Obiective TO improve the plasmid vectors in gene therapy, adeno - associated virus (AAV) basedplasmid expressing vectors containing hIL - 2 gene or mIFN-γ gene were constructed and its expression intransfected cells ...Obiective TO improve the plasmid vectors in gene therapy, adeno - associated virus (AAV) basedplasmid expressing vectors containing hIL - 2 gene or mIFN-γ gene were constructed and its expression intransfected cells was studied. Methods By means of step to step cloning, promoter CMVp was placed at thedownstream of 5’ inverted terminal repeat from AAV (AAV - ITR) of pAP, hIL - 2 gene or mIFN -γ gene insertedinto pAC between CMVp and polyA. Then intron A was inserted into pAC - hIL - 2 or pAC- mIFN-γ betweenCMVp and IL - 2 gene or IFNγ gene to construct pAI- hIL - 2 or pAI- mIFN -γ. Liposome - plasmid complexeswere formed by mixing Dosper with these AAV- based plasmids containing hIL - 2 gene or mIFN- γgene. Results High biotogical activities of IL - 2 or IFN- γ could be detected in the supernatants of NIH3T3 andMM45T Li cells after transfection. Insertion of intron A into pAC- hIL - 2 or pAC- mIFN - γ improved theexpression of IL - 2 or IFN- γ. Conclusion These data demonstrated that the constructed AAV-based plasmidexpressing vectors could ejlciently express therapeutic genes in cultured cells and could be used as a nonviral genetransfer system in human gene therapy.展开更多
Cellular immune responses,particularly those associated with CD3+CD8+ cytotoxic T lymphocytes (CTL),are critical factors in controlling viral infection.Nasopharyngeal carcinoma (NPC) is closely associated with persist...Cellular immune responses,particularly those associated with CD3+CD8+ cytotoxic T lymphocytes (CTL),are critical factors in controlling viral infection.Nasopharyngeal carcinoma (NPC) is closely associated with persistent Epstein-Barr virus (EBV) infection.NPC vaccine studies have focused on enhancing specific antiviral CTL responses.In this study,three vaccines capable of expressing the EBV-latent membrane protein 2 (LMP2) (a DNA vector,an adeno-associated virus (AAV) vector,and a replication-defective adenovirus serotype 5 (Ad5) vector) were respectively used to immunize female Balb/c mice (4-6 weeks old) at weeks 0,2 and 4,either alone or in combination.Our results suggest that combined immunization with DNA,AAV,and adenovirus vector vaccines induced specific cellular immunity more effectively than any of these vectors alone or a combination of two of the three,constituting a sound vaccine strategy for the prevention and treatment of NPC.展开更多
Objective： Although the development of trastuzumab has improved the outlook for women with human epidermal growth factor receptor 2 （HER2）-positive breast cancer, the resistance to anti-HER2 therapy is a growing cl...Objective： Although the development of trastuzumab has improved the outlook for women with human epidermal growth factor receptor 2 （HER2）-positive breast cancer, the resistance to anti-HER2 therapy is a growing clinical dilemma. We aim to determine whether HER2-specific T cells generated from dendritic cells （DCs） modified with HER2 gene could effectively kill the HER2-positive breast cancer cells, especially the trastuzumab-resistant cells. Methods： The peripheral blood mononuclear cells （PBMCs） from healthy donors, whose HLA haplotypes were compatible with the tumor cell lines, were transfected with reconstructive human adeno-association virus （rhAAV/HER2） to obtain the specific killing activities of T cells, and were evaluated by lactate dehydrogenase （LDH） releasing assay. Results： Trastuzumab produced a significant inhibiting effect on SK-BR-3, the IC50 was 100ng/ml. MDA-MB-453 was resistant to trastuzumab even at a concentration of 10,000 ng/ml in vitro. HER2-specific T lymphocytes killed effectively SK-BR-3 [（69.86±13.41）%] and MDA-MB-453 [（78.36±10.68）%] at 40：1 （effector：target ratio, E：T）, but had no significant cytotoxicity against HER2-negative breast cancer cell lines MDA-MB-231 or MCF-7 （less than 10%）. Conclusion： The study showed that HER2-specific T lymphocytes generated from DCs modified by rhAAV/HER2 could kill HER2-positive breast cancer cell lines in a HER2-dependent manner, and result in significantly high inhibition rates on the intrinsic trastuzumab-resistant cell line MDA-MB-453 and the tastuzumab-sensitive cell line SK-BR-3. These results imply that this immunotherapy might be a potential treatment to HER2-positive breast cancer.展开更多
