Applications of Virus Vector–Mediated Gene Therapy in China

Abstract

Due to the increased safety and efficiency of virus vectors, virus vector–mediated gene therapy is now widely used for various diseases, including monogenic diseases, complex disorders, and infectious diseases. Recent gene therapy trials have shown significant therapeutic benefits, and Chinese researchers have contributed significantly to this progress. This review highlights disease applications and strategies for virus vector–mediated gene therapy in preclinical studies and clinical trials in China.

Introduction

Gene therapy is a therapeutic technique that can be used to treat diseases by inserting functional genetic material into the somatic cells of patients. Based on accumulating knowledge of the genetic basis of diseases and the progression of DNA delivery technologies, primarily using virus vectors, gene therapy is being increasingly applied as clinical treatments for monogenic disorders. Although safety and effectiveness issues associated with early virus vectors once cast doubt on the usefulness of virus vector–mediated gene therapy,^1,2 these adverse events motivated researchers to develop new virus vectors that have low genotoxicity and immunogenicity, as well as high transduction efficiency. The increased safety and effectiveness of virus vectors led to clinical successes in treating monogenic disorders, and subsequent expansion of the use of these vectors to treat complex disorders including cancer, cardiovascular diseases, neurological diseases, and inflammatory diseases, as well as infectious diseases.^3,4 Treatment of these diseases with virus vectors involves various strategies such as virology, curative or toxic molecules, or cell therapy instead of the simple gene replacement approach used for monogenic diseases. By integrating genetic and pathological insights for specific diseases with effective gene therapy strategies, recent clinical trials for complex disorders and infectious diseases have achieved remarkable therapeutic effects.⁴ Together, these achievements demonstrate that gene therapy has returned to “center stage.”⁵

In China, virus vector–mediated gene therapy research and clinical trials have also been widely conducted for many diseases, particularly complex diseases. Important examples of these applications include: tumor cell death elicited by oncolytic viruses carrying immune-stimulating “cargos”⁶; antitumor cell therapy mediated by mesenchymal stem cells (MSCs) or T cells armed with chimeric antigen receptors (CARs)^7,8; secretion of growth factors and neurotrophic factors to treat nerve injury and neurodegenerative disorders^9,10; promotion of angiogenesis after myocardial infarction (MI)¹¹; removal of inflammatory cells in rheumatoid arthritis (RA)¹²; induction of hepatocyte generation in severe liver diseases¹³; and inhibition of viral replication in hepatitis B virus (HBV) infection.¹⁴ Gene therapy in China has kept pace with current advancements in virus vectors and treatment strategies, and has applied these approaches to meet medical demands, which currently benefits many Chinese people. More will benefit as new treatments are developed.

International Advances in Modifying Main Virus Vector Groups

The earliest gene therapy vectors were γ-retrovirus vectors based on murine leukemia virus that were widely used in early ex vivo gene therapy. Pseudotyping with the vesicular stomatitis virus G protein (VSV-G) has dramatically expanded the host ranges of these viruses.¹⁵ Moreover, to reduce the possibility of insertional mutagenesis, a self-inactivating (SIN) vector was developed by deleting the enhancer or promoter of the 3′ long terminal repeat, which is the most common format of current γ-retrovirus vectors.^16,17 These improvements partly addressed issues with γ-retrovirus vectors, although additional risk–benefit assessments are needed prior to their expanded clinical application.¹⁸

Most work concerning integrating vectors has focused on the development of lentiviral (LV) vectors that can infect both dividing and quiescent human cells and have a safer integrational preference relative to γ-retrovirus vectors.¹⁹ Indeed, VSV-SIN-LV vectors have superseded γ-retrovirus vectors in most ex vivo gene therapy studies. To minimize insertional mutagenesis, a non-integrating LV vector with mutant class I integrase was developed that can mediate efficient and sustained transgene expression in nondividing cells.²⁰ In addition, recent modifications such as adding targeting ligands (e.g., single-chain antibodies) to LV vectors pseudotyped with Sindbis virus and measles envelope glycoproteins enabled selective infection of various cell types by LVs.²¹

Non-integrative adenovirus vectors were first used for in vivo gene delivery. To reduce the strong immunogenicity of these viruses, helper-dependent adenovirus vectors were constructed in which all adenovirus coding genes were deleted.²² However, most current efforts to develop adenovirus vectors are now directed to applications that benefit from the immunogenicity afforded by these viruses, such as cancer therapy and vaccines. Oncolytic adenoviruses, in which adenovirus genes essential for replication in normal cells but not in tumor cells are deleted or mutated, can selectively lyse tumor cells.^23,24 Furthermore, replication-competent adenoviruses with insertion of antigenic genes in the E3 gene site or replication-defective adenoviruses displaying antigens on the capsids can induce specific immune responses as vaccines.^25,26

