient’s bone marrow are functionally normal, the current the rate of each of these steps. level of gene repair efficiency might be enough to expect The risks and benefits when applying genome editing to therapeutic effects [3]. In the liver, the HDR efficiency is therapeutic treatment vary greatly depending on the target ~10% for in vivo genome editing (direct genome editing disease. In particular, when applied to advanced cancer method in a body) in mice. In another example, an artificial treatment, the risk of the off-target mutations described nuclease called a meganuclease was used to knockout the below does not pose a problem. On the other hand, when PCSK9 gene with approximately 40% efficiency in the mon- treating genetic diseases in children, long-term therapeutic key liver [4]. In the muscle, cases of successful treatment effects and safety must be considered. The content dis- in large animals, such as the excision of mutant exon in a cussed in this paper is primarily intended for applications dog model of muscular dystrophy, have been reported. The in genetic diseases, which have higher hurdles. efficiency at the DNA level is still low, at approximately 10%. However, since muscle fibers are multinucleated cells, The Current Status of Preclinical Studies in therapeutic effects are likely to appear [5]. In addition, Gene Therapy Models reports on in vivo base editing (e.g., in post-mitotic cochlea in mice [6]) are emerging. Thus, at the animal level, the There have been numerous preclinical studies on genome effectiveness of various types of genome editing therapy editing therapy. The genome editing efficiencies reported has been demonstrated. in these studies are summarized in Fig. 2. For example, regarding ex vivo genome editing (a method in which cells Results of Previous Clinical Trials taken out of the body are subjected to genome editing and then returned to the body) in human CD34-positive For humans, clinical studies are being carried out on hematopoietic progenitor cells, primary/secondary trans- protocols that are likely to have therapeutic effects, even plantations into NOG/NSG -immunodeficient mice are with low genome editing efficiencies. Representative ex- often used to evaluate primitive progenitors. A population amples of ongoing applications include the treatment of of human-derived cells that are later detected in the mouse HIV-infection and the establishment of universal chimeric blood are regarded as progenitors that are closer to the real antigen receptor-T cells (CAR-T). In the former, immune 101 Translational and Regulatory Sciences Fig. 2. Recently reported efficiency in preclinical studies of genome editing in ex vivo hematopoietic progenitors and in vivo in the muscle and liver. The efficiency in these studies is still much lower in comparison to gene transfer efficiency because additional steps are taken. cells are made resistant to HIV infection by knocking out in adult cells. As a result, instead of adult type hemoglobin the CCR5 co-receptor gene essential for HIV infection us- with quantitative or qualitative abnormalities in these ing zinc-finger nuclease (ZFN) or CRISPR (NCT01044654, diseases, fetal hemoglobin is produced to cure the disease NCT00842634, NCT02225665, NCT02500849, NCT03164135, [11]. Both ZFN (Sangamo Therapeutics, NCT03432364 and etc.). Although it has been regarded as a highly promising NCT03653247) and CRISPR/Cas9 (CRISPR Therapeutics, treatment, the results reported in the literature thus far, show NCT03655678 and NCT03745287) are utilized in each of that the therapeutic effects have not been so remarkable [7, the clinical trials. Especially for sickle cell disease, if the 8]. The universal CAR-T is a CAR-T cell line that can be efficiency of HDR becomes higher, the ultimate gene repair commonly used in various patients without being restricted