Recent Advances in Therapeutic Genome Editing in China

Abstract

Editing of the genome to correct disease-causing mutations is a promising approach for the treatment of human diseases. Recent advances in the development of programmable nuclease-based genome editing tools have substantially improved the ability to make precise changes in the human genome. Genome editing technologies are already being used to correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional gene therapies. Chinese scientists have made remarkable breakthroughs in the field of therapeutic genome editing, particularly with the first clinical trial involving the clustered regularly interspaced short palindromic repeats-caspase 9 system that began in China. Herein, current progress toward developing programmable nuclease-based gene therapies is introduced, as well as future prospects and challenges in China.

Introduction

Gene therapy is an approach whereby an exogenous “good” gene is transferred into cells to replace a defective gene in those who suffer from genetic defects.¹ Gene therapy has made remarkable progress in recent years and is now showing promising clinical results.² However, current gene replacement therapy does not remove or modify the defective DNA. While this is fine for most recessive disorders, it is not for some diseases caused by dominant negative mutations.³ In contrast, genome editing can correct the defective DNA in its native location. Thus, genome editing based on programmable nucleases is likely to become the next generation of gene therapy, overcoming the imprecision and potentially harmful effects (e.g., insertional mutagenesis) of current gene therapy technology. Moreover, genome editing opens the possibility of curative therapy by removal or correction of deleterious mutations or insertion of protective mutations.⁴

There are four major classes of engineered nucleases: meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease caspase 9 (Cas9).⁵ Engineered nucleases can recognize a specific site in the genome and create DNA double-stranded breaks (DSBs).⁶ In the absence of a repair template, DSBs are repaired by the nonhomologous end-joining (NHEJ) pathway, which leads to a high frequency of mutagenesis and results in formation of insertions and/or deletions (indels) at the DSB site. When the DSB is induced within a gene coding region, indels often cause frameshifts. The most common application of targeted mutagenesis involves inducing frameshift mutations in knockout genes. Moreover, simultaneous introduction of two targeted DSBs has also been shown to result in targeted deletion or inversion, duplication, local indels at nuclease cleavage sites, or translocations/chromosomal rearrangements. This approach is useful for therapeutic strategies that may require removal of an entire genomic element.⁵

In the case of repair templates, DSBs will induce homology-directed repair (HDR), which occurs at lower frequencies than NHEJ. DSBs can induce precise genome editing by stimulating HDR with an exogenously supplied donor template, which is in contrast to NHEJ that can generate unpredictable mutations. Any sequence differences present in the donor template can be incorporated into the endogenous locus to correct disease-causing mutations. Alternatively, a donor template allows for site-specific DNA insertion through DSB-induced HDR using a donor template, in which the desired genetic insert is flanked by homology arms, including sequences identical to the nuclease cut site.⁷ Targeted insertion of therapeutic transgenes into predetermined sites in the genome, such as “safe harbor” loci, alleviates the risk of insertional mutagenesis and enables high levels of ubiquitous gene expression.^8,9 A wild-type copy of the disease-causing gene can be inserted into the corresponding endogenous locus, thus allowing for it to be under the control of its own promoter while still being subjected to the regulatory elements of the surrounding sequence.¹⁰ Nuclease-induced DSBs that create sequence overhangs on the donor DNA and at the endogenous site can be used as an alternative mechanism for targeted transgene insertion, thus leading to NHEJ-mediated ligation of the inserted DNA sequence directly into the target locus.¹¹