Due to their nonpathogenic nature and broad tropism, adeno-associated virus (AAV) vectors have also shown strong potential for in vivo gene therapy. Through packaging AAV2-based genomes in capsids of other AAV serotypes, AAV vectors can infect cells in different organs such as the retina, muscle, liver, heart, lung and nervous system.^27,28 Furthermore, inserting short peptides into AAV capsids by rational design and selecting satisfactory variants through directed evolution enable AAVs specifically to transduce more cell types or decrease neutralization by anti-AAV antibodies.²⁹ Self-complementary AAV (scAAV) vectors have higher transduction efficiency than conventional single-stranded AAV vectors,³⁰ and strategies involving use of split AAV vectors and reassembly of AAV genome fragments addressed limitations in AAV packaging capacity.³¹

Herpes simplex virus (HSV) vectors, which have high packaging capacity and strong tropism for neurons, are the largest virus vectors currently used in gene therapy.³² There are three types of engineered HSV vectors: (1) amplicon vectors that have the largest capacity of up to 150 kb foreign DNA; (2) replication-defective HSV vectors with decreased immunogenicity and packaging capacity of up to 40 kb exogenous genes; and (3) replication-competent HSV vectors that have the lowest capacity for exogenous genes and are widely used as oncolytic viruses and vaccines.³³

Due to their relatively low toxicity, LV and AAV vectors are currently the “main force” for ex vivo and in vivo gene therapies, respectively, for various genetic diseases. The ranges of their application are further extended by non-integrating LV vectors and genome-editing tools such as the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Moreover, oncolytic viruses and therapeutic vaccines derived from adenoviruses or HSVs play important roles in gene therapies for cancer and infectious diseases. Although virus vectors are the most popular tools for gene therapy, they nonetheless have several drawbacks, including LV genotoxicity and production of anti-AAV neutralizing antibodies. Several reviews provide detailed information about these issues and possible solutions.^4,15

Preclinical Studies of Virus Vector–Mediated Gene Therapy in China

Gene therapy was first investigated as a treatment for monogenic hereditary disorders because of the simple and well-studied genetic mechanisms of the pathogenesis of these diseases. Successful outcomes for gene therapies of monogenic diseases provided the foundation for development of virus vector–mediated gene therapy for more complex diseases, which gradually became the dominant applications. In China, gene therapy to treat various complex disorders, such as cancer, neurological diseases, cardiovascular diseases, autoimmune diseases, and liver diseases, play leading roles in preclinical research. Infectious diseases are also important targets for gene therapy. To develop effective treatments, in-depth understanding of the pathogenic mechanisms of these diseases is required. Based on this understanding, overexpression of curative factors or inhibition of toxic factors via gene therapy used to treat complex diseases, eradicate viruses, or enhance the resistance of host cells to infectious diseases have shown promising therapeutic effects in preclinical studies (Table 1).

Table 1.

Preclinical studies of virus vector–mediated gene therapy for non-cancer diseases in China

Disease type	Disease treated	Strategy	Treatment	Reference
Neurological diseases	Nerve injury (spinal cord injury and stroke)	Nerve growth/regeneration	GDNF (γ-retrovirus); Netrin-1, NGF, ERp29, Lingo-1 shRNA (LV);	9,46,47,116,117
	Nerve injury (spinal cord injury and stroke)	Anti-oxidative damage	TNF-α RNAi (LV)	118
	Alzheimer's disease	Synaptic activity	EphB2 (LV)	50
	Alzheimer's disease	Anti-oxidative damage	NRF2 (LV)	119
	Amyotrophic lateral sclerosis	Nerve growth	IGF1 (AAV)	10
	Depression	Nerve growth	BDNF-HA2TAT (AAV)	52
	Epilepsy	Nerve growth inhibition	RGMa (LV)	120
Cardiovascular diseases	Myocardial infarction (cardiac ischemia)	Angiogenesis	ILK (adenovirus); MSCs secreting survivin (LV)	11,121
		Anti-oxidative damage	MSCs expressing ecSOD (adenovirus)	55
		Calcium transport upregulation	Antisense PLB (AAV)	122
	Critical limb ischemia	Angiogenesis	MSCs expressing miR-21 (LV) or PGC-1α (adenovirus)	58,123
	Hypertension	Inhibition of vasoconstriction	Antisense CYP4A (AAV)	59
	Vascular damage	Anti-oxidative damage	Estradiol (LV)	124
Autoimmune diseases	Rheumatoid arthritis	Immunosuppression	BAFF shRNA (LV)	62
		Lymphangiogenesis	VEGF-C (AAV)	12
		Apoptosis of abnormal cells	Epirubicin plus TRAIL (AAV)	63
	Experimental allergic encephalomyelitis	Anti-apoptosis	Gelsolin (LV)	64
Liver diseases	Chronic non-virus liver injury	Liver regeneration	IGF-2 (AAV)	68
	Hepatic fibrosis	Reversion of epithelial-to-mesenchymal transition	HNF4α (adenovirus)	70
	Hepatic fibrosis	Anti-fibrogenesis in HSCs	HO-1 (AAV)	125
	Nonalcoholic steatohepatitis	Inflammatory suppression	CFLAR (AAV)	71
	Nonalcoholic steatohepatitis	Lysosome-mediated protein degradation	TMBIM1 (AAV)	126
	Liver failure	In vitro reprogramming to produce hepatocytes	Human fibroblasts expressing FOXA3, HNF1A, and HNF4A (LV)	13
Infectious diseases	HBV infection	Interference of viral replication	HBV dual-shRNA plus TGF-β shRNA (AAV); HBV S1 and P28 shRNAs (AAV)	14,74
	HIV infection
	HIV infection	Broadly neutralizing antibodies	DNA vaccines and recombinant Tiantan vaccinia vaccines expressing heterologous gp140 and gp145 env	76
Other diseases	Bone defect	Angiogenic and osteogenic differentiation	MSCs secreting HIF-1α (LV)	127
Other diseases	Tendon injury	Tenocytes proliferation and collagen production	VEGF, bFGF (AAV)	128

