EVALUATION OF AAV-SIRNA GENE THERAPY IN THE MARMOSET MODEL FOR GBV-B

GBV-B 狨猴模型中 AAV-SIRNA 基因疗法的评估

• 批准号: 7562445
• 负责人: Robert E LANFORD
• 金额: $ 0.33万
• 依托单位: TEXAS BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7562445
• 关键词: Animals; Antiviral Agents; Blood; Callithrix; Cells; Computer Retrieval of Information on Scientific Projects Database; Detection; Development; Evaluation; Funding; GB virus B; Galactosidase; Gene Transduction Agent; Grant; Hepatitis C; Hepatocyte; Infection; Institution; Laparotomy; Liver; Modeling; Monitor; Perfusion; Phase; Portal vein structure; Primates; Research; Research Personnel; Resources; Serotyping; Serum-Free Culture Media; Small Interfering RNA; Source; Staining method; Stains; System; Tissues; United States National Institutes of Health; Viral; Virus; Week; adeno-associated viral vector; beta-Galactosidase; collagenase; gene therapy; in vivo; programs; research study; small hairpin RNA; tissue culture; vector

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. This program will evaluate the suitability of the marmoset as a small primate model for the delivery of antiviral siRNA to the liver by the use of Adeno-assoicated virus gene therapy vectors. This project will be conducted in three phases; 1) development of a tissue culture system for AAV using primary marmoset hepatocytes; 2) evaluation of different AAV vectors for infection of marmoset liver in vivo using vectors that express Beta-galactosidase; 3) and evaluation of antiviral efficacy of AAV vectors encoding siRNA directed at viral sequences of GBV-B, a surrogate model of hepatitis C virus infections. Primary hepatocytes will be isolated from a marmoset using collagenase perfusion and grown in a defined serum free medium. In the initial experiments, hepatocytes will be infected with different dilutions of AAV vectors encoding ?-galactosidase for detection of infected cells with ?-gal histochemical staining. AAV vectors of three serotypes (2, 6 and 8) will be compared for infection of marmoset hepatocytes. In the second phase, marmosets will be inoculated with AAV vectors of different serotypes (2, 6 and 8) via portal vein following laparotomy. The vectors will encode ?-galactosidase for histochemical detection of infected cells. Two marmosets will be inoculated with each AAV serotype and one animal will serve as an uninfected control (7 animals). In the final phase, efficacy of silencing GBV-B replication with AAV-shRNA will be examined in marmosets. Animals will be inoculated IV with a GBV-B/HCV chimeric virus and 2 weeks later will be inoculated with the AAV-siRNA vector. Blood and tissues will be monitored to determine effect of siRNA on GBV-B replication.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 该项目将评估绒猴作为通过使用腺相关病毒基因治疗载体向肝脏递送抗病毒siRNA的小型灵长类动物模型的适用性。该项目将分三个阶段进行; 1）使用原代绒猴肝细胞开发AAV的组织培养系统; 2）使用表达β-半乳糖苷酶的载体评估不同AAV载体在体内感染绒猴肝脏的情况; 3）以及评估编码针对GBV-B病毒序列的siRNA的AAV载体的抗病毒功效，GBV-B是丙型肝炎病毒感染的替代模型。使用胶原酶灌注从绒猴中分离原代肝细胞，并在规定的无血清培养基中生长。在最初的实验中，肝细胞将感染不同稀释度的AAV载体编码？-半乳糖苷酶用于检测？- gal组织化学染色。将比较三种血清型（2、6和8）的AAV载体对绒猴肝细胞的感染。在第二阶段中，在剖腹手术后通过门静脉用不同血清型（2、6和8）的AAV载体接种绒猴。载体会编码吗？-半乳糖苷酶用于感染细胞的组织化学检测。用每种AAV血清型接种两只绒猴，一只动物用作未感染对照（7只动物）。在最后阶段，将在绒猴中检查用AAV-shRNA沉默GBV-B复制的功效。动物将用GBV-B/HCV嵌合病毒IV接种，2周后将用AAV-siRNA载体接种。将监测血液和组织以确定siRNA对GBV-B复制的影响。