Application of Genome Editing Technology in Human Gene Therapy

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

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

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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 therapy that accurately repairs the mutant gene sequence by human leukocyte-type antigens, unlike the conventional will become possible. method that requires the establishment of CAR-T for each The first example of in vivo genome editing therapy, patient. In the universal CAR-T cells, which were already which has high technical hurdles, has also been started. published, the T cell receptor α chain gene of CD19-targeted This is a protocol, in which a therapeutic gene is knocked in CAR-T cells was knocked out by TALENs. These universal at the Albumin locus of the liver of a patient with mucopoly- CAR-T cells have been used in pediatric patients with B-cell saccharidosis, an inherited metabolic disease, using ZFNs acute lymphocytic leukemia and have produced significant (NCT02702115). Low HDR efficiencies in the liver, perhaps therapeutic effects [9]. In addition to the above examples, a few percent, are compensated for by high gene activity cancer immunotherapy using T cells, gene knocked out at the knockin target Albumin locus. This strategy can be by CRISPR, was recently reported (NCT03399448) [10]. a platform that enables various diseases to be similarly In this trial, an NY-ESO-1 targeting T cell receptor gene treated, using the same ZFNs and homology arms on the was isolated from cancer-specific T cells and introduced donor DNA. This trial has been drawing much attention into patient-derived T cells with a lentiviral vector. At the because it is the firstin vivo clinical application and protocol same time, two T receptor genes (α, β) and the PD-1 gene using HDR. Recently, it has been reported that treatment were knocked out to promote a therapeutic effect. In the to knock out the causative CEP290 gene mutation has been first 3 patients (advanced multiple myeloma, n=2; myxoid started for Leber congenital amaurosis 10 (LCA10), which round cell liposarcoma, n=1), the knockout efficiency was is caused by a dominant mutation in the CEP290 gene 15–45% for each locus, and the transduction efficiencies of (NCT03872479, see reference [12] for treatment strategy). In NY- ESO-1 T cell receptors was 1.4–4.5% [10]. Although no this example, the AAV5 vector encoding SaCas9 and gRNA clear treatment effect was obtained for these terminally ill to delete that mutated exon was injected subretinally. This cancer patients, there were no noticeable side effects. is the first in vivo application of CRISPR. As in the case of Regarding hematopoietic genetic diseases, clinical trials hemoglobinopathies, a gene knockout strategy, which has of gene therapy using genome editing have begun for lower hurdles in terms of efficiency and safety than gene sickle cell disease and β-thalassemia, which are caused repair, is used. According to the American Society of Gene by an abnormal β-globin gene. The strategies for both & Cell Therapy (ASGCT) Policy Summit last November, diseases are the same, knocking out the erythroid-specific more than 30 protocols for genome editing clinical trials are enhancer sequence of the BCL11A gene, which suppresses currently in progress (type of nuclease: CRISPR, n=19; ZFNs, the expression of γ-globin that constitutes fetal hemoglobin n=9; TALENs, n=5; and other, n=2; target disease: abnormal

