We sought to construct the adenoviral vector carrying the gene encoding mouse telomerase reverse transcriptase(mTERT),as well as detect its expression and effect on the proliferation of neuronal stem cells.mTERT was...We sought to construct the adenoviral vector carrying the gene encoding mouse telomerase reverse transcriptase(mTERT),as well as detect its expression and effect on the proliferation of neuronal stem cells.mTERT was am-plified by RT-PCR and then the eukaryotic expression vector of pDC-EGFP-TERT was constructed.After DNA sequence analysis,we detected that there were 293 cells transfected with pDC-EGFP-TERT and helper adenovirus plasmid pBHG lox ΔE1,and three Cre using Lipofectamine 2000 mediation,named Ad-mTERT-GFP,to pack-age adenoviral particles.The Ad-mTERT-GFP was used to infect neuronal stem cells and then the expression and activity of mTERT were detected.In addition,Bromodeoxyuridine labeling test identified the impact of mTERT overexpression on proliferation of neuronal stem cells.The recombinant adenoviral vector confirmed that mTERT was successfully constructed.Overexpression of mTERT stimulated the proliferation of neuronal stem cells both in vitro and in vivo.mTERT overexpression via adenoviral vector carrying mTERT cDNA upregulated the ability of proliferation in neuronal stem cells.展开更多
Adenoviral pneumonia is one of the more serious types of community-acquired pneumonia in children.It easily develops into severe pneumonia with many extrapulmonary complications and a high mortality rate.However,there...Adenoviral pneumonia is one of the more serious types of community-acquired pneumonia in children.It easily develops into severe pneumonia with many extrapulmonary complications and a high mortality rate.However,there is still no effective treatment for adenovirus.Therapeutic drugs are conducive to clinical treatment.Traditional Chinese medicine often achieves satisfactory results through staged syndrome differentiation.Currently,heat,sputum,stasis,and poison are considered to be the main pathological products.Fever,phlegm,and stasis are present in its development.Analyze the progress of traditional Chinese and western medicine in children's adenoviral pneumonia,and have a clearer understanding of the law of syndrome differentiation and its prescription medication,providing ideas for clinical treatment of adenoviral pneumonia.展开更多
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) i...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.展开更多
We report here a method for the use of poly-L-lysine (PLL) to markedly improve the adenoviral transduction efficiency of primary murine osteoblasts and bone marrow stromal cells (BMSCs) in culture and in situ, whi...We report here a method for the use of poly-L-lysine (PLL) to markedly improve the adenoviral transduction efficiency of primary murine osteoblasts and bone marrow stromal cells (BMSCs) in culture and in situ, which are typically difficult to transduce. We show by fluorescence microscopy and flow cytometry that the addition of PLL to the viral-containing medium significantly increases the number of green fluorescence protein (GFP)-positive osteoblasts and BMSCs transduced with an enhanced GFP-expressing adenovirus. We also demonstrate that PLL can greatly enhance the adenoviral transduction of osteoblasts and osteocytes in situ in ex vivo tibia and calvaria, as well as in long bone fragments. In addition, we validate that PLL can improve routine adenoviral transduction studies by permitting the use of low multiplicities of infection to obtain the desired biologic effect. Ultimately, the use of PLL to facilitate adenoviral gene transfer in osteogenic cells can provide a cost-effective means of performing efficient gene transfer studies in the context of bone research.展开更多
Vaccines are an integral part of veterinary disease prevention. However there are still a significant number of veterinary diseases for which vaccines do not currently exist or where currently available vaccines do no...Vaccines are an integral part of veterinary disease prevention. However there are still a significant number of veterinary diseases for which vaccines do not currently exist or where currently available vaccines do not provide adequate immunity. Adenoviruses have transitioned from tools for gene replacement therapy to bona fide vaccine delivery vehicles because of their ability to elicit potent cell-mediated and humoral responses making them ideal for use against viruses and other intracellular pathogens. Adenoviral vector based vaccines are likely to play a significant role in overcoming these problems in the future. However, this vector is under utilized in veterinary vaccine development at this time. This review focuses on adenoviral vector based vaccines developed to date and explores the potential for veterinary vaccine development based upon this platform: advantages and potential disadvantages of this technology are discussed as well as the potential for developing efficacious commercial veterinary adenoviral vector based vaccines.展开更多
Background Human interleukin-10 (hlL-10) is a cytokine synthesis inhibitory factor,which is involved in various immune responses.The purpose of this study was to construct an adenoviral vector carrying the hlL-10 ge...Background Human interleukin-10 (hlL-10) is a cytokine synthesis inhibitory factor,which is involved in various immune responses.The purpose of this study was to construct an adenoviral vector carrying the hlL-10 gene for expression of biologically active hlL-10 in rat bone marrow mesenchymal stem cells (rMSCs).Methods A pSNAV2.0-hlL10 plasmid was used as a template to obtain a hlL-10 cDNA fragment that was subcloned by restriction enzyme digestion and ligation into a pDC316-IRES-EGFP-lacZ alpha plasmid carrying an enhanced green fluorescent protein (EGFP) marker gene.The pDC316-hlL-10-IRES-EGFP plasmid was linearized by Pmel digestion and used to transfect HEK293 packaging cells using the adenovirus packaging system AdMax.Virus particles were amplified by repeatedly infecting HEK293 cells with the seed virus and then purified by ion exchange.After the number of virus particles and titer was determined,rMSCs were infected with the adenoviral vector.The infection rate was determined by fluorescence microscopy and flow cytometry,and hlL-10 protein expression in rMSCs was measured by Western blotting.Results The virus particle concentration,OD260/280 value and virus titer of the amplified and purified recombinant adenovirus were 3.2×1011 VP/ml,approximately 2.0,and 1.1×1010 TCID50/ml,respectively.Bright green fluorescence was observed by fluorescence microscopy and flow cytometry in the recombinant adenovirus-infected rMSCs.GFP expression was considered the multiplicity of infection (MOI) and was time-dependent.The infection rate was 92.9% at 100 MOI.Conclusions A bicistrenic recombinant adenoviral vector for hlL-10 and EGFP gene expression were successfully constructed.The infection rate of rMSCs by the adenovirus was high (92.9% at 100 MOI) and the target gene hlL-10 was highly expressed in cells.The present study provides an experimental basis for further research of immunosuppressive therapy using hlL-10.The expression level of hlL-10 protein as detected by Western blotting was also MOI- and time-dependent.展开更多
