Vector-mediated Gene Research Articles

Vector-mediated cancer gene therapy: A review

Gene therapy is the transfer of genetic material to cure a disease or at least to improve the clinical status of a patient. One of the basic concepts of gene therapy is to transform viruses into genetic shuttles, which will deliver the gene of interest into the target cells. Safe methods have been devised to do this, using several viral and non-viral vectors. Two main approaches emerged: in vivo modification and ex vivo modification. Retrovirus, adenovirus, adenoassociated virus are suitable for gene therapeutic approaches which are based on permanent expression of the therapeutic gene. Non-viral vectors are far less efficient than viral vectors, but they have advantages due to their low immunogenicity and their large capacity for therapeutic DNA. The most commonly used DNA virus vectors are based on adenoviruses and adeno-associated viruses. An example of gene-knockout mediated gene therapy is the knockout of the human CCR5 gene in T-cells in order to control HIV infection. To improve the function of non-viral vectors, the addition of viral functions such as receptor mediated uptake and nuclear translocation of DNA may finally lead to the development of an artificial virus. Gene transfer protocols have been approved for human use in inherited diseases, cancers and acquired disorders. Although the available vector systems are able to deliver genes in vivo into cells, the ideal delivery vehicle has not been found. Thus, the present viral vectors should be used only with great caution in human beings and further progress in vector development is necessary.

Let-7f microRNA expression within established bladder cancer subtypes and upregulation with recombinant interferon-alpha.

462 Background: Let7f is a microRNA (miR) that may act as a tumor suppressor in multiple human malignancies. In patients with BCG-refractory non-muscle invasive bladder cancer, detectable let7f levels in the urine of patients undergoing treatment with adenoviral vector-mediated IFNα gene therapy (clinical trial setting) were found to be prognostic of treatment response at 12 months. Here we sought to define the association of let7f with clinical bladder cancer phenotypes within The Cancer Genome Atlas (TCGA) cohort and characterize the role of let7f in preclinical models. Methods: Using miRNA sequencing data from the published TCGA 2018 bladder cohort (n = 412), let7f miR expression was characterized and stratified by molecular subtype (basal-squamous, luminal, luminal-infiltrated, luminal-papillary, and neuronal). Kaplan-Meier analyses were performed to compare survival (upper vs lower median, by let7f expression level). The murine urothelial carcinoma cell line MB49 was cultured in colonies of 105 and in vitro administrations of 100U/mL of recombinant mu-IFNα were performed with cells harvested at 0, 2, 8, 12, and 24 hours. Let7f miR expression levels were then determined by qRT-PCR. Results: Within the TCGA cohort, let7f expression levels were found to be lower in the more aggressive basal-squamous (median = 12.8), luminal-infiltrated (median = 12.9), and neuronal (median = 12.4) subtypes relative to the luminal (median = 13.2) and luminal-papillary (median = 13.3) subtypes (p < 0.001). Survival analysis demonstrated a trend towards improved survival in the high let7f expressers, but this did not reach statistical significance. In MB49 cells treated with mu-IFNα, there was a time-dependent increase in let7f miR expression suggesting that IFNα may play a role in upregulation of this potential tumor suppressor. Conclusions: Let7f miR is downregulated in more aggressive subtypes of MIBC. IFNα upregulates let7f expression in MB49 cells, and the inability to do so may comprise an escape mechanism for IFNα-treated tumors. Let7f miR may serve as a novel biomarker to identify aggressive phenotypes and treatment failures for bladder tumors treated with IFNα-based gene therapy.

