Adenoviral Vector-mediated Gene Research Articles

Adenoviral Vector-Mediated NGF Gene Therapy for a Rat Model of Diabetic Bladder Dysfunction

Diabetes was a common complication occurred in aging populations. Diabetic bladder dysfunction (DBD) was one of the complications of diabetes. We used streptozocin (STZ) to induce diabetes in rat models. Cystometrography was performed to evaluate the function of bladder after NGF gene therapy. Histological analysis was used to observe the structure of bladder. Western blot and qPCR was performed to detect the expression level of NGF in bladder tissues. Diabetes could decrease the postvoid residual volume, bladder capacity and maximal detrusor pressure of Wistar rat in diabetic rats (DM) and DM injected with blank adenoviral vector (Ad) group. And there were no differences in bladder pressure among these groups. The results of western blot and polymerase chain reaction (PCR) indicated that the expression levels of Nerve Growth Factor (NGF) were decreased in DM and Ad groups, and increased in Ad-NGF group (P < 0.05). The results of morphological observation indicated that the structure of detrusor smooth muscle cells was broken in diabetes rats, including interruption of continuity and swelling of smooth muscle cells, and showed a time- depend manner. After the treatment of NGF gene, the structure and function of bladder were recovered, but the recovery effect of NGF gene therapy was reduced with the development of diabetes. These results indicated that diabetes could broke the structure and function of bladder, and the structure and function of bladder could restore under NGF-gene therapy in the early stage of diabetes but not the late stage. Funding: The experiment was supported by Project of PAP（CWJ18L004） and Family Planning Project of PLA(145BHQ090003076X) Declaration of Interest: None to declare. Ethical Approval: The experiments were approved by the Institution's Ethics Committee for Investigation of Tianjin Medical University, and all of the 40 animals were treated under humane care in compliance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals.

Advances in Alpha-1 Antitrypsin Gene Therapy

AAT (alpha-1 antitrypsin) deficiency (AATD), characterized by low levels of circulating serine protease inhibitor AAT, results in emphysematous destruction of the lung. Inherited serum deficiency disorders, such as hemophilia and AATD, have been considered ideal candidates for gene therapy. Although viral vector-meditated transduction of the liver has demonstrated utility in hemophilia, similar success has not been achieved for AATD. The challenge for AAT gene therapy is achieving protective levels of AAT locally in the lung and mitigating potential liver toxicities linked to systemically administered viral vectors. Current strategies with ongoing clinical trials involve different routes of adeno-associated virus administrations, such as intramuscular and intrapleural injections, to provide consistent therapeutic levels from nonhepatic organ sites. Nevertheless, exploration of alternative methods of nonhepatic sourcing of AAT has been of great interest in the field. In this regard, pulmonary endothelium-targeted adenovirus vector could be a key technical mandate to achieve local augmentation of AAT within the lower respiratory tract, with the potential benefit of circumventing liver toxicities. In addition, incorporation of the CRISPR/Cas9 (CRISPR-associated protein 9) nuclease system into gene-delivery technologies has provided adjunctive technologies that could fully realize a one-time treatment for sustained, lifelong expression of AAT in patients with AATD. This review will focus on the adeno-associated virus- and adenoviral vector-mediated gene therapy strategies for the pulmonary manifestations of AATD and show that endeavoring to use genome-editing techniques will advance the current strategy to one fully compatible with direct human translation.

Canine adenoviral vector-mediated gene transfer to the guinea pig brain

Pre-clinical testing of gene transfer tools is often first conducted in small animal models such as laboratory mice or rats, before extension into a larger animal such as non-human primates. The guinea pig brain may provide an intermediary model in which to examine neurodevelopmental processes given that guinea pigs are considered precocial. Here, we characterized the efficacy and biodistribution following injection of a canine adenovirus type 2 (CAV-2) vector in the young adult guinea pig brain. Despite the development of mild fevers post-treatment in the vector-treated animals, widespread transduction was evident throughout the brain parenchyma. Neutralizing antibodies against CAV-2 were not detected. Our data demonstrate that the combination of CAV-2 vectors and guinea pigs provide a clinically relevant model for evaluation of therapeutics.