Objective To prevent Graft-versus-host disease (GVHD) in rat model, we evaluated the feasibility of mesenchymal stem cells (MSCs) as a gene transfer target and studied the efficiency of recombinant adenovirus mediated...Objective To prevent Graft-versus-host disease (GVHD) in rat model, we evaluated the feasibility of mesenchymal stem cells (MSCs) as a gene transfer target and studied the efficiency of recombinant adenovirus mediated gene therapy.Methods We constructed the recombinant adenovirus containing CTLA4lg gene. Rat MSCs of passages 3-5 were infected by the adenovirus, and the transfection efficiency was monitored by GFP markers. We performed flow cytometric analysis, immunohistochemical and Western blotting analysis to identify the CTLA4lg expression. The gene transferred MSCs were tested for their ability to inhibit the allogeneic lymphocyte response in vitro and to prevent GVHD in a rat model.Results Recombinant adenovirus pAd-CTLA4lg was correctly constructed and confirmed. After MSCs were infected by the adenovirus, the CTLA4lg protein was detected not only in transgenic MSCs, but also in the culture medium. In a mixed lymphocytes response (MLR) test, the transgenic MSCs could significantly inhibit the allogeneic lymphocyte response compared with the control groups (P < 0. 05) . A model of GVHD was developed by transplanting bone marrow cells and spleen lymphocytes of F344 rats to lethally irradiated SD rats. The onset of GVHD could be ameliorated or prevented by co-administration of transgenic MSCs. All the rats in the control groups suffered severe acute GVHD. CTLA4lg expression was observed in the liver, intestine, kidney and spleen 30 days post- transplantation.Conclusions Our results indicate that adenoviral vectors could efficiently transfer CTLA4lg gene into MSCs and sustain long-term stable expression in vitro and in vivo.展开更多
There have been significant advancements in the field of retinal gene therapy in the past several years.In particular,therapeutic efficacy has been achieved in three separate human clinical trials conducted to assess ...There have been significant advancements in the field of retinal gene therapy in the past several years.In particular,therapeutic efficacy has been achieved in three separate human clinical trials conducted to assess the ability of adeno-associated viruses(AAV)to treat of a type of Leber’s congenital amaurosis caused by RPE65 mutations.However,despite the success of retinal gene therapy with AAV,challenges remain for delivering large therapeutic genes or genes requiring long DNA regulatory elements for controlling their expression.For example,Stargardt’s disease,a form of juvenile macular degeneration,is caused by defects in ABCA4,a gene that is too large to be packaged in AAV.Therefore,we investigated the ability of helper dependent adenovirus(HD-Ad)to deliver genes to the retina as it has a much larger transgene capacity.Using an EGFP reporter,our results showed that HD-Ad can transduce the entire retinal epithelium of a mouse using a dose of only 1105 infectious units and maintain transgene expression for at least 4 months.The results demonstrate that HD-Ad has the potential to be an effective vector for the gene therapy of the retina.展开更多
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 fur...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展开更多
Post-traumatic spinal cord remodeling includes both degenerating and regenerating processes,which affect the potency of the functional recovery after spinal cord injury(SCI).Gene therapy for spinal cord injury is prop...Post-traumatic spinal cord remodeling includes both degenerating and regenerating processes,which affect the potency of the functional recovery after spinal cord injury(SCI).Gene therapy for spinal cord injury is proposed as a promising therapeutic strategy to induce positive changes in remodeling of the affected neural tissue.In our previous studies for delivering the therapeutic genes at the site of spinal cord injury,we developed a new approach using an autologous leucoconcentrate transduced ex vivo with chimeric adenoviruses(Ad5/35)carrying recombinant cDNA.In the present study,the efficacy of the intravenous infusion of an autologous genetically-enriched leucoconcentrate simultaneously producing recombinant vascular endothelial growth factor(VEGF),glial cell line-derived neurotrophic factor(GDNF),and neural cell adhesion molecule(NCAM)was evaluated with regard to the molecular and cellular changes in remodeling of the spinal cord tissue at the site of damage in a model of mini-pigs with moderate spinal cord injury.Experimental animals were randomly divided into two groups of 4 pigs each:the therapeutic(infused with the leucoconcentrate simultaneously transduced with a combination of the three chimeric adenoviral vectors Ad5/35‐VEGF165,Ad5/35‐GDNF,and Ad5/35‐NCAM1)and control groups(infused with intact leucoconcentrate).The morphometric and immunofluorescence analysis of the spinal cord regeneration in the rostral and caudal segments according to the epicenter of the injury in the treated animals compared to the control mini-pigs showed:(1)higher sparing of the grey matter and increased survivability of the spinal cord cells(lower number of Caspase-3-positive cells and decreased expression of Hsp27);(2)recovery of synaptophysin expression;(3)prevention of astrogliosis(lower area of glial fibrillary acidic protein-positive astrocytes and ionized calcium binding adaptor molecule 1-positive microglial cells);(4)higher growth rates of regeneratingβIII-tubulin-positive axons accompanied by a higher number of oligodendrocyte transcription factor 2-positive oligodendroglial cells in the lateral corticospinal tract region.These results revealed the efficacy of intravenous infusion of the autologous genetically-enriched leucoconcentrate producing recombinant VEGF,GDNF,and NCAM in the acute phase of spinal cord injury on the positive changes in the post-traumatic remodeling nervous tissue at the site of direct injury.Our data provide a solid platform for a new ex vivo gene therapy for spinal cord injury and will facilitate further translation of regenerative therapies in clinical neurology.展开更多
AIM: To investigate a therapeutic method for gastrointestinal stromal tumor (GIST) based on KIT RNA interference (RNAi) with AdMax adenovirus. METHODS: KIT short hairpin RNA (shRNA), whose lateral sides were decorated...AIM: To investigate a therapeutic method for gastrointestinal stromal tumor (GIST) based on KIT RNA interference (RNAi) with AdMax adenovirus. METHODS: KIT short hairpin RNA (shRNA), whose lateral sides were decorated with restriction endonuclease sequences, was designed. T 4 DNA ligase catalyzed the joint of the KIT shRNA and the green fluorescent protein-containing PDC316-EGFP-U6 to form PDC316EGFP-U6-KIT. Homologous recombination of AdEGFPU6-KIT was performed with the AdMax system. Heterotopically transplanted GISTs were established in nude mice. AdEGFP-U6-KIT was intratumorally injected. The volume, inhibition ratio of tumor and CD117 expression of GIST graft tumor in nude mice were compared between test and control groups. RESULTS: The length of KIT shRNA was determined to be about 50bp by agarose electrophoresis. Gene se-quencing detected the designed KIT RNAi sequence in PDC316-EGFP-U6-KIT. After transfection with AdEGFPU6-KIT, 293 cells displayed green fluorescence. The physical and infective titers of AdEGFP-U6-KIT were 5 × 10 11 viral particles/mL and 5.67 × 10 7 plaque forming units/mL, respectively. The mean volume of the grafted tumor was significantly smaller in test mice than in control mice (75.3 ± 22.9 mm 3 vs 988.6 ± 30.5 mm 3 , t = -18.132, P < 0.05). The inhibition ratio of the tumors was 59.6% in the test group. CD117 positive expression was evident in two cases (20%) in the test group and 10 cases (100%) in the control group (χ 2 = 10.2083, P < 0.005). CONCLUSION: AdEGFP-U6-KIT is successfully constructed, and KIT RNAi mediated with Admax vector system can effectively inhibit the expression of the KIT gene and the growth of GIST in nude mice.展开更多