Meganucleases, ZFNs, and TALENs achieve specific DNA binding via protein–DNA interactions.¹² Because new protein design and validation for each experiment is time-consuming and often costly, widespread adoption of these earlier editing tools has been limited.⁴ The CRISPR-Cas9 system, on the other hand, uses simple base pairing rules between an engineered guide RNA (gRNA) and the target DNA site, offering simple yet effective genome editing.¹² CRISPR-Cas9 systems derive from the adaptive immunity of bacteria and archaea against viruses and plasmids by using CRISPR RNAs to guide the silencing of invading nucleic acids.¹³ This system includes two major components: the Cas9 nuclease and a gRNA that consists of a CRISPR RNA fused with a constant transactivating RNA. The 20 nucleotides at the 5′ end of the gRNA lead Cas9 to a specific target site by RNA–DNA complementarity base pairing rules, and Cas9 can cut the targeted DNA strands. These target sites should be immediately located at the 5′ end of a protospacer adjacent motif (PAM) sequence. The PAM sequence is usually a 5′-NGG, but in many species, it has been reported to have different efficiency rates.¹⁴ Type II CRISPR systems from other species of bacteria that discriminate between different PAM sequences with alternative CRISPR RNAs can be utilized for genome editing as well.¹⁵ The simplicity of CRISPR-Cas9 programming, together with a unique DNA cleaving mechanism, the capacity for multiplexed target recognition, and the existence of many natural CRISPR-Cas system variants, make it a cost-effective and easy-to-use genome editing technology.¹⁶ CRISPR-Cas9 has been widely used in research, such as with transcription control, epigenome modification, genome-wide screening, and chromosomal imaging.¹² Moreover, CRISPR-Cas systems are already being used to alleviate genetic disorders in animal models.¹⁷

Genome editing technology has been applied to different kinds of biomedical research, clinically, and for agriculture in China. Before widespread use of CRISPR-Cas9, the overwhelming majority of genome editing described in the literature employed ZFNs and TALENs. For example, ZFNs have been utilized to mutate the myostatin gene in germ cells of yellow catfish. A 4 base pair (bp) insertion in the genome was introduced by ZFN to generate the first endogenous gene knockout aquaculture fish.¹⁸ In another study, Zhang et al. targeted disruption of the myostatin gene in sheep through engineered ZFNs.¹⁹ Moreover, two research papers aimed to find a potential cure for human immunodeficiency virus (HIV) infection with the aid of ZFNs to break the expression of CCR5²⁰ or destroy proviral DNA.²¹ In the field of plant genome editing, Chen et al. established a protocol for TALEN-mediated mutagenesis in the rice genome. Rice resulting from TALEN mutagenesis could be produced within 4–5 months, which is of great significance for agricultural production.²² In addition, TALENs were also applied to generate preclinical disease animal models to help further understand the mechanisms of human-related diseases.^23,24

In recent years, China has attached great importance to gene therapy.²⁵ With the help of genome editing technologies, Chinese scientists have made remarkable breakthroughs in the fields of therapeutic genome editing. In fact, the world's first genome editing clinical trial in humans started in 2009. Researchers disabled CCR5 using ZFNs to achieve an anti-HIV effect.²⁶ However, subsequent progress in genome editing has been slow because developing a way to target each specific sequence is costly and time-consuming. Now, widespread use of CRISPR-Cas9 has greatly expanded the range and increased the developmental speed of genome editing applications.¹⁶ China has been at the forefront of this development, bringing CRISPR-Cas9 to clinical treatment.²⁷ There have also been achievements in the field of preclinical research (Table 1); in the midst of controversy, Chinese scientists made significant progress in the genetic editing of human embryos.^28

–31 Through construction of a non-retroviral pig model, clinical application of xenotransplantation has been achieved.³² The present review aims to highlight recent progress in genome editing, as well as the challenges and prospects of genome editing in China.

Table 1.

Preclinical research of genome editing in China

	Target	Therapeutic strategy	Nuclease platform	Application	Reference
1	VEGF-A	Restraining VEGF-A signaling induced angiogenesis	CRISPR-Cas9	Osteosarcoma therapy	39
2	MTH1	MTH1 gene enabled by nonviral delivery vector in SKOV3 cells	CRISPR-Cas9	Ovarian cancer therapy	41
3	OTC	Applying two AAVs in newborn mice with defective OTC	CRISPR-Cas9	Metabolic liver disease therapy	42
4	FIX Y371D mutation	Implement in situ genome editing to correct FIX mutation	CRISPR-Cas9	Hemophilia B therapy	44
5	HBB	Genome editing in iPSCs	CRISPR-Cas9	β-thalassemia therapy	49 -53
6	CCR5	Making ablation of the CCR5 gene in HSCs	CRISPR-Cas9	HIV infection therapy	69
7	CXCR4 and CCR5	Simultaneously knocking out CXCR4 and CCR5 in T cells	CRISPR-Cas9	HIV infection therapy	70
8	HBB	Genome editing in 3PN zygotes	CRISPR-Cas9	Embryo editing	28,29
9	PERVs	Knocking out all PERVs genes in a porcine primary cell line	CRISPR-Cas9	Xenotransplantation	32
10	MECP2	Knocking out MECP2 in early embryos	TALEN	Modeling Rett syndrome	23

VEGF-A, vascular endothelial growth factor A; OTC, ornithine transcarbamylase; FIX, factor IX; HBB, hemoglobin subunit β; PERV, porcine endogenous retrovirus; CRISPR-Cas9, clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease caspase 9 (Cas9); TALEN, transcription activator-like effector nucleases; HIV, human immunodeficiency virus.