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 &lt; 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.

Noninvasive and Local Delivery of Adenoviral-Mediated Herpes Simplex Virus Thymidine Kinase to Treat Glioma Through Focused Ultrasound-Induced Blood-Brain Barrier Opening in Rats.

Adenoviral-mediated gene therapy has been shown great prospects for tumor treatment. However, it is still a great challenge for its application in the glioma. A main cause is the blood-brain barrier (BBB) limits the delivery of adenoviral vectors, greatly compromising their efficacy. Here, we used focused ultrasound (FUS) induced microbubble cavitation to locally and reversibly open BBB for enhancing gene delivery. In this study, Ad-CMV-TK-IRES-EGFP (Ad-HSV-TK-EGFP) carrying the herpes simplex virus thymidine kinase (HSV-TK) and EGFP transgenes was chose as gendicine and Ad-CMV-IRES-EGFP (Ad-EGFP) as the control. The in vitro experiments showed that Ad-HSV-TK-EGFP had a high infection efficiency for C6 glioma cells, producing good tumor cell killing effects when these cells were exposed to more than 10 μg/ml ganciclovir (GCV). Taking advantage of FUS-induced BBB opening, Ad-HSV-TK-EGFP could be effectively delivered into the brain tumors, getting the overexpression of HSV-TK gene in the tumor cells. After exposure to GCV, the significantly stronger anti-tumor efficacy and longer survival time were observed in tumor-bearing mice treated with Ad-HSV-TK-EGFP + FUS than those treated with Ad-EGFP + FUS or only Ad-HSV-TK-EGFP. Histological examination indicated that the reduced expression level of Ki67 proteins and the increased apoptotic tumor cells in tumor xenografts, causing the inhibition of tumor growth. In conclusion, our study provided a new strategy to efficiently and locally deliver recombinant adenoviral vector-mediated HSV-TK gene into the brain to treat glioma.

Enhancement of adenovirus infection and adenoviral vector-mediated gene delivery by bromodomain inhibitor JQ1

Adenovirus-based vectors are among the most commonly used platforms for gene delivery and gene therapy studies. One of the obstacles for potential application is dose-related toxicity. We show here that adenovirus infection and Ad-mediated gene delivery can be enhanced by inhibitors of bromodomain and extra-terminal (BET) family proteins. We showed that JQ1, but not its inactive enantiomer (−)-JQ1, dose-dependently promoted Ad infection and Ad-mediated gene delivery in both epithelial and lymphocyte cells. Given orally, JQ1 also enhanced transgene expression in a murine tumor model. Inhibitors of histone deacetylases (HDACi) are among the commonly reported small molecule compounds which enhance Ad-mediated gene delivery. We found that JQ1 treatment did not cause histone acetylation nor expression of Ad attachment receptor CAR. Instead, JQ1 treatment induced an increase in BRD4 association with CDK9, a subunit of P-TEFb of transcription elongation. Concurrently, we showed that CDK9 inhibition blocked Ad infection and JQ1 enhancement on the infection. The study exemplifies the potentials of BET inhibitors like JQ1 in oncolytic virotherapy.

Adenoviral vector-mediated GM-CSF gene transfer improves anti-mycobacterial immunity in mice – role of regulatory T cells

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a hematopoietic growth factor involved in differentiation, survival and activation of myeloid and non-myeloid cells with important implications for lung antibacterial immunity. Here we examined the effect of pulmonary adenoviral vector-mediated delivery of GM-CSF (AdGM-CSF) on anti-mycobacterial immunity in M. bovis BCG infected mice. Exposure of M. bovis BCG infected mice to AdGM-CSF either applied on 6h, or 6h and 7days post-infection substantially increased alveolar recruitment of iNOS and IL-12 expressing macrophages, and significantly increased accumulation of IFNγpos T cells and particularly regulatory T cells (Tregs). This was accompanied by significantly reduced mycobacterial loads in the lungs of mice. Importantly, diphtheria toxin-induced depletion of Tregs did not influence mycobacterial loads, but accentuated immunopathology in AdGM-CSF-exposed mice infected with M. bovis BCG. Together, the data demonstrate that AdGM-CSF therapy improves lung protective immunity against M. bovis BCG infection in mice independent of co-recruited Tregs, which however critically contribute to limit lung immunopathology in BCG-infected mice. These data may be relevant to the development of immunomodulatory strategies to limit immunopathology-based lung injury in tuberculosis in humans.