Despite emerging contemporary biotechnological methods such as gene-and stem cell-based therapy,there are no clinically established therapeutic strategies for neural regeneration after spinal cord injury.Our previous ...Despite emerging contemporary biotechnological methods such as gene-and stem cell-based therapy,there are no clinically established therapeutic strategies for neural regeneration after spinal cord injury.Our previous studies have demonstrated that transplantation of genetically engineered human umbilical cord blood mononuclear cells producing three recombinant therapeutic molecules,including vascular endothelial growth factor(VEGF),glial cell-line derived neurotrophic factor(GDNF),and neural cell adhesion molecule(NCAM)can improve morpho-functional recovery of injured spinal cord in rats and mini-pigs.To investigate the efficacy of human umbilical cord blood mononuclear cells-mediated triple-gene therapy combined with epidural electrical stimulation in the treatment of spinal cord injury,in this study,rats with moderate spinal cord contusion injury were intrathecally infused with human umbilical cord blood mononuclear cells expressing recombinant genes VEGF165,GDNF,NCAM1 at 4 hours after spinal cord injury.Three days after injury,epidural stimulations were given simultaneously above the lesion site at C5(to stimulate the cervical network related to forelimb functions)and below the lesion site at L2(to activate the central pattern generators)every other day for 4 weeks.Rats subjected to the combined treatment showed a limited functional improvement of the knee joint,high preservation of muscle fiber area in tibialis anterior muscle and increased H/M ratio in gastrocnemius muscle 30 days after spinal cord injury.However,beneficial cellular outcomes such as reduced apoptosis and increased sparing of the gray and white matters,and enhanced expression of heat shock and synaptic proteins were found in rats with spinal cord injury subjected to the combined epidural electrical stimulation with gene therapy.This study presents the first proof of principle study of combination of the multisite epidural electrical stimulation with ex vivo triple gene therapy(VEGF,GDNF and NCAM)for treatment of spinal cord injury in rat models.The animal protocols were approved by the Kazan State Medical University Animal Care and Use Committee(approval No.2.20.02.18)on February 20,2018.展开更多
AIM: To examine the effect of intravitreal adenoviral vector-mediated tristetraprolin(Ad-TTP) on VEGF m RNA expression in a rat model of laser-induced choroidal neovascularization.METHODS: Ad-TTP was prepared using a ...AIM: To examine the effect of intravitreal adenoviral vector-mediated tristetraprolin(Ad-TTP) on VEGF m RNA expression in a rat model of laser-induced choroidal neovascularization.METHODS: Ad-TTP was prepared using a commercial kit. Retinal laser-induced photocoagulation(10 spots per eye) was performed on rats in this experimental choroidal neovascularization(CNV) model. Rats were divided into four groups: control(single intravitreal injection of balanced salt solution, n =10), laser-induced CNV(photocoagulation only, n =20), laser-induced CNV plus Ad-TTP injection(photocoagulation plus a single intravitreal Ad-TTP injection, n =20) and Ad-TTP injection only(n =10). Changes in choroidal morphology were evaluated in ten rats in the laser only and the laser plus Ad-TTP groups. Two weeks after laser injury, the size of CNV was calculated by perfusion with high-molecular-weight fluorescein isothiocyanate(FITC)-dextran. VEGF m RNA expression in retina-choroid tissue from ten rats in each group was measured by reverse transcription polymerase chain reaction(RT-PCR). RESULTS: Two weeks after treatment, the area of laser-induced CNV was reduced by approximately 60% in the rats given the Ad-TTP injection compared with that in the laser-only group. There was a tendency toward decreased VEGF m RNA expression in the Ad-TTP injection groups.CONCLUSION: A single intravitreal injection of Ad-TTP significantly suppressed CNV size in this experimental laser-induced CNV model. Ad-TTP injection also decreased VEGF m RNA expression compared with that inthe laser-induced CNV group. The present study is meaningful as the first study to investigate the effect of tristetraprolin delivered via intravitreal injection.展开更多
Dominant intermediate Charcot-Marie-Tooth disease type C(DI-CMTC) is a dominantly inherited neuropathy that has been classified primarily based on motor conduction velocity tests but is now known to involve axonal a...Dominant intermediate Charcot-Marie-Tooth disease type C(DI-CMTC) is a dominantly inherited neuropathy that has been classified primarily based on motor conduction velocity tests but is now known to involve axonal and demyelination features.DI-CMTC is linked to tyrosyl-t RNA synthetase(YARS)-associated neuropathies,which are caused by E196 K and G41 R missense mutations and a single de novo deletion(153-156 del VKQV).It is well-established that these YARS mutations induce neuronal dysfunction,morphological symptoms involving axonal degeneration,and impaired motor performance.The present study is the first to describe a novel mouse model of YARS-mutation-induced neuropathy involving a neuron-specific promoter with a deleted mitochondrial targeting sequence that inhibits the expression of YARS protein in the mitochondria.An adenovirus vector system and in vivo techniques were utilized to express YARS fusion proteins with a Flag-tag in the spinal cord,peripheral axons,and dorsal root ganglia.Following transfection of YARS-expressing viruses,the distributions of wild-type(WT) YARS and E196 K mutant proteins were compared in all expressed regions; G41 R was not expressed.The proportion of Flag/green fluorescent protein(GFP) double-positive signaling in the E196 K mutant-type mice did not significantly differ from that of WT mice in dorsal root ganglion neurons.All adenovirus genes,and even the empty vector without the YARS gene,exhibited GFP-positive signaling in the ventral horn of the spinal cord because GFP in an adenovirus vector is driven by a cytomegalovirus promoter.The present study demonstrated that anatomical differences in tissue can lead to dissimilar expressions of YARS genes.Thus,use of this novel animal model will provide data regarding distributional defects between mutant and WT genes in neurons,the DICMTC phenotype,and potential treatment approaches for this disease.展开更多
Exogenous delivery of nerve growth factor (NGF) promotes neural regeneration. However, the short half-life limits delivery efficacy. Therefore, a long-term, efficient, local delivery tool or scheme is needed. The pu...Exogenous delivery of nerve growth factor (NGF) promotes neural regeneration. However, the short half-life limits delivery efficacy. Therefore, a long-term, efficient, local delivery tool or scheme is needed. The purpose of this study was to construct a functioning, recombinant, adenoviral vector carrying human NGF-β (hNGF-β) DNA, and to measure expression of the constructed vector in vitro and in vivo. rhNGF-β adenoviral vector containing full-length hNGF-β cDNA was generated by homologous recombination in Escherichia CoIL The rhNGF-β adenovirus was packaged and amplified in human embryonic kidney HEK293 cells. Transformation efficiency, expression and function of rhNGF-β adenovirus for primary Schwann cells, Schwann cell lines, human embryonic kidney HEK 293 cells, CRH myoblasts, and NIH3T3 fibroblasts were evaluated. Subsequently, expression of rhNGF-β adenovirus at the peripheral nerve of rat was also assessed. Recombinant adenoviral vector carrying hNGF-β was successfully constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. Green fluorescent protein expression was observed in 90% of rhNGF-β adenovirus-infected cells (primary Schwann cells, Schwann cell line, human embryonic kidney HEK 293 cells, CRH myoblasts, and NIH3T3 fibroblasts) compared with non-infected cells. Total mRNA isolated from rhNGF-β adenovirus-infected cells exhibited strong expression. Maximum NGF release was induced by primary cultured Schwann cells at 4 days after infection, which steadily continued for 14 days. PC-12 cells exposed to media conditioned with rhNGF-β adenovirus-infected Schwann cells exhibited increased neurite extension. In vivo experiment revealed that the injected rhNGF-β adenovirus was transfected into the cells at the injected site and promoted expression of NGF, p75NTR and brain derived neurotrophic factor at the sciatic nerve and dorsal root ganglia.展开更多
