1. 中国科学院大学 生命科学学院 生物互作卓越创新中心，北京 100049;
2. 中国科学院生物物理研究所 脑与认知国家重点实验室，北京 100101
收稿日期：2020-03-31；接收日期：2020-07-24；网络出版时间：2020-08-12
基金项目：国家自然科学基金(No. 31571042)，北京市自然科学基金(No. 7202129)，中国科学院科教融合专项基金(Nos. KJRH2015-011, Y55201BY00)资助
摘要：阿尔茨海默症(Alzheimer's disease，AD)与帕金森症(Parkinson’s disease，PD)是人类常见的神经退行性疾病。AD与PD发病机制复杂，目前的药物和外科手术治疗无法缓解或阻止疾病进程。慢病毒载体(Lentiviral vector，LV)是一种逆转录病毒载体，由LV介导的基因治疗已成为当前疾病机制研究、临床药物开发等领域的研究热点。文中综述了近年来LV应用于AD、PD治疗的研究进展，并对今后的应用研究前景进行了展望。
关键词：阿尔茨海默症帕金森症慢病毒载体基因治疗
Progress in lentiviral vector-mediated gene therapy for Alzheimer's disease and Parkinson's disease
Derun Xu1, Li Liu2, Minglin Lang1
1. CAS Center for Excellence in Biotic Interactions, College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China;
2. State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, Chinese Academy of Sciences, Beijing 100101, China
Received: March 31, 2020; Accepted: July 24, 2020; Published: August 12, 2020
Supported by: National Natural Science Foundation of China (No.31571042), Natural Science Foundation of Beijing, China (No.7202129), Science and Education Integration Special Fund Project of Chinese Academy of Sciences (Nos.KJRH2015-011, Y55201BY00)
Corresponding author: Minglin Lang. Tel/Fax: +86-10-69672639; E-mail: langml@ucas.ac.cn.
Abstract: Alzheimer's disease (AD) and Parkinson's disease (PD) are common neurodegenerative diseases in human. The pathogenesis of AD and PD is complex, and the current drugs and surgical treatments have not successfully alleviated or terminated the progression of the diseases. The lentiviral vector (LV) is a retroviral vector. In recent years, LV mediated gene therapy has been a hotspot to study the mechanisms of human disease and clinical drug discovery. This review summarizes the recent progresses in the treatment of AD and PD by the application of LV, and offers a prospect for its application.
Keywords: Alzheimer's diseaseParkinson's diseaselentiviral vectorgene therapy