Mitigation of premature ovarian failure by over-expression of lentivirus vector-mediated Wilms tumor-suppressor gene

Purpose: To investigate the effect of WT1(Wilms tumor-suppressor gene) overexpression on premature ovarian failure (POF)-mediated ovarian dysfunction. Methods: Three mice groups were used: control group (untreated mice), POF group (mice sterilized by intravenous injection of cyclophosphamide and busulfan), and POF-LV(lentiviral vector)􀀀GFP(green fluorescent protein)􀀀WT1 group (POF mice given intra-ovarian microinjection of LV􀀀GFP􀀀WT1, a WT1􀀀overexpressing lentiviral vector, one week after sterilization). Real time-PCR was employed to analyze in vitro WT1 overexpression levels. Overall ovarian function was measured by hormonal assay, H & E staining, and immuno-histochemical techniques. Results: Overexpression of WT1 in mice models of POF alleviated ovarian granulosa cell (GC) damage, increased ovary weight, and significantly increased follicular number (p < 0.05). Radioimmunoassays revealed reduction in plasma estradiol (E2) and follicle-stimulating hormone (FSH, p < 0.05). However, results from immune-histochemical assays showed reduced Bax expression levels, and increased expression of Bcl-2 in WTI-overexpression mice, relative to POF mice. Conclusion: Overexpression of WT1 may stimulate repair of ovarian tissue while improving endocrine function by inhibiting ovarian cell apoptosis signaling pathway in POF mice. Keywords: Premature ovarian failure, Wilms tumor suppressor gene, Chemotherapy, Apoptosis, Estradiol

GW26-e0495 Impact of Myocardial Remodeling in a Rat Model of Heart Failure Transfected with Recombinant Adenovirus Vector-mediated Klotho Gene

To assess the impact of Transfection with recombinant adenovirus vector-mediated Klotho gene on myocardial remodeling in a rat model of heart failure (HF) by intraperitoneal injection of isoproterenol. In addition, we approach to the possible mechanisms how Klotho gene repair the myocardial injury

Suppression of lentiviral vector-mediated mTOR gene silencing on growth of human lens epithelial cell in vitro

Background Mammalian target of raparrmycin (mTOR) acts as a critical molecular link between growth signals and the processes underlying cell growth. The overexpression of mTOR in human lens epithelial cells (HLECs) is associated with posterior capsular opacification (PCO). Applying the mTOR shRNA transfection technique to silence mTOR in HLECs is significant for the targeting treatment of PCO. Objective This study was to investigate the function and morphology of HLECs infected by mTOR shRNA lentiviral vector. Methods 293T cells were transfected by recombinant plasmid containing mTOR shRNA, blank plasmid and PBS to prepare different lentiviral vectors. HLECs were routinely cultured and were divided into the mTOR shRNA transfected 1, 2, 3 groups, blank plasmid group and PBS group, and different lentiviral vectors were added in the medium to infect the cells. The expression of mTOR protein in HLECs was detected using Western blot to determine mTOR gene silence, and the group with the highest efficiency of silence was screened as the sequent experiment group. The proportion of HLECs in the G1 phase was analyzed by flow cotymetry. The expressions of α-smooth muscle actin (α-SMA) and E-Cadherin proteins in the cells were assayed by Western blot. The viability of HLECs was assessed by cell counting kit-8 (CCK8) test. Results Successfully packaged lentivirus were found in the supernatant after infection of mTOR shRNA in the mTOR shRNA transfected 1, 2, 3 groups with the green fluorescence in >80% cells, while green fluorescence protein (GFP) response was absent in the blank plasmid group and the PBS group. The HLECs of mTOR shRNA transfected groups and blank plasmid group grew well due to the resistance for puromycin of plasmid, however, more dead cells were found in the PBS group. Western blot revealed the weakest expression of mTOR mRNA in the cells in the mTOR shRNA transfected 3 group. The expressing bland of α-SMA in the HLECs was weaker in the mTOR shRNA transfectd 3 group than that in the blank plasmid group or PBS group, and the expression of E-Cadherin was stronger in in the mTOR shRNA transfectd 3 group than that in the blank plasmid group or PBS group. The absorbance of the mTOR shRNA transfectd 3 group was significantly declined in comparison with the blank plasmid group and the PBS group, with significant difference among the three groups in both 1 hour and 2 hours after experiment (all at P<0.05). The proportion of cells in G1 phase was (60.00±1.78)% in the mTOR shRNA transfected 3 group, which was significantly increased in comparison with (53.48±1.86)% in the blank plasmid group and (53.02±1.49)% in the PBS group (F=18.910, P=0.002). Conclusions mTOR gene is silenced after mTOR shRNA plasmid transfected into HLECs. This procedure can invert the epithelial-mesenchymal transition (EMT), inhibit cell growth and put off cellular cycle in HLECs in vitro. Key words: Lentiviral vector; Short hairpin RNA; Posterior capsule opacification; Mammalian target of raparrmycin; Epithelial-mesenchymal transition; Lens, crystal; Epithelial cells; Gene silence