Objective: To explore the effect on biological behavior of chemotherapy-resistant tumor cells by human wild-type p53, GM-CSF and B7-1 genes mediated via recombinant adenovirus. Methods: p53-abnormal KB-v200 (VCR resis...Objective: To explore the effect on biological behavior of chemotherapy-resistant tumor cells by human wild-type p53, GM-CSF and B7-1 genes mediated via recombinant adenovirus. Methods: p53-abnormal KB-v200 (VCR resistant) and KB-s (VCR sensitive) cell lines were used as model tumor cells, which are resistant and sensitive to chemotherapeutic drugs respectively. After infected with recombinant adenovirus carrying human wild-type p53, GM-CSF and B7-1 genes, changes in biological behavior (including drug sensitivity) of these two kinds of gene-transduced cancer cells were observed. Results: Both of the cell lines were susceptible to adenovirus, all of three exogenous genes (p53, GM-CSF and B7-1) could be effectively expressed in these cell lines, their growth was suppressed, and apoptosis was induced. The drug-pumping-out function of Pgp glycoprotein on the cytomembrane of drug-resistant KB-v200 cells was markedly affected 48h after transfection of the recombinant adenovirus, revealed by increase of the amount of rhodamine 123 accumulation in the cells. The MTT assay also indicated the reversal of their sensitivity to VCR drugs.In vivo experiment in nude mice it was demonstrated reduction of tumorigenicity of the KB-v200 cells or KB-s cells infected with the recombinant adenovirus, and increase of their sensitivity to VCR. Conclusion: The clinical application of this recombinant adenovirus carrying agents might be more effective in treatment of tumors with multidrug resistance(MDR).展开更多
In mammals, myeloid progenitors infiltrate the developing central nervous system (CNS), through the immature blood-brain barrier (BBB), the ventricular layer or the pial surface migrate and give rise to resident micro...In mammals, myeloid progenitors infiltrate the developing central nervous system (CNS), through the immature blood-brain barrier (BBB), the ventricular layer or the pial surface migrate and give rise to resident microglia. In the mature brain, however, the BBB hampers such recruitment from the blood-stream and long-term establishment of blood borne myeloid cells in the CNS thus appears at best limited. Hematopoietic stem cell-derived microglia, nevertheless, represents a promising tool for the correction of genetic deficits in the brain. We thus investigated the fate of primary human monocytes, and monocyte-derived macrophages, following transplantation into the adult mouse brain overpassing the BBB. Furthermore, we documented the ability of such cells to deliver a lysosomal enzyme into the brain following genetic modification with a recombinant adenoviral vector carrying the human β-glucuronidase cDNA. When implanted into the mouse striatum, the engineered primary cells survived and expressed the transgene for as much as 8 months. Moreover, the donor cells could migrate out of the grafting site and settle along blood vessels or myelin tracts although at limited distance. Migrating donor cells down-regulated the expression of CD14 andHLA DR, suggesting the adoption of a deactivated microglia-like phenotype. Our observations establish the ability of circulating mononuclear phagocytes to integrate into the brain after transplantation and express a transgene on the long term. These cells might thus be employed for autologous transplantation for the delivery of secreted therapeutic proteins in the context of a wide range of brain affections.展开更多
With rapid advances in understanding molecular pathogenesis of human diseases in the era of genome sciences and systems biology,it is anticipated that increasing numbers of therapeutic genes or targets will become ava...With rapid advances in understanding molecular pathogenesis of human diseases in the era of genome sciences and systems biology,it is anticipated that increasing numbers of therapeutic genes or targets will become available for targeted therapies.Despite numerous setbacks,efficacious gene and/or cell-based therapies still hold the great promise to revolutionize the clinical management of human diseases.It is wildly recognized that poor gene delivery is the limiting factor for most in vivo gene therapies.There has been a long-lasting interest in using viral vectors,especially adenoviral vectors,to deliver therapeutic genes for the past two decades.Among all currently available viral vectors,adenovirus is the most efficient gene delivery system in a broad range of cell and tissue types.The applications of adenoviral vectors in gene delivery have greatly increased in number and efficiency since their initial development.In fact,among over 2000 gene therapy clinical trials approved worldwide since 1989,a significant portion of the trials have utilized adenoviral vectors.This review aims to provide a comprehensive overview on the characteristics of adenoviral vectors,including adenoviral biology,approaches to engineering adenoviral vectors,and their applications in clinical and preclinical studies with an emphasis in the areas of cancer treatment,vaccination and regenerative medicine.Current challenges and future directions regarding the use of adenoviral vectors are also discussed.It is expected that the continued improvements in adenoviral vectors should provide great opportunities for cell and gene therapies to live up to its enormous potential in personalized medicine.展开更多
After two decades of ups and downs,gene therapy has recently achieved a milestone in treating patients with Leber’s congenital amaurosis(LCA).LCA is a group of inherited blinding diseases with retinal degeneration an...After two decades of ups and downs,gene therapy has recently achieved a milestone in treating patients with Leber’s congenital amaurosis(LCA).LCA is a group of inherited blinding diseases with retinal degeneration and severe vision loss in early infancy.Mutations in several genes,including RPE65,cause the disease.Using adenoassociated virus as a vector,three independent teams of investigators have recently shown that RPE65 can be delivered to retinal pigment epithelial cells of LCA patients by subretinal injections resulting in clinical benefits without side effects.However,considering the whole field of gene therapy,there are still major obstacles to clinical applications for other diseases.These obstacles include innate and immune barriers to vector delivery,toxicity of vectors and the lack of sustained therapeutic gene expression.Therefore,new strategies are needed to overcome these hurdles for achieving safe and effective gene therapy.In this article,we shall review the major advancements over the past two decades and,using lung gene therapy as an example,discuss the current obstacles and possible solutions to provide a roadmap for future gene therapy research.展开更多
基金supported by grants from the Science and Technology Foundation of Nanjing Medical University(No.09NJMUZ15)from the Natural Science Foundation of Jiangsu Province(No.10KJB31008)
文摘We sought to construct the adenoviral vector carrying the gene encoding mouse telomerase reverse transcriptase(mTERT),as well as detect its expression and effect on the proliferation of neuronal stem cells.mTERT was am-plified by RT-PCR and then the eukaryotic expression vector of pDC-EGFP-TERT was constructed.After DNA sequence analysis,we detected that there were 293 cells transfected with pDC-EGFP-TERT and helper adenovirus plasmid pBHG lox ΔE1,and three Cre using Lipofectamine 2000 mediation,named Ad-mTERT-GFP,to pack-age adenoviral particles.The Ad-mTERT-GFP was used to infect neuronal stem cells and then the expression and activity of mTERT were detected.In addition,Bromodeoxyuridine labeling test identified the impact of mTERT overexpression on proliferation of neuronal stem cells.The recombinant adenoviral vector confirmed that mTERT was successfully constructed.Overexpression of mTERT stimulated the proliferation of neuronal stem cells both in vitro and in vivo.mTERT overexpression via adenoviral vector carrying mTERT cDNA upregulated the ability of proliferation in neuronal stem cells.
