摘要: 基于基因治療作為從根本上治療疾病的技術(shù)手段, 為類風(fēng)濕性關(guān)節(jié)炎的治療帶來新的思路和方法, 綜述類風(fēng)濕性關(guān)節(jié)炎基因治療的相關(guān)研究進(jìn)展, 包括小干擾RNA(siRNA)、 微小RNA(miRNA)、 DNA, CRISPR/Cas9系統(tǒng)、 脫氧核酶以及一些其他技術(shù), 為基因治療在類風(fēng)濕性關(guān)節(jié)炎領(lǐng)域的應(yīng)用提供借鑒思路, 并為類風(fēng)濕性關(guān)節(jié)炎患者提供更有效、 更具針對性的治療方案.
關(guān)鍵詞:" 類風(fēng)濕關(guān)節(jié)炎; 基因治療; 小干擾RNA; 微小RNA; DNA折紙; CRISPR/Cas9系統(tǒng)
中圖分類號: Q527" 文獻(xiàn)標(biāo)志碼: A" 文章編號: 1671-5489(2025)01-0216-13
Research Advances of Gene TherapyTechnology for Rheumatoid Arthritis
ZHANG Hugang," JIA Jiaxin," LIU Hanyu," LI Quanshun
(Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education,
Jilin University," Changchun 130012, China)
Abstract:""" Based on gene therapy as a fundamental treatment for diseases, it brings new ideas and methods for the treatment of rheumatoid arthritis (RA). We review" the relevant research advances" of gene therapy for rheumatoid arthritis," including small interfering RNA (siRNA), micro RNA (miRNA)," DNA," CRISPR/Cas9 system," deoxyribonuclease and some other technologies,"" providing reference ideas for the application of" gene therapy in the field of RA and offering more" effective and targeted treatment plans for the patients with RA.
Keywords: rheumatoid arthritis; gene therapy; small interfering RNA; micro RNA; DNA origami; CRISPR/Cas9 system
類風(fēng)濕性關(guān)節(jié)炎(rheumatoid arthritis, RA)是一種自身免疫性疾病, 以關(guān)節(jié)炎癥、 疼痛和功能障礙為臨床特征, 影響全球0.5%~1%的人口[1-2]. 其特征表現(xiàn)為對稱性、 多關(guān)節(jié)的滑膜炎癥, 若未經(jīng)及時有效的治療, 則可逐漸導(dǎo)致關(guān)節(jié)軟骨和骨組織破壞, 進(jìn)而引發(fā)關(guān)節(jié)畸形和功能喪失, 嚴(yán)重影響患者生活質(zhì)量, 并增加社會醫(yī)療成本. 長期以來, 類風(fēng)濕性關(guān)節(jié)炎的治療處于不斷探索與發(fā)展階段. 傳統(tǒng)的治療方法主要以藥物治療為主, 如非甾體抗炎藥(NSAIDs)、 抗風(fēng)濕藥物(DMARDs)以及糖皮質(zhì)激素等, 其共同點(diǎn)是可有效緩解疼痛和炎癥, 但對疾病進(jìn)程的控制作用有限, 同時藥物也會導(dǎo)致諸多并發(fā)癥. 基因治療能通過調(diào)控與疾病相關(guān)的基因表達(dá), 從根本上糾正疾病發(fā)生發(fā)展的異常生物學(xué)過程, 為傳統(tǒng)治療效果不理想的患者提供更有效的治療方案." 常用的基因治療方法包括RNA(小干擾RNA(siRNA)和微小RNA(miRNA))、 反義寡核苷酸(ASO)、 DNA(質(zhì)粒和DNA折紙)、 基因編輯體系、 脫氧核酶(Dz)、 靶向蛋白水解嵌合體(PROTAC)技術(shù)以及疫苗等, 為類風(fēng)濕性關(guān)節(jié)炎的治療提供了多種可能.
本文綜述常用基因治療方法在類風(fēng)濕性關(guān)節(jié)炎領(lǐng)域的研究進(jìn)展, 并對該技術(shù)未來的發(fā)展前景進(jìn)行展望.