人脑是由神经元、星形胶质细胞、少突胶质细胞、伸展细胞和小胶质细胞组成的高度复杂器官，不同的细胞类型特定排列在高度复杂的网络中，共同工作并在空间上受到有序调节[1]。然而，随着个体的衰老，维持内稳态的分子调控机制出现异常，可能导致神经退行性疾病的发生，如阿尔茨海默症(Alzheimer’s disease，AD)、帕金森症(Parkinson’s disease，PD)等[1]。
AD是痴呆症最常见的一类疾病[2]。AD是以细胞外淀粉样斑块沉积及细胞内过度磷酸化的微管相关Tau蛋白积聚为主要病理特征的神经退行性疾病[3]。AD主要临床表现为进行性记忆障碍、认知功能紊乱等[4-5]。针对AD发病机制有胆碱能假说、β淀粉样蛋白假说、Tau蛋白假说、金属离子紊乱假说等[6]。目前，美国食品药品监督管理局(Food and Drug Administration，FDA)已批准4种用于治疗AD的药物，包括3种胆碱酯酶抑制剂和一种非竞争性N-甲基-D-天冬氨酸(NMDA)受体拮抗剂[2, 7]。虽然上述药物可以减轻或暂缓AD的症状，但是不能阻止和逆转AD的进程。
PD是一种复杂、慢性且异质性的神经退行性疾病[8]。PD病变主要发生在黑质(Substantia nigra，SN)纹状体通路，SN多巴胺能神经元变性和路易小体(Lewy bodies，LBs)是PD的主要病理特征[9]。α-突触核蛋白(α-synuclein，α-SYN)是LBs的主要成分，α-SYN的过度表达和聚集有细胞毒性，最终导致多巴胺能神经元死亡[9]。PD引发的运动功能障碍包括运动迟缓、僵直、静止性震颤、姿势不稳等，同时可能伴随嗅觉减退、睡眠障碍、认知功能障碍等非运动症状[8]。有****按照神经病理严重程度和疾病进展速度将PD分为缓慢进展型PD (运动症状为主)、弥漫型PD和中间型PD[10]。临床上主要采用药物(如左旋多巴)治疗PD，虽然药物疗效在3–5年内对非弥漫性PD有较好疗效，但长期使用导致“剂末现象” (又称剂末恶化，指每次用药的有效作用时间缩短，症状随血液药物浓度发生规律性波动)及“开关现象” (PD患者突然出现症状加重、僵直，但未进行任何治疗后，症状又突然消失的现象)，且目前的药物无法逆转PD进程[11-12]。
AD、PD的复杂性及异质性导致疾病的临床诊疗频频受阻，全基因组关联分析(Genome-wide associate study，GWAS)已发现数百个神经系统疾病相关基因，但不清楚这些基因在哪些细胞中活跃表达，且神经系统疾病与不同的细胞类型相关[13]。如，PD不仅与胆碱能神经元、单胺能神经元有关，而且与肠神经元、小胶质细胞及少突胶质细胞有密切联系。同时神经退行性疾病的发生因人而异，需要针对不同的患者亚群进行定制的组合治疗[13]。目前神经退行性疾病的治疗能够缓解症状，但无法解决潜在的病理问题[14]。随着疾病发展，药物治疗的效果会降低，且血脑屏障(Blood brain barrier，BBB)阻止药物进入中枢神经系统(Central nervous system，CNS)相应靶点发挥作用，药物副作用通常随着用药剂量的增大而增加[14-15]。脑内注射神经营养因子或载体介导的基因治疗是避免受BBB影响的重要途径，由于基因治疗载体可以实现良好的耐久性，受到持续关注[14]。病毒转导技术是基因治疗的重要手段，该技术应用改造后的无致病性病毒载体，将目的基因传递到靶细胞[16]。用于基因治疗的病毒载体有腺病毒(Adenovirus，Ad)、腺相关病毒(Adeno-associated virus，AAV)及慢病毒(Lentivirus，LV)[17]。Ad是一种非整合型双链DNA病毒，其外源基因装载能力约为7–10 kb。然而，Ad因具有较高的免疫原性，大大限制了该载体的临床应用[18-19]。AAV是非整合型单链DNA病毒，载体自身免疫原低，广泛应用于体内外实验研究及临床诊疗[20]。LV是属于逆转录病毒科的单链RNA病毒，免疫原性低。临床级的LV无致病性且无复制能力[16, 18, 21]。相较于AAV，LV具有如下优势：(1)整合表达。靶基因的稳定持续表达是临床实验成功的关键，LV能介导目的基因稳定整合到宿主细胞基因组中，虽然AAV某些情况下可以整合到宿主基因组中，但该载体并不是在所有条件下都能持续表达[18]。(2)包装能力。AAV的外源基因装载能力较低，目前最多容纳约5 kb的外源片段，LV相较于AAV具有较大的包装能力(8–9 kb)[22]。(3)载体应用。以水疱性口炎病毒表面糖蛋白(Vesicular stomatitis virus surface glycoprotein，VSVG)为包膜的慢病毒载体介导基因治疗不会导致轴突末端向大脑其他部位逆行转移，而AAV有超过12种不同的血清型，在不同物种中有不同的表达模式，这对解释临床前模型动物实验的结果有负面影响[18, 23]。实验表明，LV具有更强的转导神经系统终末分化细胞的能力[24]。因此，慢病毒在AD、PD的基因治疗中更具优势。此篇综述将主要讨论慢病毒载体介导基因治疗AD、PD的研究进展。
