Targeted delivery of anti HIV sRNAs/shRNAs to T cells

将抗 HIV sRNA/shRNA 靶向递送至 T 细胞

• 批准号: 7683238
• 负责人: Premlata Shankar
• 金额: $ 35.03万
• 依托单位: TEXAS TECH UNIVERSITY HEALTH SCIS CENTER
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-15 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7683238
• 关键词: Address; Aftercare; Animals; Antibodies; Base Sequence; Binding; CD34 gene; CD4 Positive T Lymphocytes; CD7 gene; Cells; Charge; Chimeric Proteins; Chronic; Clinic; Clinical; Encapsulated; Engraftment; Gene Silencing; Genes; Genome; Goals; Guide RNA; HIV; HIV Envelope Protein gp120; HIV Infections; HIV-1; Hematopoietic stem cells; Human; Human Cell Line; Human Development; Immune system; Immunoliposome; In Vitro; Infection; Interferons; Interleukin 2 Receptor Gamma; Intervention; Knockout Mice; Laboratories; Lentivirus Vector; Leukocytes; Life Cycle Stages; Mediating; Methods; Modeling; Molecular Conformation; Mouse Strains; Mus; Mutation; Nucleotides; Pan Genus; Peripheral Blood Mononuclear Cell; Pharmaceutical Preparations; Protamines; Proteins; RNA; RNA Interference; Reagent; Research Personnel; Resistance; Rest; SCID-hu Mice; Subfamily lentivirinae; System; T-Lymphocyte; Technology; Testing; Therapeutic; Transfection; Translating; Translations; Transplantation; Umbilical Cord Blood; Viral; Viral Genes; Virus; Virus Replication; base; cell type; cellular targeting; efficacy testing; gene discovery; in vivo; lentivirally transduced; mRNA Transcript Degradation; macrophage; mouse model; new technology; novel; novel therapeutics; polypeptide; reconstitution; response; small hairpin RNA; targeted delivery

DESCRIPTION (provided by applicant): RNA interference (RNAi) is a recently discovered gene-silencing phenomenon mediated by double stranded short interfering RNA (siRNA) that guide mRNA degradation in a sequence specific fashion. We have shown that siRNAs targeting cellular and viral genes involved in HIV life cycle can dramatically suppress virus replication in human cell lines and primary cells. Since RNAi is critically dependent on nucleotide sequence match, the propensity of HIV for sequence mutations is an impediment for using the technology as therapy against the virus. To overcome this limitation, we have identified optimal siRNA targets in highly conserved regions of the HIV genome and showed that these shRNAs can protect primary CD4 T cells from HIV-1 primary isolates within clade B or across multiple clades. In vivo delivery of siRNA/shRNA is the other major hurdle for translation of this promising new technology into the clinic. To use siRNA as a drug it will be critical to develop a method to introduce siRNA into primary T cells and macrophages. This is a major challenge because T cells take up siRNA very poorly even by in vitro transfection. We have recently shown that targeted delivery of siRNA to this difficult cell type can be accomplished by using a single chain antibody fused to protamine, which is a highly basic polypeptide that can bind siRNA. Further, we have also used a novel immunoliposome conjugated to 2 different antibodies that recognize LFA-1 either in the closed or open activated conformation to differentially deliver siRNA to all T cells irrespective of the activation status or specifically to activated T cells only. One other problem in a chronic infection like HIV is the need for long-term therapy. To this end, we have developed lentiviral vectors to endogenously express shRNAs and showed its potential to derive HIV-resistant progeny by transduction of CD34+ hematopoietic stem cells (HSCs). We now propose to optimize delivery methods for potential RNAi-based interventions in HIV infection in a humanized mouse model, using the novel SCID/NODIL2r?c-/- mouse strain developed by our collaborator Dr. Leonard Shultz at the Jackson Laboratory. The common gamma chain null SCID/Hu mice support complete development of human immune system after engraftment with purified CD34+ hematopoietic stem cells and reconstituted animals infected with HIV-1 show sustained and high levels of viral replication. Thus, the model provides us with a unique experimental system in which to optimize our delivery strategies. Our specific objectives are first to further refine the antibody/protamine fusion protein- and immunoliposome- mediated naked siRNA delivery to resting and/or activated T cells using antibodies to CD7 (a pan T cell molecule) or LFA-1 in open or closed conformation. Our next goal will be to test the in vivo efficacy of both delivery strategies in NOD/Lt-scid IL2r?null mice humanized either by PBL transplantation (SCID/hu-PBL) or by engrafting with cord blood CD34+ hematopoietic stem cells. Finally, we will reconstitute NOD/Lt-scid IL2r?null mice with lentivirus-transduced CD34+ human HSCs and test establishment of long-term HIV resistance.