P1213 : Adeno-associated virus vector-mediated liver gene therapy for Crigler-Najjar syndrome

P1213 : Adeno-associated virus vector-mediated liver gene therapy for Crigler-Najjar syndrome

PD20-05 HERPES SIMPLEX VIRUS (HSV) VECTOR-MEDIATED GENE DELIVERY OF PROTEIN PHOSPHATASE 1α REDUCES BLADDER OVERACTIVITY AND

INTRODUCTION AND OBJECTIVES: Increased afferent excitability has been proposed as an important pathophysiological basis of overactive bladder (OAB) and hypersensitive bladder disorders such as interstitial cystitis/bladder pain syndrome (IC/BPS). Previous studies reported that transient receptor potential vanilloid-1 (TRPV1) receptors greatly contribute to afferent sensitization. We recently found Protein Phosphatase 1a (PP1a) as a modulator of TRPV1 receptor activity using an HSV vector cDNA library screen method to identify genes that antagonize TRPV1 activation. HSV vector-mediated PP1a expression significantly reduced capsaicin-induced thermal hyperalgesia. Therefore, we investigated the effect of HSV vectors-mediated gene delivery of PP1a on bladder overactivity and pain-related behavior in rats. METHODS: Replication-deficient HSV vectors encoding PP1a or green fluorescent protein (GFP) as control were injected into the bladder wall of adult female Sprague-Dawley rats. Cystometry (CMG) under urethane anesthesia was performed 1 week after viral injection to evaluate bladder overactivity induced by resiniferatoxin (RTX, a TRPV1 agonist). RTX-induced nociceptive behavior such as licking (lower abdominal licking) and freezing (motionless headturning) was observed 2 weeks after viral innoculation. GFP expression in the L6/S1 DRG and the bladder as well as c-Fos positive cells in the L6 spinal cord dorsal horn were evaluated. The expression of p-ERK, p-Inositol trisphosphate receptor (p-IP3R), and p-TRPV1 in the bladder was estimated by Western blot. RESULTS: GFP expression was seen in L6/S1 DRG as well as in the bladder. In CMG, the PP1a group showed a significantly (p1⁄40.03) smaller reduction (46 1%) in intercontraction intervals after RTX infusion than the GFP group (64 2%). The number of RTX-induced freezing behavior, which is correlated with bladder pain sensation, was significantly (p<0.001) decreased in the PP1a vs. GFP groups. The number of c-Fos positive cells in the L6 spinal dorsal horn was significantly (p<0.01) smaller in the PP1a vs. the GFP group (33 4 vs. 59 6 cells per section). Western blot revealed that p-ERK, p-IP3R, and p-TRPV1 was less expressed in the PP1a vs. the GFP group (p1⁄40.07, 0.01, and 0.04, respectively) CONCLUSIONS: These results indicate that HSV vectorsmediated gene delivery of PP1a can suppress TRPV1-mediated bladder overactivityandpainbehavior, possibly due todecreasedphosphorylation of TRPV1 channels. Thus, PP1a gene thetapy could be a novel treatment of OAB and/or hypersensitive bladder disorders such as IC/BPS.

Impact of transfection with recombinant adenovirus vector-mediated Klotho gene on myocardial remodeling in a rat model of heart failure