Genome Editing in Cancer Gene Therapy

Cancer is a major disease that threatens human health, and recently, cancer gene therapy has become one of the hottest areas.³³ Genetic engineering of T cells can be an ideal therapeutic method.³⁴ As an important immunosuppressor, programmed death-1 (PD-1) was confirmed to be associated with tumor escape from immune surveillance.³⁵ In October 2016, a metastatic non-small-cell lung cancer patient in China became the first person in the world to receive clinical CRISPR treatment at West China Hospital (Chengdu, China).²⁷ In this clinical trial, T cells were extracted from patient blood and modified with CRISPR-Cas9 to disable PD-1. Then, the edited immune cells were returned to the patient. Researchers hypothesized that edited immune cells could destroy cancer cells in the body. This clinical study is estimated to be completed in 2018. Although routine clinical use of CRISPR is years away, CRISPR is still seen as an ideal strategy for patients who have no other choices after standard therapy.

Two other nuclease platforms, ZFNs and TALENs, reached the clinical stage before CRISPR-Cas9. However, because targeting a new site requires designing and cloning a new protein, these tools are precluded from being used for high-throughput clinical applications. Due to its ease of engineering, the CRISPR system could be a preferred therapeutic platform. For clinical applications, this powerful editing tool could improve the therapeutic effects of treatments editing T cells and substantially eliminate some of their vulnerabilities. Recently, more and more institutions have brought CRISPR into the clinic, both in China and around the world²⁷ (Table 2). Most of these clinical trials are in the patient recruitment stage, and six of these trials are knocking out PD-1 in T cells. Another promising approach for cancer treatment is chimeric antigen receptor T-cell (CAR-T) therapy.³⁶ T cells can be genetically modified to express CARs, which are fusion proteins consisting of antigen-recognition moieties and T-cell activation domains.³⁷ CRISPR-Cas9-mediated genome editing has been applied to CAR-T cells, and a protocol for achieving multiplex genome editing in CAR-T cells has been designed.³⁸

Table 2.

Clinical trials of CRISPR in China and the United States

	Disease	Intervention	Status	Country
1	HPV-related cervical intraepithelial neoplasia	TALEN, CRISPR-Cas9	Not yet recruiting	China
2	AIDS and hematological malignancies	CCR5 gene ablated by CRISPR-Cas9	Recruiting	China
3	Sickle-cell disease	CRISPR-Cas9	Not yet recruiting	United States
4	Relapsed or refractory CD19⁺ leukemia and lymphoma	CAR-T	Recruiting	China
5	Advanced esophageal cancer	PD-1 knockout T cells	Recruiting	China
6	Muscle-invasive bladder cancer	PD-1 knockout T cells	Not yet recruiting	China
7	Castration-resistant prostate cancer	PD-1 knockout T cells	Not yet recruiting	China
8	Metastatic renal-cell carcinoma	PD-1 knockout T cells	Not yet recruiting	China
9	Metastatic non-small-cell lung cancer	PD-1 knockout T cells	Recruting	China
10	Advanced-stage EBV-associated malignancies	PD-1 knockout T cells	Recruting	China

Data derived from https://clinicaltrials.gov/

HPV, human papillomavirus; AIDS, acquired immune deficiency syndrome; EBV, Epstein–Barr virus; PD-1, programmed death-1.

