Virus-mediated gene transfer for cutaneous gene therapy

Cutaneous gene therapy offers unique opportunities and limitations in the use of viral vectors for corrective gene transfer. Skin presents a formidable barrier to microbial invasion and is nourished by small blood vessels, thus ruling out the possibility of directed virus delivery through cannulated blood vessels. However, skin is physically accessible and its resident keratinocyte stem cell population is susceptible to direct in vivo transduction with retroviral vectors. Furthermore, keratinocyte stem cells transduced in culture have been shown to persist and to express the encoded transgene when grafted to immunocompromised mice. Cutaneous gene therapy trials are likely to involve virus-mediated transduction as a principal means of gene transfer.     

金黄色葡萄球菌耐消毒剂基因及5类抗生素耐药相关基因检测

目的了解本地区临床分离的金黄色葡萄球菌（SA）的耐药特点,探讨SA中耐消毒剂基因（qacA）、β-内酰胺类、氨基糖苷类、大环内酯-林可霉素-链阳菌素B类（MLSB）、四环素类、糖肽类药物耐药基因的存在情况。方法采用聚合酶链反应（PCR）法对20株SA进行β-内酰胺酶基因、氨基糖苷类修饰酶基因、MLSB类基因、四环素类基因、糖肽类基因、耐消毒剂基因检测。结果 PCR结果显示20株SA中13种耐药相关基因检出率分别为mecA75%、TEM90%、aac（6＇）/aph（2″）70%、ant（4＇,4″）15%、ant（6）-Ⅰ25%、ermA100%、ermC55%、msrA20%、msrB55%、tetM65%、qacA60%,vanA、vanB基因均为阴性。结论多数SA菌株具有耐多药特征,存在耐β-内酰胺类、氨基糖苷类、MLSB类、四环素类等多种抗生素耐药基因,且对胺类、胍类消毒剂耐受。     

klotho gene

The klotho gene, originally identified by insertional mutagenesis in mice, suppresses multiple aging phenotypes(e.g. arteriosclerosis, pulmonary emphysema, osteoporosis, infertility, short lifespan). We have shown that mice deficient for the klotho gene show endothelial dysfunction as manifested by an attenuated response of aortic relaxation in response to acetylcholine stimulation. Nitric oxide production was also significantly reduced in klotho deficient mice. A decrease in klotho gene expression in animals under sustained circulatory and metabolic stress(e.g. atherosclerosis). The klotho gene delivery improves endothelial dysfunction through a pathway involving nitric oxide, and is involved in modulating vascular function(e.g. hypertension, vascular remodeling). Our findings establish the basis for the therapeutic potential of klotho gene delivery in atherosclerotic disease.     

炭疽芽孢杆菌基因芯片探针的制备

目的制备炭疽芽孢杆菌(BA)基因芯片探针,应用于BA基因芯片的检测。方法采用PCR方法,以BA疫苗株A16R基因组DNA为模板,扩增pXO1质粒上的8个特异性片段,连接至pMD19-T Simple载体,构建分别包含8个探针序列的重组质粒,以其为PCR扩增模板获取探针DNA。结果基因测序证明,成功获得BA的3个探针。结论 BA基因探针的成功获取,为炭疽芽孢杆菌基因芯片的制备奠定了基础。     

An episomally maintained MDR1 gene for gene therapy

Potential applications of the MDR1 multidrug transporter in gene therapy include protecting sensitive bone marrow cells against cytotoxic drugs during cancer chemotherapy and serving as a dominant selectable marker when coexpressed with a corrective passenger gene. To address safety concerns associated with integrating viral systems, such as retroviruses, we tested the feasibility of maintaining a nonvirally delivered MDR1 gene (pEpiHaMA) episomally. An MDR1 vector containing the Epstein-Barr virus (EBV) origin of replication (OriP) and its nuclear retention protein (EBNA-1) was transfected into human (KB-3-1) cells. MDR1 was expressed at a higher level in cells carrying the episomal vector, pEpiHaMA, compared with the vector lacking sequences needed for episomal maintenance (pHaMA). Furthermore, more drug-resistant KB-3-1 colonies were obtained on selection after transfection with pEpiHaMA. These observations correlated with longer maintenance of episomes in cells transfected with pEpiHaMA. In addition, episomes could still be recovered for more than 1 month from tumor explants in nude mice that were injected with pEpiHaMA-liposome complexes after drug selection, suggesting that these constructs can be maintained extrachromosomally in vivo.     

