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

艾滋病毒/艾滋病仍然是对人类健康的重大威胁。小分子药物联合用药的应用 在高度活跃的重组病毒疗法(HAART)中，阻止或阻止艾滋病毒传播对 延缓从艾滋病毒-1感染到艾滋病的发展。尽管取得了这些进展，但仍有 与终生抗病毒小剂量药物治疗相关的问题，包括毒性，出现 病毒对多种药物的抵抗力，以及每天、终身服药的费用。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 能够破坏基因功能。内切酶正在这个项目的其他项目中开发 将针对HIV CCR5趋化因子共同受体以及整合前病毒的计划 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交付 编码HIV-1感染细胞的酶或酶序列。由于长期表达的 核酸内切酶可导致二次非靶向切割和基因破坏，瞬时表达是一种 如果这种方法要在临床上有用，就有必要这样做。拟议中的研究利用了两部小说 将锌指和归巢核酸内切酶编码序列输送到HIV(和SIV)的平台 可感染和受感染的细胞。第一种方法使用已证实有能力与HIV-1结合的RNA适配子 表达于人类疱疹病毒感染细胞表面并内化为功能性递送的附着物 SiRNA。我们现在将测试这些适配子以及内化的CD4特异性适配子的能力 将编码核酸内切酶的骨架修饰的、抗核酸酶的mRNAs传递给未感染和SHIV 被感染的细胞。核酸酶的靶点是CCR5共受体基因和Shiv ltrs。作为一种 替代交付方法我们将测试灵活的Tamam树枝状分子，我们已经演示了 在细胞培养和体内传递siRNA的有效试剂。我们将利用这种无毒但不具针对性的 递送平台作为递送主干修饰的mRNAs和/或cDNA的替代方法 编码抗病毒和抗CCR5内切酶。由于这两种方法都已成功测试 在活体内，在人源化的小鼠模型中，我们将继续开发这些药物，为未来 在本方案其他项目中使用的Shiv-辫尾猕猴模型中的应用。获得的知识 将产生新的体内传递DNA靶向核酸内切酶的方法 用于治疗，并可能清除感染艾滋病毒-1的人。

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.