Instead of triggering T cells, restraining vascular endothelial growth factor A signaling has been shown to suppress angiogenesis in osteosarcoma.³⁹ Use of an osteosarcoma cell-specific aptamer for in vivo delivery of CRISPR-Cas9 has been shown to restrain autocrine and paracrine vascular endothelial growth factor A signaling in tumor cells simultaneously. In another study, a CRISPR-Cas9-based “signal conductor” that regulated transcription of endogenous genes was created and applied to redirect oncogenic signal transduction, enabling reprogramming of cancer cell fate.⁴⁰ By constructing a multifunctional “core-shell” artificial virus, CRISPR-Cas9 was delivered into the nucleus without an additional nuclear localization signal. Furthermore, when loaded with CRISPR-Cas9 targeting MTH1, the system led to MTH1 disruption in a mouse cancer model, providing a new way to treat ovarian cancer.⁴¹

Genome Editing in Hereditary Disease Therapy

Many diseases are related to genetic variations. Based on programmable nucleases, genome editing technologies can remove or correct mutations within genomic DNA in situ or insert protective mutations. Therapeutic genome modifications can be classified into four types: gene destruction, NHEJ-mediated gene correction, HDR-mediated gene correction, and HDR-mediated gene addition. These classes of therapeutic strategies are undoubtedly very beneficial for treating a variety of genetic diseases, not merely for monogenic disease. They could also be applied to diseases with more complex genetic backgrounds.⁵

Gene therapy using non-integrating delivery vectors, such as an adeno-associated virus (AAV), is not optimal in newborns because the non-integrating genome is lost as developing hepatocytes proliferate. CRISPR-Cas9-mediated gene correction delivered by AAV vectors was tested in newborn liver. In these studies, the CRISPR-Cas9 system was incorporated into two AAV8 vectors, with one expressing Cas9 from Staphylococcus aureus and the other containing gRNA and a donor ornithine transcarbamylase (OTC) DNA template, to correct a splicing mutation in OTC that encodes a key enzyme in the urea cycle. After systemic injection into newborn mice with partial OTC deficiency caused by a splicing mutation, 10% (6.7–20.1%) of OTC-defective hepatocytes were reverted to normal, and survival of mice was increased when given a high-protein diet. However, HDR-mediated gene correction in adult OTC-deficient mice was lower and accompanied by larger deletions that extended to the adjacent exon and ablated residual expression from endogenous OTC, leading to diminished protein tolerance and lethal hyperammonemia on a chow diet.⁴² The results demonstrated that liver OTC enzyme activity was recovered by clonal expansion in the growing liver. Building on this approach, a universal CRISPR-Cas9 gene targeting approach was developed in which the vector system could be applied to the majority of patients with a specific disease. Co-injection of the dual AAV vector system in factor IX (FIX)-knockout mice resulted in stable FIX activity at or above normal levels for 8 months.⁴³ Thus, delivery of CRISPR-Cas9 components targeting the FIX Y371D mutation in adult mice was used to implement in situ genome editing and correct the FIX mutation.⁴⁴

Depending on the cells being edited, ex vivo is another important way in which to treat genetic diseases. One of the most common genetic diseases worldwide, β-thalassemia, is an inherited blood disorder characterized by reduced or absent synthesis of hemoglobin subunit β (HBB).⁴⁵ In southern China, the average carrier rate of β-thalassemia in the population is high (∼2.8%).⁴⁶ Based on homologous recombination gene correction, Song's team repaired HBB by editing patient-specific induced pluripotent stem cells (iPSCs)⁴⁷ through CRISPR-Cas9. They showed that subsequently derived hematopoietic stem cells from gene-corrected iPSCs restored expression of HBB.⁴⁸ There have also been other studies on editing iPSCs to correct the mutations associated with HBB.^49

–53 Targeting modification of the iPSC genome by CRISPR-Cas9, Chinese researchers have also made advances in the treatment of hemophilia B,⁵⁴ hearing loss,^55,56 cancer,⁵⁷ oculopathy,^58,59 and other genetic diseases.^60
–62

Genome Editing for Viral Infection Therapy

Genome editing technologies aiming to disrupt proviruses are an attractive approach to eliminating viral genomes from infected individuals and curing viral infections.⁵ China has a high incidence of hepatitis B virus infection.⁶³ By targeting disruption of the hepatitis B virus genome, the replication activity of this virus can be destroyed using the CRISPR-Cas9 system.^64

