A novel fiber chimeric conditionally replicative adenovirus-Ad5/F35 for tumor therapy

ABSTRACT

Significant progress has been made in the diagnosis and treatment of cancer; however, significant challenges remain. Conditionally replicating adenoviruses (CRAds), which not only kill cancer cells, but also serve as vectors to express therapeutic genes, are a novel and effective method to treat cancer. However, most adenoviruses are Ad5, which infect cells through the coxsackie and adenovirus receptor (CAR). The transduction efficacy of Ad5 is restricted because of the absent or low expression of CAR on several cancer cells. Ad serotype 35 has a different tropism pattern to Ad5. Ad35 attaches to cells via a non-CAR receptor, CD46, which is expressed widely on most tumor cells. Thus, chimeric adenoviral vectors consisting of the knob and shaft of Ad35 combined with Ad5 have been constructed. The chimeric fiber adenoviral vectors can transduce CAR-positive and CAR-negative cell lines. In this review, we explore the application of the novel fiber chimeric conditionally replicative adenovirus-Ad5/F35 in tumor therapy in terms of safety, mechanism, transduction efficacy, and antitumor effect.

Introduction

With advances in imaging and diagnostic techniques, we have obtained a more comprehensive understanding of the molecular processes leading to cancer. In addition, using a combination of chemotherapy and radiotherapy, overall survival rates have improved significantly in patients with cancer. However, cancer remains the second leading cause of death, mostly because of the limited efficacy of chemotherapeutic agents in killing tumors.^1,2 Traditional research and development has focused on the development of a curative agent for each type of cancer; however, tolerance to chemotherapy drugs greatly reduces the effectiveness of cancer treatment. Combination therapy, as the most effective method to improve the survival rate, has attracted much attention.

An effective and promising approach to treat malignant tumors is the application of conditionally replicating adenoviruses (CRAds),³ which can replicate in, and lyse, tumor cells, but do not affect normal cells. When infected cancer cells are lysed, CRAds are released, and then adjacent tumor cells are infected. This characteristic is superior to non-replicating adenoviruses (Ads).⁴ In addition, CRAds may be used as a vehicle to deliver a therapeutic transgene to further mediate gene therapy by targeting tumor cells. CRAds not only selectively replicate in and kill cancer cells, but also enhance therapeutic gene expression and function in the tumor microenvironment.⁵

Some laboratories have found that CRAds induce an enhanced antitumor activity when armed with antiproliferative and proapoptotic transgenes, for example, second mitochondria-derived activator of caspases (Smac).⁶ tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL).⁷ melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24).⁸ a short interfering RNA (siRNA) against the antiapoptotic factor Apollon.⁹ and the antioxidant enzyme, manganese superoxide dismutase (MnSOD).¹⁰ In addition, CRAds armed with immunostimulatory cytokines, such as granulocyte-macrophage colony-stimulating factor¹¹ heat shock protein 21.¹² and interleukin-12 (IL-12)¹³ can recruit effector cells and enhance host antitumor immunity. Furthermore, CRAds can also mediate suicide genes, such as the herpes simplex virus thymidine kinase suicide gene (HSV-TK).¹⁴

The antitumor efficacy of CRAds is associated with the ability of adenoviruses to infect tumor cells. However, many factors influence the infection capability of CRAds. One of the most critical factors is the expression level of the adenovirus receptor on the surface of tumor cells.^15,16 Adenoviruses infect tumor cells by binding to their target receptor on the cell surface. Most of traditional adenoviral vectors are serotype 5 adenovirus (Ad5) of subgroup C, whose receptor on the cell membrane is coxsackie and adenovirus receptor (CAR).¹⁷ However, the ability of Ad5 to infect a variety of primary tumor cells is often limited by the absent or low expression of CAR on several cancer cells.¹⁸ To improve adenoviral infectivity, many researchers considered using other Ad serotypes that infect cells via non-CAR receptors. Certain subgroup B adenoviruses can infect tumor cells via the non-CAR receptor, CD46, which is a widely expressed membrane cofactor protein. To make CRAds that can exert an anti-tumor effect on various tumors, chimeric adenoviruses that can bind to CAR and CD46 have been constructed. Chimeric adenoviruses comprise the knob and shaft of adenovirus of serotype 35 (replacing the knob and shaft of Ad5), such that the chimeric adenovirus inherits the infection characteristics of Ad35, which can transduce many CAR-negative tumor cells.¹⁹ Both Ad3 and Ad35 attach to tumor cells via the non-CAR receptor, CD46. However, compared with Ad3, Ad35 has more cell membrane receptors, closer integration with the CD46 molecules, and can infect a variety of tumor cells efficiently.²⁰ Thus, researchers have constructed a novel fiber chimeric adenovirus-Ad5/F35 that can transduce cell lines with absent or low expression of CAR more efficiently than Ad5 vectors. The novel fiber chimeric adenoviruses can infect both CAR-positive and CAR-negative tumor cells. Fig. 1 shows a diagram comparing the fiber structures of Ad5 and Ad5/Ad35.

