Specific targeting of a neuronal subset using a novel chimeric vector pseudotype

使用新型嵌合载体假型特异性靶向神经元子集

• 批准号: 7394664
• 负责人: Christopher Trimby
• 金额: $ 2.83万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-01-01 至 2009-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7394664
• 关键词: Afferent Neurons; Amino Acid Sequence; Amino Acids; Animals; Antibodies; Binding; Brain; Cell Fraction; Cell Line; Cell surface; Cells; Cercopithecine Herpesvirus 1; Characteristics; Chimera organism; Clinical; Cutaneous; Disease; Endoplasmic Reticulum; Engineering; Figs - dietary; Future; Gel; Gene Transduction Agent; Genes; Glutamate Decarboxylase; Glycoproteins; Green Fluorescent Proteins; Hela Cells; Immunohistochemistry; In Vitro; Injection of therapeutic agent; Interphase Cell; Intramuscular; Invasive; Lentivirus Vector; Limb structure; Membrane; Mus; Nerve Growth Factors; Nervous system structure; Neurons; Neurotrophic Tyrosine Kinase Receptor Type 1; Neurotrophin 3; Nociception; Numbers; Pain; Pain management; Pattern; Peripheral; Population; Population Heterogeneity; Proteins; Publishing; Rabies virus; Rattus; Receptor Signaling; Reporter; Reporting; Research; Resistance; Route; Running; Site; Specificity; Spinal Cord; Spinal Ganglia; Spinal Injections; Sprague-Dawley Rats; Staining method; Stains; Surface; Testing; Therapeutic; Time; Tissues; Toxin; Transfection; Tropism; Viral; Virion; Virus; Virus Diseases; base; chronic pain; clinical application; fluorophore; gene therapy; immunocytochemistry; in vivo; intradermal injection; nervous system disorder; neurotropic; novel; proenkephalin; rabies virus glycoprotein G; receptor; receptor binding; spinal cord regeneration; spinal nerve posterior root; success; tool; trafficking; vector; virus envelope

DESCRIPTION (provided by applicant): This proposal will investigate the hypothesis that the rabies virus envelope glycoprotein (RvG) can be used as a base for making a novel chimeric envelope glycoprotein, to be called RvGN2, by inserting Loop 2 from Nerve Growth Factor (NGF) in place of the "toxin-like" loop of RvG. Loop 2 confers the ability of NGF to bind the TrkA receptor, which is a receptor that is primarily expressed on sensory neurons including those projecting from the dorsal root into lamina I & II of the spinal cord. The RvGN2 chimera will still maintain the ability to be retrogradely transported, making the vector more attractive for clinical applications by allowing the potential for a less invasive delivery strategy. The more specific aims of this project will be to test the hypothesis that the RvG can act as a functionally viable basis for engineered retargeting. This will be assessed by immunocytochemistry to detect RvGN2 expression on the surface of transfected cells. The next step will be to test the hypothesis that RvGN2 reporter viruses will preferentially infect cells in vitro that express TrkA over cells that are permissive to wild- type Rabies virus infection. Transient transfection of the TrkA receptor will also be used to "rescue" resistant cell lines. The last aim will test the hypothesis that the in vitro targeting profile will be maintained when RvGN2 reporter virus is administered to rats in vivo, by injection into the spinal cord. The second part of this aim is to show that peripheral delivery by intramuscular or intradermal injection will preserve this targeting profile. Immunohistochemistry will then be used on tissue sections with antibodies to detect different markers indicative of separate subsets of neurons. This staining pattern will be compared to the transduction pattern of the reporter viruses to assess which neuronal subpopulations were transduced. At this time viral gene therapy vectors cannot differentiate among subsets of neurons, making them impractical to treat many disorders. The capability to selectively target subsets of neurons will allow the basis for treatment of a multitude of disorders, from chronic pain to aiding in spinal cord regeneration. And, the ability to be retrogradely transported will allow for less invasive delivery of these vectors as opposed to direct injections into the spinal cord.

描述(申请人提供)：这项建议将调查狂犬病病毒包膜糖蛋白(RVG)可以作为基础的假说，通过插入神经生长因子(NGF)的环2来代替RVG的“毒素样”环，可以制造一种新的嵌合包膜糖蛋白，称为RvGN2。环路2赋予NGF结合TrkA受体的能力，TrkA受体是一种主要在感觉神经元上表达的受体，包括那些从背根投射到脊髓I和II层的感觉神经元。RvGN2嵌合体仍将保持逆行运输的能力，通过允许一种侵入性更小的递送策略的可能性，使载体对临床应用更具吸引力。这个项目更具体的目标将是测试RVG可以作为工程化重定目标的功能可行基础的假设。这将通过免疫细胞化学检测RvGN2在转基因细胞表面的表达来进行评估。下一步将检验这一假设，即RvGN2报告病毒将优先感染体外表达TrkA的细胞，而不是允许野生型狂犬病病毒感染的细胞。TrkA受体的瞬时转基因也将被用来“拯救”耐药细胞系。最后一个目的将检验这样一个假设，即当RvGN2报告病毒通过注射到脊髓体内给大鼠时，体外靶向性将保持不变。这一目标的第二部分是为了表明，通过肌肉内或皮内注射的外周给药将保留这种靶向特征。然后，免疫组织化学将用于带有抗体的组织切片，以检测指示不同神经元亚群的不同标记。这种染色模式将与报告病毒的转导模式进行比较，以评估哪些神经元亚群被转导。目前，病毒基因治疗载体不能区分神经元亚群，这使得它们在治疗许多疾病方面不切实际。选择性地针对神经元亚群的能力将为治疗从慢性疼痛到帮助脊髓再生的多种疾病奠定基础。而且，逆行运输的能力将使这些载体的侵入性更小，而不是直接注入脊髓。