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hemoglobinosis, n=5; cancer, n=16; infectious disease, n=7; site. However, the detection sensitivity is approximately ophthalmic disease, n=1; and other, n=8). This number is 0.1%, based on the error rate of next-generation sequencing. expected to increase steadily. Many of these protocols, like As with the above-mentioned example, when genome edit- conventional gene therapy, use viral vectors to achieve high ing is performed on 108 cells, mutations with a frequency delivery efficiency and high nuclease expression levels. of <105 cannot be detected at this error rate. In addition, it However, a stable gene expression, which is advantageous is difficult to analyze off-target sites when using a genome in conventional gene therapy, may be disadvantageous editing tool such as base editing, which does not introduce in the case of the expression of nuclease because of im- double-strand breaks into chromosomal DNA. Recently, the munogenicity and off-target effects. There will be a high relationship between the p53 gene and genome editing has demand for the development of technologies capable of been reported. Cells with a normal p53 gene are likely to transiently and strongly expressing a nuclease, which has die due to DNA double-strand breaks, suggesting that cells received relatively little attention in the development of with successful genome editing are likely to have an abnor- gene therapy vectors. mality in the p53 pathway [18]. However, at least in human hematopoietic stem progenitor cells, this problem can be Off-target Mutation by Artificial Nuclease avoided by selecting targets with low off-target mutations [19]. In addition, DNA double-strand break repair errors Establishing a protocol to obtain a therapeutic effect is were reported to result in deletions of several kilobase pairs the highest priority when developing a therapeutic strategy. or larger in size [20], suggesting that frequent chromosomal However, since it is used in humans, “safety” is the most translocations may occur. The most problematic result of important priority. Problems in gene therapy as a whole, off-target mutations may be oncogenesis, but the above include immunogenicity and cytotoxicity derived from methods only analyze mutations at the DNA level. It is diffi- the vector. In addition, when chromosomally integrating cult to distinguish oncogenic and non-oncogenic mutations. vectors, such as retrovirus or lentivirus vectors are used, Off-target mutations by the nucleases are not the only genotoxicity (the adverse effect on chromosomal DNA) sources of DNA mutations. When using donor DNA becomes a problem. Additional problems associated with (double-stranded DNA or AAV vector), it is known that the genome editing technique include immunogenicity, they are incorporated into on-target and off-target sites as cytotoxicity, and genotoxicity (so-called off-target muta- frequently as 50% (0.5 sites per cell) of some cells (Fig. 3) tion) of artificial nucleases. Furthermore, when donor DNA [21]. In addition, mutations that occur during DNA replica- for HDR is used, genotoxicity due to the integration of the tion during cell division. Assuming that replication errors donor DNA into the chromosome becomes an issue. occur at a frequency of 1 per 1010 bases per DNA replication Off-target mutations by nucleases can be analyzed in [22], it is calculated that there are approximately 20 random various ways [13]. The most accurate method, in theory, mutations per cell after about 30 cell divisions (correspond- would be the analysis of the whole-genome sequence of ing to expansion from 1 to 109 cells) (Fig. 3). This is orders of gene-edited cells. However, even if one mutation is found magnitude higher than the off-target mutation frequencies with ×100 coverage of the whole genome, the detection of artificial nucleases, which are considered to be less than sensitivity will be at most 1%. Considering that the num- 0.1% per cell. Therefore, stem cell therapy using iPS cells, ber of target cells exceeds the order of 108 cells in many after the process of iPSC induction, has mutation rates at clinical applications, at this sensitivity, mutations cannot be an order of magnitude higher than in vivo genome editing. detected in nearly 106 cells. Considering the possibility that For this reason, cell cultures should be minimized, even in only one mutant cell eventually causes cancer, this detection ex vivo genome editing. Finally, there are approximately sensitivity is insufficient. On the other hand, regarding the 0.1% DNA sequence variants on genomic DNA among prediction of the off-target sites, a method of identifying individuals (106 locations per cell) [22], making it difficult genome sequences similar to the target sequence by a to find and interpret off-target mutations (Fig. 3). To our computer analysis is convenient. However, it is recognized knowledge, there have been no reports on cellular trans- that many of the sites predicted in silico do not match the formation/tumorigenesis caused by off-target mutations actual off-target site [14]. As a comprehensive and unbi- in preclinical or clinical genome editing therapy. For this ased off-target prediction, methods have been developed reason, it is recently considered that a carefully designed to express the artificial nuclease in cultured cells to detect artificial nuclease would be associated with a very low risk the cleavage site (BLESS [15], GUIDE-seq [14], etc.), or cut of carcinogenesis due to off-target mutations, when the the DNA extracted from the target cells with the artificial above-described comprehensive and unbiased screening nuclease in a test tube (Digenome-seq [16], CIRCLE-seq [17], methods are used. etc.). In particular, the latter can detect differences due to The safety of the ex vivo method may be similar to the SNP in the genomic sequence between individuals. These case of using ES cells or iPS cells for therapeutic purposes. methods are only applicable in pre-screening of off-target In 2013, the Cell Tissue Processing Product Special Com- sites. After using these methods, nucleases or guide RNAs mittee of the Pharmaceuticals and Medical Devices Agency that have the smallest number of off-target sites, especially (PMDA) in Japan issued a report “Current Perspective on those that are not located near cancer-related genes, will be Evaluation of Tumorigenicity of Cellular and Tissue-based chosen for further use. In order to examine the frequency of Products Derived from Induced Pluripotent Stem Cells off-target mutations in cells after the actual genome editing (iPSCs) and iPSCs as Their Starting Materials”. According process, deep sequencing is performed for each predicted to this report, the criteria to evaluate the safety of stem

