Aptamer &Dendrimer Delivery of Zn Finger Nuclease &Homing Endonuclease mRNA &cDNA

适体

• 批准号: 8202343
• 负责人: John Joseph Rossi
• 金额: $ 21.78万
• 依托单位: FRED HUTCHINSON CANCER RESEARCH CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-07-08 至 2016-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8202343
• 关键词: AIDS/HIV problem; Acquired Immunodeficiency Syndrome; Affinity; Allogenic; Anti-HIV Agents; Binding; CCR5 gene; CD4 Positive T Lymphocytes; CXCR4 gene; Cell Culture Techniques; Cells; Chemokine Receptor Gene; Code; Complement component C5; Complementary DNA; Cytoplasm; DNA; DNA delivery; Dendrimers; Development; Diagnostic; Disease; Drug Costs; Drug resistance; Drug toxicity; Enzymes; Fingers; Future; Gene Targeting; Genes; Genetic; Goals; HIV; HIV Envelope Protein gp120; HIV Infections; HIV Receptors; HIV-1; Health; Hematopoietic System; Hematopoietic stem cells; Highly Active Antiretroviral Therapy; Homing; Human; Image; In Vitro; Individual; Infection; Knowledge; Lead; Macaca nemestrina; Mediating; Messenger RNA; Modeling; Multi-Drug Resistance; Mus; Mutate; Nucleic Acid Regulatory Sequences; Nucleic Acids; Patients; Pharmaceutical Preparations; Pharmacotherapy; Population; RNA; Receptor Gene; Research; Resistance; Secondary to; Site-Directed Mutagenesis; Small Interfering RNA; Specificity; Stem cell transplant; Surface; T-Lymphocyte; Technology; Testing; Therapeutic; Toxic effect; Translations; Treatment Efficacy; Tropism; Vertebral column; Viral; Virus; Virus Diseases; Work; Zinc Fingers; aptamer; cell type; chemokine; combat; combinatorial; design; endonuclease; flexibility; gene function; gene therapy; genetic element; in vivo; inhibitor/antagonist; leukemia; mouse model; nanoparticle; nonhuman primate; novel; novel strategies; nuclease; prevent; programs; purge; receptor; simian human immunodeficiency virus; small molecule; tool

HIV/AIDS continues to be a major threat to human health. The use of combinations of small molecule drugs in highly active any-retoviral therapy (HAART) to stop or thwart HIV propagation has had a major impact on delaying the progression from HIV-1 infection to the development of AIDS. Despite this progress, there are problems associated with a lifetime of anti-viral small drug therapy which include toxicity, the emergence of virus resistant to multiple drugs, and the cost of a daily, lifelong medication. The proposed studies take advantage of recent advances in the functionality of sequence specific Zn finger and homing endonucleases are capable of destroying gene function. The endonucleases are being developed in other projects of this program which will target both the chemokine co-receptor for HIV CCR5 as well as integrated proviral regulatory regions. The major challenge in using these endonucleases in a therapeutic setting is delivery of the enzymes or sequences encoding the enzymes to HIV-1 infectible cells. Since long term expression of the endonucleases could lead to secondary non-target cleavage and gene destruction, transient expression is a necessity if this approach is to be clinically useful. The proposed studies take advantage of two novel platforms for delivery of Zn finger and homing endonuclease encoding sequences into HIV (and SHIV) infectible and infected cells. The first approach uses RNA aptamers with proven ability to bind to the HIV-1 envelope expressed on the surface of HUV infected cells and internalize for functional delivery of attached siRNAs. We now will test the ability of these aptamers along with an internalizing CD4 specific aptamer to deliver backbone modified, nuclease resistant mRNAs encoding the endocucleases to uninfected and SHIV infected cells. The targets for the nucleases are the CCR5 co-receptor gene and the SHIV LTRs. As an alternative delivery approach we will test flexible TAMAM dendrimers which we have demonstrated as effective agents for siRNA deliverty in cell culture and in vivo. We will utilize this non-toxic but non-targeted delivery platform as an alternative approach for delivering backbone modified mRNAs and/or cDNAs encoding the anti-viral and anti-CCR5 endonucleases. Since both approaches have been successfully tested in vivo in a humanized mouse model for HIV infection, we will proceed to develop these agents for future application in the SHIV-pigtail macaque model utilized in other projects of this proposal. Knowledge gained from the proposed studies will result in new in vivo approaches for delivery of DNA targeting endonucleases for the treatment, and potential purging of HIV-1 infected.

艾滋病毒/艾滋病仍然是对人类健康的主要威胁。使用小分子药物的组合 在高活性抗逆转录病毒治疗（HAART）中，阻止或阻止HIV传播对 延缓从HIV-1感染到艾滋病发展的进程。尽管取得了这些进展， 与抗病毒小药物治疗的寿命相关的问题，包括毒性， 对多种药物耐药的病毒，以及每日、终身用药的费用。拟议的研究需要 序列特异性锌指和归巢核酸内切酶功能的最新进展的优势 能够破坏基因功能。核酸内切酶正在开发的其他项目， 该计划将靶向HIV CCR5的趋化因子共受体以及整合的前病毒 监管区域。在治疗环境中使用这些核酸内切酶的主要挑战是递送核酸内切酶。 所述酶或编码所述酶的序列至HIV-1可感染细胞。由于长期表达的 核酸内切酶可导致二次非靶切割和基因破坏，瞬时表达是一种 如果这种方法是临床上有用的必要性。拟议的研究利用了两个新的 用于将锌指和归巢核酸内切酶编码序列递送到HIV（和SHIV）中的平台 可感染和受感染的细胞。第一种方法使用RNA适体，其已被证明能够结合HIV-1 包膜表达在HUV感染细胞的表面上，并内化用于功能性递送附着的 siRNA。我们现在将测试这些适体沿着内化CD4特异性适体的能力， 将编码核酸内切酶的主链修饰的核酸酶抗性mRNA递送至未感染的SHIV和SHIV 被感染的细胞核酸酶的靶标是CCR5共受体基因和SHIV LTR。作为 我们将测试灵活的TAMAM树枝状聚合物，我们已经证明， 用于在细胞培养物和体内递送siRNA的有效试剂。我们将利用这种无毒但无针对性的 作为递送骨架修饰的mRNA和/或cDNA的替代方法的递送平台 编码抗病毒和抗CCR 5核酸内切酶。由于这两种方法都已成功测试 在HIV感染人源化小鼠模型体内，我们将继续为将来开发这些药物 在本提案的其他项目中使用的SHIV猪尾猕猴模型中的应用。获得的知识 将导致新的体内方法用于递送DNA靶向核酸内切酶 用于治疗和清除HIV-1感染者。