Targeting Entry of Retroviral/Lentiviral Vectors

逆转录病毒/慢病毒载体的靶向进入

• 批准号: 7533233
• 负责人: MONICA J ROTH
• 金额: $ 28.64万
• 依托单位: UNIV OF MED/DENT NJ-R W JOHNSON MED SCH
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-01-01 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7533233
• 关键词: ABCB1 gene; ABCG2 gene; Address; Amino Acids; Animal Model; Animals; Biological Models; Canis familiaris; Cell Line; Cell Surface Proteins; Cell Surface Receptors; Cell surface; Cells; Class; Clinical; Complex; Condition; Dependence; Development; Drug Delivery Systems; Drug resistance; Effectiveness; Expression Library; Feline Leukemia Virus; Funding; Gene Delivery; Gene Targeting; Gene-Modified; Goals; Human; Immunocompromised Host; In Vitro; Infection; Integral Membrane Protein; Interphase Cell; Laboratories; Lentivirus Vector; Libraries; Life; Luc Gene; Membrane; Methods; Molecular Conformation; Monitor; Mus; Numbers; P-Glycoprotein; P-Glycoproteins; Process; Protein Overexpression; Proteins; Protocols documentation; Randomized; Range; Receptor Cell; Research; Retroviral Vector; Safety; Screening procedure; Selection Bias; Specificity; Structure; System; Technology; Testing; Tissues; Vertebral column; Viral; Viral Interference; Virus; Virus Diseases; advanced system; base; cDNA Expression; cancer therapy; cell type; charge coupled device camera; concept; desire; env Gene Products; improved; in vivo; novel; osteosarcoma; particle; receptor; receptor binding; research study; subcutaneous; success; tissue culture; tumor; tumor xenograft; vector

DESCRIPTION (provided by applicant): The ability to target gene and drug delivery to defined and limited tissues is one of the key hurdles for effective therapies. This application builds on the system developed in the laboratory that has successfully screened for productive retroviral entry into targeted cells through selection of novel Env/receptor conjugate pairs. Through the use of random libraries generated within the retroviral Env receptor-binding domain, productive infection using alternative host-cell receptors is rapidly obtained. Two major benefits of this system is that the random library is generated within the context of the Env backbone, guaranteeing that the presentation of the novel receptor binding region is in the conformation required for viral entry and that the target cell can essentially be uncharacterized. The experiments in this proposal move this system beyond the basic proof-of- principal, to address the next critical set of milestones. Questions include how many unique, high titer isolates can be obtained from a single cell line? What classes of receptors are selected for productive infection? Can receptor selection be biased towards specific cell surface proteins? Are these vectors suitable for delivery in animal models and what is the most effective means of delivery? Can these novel Env isolates be incorporated into vectors capable of infecting quiescent cells? These questions are addressed focusing on human osteosarcoma (OS) cells as the model system. Two novel Env isolates (L1 and CP) with distinct specificity towards OS cells have been isolated. Experiments aim at defining the range as well as the limitations of the random library screen through characterizing a panel of OS targeting Env isolates with respect tissue specificity, viral interference groups and receptor usage. Protocols to bias the receptor selection through overexpression of the putative receptor protein on the target cell are tested. Experiments that move the test system from in vitro tissue culture systems to in vivo tumor animal models are developed. To date, the known receptors for Env proteins within the MuLV and FeLV Env backbones have utilized multiple membrane-pass proteins. To broaden the class of potential receptors to include single-membrane pass protein receptors, as well as to extend the system to include non-dividing cells, the random Env library will be incorporated into a modified Sindbis Env protein pseudotyped onto lentiviral particles. Collectively, these experiments improve the technology with the goal of identifying novel viral Env vectors targeting entry to specific cells for a broad range of gene delivery protocols. Project Narrative: The safety and effectiveness of retroviral gene delivery would be greatly enhanced with the ability to direct viral infection to limited and targeted cells. This application develops methods to selectively screen for retroviral entry into targeted cells, using human osteosarcoma cells as the model system. The development of retroviral vectors capable of specific entry into targeted cell types is key for the clinical use of retroviral vectors for gene delivery and cancer therapies.

描述（由申请人提供）：将基因和药物输送到定义和有限的组织的能力是有效疗法的关键障碍之一。该应用建立在实验室中开发的系统上，该系统通过选择新型的Env/受体共轭对，成功筛选了逆转录病毒进入靶向细胞。通过使用逆转录病毒ENV受体结合结构域中产生的随机文库，迅速获得了使用替代宿主细胞受体的生产感染。该系统的两个主要好处是，随机文库是在ENV主链的背景下生成的，确保新型受体结合区域的呈现是病毒进入所需的构象所需要的，并且靶细胞基本上可以未经表征化。该提案中的实验使该系统超出了基本的校长证明，以解决下一个关键的里程碑。问题包括可以从单个单元线获得多少个独特的高滴度分离株？选择哪些类别的受体进行生产感染？受体选择可以偏向特定的细胞表面蛋白吗？这些向量是否适合在动物模型中交付？最有效的交付方式是什么？这些新颖的环境能够被纳入能够感染静态细胞的载体中吗？这些问题的重点是人类骨肉瘤（OS）细胞作为模型系统。已经分离出了两个对OS细胞的特异性不同的新颖的Envies（L1和CP）。实验旨在通过表征OS靶向ENV分离物，尊重组织特异性，病毒干扰组和受体用法来定义随机库筛选的范围和局限性。测试了通过推定受体蛋白在靶细胞上的过表达偏向受体选择的方案。开发了将测试系统从体外组织培养系统转移到体内肿瘤动物模型的实验。迄今为止，MULV和FELV ENV骨架内的ENV蛋白的已知受体已经利用了多种膜通蛋白。为了扩大潜在受体的类别，以包括单膜通过蛋白受体以及扩展系统以包含非分散细胞，随机env库将被纳入经过修改的Sindbis Env蛋白假蛋白化的疾病颗粒上。总的来说，这些实验改进了技术，目的是识别针对特定细胞的新型病毒end vector，以进行广泛的基因递送方案。项目叙述：逆转录病毒基因递送的安全性和有效性将大大增强，并能够将病毒感染引向有限和靶向细胞。该应用程序开发了使用人骨肉瘤细胞作为模型系统选择性筛选逆转录病毒进入目标细胞的方法。逆转录病毒载体的发展能够特异性进入靶向细胞类型是临床使用逆转录病毒载体进行基因递送和癌症疗法的关键。