1 慢病毒载体结构及特点1.1 慢病毒载体的种类慢病毒属于逆转录病毒科的RNA病毒家族[25]。早期慢病毒载体，由人类免疫缺陷病毒(Human immunodeficiency virus，HIV)改装而来[22]，随后，猫免疫缺陷病毒(Felines immunodeficiency virus，FIV)[26]、猿类免疫缺陷病毒(Simian immunodeficiency virus，SIV)[27]、牛免疫缺陷病毒(Bovine immunodeficiency virus，BIV)[28]、绵羊髓鞘脱落病毒(Maedi-visna virus，MVV)[29]等载体相继研发。
1.2 慢病毒载体的结构以应用最广泛的HIV-1型慢病毒载体为例，目前开发的第3代质粒系统为四质粒系统，包括Rev (编码辅助蛋白)、Gag (编码结构蛋白)/Pol (编码病毒复制相关酶)、Env (编码病毒包膜蛋白)和目的基因序列载体[30]。四质粒系统大大降低体系产生意外重组的可能性，且未对载体感染效率产生负面影响[29-30]。
1.3 慢病毒载体的应用LV的趋向性和转导宿主类型主要取决于使用的包膜蛋白[22]。VSVG是一种三聚体蛋白，与其受体低密度脂蛋白受体(LDLR)的结合可以促进细胞内吞作用[31]。VSVG-LV较稳定，可以承受超速离心，从而产生高滴度的LV制剂[31]。VSVG-LV具有高度的稳定性和广泛的趋向性，可以转导干细胞、神经元等较难转导的细胞[31-32]。目前，使用异源包膜蛋白的慢病毒载体已被广泛使用，淋巴细胞性脉络丛脑膜炎病毒(Lymphocytic choriomeningitis virus，LCMV)糖蛋白包装LV可实现大鼠星形胶质细胞和黑质神经元优先转导[33]；VSV相关病毒金迪普拉(Chandipura virus glycoprotein，CNVG)相对于VSVG转导神经母瘤细胞的能力更强[31]；狂犬病毒(Rabies Virus，RabV)包膜的糖蛋白受体是神经营养素受体(P75NTR)、神经细胞黏附分子(NCAM)和烟碱型乙酰胆碱受体(NAChR)，RabV-LV可高效转导神经细胞[34]，且能够通过动力蛋白复合体介导从轴突表面到细胞核的逆向运输[25]。目的基因在时空上的可控性是基因治疗成功与否的关键因素，如四环素诱导调控表达系统具有灵敏、机理清楚等优势，广泛应用于体内外研究，该系统可用于LV实现目的基因的条件性表达[35]。因此，慢病毒载体介导的基因治疗在临床实验中发挥着举足轻重的作用。
2 慢病毒介导基因治疗PD进展2.1 PD诊疗现状目前，临床治疗PD主要通过药物治疗和手术治疗。治疗药物可分为抗胆碱能药物、多巴胺替代疗法药物、多巴胺受体激动剂、单胺氧化酶-B抑制剂、金刚烷胺、儿茶酚-O-甲基转移酶(COMT)抑制剂等[12, 36-37]。抗胆碱能药物和多巴胺受体激动剂常作为PD辅助治疗药物，但有多种不良反应[8, 36]。针对PD患者中脑黑质多巴胺神经元变性死亡、纹状体多巴胺水平明显降低的病理特征，左旋多巴是改善PD症状的现有最佳药物[12, 38]。左旋多巴能有效治疗僵直及运动迟缓[36]。卡比多巴(Carbidopa)是外周多巴脱羧酶抑制剂，与左旋多巴合用时，减少外周组织生成多巴胺，从而减轻恶心等不良反应[8]。然而，随着PD的进展，内源性多巴胺储存和释放能力降低可能导致左旋多巴疗效下降，出现运动性波动和异动症[36]。PD手术治疗包括经皮内镜下空肠造口术(Percutaneous endoscopic gastrostomy with jejunal extension，PEG-J)提供持续的左旋多巴/卡比多巴肠凝胶，该方式较口服左旋多巴/卡比多巴虽能更长时间控制PD症状，但并不能避免药物治疗后运动性波动和异动症的发生[39]。脑深部电刺激疗法(Deep brain stimulation，DBS)是通过脑立体定向手术在脑深部植入高频刺激电极，改善PD患者运动症状与非运动症状，从而提高生活质量的技术。然而，手术过程中脑脊液(Cerebral spinal fluid，CSF)丢失、颅内压下降可能导致脑部移位现象，术后并发症包括心肌梗死、脑血肿、中风、癫痫等[8, 40]。PD现有药物的治疗只能暂时改善患者部分运动性症状，手术治疗也需要权衡疾病进程与术后并发症的利弊，且非运动性症状还无法得到很好的改善。因此，基因治疗也许是延缓、阻止甚至逆转PD疾病进程的可行方式。
