Lentiviral Vector-Based Gene Therapy and The Host Genetic Background

基于慢病毒载体的基因治疗和宿主遗传背景

• 批准号: 9302512
• 负责人: TAL KAFRI
• 金额: $ 75.81万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-09-02 至 2019-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9302512
• 关键词: Activated Partial Thromboplastin Time measurement; Adverse effects; Affect; Animal Model; Animals; Antibodies; Biological Assay; Breeding; Candidate Disease Gene; Cell Line; Cells; Cessation of life; Clinical Trials; Data; Development; Disease; Dose; Embryo; Enzyme-Linked Immunosorbent Assay; Epitopes; Factor IX; Fibrin fragment D; Fibroblasts; Firefly Luciferases; Future; Gene Delivery; Gene Expression; Genes; Genetic; Genetic Variation; Genome; Goals; Hepatic; Hepatocyte; Hereditary Disease; Human; Human Genetics; Image; Immune response; In Vitro; Inbreeding; Inheritance Patterns; Kinetics; Lentivirus Vector; Life Cycle Stages; Liver; Luc Gene; Luciferases; Maintenance; Mediating; Modeling; Mouse Strains; Mus; Nuclear Import; Oryctolagus cuniculus; Outcome; Pathology; Patients; Pattern; Pharmaceutical Preparations; Phenotype; Plasma; Process; Protocols documentation; RNA; Recombinants; Regimen; Resistance; Rodent Model; Role; Safety; System; T-Lymphocyte; Testing; Therapeutic; Variant; Viral Vector; Virus Integration; aptamer; base; design; expression vector; fusion gene; gene delivery system; gene replacement therapy; gene therapy; gene therapy clinical trial; genetic variant; in vivo; individual patient; integration site; knock-down; mouse genome; mouse model; novel; outcome forecast; outcome prediction; overexpression; preclinical study; preclinical trial; promoter; public health relevance; response; screening; trait; transduction efficiency; transgene expression; vector; vector biodistribution; vector genome

 DESCRIPTION: In contrast to conventional medications, therapeutic viral vectors are given as a single dose and their levels cannot be adjusted to meet the therapeutic needs of an individual patient. Thus, one cannot overestimate the importance of animal model-based preclinical studies as a means to characterize the efficacy and safety of gene therapy regimens. Most preclinical trials are based on a rodent model comprising a large number of animals of the same strain, and a large animal model involving a smaller number of animals. Importantly, the effects of the genetic background of mouse strains employed in preclinical studies on the safety and efficacy of viral vectors has not been characterized. Similarly the effects of the patient's geneti background on the outcome of gene therapy regimens cannot be evaluated. Consequently, several gene therapy clinical trials have resulted in major adverse effects, which could not be observed in relevant earlier preclinical studies. The overall goal of the proposed studies is to characterize the effects of the host genetic variation on the efficacy and safety of lentiviral vector-based gene replacement therapy. Our experimental approach is premised on the Collaborative Cross (CC) mouse strains, which were derived by a funnel breeding of 8 genetically diverse, inbred founder strains. The genomes of these mouse strains are well defined, and will be utilized to study the overall effects of the host genetic background on the overall efficacy and safety of lentiviral vectors, as well as to identify putative genetic loci affecting these processes, test these variants' effects on the safety and efficiency of transducing human cells, and to evaluate the approach of employing an in vitro strain (potentially patient)-specific mouse embryo fibroblast (MEF's)-based system as a means to predict the safety and efficacy of hepatic gene delivery. In Aim 1, we will focus on characterizing the effects of the hos genetic background on the ability of lentiviral vectors to deliver and maintain long-term hepatic transgene expression, as well as lentiviral vector pattern of integration. F2 intercrosses of CC strains with contrasting phenotypes will be employed to identify genetic loci and the putative candidate genes involved in these processes. The optimal mouse strains for lentiviral vector-based hepatic gene delivery (demonstrating highest long-term transgene expression) will be identified. In Aim 2, we will employ MEF's from the above CC strains to study the effects of the host genetic background on the early steps of, as well as overall lentiviral vector transduction. Gene loci involved in these processes will be identified and their effects on safety and efficacy of vector transduction of human cells will be determined. The ability of the MEF-based model to predict in vivo transduction efficiency will be determined, as will this prediction's efficacy acros genetically diverse strains. In Aim 3, we will characterize the effects of the host genetic background on: the ability of lentiviral vectors to maintain therapeutic levels of factor IX (FIX) n vivo, the immune responses to FIX and vector- transduced hepatocytes, and on the development of thrombotic pathologies in the presence of high levels FIX.

 产品说明：与常规药物相反，治疗性病毒载体作为单剂量给予，并且其水平不能被调整以满足个体患者的治疗需求。因此，我们不能高估基于动物模型的临床前研究作为表征基因治疗方案的有效性和安全性的手段的重要性。大多数临床前试验是基于啮齿动物模型，包括大量相同品系的动物，以及涉及较少数量动物的大型动物模型。重要的是，尚未表征临床前研究中使用的小鼠品系的遗传背景对病毒载体安全性和有效性的影响。同样，患者基因背景对基因治疗方案结果的影响也无法评估。因此，一些基因治疗临床试验导致了严重的不良反应，这在相关的早期临床前研究中无法观察到。拟议研究的总体目标是表征宿主遗传变异对基于慢病毒载体的基因替代疗法的疗效和安全性的影响。我们的实验方法是在协作杂交（CC）小鼠品系上进行的，这些小鼠品系是由8个遗传多样性的近交创始品系的漏斗育种得到的。这些小鼠品系的基因组是明确定义的，并且将用于研究宿主遗传背景对慢病毒载体的总体功效和安全性的总体影响，以及鉴定影响这些过程的推定遗传位点，测试这些变体对转导的安全性和效率的影响。 人细胞，并评估采用体外菌株（潜在患者）特异性小鼠胚胎成纤维细胞（MEF）为基础的系统作为预测肝脏基因递送安全性和有效性的手段的方法。在目标1中，我们将集中于表征宿主遗传背景对慢病毒载体递送和维持长期肝转基因表达的能力的影响，以及慢病毒载体整合模式。将采用具有对比表型的CC菌株的F2互交来鉴定遗传位点和参与这些过程的推定候选基因。将鉴定用于基于慢病毒载体的肝脏基因递送的最佳小鼠品系（证明最高的长期转基因表达）。在目的2中，我们将使用来自上述CC株的MEF来研究宿主遗传背景对早期步骤以及整个慢病毒载体转导的影响。将鉴定参与这些过程的基因位点，并确定其对人细胞载体转导的安全性和有效性的影响。将确定基于MEF的模型预测体内转导效率的能力，以及该预测在遗传多样性菌株中的功效。在目标3中，我们将表征宿主遗传背景对以下方面的影响：慢病毒载体在体内维持治疗水平的因子IX（FIX）的能力、对FIX和载体转导的肝细胞的免疫应答，以及在高水平FIX存在下对血栓形成病理学的发展。