GDNF, glial cell–derived neurotrophic factor; NGF, nerve growth factor; ERp29, endoplasmic reticulum protein 29; shRNA, short hairpin RNA; TNF-α, tumor necrosis factor-α; RNAi, RNA interference; EphB2, ephrin type-B receptor 2; NRF2, nuclear factor erythroid 2-related factor 2; IGF1, insulin-like growth factor 1; BDNF, brain-derived neurotrophic factor; RGMa, repulsive guidance molecule a; ILK, integrin-linked kinase; MSCs, mesenchymal stem cells; ecSOD, extracellular superoxide dismutase; PLB, phospholamban; miR-21, microRNA-21; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator-1α; CYP4A, arachidonic acid cytochrome P-450 (CYP) hydroxylase 4A; BAFF, B-cell-activating factor; VEGF-C, vascular endothelial growth factor C; TRAIL, tumor necrosis factor–related apoptosis-inducing ligand; HNF, hepatocyte nuclear factor; HSCs, hepatic stellate cells; HO-1, hemeoxygenase-1; CFLAR, caspase 8 and Fas-associated protein with death domain-like apoptosis regulator; TMBIM1, transmembrane BAX inhibitor motif-containing 1; FOXA3, forkhead box protein A3; HBV, hepatitis B virus; TGF-β, transforming growth factor-β; HIV, human immunodeficiency virus; HIF-1α, hypoxia-inducible factor-1α; bFGF, basic fibroblast growth factor.

Cancer

The estimated numbers of new cancer cases and cancer deaths in China for 2015 were 4,292,000 and 2,814,000, respectively.³⁴ Furthermore, China's cancer profile differs significantly from that of developed countries.³⁴ These differences partly determine priorities for cancer gene therapy efforts in China, which focus on cancers that are common in Chinese populations, such as hepatocellular carcinoma (HCC) and colorectal cancer.

Strategies for cancer gene therapy involve either attacking the tumor directly or increasing host resistance to disease or treatment-related side effects.³⁵ Previous therapeutic strategies used replication-defective viruses to deliver corrective or destructive genes, including oncogene inhibitors, tumor-suppressor genes, suicide genes, or immune stimulating genes, to tumor tissues. For example, Li et al. delivered small interfering RNAs (siRNAs) targeting the oncoprotein p28GANK (also called Psmd10, proteasome 26S subunit, non-ATPase 10) to HCC cells via a recombinant adenovirus and successfully suppressed the growth of HCC tumors in a mouse model. This study showed that p28GANK could be a potential therapeutic target for HCC treatments.³⁶

However, because replication-defective virus vectors cannot transduce and distribute throughout a tumor mass, such strategies could not achieve satisfactory outcomes in cancer treatments.³⁵ Given the need for greater efficacy, oncolytic adenoviruses are becoming increasingly popular tools for preclinical studies of cancer gene therapy in China. The first oncolytic adenovirus, ONYX-015, was safe in patients with recurrent head and neck cancer (HNC), but did not elicit sufficiently potent and durable antitumoral responses unless combined with chemotherapy.³⁷ Thus, Chinese researchers constructed a novel oncolytic adenovirus system that, similar to ONYX-015, has a deletion in E1B 55kD gene, but includes an additional antitumor gene. Compared to each individual component, this oncolytic adenovirus had greatly enhanced antitumor efficacy in a mouse xenograft model of colon cancer.³⁸ In a subsequent study from the same team, the ZD55-gene system equipped with an isoform of an vascular endothelial cell growth inhibitor (VEGI-251), which combines the antiangiogenic effect of VEGI-251 and the oncolytic activity of the ZD55-gene system, significantly inhibited tumor growth in mouse models of colorectal tumors.³⁹ These results support the use of oncolytic adenoviruses expressing antitumor factors as simple and efficient methods for treating solid tumors.