–67 A similar challenge worldwide is HIV infection.⁶⁸ Ablation of CCR5 in hematopoietic stem cells with CRISPR-Cas9 to induce gene knockout might be a way to block HIV infection.⁶⁹ Moreover, co-delivery of two single-gRNAs and simultaneous knockout of CXCR4 and CCR5 in CD4⁺ T cells via CRISPR-Cas9 was shown to be a potential functional cure for HIV-1 infection.⁷⁰ Furthermore, CRISPR-Cas9 genome editing could also be used against other types of viral infections, such as H5N1 avian influenza virus⁷¹ and herpes simplex virus type 1.⁷²

Controversial Embryo Editing

Injecting genome editing components into zygotes or early stage embryos results in permanent genome modification. Because these changes can be passed on to offspring, embryo editing offers a possible way to eliminate genetic disorders that are familial. However, human germline genome editing carries with it significant ethical and social issues.

In 2015, Liang et al. took the lead in investigating CRISPR-Cas9-mediated gene editing in abnormal human embryos. They experimented on tripronuclear (3PN) zygotes and found CRISPR-Cas9 could effectively cleave endogenous HBB, but the efficiency of HDR of HBB was low and accompanied with significant off-target effects. Moreover, the edited embryos were mosaic.²⁸ At the same time, their achievement brought human embryo genome editing to the center of a debate over the ethical implications of such work.⁷³ Though the argument continued, a second study demonstrated that CRISPR-Cas9 editing resulted in four 3PN zygotes with a disabled CCR5 that successfully conferred immunity against HIV.³¹ In 2017, Liang et al. presented another achievement. They showed that a single base editing system could be accurately used to repair a specific type of single base mutation within the human embryonic genome. This major breakthrough had immense clinical significance for the repair of the β-thalassemia HBB-28 (A>G) point mutation.²⁹ Recently, Zhou et al. carried out base editing in human 3PN zygotes by microinjection of base editor 3 and gRNAs and obtained a single nucleotide substitution in HBB.³⁰ Except for editing human embryos, Chinese scientists have also focused on genome editing in animal embryos. A protocol was established for the generation of gene knockout and knock-in rats via CRISPR-Cas9 that was a good guideline for design and construction of gene mutant models.⁷⁴ Another team successfully obtained complete single and multiple gene knockout mice and monkeys.⁷⁵ Some essential genes are important in developmental biology. By injecting both Cre mRNA and CRISPR-Cas9 into mouse embryos and analyzing the mutant cells, some novel functions of the necessary gene Tet3 were revealed; this gene was identified to be important for regulating neocortical development of mice.⁷⁶

Breakthroughs in Xenotransplantation

Xenotransplantation involves transplanting animal organs into the human body, which is a promising way to alleviate severe organ shortages for human transplants.⁷⁷ Since the sizes of porcine organs are similar to those of humans, pigs have become an ideal animal model for xenotransplantation. However, porcine endogenous retroviruses (PERVs)⁷⁸ limit their use. In 2015, Yang et al. showed that PERVs could be inactivated by a CRISPR-Cas9 system.⁷⁹ Recently, Niu et al. used CRISPR-Cas9 to knock out all PERV genes in the original fibroblasts of pigs, and through somatic cell nuclear transfer technology, they successfully developed energetic pig embryos excluding PERVs. Subsequent transplantation of those embryos into surrogate mothers confirmed that these newborn pigs were not infected with PERVs again. This research representatively made significant progress in addressing the safety issues of cross-species viral transmission and was a major breakthrough in the development of safe and effective xenotransplantation.³²

Genome Editing for Constructing Disease Models

A deeper understanding of the mechanisms of diseases is of great benefit to the improvement of treatment methods. Therefore, establishing animal disease models can verify therapeutic efficacy before human clinical trials and help us better understand the pathogenesis of disease. In turn, clinical treatment plans would also be improved.⁸⁰ Some recent studies have described successful deployment of genome editing tools to establish disease models in China.¹⁷ Rett syndrome (RTT) is a monogenic abnormity without cure that can lead to progressive neurodevelopmental maladjustment. RTT is largely caused by MECP2 mutations.⁸¹ Although RTT rodent models have been established, their neurological phenotypes are different from that in humans. Thus, genome editing in nonhuman primates may be a good choice.⁸² In Chen's study, TALENs were used to cause mutational deficiency of MECP2 in cynomolgus monkeys without causing off-target mutations. In MECP2-mutant males, embryonic lethality was observed, while physiological and behavioral disorders were found in mutant females. Importantly, these abnormalities are seen in human RTT patients. Magnetic resonance imaging of the brain and other tests on MECP2 mutant monkeys indicated that this RTT animal model could sufficiently imitate human clinical manifestations, thereby providing more opportunities to discover disease mechanisms and evaluate possible treatment options.²³