Protective role of adenovirus vector-mediated interleukin-10 gene therapy on endogenous islet β-cells in recent-onset type 1 diabetes in NOD mice.

The aim of the present study was to provide an animal experimental basis for the protective effect of the adenoviral vector-mediated interleukin-10 (Ad-mIL-10) gene on islet β-cells during the early stages of type 1 diabetes (T1D) in non-obese diabetic (NOD) mice. A total of 24 female NOD mice at the onset of diabetes were allocated at random into three groups (n=8 per group): Group 1, intraperitoneally injected with 0.1 ml Ad-mIL-10; group 2, intraperitoneally injected with 0.1 ml adenovirus vector; and group 3, was a diabetic control. In addition to groups 1, 2 and 3, 8 age- and gender-matched NOD mice were intraperitoneally injected with 0.1 ml PBS and assigned to group 4 as a normal control. All mice were examined weekly for body weight, urine glucose and blood glucose values prior to onset of diabetes, and at 1, 2 and 3 weeks after that, and all mice were sacrificed 3 weeks after injection. Serum levels of interleukin (IL)-10, interferon (IFN)-γ, IL-4, insulin and C-peptide were evaluated, and in addition the degree of insulitis and the local expression of IL-10 gene in the pancreas were detected. The apoptosis rate of pancreatic β-cells was determined using a TUNEL assay. Compared with groups 2 and 3, IL-10 levels in the serum and pancreas were elevated in group 1. Serum IFN-γ levels were decreased while serum IL-4 levels and IFN-γ/IL-4 ratio were significantly increased in group 1 (P<0.01). C-peptide and insulin levels were higher in group 1 compared with groups 2 and 3, (P<0.01). Furthermore, compared with groups 2 and 3, the degree of insulitis, islet β-cell apoptosis rate and blood glucose values did not change significantly (P>0.05). The administration of the Ad-mIL-10 gene induced limited immune regulatory and protective effects on islet β-cell function in NOD mice with early T1D, while no significant reduction in insulitis, islet β-cell apoptosis rate and blood glucose was observed.

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

Efficient Gene Transduction of Dispersed Islet Cells in Culture Using Fiber-Modified Adenoviral Vectors.

To establish novel islet-based therapies, our group has recently developed technologies for creating functional neo-islet tissues in the subcutaneous space by transplanting monolithic sheets of dispersed islet cells (islet cell sheets). Improving cellular function and viability are the next important challenges for enhancing the therapeutic effects. This article describes the adenoviral vector-mediated gene transduction of dispersed islet cells under culture conditions. Purified pancreatic islets were obtained from Lewis rats and dissociated into single islet cells. Cells were plated onto laminin-5-coated temperature-responsive polymer poly(N-isopropylacrylamide)-immobilized plastic dishes. At 0 h, islet cells were infected for 1 h with either conventional type 5 adenoviral vector (Ad-CA-GFP) or fiber-modified adenoviral vector (AdK7-CA-GFP) harboring a polylysine (K7) peptide in the C terminus of the fiber knob. We investigated gene transduction efficiency at 48 h after infection and found that AdK7-CA-GFP yielded higher transduction efficiencies than Ad-CA-GFP at a multiplicity of infection (MOI) of 5 and 10. For AdK7-CA-GFP at MOI = 10, 84.4 ± 1.5% of islet cells were found to be genetically transduced without marked vector infection-related cellular damage as determined by viable cell number and lactate dehydrogenase (LDH) release assay. After AdK7-CA-GFP infection at MOI = 10, cells remained attached and expanded to nearly full confluency, showing that this adenoviral infection protocol is a feasible approach for creating islet cell sheets. We have shown that dispersed and cultured islet cells can be genetically modified efficiently using fiber-modified adenoviral vectors. Therefore, this gene therapy technique could be used for cellular modification or biological assessment of dispersed islet cells.