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Fig. 3. The frequency of mutations and variants, which potentially arise after therapeutic genome editing. In general, the frequency of off-target mutations by nucleases is not much higher than other types of mutations/variants.

cell products are genomic instability and mutations in a similar results in large animals or humans, because they panel of cancer-related genes [23]. To evaluate the safety of are often outbred and have a more complicated immune gene-edited hematopoietic progenitors, a group in the U.S. system. The difficulty in producing gene therapy drugs transplanted as many genome-edited cells as planned for in high quantities is another challenge. The development transplantation into patients into 100 or more NSG mice, of nucleases with lower immunogenicity and a method of which were observed for 5 months [24]. As mentioned efficiently introducing them as protein products rather than above, only off-target mutations associated with the trans- as a gene are desired to circumvent these issues [27]. In formation of genome-edited cells are problematic. Thus, it any case, it is difficult to completely prevent the immune is more realistic and sensitive to transplant the edited cells response, and it is necessary to consider whether there are into animals than to analyze the off-target candidate sites advantages in choosing a genome-editing treatment—even of unknown biological significance by deep sequencing. with a strong immunosuppressive drug—in comparison to However, for in vivo genome editing (e.g., liver and muscle), other therapies. it is almost impossible to transplant the patient-equivalent number of genome-edited cells into animals, as in the case Target Diseases to be Considered According of hematopoietic progenitors. Furthermore, as described to Risks and Benefits above, most in vivo genome editing performed thus far involves the continuous expression of artificial nucleases Almost any therapy is accompanied by various degrees of using viral vectors, which might result in the accumulation risk. Thus, it is necessary to compare the risks and benefits of off-target mutations. The development of assay systems between genome editing and existing treatment methods, to assess the safety of in vivo genome editing is a major including gene (addition) therapy. Of course, there is a large issue for the future. benefit in diseases that can be treated by genome editing alone, and dominant genetic diseases fall into this category. Immune Response Immune diseases, such as CD40 ligand deficiency, which require a controlled gene expression, are also target diseases Increasing the number of emerging high-fidelity for which genome editing can have large benefits [28]. Con- nucleases might eventually solve the problem of off-target sidering the risk, ex vivo genome editing for hematological mutations. However, the main problem will be the prob- malignancies and infectious diseases have far lower hurdles lem of immune response. As mentioned above, one of the in comparison to in vivo treatment of genetic diseases. In most important themes in gene therapy research has been addition, disorders, in which normal cells (gene repair cells) the immune response against gene therapy products by have a growth advantage among mutant cells would be good the host. Immunogenicity is unavoidable when artificial candidates because of the relatively low efficiency of gene nucleases are non-human proteins, especially those of repair at the present time. In addition, disorders, such as bacterial origin. In a study that actually examined the hemophilia, in which therapeutic efficacy is expected, even prevalence of anti-Cas9 antibodies and anti-Cas9 T cells with low gene expression levels, are considered appropriate in normal individuals, more than half already possessed targets. However, in many of these diseases, conventional humoral and cellular immunity to Cas9, depending on the gene addition therapy has already shown effective results. target population [25]. On the other hand, recent results There is a need for research that shows that genome edit- of preclinical in vivo genome editing of muscle showed ing is safer and more effective than conventional therapy. that an immune response was elicited in adult mice but Nonetheless, with further advancements in genome editing not neonatal mice [26]. Looking back at the history of gene technologies, there is no doubt that genome editing will be therapy research, even if successful treatment with inbred increasingly applied in clinical areas. mouse strains is obtained, it is quite challenging to obtain

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