基金National administration of traditional Chinese medicine(No.2019xzzx-ek004)Shandong province development plan of TCM science and technology(No.2019-0972)
文摘Adenoviral pneumonia is one of the more serious types of community-acquired pneumonia in children.It easily develops into severe pneumonia with many extrapulmonary complications and a high mortality rate.However,there is still no effective treatment for adenovirus.Therapeutic drugs are conducive to clinical treatment.Traditional Chinese medicine often achieves satisfactory results through staged syndrome differentiation.Currently,heat,sputum,stasis,and poison are considered to be the main pathological products.Fever,phlegm,and stasis are present in its development.Analyze the progress of traditional Chinese and western medicine in children's adenoviral pneumonia,and have a clearer understanding of the law of syndrome differentiation and its prescription medication,providing ideas for clinical treatment of adenoviral pneumonia.
基金the National Natural Science Foundation of China, No. 39900048the Natural Science Foundation of Guangdong Province, No.010721
文摘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.
基金supported by grants, R01-AR063631 (JPS) and F31-AR064673 (AMB), from the National Institutes of Health/National Institute for Arthritis, Musculoskeletal and Skin Diseases
文摘We report here a method for the use of poly-L-lysine (PLL) to markedly improve the adenoviral transduction efficiency of primary murine osteoblasts and bone marrow stromal cells (BMSCs) in culture and in situ, which are typically difficult to transduce. We show by fluorescence microscopy and flow cytometry that the addition of PLL to the viral-containing medium significantly increases the number of green fluorescence protein (GFP)-positive osteoblasts and BMSCs transduced with an enhanced GFP-expressing adenovirus. We also demonstrate that PLL can greatly enhance the adenoviral transduction of osteoblasts and osteocytes in situ in ex vivo tibia and calvaria, as well as in long bone fragments. In addition, we validate that PLL can improve routine adenoviral transduction studies by permitting the use of low multiplicities of infection to obtain the desired biologic effect. Ultimately, the use of PLL to facilitate adenoviral gene transfer in osteogenic cells can provide a cost-effective means of performing efficient gene transfer studies in the context of bone research.
文摘Vaccines are an integral part of veterinary disease prevention. However there are still a significant number of veterinary diseases for which vaccines do not currently exist or where currently available vaccines do not provide adequate immunity. Adenoviruses have transitioned from tools for gene replacement therapy to bona fide vaccine delivery vehicles because of their ability to elicit potent cell-mediated and humoral responses making them ideal for use against viruses and other intracellular pathogens. Adenoviral vector based vaccines are likely to play a significant role in overcoming these problems in the future. However, this vector is under utilized in veterinary vaccine development at this time. This review focuses on adenoviral vector based vaccines developed to date and explores the potential for veterinary vaccine development based upon this platform: advantages and potential disadvantages of this technology are discussed as well as the potential for developing efficacious commercial veterinary adenoviral vector based vaccines.
基金This study was supported by grants from the Natural Science Foundation of Fujian Province (No. 2008J0091 and No. 080916).
文摘Background Human interleukin-10 (hlL-10) is a cytokine synthesis inhibitory factor,which is involved in various immune responses.The purpose of this study was to construct an adenoviral vector carrying the hlL-10 gene for expression of biologically active hlL-10 in rat bone marrow mesenchymal stem cells (rMSCs).Methods A pSNAV2.0-hlL10 plasmid was used as a template to obtain a hlL-10 cDNA fragment that was subcloned by restriction enzyme digestion and ligation into a pDC316-IRES-EGFP-lacZ alpha plasmid carrying an enhanced green fluorescent protein (EGFP) marker gene.The pDC316-hlL-10-IRES-EGFP plasmid was linearized by Pmel digestion and used to transfect HEK293 packaging cells using the adenovirus packaging system AdMax.Virus particles were amplified by repeatedly infecting HEK293 cells with the seed virus and then purified by ion exchange.After the number of virus particles and titer was determined,rMSCs were infected with the adenoviral vector.The infection rate was determined by fluorescence microscopy and flow cytometry,and hlL-10 protein expression in rMSCs was measured by Western blotting.Results The virus particle concentration,OD260/280 value and virus titer of the amplified and purified recombinant adenovirus were 3.2×1011 VP/ml,approximately 2.0,and 1.1×1010 TCID50/ml,respectively.Bright green fluorescence was observed by fluorescence microscopy and flow cytometry in the recombinant adenovirus-infected rMSCs.GFP expression was considered the multiplicity of infection (MOI) and was time-dependent.The infection rate was 92.9% at 100 MOI.Conclusions A bicistrenic recombinant adenoviral vector for hlL-10 and EGFP gene expression were successfully constructed.The infection rate of rMSCs by the adenovirus was high (92.9% at 100 MOI) and the target gene hlL-10 was highly expressed in cells.The present study provides an experimental basis for further research of immunosuppressive therapy using hlL-10.The expression level of hlL-10 protein as detected by Western blotting was also MOI- and time-dependent.
基金China-France Collaboration Fund ( PRA B01 - 07 ) and Natural Science Fund of Guangdong Province, China (A101689).