Background and aim Hepatic ischemia–reperfusion injury(IRI)is a significant challenge in liver transplantation,trauma,hypovolemic shock,and hepatectomy,with limited effective interventions available.This study aimed ...Background and aim Hepatic ischemia–reperfusion injury(IRI)is a significant challenge in liver transplantation,trauma,hypovolemic shock,and hepatectomy,with limited effective interventions available.This study aimed to investigate the role of leukocyte cell-derived chemotaxin 2(LECT2)in hepatic IRI and assess the therapeutic potential of Lect2-short hairpin RNA(shRNA)delivered through adeno-associated virus(AAV)vectors.Materials and methods This study analyzed human liver and serum samples from five patients undergoing the Pringle maneuver.Lect2-knockout and C57BL/6J mice were used.Hepatic IRI was induced by clamping the hepatic pedicle.Treatments included recombinant human LECT2(rLECT2)and AAV-Lect2-shRNA.LECT2 expression levels and serum biomarkers including alanine aminotransferase(ALT),aspartate aminotransferase(AST),creatinine,and blood urea nitrogen(BUN)were measured.Histological analysis of liver necrosis and quantitative reverse-transcription polymerase chain reaction were performed.Results Serum and liver LECT2 levels were elevated during hepatic IRI.Serum LECT2 protein and mRNA levels increased post reperfusion.Lect2-knockout mice had reduced weight loss;hepatic necrosis;and serum ALT,AST,creatinine,and BUN levels.rLECT2 treatment exacerbated weight loss,hepatic necrosis,and serum biomarkers(ALT,AST,creatinine,and BUN).AAV-Lect2-shRNA treatment significantly reduced weight loss,hepatic necrosis,and serum biomarkers(ALT,AST,creatinine,and BUN),indicating therapeutic potential.Conclusions Elevated LECT2 levels during hepatic IRI increased liver damage.Genetic knockout or shRNA-mediated knockdown of Lect2 reduced liver damage,indicating its therapeutic potential.AAV-mediated Lect2-shRNA delivery mitigated hepatic IRI,offering a potential new treatment strategy to enhance clinical outcomes for patients undergoing liver-related surgeries or trauma.展开更多
Background Spinal cord injury （SCI） is a serious neurological injury that often leads to permanent disabilities for the victims. The aim of this study was to determine the effects of glial-derived neurotrophic fact...Background Spinal cord injury （SCI） is a serious neurological injury that often leads to permanent disabilities for the victims. The aim of this study was to determine the effects of glial-derived neurotrophic factor （GDNF） mediated by recombinant adeno-associated virus type 2 （rAAV2） alone or in combination with early rehabilitation training on SCI. Methods SCI was induced on the T8-9 segments of the spinal cord by laminectomy in adult male Sprague-Dawley rats. Then besides the sham operation group, the SCI rats were randomly divided into four groups： natural healing group, gene therapy group, rehabilitation training group, and combination therapy group （gene therapy in combination with rehabilitation training）. Motor dysfunction, protein expression of GDNF, edema formation, and cell injury were examined 7, 14, and 21 days after trauma. Results The topical application of rAAV-GDNF-GFP resulted in strong expression of GDNF, especially after the 14th day, and could protect the motor neuron cells. Early rehabilitative treatment resulted in significantly improved motor function, reduced edema formation, and protected the cells from injury, especially after the 7th and 14th days, and increased the GDNF expression in the damaged area, which was most evident after Day 14. The combined application of GDNF and early rehabilitative treatment after SCI resulted in a significant reduction in spinal cord pathology and motor dysfunction after the 7th and 14th days. Conclusion These observations suggest that rAAV2 gene therapy in combination with rehabilitation therapy has potential clinical value for the treatment of SCI.展开更多