Figure 1.

Schematic diagrams of the Ad5 vectors and Ad5/F35.

In this review, we provide an overview of the novel fiber chimeric adenoviruses for tumor therapy.

The safety of the novel fiber chimeric adenovirus

The anti-tumor effect of Ad5-based vectors on various tumor cells has been observed. Ad5-based vectors are well tolerated when injected directly into tumors; however, toxicity associated with non-oncolytic Ad5 administration has been observed at high systemic doses, and might induce complement activation, cytokine release, and consequent vascular damage, which leads to systemic inflammatory responses. The uptake of Ad5 by its target cells can induce elevation of liver transaminases, tissue damage, and release of cytokines and chemokines, which in turn lead to inflammation and the induction of an antiviral adaptive immune response.^21–24 Thus, the safety of systemic adenovirus injection has received significant research attention.

The safety of Ad5/F35 vectors should be considered seriously if Ad5/F35 vectors are to be applied in clinical trials. Compared with animals injected with Ad5 vectors, animals injected with Ad5/F35 vectors had lower serum proinflammatory cytokine levels,^25–27 lower toxicity,^28,29 lower liver enzyme levels,^27,30 a higher survival rate, and less weight loss.²⁷ Recently, a study³¹ evaluating the effects of Ad5/F35 vectors on the heart found that Ad5 vectors evoked more severe tissue damage, with large areas of interstitial inflammatory cell infiltration and myocyte necrosis, compared with Ad5/F35 vectors. Taken together, these studies suggested that the chimeric adenovirus Ad5/F35 has a better safety profile than Ad5.

Possible mechanisms of the antitumor effect of Ad5/F35 mediated therapy

When CRAds are administrated into the body, they initially pass through the connective tissue and extracellular matrix (ECM), and then enter cells through a series of complex processes to kill tumor cells. A schematic diagram of these processes is shown in Fig. 2. The Ad35 fiber knob in Ad5/F35 attaches to CD46, and once attached, an Arg-Gly-Asp (RGD) motif in the penton base interacts with integrin which is a secondary receptor, resultly in promotion of endocytosis and internalization of the adenovirus.^32–34 After cell internalization, viruses escape from the endosome into the cytosol. Viral escape is one of the most important steps of infection and one of the least understood. CRAds then traffic toward the nucleus along microtubules, via interactions with the motor protein dynein.³⁵ The viral genome is then delivered to the cell nucleus. Ad5/F35 then selectively replicates in tumor cells, and lyses then via apoptosis. The released adenovirus then infects adjacent tumor cells.

Figure 2.

The mechanism of the antitumor effect of vectors incorporating the novel chimeric fiber Ad5/F35 adenovirus. Ad5/F35 first attaches to cells via CD46 or CAR. Once attached, the RGD motif in the penton base interacts with integrin to promote endocytosis and internalization of the adenovirus. Ad5/F35 then lyses the tumor cells via apoptosis. The released adenoviruses infect adjacent tumor cells.

Factors influencing the transduction efficacy of the novel fiber chimeric adenovirus Ad5/F35

The transduction efficacy of adenoviruses depends mainly on the capacity of the adenovirus to enter the targeted cells and whether they successfully transfer their genome into the host nucleus, which is largely dependent on the intracellular route by which the virus travels within the host cells. Hence, all factors that participate in this process can influence the transduction efficacy of adenoviruses (Table 1).

Table 1.

Factors influencing the transduction efficacy of Ad5/F35 vectors.