Another promising strategy is CAR T-cell immunotherapy, in which autologous T cells are transduced with synthetic CARs that recognize specific tumor-associated antigens in vitro and then are infused back into the patient.⁴⁰ In several international trials, T cells armed with CARs directed against the B-cell surface marker CD19 showed durable, complete tumor regression and controllable toxicity in patients with B-cell malignancies.^41,42 Although CAR T-cell immunotherapy is a powerful tool to treat metastatic cancers,⁴³ the finite replicative life-span of CAR-modified T cells impedes long-term curative benefits of this strategy. To prolong the persistence of these therapeutic T cells in vivo, Bai et al. transduced human T cells not only with LV vectors encoding anti-CD19 CARs but also with mRNAs encoding human telomerase reverse transcriptase, which could transiently increase telomerase activity in these T cells. These modified anti-CD19-CAR T cells had more enduring persistence and antitumor effects in mouse models of human B-cell malignancies compared to conventional anti-CD19-CAR T cells.⁸ An oncolytic virus coupled with CAR T-cell immunotherapy may thus be a promising approach to improve therapeutic outcomes for cancer patients further.

Several Chinese preclinical studies that exploited the tumor tropism of MSCs have also shown encouraging results. In one study, human umbilical cord-derived MSCs (HUMSCs) engineered with LV vectors to encode tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were intravenously injected into a mouse model of hepatocarcinoma. The engineered HUMSCs localized exclusively at tumor sites and showed significant antitumor activities.⁷ Another study combined advantages of both MSCs and oncolytic viruses by engineering HUMSCs to produce a novel conditionally oncolytic adenovirus. In this system, hepatic differentiation of HUMSCs in the tumor microenvironment drove the packaging and release of oncolytic viruses, which eventually resulted in specific elimination of HCC cells in mice.⁴⁴ This combination not only exhibited dramatic tumor inhibition with less toxicity toward normal organs, but also bypassed the obstacle of intravenous or intratumor injection of adenoviruses.⁴⁴ These successful results highlight the potential of MSCs as vehicles to deliver antitumor agents in cancer therapy.

In parallel, Lv et al. demonstrated that an AAV-mediated anti-DR5 (death receptor 5) chimeric antibody expressed in muscle cells of nude mice burdened with human liver and colon cancer conferred durable and significant suppression of tumor growth,⁴⁵ suggesting that antibody gene therapy can be an alternative treatment strategy for a variety of cancers.

Neurological Diseases

Virus vector–mediated gene therapy has recently been widely used to treat various diseases of the nervous system that affect the movement, cognition, or mental status of patients to varying degrees. Chinese researchers have focused mainly on three types of nervous system disorders: nerve injuries, neurodegenerative diseases, and mental disorders. In China, early attempts to treat spinal cord injury, involving genetic modification of olfactory ensheathing cells with a retrovirus vector to produce high levels of glial cell line–derived neurotrophic factor in vivo,⁴⁶ showed the potential therapeutic benefits of neurotrophic factors. Two recent studies in China showed that LV-mediated overexpression of Netrin-1 (a neuron growth-promoting factor) or gene silencing of Lingo-1 (a nerve regeneration inhibitor) in injured sites markedly promoted motor and sensory function recovery after spinal cord injuries.^9,47 These research outcomes add several neurotrophic factors and nerve growth factors to the toolkit for gene therapies for nerve injury.

Alzheimer's disease (AD) is a neurodegenerative disease that severely damages the cognitive levels of patients. Previous treatment strategies aimed at reducing levels of amyloid-beta, which has been implicated in AD pathology, were unsuccessful.⁴⁸ So far, no effective methods are available to reverse AD progression. A study by Hu et al. showed that regulation of N-methyl-D-aspartic acid (NMDA) receptor trafficking, which is important for synaptic activity and cognition but is reduced by amyloid-beta,⁴⁹ may be a promising AD treatment strategy. In this study, LV vector–mediated overexpression of ephrin type-B receptor 2, a key protein that regulates synaptic localization of NMDA receptors, in the dorsal hippocampus ameliorated diminishment of cognitive functions in a mouse model of AD by rescuing NMDA receptor trafficking.⁵⁰ In another Chinese study, intramuscular injection of scAAV9, an efficient AAV serotype that promotes gene delivery to the central nervous system (CNS), resulted in expression of human IGF1 in lumbar spinal cords, which reduced motor neuron apoptosis, delayed disease onset, and prolonged the life-span of amyotrophic lateral sclerosis model mice.¹⁰