To assess the impact of transfection with recombinant adenovirus vector-mediated Klotho gene on myocardial remodeling in a rat model of heart failure (HF) by intraperitoneal injection of isoproterenol. Rats were divided into 5 groups by table of exponential random numbers: normal control group, HF group, saline-control HF group, recombinant adenovirus vector transfection group (Ad.EGFP group, 2 × 10¹⁰ pfu, 0.5 ml/rat), pDC316-CMV-EGFP-rKlotho transfection group (Ad.Klotho group, n=5 each). Left ventricular ejection fraction (LVEF) was obtained by echocardiography, hemodynamic parameters obtained by multi-channel physiological recorder, myocardial tissue underwent pathohistological examination. Additionally, the green fluorescin expression was observed on frozen heart section. Myocardial fibrosis correlated gene expression including Klotho gene, collagen I and III was detected by real time-PCR. Moreover, plasma levels of B-type natriuretic peptide (BNP) were measured with ELISA. Compared to saline control HF group, LVEF, LVSP and ±dp/dtmax were significantly increased, myocardial fibrosis and myocardial remodeling were significantly attenuated in the Ad. Klotho group and there was green fluorescin distribution in myocardial tissues of Ad. Klotho group. Klotho expression was down-regulated and collagen I and III expression was upregulated in HF rats compared to normal control group (all P<0.05) and these changes could be significantly reversed in Ad. Klotho group (all P<0.05). Plasma BNP level was also significantly lower in Ad. Klotho group than in HF group (P<0.05). Klotho gene transfection could improve cardiac function and attenuate cardiac remodeling and reducing myocardial fibrosis.

A novel hybrid baculovirus-adeno-associated viral vector-mediated radionuclide reporter gene imaging system for stem cells transplantation monitoring.

Hybrid baculovirus-adeno-associated virus (BV-AAV) containing enhanced green fluorescent protein (eGFP) reporter gene or human sodium-iodide symporter (hNIS) reporter gene flanked by inverted terminal repeats (ITRs) derived from AAV (BV-CMV-eGFP-ITR and BV-CMV-hNIS-ITR) were constructed and used to investigate the feasibility of using hybrid BV-AAV transgenic vector to mediate hNIS reporter gene imaging for monitoring bone marrow-derived mesenchymal stem cells (BM-MSCs) transplantation therapy as a novel biotechnological platform in radionuclide reporter gene imaging. The results showed that the infection efficiency of BV-CMV-eGFP-ITR in BM-MSCs reached 84.25 ± 1.38%, and there were no obvious adverse effects on BM-MSCs. The (125)I(-) and (99m)TcO₄(-) uptake assays showed that the radionuclide accumulation induced by BA-AAV-mediated hNIS was highly efficient in infected BM-MSCs. Furthermore, there was a robust correlation between the infected BM-MSCs cell number and the (125)I(-) accumulation amount (R(2) = 0.9026). The micro-SPECT/CT imaging showed that BV-CMV-hNIS-ITR-infected BM-MSCs accumulated radioiodine efficiently in vivo, exhibiting obvious radiotracer accumulation in transplantation sites. Further quantitative analysis revealed that 30 min might be the optimal imaging time point. Moreover, the revealed high target/individual organ background ratios also supported the feasibility of BV-AAV-mediated hNIS reporter gene imaging for monitoring BM-MSCs transplantation in most of commonly used transplantation sites, thus highlighting this promise biotechnological platform in radionuclide reporter gene imaging for stem cell transplantation therapy.

Intrastriatal gene delivery of GDNF persistently attenuates methamphetamine self-administration and relapse in mice

Relapse of drug abuse after abstinence is a major challenge to the treatment of addicts. In our well-established mouse models of methamphetamine (Meth) self-administration and reinstatement, bilateral microinjection of adeno-associated virus vectors expressing GDNF (AAV-Gdnf) into the striatum significantly reduced Meth self-administration, without affecting locomotor activity. Moreover, the intrastriatal AAV-Gdnf attenuated cue-induced reinstatement of Meth-seeking behaviour in a sustainable manner. In addition, this manipulation showed that Meth-primed reinstatement of Meth-seeking behaviour was reduced. These findings suggest that the AAV vector-mediated Gdnf gene transfer into the striatum is an effective and sustainable approach to attenuate Meth self-administration and Meth-associated cue-induced relapsing behaviour and that the AAV-mediated Gdnf gene transfer in the brain may be a valuable gene therapy against drug dependence and protracted relapse in clinical settings.

