经Hutat2:Fc基因修饰的单核细胞干预HIV相关神经认知紊乱的实验研究

HIV-1 Tat is a well-known potent neurotoxin causing HIV-1 associated neurocognitive disorders (HAND). Currently, combined antiretroviral therapy cannot prevent the production of early viral proteins, especially Tat, and completely stop the progression of the disease. One potential adjuvant therapy for HAND is to block the neurotoxicity induced by Tat in the central neural system (CNS). The core aim of this proposal is to establish an efficient gene delivery system using the natural homing properties of monocytes as cell vehicles to deliver potentially neuroprotective factors, anti-Tat Hutat2:Fc fusion antibodies into the CNS, in a non-invasive manner. In this proposal, CRISPR/Cas9-mediated knock-in approach of DNA cassettes into primary mouse bone marrow-derived monocytes will be established. The neuroprotective molecule, DNA cassette of anti-Tat Hutat2:Fc, will be inserted into the “safety harbor” site of host cells' genomes and expressed in high efficiency. Dot-immunobinding assay, Western blotting, neuroprotection assays will be performed to assess the binding ability and neuroprotective effect of Hutat2:Fc fusion antibodies to Tat. Potential adverse effects for CRISPR/Cas9-mediated transfection of cells will be evaluated in vitro by assessing the change of cell morphology, proliferation, and cellular activation in transcriptional profiling of monocyte-related functional and regulatory genes, and expressing of cell surface markers. Then, the distribution and differentiation of genetically modified monocytes intra-CNS following peripheral infusions will be evaluated by the use of a mouse Tat-induced encephalitis model. Finally, whether transgenic monocytes expressing Hutat2:Fc could ameliorate disease progression or induce potential adverse effects will be assessed by TUNEL assay for neurons and immunofluorescent staining for neuroglia in the same mouse model.

抗逆转录病毒治疗不能阻止HAND的重要致病因子Tat的产生，不能完全控制HAND的进展。如设法在脑内拮抗Tat将可能阻断Tat的神经毒性作用，协同治疗HAND。本课题拟建立原代小鼠单核细胞CRISPR/Cas9基因定点敲入系统，将构建的抗HIV Tat融合抗体Hutat2:Fc的编码DNA敲入细胞“安全港”位点，使其整合并持续表达，体外验证小鼠转基因单核细胞表达的Hutat2:Fc融合抗体与Tat的结合能力和神经保护效果，评价CRISPR/Cas9介导Hutat2:Fc基因敲入对原代小鼠单核细胞功能的潜在影响；利用单核细胞可穿越血脑屏障的特性，用Tat诱导的小鼠脑炎模型评估小鼠Hutat2:Fc转基因单核细胞在脑内迁徙、分化情况，体内验证转基因单核细胞的神经保护作用及潜在的副作用。最终建立转基因单核细胞携带治疗性基因迁徙至脑内炎症部位并持续表达Hutat2:Fc的中枢神经系统细胞转运系统。

HIV-1 Tat在HIV相关神经认知功能紊乱（HIV-associated neurocognitive disorder, HAND）的发病过程中发挥重要作用，拮抗脑内的Tat对于HAND的治疗具有重要意义。人源化抗Tat单链抗体-Fc段融合蛋白Hutat2:Fc具有高效的抗Tat活性。然而，血脑屏障是治疗性抗体进入中枢神经系统的障碍。外周血单核细胞可以跨越血脑屏障，被募集在脑内炎症部位周围，单核细胞的这种特性为治疗性抗体的脑内运送提供了潜在的细胞载体。因此我们拟利用CRISPR/Cas9技术构建Hutat2:Fc基因整合的单核细胞以抵抗HIV-Tat引起的神经毒性作用。首先，我们选取人基因组“安全港”AAVS1位点设计6条单链引导RNA序列（single guide RNA, sgRNA），并确定最佳打靶序列以及sgRNA和Donor质粒最适共转染比例。用所建立的CRISPR/Cas9技术在未经筛选的原代人单核细胞中可达到~10%的基因敲入效率。其次，通过免疫荧光染色、Western bloting和ELISA分别检测Hutat2:Fc基因编辑的细胞系和人原代单核细胞Hutat2:Fc蛋白的细胞内分布、表达和分泌水平。证明以上转基因细胞可以稳定的表达Hutat2:Fc并释放出胞，分泌的Hutat2:Fc蛋白可以在培养上清中积累，逐渐达到峰值浓度。再次，我们用小鼠原代神经元、HIV抵抗实验、PBMC淋巴细胞亚群的比例变化和激活抑制分子的表达变化，分别验证了各种细胞培养上清中的Hutat2:Fc蛋白均可不同程度的抵抗HIV-Tat引起的神经毒性作用，抑制HIVBA-L的复制和扩散，维持淋巴细胞稳态，且这些保护作用同上清中的Hutat2:Fc蛋白的浓度正相关。最后，我们通过比较Hutat2:Fc蛋白表达量、单核细胞相关功能因子基因表达差异、炎症因子分泌的差异以及单核细胞迁徙实验的检测发现，CRISPR/Cas9基因整合技术虽然在插入效率和蛋白表达量上稍逊于传统的慢病毒转导技术，但其对单核细胞正常功能、尤其是单核细胞的跨膜功能影响较小。综上所述，本研究成功建立了以CRISPR/Cas9介导的Hutat2:Fc单核细胞基因整合系统，为保存单核细胞生物学功能创造了条件，为HAND的细胞治疗提供了初步的理论和实验基础。