In addition to neuropathy, neurotrophic factor therapy was also applied to treat mental disorders. Depression is a widespread psychiatric disease that lacks reliable therapeutics. Although brain-derived neurotrophic factor (BDNF) was shown to have potential as an antidepressant agent,⁵¹ there are few methods for effective delivery of BDNF to the CNS without tissue damage.⁵² In a recent study, Ma et al. demonstrated that intranasal injection of AAV vectors expressing BDNF-HA2TAT (BDNF fused with cell-penetrating peptides, influenza hemagglutinin 2 and transactivator of transcription) successfully delivered BDNF-HA2TAT to the CNS through the nose–brain pathway and alleviated depression-like behaviors in mice subjected to chronic mild stress. This study thus identified a feasible administration route for clinical BDNF delivery.⁵² In general, Chinese researchers have showed potential uses of several curative factors and improved administration approaches for gene therapies for nervous system diseases.

Cardiovascular Diseases

Among cardiovascular diseases in China, MI, critical limb ischemia (CLI), and hypertension have attracted the most attention. In the last decade, Chinese researchers have investigated several potential targets to treat ventricular dysfunction after MI. Integrin-linked kinase (ILK), which plays an important role in recruiting endothelial progenitor cells to ischemic regions,⁵³ was an early example of such a treatment. Adenovirus vector–mediated overexpression of ILK in rat peri-infarct myocardium reduced infarct size and attenuated cardiac remodeling by enhancing angiogenesis, reducing apoptosis, and increasing cardiomyocyte proliferation.¹¹ Moreover, considering the important but harmful role of reactive oxygen species (ROS) in myocardial ischemic injury,⁵⁴ Pan et al. injected bone-marrow MSCs (BMSCs) transfected with adenovirus-extracellular superoxide dismutase (ecSOD, an enzyme scavenging superoxide anion in extracellular space) into the infarction regions of mice model and saw decreases in the size of MI and improved cardiac function, as well as reduced oxidative stress and prolonged BMSC survival in mouse hearts.⁵⁵ All these results suggest that promoting angiogenesis and decreasing oxidative stress are effective approaches to restore cardiac function after MI.

CLI is severe blockage in arteries of the lower extremities that has crippled many people. Delivering proangiogenic genes or cells have been demonstrated as potential methods to mitigate symptoms of CLI.⁵⁶ Thus, the combination of microRNA-21, a molecule that has potent revascularization effects,⁵⁷ and proangiogenic human umbilical cord blood-derived MSCs (UCBMSCs) hold promise for improved treatment of CLI. In one Chinese study, UCBMSCs transduced with LV-miR-21 injected into the thigh muscle of mice with CLI promoted significant neovascularization and perfusion recovery in ischemic limbs.⁵⁸

Gene therapy can also be used to alleviate hypertension. Zhang et al. reported that AAV-mediated antisense knockdown of CYP4A1 (arachidonic acid cytochrome P-450 hydroxylase 4A1), an enzyme catalyzing the production of vasoactive 19/20-hydroxyeicosatetraenoic acids, markedly reduced high blood pressure of spontaneously hypertensive rats and maintained normal blood pressure for at least 2 years.⁵⁹ This study thus provides a novel therapeutic target for hypertension.⁶⁰

Autoimmune Diseases

To function properly, the adaptive immune system must precisely distinguish a wide variety of pathogens from self-antigens. Errors in antigen recognition often result in autoimmune disorders that involve damage to health tissues resulting from aberrant immune responses. Rheumatoid arthritis (RA) is a common autoimmune disease in China that has an incompletely understood pathogenesis. Researchers have made many efforts to develop effective RA treatments that have limited side effects. Recently, long-term immunological tolerance induced by intraarticular injection of AAV2 vectors carrying the human tumor necrosis factor receptor-immunoglobulin Fc fusion gene were identified as an efficient and safe method to diminish immune responses in patients with inflammatory arthritis.⁶¹

Chinese researchers have also contributed several potential and novel treatment approaches to autoimmune disorders. For example, inhibiting activation of immune cells by LV-mediated delivery of short hairpin RNAs (shRNAs) targeting B cell-activating factor to afflicted joints efficiently inhibited synovial inflammation, alleviated joint damage, and did not induce systemic immunosuppression in collagen-induced arthritis mice.⁶² In addition to methods that directly affect immune response regulators, indirect regulation of the immune system is also useful. Zhou et al. found that AAV-mediated intraarticular overexpression of the lymphatic growth factor human vascular endothelial growth factor C could increase lymphangiogenesis and remove excess lymphocytes from inflamed joints, which eventually attenuated joint tissue damage in mice with chronic inflammatory arthritis.¹² This result suggests that enhancing lymphatic drainage by lymphangiogenesis is also an effective method to treat inflammation that accompanies autoimmune diseases. Furthermore, a treatment that targeted abnormal proliferation of synovial cells in rheumatoid synovium using epirubicin and AAV2/5-TRAIL to induce synovial cell apoptosis showed remarkable anti-arthritic effects without causing obvious harmful side effects,⁶³ which provides another possible application of gene therapy to treat autoimmune arthritis.