In another example, editing microcephalin 1 with TALENs produced genetic mutations in cynomolgus monkeys, allowing simulation of human microcephaly.²⁴ Guided by gRNA, CRISPR-Cas9 can cut the genome of pigs but not of primates. After creating site-specific indels, cell colonies with bi-allelic low-density lipoprotein receptor and apolipoprotein E mutations were obtained. Finally, apolipoprotein E and low-density lipoprotein receptor double gene knockout pigs were generated. This model was of great significance for the study of human cardiovascular diseases.⁸³ With the combination of genome editing and somatic cloning technology, the world's first genome edited, cloned dog was successfully bred in China.

Challenges and Prospects

As an important auxiliary method of gene therapy, genome editing provides opportunities for curing diseases by correcting genetic mutations or inserting protective mutations.⁵ Thanks to its powerful function, the prospect of applying genome editing technology clinically is very good in China. Translating genome editing technology into the clinic still faces challenges, however, mainly related to its safety and efficacy. Furthermore, policy guarantee is equally important.

Improving Editing Specificity and Efficiency

Various assessments of genome editing specificity have shown that off-target mutagenesis could be a major concern in therapeutic applications.⁸⁴ For clinical applications, identification of even low-frequency off-target efficiency will be critically important because ex vivo and in vivo therapeutic strategies using genome editing are expected to require the modification of very large cell populations. The induction of oncogenic transformation in even the rarest clones (e.g., inactivating a tumor suppressor gene or activating an oncogene) is of particular concern, as such alteration could lead to unfavorable clinical outcomes.⁵ Therefore, many studies have attempted to evaluate the specificity of genome editing tools, especially aimed at optimizing CRISPR-Cas9. In Chen's research, changing the domain within the Cas9 recognition-3 domain could enhance proofreading governing the targeting accuracy of CRISPR-Cas9.⁸⁵ C2c1 is a dual-RNA-guided DNA endonuclease. When combined with a chimeric single-molecule gRNA, this system is highly sensitive to single nucleotide mismatches between gRNA and the target site.⁸⁶ A high-fidelity CRISPR-Cas9 variant, Streptococcus pyogenes Cas9-HF1, was created that reportedly did not have any detectable genome-wide off-target effects.⁸⁷ Editing efficiency determines whether a gene editing tool can effectively edit a given region of interest. Some small molecules that inhibit HDR have been found to enhance the possibilities of indels mediated by NHEJ.⁸⁸

Finding New Editing Tools

Looking for other genome editing tools may be another way to improve editing effects and supply more options for clinical adhibition. Some altered PAM-specific variants have been shown to enable increased editing efficiency of endogenous gene sites that are not convenient for S. pyogenes Cas9.¹⁵ Except for Cas9, the putative Class 2 CRISPR effector Cpf1 was also verified to have efficient genome editing activity.⁸⁹ In 2016, Chinese scientist Feng Gao claimed that he had found another new and efficient gene editing tool: the DNA-guided endonuclease Natronobacterium gregoryi Argonaute.⁹⁰ However, soon afterwards, an increasing number of scientists reported that they could not replicate Han's results,⁹¹ and this controversy remains.

Genome Editing Delivery Systems

Successful clinical translation will depend on appropriate and efficacious genome editing delivery systems to target specific disease tissues. Viral and nonviral delivery methods can be used to deliver genome editing tools to target cells or tissues. Current viral delivery systems of genome editing vectors include retroviruses, adenoviruses, and AAV.