1 RNA治療
1.1 小干擾RNA
siRNA作為一類長度約為21~23個核苷酸的雙鏈RNA分子, 基于RNA干擾 (RNA interference) 在生物體內(nèi)發(fā)揮重要的基因調(diào)控作用[3], 具體作用過程如下: 1) 起始階段." Dicer酶作為具有RNase Ⅲ活性的核酸內(nèi)切酶, 特異性識別并切割細(xì)胞內(nèi)長鏈dsRNA以形成多個雙鏈siRNA分子. 2) 效應(yīng)階段. RNA誘導(dǎo)沉默復(fù)合物 (RNA-induced silencing complex," RISC) 內(nèi)包含的Argonaute 蛋白家族成員識別、 結(jié)合siRNA的引導(dǎo)鏈并降解反義鏈. 引導(dǎo)鏈通過堿基互補(bǔ)配對結(jié)合靶mRNA和Argonaute 蛋白作為核酸內(nèi)切酶切割并降解靶mRNA, 阻止翻譯為功能蛋白進(jìn)而實(shí)現(xiàn)基因沉默. siRNA以其高度的底物特異性, 可實(shí)現(xiàn)精準(zhǔn)基因調(diào)控避免脫靶效應(yīng), 以高效沉默活性快速結(jié)合靶mRNA完成基因沉默, 在基因功能研究、 疾病治療及農(nóng)業(yè)生物技術(shù)領(lǐng)域具有廣闊的應(yīng)用前景[4]. 目前, siRNA已成功應(yīng)用于類風(fēng)濕性關(guān)節(jié)炎基因治療領(lǐng)域, 通過干預(yù)NF-κB和MAPK等經(jīng)典炎癥信號通路以抑制滑膜炎癥、 降低骨損傷以及減緩類風(fēng)濕性關(guān)節(jié)炎小鼠的病理學(xué)進(jìn)程[5]. 如 Liu等[6]將HIF-1α siRNA裝載至以重組高密度脂蛋白(rHDL)為核心、 摻入磷酸鈣(CaP)及載脂蛋白E3(apoE3)形成的HIF-CaP-rHDL納米復(fù)合物中, 將納米復(fù)合物轉(zhuǎn)染至脂多糖誘導(dǎo)的炎癥巨噬細(xì)胞模型, 可特異性降低HIF-1α表達(dá)水平, 抑制NF-κB和 MAPK信號通路p-p65和p-IκBα等因子的表達(dá), 有效減少NF-κB受體活化因子配體(RANKL)誘導(dǎo)的破骨細(xì)胞生成. 將CaP-rHDL/siHIF納米復(fù)合物經(jīng)尾靜脈注射至膠原誘導(dǎo)的類風(fēng)濕性關(guān)節(jié)炎小鼠模型(CIA模型)中, 在實(shí)驗(yàn)終點(diǎn)第46天時, 納米復(fù)合物高效降低CIA小鼠爪部腫脹至3.0 mm以下, 并降低血清炎癥因子表達(dá)水平、 緩解炎癥滑膜細(xì)胞浸潤及軟骨損傷(圖1).
Guo等[7]將TNF-α siRNA裝載至由三價鐵(Fe Ⅲ)和單寧酸(TA) 配位形成的抗氧化金屬有機(jī)框架材料中, 再經(jīng)牛血清白蛋白 (BSA) 修飾后得到TFSB納米復(fù)合物. 該納米復(fù)合物能主動靶向M1型巨噬細(xì)胞受體實(shí)現(xiàn)高效基因遞送, 可顯著降低炎癥巨噬細(xì)胞內(nèi)TNF-α,IL-1β和IL-6等炎癥因子水平." 此外, 納米復(fù)合物轉(zhuǎn)染后炎癥巨噬細(xì)胞內(nèi)M1型巨噬細(xì)胞蛋白標(biāo)識物CD68和iNOS表達(dá)量下降, M2型巨噬細(xì)胞標(biāo)識物Arg-1和CD206等表達(dá)量升高, 實(shí)現(xiàn)巨噬細(xì)胞復(fù)極化. 經(jīng)尾靜脈注射熒光標(biāo)記TNF-α siRNA后, 可蓄積至CIA小鼠發(fā)炎關(guān)節(jié), 降低局部炎癥因子基因及蛋白表達(dá)水平, 促使關(guān)節(jié)炎癥巨噬細(xì)胞復(fù)極化, 抑制金屬基質(zhì)蛋白酶2 (MMP2) 含量的增加以預(yù)防軟骨損傷, 減緩CIA炎癥疾病進(jìn)程(圖2).
Nasra等[8]將靶向NF-κB的REL-A siRNA和甲氨蝶呤 (MTX) 裝載至葉酸修飾的脂質(zhì)體中得到FOL-脂質(zhì)體納米復(fù)合物以實(shí)現(xiàn)對RA的協(xié)同治療. FOL-脂質(zhì)體表面修飾的葉酸能主動靶向至M1型炎癥巨噬細(xì)胞表面的葉酸受體, 實(shí)現(xiàn)藥物在巨噬細(xì)胞內(nèi)的高效富集; 在膠原誘導(dǎo)的類風(fēng)濕性關(guān)節(jié)炎大鼠模型中, 納米復(fù)合物有效降低滑膜炎癥并改善動物活動能力;" FOL-脂質(zhì)體納米復(fù)合物可抑制IL-6等炎癥因子、 類風(fēng)濕因子(RF)以及C反應(yīng)蛋白(CRP)的表達(dá)水平(圖3). 該研究高效抑制RA病灶部位炎癥反應(yīng)級聯(lián)放大, 實(shí)現(xiàn)巨噬細(xì)胞去極化, 恢復(fù)關(guān)節(jié)免疫微環(huán)境, 為RA的納米基因治療體系構(gòu)建提供了實(shí)驗(yàn)依據(jù).
