Reversing HIV T cell dysfunction by aptamer targeting of therapeutic siRNAs

 描述：HIV特异性CD8+T细胞是抗病毒免疫反应的关键环节，表现为进行性功能障碍，称为“衰竭”。免疫衰竭的严重程度与疾病的进展有关，在药物滥用患者中尤其普遍，他们经常推迟控制艾滋病毒复制的治疗。PD-1是一种免疫抑制受体，在慢性感染和癌症的背景下是疲劳的介质，它在衰竭的HIV特异性CD8+T细胞上上调，并在体外用抗PD-L1抗体阻断其配体，恢复它们的免疫功能，尽管是部分恢复。鉴于衰竭的T细胞表达多种免疫抑制受体，两种受体共同阻断的协同作用，以及PD-L1抗体在体外的有限作用，强烈认为仅阻断PD-1不足以挽救衰竭的HIV特异性CD8+T细胞的全部功能。与 我们的目标是开发一种临床上可行的、经济有效的、广泛适用的治疗方法来逆转艾滋病毒感染患者的免疫功能障碍，我们的目标是开发一种单一的、化学合成的、基于核酸的药物，它可以阻断多种抑制受体的作用。 同样的细胞，并可以有效和特异性地输送到患者耗尽的T细胞。所提出的方法是通过以siRNAs的形式使用RNAi来抑制衰竭的下游介质，如Blimp-1，eome，VHL或Sprouty-2，通过与衰竭的T细胞上表达的受体(如PD-1，LAG3或Tim-3)结合的寡核苷酸适配子配体，靶向于衰竭的HIV特异性CD8+T细胞。化学合成的适体是一种新型的具有工程特异性的配体平台，在制造、成本和降低免疫原性方面比单抗平台具有显著的优势。我们的实验室率先使用适体作为治疗剂和靶向配体来调节癌症背景下的免疫，最近还证明了适体靶向siRNA向小鼠循环CD8+T细胞传递的显著效率。这一建议的中心假设是，PD-1、LAG3或Tim-3适配子靶向siRNA抑制Blimp-1、Eome、VHL或Sprout-2等耗竭介质将在一定程度上逆转HIV特异性CD8+T细胞的功能障碍，其效果优于以PD-1等抑制性受体为靶标的单抗阻断。研究计划是为上述抑制性受体产生高亲和力的适体，构建适体-siRNA结合物，并与抗体阻断相比，评估它们在(I)从HIV感染者体内培养的耗尽的T细胞，(Ii)完全与人类免疫系统重组的HIV感染的小鼠，以及(Iii)SIV/猕猴模型中恢复免疫功能的能力。该计划将开发出一种成本效益高、适用范围广的先导化合物，用于在慢性感染艾滋病毒的患者身上进行测试。

 DESCRIPTION: HIV specific CD8+ T cells, a pivotal arm of the antiviral immune response, exhibit progressive dysfunction, known as "exhaustion". Severity of immune exhaustion correlates with disease progression, and is especially prevalent among drug abuse patients who often delay treatments that control HIV replication. PD-1, an immune inhibitory receptor and mediator of exhaustion in the setting of chronic infections and cancer, is upregulated on exhausted HIV-specific CD8+ T cells and in vitro blockade of its ligand with anti-PD- L1 antibodies restores, albeit partially, their immune functions. Given that exhausted T cells express multiple immune inhibitory receptors, the synergism of co-blockade of two receptors, and the limited effect of PD-L1 antibodies in vitro, strongly argues that PD-1 blockade alone will not be sufficient to rescue the full functionality of exhausted HIV specific CD8+ T cells. With the aim of developing a clinically feasible, cost-effective, and broadly applicable, treatment to reverse immune dysfunction in HIV-infected patients, our goal is to develop a single, chemically synthesized, nucleic acid based agent that can block the action of multiple inhibitory receptors in the same cell, and can be efficiently and specifically delivered to the exhausted T cells of the patient. The proposed approach is to inhibit downstream mediators of exhaustion like Blimp-1, Eomes, VHL or Sprouty-2, using RNAi in the form of siRNAs that are targeted to exhausted HIV-specific CD8+ T cells by conjugation to an oligonucleotide aptamer ligand that binds to receptors expressed on exhausted T cells, like PD-1, LAG3, orTim-3. The chemically synthesized aptamers, a novel platform for ligands with engineered specificity, offer significant advantages over the monoclonal antibody platform in terms of manufacture, cost, and reduced immunogenicity. Our lab has pioneered the use of aptamers as therapeutic agents and targeting ligands to modulate immunity in the setting of cancer, and have recently demonstrated the remarkable efficiency of aptamer-targeted siRNA delivery to circulating CD8+ T cells in mice. The central hypothesis of this proposal is that PD-1, LAG3, or Tim-3 aptamer targeted siRNA inhibition of mediators of exhaustion like Blimp- 1, Eomes, VHL or Sprout-2 will reverse the dysfunction of HIV-specific CD8+ T cells to an extent that is superior to that of blockade with monoclonal antibodies targeting inhibitory receptors like PD-1. The research plan is to generate high affinity aptamers to the aforementioned inhibitory receptors, construct aptamer-siRNA conjugates, and assess their ability, in comparison to blockade with antibodies, to restore immune functions in (i) Exhausted T cell cultured from HIV infected patients, (ii) HIV infected mice that are fully reconstituted with a human immune system, and (iii) an SIV/macaque model. This program will have developed a cost-effective and broadly applicable lead compound to test in patients chronically infected with HIV.