Inhibition of Lnk in Mouse Induced Pluripotent Stem Cells Promotes Hematopoietic Cell Generation

Embryonic stem (ES) cell- and induced pluripotent stem (iPS) cell-derived hematopoietic stem/progenitor cells (HSPCs) are considered as an unlimited source for HSPC transplantation. However, production of immature hematopoietic cells, especially HSPCs, from ES and iPS cells has been challenging. The adaptor protein Lnk has been shown to negatively regulate HSPC function via the inhibition of thrombopoietin (TPO) and stem cell factor signaling, and Lnk-deficient HSPCs show an enhanced self-renewal and repopulation capacity. In this study, we examined the role of Lnk on the hematopoietic differentiation from mouse ES and iPS cells by the inhibition of Lnk using a dominant-negative mutant of the Lnk (DN-Lnk) gene. We generated mouse ES and iPS cells stably expressing a DN-Lnk, and found that enforced expression of a DN-Lnk in ES and iPS cells led to an enhanced generation of Flk-1-positive mesodermal cells, thereby could increase in the expression of hematopoietic transcription factors, including Scl and Runx1. We also showed that the number of both total hematopoietic cells and immature hematopoietic cells with colony-forming potential in DN-Lnk-expressing cells was significantly increased in comparison with that in control cells. Furthermore, Lnk inhibition by the overexpression of the DN-Lnk gene augmented the TPO-induced phosphorylation of Erk1/2 and Akt, indicating the enhanced sensitivity to TPO. Adenovirus vector-mediated transient DN-Lnk gene expression in ES and iPS cells could also increase the hematopoietic cell production. Our data clearly showed that the inhibition of Lnk in ES and iPS cells could result in the efficient generation and expansion of hematopoietic cells.

Retroviral vector-mediated hypoxia-regulated neurotrophin-3 gene transfer reduces apoptosis induced by hypoxia in PC12 cells

Background Gene therapy for ischemic diseases is a prospective strategy. However, excessive expression of therapeutic genes may produce undesired side effects. Recently, multiple copies hypoxia response elements (HRE) were developed to conditionally regulate gene expression under hypoxia. As a nerve growth factor, Neurotrophin3 (NT-3) possesses neural protect effects either in vitro or in vivo. To explore hypoxia-controlled NT-3 expression, we constructed a recombinant retrovirus vector with 5HRE and NT-3, and generated a gene transferred cell line PC12-5HRE-NT3 to determine effects of conditionally expressed NT-3 on apoptosis induced by hypoxia in PC12 cells.

Reconstruction of corneal surface layer using human amniotic epithelial cells modified by lentiviral vector-mediated EGFP gene

Background Studies demonstrated that human amniotic epithelial cells (AECs) have some characteristics of embryonic stem cells and they were used to re-establish the surface of eyes. Human AECs may serve as new seed cells in tissue engineering for corneal epithelium reconstitution in the future. Objective The present study was to investigate the application value of human amniotic epithelium cells transfected by lentiviral vectormediated enhanced green fluorescent protein (EGFP) gene as new seed cell source for engineering the corneal surfacelayer. Methods Lentiviral vector carrying the objective gene EGFP was transfected into human amniotic epithelial cells (pLenti6/V5-DEST),and the transient expression of the transgene in the human amniotic epithelial cells was observed under the fluorescence microscope. Flow cytometry was used to detect the positive expression rates of EGFP in transfected cells. The transfected human amniotic epithelial cells were seeded onto the fresh corneal stromal surface of New Zealand white rabbit and cultured in vitro. The stem cell deficiency ( SCD ) models were established by cutting off the limbus of cornea in 20 eyes of New Zealand white rabbits, and the model rabbits were then divided into 2 groups randomly. The transplanted grafts carrying the pLenti6/V5-DEST-EGFP gene-transferred human amniotic epithelium cells were regarded as the pLenti6/V5-DEST-EGFP group, and the corneal stroma graft without any epithelial cell served as the control group. The opacity of stroma and corneal conjunctivalization and vascularization were observed daily. The rabbits' eyes were extracted one month after operation. The expression of EGFP in the cornea was detected under the fluorescence microscope, and the expression of CK8, CK18 and CK12 in cornea was detected by immunohistochemical staining. Results The shape of the transferred human amniotic epithelial cells resembled normal human amniotic epithelial cells. 48 hours after the transient transfection of EGFP presented with the highest expression level throughout the observation duration, with a positive expression rate of EGFP of 61.5% ,showing significant differences in comparison with that of 12 ( 5.24% ) , 24 ( 38.27% ) or 96 ( 39. 10% ) hours ( P ＜0. 05) post-transfection; but no obvious difference was found in the positive rate of transiently transfected EGFP between 48 hours and 72 hours ( 58.36% ) ( P＞0. 05 ). Six cornea grafts were clear in 1 month and two corneas were rejected during the observation period in the pLenti6/V5-DEST-EGFP group. A few new blood vessels were seen around the graft. Ten corneas of the control group became opaque and cloudy with new blood vessels growth around the grafts. Imunohistochemistry revealed the positive expressions of CK8, CK1 8 and CK12 in the corneal epithelial layer in the pLenti6/V5-DEST-EGFP group. However,the expression of CK12 was absent in the control group. Conclusion Human amniotic epithelium cells transfected with the pLenti6/V5-DEST-EGFP gene is a new and ideal feed cell type to reconstruct the corneal surface layer. Lentivirus is a relatively safe gene transfection vector. Key words: Lentiviral vector; Enhanced green fluorescent protein; Gene transfection; Human amniotic epithelial cells; Corneal transplantation

