FIV Vectors for the Treatment of Hemophilia A

用于治疗 A 型血友病的 FIV 载体

• 批准号: 7910804
• 负责人: WILLIAM C RASCHKE
• 金额: $ 90.58万
• 依托单位: VIROGENICS, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-22 至 2013-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7910804
• 关键词: A Mouse; Acute; Address; Advanced Development; Affect; Animal Model; Antibodies; Apoptosis; Asparagine; B-domain-deleted factor VIII; Baculoviruses; Biomedical Engineering; Blood Clot; Blood Coagulation Disorders; Blood coagulation; Canis familiaris; Cells; Clinic; Clinical; Clinical Research; Clinical Trials; Code; Complementary DNA; Development; Elements; Endoplasmic Reticulum; Factor VIII; Feline Immunodeficiency Virus; Frequencies; Funding; Future; Gene Transfer; Genes; Genetic; Goals; Golgi Apparatus; Hemophilia A; Hemorrhage; Hepatocyte; Human; Immune response; Immunologics; In Vitro; Incidence; Length; Lentivirus Vector; Life; Link; Liver; Mannose Binding Lectin; Mannose-Binding Lectins; Maps; Measures; Messenger RNA; Methods; Modeling; Modification; Molecular Chaperones; Morbidity - disease rate; Mus; Oligosaccharides; Operative Surgical Procedures; Outcome; Patients; Phase; Phenotype; Plasma; Positioning Attribute; Production; Protein Secretion; Proteins; Series; Shapes; Site; Staging; System; Testing; Therapeutic; Time; Tissues; Toxic effect; Vesicular stomatitis Indiana virus; Virus; Whole Blood; base; cDNA Expression; cellular transduction; clinical application; cytokine; cytotoxicity; design; endoplasmic reticulum stress; enhancing factor; expression vector; gene delivery system; gene replacement; gene therapy; glycosylation; human disease; improved; in vivo; liver biopsy; male; method development; phase 2 study; promoter; protein expression; public health relevance; research study; response; therapeutic protein; transgene expression; vector

DESCRIPTION (provided by applicant): Although several labs including ours have demonstrated the feasibility of gene therapy for hemophilia A in animal models, long-term expression of the transgene at therapeutic levels was not observed in the clinic. The lack of sufficient expression levels Factor VIII (FVIII) in hemophilic patients indicates that further improvements of the current gene delivery systems are needed to generate a therapeutic product. In the Phase I of this project we initiated a study to address the issues of FVIII expression level and duration based on findings showing that FVIII expression is limited by unstable mRNA, interaction with endoplasmic reticulum (ER) chaperones, and a requirement for facilitated ER to Golgi transport through interaction with the mannose- binding lectin LMAN1. Vectors for FVIII expression in gene therapy applications typically have used a B domain deleted (BDD) cDNA in which the coding sequence for this domain is removed to yield a shorter construct for protein production and gene transfer. Of note, the expression of BDD FVIII within the cell has the same limitations as the whole FVIII molecule. However, the inclusion of several asparagine-linked oligosaccharides within a short B-domain spacer increased ER to Golgi transport resulting in secretion of functional FVIII at levels 15- to 25-fold higher than full-length or B domain deleted FVIII both in vitro and in vivo. Phase I initiated the testing of the hypothesis that therapeutic FVIII production can be significantly enhanced by addition of parts of the B domain in hemophilic dogs, the most relevant animal model to the human disease and a prerequisite test for clinical studies, Two FIV vectors were constructed and tested which contain canine FVIII with different lengths of the B domain added. The constructs were prepared with the canine FVIII genetic sequence to reduce complicating cross-species factors when introduced into the hemophilic dog. Also, in Phase I the parameters for preparing high titer virus were optimized and the innate immune response to VSV- G and GP64 pseudotyped vectors were analyzed. The completion of the Phase I aims puts the project in position to conduct the FVIII expression level and duration studies in hemophilic mice and dogs and to select the most favorable FVIII cDNA for safe and long term expression. In this Phase II study the aims are to evaluate expression level, duration and clinical benefit from FIV delivery of canine FVIII with no B domain or with two different lengths of the B domain included. Also, additional elements that may further enhance FVIII production significantly from transduced cells will be incorporated into the transfer vector and tested. This Phase II also includes the development of methods to provide increased production of the virus vector for these and subsequent studies, which will require large amounts of the vector. PUBLIC HEALTH RELEVANCE: Clinical application of Factor VIII gene therapies to hemophilia A patients have suffered from insufficient production of Factor VIII. Using an FIV lentiviral vector capable of long-term expression in host cells, Factor VIII gene sequences with improvements designed to overcome the problem of poor expression will be tested in hemophilic dogs, the animal model which closely resembles the human disease. The goal is to advance the best product as determined from this study toward application in the clinic to human patients.