With the approval of the first gene therapy product (Glybera, UniQure) by the European Medicine Agency, an AAV-based product finally reached the market. This has made AAV a prime candidate vector for bench-to-bedside therapeutic genome editing. However, numerous hurdles still need to be overcome for AAV to reach its full potential for therapeutic genome engineering. For example, the naturally evolved infectious properties of AAVs are mismatched with the delivery needs for most therapeutic applications, which preclude its utility for a large number of diseases. Moreover, widespread natural exposure to AAVs has resulted in a large portion of the population with capsid-specific neutralizing antibodies in their blood and other body fluids, which markedly limits AAV gene delivery efficacy.^92,93 Evasion of neutralizing antibodies and overcoming other barriers to targeted delivery are needed in order to realize the potential of AAV delivery fully. Moreover, the restrictive cargo size (4.5 kb, excluding the inverted terminal repeats) of AAV presents an obstacle for packaging the commonly used S. pyogenes Cas9 (4.2 kb) and its single-gRNA in a single vector. New approaches, such as rational design and directed evolution of AAV capsids, have been harnessed to engineer and improve AAV vectors to overcome these barriers.⁹⁴

Based on cationic lipids or polymers, nonviral vectors could overcome the limitations associated with delivering large payloads to a certain degree.⁹⁵ Nonviral vectors can be categorized into multifunctional hybrid nucleic acid, novel membrane/core nanoparticles for nucleic acids and ultrasound-responsive nucleic acids.⁹⁶ Nonviral vectors have the advantage of having low immunogenicity, absence of endogenous viral recombination, and fewer limitations with delivering large gene payloads. By constructing an artificial virus loaded with CRISPR-Cas9 technology, Chinese scientists have applied nonviral vectors to cancer gene therapy.⁴¹ However, nonviral delivery systems have low delivery efficiency.⁹⁵ At present, researchers have designed and synthesized many potential nonviral delivery materials to improve this efficiency.⁹⁶ With the development of more and widely available varieties of delivery methods, genome editing platforms can be better applied for therapeutic purposes.

Assurance of Supervision

Although CRISPR was invented in the West, Chinese scientists are zealously popularizing its applications in many fields.²⁷ As the world's first CRISPR clinical trial is under way in China, more are on the way,²⁷ and though controversial, Chinese scientists have taken the lead in embryonic genome editing of human 3PN zygotes by CRISPR-Cas9.^28

–31 Recently, researchers in the United Kingdom revealed the role of OCT4 in human embryogenesis by genome editing.⁹⁷ In the United States, scientists have tried to edit human preimplantation embryos.⁹⁸ While considering the scientific, ethical, and policy-related questions, many workgroups around the world have called for caution in the use of genome editing in human germlines.⁹⁹ As a new scientific topic, human genome editing is developing, along with public expectation and anxiety. A pressing problem is not only ensuring that regulatory agencies can accept, review, and approve such an application, but also that they can legally ensure its availability.¹⁰⁰ A recent report entitled “Human Genome Editing: Science, Ethics, and Governance” has provided the first set of guidelines for the application of human genome editing.²⁵ In China, using human egg plasma and nuclear transfer technology and manipulation of genes in human gametes, zygotes, or embryos for the purposes of reproduction are prohibited. There have been several policies and national regulations related to human genetic research, especially for stem-cell research. Nevertheless, no specific formal, legal regulations on the use of genome editing are available thus far. Chinese scientists have organized meetings with panel discussions and coordinated with related government agencies to discuss regulatory policies related to this issue, and the “Genome Editing branch of the Chinese Society of Genetics” was established in September 2017. Members of this community have supplied a number of instructional suggestions, and now, government agencies are collecting suggestions and comments to help guide development of regulatory policies for genome editing. With more and more scientific projects being funded, the number of genome editing publications is quickly rising in China. Moreover, the immense long-term prospects of genome editing cannot be denied. Hence, the Chinese government and relevant institutions should develop a reasonable framework for mechanistic research and clinical application of genome editing technology to ensure its lawful, orderly, and timely development.

Conclusion

Genome editing technology has been widely used in many fields. Specifically, the emergence of CRISPR-Cas9 has greatly expanded the application of genome editing technologies. Chinese scientists are actively involved in the field of gene therapy and first brought CRISPR into the clinic. In conclusion, China's achievements in genome editing research clear and significant. With continued technological innovation and implementation of effective supervisory/regulatory mechanisms, the future of genome editing is very bright in China.

Footnotes

Acknowledgments

This work was supported by the National Natural Science Foundation of China (81402302, 81502677, and 81602699) and the China Postdoctoral Science Foundation Funded Project (2017M612968).

Author Disclosure

The authors declare no conflicts of interest.

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