Factors		References
Neutralizing antibodies (nAbs)		36,38,39,
	CD46	31,40–43
Receptors	Other receptors	44,45
	Integrin (RGD motif)	46
Insertion of foreign peptides		47,20
The affinity to CD46		51,52
ECM	Hyaluronidase	57
	Matrix metalloproteinases (Relaxin)	64
Factor X (FX)		67
X-binding protein (X-bp)		71,72
	Cell type	77
Features of cells	Regulation and apoptosis	79,80
	The metabolic activity of the host cell	81
	survival potential	82

When chimeric adenoviruses are injected into the body, they are first cleared by neutralizing antibodies (nAbs) in the humoral immune system. nAbs against type 5 adenovirus are commonly ecountered.³⁶ and greatly weaken the effect of Ad5.³⁷ However, less than 10% of patients can produce nAbs to Ad35^36,38,39 because people are exposed to Ad5 more frequently than to Ad35. However, nAbs are not the most critial factor influencng transduction efficiency. The adenovirus receptor is one of the most important factors that influence transduction efficiency.^15,16 CAR and integrins are the main factors that influence the transduction efficacy of Ad5. CAR and integrins are low in most tumor cells. However, in contrast Ad5, the expression level of CD46 is a vital factor that influences the transduction efficacy of Ad5/F35. Ad5/F35 can improve the systemic gene delivery properties of the adenovirus vectors in human and rodent cells, especially in cells lacking in sufficient CAR expression.⁴⁰ Furthermore, Koizumia et al.⁴¹ and Acharya et al.⁴² found that the transduction efficacy of Ad5/F35 vector was significantly higher in trophoblast cell lines and renal carcinoma cells with high expression CD46, compared with that of Ad5 vectors. However, in both human dilated cardiomyopathy (DCM) hearts and non-DCM hearts with higher CAR expression and lower CD46 expression, the transduction efficacy of Ad5 was higher than that of Ad5/F35.^31,43 Thus, the level of CD46, not CAR, mainly affects the transduction efficacy of Ad5/F35 vectors However, interestingly, Yu et al. found that in esophageal and oral tumor cells⁴⁴ and pancreatic and breast cancer cells.⁴⁵ the transduction efficacy of Ad5/F35 was influenced by other receptors, and did not correlate with CD46.

Integrin is the second receptor for Ad5/F35 binding to targeted cells. The interaction between integrin and the RGD motif can increase the rate of adenovirus entry into cells and contributes to adenovirus escaping from endosomes, eventually improving the transduction efficacy. For example, Shayakhmetov et al.⁴⁶ found that deletion of RGD motify significantly reduced the rate of adenovirus internalization for Ad5/F35 vectors.

Binding to CD46 is significantly inhibited by inserting foreign peptides into HI, IJ, or FG loops in the fiber knob of Ad35, especially the HI loop, because the HI loop is the most crucial region for CD46 binding. However, in CD46-negative cells, insertion of foreign peptides into the HI, IJ, or FG loop can significantly increase the transduction efficacy of Ad5/F35 vectors.⁴⁷ Another study²⁰ showed that insertion of foreign peptides into the HI or FG loops mediated the highest transduction efficacy in CD46-negative cells. In CD46-positive cells, the transduction efficacy of Ad5/F35 vectors containing two copies of the foreign peptide was higher than that of that of Ad5/F35 containing one copy of the foreign peptide, although lower than that of parent Ad5/F35.²⁰ These observations demonstrated that insertion of two copies of the foreign peptides would reduce the affinity to CD46, but not by as much as inserting one copy of the foreign peptide, because multimeric foreign peptides have a higher binding affinity for integrins than monomeric foreign peptides.^48–50 Thus, the affinity of adenovirus for CD46 also influences the transduction efficacy of Ad5/F35.^51,52  Wang et al.⁵¹ found that the affinity of Ad5/F35 for CD46 produced different transduction efficiencies in vitro and in vivo. In vitro, regardless of the CD46 receptor density on the cells, the higher affinities of Ad5/35+ and Ad5/35++ to CD46 did not lead to correspondingly higher transduction efficiency. Whereas, in vivo, Ad5/F35 vectors with increased affinity for CD46 showed higher transduction efficiency. This is because Ad5/35 vectors with increased affinity to CD46 can be sequestered less efficiently by blood cells and tissue macrophages after intravenous injection. Similarly, Shayakhmetov et al.⁵² found that the transduction efficiency of Ad5/F35 in vivo was not only dependent on the high affinity for CD46, but also was influenced strongly by the surrounding ECM.