T-cell-mediated inflammatory demyelinating disease is a severe and potentially life-threatening clinical condition that can damage the nervous system. Gao et al. showed that gelsolin (GSN) could delay the onset and decrease the severity of experimental allergic encephalomyelitis (EAE) by injecting GSN-overexpressed LV vectors into EAE animals. Combining GSN overexpression with vitamin D treatment further retarded disease progression.⁶⁴ These results suggest that GSN can be an adjuvant of current vitamin D therapy to achieve better therapeutic effects in EAE.

Liver Diseases

China has >300 million patients with liver disease,⁶⁵ so significant attention has been paid to various critical and chronic liver diseases in China, including HCC, liver cirrhosis, liver failure, and steatohepatitis.

For severe liver diseases such as liver cirrhosis and liver failure, autologous hepatocyte transplantation may be a promising treatment. Huang et al. reported an efficient in vitro reprogramming approach that generates human-induced hepatocytes (hiHeps) from human fibroblasts by LV-mediated delivery of genes for the transcription factors forkhead box protein A3, hepatocyte nuclear factor 1A, and hepatocyte nuclear factor 4A (FOXA3, HNF1A, and HNF4A). After transplantation into mice with metabolic liver disease or acute liver failure, hiHeps restored liver function and prolonged the survival time of treated mice.¹³ Considering recent international advancements in in vivo reprogramming to produce hepatocytes,^66,67 this treatment for liver diseases is nearing practical clinical application.

Since chronic hepatic diseases can eventually develop into severe liver diseases, inducing liver regeneration or inhibiting pathologic reactions are most the common gene therapy strategies in China. For example, using AAV vectors to induce overexpression of IGF-2 in mice hepatocytes, Liu et al. successfully induced hepatocyte proliferation and repaired liver tissue damage in two different mouse models of chronic liver injuries.⁶⁸ In various types of liver damage, hepatic fibrosis is a common pathological state and a feature of early stage cirrhosis. Previous studies indicated that HNF4α could induce mesenchymal-to-epithelial transition in fibroblasts,⁶⁹ in contrast to the epithelial–mesenchymal transition (EMT) of hepatocytes during hepatic fibrosis development. A Chinese preclinical study exploited this characteristic of HNF4α in adenovirus vector-mediated overexpression of HNF4α that successfully inhibited hepatocyte EMT and ameliorated liver fibrosis in a rat model of liver fibrosis.⁷⁰ This year, Wang et al. reported that the innate immune signaling regulator, caspase 8 and Fas-associated protein with death domain-like apoptosis regulator (CFLAR), is a key suppressor of steatohepatitis, and AAV8-mediated delivery of a small peptide segment of CFLAR to the liver effectively attenuated progression of steatohepatitis and metabolic disorders in both mice and monkeys by inhibiting inflammatory reactions.⁷¹ This study demonstrated the CFLAR could be a novel target for treating steatohepatitis and associated metabolic disorders.

Infectious Diseases

Infectious diseases harm and kill large numbers of individuals each year. In China, virus vector–mediated gene therapy is mainly applied to treat HBV and human immunodeficiency virus (HIV) infection. There are currently about 100 million HBV carriers in China, or about one-third of global HBV carriers.⁷² HBV infection causes acute and chronic liver disease and may progress to liver cirrhosis and HCC. Although HBV vaccines can effectively protect uninfected individuals, there are still no reliable treatments for infected patients. To cure HBV infection, silencing or eradicating covalently closed circular DNA (cccDNA) to suppress HBV replication in infected hepatocytes is crucial.⁷³

RNA interference (RNAi) is a potent method to silence HBV cccDNA, but HBV mutants with mutations in target sequences can escape the antiviral activity of RNAi. An interesting study that evaluated the efficiency of shRNA therapy toward a pre-existing shRNA-resistant HBV variant showed that treating HBV infection with highly potent shRNA alone had little antiviral efficacy due to escaped mutants. However, the combination of two shRNAs could reduce titers of all HBV variants.⁷⁴ Another study using a dual-shRNA strategy also exhibited stronger antiviral effects than a single shRNA.¹⁴ Moreover, a combination of HBV dual-shRNA and transforming growth factor-β (TGF-β) shRNA could further reduce virus titer and improve liver morphology in HBV-persistent mice.¹⁴ These findings from Chinese researchers indicate that combinatorial RNAi therapy should be a viable treatment for human HBV infection.