文摘Objective To prevent Graft-versus-host disease (GVHD) in rat model, we evaluated the feasibility of mesenchymal stem cells (MSCs) as a gene transfer target and studied the efficiency of recombinant adenovirus mediated gene therapy.Methods We constructed the recombinant adenovirus containing CTLA4lg gene. Rat MSCs of passages 3-5 were infected by the adenovirus, and the transfection efficiency was monitored by GFP markers. We performed flow cytometric analysis, immunohistochemical and Western blotting analysis to identify the CTLA4lg expression. The gene transferred MSCs were tested for their ability to inhibit the allogeneic lymphocyte response in vitro and to prevent GVHD in a rat model.Results Recombinant adenovirus pAd-CTLA4lg was correctly constructed and confirmed. After MSCs were infected by the adenovirus, the CTLA4lg protein was detected not only in transgenic MSCs, but also in the culture medium. In a mixed lymphocytes response (MLR) test, the transgenic MSCs could significantly inhibit the allogeneic lymphocyte response compared with the control groups (P < 0. 05) . A model of GVHD was developed by transplanting bone marrow cells and spleen lymphocytes of F344 rats to lethally irradiated SD rats. The onset of GVHD could be ameliorated or prevented by co-administration of transgenic MSCs. All the rats in the control groups suffered severe acute GVHD. CTLA4lg expression was observed in the liver, intestine, kidney and spleen 30 days post- transplantation.Conclusions Our results indicate that adenoviral vectors could efficiently transfer CTLA4lg gene into MSCs and sustain long-term stable expression in vitro and in vivo.
文摘There have been significant advancements in the field of retinal gene therapy in the past several years.In particular,therapeutic efficacy has been achieved in three separate human clinical trials conducted to assess the ability of adeno-associated viruses(AAV)to treat of a type of Leber’s congenital amaurosis caused by RPE65 mutations.However,despite the success of retinal gene therapy with AAV,challenges remain for delivering large therapeutic genes or genes requiring long DNA regulatory elements for controlling their expression.For example,Stargardt’s disease,a form of juvenile macular degeneration,is caused by defects in ABCA4,a gene that is too large to be packaged in AAV.Therefore,we investigated the ability of helper dependent adenovirus(HD-Ad)to deliver genes to the retina as it has a much larger transgene capacity.Using an EGFP reporter,our results showed that HD-Ad can transduce the entire retinal epithelium of a mouse using a dose of only 1105 infectious units and maintain transgene expression for at least 4 months.The results demonstrate that HD-Ad has the potential to be an effective vector for the gene therapy of the retina.
文摘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
基金supported by a grant from the Russian Science Foundation,No. 16-15-00010 (to RRI)funded by government assignment for FRC Kazan Scientific Center of RAS
文摘Post-traumatic spinal cord remodeling includes both degenerating and regenerating processes,which affect the potency of the functional recovery after spinal cord injury(SCI).Gene therapy for spinal cord injury is proposed as a promising therapeutic strategy to induce positive changes in remodeling of the affected neural tissue.In our previous studies for delivering the therapeutic genes at the site of spinal cord injury,we developed a new approach using an autologous leucoconcentrate transduced ex vivo with chimeric adenoviruses(Ad5/35)carrying recombinant cDNA.In the present study,the efficacy of the intravenous infusion of an autologous genetically-enriched leucoconcentrate simultaneously producing recombinant vascular endothelial growth factor(VEGF),glial cell line-derived neurotrophic factor(GDNF),and neural cell adhesion molecule(NCAM)was evaluated with regard to the molecular and cellular changes in remodeling of the spinal cord tissue at the site of damage in a model of mini-pigs with moderate spinal cord injury.Experimental animals were randomly divided into two groups of 4 pigs each:the therapeutic(infused with the leucoconcentrate simultaneously transduced with a combination of the three chimeric adenoviral vectors Ad5/35‐VEGF165,Ad5/35‐GDNF,and Ad5/35‐NCAM1)and control groups(infused with intact leucoconcentrate).The morphometric and immunofluorescence analysis of the spinal cord regeneration in the rostral and caudal segments according to the epicenter of the injury in the treated animals compared to the control mini-pigs showed:(1)higher sparing of the grey matter and increased survivability of the spinal cord cells(lower number of Caspase-3-positive cells and decreased expression of Hsp27);(2)recovery of synaptophysin expression;(3)prevention of astrogliosis(lower area of glial fibrillary acidic protein-positive astrocytes and ionized calcium binding adaptor molecule 1-positive microglial cells);(4)higher growth rates of regeneratingβIII-tubulin-positive axons accompanied by a higher number of oligodendrocyte transcription factor 2-positive oligodendroglial cells in the lateral corticospinal tract region.These results revealed the efficacy of intravenous infusion of the autologous genetically-enriched leucoconcentrate producing recombinant VEGF,GDNF,and NCAM in the acute phase of spinal cord injury on the positive changes in the post-traumatic remodeling nervous tissue at the site of direct injury.Our data provide a solid platform for a new ex vivo gene therapy for spinal cord injury and will facilitate further translation of regenerative therapies in clinical neurology.
基金Supported by Grants from the Chinese Ministry of Education Funds, No. 20070558266Guangdong Provincial Funds for Science and Technology, No. 2007B 031515004
文摘AIM: To investigate a therapeutic method for gastrointestinal stromal tumor (GIST) based on KIT RNA interference (RNAi) with AdMax adenovirus. METHODS: KIT short hairpin RNA (shRNA), whose lateral sides were decorated with restriction endonuclease sequences, was designed. T 4 DNA ligase catalyzed the joint of the KIT shRNA and the green fluorescent protein-containing PDC316-EGFP-U6 to form PDC316EGFP-U6-KIT. Homologous recombination of AdEGFPU6-KIT was performed with the AdMax system. Heterotopically transplanted GISTs were established in nude mice. AdEGFP-U6-KIT was intratumorally injected. The volume, inhibition ratio of tumor and CD117 expression of GIST graft tumor in nude mice were compared between test and control groups. RESULTS: The length of KIT shRNA was determined to be about 50bp by agarose electrophoresis. Gene se-quencing detected the designed KIT RNAi sequence in PDC316-EGFP-U6-KIT. After transfection with AdEGFPU6-KIT, 293 cells displayed green fluorescence. The physical and infective titers of AdEGFP-U6-KIT were 5 × 10 11 viral particles/mL and 5.67 × 10 7 plaque forming units/mL, respectively. The mean volume of the grafted tumor was significantly smaller in test mice than in control mice (75.3 ± 22.9 mm 3 vs 988.6 ± 30.5 mm 3 , t = -18.132, P < 0.05). The inhibition ratio of the tumors was 59.6% in the test group. CD117 positive expression was evident in two cases (20%) in the test group and 10 cases (100%) in the control group (χ 2 = 10.2083, P < 0.005). CONCLUSION: AdEGFP-U6-KIT is successfully constructed, and KIT RNAi mediated with Admax vector system can effectively inhibit the expression of the KIT gene and the growth of GIST in nude mice.
