Constructing recombinant replication-defective adenoviral vectors that express glucose transporter-1 through in vitro ligation

BACKGROUND： We constructed a homologous recombination bacterial method based on the pAdEasy system, a widely used system, for generating recombinant adenoviral vectors that express glucose transporter- 1 （GLUT 1） in rats, OBJECTIVE： This study was designed to investigate the feasibility of generating recombinant replication-defective adenoviral vectors that express GLUT1 in rats by in vitro ligation based on the Adeno-X^TM system. DESIGN： An in vitro cell-based experiment. SETTING： This study was performed at the Linbaixin Medical Research Center of the Second Hospital Affiliated to Sun Yat-sen University and Central Laboratory for Prevention and Treatment of Tumor, Sun Yat-sen University between January and August 2004. MATERIALS： Male, adult, Sprague Dawley rats were used to extract total RNA from brain tissue. E. coli DH5 a and human embryonic kidney 293 cells （HEK293 cells） used in the present study were cryo-preserved by the Second Hospital Affiliated to Sun Yat-sen University. Rabbit anti-rat GLUT1 polyclonal antibody （Chemicon, U.S.A.） and primers （Shanghai Boya Bioengineering Co., Ltd） were also used. METHODS： E1/E3-deleted replication-defective adenoviral vectors were used. Using in vitro ligation, the target gene was first sub-cloned into a shuttle vector plasmid to obtain the fragment containing target gene expression cassettes by enzyme digestion. Subsequently, the fragment was co-transformed with linearized adenoviral backbone vector into the E. coli strain. The recombinant adenoviral plasmid was transfected into HEK293 cells to assembly recombinant adenoviral vectors with replication capabilities. The procedure was repeated several times for recombinant adenoviral vectors amplification. MAIN OUTCOME MEASURES： Efficiency of recombinant adenoviral vectors to express the target gene was measured by gene and protein expression through polymerase chain reaction and Western Blot assays, respectively. RESULTS： Results demonstrated that recombinant adenoviral vectors successfully expressed GLUT1 protein, with a relative molecular mass of 55000 in HEK293 cells. These results suggest that recombinant adenoviral vectors obtained by homologous bacterial recombination feature high efficiency, rapidness, and simplicity. CONCLUSION： We successfully amplified the rat GLUT1 gene and constructed replication-defective adenoviral vectors expressing GLUT1. The replication-defective adenoviral vectors proved to successfully express the target gene in HEK293 cells.

Cloning and analysis of a new 4CL-like gene in Populus tomentosa

The phenylpropanoid enzyme 4-coumarate： coenzyme A ligase （4CL）, plays a key role in general phenylpropanoid metabolism. Our investigation involves a new 4CL-like gene cloned from Populus tomentosa. This new 4CL- like gene is 1,692 bp and encodes a protein of 552 aa. After analysis of its domain, we found a highly conserved region of a 4CL gene family in this 4CL-like gene. Based on our results, we believe that this gene belongs to the family of 4CL. A recombinant vector, referred to as pET30a-4CL-like, was constructed by connecting this 4CL-like gene fragment to pET30a. After inducing it with IPTG for 3 h, the 4CL-like protein was purified by a Ni-NTA method. This 4CL-like protein has a calculated molecular weight of 60 kDa by SDS-PAGE.

Antiapoptotic Effect of Gene Therapy with Recombinant Adenovirus Vector Containing Hypoxia-inducible Factor-1α after Cerebral Ischemia and Reperfusion in Rats