Contributions of gene marking to cell and gene therapies

The first human genetic modification studies used replication-incompetent integrating vector vectors to introduce marker genes into T lymphocytes and subsequently into hematopoietic stem cells. Such studies have provided numerous insights into the biology of hematopoiesis and immune reconstitution and contributed to clinical development of gene and cell therapies. Tracking of hematopoietic reconstitution and analysis of the origin of residual malignant disease after hematopoietic transplantation has been possible via gene marking. Introduction of selectable marker genes has enabled preselection of specific T-cell populations for tumor and viral immunotherapy and reduced the threat of graft-versus-host disease, improving the survival of patients after allogeneic marrow transplantation. Marking studies in humans, murine xenografts, and large animals have helped optimize conditions for gene transfer into CD34(+) hematopoietic progenitors, contributing to the achievement of gene transfer efficiencies sufficient for clinical benefit in several serious genetic diseases such as X-linked severe combined immunodeficiency and adrenoleukodystrophy. When adverse events linked to insertional mutagenesis arose in clinical gene therapy trials for inherited immunodeficiencies, additional animal studies using gene-marking vectors have greatly increased our understanding of genotoxicity. The knowledge gained from these studies is being translated into new vector designs and clinical protocols, which we hope will continue to improve the efficiency, effectiveness and safety of these promising therapeutic approaches.     

基因转染新方法--声化学基因转染

目的 基因转染效率低下一直是限制基因研究与基因治疗进一步发展和广泛应用的瓶颈问题。笔者将生物技术和超声技术有机结合，提出一种基因转染新方法即声化学基因转染或声化学内在化，为提高基因转染效率提供新的思路和新手段。     

Liposomal gene transfer of multiple genes is more effective than gene transfer of a single gene

Liposomal gene transfer is an effective therapeutic approach for the treatment of several pathophysiologic states. The purpose of the present study was to define whether gene transfer of multiple genes is a feasible approach and whether this approach would be more effective than the single transfer of cDNA. Rats were inflicted an acute wound and divided into four groups to receive weekly subcutaneous injections of liposomes plus the Lac-Z gene (0.22 microg, vehicle), or liposomes plus the insulin like-growth factor-I (IGF-I)cDNA (2.2 microg) and Lac Z gene (0.22 microg), or liposomes plus the keratinocyte growth factor (KGF) cDNA (2.2 microg) and Lac Z gene (0.22 microg), or liposomes plus the IGF-I/KGF cDNA (2.2 microg) and Lac Z gene (0.22 microg). Planimetry, immunological assays, histological and immunohistochemical techniques were used to determine molecular mechanisms after gene transfer, protein expression, dermal and epidermal regeneration. IGF-I/KGF cDNA transfer increased IGF-I and KGF protein concentration and caused concomitant cellular responses, for example,by increasing IGFBP-3, P<0.05. IGF-I/KGF cDNA gene transfer improved epidermal regeneration by exhibiting the most rapid area and linear wound re-epithelization by almost 250% compared to control and each growth factor given individually, P<0.001, which was probably due to promitogenic and antiapoptotic effects on basal keratinocytes when compared to controls, P<0.001. Dermal regeneration was improved in IGF-I/KGF cDNA-treated animals by an increased collagen deposition and morphology when compared with vehicle, IGF-I and KGF, P<0.001. IGF-I/KGF cDNA increased VEGF concentrations and thus neovascularization when compared with vehicle, IGF-I and KGF, P<0.001. In the present study, we showed that exogenous gene transfer of multiple cDNA sequences have an additive effect on intracellular and biological responses when compared to the same gene administered as a single cDNA sequence. Our findings demonstrate that gene therapy with multiple genes is feasible, and that the gene transfer of multiple genes can enhance and accelerate physiologic and biological effects.