Kim等[9]將靶向跨膜受體蛋白Notch1的siRNA封裝至硫醇化殼聚糖納米顆粒中形成siRNA(Notch1)-NPs納米復(fù)合物, 將該納米復(fù)合物轉(zhuǎn)染至炎癥巨噬細(xì)胞中, 可降低細(xì)胞內(nèi)Notch1的表達(dá)水平. 利用Flamma FPR-675標(biāo)記siRNA并基于活體成像檢測核酸分子的生物分布, 結(jié)果表明, 納米復(fù)合物經(jīng)大鼠尾靜脈注射10 h后高效蓄積在炎癥關(guān)節(jié)部位, 進(jìn)而促使Notch1 siRNA在關(guān)節(jié)部位更好地發(fā)揮作用. 納米復(fù)合物在建模26 d后降低了關(guān)節(jié)炎臨床評分, 緩解了大鼠爪部腫脹(圖4); 病理學(xué)切片結(jié)果顯示, 納米復(fù)合物能緩解軟骨損傷, 降低滑膜炎癥細(xì)胞的浸潤, 同時未產(chǎn)生顯著的副作用, 為類風(fēng)濕性關(guān)節(jié)炎基因治療提供了一種安全有效的方法.
1.2 微小核糖核酸
miRNA是一類內(nèi)源性的非編碼單鏈RNA分子, 長度約為19~25個核苷酸, 在生物體內(nèi)廣泛存在, 且在不同組織和細(xì)胞類型中均具有特定的表達(dá)模式[10]. 其作用機(jī)制是通過與靶mRNA的3′端非翻譯區(qū)(3′-UTR)的互補(bǔ)序列結(jié)合, 進(jìn)而調(diào)節(jié)mRNA的穩(wěn)定性和翻譯, 導(dǎo)致靶向mRNA 降解或抑制其翻譯過程, 是細(xì)胞發(fā)育、 增殖、 分化和凋亡等基本生物過程的重要調(diào)控器[11-14]. 目前, miRNA已廣泛應(yīng)用于疾病診斷、 治療、 藥物研發(fā)和農(nóng)業(yè)等領(lǐng)域, 在RA的治療中也具有廣闊的應(yīng)用前景. Han等[15]利用氟化修飾聚酰胺-胺樹枝狀大分子(FP)實(shí)現(xiàn)miR-23b高效、 穩(wěn)定的遞送. 研究表明, FP/miR-23b能有效激活線粒體凋亡途徑以誘導(dǎo)巨噬細(xì)胞凋亡, 并通過靶向IKK-α,TAB2和TAB3抑制NF-κB信號通路, 從而降低促炎癥因子TNF-α,IL-1β和IL-6的表達(dá)(圖5(A)). 在佐劑誘導(dǎo)關(guān)節(jié)炎大鼠模型(AIA模型)和CIA小鼠模型中, 發(fā)現(xiàn)納米復(fù)合物能通過ELVIS效應(yīng)滯留在患病關(guān)節(jié)處, 提高關(guān)節(jié)腔內(nèi) miR-23b 的水平. 在已給藥處理的動物體內(nèi), 滑膜組織浸潤的現(xiàn)象得到明顯抑制, 患病關(guān)節(jié)及血清內(nèi)促炎因子的表達(dá)量均下降或恢復(fù)到正常水平(圖5(B)), 證明FP/miR-23b納米復(fù)合物具有減緩炎癥和抑制骨組織侵蝕的功能, 同時該納米復(fù)合物具有良好的生物相容性.
韓玲玲等[16]在初發(fā)RA患者miRNA表達(dá)中發(fā)現(xiàn), 患者的miR-21表達(dá)水平降低, 并與轉(zhuǎn)錄激活因子3(STAT3)mRNA的表達(dá)呈負(fù)相關(guān), 推測其可能參與RA的發(fā)生發(fā)展. Deng等[17]設(shè)計(jì)納米復(fù)合物用于介導(dǎo)miR-21和白細(xì)胞介素4(IL-4)的分層共遞送, 用以調(diào)控類風(fēng)濕性關(guān)節(jié)炎中的免疫微環(huán)境. 結(jié)果表明, 具有抗炎效果的miR-21和IL-4通過協(xié)同作用抑制NF-κB信號通路以減輕炎癥, 并促使巨噬細(xì)胞極化為M2表型, 實(shí)現(xiàn)類風(fēng)濕性關(guān)節(jié)炎的損傷組織修復(fù). 該研究也為基于基因治療的聯(lián)合機(jī)制設(shè)計(jì)推進(jìn)炎癥性疾病的治療提供了新思路.