Although HIV is relatively less common in China than in many nations, controlling HIV infection remains an important task. Current gene therapy strategies for HIV infection include interfering with HIV replication by RNAi or mutant HIV proteins, increasing HIV-resistance of T cells, and anti-HIV vaccines that elicit broadly neutralizing antibodies.⁷⁵ In recent years, the latter one has become popular given its ability to treat and prevent infection by many HIV variants through induction of both cellular and humoral immune responses. A Chinese group provided a novel immunization approach to elicit broadly neutralizing antibodies efficiently by constructing several DNA vaccines and recombinant Tiantan vaccinia vaccines that carry heterologous gp140 and gp145 env genes. Injection of these engineered viruses into guinea pigs successfully elicited broad HIV-1 neutralizing antibody responses and neutralized a variety of HIV-1 variants. However, the ability of this method to induce potent anti-HIV T-cell response requires additional enhancement.⁷⁶

Clinical Applications of Gene Therapy With Virus Vectors in China

Compared to other treatments for diseases, gene therapy raises more ethical concerns because of its unique ability to engineer human genetic material. Therefore, gene therapy is currently largely used as a last resort to treat severely harmful even fatal diseases that lack other treatment options. Nonetheless, the therapeutic potential of gene therapy has been evaluated for many chronic diseases in China. As with other countries, clinical testing of virus vector–mediated gene therapy in China was initially applied to monogenic diseases. Subsequently, cancer, a complex class of diseases with high incidence, high mortality, and significant unmet medical need, rapidly became the focus of gene therapy clinical trials in China.

Monogenic Diseases

The world's first successful gene therapy trial was performed in 1990, wherein normal adenosine deaminase (ADA) genes were delivered into the T cells of patients with ADA deficiency by γ-retroviral vectors.^77,78 In 1991, China conducted its first gene therapy trial to examine treatment of two hemophilia B patients transplanted with autologous fibroblasts engineered by a retrovirus expressing clotting factor IX (hFIX). After the treatment, the plasma hFIX protein levels and blood clotting activity of both patients significantly increased, and both showed partial correction of hemorrhagic tendencies without treatment-related side effects for >420 days.⁷⁹

Because of safety concerns for virus vectors, there were few trials to treat monogenic diseases in China, and efforts instead shifted to cancer treatments. However, due to the increasing safety and transduction efficiency of virus vectors, especially LV and AAV vectors, clinical experiments for monogenic diseases have been revived in China. In a Phase II trial, AAV2 vectors expressing NADH dehydrogenase subunit 4 were used to cure Leber's hereditary optic neuropathy patients. In this trial, following intravitreal injection of the AAV2 vectors, two-thirds of the patients had improved visual acuity of at least 0.3 logarithm of the minimum angle of resolution, and no adverse events were seen.⁸⁰ Moreover, referencing successful clinical treatments for rare fatal disorders (e.g., X-linked adrenoleukodystrophy,⁸¹ metachromatic leukodystrophy⁸²) and hemoglobinopathies (e.g., β-thalassaemia⁸³) used in other countries, trials using LV or AAV vector–mediated gene therapy to treat these diseases and X-linked severe combined immunodeficiency are also underway in China; these trials have been recorded in the ClinicalTrials.gov database.

Cancer

According to the Journal of Gene Medicine clinical trial database (Wiley), ClinicalTrials.gov database and retrieved articles,^{84

–113} 90.7% of gene therapy trials in China are for cancer and involve a variety of therapeutic strategies (Fig. 1). Among these cancers, HCC, HNC, non-small-cell lung cancer (NSCLC), and lymphoma are the diseases most frequently targeted for gene therapies.

Figure 1.

A survey of virus vector–mediated gene therapy clinical trials in China. This review contains 129 clinical trials of virus vector–mediated gene therapy in China, including completed, ongoing, and pending subjects. (A) Most clinical trials of gene therapy adopting virus vectors focus on cancer treatments. (B) For cancer gene therapy, T-cell immunotherapy and recombinant adenovirus delivering p53 gene are most commonly used strategies in clinical trials. Oncolytic viruses are also evaluated in about 11.1% of cancer gene therapy trials in China. rAd, recombinant adenovirus.