Transient blocking of NK cell function with small molecule inhibitors for helper dependant adenoviral vector-mediated gene delivery

One major challenge in gene therapy is the host immune responses against viral vectors. Previous studies indicate the involvement of NK cells in stunted gene expression in viral vector mediated gene therapy. To understand the problem of the immune responses, we have developed an in-vitro co-culture system with human NK cell line, macrophages and airway epithelial cells. We showed that small molecule blockers, CAPE and ruxolitinib, for NF-κB and JAK-STAT pathways, respectively, significantly inhibited cytokine secretion by macrophages. When NK cells are co-cultured with helper-dependent adenoviral (HD-Ad) vector activated macrophages, IFN-γ cytokine expression by NK cells increased significantly, which was inhibited effectively by ruxolitinib and CAPE, and there was an additive effect when both inhibitors were used. We demonstrated that NK cells activated by cytokines produced by HD-Ad-activated macrophages kill HD-Ad vector transduced bronchial epithelial cells. This cell killing activity was significantly reduced by CAPE and ruxolitinib. Combination of these two inhibitors had an additive effect on inhibiting NK cell mediate killing of gene transduced cells. Transient inhibition of NK cell response at its peak may enhance sustained gene expression. Our data suggest that combination of CAPE and ruxolitinib may help in protecting gene transduced airway epithelial cells to prolong transgene expression.Electronic supplementary materialThe online version of this article (doi:10.1186/s13578-015-0023-0) contains supplementary material, which is available to authorized users.

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.

Formulation and in vitro and in vivo evaluation of a cationic emulsion as a vehicle for improving adenoviral gene transfer

Advancements in the use of adenoviral vectors in gene therapy have been limited by the need for specific receptors on targeted cell types, immunogenicity and hepatotoxicity following systemic administration. In an effort to overcome the current limitations of adenovirus-mediated gene transfer, cationic emulsions were explored as a vehicle to improve adenoviral vector-mediated gene transfer. Complexation of adenovirus with emulsions containing the cationic lipid 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) enhanced the potency of adenoviral gene transfer as compared to DOTAP liposomes. Among the various emulsion formulations examined, those containing the iodized oil, Lipiodol, as an inner core and stabilized by DOTAP/cholesterol/1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy(poly-ethylene glycol)-5000 most efficiently enhanced adenovirus-mediated gene transfer. Optimized Lipiodol-containing emulsions appear to be more strongly associated with adenoviral particles, exhibiting higher complex stability compared to other formulations. They provide the adenovirus with an additional cellular entry mechanism through caveolae-dependent endocytosis, thereby increasing adenovirus entry into cells. Furthermore, adenovirus–emulsion complexation significantly reduced transgene expression in the liver following systemic administration. These findings indicate that emulsion complexation may be a promising strategy for overcoming many of the challenges associated with the use of adenoviruses in gene therapy. Additionally, the observation of increased transgene expression in lung together with reduced expression in liver demonstrates that the adenovirus–emulsion complex may act as a lung-targeting adenoviral gene delivery system.

507. Adenoviral Vector-Mediated CFTR Gene Transfer and Anion Transport Correction of CF Pig Airways In Vivo

507. Adenoviral Vector-Mediated CFTR Gene Transfer and Anion Transport Correction of CF Pig Airways In Vivo

Challenges and Prospects for Helper-Dependent Adenoviral Vector-Mediated Gene Therapy.

Helper-dependent adenoviral (HDAd) vectors that are devoid of all viral coding sequences are promising non-integrating vectors for gene therapy because they efficiently transduce a variety of cell types in vivo, have a large cloning capacity, and drive long-term transgene expression without chronic toxicity. The main obstacle preventing clinical applications of HDAd vectors is the host innate inflammatory response against the vector capsid proteins that occurs shortly after intravascular vector administration and result in acute toxicity, the severity of which is dose dependent. Intense efforts have been focused on elucidating adenoviral vector–host interactions and the factors involved in the acute toxicity. This review focuses on the recent acquisition of data on such interactions and on strategies investigated to improve the therapeutic index of HDAd vectors.