Zhou等[18]在探究維生素D(VD)調(diào)節(jié)T細(xì)胞的分子機(jī)制中發(fā)現(xiàn), VD可通過抑制miR-124介導(dǎo)的IL-6信號轉(zhuǎn)導(dǎo)以減輕Th17細(xì)胞分化, 在RA治療中具有指導(dǎo)意義. 為進(jìn)一步增強(qiáng)抗RA活性, Yu等[19]將miR-124和酮洛芬(KMS)摻雜到PLGA微球(KMMS)中, 與僅擔(dān)載miR-124的微球相比, KMMS中miR-124誘導(dǎo)的受體激活劑NF-κB配體(RANKL)表達(dá)下調(diào)與KMS介導(dǎo)的鎮(zhèn)痛發(fā)揮協(xié)同作用, 對AIA大鼠有更好的治療效果(圖6(A)). 將酮洛芬更換為抗風(fēng)濕藥物甲氨蝶呤(MTX)[20]后, 抗RA活性得到進(jìn)一步改善, 該體系中miR-124可下調(diào)活化T細(xì)胞核因子1(NFATc1)的水平, 而納米復(fù)合物在AIA大鼠發(fā)炎關(guān)節(jié)中的蓄積則有效抑制了炎癥病癥(圖6(B)).
Ammari等[21]對miR-146a在RA中的作用機(jī)制進(jìn)行了深入探究. 與健康小鼠相比, CIA小鼠的Ly6Chigh亞群和RA患者的類單核細(xì)胞亞群(CD14+CD16-)中miR-146a的表達(dá)顯著下調(diào), 小鼠中miR-146a的耗竭促使關(guān)節(jié)炎嚴(yán)重惡化, 體外表現(xiàn)為破骨細(xì)胞分化, 體內(nèi)則表現(xiàn)為骨侵蝕增加. 在CIA小鼠體內(nèi)將miR-146a遞送至Ly6Chigh單核細(xì)胞, 可抑制miR-146a基因敲除小鼠所導(dǎo)致的骨組織侵蝕, 減少miR-146a+/+小鼠關(guān)節(jié)的致病性骨侵蝕, 但對炎癥并無顯著的治療效果. 該研究進(jìn)一步證實(shí)經(jīng)典單核細(xì)胞在RA發(fā)生發(fā)展中的關(guān)鍵作用以及miR-146a抑制關(guān)節(jié)炎的治療潛力.
Liu等[22]對miR-125在RA發(fā)展方面的影響進(jìn)行了深入探討." miR-125與多種疾病的發(fā)生發(fā)展密切相關(guān), 且被認(rèn)為是一種重要的腫瘤抑制因子. 與正常組相比, RA大鼠模型滑膜組織中miR-125表達(dá)下調(diào), PARP2表達(dá)上調(diào)(圖7); 熒光素酶報(bào)告基因檢測證實(shí)PARP2被miR-125直接抑制, 進(jìn)而調(diào)節(jié)PI3K/Akt/mTOR信號通路的活性, 最終減弱RA的發(fā)病進(jìn)程. Liu等[23]證明miR-125能靶向SPDEF抑制過敏性氣道炎癥中的杯狀細(xì)胞分化: Duroux-Richard等[24]證明miR-125可通過線粒體代謝和動力學(xué)控制單核細(xì)胞對炎癥的適應(yīng)性. 以上研究均為深入理解RA的作用機(jī)制提供了前期基礎(chǔ).
2 DNA治療
DNA 治療是指將含有特定治療基因的 DNA 序列導(dǎo)入患者體內(nèi), 通過表達(dá)治療性蛋白質(zhì)或調(diào)節(jié)基因表達(dá)治療疾病的方法[25]. 在RA治療中, DNA 治療的原理主要是通過導(dǎo)入編碼抗炎細(xì)胞因子、 免疫調(diào)節(jié)分子或關(guān)節(jié)修復(fù)因子等基因[26]調(diào)節(jié)免疫系統(tǒng)、 減輕炎癥反應(yīng)和促進(jìn)關(guān)節(jié)修復(fù).
