PROTOTYPE GENE THERAPY DELIVERY SYSTEM

原型基因治疗输送系统

• 批准号: 2223000
• 负责人: ALLAN D. CALLOW
• 金额: $ 22.08万
• 依托单位: WASHINGTON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-01 至 1994-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2223000
• 关键词: biological products; biomarker; calcium metabolism; cardiovascular prosthesis; chemical structure function; computer assisted sequence analysis; dogs; drug delivery systems; flow cytometry; gene therapy; genetic manipulation; genetic transduction; hollow fiber separation technique; hypocalcemia; implant; parathyroid hormones; peptide hormone biosynthesis; protein engineering; radioimmunoassay; synthetic peptide; tissue /cell culture; transposon /insertion element; vascular endothelium

Knowledge in the field of gene transfer to mammalian somatic cells is increasing, and a protocol for a pilot of gene transfer for the study of human disease has recently been approved. Major questions exist about effective ways to insure that products of transferred genes reach target organs to correct pathologic deficiencies. We propose to test three hypotheses regarding the role of genetically modified vascular endothelial cells (GMEC) as carriers for transferred genes, and as sources of gene product, in a model for development of a therapeutically applicable gene therapy delivery system. Hypothesis 1 That autologous canine external jugular vein endothelial cells (CEJVEC), transduced with retroviral vectors contain the gene sequence for human preproparathyroid hormone, synthesize and secrete human parathyroid hormone (hPTH) in cell culture and continue to do so after seeding on vascular grafts and subsequent implantation in canine carotid arteries. We will transfer genes into CEJVEC with either of two recombinant retroviral vectors: pMSV-hPTH-gpt, or pBGCP. Selection with mycophenolic acid (gpt) or by fluorescence-activated cell sorting (lacZ in pBGCP) will produce cultures containing high percentages of genetically modified cells. Hormones and related peptides secreted by GMEC will be characterized from culture medium by amino acid sequence analysis. Production of hPTH by cultures of GMEC will be calculated for each vector. GMEC seeded on vascular grafts will be reharvested after graft explantation, and characterized for endothelial-specific cytochemical markers, for gene transfer markers and for hPTH production in culture. Hypothesis 2 That the hPTH synthesized and secreted by transduced autologous CEJVEC exhibits biological activity in vitro and in vivo. hPTH isolated from culture medium will be assayed for its ability to stimulate adenylate-cyclase-coupled receptors for PTH on the surface of cultured ROS 17/2.8 osteosarcoma cells. In vivo activity of autologous GMEC culture medium will be evaluated by its ability to correct hypocalcemia in parathyroidectomized dogs. Vascular grafts seeded with GMEC will also be evaluated for the serum levels of hPTH they produce, and for their ability to correct surgically created hypocalcemia. Hypothesis 3 That the amount of gene product produced in vivo can be amplified by increasing the capacity of a carrier system from a single vascular tube with limited surface area to a device of greatly increased surface area such as a hollow fiber array. We will use the increased surface area to maximize the number of GMEC available for hPTH production. Conditions for optimum growth of GMEC on fiber material will be investigated. In anticipation of vascular implants, we will measure hPTH serum levels produced by GMEC-seeded devices in hypocalcemic dogs, using an ex vivo arteriovenous shunt preparation. Correction of hypocalcemia by an ex vivo, GMEC-seeded device will also be evaluated.

基因转移到哺乳动物体细胞的知识是 增加，以及用于研究基因转移试点的方案 人类疾病最近已获批准。 存在主要问题 确保转移基因产物达到目标的有效方法 器官纠正病理缺陷。 我们建议测试三个 关于基因修饰的血管内皮作用的假设 细胞（GMEC）作为转移基因的载体，作为基因来源 产品，用于开发治疗性基因的模型 治疗输送系统。 假设1自体犬外颈静脉内皮 用逆转录病毒载体转导的细胞（cejvec）包含基因 人类前脊髓运动激素的序列，合成和分泌人类 细胞培养中的甲状旁腺激素（HPTH），并在此之后继续这样做 在血管移植物上播种，随后植入犬颈 动脉。 我们将以两个中的任何一个将基因转移到cejvec 重组逆转录病毒载体：PMSV-HPTH-GPT或PBGCP。 与 霉酚酸（GPT）或通过荧光激活的细胞分选（LACZ中的lacz PBGCP）将产生含有高百分比遗传学的培养物 修饰的细胞。 GMEC分泌的激素和相关肽将是 通过氨基酸序列分析从培养基中进行表征。 通过GMEC培养物的HPTH生产将为每个载体计算。 在移植后将重新进行血管移植物播种的GMEC 露天，并为内皮特异性细胞化学特征 标记物，用于基因转移标记和培养物中的HPTH产生。 假设2是HPTH合成并分泌的转导 自体CEJVEC在体外和体内表现出生物活性。 HPTH 从培养基中分离出来的刺激能力将被分析 在培养的ROS表面上的PTH的腺苷酸周期酶偶联受体 17/2.8骨肉瘤细胞。 自体GMEC培养的体内活性 培养基将通过其纠正低血钙血症的能力来评估 甲状旁腺切除犬。 用GMEC播种的血管移植也将是 评估它们产生的HPTH的血清水平以及其能力 纠正外科手术产生的低钙血症。 假设3可以是体内产生的基因产物量 通过增加单个载体系统的容量来放大 具有有限表面积的血管管大大增加 表面积，例如空心纤维阵列。 我们将使用增加 表面积可最大化可用于HPTH生产的GMEC数量。 GMEC在纤维材料上的最佳生长条件将是 调查。 预期血管植入物，我们将测量HPTH 使用GMEC种子在低钙犬中产生的血清水平，使用 离体动力分流器准备。 通过 外体，GMEC种子设备也将进行评估。