2.2 PD的基因治疗基因治疗PD的想法是由基底神经节神经元的基因修饰能弥补多巴胺释放不足，并挽救运动起始通路的活性而优化的[11]。慢病毒载体已被证明能高效地在黑质和纹状体中传递基因[21]。目前临床基因治疗PD主要通过增加多巴胺合成、调节神经营养因子表达和促进神经调节弥补基底节多巴胺信号缺失，挽救PD运动性症状[11, 41]。ProSavin是携带酪氨酸羟化酶(Tyrosine hydroxylase，TH)、GTP环化水解酶1 (GTP cyclization hydrolase 1，GCH1)和芳香族氨基酸脱羧酶(Aromatic acid decarboxylase，AADC)序列的慢病毒载体，这3种酶是酪氨酸合成多巴胺所必需的[42]。一项临床1/2期开放标签实验证明，ProSavin对PD患者是安全的，并且改善了运动能力，但并没有达到多巴胺替代疗法的最佳水平[43]。OXB-102慢病毒载体与ProSavin基因组相似，但3个外源基因顺序不同，在1-甲基-4-苯基-1, 2, 3, 6-四氢吡啶(1-methy-4-phenyl-1, 2, 3, 6-tetrahydropyridine，MPTP)猕猴模型中，多巴胺产量较ProSavin高，在载体注射6个月后，治疗动物临床评分和自发活动都有显著改善，动物未出现载体相关的临床或行为异常，目前正进行进一步临床开发[44]。研究表明，外源性单唾液酸四己糖神经节苷脂(Trisialoganglioside- GT1b，GM1)具有广泛的神经保护作用，抑制神经节苷脂GD3合酶(Ganglioside biosynthetic enzyme GD3 synthase，GD3S)能增加内源性GM1水平，近期有实验室应用慢病毒载体介导靶向GD3S的短发夹RNA (Short hairpin RNA，shRNA)抑制其表达，结果表明抑制GD3S可以保护小鼠与MPTP给药相关的黑质、纹状体损伤，并能减轻运动迟缓等症状[45]。Persephin蛋白(PSPN)与胶质细胞源性神经营养因子(Glial cell line-derived neurotrophic factor，GDNF)均具有神经营养作用。将带有PSPN的LV载体转染大鼠黑质多巴胺能神经元，处理1周后，将6-羟基多巴胺(6-hydroxydopamine，6-OHDA)注入大鼠内侧前脑束建立PD模型。PSPN预处理组黑质多巴胺能神经元数量增加，纹状体多巴胺及其代谢产物浓度升高，表明PSPN对6-OHDA诱导的PD具有神经保护作用[46]。硫化氢(H2S)在神经系统中起重要作用，利用带有胱硫醚-β-合成酶(Cystathionine-β-synthase，CBS) (脑内合成H2S合成酶)的LV载体转染大鼠左侧纹状体，显著抑制6-OHDA诱导的氧化应激损伤，减少黑质多巴胺能神经元的丢失，证明CBS过表达对PD大鼠具有神经保护作用[47]。LV-GDNF也应用于多种动物模型，GDNF过表达对多巴胺能神经元有显著保护作用[48]。基因治疗通常对孟德尔遗传病有较好的治疗效果[16]，家族性PD约占PD患者的10%[11]，PINK1 (PTEN induced putative kinase 1)、β-葡糖脑苷脂酶(β-glucocerebrosidase，GBA1)、LRRK2 (Leucine-rich repeat kinase 2)等基因突变是造成PD的高风险因素[49-50]，虽然其中的机制还未明确，但也为基因治疗提供相应的靶点。目前，除了前述PD具有复杂性、异质性外，PD的临床诊疗同样面临如下挑战：(1)未知PD确切的病因和发病机制，大部分研究仅仅针对单一靶点，通常也只假设单一的主要致病机制。(2)临床前细胞、动物模型可能不能很好地反映人类PD发病机制。(3)尚不清楚所使用的结果衡量标准或临床实验设计是否适用于评估疾病状态，缺乏可靠的生物标志物反映疾病的存在和严重程度。上述慢病毒介导基因治疗PD在模型动物实验中有显著改善，为临床实验打下了良好基础。同时LV因其转导效率高、重复性好、包装能力较强(可承载多个目的基因，如ProSavin)等优势，具有良好前景。
3 慢病毒介导基因治疗AD进展3.1 AD诊疗现状如前所述，FDA批准用于治疗AD的药物能减缓疾病发展，但无法阻止或逆转AD的进程。轻度认知障碍患者使用胆碱酯酶抑制剂(Cholinesterase inhibitors，ChEIs)甚至可能恶化认知[51]。目前，AD的非药物疗法如记忆训练、音乐疗法、有氧运动、认知行为疗法等可以提高部分患者生活质量但同药物治疗相同，并不能阻止AD引发的神经元损伤、破坏[52]。