2.1 質(zhì) 粒
質(zhì)粒是一種存在于細(xì)菌細(xì)胞質(zhì)中的環(huán)狀雙鏈 DNA分子, 具有獨(dú)立于染色體之外進(jìn)行自我復(fù)制的能力[27]. 在RA治療中, 攜帶治療基因的質(zhì)粒導(dǎo)入患者體內(nèi)后, 質(zhì)粒使細(xì)胞表達(dá)相應(yīng)的治療性產(chǎn)物, 從而發(fā)揮治療作用. Zhang等[28]構(gòu)建了一種創(chuàng)新型IL-10質(zhì)粒遞送體系, 該體系利用核定位信號肽(NLS)以及谷胱甘肽響應(yīng)性的聚合物(bPEI-SS-PEG-T)為載體, 通過靜電相互作用形成bPEI-SS-PEG-T/NLS/DNA納米復(fù)合物, IL-10通過誘導(dǎo)哺乳動物雷帕霉素靶蛋白(mTOR)活性, 進(jìn)而抑制糖酵解并維持線粒體的完整和功能. 同時, 該體系可正向調(diào)節(jié)線粒體精氨酸酶-1(Arg-2)的表達(dá)(圖8(A)), 導(dǎo)致線粒體呼吸增加以及琥珀酸和HIF-1α下調(diào), 炎癥介質(zhì)的減少進(jìn)一步負(fù)向調(diào)節(jié)IL-1β的產(chǎn)生和糖酵解活性. 線粒體異常功能的消失和促炎巨噬細(xì)胞中氧化代謝的增加, 進(jìn)一步強(qiáng)化了細(xì)胞抗炎表型(圖8(A))." 在動物水平上, 該納米復(fù)合物可實(shí)現(xiàn)在炎癥部位有效蓄積, 治療效果良好(圖8(B)), 為基于炎癥微環(huán)境角度出發(fā)構(gòu)建RA治療和遞送體系提供了新的策略.
2.2 DNA折紙
DNA 折紙是一項(xiàng)基于DNA分子自組裝特性以構(gòu)建納米結(jié)構(gòu)的先進(jìn)技術(shù). 通過精心設(shè)計(jì)特定的DNA序列, 將DNA鏈折疊成如納米盒、 納米管和納米球等各種復(fù)雜的二維或三維結(jié)構(gòu)[29]. DNA折紙具有高度的可編程性、 精確的結(jié)構(gòu)控制能力以及良好的生物相容性, 在生物醫(yī)學(xué)領(lǐng)域具有廣闊的應(yīng)用前景.
Li 等[30]以跨膜CD95受體為研究對象, 設(shè)計(jì)出納米級柔性可編程DNA折紙機(jī)器, 用于調(diào)節(jié)炎癥滑膜組織中活化免疫細(xì)胞的CD95致死誘導(dǎo)信號, 進(jìn)而建立局部免疫耐受以實(shí)現(xiàn)對RA的逆轉(zhuǎn)(圖9). 該 DNA 折紙將CD95L陣列以二維六邊形模式呈現(xiàn), 分子間距約為10 nm, 與跨膜CD95受體簇的幾何排列幾乎完全吻合(圖9(A)).
將i-motif DNA 序列進(jìn)一步耦合至DNA折紙, 可響應(yīng)pH觸發(fā)實(shí)現(xiàn)可逆的構(gòu)象關(guān)閉和開關(guān)轉(zhuǎn)變. 所設(shè)計(jì)的DNA折紙?jiān)?pH值為中性條件下保持閉合構(gòu)型, 而在弱酸性環(huán)境下轉(zhuǎn)變?yōu)殚_放構(gòu)型, 從而暴露 CD95L陣列的六邊形圖案. 該顯著特征極大增強(qiáng)了炎癥滑膜組織(pH≈6.5)中活化免疫細(xì)胞CD95誘導(dǎo)死亡信號的選擇性激活, 同時又保留了肝臟中表達(dá)低水平CD95受體的健康肝細(xì)胞(pH≈7.4), 最大限度減少了肝毒性. 研究表明, 在CIA小鼠模型中, 柔性DNA折紙?jiān)谘装Y滑膜組織中的活化免疫細(xì)胞中, 激發(fā)了強(qiáng)大且具有選擇性的CD95死亡誘導(dǎo)信號, 從而顯著緩解慢性炎癥, 促進(jìn)局部免疫耐受, 改善關(guān)節(jié)損傷(圖9(B)~(C)). 因此, 該研究所開發(fā)的納米 DNA折紙可對細(xì)胞信號進(jìn)行精確的空間控制, 拓展了配體-受體相互作用的理解, 為開發(fā)針對這些相互作用的藥理干預(yù)提供了一個有廣闊前景的平臺.
3 反義寡核苷酸
ASO是由十幾個到幾十個核苷酸組成的短鏈核酸分子, 通常為單鏈 DNA 或 RNA[31]. 它們的序列通過堿基配對原則與靶 mRNA的特定區(qū)域互補(bǔ), 從而干擾基因表達(dá).
ASO的長度既可保證其與靶mRNA的特異性結(jié)合, 又能避免被核酸酶快速降解. ASO的作用機(jī)制包括: 誘導(dǎo)RNase H介導(dǎo)的靶mRNA降解、 抑制mRNA翻譯和改變mRNA剪接等[32].