Extending the Activities of Human Nucleus Pulposus Cells with Recombinant Adeno-Associated Virus Vector-Mediated Human Telomerase Reverse Transcriptase Gene Transfer

Even though human nucleus pulposus (HNP) cells have a limited replicative lifespan in vitro culture, the lifespan of ovine nucleus pulposus cells transferred by lipofectamine with human telomerase reverse transcriptase (hTERT) gene could be prolonged. However, the lipofectamine is a transient expression system with an expressed duration of hTERT for less than 73 days. Here we present a viral vector system to investigate whether recombinant adenoassociated virus vector-mediated hTERT gene (rAAV-hTERT) could safely prolong the activities of HNP cells. Our hypothesis was that the activities of HNP cells could be extended with rAAV-hTERT gene transfer. Therefore, we designed an in vitro and in vivo evaluation of a HNP cells gene transfectant. Second-generation HNP cells were transfected with rAAV-hTERT and the expression of hTERT determined by reverse transcription-polymerase chain reaction (RT-PCR) and western blot. Cumulative population doubling, senescence assay, real-time RT-PCR, and enzyme-linked immunosorbent assay were used to determine cellular activities. Genetic phenotype was validated by karyotypic analysis and ontogenicity by nude mice injected in vivo. The continuing expression of hTERT gene and production of extra cellular matrix for 120 days were found in the transfected HNP cells, and karyotypic instability was detected, but there was no evidence of polyploidy or oncogenicity in nude mouse. The results showed that this transfection model might provide a resource cells necessary for the biological treatment of degenerative disc disease.

Effect of short-hairpin RNA expression vector-mediated osteopontin RNA interference on proliferation and invasion of prostate cancer PC-3 cells

Studies showed that osteopontin (OPN) regulates cell migration and invasion in a variety of cancers, which associates with the activities of matrix metalloproteinase (MMP)-2 and MMP-9. This study was to investigate the role of OPN in the proliferation and invasion of human prostate cancer PC-3 cells and the possible functions of IgammaB kinase (IKK) in nuclear factor kappa B (NF-kappaB)-mediated signaling pathways. OPN short-hairpin RNA (shRNA) recombinant plasmids were transfected into PC-3 cells and different concentrations of IKK inhibitors were used to inhibit the activities of IKKalpha and IKKbeta. The mRNA and protein expression levers of OPN, MMP-2, and MMP-9 were detected by real-time polymerase chain reaction (PCR) and Western blot. Cell cycle was detected by flow cytometry, cell proliferation by MTT assay, and cell invasion by Transwell chamber assay. Compared with untreated cells, the protein levers of OPN, MMP-2, and MMP-9 in OPN shRNA-transfected PC-3 cells were reduced by 55.22%, 51.71%, and 28.35%, respectively, and the ability of cell migration and invasion were decreased by 45.48% and 51.96%, respectively (P<0.05). Moreover, the inhibition of IKKbeta inhibited the expressions of MMP-2 and MMP-9. A shRNA expression vector-mediated OPN gene silencing can inhibit the malignant biological behaviors of PC-3 cells. IKKbeta may play a crucial role in the OPN-induced activation of MMP-2 and MMP-9 via NF-kappaB-mediated IkappaB/IKKbeta pathways.