基金supported by the grant of Russian Science Foundation,No.16-15-00010(to RRI)supported by the Russian Government Program of Competitive Growth of Kazan Federal University。
文摘Despite emerging contemporary biotechnological methods such as gene-and stem cell-based therapy,there are no clinically established therapeutic strategies for neural regeneration after spinal cord injury.Our previous studies have demonstrated that transplantation of genetically engineered human umbilical cord blood mononuclear cells producing three recombinant therapeutic molecules,including vascular endothelial growth factor(VEGF),glial cell-line derived neurotrophic factor(GDNF),and neural cell adhesion molecule(NCAM)can improve morpho-functional recovery of injured spinal cord in rats and mini-pigs.To investigate the efficacy of human umbilical cord blood mononuclear cells-mediated triple-gene therapy combined with epidural electrical stimulation in the treatment of spinal cord injury,in this study,rats with moderate spinal cord contusion injury were intrathecally infused with human umbilical cord blood mononuclear cells expressing recombinant genes VEGF165,GDNF,NCAM1 at 4 hours after spinal cord injury.Three days after injury,epidural stimulations were given simultaneously above the lesion site at C5(to stimulate the cervical network related to forelimb functions)and below the lesion site at L2(to activate the central pattern generators)every other day for 4 weeks.Rats subjected to the combined treatment showed a limited functional improvement of the knee joint,high preservation of muscle fiber area in tibialis anterior muscle and increased H/M ratio in gastrocnemius muscle 30 days after spinal cord injury.However,beneficial cellular outcomes such as reduced apoptosis and increased sparing of the gray and white matters,and enhanced expression of heat shock and synaptic proteins were found in rats with spinal cord injury subjected to the combined epidural electrical stimulation with gene therapy.This study presents the first proof of principle study of combination of the multisite epidural electrical stimulation with ex vivo triple gene therapy(VEGF,GDNF and NCAM)for treatment of spinal cord injury in rat models.The animal protocols were approved by the Kazan State Medical University Animal Care and Use Committee(approval No.2.20.02.18)on February 20,2018.
基金Supported by Biomedical Research Institute Fund (GNUHBRIF-2013-0002) from the Gyeongsang National University Hospital
文摘AIM: To examine the effect of intravitreal adenoviral vector-mediated tristetraprolin(Ad-TTP) on VEGF m RNA expression in a rat model of laser-induced choroidal neovascularization.METHODS: Ad-TTP was prepared using a commercial kit. Retinal laser-induced photocoagulation(10 spots per eye) was performed on rats in this experimental choroidal neovascularization(CNV) model. Rats were divided into four groups: control(single intravitreal injection of balanced salt solution, n =10), laser-induced CNV(photocoagulation only, n =20), laser-induced CNV plus Ad-TTP injection(photocoagulation plus a single intravitreal Ad-TTP injection, n =20) and Ad-TTP injection only(n =10). Changes in choroidal morphology were evaluated in ten rats in the laser only and the laser plus Ad-TTP groups. Two weeks after laser injury, the size of CNV was calculated by perfusion with high-molecular-weight fluorescein isothiocyanate(FITC)-dextran. VEGF m RNA expression in retina-choroid tissue from ten rats in each group was measured by reverse transcription polymerase chain reaction(RT-PCR). RESULTS: Two weeks after treatment, the area of laser-induced CNV was reduced by approximately 60% in the rats given the Ad-TTP injection compared with that in the laser-only group. There was a tendency toward decreased VEGF m RNA expression in the Ad-TTP injection groups.CONCLUSION: A single intravitreal injection of Ad-TTP significantly suppressed CNV size in this experimental laser-induced CNV model. Ad-TTP injection also decreased VEGF m RNA expression compared with that inthe laser-induced CNV group. The present study is meaningful as the first study to investigate the effect of tristetraprolin delivered via intravitreal injection.
基金supported by the National Research Foundation(NRF)of Korea grant funded by Korean Government(MEST)(No.2011-0030072)
文摘Dominant intermediate Charcot-Marie-Tooth disease type C(DI-CMTC) is a dominantly inherited neuropathy that has been classified primarily based on motor conduction velocity tests but is now known to involve axonal and demyelination features.DI-CMTC is linked to tyrosyl-t RNA synthetase(YARS)-associated neuropathies,which are caused by E196 K and G41 R missense mutations and a single de novo deletion(153-156 del VKQV).It is well-established that these YARS mutations induce neuronal dysfunction,morphological symptoms involving axonal degeneration,and impaired motor performance.The present study is the first to describe a novel mouse model of YARS-mutation-induced neuropathy involving a neuron-specific promoter with a deleted mitochondrial targeting sequence that inhibits the expression of YARS protein in the mitochondria.An adenovirus vector system and in vivo techniques were utilized to express YARS fusion proteins with a Flag-tag in the spinal cord,peripheral axons,and dorsal root ganglia.Following transfection of YARS-expressing viruses,the distributions of wild-type(WT) YARS and E196 K mutant proteins were compared in all expressed regions; G41 R was not expressed.The proportion of Flag/green fluorescent protein(GFP) double-positive signaling in the E196 K mutant-type mice did not significantly differ from that of WT mice in dorsal root ganglion neurons.All adenovirus genes,and even the empty vector without the YARS gene,exhibited GFP-positive signaling in the ventral horn of the spinal cord because GFP in an adenovirus vector is driven by a cytomegalovirus promoter.The present study demonstrated that anatomical differences in tissue can lead to dissimilar expressions of YARS genes.Thus,use of this novel animal model will provide data regarding distributional defects between mutant and WT genes in neurons,the DICMTC phenotype,and potential treatment approaches for this disease.
基金the Korea Health R&D Project,A080863,a Grant by Ministry of Health and Welfare,Republic of Korea
文摘Exogenous delivery of nerve growth factor (NGF) promotes neural regeneration. However, the short half-life limits delivery efficacy. Therefore, a long-term, efficient, local delivery tool or scheme is needed. The purpose of this study was to construct a functioning, recombinant, adenoviral vector carrying human NGF-β (hNGF-β) DNA, and to measure expression of the constructed vector in vitro and in vivo. rhNGF-β adenoviral vector containing full-length hNGF-β cDNA was generated by homologous recombination in Escherichia CoIL The rhNGF-β adenovirus was packaged and amplified in human embryonic kidney HEK293 cells. Transformation efficiency, expression and function of rhNGF-β adenovirus for primary Schwann cells, Schwann cell lines, human embryonic kidney HEK 293 cells, CRH myoblasts, and NIH3T3 fibroblasts were evaluated. Subsequently, expression of rhNGF-β adenovirus at the peripheral nerve of rat was also assessed. Recombinant adenoviral vector carrying hNGF-β was successfully constructed and confirmed by restriction endonuclease analysis and DNA sequence analysis. Green fluorescent protein expression was observed in 90% of rhNGF-β adenovirus-infected cells (primary Schwann cells, Schwann cell line, human embryonic kidney HEK 293 cells, CRH myoblasts, and NIH3T3 fibroblasts) compared with non-infected cells. Total mRNA isolated from rhNGF-β adenovirus-infected cells exhibited strong expression. Maximum NGF release was induced by primary cultured Schwann cells at 4 days after infection, which steadily continued for 14 days. PC-12 cells exposed to media conditioned with rhNGF-β adenovirus-infected Schwann cells exhibited increased neurite extension. In vivo experiment revealed that the injected rhNGF-β adenovirus was transfected into the cells at the injected site and promoted expression of NGF, p75NTR and brain derived neurotrophic factor at the sciatic nerve and dorsal root ganglia.