ASO不僅特異性高、 效率高、 設(shè)計(jì)性強(qiáng)及應(yīng)用范圍廣, 且在感染性疾病和自身免疫性疾病中均表現(xiàn)出良好的治療效果. Makalish等[33]使用反義寡核苷酸Cytos-11進(jìn)行RA的治療效果探究, 在大鼠模型中, Cytos-11可抑制關(guān)節(jié)炎癥狀, 表現(xiàn)為關(guān)節(jié)周圍腫脹減少, 外周血中TNF-α表達(dá)降低. 與經(jīng)典抗風(fēng)濕藥物阿達(dá)木單抗(Humira)相比, Cytos-11具有類似的療效, 兩種藥物在治療14 d后均表現(xiàn)出良好的治療效果(表1和圖10), 為基于ASO技術(shù)的RA治療藥物設(shè)計(jì)和評價提供了良好的研究基礎(chǔ).
4 CRISPR/Cas9系統(tǒng)
基因編輯技術(shù)是一種對生物體基因組特定目標(biāo)基因進(jìn)行精確修飾的技術(shù)[34]. 目前廣泛應(yīng)用的基因編輯技術(shù)主要是CRISPR/Cas9系統(tǒng), 該系統(tǒng)由引導(dǎo)RNA(gRNA)和Cas9 蛋白組成. gRNA可特異性識別目標(biāo) DNA 序列, Cas9蛋白如分子剪刀, 在特定位置切割DNA雙鏈, 引發(fā)細(xì)胞自身的DNA修復(fù)機(jī)制. 這種修復(fù)機(jī)制主要有兩種方式: 1) 非同源末端連接, 這種方式通常會引入一些隨機(jī)插入或缺失突變; 2) 同源重組修復(fù), 若提供合適的模板DNA, 則可實(shí)現(xiàn)對目標(biāo)基因的精確編輯, 如插入特定的基因片段或進(jìn)行點(diǎn)突變等[35]. 在基礎(chǔ)生命科學(xué)研究中, 基因編輯技術(shù)有助于科學(xué)家深入了解基因的功能和調(diào)控機(jī)制, 構(gòu)建各種疾病模型; 在農(nóng)業(yè)領(lǐng)域中, 該技術(shù)可用于培育優(yōu)良品種, 提高農(nóng)作物的產(chǎn)量和抗逆性[36]; 在醫(yī)學(xué)領(lǐng)域, 該技術(shù)有望成為多種遺傳疾病的治療方法, 如通過糾正致病基因突變治療囊性纖維化[37]和地中海貧血[38]等疾病, 或通過增強(qiáng)免疫系統(tǒng)對腫瘤細(xì)胞的識別和攻擊能力用于癌癥治療.
如圖11所示, Choi等[39]針對RA中的炎性細(xì)胞因子和通路, 利用CRISPR/Cas9基因編輯技術(shù)改造誘導(dǎo)多能干細(xì)胞(iPSCs), 構(gòu)建了稱為“SMART”的軟骨干細(xì)胞. 在細(xì)胞中植入受IL-1調(diào)控以生產(chǎn)IL-1受體拮抗劑(IL-1Rα)的合成基因回路, 當(dāng)炎癥發(fā)生時, 細(xì)胞內(nèi)的基因回路會感知內(nèi)源性IL-1細(xì)胞因子水平的變化而被激活, 從而分泌對應(yīng)治療水平的IL-1Rα(圖11(A)). 但考慮到IL-1Rα藥物的半衰期較短, 在RA中的療效較差, 因此將干細(xì)胞接種至生物支架, 形成軟骨植入物后植入皮下, 以保證細(xì)胞在體內(nèi)長期存活和分泌相應(yīng)比例的藥物治療RA. 結(jié)果表明, 該軟骨構(gòu)建體在體外和體內(nèi)均表現(xiàn)為快速激活和恢復(fù)(圖11(B)), 在炎癥性關(guān)節(jié)炎的K/BxN小鼠模型中, 生物工程植入物在關(guān)節(jié)疼痛、 結(jié)構(gòu)損傷及全身和局部炎癥中發(fā)揮功效, 減輕疾病的炎癥程度(圖11(C),(D)). 由此可見, 組織工程和合成生物學(xué)相結(jié)合有望通過定制設(shè)計(jì)的細(xì)胞, 響應(yīng)動態(tài)變化的生物信號而表達(dá)治療性基因, 進(jìn)而推進(jìn)慢性病的治療.
Huang等[40]基于CRISPRa的基因編輯技術(shù)與過繼細(xì)胞療法相結(jié)合, 構(gòu)建持久表達(dá)IL-10的工程化M2型巨噬細(xì)胞(Elite MΦs)用于RA治療(圖12). CRISPRa通過特定的核酸酶缺陷型Cas9(dCas9)和sgRNA特異性激活目標(biāo)基因. Elite MΦs 表現(xiàn)出強(qiáng)大的抗炎能力, 代表了M2 MΦs 在體外的預(yù)激活狀態(tài), 對M2誘導(dǎo)劑更敏感, 而對 M1 誘導(dǎo)劑具有抵抗能力. 結(jié)果表明, Elite MΦs可在炎癥部位蓄積, 通過恢復(fù)巨噬細(xì)胞M1/M2的平衡以緩解RA小鼠模型的炎癥、 滑膜增生和關(guān)節(jié)破壞.