文摘Obiective TO improve the plasmid vectors in gene therapy, adeno - associated virus (AAV) basedplasmid expressing vectors containing hIL - 2 gene or mIFN-γ gene were constructed and its expression intransfected cells was studied. Methods By means of step to step cloning, promoter CMVp was placed at thedownstream of 5’ inverted terminal repeat from AAV (AAV - ITR) of pAP, hIL - 2 gene or mIFN -γ gene insertedinto pAC between CMVp and polyA. Then intron A was inserted into pAC - hIL - 2 or pAC- mIFN-γ betweenCMVp and IL - 2 gene or IFNγ gene to construct pAI- hIL - 2 or pAI- mIFN -γ. Liposome - plasmid complexeswere formed by mixing Dosper with these AAV- based plasmids containing hIL - 2 gene or mIFN- γgene. Results High biotogical activities of IL - 2 or IFN- γ could be detected in the supernatants of NIH3T3 andMM45T Li cells after transfection. Insertion of intron A into pAC- hIL - 2 or pAC- mIFN - γ improved theexpression of IL - 2 or IFN- γ. Conclusion These data demonstrated that the constructed AAV-based plasmidexpressing vectors could ejlciently express therapeutic genes in cultured cells and could be used as a nonviral genetransfer system in human gene therapy.
基金supported by the National High Technology Research and Development Program of China(Grant No. 2006AA02A229)
文摘Cellular immune responses,particularly those associated with CD3+CD8+ cytotoxic T lymphocytes (CTL),are critical factors in controlling viral infection.Nasopharyngeal carcinoma (NPC) is closely associated with persistent Epstein-Barr virus (EBV) infection.NPC vaccine studies have focused on enhancing specific antiviral CTL responses.In this study,three vaccines capable of expressing the EBV-latent membrane protein 2 (LMP2) (a DNA vector,an adeno-associated virus (AAV) vector,and a replication-defective adenovirus serotype 5 (Ad5) vector) were respectively used to immunize female Balb/c mice (4-6 weeks old) at weeks 0,2 and 4,either alone or in combination.Our results suggest that combined immunization with DNA,AAV,and adenovirus vector vaccines induced specific cellular immunity more effectively than any of these vectors alone or a combination of two of the three,constituting a sound vaccine strategy for the prevention and treatment of NPC.
基金supported by the grant from the National"973"Basic Research Program of China(No.2009CB521703)Medical Oncology Leadership of Beijing Municipal Government Health Burean(No.2009-2-16)
文摘Objective： Although the development of trastuzumab has improved the outlook for women with human epidermal growth factor receptor 2 （HER2）-positive breast cancer, the resistance to anti-HER2 therapy is a growing clinical dilemma. We aim to determine whether HER2-specific T cells generated from dendritic cells （DCs） modified with HER2 gene could effectively kill the HER2-positive breast cancer cells, especially the trastuzumab-resistant cells. Methods： The peripheral blood mononuclear cells （PBMCs） from healthy donors, whose HLA haplotypes were compatible with the tumor cell lines, were transfected with reconstructive human adeno-association virus （rhAAV/HER2） to obtain the specific killing activities of T cells, and were evaluated by lactate dehydrogenase （LDH） releasing assay. Results： Trastuzumab produced a significant inhibiting effect on SK-BR-3, the IC50 was 100ng/ml. MDA-MB-453 was resistant to trastuzumab even at a concentration of 10,000 ng/ml in vitro. HER2-specific T lymphocytes killed effectively SK-BR-3 [（69.86±13.41）%] and MDA-MB-453 [（78.36±10.68）%] at 40：1 （effector：target ratio, E：T）, but had no significant cytotoxicity against HER2-negative breast cancer cell lines MDA-MB-231 or MCF-7 （less than 10%）. Conclusion： The study showed that HER2-specific T lymphocytes generated from DCs modified by rhAAV/HER2 could kill HER2-positive breast cancer cell lines in a HER2-dependent manner, and result in significantly high inhibition rates on the intrinsic trastuzumab-resistant cell line MDA-MB-453 and the tastuzumab-sensitive cell line SK-BR-3. These results imply that this immunotherapy might be a potential treatment to HER2-positive breast cancer.
