Exogenous gene suture was used to achieve peripheral nerve anastomoses to probe into the feasibility that the sites of anastomoses of nerves directly transfer gene and thus enable gene to be expressed at the sites of...Exogenous gene suture was used to achieve peripheral nerve anastomoses to probe into the feasibility that the sites of anastomoses of nerves directly transfer gene and thus enable gene to be expressed at the sites of anastomoses under the condition that perfect nerve anastomoses are ensured. PCMVβ plasmid containing cytomegalovirus promoter (CMV promoter) and Escherichia coli (E.coli) β Galactosidase (β Gal) structural gene (lacZ gene) was conducted. A soaked medical 8 0nylon suture was used to perform epineurial repair of rabbit sciatic nerve. In the control group a suture soaked in sucrose PBS was used, while in the experimental group a suture soaked in PCMVβ plasmid solution was applied. The sites of anastomoses of nerves by stages were taken out, and β Gal histochemical staining was performed and β Gal enzyme activity was assayed with 5 bromo 4 chloro 3 indolyl β D galactoside. Results showed that the sites of anastomoses of nerves were taken out 2 days, 7 days, 14 days and 30 days respectively after the operation. The β Gal histochemical stains at the sites of anastomoses showed no indigo positive cells at different stages in the control group, whereas displayed indigo positive cells in the experimental group. In the control group, no β Gal enzyme activity was detected at different stages after operation, but in the experimental group, β Gal enzyme activity could be detected from the 3rd day to the 30th day after operation. It was concluded that by using exogenous gene suture, exogenous gene could be transferred to the sites of peripheral nerve and expressed the exogenous gene expression products with bioactivity, which provided the feasibility of using gene therapy to accelerate the recovery of nerve function.展开更多
OBJECTIVE: To determine the efficacy of adenovirus mediated suicide gene transduction combined with prodrug 5-fluorocytosine (5FC) as a therapeutic protocol for pancreatic cancer. METHODS: Cytosine Deaminase(CD) gene...OBJECTIVE: To determine the efficacy of adenovirus mediated suicide gene transduction combined with prodrug 5-fluorocytosine (5FC) as a therapeutic protocol for pancreatic cancer. METHODS: Cytosine Deaminase(CD) gene was cloned into pAdTrack-CMV-CD, pAdTrack-CMV-CD and pAdEasy-1 were recombined in bacteria. The newly recombined adenovirus (Ad)-CD containing green fluorescent protein (GFP) were packaged and propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic carcinoma cell line-Patu8988 was infected with this virus, then 5FC was added. XTT assay was used to estimate relative numbers of viable cells. In vivo model of pancreatic cancer was established by injecting 1.0 x 10(7) Patu8988 cells subcutaneously in Balb/c nude mice. When tumors were palpable, Ad-CD was injected into each tumor and 5FC was administered. RESULTS: Positive clones were selected using endonuclease to digest the recombinants and the concentration of viral liquids containing the CD gene was 2 x 10(11) pfu /ml. Significant cytotoxic activity as shown for 5FC in the CD gene transduced 8988 cell line, while little effect was found in the nontransduced pancreatic carcinoma cells. Antitumor effect was observed in Patu8988 xenograft nude mice with in situ CD gene transduction. CONCLUSIONS: CD gene mediated by adenovirus has high infectivity and may be useful for gene therapy in pancreatic carcinoma. These data demonstrate the use of an enzyme prodrug strategy in experimental pancreatic cancer.展开更多
In past two decades the gene therapy using genetic modified autologous hematopoietic stem cells(HSCs)transduced with the viral vector has become a promising alternative option for treating primary immunodeficiency dis...In past two decades the gene therapy using genetic modified autologous hematopoietic stem cells(HSCs)transduced with the viral vector has become a promising alternative option for treating primary immunodeficiency diseases(PIDs).Despite of some pitfalls at early stage clinical trials,the field of gene therapy has advanced significantly in the last decade with improvements in viral vector safety,preparatory regime for manufacturing high quality virus,automated CD34 cell purification.Hence,the overall outcome from the clinical trials for the different PIDs has been very encouraging.In addition to the viral vector based gene therapy,the recent fast moving forward developments in genome editing using engineered nucleases in HSCs has provided a new promising platform for the treatment of PIDs.This review provides an overall outcome and progress in gene therapy clinical trials for SCID-X,ADA-SCID,WAS,X-CGD,and the recent developments in genome editing technology applied in HSCs for developing potential therapy,particular in the key studies for PIDs.展开更多
The adenosine deaminase(ADA) activities in blood lymphocytes of 41normal children and 17 with recurrent respiratory tract infections were examined,and the T-lymphocytes of two children whose ADA activities were obviou...The adenosine deaminase(ADA) activities in blood lymphocytes of 41normal children and 17 with recurrent respiratory tract infections were examined,and the T-lymphocytes of two children whose ADA activities were obviously lower than those of others were cultared in vilro. Then the exogenous human ADA gene was transfected into these cells by means of lipofectin mediated gene transfer. The results showed that the ADA activities in cultured T-lymphocytes were raised and the immunological were also improved.展开更多
免疫出生错误(inborn errors of immunity,IEI)是由遗传因素导致免疫结构或功能障碍所致的一类疾病,可累及固有免疫和适应性免疫。2022年IEI新分类包含485种IEI,分为十大类疾病。近年来随着分子生物学的快速发展,许多IEI的具体发病机制...免疫出生错误(inborn errors of immunity,IEI)是由遗传因素导致免疫结构或功能障碍所致的一类疾病,可累及固有免疫和适应性免疫。2022年IEI新分类包含485种IEI,分为十大类疾病。近年来随着分子生物学的快速发展,许多IEI的具体发病机制得以揭示,使得基因治疗在该类疾病的临床前和临床研究成为可能。该文综述基因治疗在IEI中的研究和应用,以进一步提高临床医生对IEI诊治的认知。展开更多