描述（由申请人提供）：RNA干扰（RNAi）是最近发现的由双链短干扰RNA（siRNA）介导的基因沉默现象，其以序列特异性方式引导mRNA降解。我们已经证明，针对 HIV 生命周期中涉及的细胞和病毒基因的 siRNA 可以显着抑制人类细胞系和原代细胞中的病毒复制。由于 RNAi 严重依赖于核苷酸序列匹配，因此 HIV 的序列突变倾向是使用该技术作为病毒治疗的障碍。为了克服这一限制，我们在 HIV 基因组的高度保守区域中确定了最佳 siRNA 靶标，并表明这些 shRNA 可以保护初级 CD4 T 细胞免受进化枝 B 内或跨多个进化枝的 HIV-1 原代分离株的侵害。 siRNA/shRNA 的体内递送是将这一有前途的新技术转化为临床的另一个主要障碍。为了使用 siRNA 作为药物，开发一种将 siRNA 引入原代 T 细胞和巨噬细胞的方法至关重要。这是一个重大挑战，因为即使通过体外转染，T 细胞对 siRNA 的吸收也很差。我们最近表明，可以通过使用与鱼精蛋白融合的单链抗体来将 siRNA 靶向递送到这种困难的细胞类型，鱼精蛋白是一种可以结合 siRNA 的高碱性多肽。此外，我们还使用了一种与 2 种不同抗体缀合的新型免疫脂质体，这些抗体可识别闭合或开放激活构象的 LFA-1，以差异化地将 siRNA 递送至所有 T 细胞，无论激活状态如何，或仅特异性递送至激活的 T 细胞。艾滋病毒等慢性感染的另一个问题是需要长期治疗。为此，我们开发了内源表达 shRNA 的慢病毒载体，并展示了其通过转导 CD34+ 造血干细胞 (HSC) 获得 HIV 抗性后代的潜力。我们现在建议使用我们的合作者 Leonard Shultz 博士在杰克逊实验室开发的新型 SCID/NODIL2r?c-/- 小鼠品系，优化人源化小鼠模型中基于 RNAi 的潜在 HIV 感染干预的递送方法。常见的γ链缺失SCID/Hu小鼠在植入纯化的CD34+造血干细胞后支持人类免疫系统的完全发育，并且感染HIV-1的重组动物显示出持续和高水平的病毒复制。因此，该模型为我们提供了一个独特的实验系统来优化我们的交付策略。我们的具体目标首先是使用开放或封闭构象的 CD7（泛 T 细胞分子）或 LFA-1 抗体，进一步完善抗体/鱼精蛋白融合蛋白和免疫脂质体介导的裸 siRNA 向静息和/或活化 T 细胞的递送。我们的下一个目标是测试两种递送策略在通过 PBL 移植 (SCID/hu-PBL) 或通过移植脐带血 CD34+ 造血干细胞人源化的 NOD/Lt-scid IL2r?null 小鼠中的体内功效。最后，我们将用慢病毒转导的 CD34+ 人 HSC 重建 NOD/Lt-scid IL2r?null 小鼠，并测试长期 HIV 耐药性的建立。