3.2 AD的基因治疗3.2.1 Aββ分泌酶1 (Beta-secretase，BACE1)是水解淀粉样前体蛋白(Amyloid precursor protein，APP)生成β-淀粉样蛋白(Amyloid β-protein，Aβ)的限速酶。鉴于BACE1在产生Aβ中的关键作用，使用LV载体表达BACE1-siRNA转导AD小鼠，可以减少模型动物Aβ生成并改善小鼠的行为缺陷[53]。然而有研究表明，抑制BACE1对突触可塑性及认知功能有不良影响，有证据显示低水平Aβ对认知水平有积极作用[54]。Aβ的沉积影响cAMP反应元件结合蛋白(cAMP-response element binding protein，CREB)的活性，LV过表达CREB转导APP/PS1/Tau小鼠，能改善模型的学习和记忆障碍[55]。
3.2.2 神经营养因子神经营养因子通过诱导不同信号通路调控神经元的存活、分化等，与AD密切相关。脑源性神经营养因子(Brain-derived neurotrophic factor，BDNF)在AD中表达水平下降[3]，将LV过表达BDNF载体转导APP小鼠，可以改善学习记忆、增加突触蛋白突触素(Synaptophysin，Syn)的表达，防止内嗅皮层神经元死亡[56]。该实验表明，BDNF通过与淀粉样蛋白无关的机制(实验不伴随淀粉样蛋白的减少)对AD相关的神经回路发挥保护作用[56]。LV过表达GDNF载体转导APP/PS1/Tau小鼠，与绿色荧光蛋白载体转导组相比，实验组小鼠学习和记忆功能相对完好，GDNF并没有显著缓解淀粉样蛋白和Tau蛋白病理学变化，却可以诱导BDNF上调，可能协同GDNF发挥神经保护作用[57]。一项临床1/2期研究将AAV介导的神经生长因子(Nerve growth factor，NGF)转导AD患者前脑基底大细胞核。结果表明，AAV-NGF的治疗在24个月内是安全的，且AAV-NGF可以使表现有Tau病理的神经元及正常神经元表达NGF，但AD评定量表-认知量表结果显示，治疗组和对照组间没有显著差异[58-59]。
3.2.3 神经炎症神经炎症也是AD发病重要原因之一，小胶质细胞是AD致炎细胞因子的主要来源[60]。髓样细胞触发受体2 (Triggering receptor expressed on myeloid cells 2，TREM2)通过抑制小胶质细胞介导的细胞因子的产生和分泌来抑制炎症反应，并参与吞噬途径的调节[61]。在APP/PS1小鼠脑内，LV介导TREM2的过表达能改善Aβ的沉积、神经炎症、神经元和突触丢失，并且伴随着空间认知功能的改善。可能的机制为TREM2过表达增强了小胶质细胞吞噬作用[61]。此研究者运用LV介导TREM2在P301S (Tau转基因)小鼠的小胶质细胞过表达，模型小鼠空间认知损伤得到挽救，并改善了Tau过度磷酸化及神经元和突触的丢失。这种保护作用可能与抑制神经炎症和Tau磷酸化相关激酶(白细胞介素1、白细胞介素6)活性相关。同时，M2型小胶质细胞标志物白细胞介素4、白细胞介素10的表达明显增加，提示TREM2诱导小胶质细胞活化可能是其抑制神经炎症的机制之一[62]。
3.2.4 氧化应激作用氧化应激作用是AD的重要机制，其中Nrf2 (NF-E2-related factor 2)可通过与抗氧化反应元件ARE (Antioxidant response element)相互作用调节抗氧化蛋白的表达[63]。Kanninen等利用LV过表达Nrf2转染APP/PS1小鼠明显改善了小鼠空间学习能力[64]。
3.2.5 自噬作用自噬作用是维持神经元正常生理功能的重要细胞机制[65]，Beclin1基因也称BECN1基因，是哺乳动物参与自噬的特异性基因，该基因的表达可激活自噬作用、降低淀粉样蛋白聚集并改善认知水平[66]。实验表明，LV介导Beclin1在海马区过表达，降低了淀粉样蛋白的积累[1]。
目前，由于LV安全性以及AD发病的具体机制了解还不够深入等原因，LV介导基因治疗AD距离临床实验仍有不小的距离。表 1总结了近年来LV应用于AD动物模型的实验进展。
表 1 LV基因治疗AD动物模型Table 1 LV-mediated gene therapy in AD models
| Gene | Expression type | Target/Mechanism | Animal model | Phenotypes | Reference |
| Klotho | OE | Microglia autophagy | APP/PS1 mouse | Ameliorated cognitive deficit and AD-like pathology | [67] |
| NPY | OE | Neuroprotection | APP mouse | Reversed neurodegenerative pathology and behavioral deficits | [68] |