文摘Background and aim Hepatic ischemia–reperfusion injury(IRI)is a significant challenge in liver transplantation,trauma,hypovolemic shock,and hepatectomy,with limited effective interventions available.This study aimed to investigate the role of leukocyte cell-derived chemotaxin 2(LECT2)in hepatic IRI and assess the therapeutic potential of Lect2-short hairpin RNA(shRNA)delivered through adeno-associated virus(AAV)vectors.Materials and methods This study analyzed human liver and serum samples from five patients undergoing the Pringle maneuver.Lect2-knockout and C57BL/6J mice were used.Hepatic IRI was induced by clamping the hepatic pedicle.Treatments included recombinant human LECT2(rLECT2)and AAV-Lect2-shRNA.LECT2 expression levels and serum biomarkers including alanine aminotransferase(ALT),aspartate aminotransferase(AST),creatinine,and blood urea nitrogen(BUN)were measured.Histological analysis of liver necrosis and quantitative reverse-transcription polymerase chain reaction were performed.Results Serum and liver LECT2 levels were elevated during hepatic IRI.Serum LECT2 protein and mRNA levels increased post reperfusion.Lect2-knockout mice had reduced weight loss;hepatic necrosis;and serum ALT,AST,creatinine,and BUN levels.rLECT2 treatment exacerbated weight loss,hepatic necrosis,and serum biomarkers(ALT,AST,creatinine,and BUN).AAV-Lect2-shRNA treatment significantly reduced weight loss,hepatic necrosis,and serum biomarkers(ALT,AST,creatinine,and BUN),indicating therapeutic potential.Conclusions Elevated LECT2 levels during hepatic IRI increased liver damage.Genetic knockout or shRNA-mediated knockdown of Lect2 reduced liver damage,indicating its therapeutic potential.AAV-mediated Lect2-shRNA delivery mitigated hepatic IRI,offering a potential new treatment strategy to enhance clinical outcomes for patients undergoing liver-related surgeries or trauma.
基金The study was supported by the National Natural Science Foundation of China （No. 30970880）, the Natural Science Foundation of the Jiangsu Province （No. BK20130342）, and the Jiangsu Planned Projects for Postdoctoral Research Funds （No. 1301069C）.
文摘Background Spinal cord injury （SCI） is a serious neurological injury that often leads to permanent disabilities for the victims. The aim of this study was to determine the effects of glial-derived neurotrophic factor （GDNF） mediated by recombinant adeno-associated virus type 2 （rAAV2） alone or in combination with early rehabilitation training on SCI. Methods SCI was induced on the T8-9 segments of the spinal cord by laminectomy in adult male Sprague-Dawley rats. Then besides the sham operation group, the SCI rats were randomly divided into four groups： natural healing group, gene therapy group, rehabilitation training group, and combination therapy group （gene therapy in combination with rehabilitation training）. Motor dysfunction, protein expression of GDNF, edema formation, and cell injury were examined 7, 14, and 21 days after trauma. Results The topical application of rAAV-GDNF-GFP resulted in strong expression of GDNF, especially after the 14th day, and could protect the motor neuron cells. Early rehabilitative treatment resulted in significantly improved motor function, reduced edema formation, and protected the cells from injury, especially after the 7th and 14th days, and increased the GDNF expression in the damaged area, which was most evident after Day 14. The combined application of GDNF and early rehabilitative treatment after SCI resulted in a significant reduction in spinal cord pathology and motor dysfunction after the 7th and 14th days. Conclusion These observations suggest that rAAV2 gene therapy in combination with rehabilitation therapy has potential clinical value for the treatment of SCI.