Background：Mounting evidence has demonstrated that hypoxia-inducible factor-1α （HIF-1α） could attenuate brain injuries after cerebral ischemia and reperfusion （CIR）.However,few reports have addressed the therapeutic efficacies of a recombinant adenovirus vector containing HIF-1o （AdHIF-1o） gene after ischemia and reperfusion.The aim of this study was to examine the antiapoptotic and neuroprotective effects ofAdHIF-1o gene for cerebral injuries after ischemia and reperfusion in rats.Methods：From February to December 2016,male Sprague-Dawley rats were randomly divided into normal,sham,CIR,AdHIF-1α,and recombinant adenovirus （Ad） groups.Middle cerebral artery occlusion model was established by Longa＇s method and reperfusion resumed at 2 h postocclusion.AdHIF-1α solution,Ad solution,and phosphate-buffered saline were injected into the right lateral ventricle of rats in AdHIF-lα,Ad,and CIR groups.Brain tissue sections were observed under fluorescent microscope to confirm the definite expression of recombinant adenovirus in Ad and AdHIF-1o groups.The expressions of HIF-lα protein were analyzed by immunohistochemical staining at 6 h,24 h,and 72 h postreperfusion.Brain water content and neurological deficit scores were evaluated at 6 h,24 h,and 72 h postreperfusion.Pathological brain injuries were examined after hematoxylin and eosin stain and nerve cell apoptosis was measured after terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling （TUNEL） stain at 72 h postreperfusion.Comparisons were conducted with one-way analysis of variance by post hoc Scheffe＇s test among different experimental groups.Results：Green fluorescent protein was successfully expressed in brain tissue ofAd andAdHIF-1α groups from 24 h to 21 days postinjection.As detected by immunohistochemical staining,the expressions of HIF-lα protein were obviously enhanced in AdHIF-1o group than those in CIR and Ad groups at 24 h and 72 h postreperfusion,respectively.There were significant reductions of brain water content （78.83% ± 0.34% vs.83.21% ± 0.50% and 83.35% ± 0.32%;84.13% ± 0.24% vs.89.76% ± 0.34% and 89.70% ± 0.18%;respectively;all P 〈 0.05） and neurological deficit scores （2.90 ± 0.74 vs.3.50 ± 0.52 and 3.60 ± 0.53 at 24 h;2.40 ± 0.84 vs.3.60 ± 0.52 and 3.50 ± 0.53 at 72 h;respectively;all P 〈 0.05） in AdHIF-1 α group versus CIR and Ad groups at 24 h and 72 h postreperfusion,respectively.The pathologic changes ofAdHIF-1 α group were milder than those in CIR and Ad groups at 72 h postreperfusion.The percentage of TUNEL-positive cells in cerebral subcortex decreased significantly in AdHIF-1α group versus CIR and Ad groups at 72 h postreperfusion （P 〈 0.05）.Conclusion：AdHIF-1α has an obvious neuroprotective effect on ischemia and reperfusion in rat brains possibly through inhibiting the apoptosis of nerve cells.

Transfection of the glial cell line-derived neurotrophic factor gene promotes neuronal differentiation

Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, micro- tubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the su- pernatant were significantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mes- enchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein743 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesen- chymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.

Protective effects of ciliary neurotrophic factor on the retinal ganglion cells by injure of hydrogen peroxide

AIM： To explore the effect of ciliary neurotrophic factor （CNTF） on retinal ganglion cell （RGC）-5 induced by hydrogen peroxide （H2O2）. METHODS： After cell adherence, RGC-5 culture medium was changed to contain different concentrations of H2O2 from 50 to 150 μmol/L at four time points （0.5, 1, 1.5 and 2h） to select the concentration and time point for H2O2 induced model. Two different ways of interventions for injured RGC-5 cells respectively were CNTF as an addition in the culture medium or recombinant lentiviral plasmid carrying CNTF gene transfecting bone mesenchymal stem cells （BMSCs） for co-culture with RGC-5. RESULTS： Compared to the control group, H2O2 led to RGC-5 death closely associated with concentrations and action time of H2O2 and we chose 125 μmol/L and 2h to establish the H2O2-induced model. While CNTF inhibited the loss of RGC-5 cells obviously with a dose-dependent survival rate. Nevertheless two administration routes had different survival rate yet higher rate in recombinant lentiviral plasmid group but there were no statistically significant differences. CONCLUSION： Both the two administration routes of CNTF have effects on RGC-5 cells induced by H2O2. If their own advantages were combined, there may be a better administration route.

Inhibition of vascular smooth muscle cell proliferation by in troduction of retinoblastoma gene via a recombinant adenovirus vector

This study was supported in part by grant from National Natural Science Foundation of China (No. 39570775). Objective To investigate the vascular smooth muscle cell (SMC) growth suppression by recombinant adenovirus vector expressing a retinoblastoma (Rb) protein and to explore a gene therapy approach for vascular proliferative disorders including atherosclerosis and artery restenosis. Methods A replication deficient adenovirus vector encoding a wild type Rb and AdCMVRb, was constructed and transfected into cultured rabbit aortic SMC. The efficiency of gene transfection and expression was detected by immunochemical staining and polymerase chain reaction. The role of Rb in regulating vascular SMC proliferation was observed by cell counting, thymidine incorporation, and flow cytometry. Results Wild type Rb gene transfected effectively into the cultured SMC with AdCMVRb can suppress growth factor stimulated cell proliferation through regulation of DNA synthesis and cell cycle progression. Conclusion The results demonstrate the potential of adenovirus mediated Rb gene therapy for atherosclerosis and artery restenosis after balloon angioplasty.