Recent evidence has demonstrated that connective tissue and ECM components may play an important role in inhibiting viral spread following administration of adenovirus.^53–55 Therefore, wider viral distribution is observed when the ECM is degraded. Viral spread can be improved by proteolytic enzymes⁵⁶ including hyaluronidase and matrix metalloproteinases. Ganesh et al.⁵⁷ observed that the transduction efficiency increased using a combination of Ad5/F35 and hyaluronidase, which degraded the hyaluronan-rich matrix and depolymerized viscoelastic ECM components.^58,59 The peptide hormone Relaxin could upregulate matrix metalloproteinases^58,60–63 and increase the spread of adenoviruses within tumors when combined with Ad5/F35.⁶⁴

The anti-tumor effect of CRAd-mediated therapy is significantly hindered by coagulation factor X (FX), which binds to CRAds, especially Ad5.^65,66 However, transduction with an Ad5/F35 vector in epithelial ovarian cancer cells (EOCs) in vitro was not affected by FX⁶⁷ because binding of Ad35 to FX is either not detectable.⁶⁸ or much lower than that of Ad5.⁶⁹ X-binding protein (X-bp), a snake venom-derived protein that binds to FX with high affinity, can inhibit the binding of Ad5 to FX.⁷⁰ In addition, X-bp significantly reduced liver transduction after injection of Ad5 vectors.⁶⁹ Similarly, Liu et al.⁷¹ and Greig et al.⁷² showed that X-bp improved the transduction of Ad5/F35 in liver metastases and increased the antitumor efficacy of Ad5/35-based vectors by reducing hepatocyte transduction and increasing the circulation time after intravenous injection of Ad5/F35 vectors.

In addition, features of cells, such as endocytosis, trafficking, and sorting mechanisms for animal viruses, have a critical influence on the fate of the infecting virus.^73–78 Drouin et al.⁷⁷ found that the selection of intracellular trafficking routes is not only determined by the fiber knob domain and the cellular receptor, but also by the targeted cell type. For efficiently transduced cells, adenoviruses were localized in early endosomes or the cytosol. By contrast, in poorly transduced cells, they were localized within late endosomes/lysosomes. Phosphatase inhibitors (PSPs) can later the intracellular entry routes. PSPs are involved in cell cycle regulation and apoptosis⁷⁸ and treatment with PSPs, such as okadaic acid, can increase caveolae endocytosis.^79,80 In addition, Cayer et al.⁸¹ found that the only PSPs that could enhance the transduction efficacy of Ad5/F35 were those specific to protein phosphatase 2 phosphatase activator (PP2A). Thus, PSPs, especially those targeting PP2A, have a major effect on the transduction efficiency of Ad5/F35. Interestingly, Samson et al.⁷⁸ found that there was a link between virus endocytic trafficking and the metabolic activity of the host cell. Ad5/F35 showed higher transduction efficiency in cells displaying strong transcriptional activity, which results in differences in intracellular trafficking. Kim et al.⁸² showed that the transduction efficiency of Ad5/F35 specifically correlates with the survival potential of the cell type. The transduction efficacy of Ad5/F35 was enhanced in certain cancer cells that were exposed to rapamycin, an autophagy inducer.

Thus, we concluded that many factors can influence the transduction of Ad5/F35, and that Ad5/F35-mediated therapy for cancers is a complex process.

The application of the fiber chimeric adenovirus Ad5/F35 in tumor therapy

Although CRAds can be used to treat cancer, they still cannot completely eradicate tumors. To achieve a more effective anti-tumor effect, combination therapies, such as CRAds with chemotherapy, CRAds with radiotherapy, CRAds-mediated gene therapy, and CRAds-mediated gene therapy in combination with chemotherapy or radiotherapy, have been developed. The following text comprises a review of Ad5/F35 vectors in cancer therapy.

Ad5/F35 alone for cancer treatment

The novel oncolytic adenovirus-Ad5/F35 can produce an oncolytic effect on most cancers (Table 2). For example, in head and neck cancer in vitro and in vivo,^83,84 melanoma cells in vitro and in vivo,⁸³ glioblastoma in vitro and in vivo,⁸⁵ B-lymphocytic malignancies in vitro and in vivo⁸⁶ and renal cancer cells in vitro.⁴² Ad5/F35 administration into tumors significantly inhibit growth and enhance apoptosis. These studies demonstrated that the anti-tumor effect of Ad5/F35 is higher than that of Ad5 in most tumors with high expression of CD46.