In a Phase II trial, HCC patients who had undergone liver transplantation (LT) were treated with adenovirus containing HSV thymidine kinase as an adjuvant treatment. These patients had a significantly higher survival rate over the ensuing 3 years compared to patients that received LT alone.¹¹⁴ In a Phase III trial, intratumor injection of the oncolytic adenovirus H101 combined with a cisplatin plus 5-fluorouracil (PF) regimen produced nearly twofold higher antitumoral efficacy than PF alone in HNC or esophagus squamous-cell cancer patients, and more importantly, this treatment was relatively safe.¹⁰³ In another recurrent HNC trial, patients injected with KH901 (an oncolytic adenovirus expressing granulocyte-macrophage colony-stimulating factor) showed good tolerance for the treatment. Furthermore, 12/19 patients remained stable, and two showed disintegration of injected tumors.⁶ A Phase II clinical trial involving NSCLC patients indicated that trans-tracheal injection of adenoviruses expressing p53 plus docetaxel did not increase overall survival or efficacy, although this treatment had tolerable levels of toxicity and slightly enhanced the survival time of patients compared to docetaxel treatment only.¹⁰⁰ Thus, delivering the p53 gene by a replication-defective adenovirus vector is not an effective NSCLC treatment, and more efficient gene therapy strategies should be evaluated or developed to treat this cancer. In addition to the above strategies, T-cell immunotherapy has gained popularity in recent years for its almost complete tumor clearance of B-cell lymphoma in preclinical studies. In an earlier clinical trial, infusion of cytotoxic T lymphocytes induced by transfected dendritic cells with recombinant AAV vectors carrying carcinoembryonic antigen complementary DNA induced improvements without adverse reactions in more than half of treated cancer patients.⁸⁶ This result demonstrates the promising role of T-cell therapy in cancer treatments. According to the ClinicalTrials.gov database, >40 clinical trials are in the preparatory or recruitment stage to evaluate the safety and efficacy of CAR-modified T-cell infusions, such as anti-CD19-CAR T cells and anti-CD28-CD137-CAR T cells, to treat lymphoma or lymphoblastic leukemia patients.

Other Diseases

According to the above-mentioned databases, gene therapy clinical trials for several complex disorders and infectious diseases have also been initiated in recent years. These include adenovirus-mediated delivery of hepatocyte growth factor to treat ischemic heart disease, transplanting anti-CD19-CAR T cells with optimized hinge and transmembrane domain to patients with systemic lupus erythematosus, and treating HIV-infected patients with a replication-defective adenovirus expressing HIV-1 gag or a DNA vaccine coupled with a replication-defective Ankara vaccinia virus expressing HIV-1 gag-pol and env.

Conclusion

Based on insights into disease pathology and the development of virus vectors with lower toxicity, higher transduction efficiency, and wider applications, virus vector–mediated gene therapy has now been extended to treat a variety of diseases. To some extent, gene replacement therapy for monogenic diseases has reached sufficient maturity. Furthermore, gene editing tools such as the CRISPR/Cas9 system can restrict these replacements to single or several nucleotides. Although the pathogenic mechanisms of many complex diseases remain unclear, general gene therapy strategies involving addition of curative and protective genetic materials or elimination of harmful factors have shown excellent therapeutic benefits for some diseases. For infectious diseases, genetic vaccines that induce potent cellular and humoral immune responses have promise for both treatment and prevention. Chinese researchers have contributed significantly to the progress of gene therapy by identifying many therapeutic targets as well as characterizing factors and novel strategies to treat these diseases, such as promoting lymphangiogenesis or inducing apoptosis of abnormal synovial cells to cure arthritis. Moreover, China shows an obvious interest in treating various liver diseases as evidenced by the development of several innovative methods to generate hepatocytes and identification of novel targets to suppress liver inflammation or fibrosis.

For clinical trials in China, cancer treatments are dominant, with adenovirus-mediated antitumor gene transfer and CAR T-cell immunotherapy being the main strategies. Importantly, China has been a leader in developing CRISPR-based therapies, as 90% of clinical trials involving CRISPR clinical trials are being conducted in China.¹¹⁵ These efforts will benefit cancer patients in China and around the world. Clinical trials for gene therapies for monogenic diseases, heart diseases, autoimmune diseases, and HIV infection are also underway in China, although the progress in these areas is less pronounced.

In China, additional well-designed clinical trials concerning non-cancer diseases should soon be underway to evaluate promising gene therapy approaches identified in preclinical studies. The cogent information gathered from clinical trials can impact and guide further development of gene therapy. Additionally, more critical therapeutic targets, more innovative strategies, and more powerful vectors, such as the newer generation of oncolytic viruses, are still required to address many unmet medical demands.

Footnotes

Acknowledgments

This review was supported by grants from the National Key Research and Development Plan (2016YFC0903900), PUMC Youth Fund (3332016047, 2017310001), and Chinese Academy of Medical Sciences Innovation Fund for Medical Sciences (CIFMS, 2016-I2M-1-011, 2017-I2M-1-008). We thank Yan Xie for assistance in revising the text.

Author Disclosure

The authors have no conflicts of interest to disclose.

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