Preparation of a recombinant adeno-associated viral vector with a mutation of human factor-Ⅸin large scale and its expression in vitro and in vivo

A series of adeno-associated viral vectors containing a mutation of human factor Ⅸ (hFⅨR338A) with different regulation elements were constructed and used to transduce cell lines. The plasmids and the stable transduction cell clones with high expression level of hFⅨR338A were obtained by selecting and optimizing, and then, the recombinant adeno-associated viral vector with hFⅨR338A was prepared via novel rHSV/AAV hybrid virus packaging system on a large scale, which contained the capsid protein genes. A method for producing rAAV-hFⅨR338A viral stocks on a large scale and higher titer was established, which can be used for industrial purpose. The titer of rAAV-hFⅨR338A was more than 1.25?012 particle/mL, and then, a mammalian cell line, C2C12 and the factor Ⅸ knock-out mice were transfected with the rAAV-hFⅨR338A in vitroand in vivo. The results show that the high-level expression of rAAV-hFⅨR338A was achieved in cell line and hemophilia B mice. It reached at (2551.32±92.14) ng·(106 cells)-1·(24h)-1 in C2C12 cell in vitro and had a peak concentration of 463.28 ng/mL in mice treated with rAAV-hFⅨR338A, which was as high as the expression of rAAV-hFⅨ-wt(2565.76?4.36) ng·(106 cells) -1·(24 h)-1 in C2C12 and453.92 ng/mL in the mice treated with rAAV-hFⅨ-wt) in vitro and in vivo, there is no any difference between two groups, but the clotting activity of hFⅨR338A is about 2.46 times higher than that of hFⅨ-wt. It was first reported that a mutation of human factor Ⅸ was used into gene therapyresearch for hemophilia B, meanwhile, a novel packagingsystem, rAAV/HSV was used for preparation of rAAV-hFⅨR338A on a large scale, which laid the foundation ofindustrial production for applying rAAV viral stocks to gene therapy clinical trial for hemophilia B mediated withrAAV-hFⅨ.

Isolation and cultivation of murine hematopoietic stem cells and expression of hFIX mediated by recombinant lentiviral vectors in vitro

Hematopoietic stem cells(HSCs)are an attractive target for gene therapy,especially for inherited blood diseases.Moreover,recombinant lentiviral vectors are considered to be prospective in HSCs gene therapy for the high efficiency of infection.In this study,murine mononuclear cells(MNCs)were isolated from bone marrow and cultured in suspension,and then Lin−CD117+HSCs were isolated by immunomagnetic beads.During culturing,cells and colonies increased in HSCs supplied with cytokines while no change was observed in the control group without cytokines.FUXW recombinant lentiviral vectors were produced by calcium phosphate-mediated transient cotransfection infected MNCs from ICR and C57 mice.The hFIX expressions were 41.7±4.2 ng/mL and 34.5±6.6 ng/mL in supernatant on 7d.The hFIX expressions of HSCs infected by FUXW recombinant lentiviral vectors were 46.6±5.7 ng/mL(with cytokines)and 33.3±4.8 ng/mL(without cytokines)in supernatant on 7d.Results indicate that recombinant lentiviral vectors can infect murine MNCs and Lin−CD117+HSCs efficiently,and expression of the transgene can be improved when supplied with cytokines.

Experimental study of human umbilica cord blood stromal cells transfected with recombinant adenoviral vector co-expressing VCAM-1

This work was funded from National Natural Science Foun-dation of China(No.39770335 and No.30070327)andChongqing Medical Key construction Foundation.Abstract:Ob-jective To establish the experimental foundation for furtherstudying the effect of VCAM-1 gene modified HUCBSCs

Prevalence of neutralizing antibodies against liver-tropic adeno-associated virus serotype vectors in 100 healthy Chinese and its potential relation to body constitutions

Recombinant adeno-associated virus （rAAV） serotype 2, 3 and 8 vectors are the most promising liver- tropic AAV serotype vectors. Liver diseases are significant problems in China. However, to date, few studies on AAV neutralizing antibodies （Nabs） were working with the Chinese population or with the rAAV3 vectors. The present study aimed to determine the prevalence of Nabs in Chinese population against wild-type AAV2, AAV3 and AAV8 capsids as well as additional two AAV3 variants. In addition, we performed a preliminary analysis to investigate the potential influence of traditional Chinese medicine body constitutions on AAV Nabs. Our work demonstrated that the majority of healthy Chinese subjects were positive for AAV Nabs, with the order of AAV2 〉 AAV3 = AAVLK03 〉 AAV8. There was no difference between： 1）AAV3 and its variants; 2） male and female subjects; and 3） different age cohorts （〈 35, 36- 50, and 〉 51 years old）. People in the Qi-deficiency constitution had significantly increased AAV8 Nabs than people in the Gentleness constitution. Our studies may have impact on the future clinical design of AAV-based gene therapy in the Chinese population.