Table 2.

Studies using Ad5/F35-based vectors in various cancers.

Ad5/F35 vectors alone		Tumor targeted	Effect compared to Ad5 vectors alone	Studies	References
		Head and neck cancer	Effective	In vitro/in vivo	83,84
		Melanoma cells	Effective	In vitro/in vivo	83
		Glioblastoma	Effective	In vitro/in vivo	85
		B-lymphocytic Malignancies	Effective	In vitro/in vivo	86
		Renal cancer cells	Effective	In vitro	42
Ad5/F35 mediated gene therapy	gene	Tumor targeted	Effect compared to Ad5 mediated genetherapy	Studies	References
	TERT	Head and neck cancer	Enhanced	In vitro/in vivo	87
	TRAIL	1) Glioblastoma	Enhanced	In vitro/in vivo	88
		2) Leukemia	Enhanced	In vitro/in vivo	89
	XAF1	Hepatocellular carcinoma	Enhanced	In vitro/in vivo	90
	p53	1)Hepatocellular carcinoma	Enhanced	In vitro/in vivo	91
		2)Breast cancer	Enhanced	In vitro/in vivo	92
		1) Lung cancer	Enhanced	In vitro	93
	MKp-E1	2) Mesothelioma	similar	In vitro	94
			Enhanced/similar	In vitro	95
			enhanced	in vivo	95
		3) Bladder cancer	Enhanced	In vitro	96
		4) Osteosarcoma	Enhanced	In vitro/in vivo	97
	Hep27	Renal cancer	Enhanced	In vitro/in vivo	98
Ad5/F35 with chemotherapy	Drugs	Tumor targeted	Effect compared to Ad5 with chemotherapy	Studies	References
Ad5/F35-M/V-HF	DTIC	Melanoma	Enhanced	In vitro/in vivo	99
Ad5/F35-tk	GCV	Colorectal cancer	Enhanced	In vitro/in vivo	100
Ad5/F35	Cisplatin	Head and neck cancer	Enhanced	in vivo	101
Ad5/F35 with radiation	Radiation	Tumor targeted	Effect compared to Ad5 with radiation	Studies	References
Ad5/F35		Head and neck cancer	Enhanced	in vivo	101
Ad5/F35-APE1 siRNA		Colorectal cancer	Enhanced	In vitro/in vivo	102

Ad5/F35-mediated gene therapy

As an oncolytic transgene delivery system, CRAds can amplify therapeutic gene expression and function in the tumor microenvironment.⁵ This makes CRAds- mediated gene therapy a promising method for tumor therapy. To date, some laboratories have constructed CRAds armed with various genes, such as proapoptotic transgenes, immunostimulatory cytokines, and suicide genes. Several studies have demonstrated that Ad5/F35 vectors-mediated gene therapy has significant anti-tumor effect in a variety of cancers (Table 2), such as glioblastoma⁸⁷ and leukemia.⁸⁸ in vivo and in vitro via TRAIL; hepatocellular carcinoma in vivo and in vitro by mediating XAF1 (XIAP associated factor 1)⁸⁹ which induces apoptosis or p53⁹⁰ breast cancer in vivo and in vitro via p53⁹¹ and renal cancer in vitro and in vivo by mediating Hep27 (encoding dehydrogenase/reductase 2)⁹² which can inhibit the degradation of p53 by binding to Mdm2 (mouse double minute 2, human homolog of; p53-binding protein).

In addition, the replication capacity of CRAds can be expanded by including a promoter that controls E1 gene expression, which is the necessary for CRAd replication in cells. Table 2 summarizes studies demonstrating the effect of Ad5/F35 vectors containing other promoters, such as TERT, which is the telomerase reverse transcriptase promoter in head and neck cancer in vivo and in vitro.⁹³ Other studies used MKp, which is a midkine minimal promoter, in lung cancer in vitro.⁹⁴ in mesothelioma in vitro⁹⁵ or both in vitro and in vivo,⁹⁶ in bladder cancer in vitro.⁹⁷ and in osteosarcoma in vivo and in vitro.⁹⁸

Those studies demonstrated that Ad5/F35-mediated gene therapy produce stronger anti-tumor effect than Ad5/F35 alone or Ad5-mediated gene therapy.^{89–91,94,97–98} However, some studies^95,96 found Ad5/F35-mediated gene therapy produces a similar anti-tumor effect compared with Ad5-mediated gene therapy in vitro, mainly because the mesothelioma cells in the study expressed a high level of CAR. Interestingly, another study⁹⁶ showed that in high CAR-expressing mesothelioma cell lines, Ad5/F35 mediated gene therapy resulted in similar or even higher levels compared with Ad5 mediated gene therapy.