Modulation of Treg function improves adenovirus vector-mediated gene expression in the airway

Virus vector -mediated gene transfer has been developed as a treatment for cystic fibrosis (CF) airway disease, a lethal inherited disorder caused by somatic mutations in the CFTR gene. The pathological pro-inflammatory environment of CF as well as the naïve and adaptive immunity induced by the virus vector itself limit the effectiveness of gene therapy for CF airway. Here, we report the use of an HDAC inhibitor, valproic acid (VPA), to enhance the activity of the regulatory T cells (Treg) and improve the expression of virus vector-mediated gene transfer to the respiratory epithelium. Our study demonstrates the potential utility of VPA, a drug used for over 50 years in humans as an anticonvulsant and mood-stabilizer, in controlling inflammation and improving the efficacy of gene transfer in CF airway.

Switching a Replication-Defective Adenoviral Vector into a Replication-Competent, Oncolytic Adenovirus

The adenovirus immediate early gene E1A initiates the program of viral gene transcription and reprograms multiple aspects of cell function and behavior. For adenoviral (Ad) vector-mediated gene transfer and therapy approaches, where replication-defective (RD) gene transfer is required, E1A has thus been the primary target for deletions. For oncolytic gene therapy for cancer, where replication-competent (RC) Ad viral gene expression is needed, E1A has been either mutated or placed under tumor-specific transcriptional control. A novel Ad vector that initially infected target tumor cells in an RD manner for transgene expression but that could be "switched" into an RC, oncolytic state when needed might represent an advance in vector technology. Here, we report that we designed such an Ad vector (proAdΔ24.GFP), where initial Ad replication is silenced by a green fluorescent protein (GFP) transgene that blocks cytomegalovirus (CMV)-mediated transcription of E1A. This vector functions as a bona fide E1A-deleted RD vector in infected tumor cells. However, because the silencing GFP transgene is flanked by FLP recombination target (FRT) sites, we show that it can be efficiently excised by Flp recombinase site-specific recombination, either when Flp is expressed constitutively in cells or when it is provided in trans by coinfection with a second RD herpes simplex virus (HSV) amplicon vector. This switches the RD Ad, proAdΔ24.GFP, into a fully RC, oncolytic Ad (rAdΔ24) that lyses tumor cells in culture and generates oncolytic progeny virions. In vivo, coinfection of established flank tumors with the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon leads to generation of RC, oncolytic rAdΔ24. In an orthotopic human glioma xenograft tumor model, coinjection of the RD proAdΔ24.GFP and the RD Flp-bearing HSV1 amplicon also led to a significant increase in animal survival, compared to controls. Therefore, Flp-FRT site-specific recombination can be applied to switch RD Ad into fully oncolytic RC Ad for tumor therapy and is potentially applicable to a variety of gene therapy approaches.

Inhibition of nuclear factor-κB enhances the antitumor effect of tumor necrosis factor-α gene therapy for hepatocellular carcinoma in mice

Hepatocellular carcinoma (HCC) is often resistant to chemotherapy. Gene therapy using an adenoviral vector-expressing tumor necrosis factor (TNF)-α is a new therapeutic approach for chemoresistant malignancies. The efficacy of TNF-α, however, is limited, because it leads to the activation of nuclear factor (NF)-κB. We hypothesized that the NF-κB inhibitor nafamostat mesilate would enhance the antitumor effect of adenovirus vector-mediated TNF-α gene therapy for HCC. Invitro, we assessed the inhibitory effect of nafamostat mesilate on TNF-α-induced NF-κB activation and enhanced apoptosis in human HCC cell lines (Huh-7 and Hep3B). Invivo, we established a xenograft HCC model in mice by subcutaneous injection of Huh-7 and Hep3B cells. The animals received intraperitoneal (IP) injections of nafamostat mesilate 3 times a week (nafamostat mesilate group), intratumoral (IT) injections of the human TNF-α-expressing adenoviral vector (AxCAhTNF-α) once a week (TNF-α group), IT injections of AxCAhTNF-α once a week, or IP injections of nafamostat mesilate 3 times a week (combination group). In the combination group, TNF-α-induced NF-κB activation was inhibited and TNF-α-induced apoptosis was enhanced in comparison with the other groups both invitro and invivo. In the combination group, tumor growth was significantly slower and the apoptotic cell numbers were significantly greater than those of the TNF-α group. Inhibition of NF-κB by nafamostat mesilate enhances the antitumor effect of adenoviral vector-mediated TNF-α gene therapy for HCC in mice.