5 脫氧核酶
脫氧核酶(DNAzyme, Dz)是一類具有催化活性的DNA分子[41], 它的發(fā)現(xiàn)打破了人們以為只有蛋白質(zhì)才能作為酶分子發(fā)揮催化作用的觀念, 為生物化學(xué)領(lǐng)域開辟了新的研究方向[42]. Dz的催化作用基于其特定的DNA序列結(jié)構(gòu), 它通過與底物分子進(jìn)行特異性的堿基互補(bǔ)配對結(jié)合, 然后在合適的環(huán)境條件下(如特定的離子濃度和pH值等)引發(fā)化學(xué)反應(yīng)[43]. 以常見的具有RNA切割活性的Dz為例, 其作用機(jī)制為: 首先, 脫氧核酶通過自身特定的 DNA 序列與目標(biāo)RNA分子進(jìn)行互補(bǔ)配對, 形成一種類似酶-底物復(fù)合物結(jié)構(gòu); 其次, 在特定金屬離子(如鎂離子)的協(xié)助下, Dz發(fā)揮其催化活性, 對目標(biāo)RNA分子進(jìn)行切割, 將其斷裂成兩個或多個片段, 從而實(shí)現(xiàn)對RNA的修飾或降解作用[44]. Dz的特點(diǎn)包括穩(wěn)定性高和可設(shè)計(jì)性強(qiáng)等, 在基因治療、 傳感器開發(fā)及核酸藥物研發(fā)等領(lǐng)域具有廣闊的應(yīng)用前景.
Fahmy等[45]借助DNA分子Dz13敲低堿性區(qū)域亮氨酸拉鏈蛋白c-Jun, 抑制血管通透性和白細(xì)胞的跨內(nèi)皮遷移. 在體外, Dz13消除了單核細(xì)胞-內(nèi)皮細(xì)胞黏附, 消除了小鼠炎癥模型中的白細(xì)胞滾動、 黏附和外滲. 機(jī)制研究表明, Dz13 阻斷細(xì)胞因子誘導(dǎo)的內(nèi)皮 c-Jun、 E-選擇素、 ICAM-1、 VCAM-1和血管內(nèi)皮鈣黏蛋白表達(dá), 最終減少RA小鼠模型中的關(guān)節(jié)腫脹、 炎性細(xì)胞浸潤和骨侵蝕(圖13).
6 其他基因治療策略
6.1 靶白蛋白水解嵌合體
PROTAC技術(shù)作為一種創(chuàng)新的蛋白質(zhì)降解技術(shù), 近年來在生物醫(yī)藥領(lǐng)域備受關(guān)注. PROTAC分子是一種雙功能小分子化合物, 由3部分構(gòu)成:" 1) 靶蛋白結(jié)合配體, 它可特異性與目標(biāo)蛋白質(zhì)結(jié)合, 精準(zhǔn)鎖定需降解的蛋白;" 2) E3泛素連接酶結(jié)合配體, 其作用是與細(xì)胞內(nèi)的E3泛素連接酶相連, E3 泛素連接酶在泛素-蛋白酶體系統(tǒng)中負(fù)責(zé)將泛素分子連接到目標(biāo)蛋白上;" 3) 連接鏈將二者連接形成完整的PROTAC分子. 當(dāng) PROTAC分子通過兩端配體分別與目標(biāo)蛋白質(zhì)和E3泛素連接酶結(jié)合后, 形成一個三元復(fù)合物. 在該復(fù)合物中, E3泛素連接酶將泛素分子依次連接到目標(biāo)蛋白質(zhì)上, 形成多聚泛素鏈, 若目標(biāo)蛋白質(zhì)被標(biāo)記上足夠數(shù)量的泛素分子, 則會被細(xì)胞內(nèi)的蛋白酶體識別并降解, 從而實(shí)現(xiàn)對目標(biāo)蛋白質(zhì)的靶向清除[46]. PROTAC技術(shù)的優(yōu)勢包括高度靶向特異性和可調(diào)節(jié)性, 可應(yīng)用于治療腫瘤[47]、 神經(jīng)退行性疾?。?8]以及炎癥性疾?。?9]等.
