Autologous HIV-1 resistant T cells through accelerated CCR5 gene disruption

描述（由申请人提供）：治疗感染人类免疫缺陷病毒-1 （HIV-1）的患者是一项重大的临床挑战，需要不断发展的药物和剂量选择。治疗应通过减少病毒血症和保持免疫功能来改善艾滋病毒感染者的健康和生活质量。目前的治疗方法利用抗逆转录病毒药物联合治疗方案（cARV，历史上称为高活性抗逆转录病毒治疗或HAART）来减少正在进行的病毒复制并改善患者预后。然而，cARV疗法不能治愈，而且价格昂贵，与药物相关的毒性发生率高，并且极易通过赋予耐药性的突变逃避病毒。基因靶向CCR5是一种很有前途的替代cARV治疗方法。HIV-1利用CCR5基因进入细胞，那些自然缺乏CCR5表达的个体完全抵抗HIV-1感染。在一项重要的临床病例研究中，来自ccr5缺陷供体的骨髓移植产生了首例功能性治愈持续HIV-1感染的病例。这促使治疗策略使用基于核酸酶的试剂在CCR5基因中产生破坏性突变，从而产生HIV-1抗性患者来源的T细胞。尽管在HIV-1患者中提供了关键的概念证明，但由于这些第一代核酸酶技术的基因破坏活性较低，目前在临床试验中基因靶向治疗的成功似乎受到限制。Pregenen利用其表面展示工程平台生产了高度特异性的靶向CCR5的LAGLIDADG归巢内切酶（LHE），最近重组为非常强大的MegaTAL核酸酶，并结合了SCRI开发的另一层速率增强技术-外切酶加速基因组编辑（X-AGE）。拟议的治疗方法将通过使用简化和可扩展的基于mrna的方法提供这些增强试剂来分离和处理患者来源的t细胞。这一制造过程将用于制造抗hiv、缺乏ccr5的自体CD4+ T细胞。自体ccr5破坏的T细胞具有持久抑制HIV-1感染的潜力，并在减少对抗逆转录病毒药物依赖的同时重建CD4+细胞室的全部功能。本提案中描述的实验是将ccr5靶向核酸酶疗法推向临床的综合计划的一部分。我们计划分析ccr5靶向试剂处理的原代人T细胞的安全性、生物分布和效力。性能最好的试剂和方案将用于产生缺乏ccr5的人类CD4+ T细胞，以测试这些细胞在体外和体内抵抗HIV感染的能力。我们将使用这些数据集来支持关键的临床前实验和随后的IND申请，以继续我们的细胞疗法的临床开发。

DESCRIPTION (provided by applicant): Treating patients infected with Human Immunodeficiency Virus-1 (HIV-1) is a substantial clinical challenge with evolving pharmaceutical drug and dosing options. Treatments should improve the health and quality of life for HIV-infected individuals by reducing viremia and preserving immunological function. Currents therapeutic approaches utilize combination antiretroviral drug regiments (cARV, historically referred to highly active antiretroviral therapy, or HAART) to reduce ongoing viral replication and improve the patient's prognosis. However, cARV therapies are not curative and furthermore they are expensive, are associated with high rates of drug-related toxicity, and are highly susceptible to viral evasion through resistance-conferring mutation. Genetic targeting CCR5 is a promising alternative approach to cARV therapy. The CCR5 gene is used by HIV-1 for cellular entry and those individuals who naturally lack CCR5 expression are fully resistant to HIV-1 infection. In an important clinical case study, bone marrow transplantation from a CCR5-deficient donor has produced the first recorded case of functional cure of an ongoing HIV-1 infection. This has prompted therapeutic strategies to create HIV-1 resistant patient-derived T cells using nuclease- based reagents to create disruptive mutations in the CCR5 gene. Though providing critical proof-of- concept in HIV-1 patients, the success of the gene-targeting therapy currently in clinical trials appears to be limited due to low gene disruption activity of these firt-generation nuclease technologies. Pregenen has used its surface display engineering platform to produce highly specific CCR5- targeting LAGLIDADG homing endonucleases (LHE), recently reformatted as extremely powerful MegaTAL nucleases and combined with an additional layer of rate-enhancement technology - exonuclease-accelerated genome editing (X-AGE) developed at SCRI. The proposed therapy will isolated and process patient-derived T-cells by delivering these enhanced reagents using a simplified and scalable mRNA-based method. This manufacturing process will be used to create HIV-resistant, CCR5-deficient autologous CD4+ T cell. Autologous CCR5-disrupted T cells have the potential to durably suppress HIV-1 infection and reconstitute the full functionality of the CD4+ compartment while reducing dependence on antiretroviral drugs. The experiments described in this proposal are part of a comprehensive plan to bring the CCR5-targeting nuclease therapy to the clinic. We plan to profile the safety, biodistribution, and potency of primary human T cells treated with CCR5-targeting reagents. The best-performing reagents and protocols will be used to generate CCR5-deficient human CD4+ T cells to test the capacity of these cells to withstand HIV infection in vitro and in vivo. We will se this data-set to support pivotal preclinical experiments and subsequent IND submission for the continued clinical development of our cellular therapy.