描述（由申请人提供）：尽管包括我们的一些实验室证明了基因治疗在动物模型中的可行性，但在诊所未观察到转基因在治疗水平上的长期表达。血友病患者缺乏足够的表达水平因子VIII（FVIII）表明，需要进一步改善当前基因递送系统来产生治疗产物。在该项目的第一阶段中，我们开始了一项研究，以解决FVIII表达水平和持续时间的问题，该发现表明FVIII表达受到不稳定的mRNA的限制，与内质网（ER）伴侣的相互作用，以及对促进Golgi促进GOLGI运输的曼纳索蛋白结合型Lectin-Lectin-Lectin-Lectin-Lectin-Lectin Lectin Lectin Lectin Lectin Lectin Lectin Lectin Lectin lectin 1的互动。基因治疗应用中FVIII表达的载体通常使用了B结构域已删除（BDD）cDNA，其中除去该结构域的编码序列以产生用于蛋白质产生和基因转移的较短构建体。值得注意的是，细胞内BDD FVIII的表达与整个FVIII分子具有相同的局限性。然而，将几种天冬酰胺连接的寡糖纳入短的B域间隔物中，将ER增加到高尔基传输中，导致功能性FVIII分泌在比全长或B域DERETED FVIII的水平高15至25倍的水平上分泌。第一阶段启动了通过在血友病犬中添加B结构域的一部分，可以显着增强治疗性FVIII产生的假设，这是人类疾病中最相关的动物模型以及对临床研究的先决条件测试，对临床研究进行了前提测试，两个FIV载体构建和测试构建和测试，其中包含添加了ban fviii fviii bengement bengement bengement bengement bementain domain domain domain domain domain的长度。用犬FVIII遗传序列制备构建体，以减少跨物种的复杂因素，当时引入了血友病的狗。同样，在I期中，优化了用于制备高滴度病毒的参数，并分析了对VSV-G和GP64伪型载体的先天免疫反应。 I阶段目标的完成使该项目有能力在血友病小鼠和狗中进行FVIII表达水平和持续时间研究，并选择最有利的FVIII cDNA以进行安全和长期表达。在这项II阶段研究中，目的是评估犬FVIII的FIV输送的表达水平，持续时间和临床益处，而没有B结构域或包括两个不同长度的B结构域。同样，可能会从转导的细胞中显着增强FVIII产生的其他元素将被纳入转移载体并进行测试。该阶段II还包括开发为这些和随后的研究提供增加病毒载体产生的方法，这将需要大量载体。 公共卫生相关性：VIII因子基因疗法对血友病的临床应用A患者的产生不足，VIII因子的产生不足。使用能够在宿主细胞中长期表达的FIV慢病毒载体，将在旨在克服较差表达问题的改进的因子VIII基因序列中进行测试，这是血友病犬，这是与人类疾病非常相似的动物模型。目的是从这项研究确定的最佳产品推向诊所对人类患者的应用。