不用接头进行异末端DNA重组的方法

用过渡载体通过两次重组进行不同粘性末端之间的ＤＮＡ重组．该方法不需要替换接头，从而省去昂贵的试剂和平末端连接这种较难的操作，既经济，简单又有效，易于操作．

Cytotoxic genes from traditional Chinese medicine inhibit tumor growth both in vitro and in vivo

OBJECTIVE： Little effort has been made to study the protein-encoding genes isolated from traditional Chinese medicine（TCM） drugs, and the delivery of these genes into malignant cells through recombinant adeno-associated virus（r AAV） vectors has not been attempted. METHODS： We synthesized the c DNAs of five known cytotoxic proteins isolated from TCM drugs and the FLAG epitope-tagged c DNAs were subcloned into a r AAV plasmid vector. The protein expression was confi rmed by Western blot assay. Various cancer cell lines were transfected with the above plasmids and cell growth was monitored both in vitro and in vivo. The best cytotoxic gene was further packaged into r AAV vectors, under the control of a liver cancer-specifi c promoter. The liver tumor growth was then monitored following intratumor administration of the r AAV vectors.RESULTS： The expression plasmids, encoding individual potential cytotoxic genes tagged with FLAG epitope, were successfully generated and sequenced. Among these genes, trichosanthin（TCS） gene yielded the most promising results for the inhibition of cancer cell growth in vitro. The over-expressed TCS functioned as a type I ribosome-inactivating protein, followed by inducing apoptosis that is associated with the Bcl-PARP signaling pathway. Furthermore, intratumor injection of r AAV vectors containing the TCS gene signifi cantly inhibited the growth of human hepatocellular carcinoma tumors in a murine xenograft model.CONCLUSION： Our studies suggest that the use of TCM cytotoxic genes is a useful therapeutic strategy for treating human cancers in general, and liver tumors in particular.

The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy

Objective:Plant-derived cytotoxic transgene expression,such as trichosanthin(tcs),regulated by recombinant adeno-associated virus(r AAV)vector is a promising cancer gene therapy.However,the cytotoxic transgene can hamper the vector production in the r AAV producer cell line,human embryonic kidney(HEK293)cells.Here,we explored micro RNA-122(miR122)and its target sequence to limit the expression of the cytotoxic gene in the r AAV producer cells.Methods:A miR122 target(122 T)sequence was incorporated into the 30 untranslated region of the tcs c DNA sequence.The firefly luciferase(fluc)transgene was used as an appropriate control.Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells.The effects of miR122 overexpression on cell growth,transgene expression,and r AAV production were determined.Results:The presence of 122 T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line(in vitro),fresh human hepatocytes(ex vivo),and mouse liver(in vivo).Also,the normal liver physiology was unaffected by delivery of 122 T sequence by r AAV vectors.Compared with the parental cells,the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122 T,as well as the ability to produce liver-targeting r AAV vectors.Fascinatingly,the yield of r AAV vectors carrying the tcs-122 T gene was increased by 77.7-fold in HEK293-mir122 cells.Moreover,the tcs-122 T-containing r AAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells.Conclusion:HEK293-mir122 cells along with the 122 T sequence provide a potential tool to attenuate the cytotoxic transgene expression,such as tcs,during r AAV vector production.

Recent development and gene therapy for glycogen storage disease type Ia

Glycogen storage disease type Ia(GSD-Ia)is an autosomal recessive metabolic disorder caused by a deficiency in glucose-6-phosphatase-α(G6Pase-αor G6PC)that is expressed primarily in the liver,kidney,and intestine.G6Pase-αcatalyzes the hydrolysis of glucose-6-phosphate(G6P)to glucose and phosphate in the terminal step of gluconeogenesis and glycogenolysis,and is a key enzyme for endogenous glucose production.The active site of G6Pase-αis inside the endoplasmic reticulum(ER)lumen.For catalysis,the substrate G6P must be translocated from the cytoplasm into the ER lumen by a G6P transporter(G6PT).The functional coupling of G6Pase-αand G6PT maintains interprandial glucose homeostasis.Dietary therapies for GSD-Ia are available,but cannot prevent the long-term complication of hepatocellular adenoma that may undergo malignant transformation to hepatocellular carcinoma.Animal models of GSD-Ia are now available and are being exploited to both delineate the disease more precisely and develop new treatment approaches,including gene therapy.