Thus, Ad5/F35-mediated gene therapy may be a promising antitumor strategy, with dramatic antitumor effects on most cancers, especially those with low levels of CAR.

Ad5/F35 vectors with chemotherapy

Despite the emergence of many new and effective chemotherapeutic agents, a radical cure for cancer remains a great challenge. CRAds combined with chemotherapy have shown promising results, which are summarized in Table 2. Ad5/F35 vectors in combination with chemotherapy produce a more effective anti-tumor effect than Ad5/F35 vectors alone.^99,100 In addition, Ad5/F35 vectors combined with chemotherapy are superior to Ad5 vectors combined with chemotherapy.^99–101 These studies implied that Ad5/F35 vectors combined with chemotherapy are a promising method to treat cancer without increased toxicity because of the lack of cross-resistance between Ad5/F35 vectors and chemotherapeutic drugs.

Ad5/F35 vectors with radiotherapy

The mechanism by which CRAds kill cancer cells is different from that of radiotherapy. This has prompted researchers to study CRAds in combination with radiation. The results of studies on Ad5/F35 vectors combined with radiation are shown in Table 2. In head and neck cancer, Ad5/F35 vectors combined with radiation significantly delayed tumor progression and increased the survival of mice bearing highly aggressive tumors in vivo.¹⁰¹ Another study demonstrated that a combination of Ad5/F35 expressing an APE1 (apurinic/apyrimidinic endodeoxyribonuclease 1) siRNA with radiation significantly enhanced the sensitivity of colorectal cancer cells to irradiation in vitro, and enhanced the inhibition of tumor growth by irradiation in vivo.¹⁰² The above studies also showed that Ad5/F35 vectors combined with radiation produce an enhanced anti-tumor effect compared with than Ad5 vectors combined with radiation. Thus, Ad5/F35 vectors combined with radiation might be an effective method to overcome the resistance of tumor cells to radiation.

Conclusions and perspectives

Compared with traditional Ad5, the novel fiber chimeric adenovirus Ad5/F35 not only binds to CAR-positive cells, but also binds to CAR-negative cells by binding to the Ad35 components, CD46, which is expressed on the surface of most cancer cells. Importantly, Ad5/F35-based vectors have higher safety and lower toxicity compared with Ad5-based vectors. Therefore, Ad5/F35 could be considered as a better choice for tumor therapy compared with Ad5-based vectors. By analyzing the mechanism of the transfer of Ad5/F35-based vectors into cancer cells, we could eliminate the factors that influence the transduction efficacy negatively, thereby increasing the antitumor effect of Ad5/F35-based vectors.

CRAds are a novel anti-tumor approach with confirmed efficacy. As novel antitumor agents, CRAds not only selectively replicate in and lyse cancer cells, but also can enhance the expression and efficacy of therapeutic genes. A number of studies have demonstrated that Ad5/F35-mediated gene therapy has greater anti-tumor effects than Ad5/F35 alone. Chemotherapy agents and radiation are important therapies for cancers. However, resistance to chemotherapeutic agents or radiation restricts their effects. Emerging evidence from preclinical and clinical trials show that Ad5/F35-mediated gene therapy, combined with chemotherapy or radiation, might have complementary or synergistic effects, leading to a greater antitumor effect than either treatment alone.

Thus, considering the characteristics of Ad5/F35-based vectors, we believe that these vectors represent a better therapeutic approach to treat cancer.

Conflict of interest

The authors have no conflicts of interest to declare.

Funding

This work was supported by the National Natural Science Foundation of China [grant numbers 81372916, 81572976], the Science and Technology Department of Jiangsu Province [grant number BK20141142], the China Postdoctoral Science Foundation [grant numbers 2017T100407, 2016M590505], and the Jiangsu Provincial Medical Talent Foundation.