6.2 治療性適配體
適配體是單鏈DNA或RNA, 可高親和力特異性結(jié)合其靶標(biāo), 通常通過指數(shù)富集(SELEX)技術(shù)對配體進(jìn)行系統(tǒng)進(jìn)化而開發(fā)[50]. 利用高結(jié)合力和易編程特性能將DNA或RNA偶聯(lián)至藥物載體表面, 用以改善治療或診斷目的的靶向特性. 此外, 一些適配體可與受體結(jié)合, 激活下游信號通路, 因此也可被用作治療劑, 如DNA適配體SL2B和C2NP等用于癌癥治療. 受上述發(fā)現(xiàn)的啟發(fā), 已開發(fā)出多種用于類風(fēng)濕關(guān)節(jié)炎治療的適配體, 如Cao等[51]利用微針作為載體經(jīng)皮遞送抗DEK適配子(DTA), 可顯著降低炎性巨噬細(xì)胞中DEK的表達(dá), 在CIA小鼠體內(nèi)降低炎癥因子水平并抑制關(guān)節(jié)軟骨的損傷.
6.3 疫 苗
疫苗是目前預(yù)防疾病最有效、 最經(jīng)濟(jì)的手段. 已開發(fā)了兩代疫苗: 第一代是減毒活疫苗和滅活疫苗; 第二代的主要代表是亞單位疫苗和重組基因疫苗. 隨著生物納米技術(shù)的發(fā)展, 第三代疫苗應(yīng)運(yùn)而生, 即DNA和mRNA疫苗[52], 其技術(shù)是將編碼某種抗原蛋白的病毒基因片段DNA或mRNA直接引入人體, 抗體蛋白由宿主細(xì)胞表達(dá), 進(jìn)一步調(diào)節(jié)全身免疫反應(yīng). DNA疫苗需轉(zhuǎn)錄成mRNA進(jìn)行抗原蛋白表達(dá). 與傳統(tǒng)疫苗相比, DNA疫苗更經(jīng)濟(jì)穩(wěn)定、 特異性高、 易于制備, 并可靈活編輯進(jìn)而實(shí)現(xiàn)免疫療法的個性化治療[53]. Song等[54]構(gòu)建CCOL2A1特異性耐受DNA疫苗(pcDNA-CCOL2A1)用于RA治療, 單次靜脈注射pcDNA-CCOL2A1可誘導(dǎo)CIA小鼠有效的免疫耐受, 疫苗的治療效果與 MTX 相當(dāng), 同時該疫苗未引起任何異常臨床癥狀或正常生理功能的副作用, 在最大劑量下不具有免疫原性. Zhao等[55]研究表明, pcDNA-CCOL2A1也可作為預(yù)防性疫苗, 接種該疫苗14 d后, CIA的發(fā)生率、 嚴(yán)重程度和發(fā)病顯著下降. 機(jī)制研究表明, 該疫苗通過降低抗Ⅱ型膠原免疫球蛋白G(IgG)的水平, 減少Th17和CD4/CD29 T細(xì)胞和細(xì)胞因子的數(shù)量, 表現(xiàn)出良好的保護(hù)效果. 以上研究充分證實(shí)了pcDNA-CCOL2A1 DNA 疫苗不僅在治療RA方面的巨大潛力, 同時能有效實(shí)現(xiàn)RA的預(yù)防.
除DNA疫苗外, mRNA疫苗可直接進(jìn)行抗原蛋白表達(dá), 在細(xì)胞質(zhì)中發(fā)揮作用, 沒有基因組整合和突變的風(fēng)險. 此外, mRNA的壽命較短, 避免了抗原蛋白的連續(xù)表達(dá)[56]. 目前mRNA疫苗用于RA的預(yù)防和治療尚未見文獻(xiàn)報(bào)道, 但Krienke等[57]已開發(fā)出治療實(shí)驗(yàn)性自身免疫性腦脊髓炎的mRNA疫苗, 為自身免疫性疾病疫苗的開發(fā)提供了借鑒思路.
7 結(jié)論與展望
本文介紹的基因治療手段在治療RA中已有較多報(bào)道, 但目前該疾病的治療仍通過抑制炎癥以緩解腫脹和疼痛, 均未能從根源上解決RA的發(fā)生發(fā)展. 盡管RA的基因治療距臨床應(yīng)用仍有較大距離, 但其在疾病治療中的原理決定了其未來應(yīng)用有很大的可能性. 隨著基因編輯技術(shù)的進(jìn)一步完善, 更精準(zhǔn)、 高效和無脫靶效應(yīng)的基因操作將成為可能, 為RA關(guān)鍵靶點(diǎn)的編輯及表達(dá)水平的調(diào)控提供了技術(shù)支撐. 此外, 將基因治療與現(xiàn)有的治療方法相結(jié)合, 既能從根本上糾正基因異常, 又能及時減輕患者的痛苦, 通過各自優(yōu)勢的發(fā)揮取得更
理想的治療效果. 基因治療作為從根本上治療疾病的策略, 有望成為未來RA及其他自身免疫性疾病治療的新策略.
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(責(zé)任編輯: 單 凝)