| ANP32A | KD | Histone acetylation | Tau mouse | Ameliorated synaptic plasticity and memory ability | [69] |
| SHIP2 | KD | Tau | APP/PS1/Tau mouse | Rescued tau hyperphosphorylation and memory impairments | [70] |
| EphB2 | OE | NMDA receptor | APP/PS1 mouse | Ameliorated impaired memory deficits and anxiety or depression-like behaviors | [71] |
| MCT4 | KD | Astrocyte | APP/PS1 mouse | Improved cognitive ability, reduced neuronal apoptosis and γ-secretase expression | [72] |
| S1T | OE | ER stress | APP/PS1/Tau mouse | Enhanced the β-secretase expression and triggered neuroinflammation | [73] |
| BACE1-AS | KD | BACE1 | SAMP8 mouse | Improved the memory and learning behaviors, inhibited BACE1 production, and rescued tau hyperphosphorylation | [74] |
| NLRP1 | KD | ROS | DEX mouse | Alleviated neuronal degeneration | [75] |
| GSK-3β | KD | Tau | APP/PS1 mouse | Ameliorated memory ability and rescued tau hyperphosphorylation | [76] |
| HDAC3 | KD | Aβ | APP/PS1 mouse | Attenuated spatial memory deficits and decreased Aβ levels | [77] |
| ABCA7 | OE | Aβ, ER stress | Aβ inject mouse | Improved cognitive behavior and decreased Aβ levels | [78] |
| NPY: neuropeptide Y; ANP32A: recombinant acidic nuclear phosphoprotein 32 family member A; SHIP2: Src homology 2 domain containing inositol polyphosphate; EphB2: Erythropoietin-producing hepatocyte receptor B2; MCT4: monocarboxylate transporter 4; S1T: sarco-endoplasmic reticulum Ca 2+ ATPase1; BACE1-AS: BACE1 antisense transcript; NLRP1: nucleotide-binding oligomerization domain-like receptor protein 1; DEX: Dexamethasone; GSK-3β: Glycogen synthase kinase 3β; HDAC3: histone deacetylase 3; ABCA7: ATP-binding cassette transporter A7. | |||||
表选项
4 展望目前，AD与PD发病机制了解得还不够深入。LV介导治疗PD的临床实验显示，治疗效果并不比左旋多巴等药物治疗有效，同时出于LV自身安全性考虑，载体介导基因治疗AD与PD距离临床应用还有一定距离。值得肯定的是，LV转导人CD34+细胞是一项成熟的技术，已经在一些患有联合免疫缺陷症的患者中产生了疗效，迄今已被证明是安全的[79]。综合上述进展，未来应着手以下研究。
(1) 深入研究神经退行性疾病机制。AD临床药物研发中，不乏众多靶向Aβ低聚物、Aβ斑块、γ-分泌酶、β分泌酶、Tau、线粒体代谢、过氧化物酶体增殖物激活受体(Peroxisome proliferator- activated receptor，PPAR)等，但大多对病患认知及ADL没有改善作用[80]。PD临床药物中虽有特效药左旋多巴，但仍无法避免药物带来的“开关现象”、“剂末现象”。随着AD、PD机制研究更加深入，可能为药物研发提供合适的靶点。