Chimeric adenoviral vector Ad5F35L containing the Ad5 natural long-shaft exhibits efficient gene transfer into human T lymphocytes

Adoptive therapy using T cells modified with tumour antigen-specific T cell receptor (TCR) genes has become a popular area of research in tumour biotherapy research. However, the efficiency of this treatment is low. To increase the efficiency of this therapy, the antigen specific TCR expression in the T cells needs to be improved. Adenoviral vector-mediated gene expression is an attractive approach to bypass the issue of TCR gene modification. The efficiency of adenovirus vector serotype 5 (Ad5) infection is low due to the absence of coxsackievirus B-adenovirus receptor (CAR) expression in T cells. In the present study, a chimeric adenoviral vector (Ad5F35L) was generated; this construct contained both the natural long-shaft of Ad5 and the Ad35 knob. A transduction study showed that the Ad5F35L vector exhibited a higher transduction efficiency in human primary T lymphocytes than the Ad5 vector and the Ad5F35S vector, which contained the Ad35 natural short-shaft and the Ad35 knob. Similar transduction efficiencies were observed for both CD4+ T lymphocytes and CD8+ T lymphocytes and the transfection was independent of the expression of cell surface receptors. The activation of T lymphocytes resulted in an improvement of the Ad5F35L transduction efficiency in CD4+ T cells and a decrease in Ad5F35L transduction efficiency in CD8+ T cells. The results demonstrate that Ad5F35L is a promising viral vector and will facilitate the clinical application of tumour antigen-specific TCR gene therapy.

Effect of human 15-lipoxygenase-1 metabolites on vascular function in mouse mesenteric arteries and hearts

Lipoxygenases regulate vascular function by metabolizing arachidonic acid (AA) to dilator eicosanoids. Previously, we showed that endothelium-targeted adenoviral vector-mediated gene transfer of the human 15-lipoxygenase-1 (h15-LO-1) enhances arterial relaxation through the production of vasodilatory hydroxyepoxyeicosatrienoic acid (HEETA) and trihydroxyeicosatrienoic acid (THETA) metabolites. To further define this function, a transgenic (Tg) mouse line that overexpresses h15-LO-1 was studied. Western blot, immunohistochemistry and RT-PCR results confirmed expression of 15-LO-1 transgene in tissues, especially high quantity in coronary arterial wall, of Tg mice. Reverse-phase HPLC analysis of [14C]-AA metabolites in heart tissues revealed enhanced 15-HETE synthesis in Tg vs. WT mice. Among the 15-LO-1 metabolites, 15-HETE, erythro-13-H-14,15-EETA, and 11(R),12(S),15(S)-THETA relaxed the mouse mesenteric arteries to the greatest extent. The presence of h15-LO-1 increased acetylcholine- and AA-mediated relaxation in mesenteric arteries of Tg mice compared to WT mice. 15-LO-1 was most abundant in the heart; therefore, we used the Langendorff heart model to test the hypothesis that elevated 15-LO-1 levels would increase coronary flow following a short ischemia episode. Both peak flow and excess flow of reperfused hearts were significantly elevated in hearts from Tg compared to WT mice being 2.03 and 3.22 times greater, respectively. These results indicate that h15-LO-1-derived metabolites are highly vasoactive and may play a critical role in regulating coronary blood flow.