基金a grant from the ScientificCommittee of Hebei Province(No.982 76 110 1)
文摘Exogenous gene suture was used to achieve peripheral nerve anastomoses to probe into the feasibility that the sites of anastomoses of nerves directly transfer gene and thus enable gene to be expressed at the sites of anastomoses under the condition that perfect nerve anastomoses are ensured. PCMVβ plasmid containing cytomegalovirus promoter (CMV promoter) and Escherichia coli (E.coli) β Galactosidase (β Gal) structural gene (lacZ gene) was conducted. A soaked medical 8 0nylon suture was used to perform epineurial repair of rabbit sciatic nerve. In the control group a suture soaked in sucrose PBS was used, while in the experimental group a suture soaked in PCMVβ plasmid solution was applied. The sites of anastomoses of nerves by stages were taken out, and β Gal histochemical staining was performed and β Gal enzyme activity was assayed with 5 bromo 4 chloro 3 indolyl β D galactoside. Results showed that the sites of anastomoses of nerves were taken out 2 days, 7 days, 14 days and 30 days respectively after the operation. The β Gal histochemical stains at the sites of anastomoses showed no indigo positive cells at different stages in the control group, whereas displayed indigo positive cells in the experimental group. In the control group, no β Gal enzyme activity was detected at different stages after operation, but in the experimental group, β Gal enzyme activity could be detected from the 3rd day to the 30th day after operation. It was concluded that by using exogenous gene suture, exogenous gene could be transferred to the sites of peripheral nerve and expressed the exogenous gene expression products with bioactivity, which provided the feasibility of using gene therapy to accelerate the recovery of nerve function.
基金ThisworkwassurpportedbyScientificCommitteeFoundationofShanghai (No 9941190 44 )
文摘OBJECTIVE: To determine the efficacy of adenovirus mediated suicide gene transduction combined with prodrug 5-fluorocytosine (5FC) as a therapeutic protocol for pancreatic cancer. METHODS: Cytosine Deaminase(CD) gene was cloned into pAdTrack-CMV-CD, pAdTrack-CMV-CD and pAdEasy-1 were recombined in bacteria. The newly recombined adenovirus (Ad)-CD containing green fluorescent protein (GFP) were packaged and propagated in 293 cells and purified by cesium chloride gradient centrifugation. Human pancreatic carcinoma cell line-Patu8988 was infected with this virus, then 5FC was added. XTT assay was used to estimate relative numbers of viable cells. In vivo model of pancreatic cancer was established by injecting 1.0 x 10(7) Patu8988 cells subcutaneously in Balb/c nude mice. When tumors were palpable, Ad-CD was injected into each tumor and 5FC was administered. RESULTS: Positive clones were selected using endonuclease to digest the recombinants and the concentration of viral liquids containing the CD gene was 2 x 10(11) pfu /ml. Significant cytotoxic activity as shown for 5FC in the CD gene transduced 8988 cell line, while little effect was found in the nontransduced pancreatic carcinoma cells. Antitumor effect was observed in Patu8988 xenograft nude mice with in situ CD gene transduction. CONCLUSIONS: CD gene mediated by adenovirus has high infectivity and may be useful for gene therapy in pancreatic carcinoma. These data demonstrate the use of an enzyme prodrug strategy in experimental pancreatic cancer.
基金We thank Dr Alessia Cavazza for helping in the manuscript correction.FZ is supported by the Wellcome Trust(104807/Z/14/Z)ZYZ is supported by National Natural Science Foundation of China(NO.81202316)+1 种基金Foundation from Children’s Hospital of Chongqing Medical University.AJT is supported by both the Wellcome Trust(104807/Z/14/Z)by the National Institute for Health Research Biomedical Research Centre at Great Ormond Street Hospital for Children NHS Foundation Trust and University College London.
文摘In past two decades the gene therapy using genetic modified autologous hematopoietic stem cells(HSCs)transduced with the viral vector has become a promising alternative option for treating primary immunodeficiency diseases(PIDs).Despite of some pitfalls at early stage clinical trials,the field of gene therapy has advanced significantly in the last decade with improvements in viral vector safety,preparatory regime for manufacturing high quality virus,automated CD34 cell purification.Hence,the overall outcome from the clinical trials for the different PIDs has been very encouraging.In addition to the viral vector based gene therapy,the recent fast moving forward developments in genome editing using engineered nucleases in HSCs has provided a new promising platform for the treatment of PIDs.This review provides an overall outcome and progress in gene therapy clinical trials for SCID-X,ADA-SCID,WAS,X-CGD,and the recent developments in genome editing technology applied in HSCs for developing potential therapy,particular in the key studies for PIDs.
文摘The adenosine deaminase(ADA) activities in blood lymphocytes of 41normal children and 17 with recurrent respiratory tract infections were examined,and the T-lymphocytes of two children whose ADA activities were obviously lower than those of others were cultared in vilro. Then the exogenous human ADA gene was transfected into these cells by means of lipofectin mediated gene transfer. The results showed that the ADA activities in cultured T-lymphocytes were raised and the immunological were also improved.
文摘免疫出生错误(inborn errors of immunity,IEI)是由遗传因素导致免疫结构或功能障碍所致的一类疾病,可累及固有免疫和适应性免疫。2022年IEI新分类包含485种IEI,分为十大类疾病。近年来随着分子生物学的快速发展,许多IEI的具体发病机制得以揭示,使得基因治疗在该类疾病的临床前和临床研究成为可能。该文综述基因治疗在IEI中的研究和应用,以进一步提高临床医生对IEI诊治的认知。