文摘Objective: To explore the effect on biological behavior of chemotherapy-resistant tumor cells by human wild-type p53, GM-CSF and B7-1 genes mediated via recombinant adenovirus. Methods: p53-abnormal KB-v200 (VCR resistant) and KB-s (VCR sensitive) cell lines were used as model tumor cells, which are resistant and sensitive to chemotherapeutic drugs respectively. After infected with recombinant adenovirus carrying human wild-type p53, GM-CSF and B7-1 genes, changes in biological behavior (including drug sensitivity) of these two kinds of gene-transduced cancer cells were observed. Results: Both of the cell lines were susceptible to adenovirus, all of three exogenous genes (p53, GM-CSF and B7-1) could be effectively expressed in these cell lines, their growth was suppressed, and apoptosis was induced. The drug-pumping-out function of Pgp glycoprotein on the cytomembrane of drug-resistant KB-v200 cells was markedly affected 48h after transfection of the recombinant adenovirus, revealed by increase of the amount of rhodamine 123 accumulation in the cells. The MTT assay also indicated the reversal of their sensitivity to VCR drugs.In vivo experiment in nude mice it was demonstrated reduction of tumorigenicity of the KB-v200 cells or KB-s cells infected with the recombinant adenovirus, and increase of their sensitivity to VCR. Conclusion: The clinical application of this recombinant adenovirus carrying agents might be more effective in treatment of tumors with multidrug resistance(MDR).
基金funded by the CNRS,The INSERM and Vaincre les Maladie Lysosomiales(vml-asso.org).
文摘In mammals, myeloid progenitors infiltrate the developing central nervous system (CNS), through the immature blood-brain barrier (BBB), the ventricular layer or the pial surface migrate and give rise to resident microglia. In the mature brain, however, the BBB hampers such recruitment from the blood-stream and long-term establishment of blood borne myeloid cells in the CNS thus appears at best limited. Hematopoietic stem cell-derived microglia, nevertheless, represents a promising tool for the correction of genetic deficits in the brain. We thus investigated the fate of primary human monocytes, and monocyte-derived macrophages, following transplantation into the adult mouse brain overpassing the BBB. Furthermore, we documented the ability of such cells to deliver a lysosomal enzyme into the brain following genetic modification with a recombinant adenoviral vector carrying the human β-glucuronidase cDNA. When implanted into the mouse striatum, the engineered primary cells survived and expressed the transgene for as much as 8 months. Moreover, the donor cells could migrate out of the grafting site and settle along blood vessels or myelin tracts although at limited distance. Migrating donor cells down-regulated the expression of CD14 andHLA DR, suggesting the adoption of a deactivated microglia-like phenotype. Our observations establish the ability of circulating mononuclear phagocytes to integrate into the brain after transplantation and express a transgene on the long term. These cells might thus be employed for autologous transplantation for the delivery of secreted therapeutic proteins in the context of a wide range of brain affections.
基金Research in the authors’laboratories was supported in part by research grants from the National Institutes of Health(AT004418,DE020140 to TCH and RRR)the US Department of Defense(OR130096 to JMW)+3 种基金the Scoliosis Research Society(TCH and MJL)the 973 Program of the Ministry of Science and Technology(MOST)of China(#2011CB707906 to TCH)The reported work was also supported in part by The University of Chicago Cancer Center Support Grant(P30CA014599)the National Center for Advancing Translational Sciences of the National Institutes of Health through Grant Number UL1 TR000430.
文摘With rapid advances in understanding molecular pathogenesis of human diseases in the era of genome sciences and systems biology,it is anticipated that increasing numbers of therapeutic genes or targets will become available for targeted therapies.Despite numerous setbacks,efficacious gene and/or cell-based therapies still hold the great promise to revolutionize the clinical management of human diseases.It is wildly recognized that poor gene delivery is the limiting factor for most in vivo gene therapies.There has been a long-lasting interest in using viral vectors,especially adenoviral vectors,to deliver therapeutic genes for the past two decades.Among all currently available viral vectors,adenovirus is the most efficient gene delivery system in a broad range of cell and tissue types.The applications of adenoviral vectors in gene delivery have greatly increased in number and efficiency since their initial development.In fact,among over 2000 gene therapy clinical trials approved worldwide since 1989,a significant portion of the trials have utilized adenoviral vectors.This review aims to provide a comprehensive overview on the characteristics of adenoviral vectors,including adenoviral biology,approaches to engineering adenoviral vectors,and their applications in clinical and preclinical studies with an emphasis in the areas of cancer treatment,vaccination and regenerative medicine.Current challenges and future directions regarding the use of adenoviral vectors are also discussed.It is expected that the continued improvements in adenoviral vectors should provide great opportunities for cell and gene therapies to live up to its enormous potential in personalized medicine.
基金Research in our laboratories was supported by Operating Grants from the Canadian Institutes of Health Research,the Canadian Cystic Fibrosis Foundation,and the Foundation Fighting Blindness-Canada.
文摘After two decades of ups and downs,gene therapy has recently achieved a milestone in treating patients with Leber’s congenital amaurosis(LCA).LCA is a group of inherited blinding diseases with retinal degeneration and severe vision loss in early infancy.Mutations in several genes,including RPE65,cause the disease.Using adenoassociated virus as a vector,three independent teams of investigators have recently shown that RPE65 can be delivered to retinal pigment epithelial cells of LCA patients by subretinal injections resulting in clinical benefits without side effects.However,considering the whole field of gene therapy,there are still major obstacles to clinical applications for other diseases.These obstacles include innate and immune barriers to vector delivery,toxicity of vectors and the lack of sustained therapeutic gene expression.Therefore,new strategies are needed to overcome these hurdles for achieving safe and effective gene therapy.In this article,we shall review the major advancements over the past two decades and,using lung gene therapy as an example,discuss the current obstacles and possible solutions to provide a roadmap for future gene therapy research.