(2) 模型生物的合理选择。近年来转基因小鼠是AD研究的常用模型，但是小鼠与人类在基因组、神经解剖学、免疫系统及行为学等方面具有差异，并且不同实验室间小鼠研究结果缺乏可重复性，导致该模型难以完全模拟人类AD[81]。目前，一些实验室使用诱导多功能干细胞(Induced pluripotent stem cells，iPSCs)建立细胞模型用于研究AD发病机制，使用遗传学和基因编辑的手段进行2D或3D建模，但是同一疾病的不同遗传背景和个体差异也是iPSCs在AD研究中面临的挑战之一[82]。
(3) LV的生物安全性及载体系统的优化。CRISPR/Cas9介导的基因编辑可以激活转录或实现基因沉默，与RNAi相比具有独到优势。LV-CRISPR/Cas9系统已广泛应用于多种疾病模型，这一技术在基础研究及临床实验应用中具有巨大潜力[1]。研究表明，胶质细胞成熟因子(Glia maturation factor，GMF)是一种促炎分子，在AD大脑的不同区域显著上调。表达化脓性链球菌Cas9和GMF-sgRNAs的LV转导细胞显示GMF表达量降低，且相较于AAV介导的基因编辑具有更高的效率[83]。虽然LV载体经过多代改造后安全性增高，但是病毒载体的某些成分(如编码结构蛋白的基因)仍有引发人体免疫排斥反应的可能[17]。针对LV引发的免疫反应可以通过免疫抑制药物、载体加入破坏先天免疫系统的单克隆抗体缓解。LV载体在转染细胞时，目的片段随机插入基因组有可能会造成基因突变等严重后果，整合酶缺陷型慢病毒包装系统具有更高的生物安全性[35]。另外，加入可对外源基因进行转录调节的调控元件，如四环素调节系统，是基因治疗安全性有效性的重要保障[84]。尽管理论上LV具有致癌的可能，但尚无相关的病例报道。对已接受LV为基础的基因治疗患者，定期随访对于了解载体安全性及有效性是必要的[85]。
(4) 生物标志物与诊断方式。AD、PD现有的治疗方式只能在患者确诊后才能实施，此时的大脑已受到不可逆损伤[86]。多种临床实验药物无法很好地改善ADL、认知障碍、运动障碍可能与临床实验患者症状较重有关。因此，探索生物标志物，在疾病早期确诊并优化诊断方式迫在眉睫。最近有研究发现，线粒体动力蛋白相关蛋白1 (Dynamin related protein，DRP1)的缺失会引发多条应激反应通路，刺激神经来源的成纤维生长因子21 (Fibroblast growth factor 21，FGF21)释放，导致线粒体损伤。该现象在神经退行性疾病中，发生在神经元死亡之前，有望成为新的生物标志物[87]。Aβ在大脑中广泛积累，甚至在没有任何症状的人群中也是如此[88]。相反，Tau恰好集中在脑萎缩最严重的位置(有助于解释患者症状差异的区域)，新型可注射分子Flortaucipir能与大脑中错误折叠的Tau结合，并通过正电子发射型计算机断层显像(Positron emission computed tomography，PET)技术扫描成像有助于诊断及预测AD的进程[88]。
(5) 靶向多靶点治疗。AD、PD等神经退行性疾病进程复杂，涉及到的基因、通路众多，单一增强或抑制某个靶点，可能无法阻止疾病进程。研究表明，利用过表达Ascl1 (Achaete-scute complex homolog 1)、Brn2 (POU class homeobox 2)、Ngn2 (Neurogenin 2)三个转录因子的LV注射小鼠(Aβ处理)海马区，可明显改善AD小鼠的空间学习记忆障碍[89]。LV具有较大的克隆容量，携带多个序列进行多靶点治疗可能阻止或逆转疾病进程。
(6) 病患生活质量。临床根据AD、PD患者症状进行医治，往往忽视疾病并发症的诊疗。关注病患生活质量，如PD患者非运动性症状(便秘、失眠、抑郁、焦虑)[8]，提高生活幸福感也是完善诊疗方法的重要环节。
5 结语AD、PD两种神经退行性疾病长时间以来给患者、患者家庭、社会带来沉重的负担。关于AD、PD诊疗，尽管全球在机制研究、疾病诊断、临床治疗、生物制药等方面作出努力，但距离明确疾病具体机制、临床药物成功开发、病患成功治疗仍有很长的距离。本文旨在综述目前LV介导基因治疗AD、PD进展，为相关研究提供借鉴与参考。长期研究表明，LV具有转导效率高、免疫原性较低、克隆容量大等无可比拟的优势，CRISPR/Cas9等技术又为其发展注入新的可能，为治疗AD、PD提供巨大的潜力与希望。
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