Adenovirus-mediated vascular endothelial growth factor165 gene therapy in treatment of hypoxic-ischemic brain damage: experiment with rats

To investigate the therapeutical effect of adenovirus-mediated vascular endothelial growth factor (VEGF)165 gene transplantation in treatment of hypoxic-ischemic brain damage. Recombinant vector of adenovirus-mediated VEGF165 gene (Ad-VEGF) was constructed by bacterial homologous recombination technology. Sixty 7-day-old Sprague-Dawley rats were randomly divided into 3 equal groups: hypoxic-ischemic brain damage (HIBD) group undergoing ligation of the left common carotid artery and inhalation of 8% oxygen for 2 hours, Ad-VEGF group undergoing injection of Ad-VEGF into the left sensorimotor cortex with the help of stereo-positioner 3 days after hypoxia-ischemia (HI), and sham operation group. Seven days after transplantation, 5 rats from each group were killed with their left brains taken out. The VEGF protein expression was detected by immunohistochemistry, and the neuron apoptosis was detected by terminal deoxynucleotidyl transferase-mediated biotinylated deoxyuridine triphosphate nickel end labeling (TUNEL). Since the age of 28 days T-maze foraging test was conducted. At the age of 34 days, sensorimotor tests were performed. After the behavioral tests all the rats were killed. The number of neurons in the CA1 region of the hippocampus and cerebral cortex was detected by Nissl's staining. Immunohistochemistry showed that the density levels of VEGF positive cells in cerebral cortex and hippocampus of the Ad-VEGF group were (68.09 +/- 3.37) and (68.37 +/- 3.17) respectively, both significantly higher than those of the HIBD group [(24.65 +/- 3.14) and (25.14 +/- 1.86) respectively, both P < 0.05]. TUNEL showed that the number of apoptotic neurons of the Ad-VEGF group was (151.4 +/- 21.7), significantly lower than that of the HIBD group [(264.4 +/- 16.3), P < 0.05]. Behavioral tests showed the percentages of accuracy on day 4 of the Ad-VEGF and sham operation groups were both significantly higher than that of the HIBD group (both P < 0.01). Nissl's staining showed that the numbers of neurons per unit area in the hippocampal CA1 area and cortex of the Ad-VEGF group were (70.6 +/- 2.3) and (95.1 +/- 2.8) respectively, both significantly higher than those of the HI group [(55.3 +/- 2.1) and (70.1 +/- 2.7) respectively, both P < 0.05]. Adenovirus vector-mediated VEGF gene therapy increases the VEGF protein expression, decreases neuron apoptosis, improves the long-term behavioral function after brain damage, and reduces hypoxic ischemic brain injury, thus possessing neuroprotective effects.

48. MicroRNA-Regulated Transgene Expression System Reduces Unwanted Side Effects in Adenovirus Vector-Mediated Suicide Gene Therapy

48. MicroRNA-Regulated Transgene Expression System Reduces Unwanted Side Effects in Adenovirus Vector-Mediated Suicide Gene Therapy

Gamma-globin lentiviral vector-mediated stem cell gene therapy of beta-thalassemia and sickle cell disease

Gamma-globin lentiviral vector-mediated stem cell gene therapy of beta-thalassemia and sickle cell disease

Anterograde Tracing of A1 and A5 Efferents Using Phenotypically Restricted Lentivirus Vector Mediated Reporter Gene Expression

Anterograde Tracing of A1 and A5 Efferents Using Phenotypically Restricted Lentivirus Vector Mediated Reporter Gene Expression

Lentivirus vector-mediated SERCA2 gene transfer ameliorates heart failure induced by myocardial infarction in rat

Lentivirus vector-mediated SERCA2 gene transfer ameliorates heart failure induced by myocardial infarction in rat