吳國玖 查旭 張遠(yuǎn)平 曹霞 馬林昆
視神經(jīng)是由視網(wǎng)膜神經(jīng)節(jié)細(xì)胞(retinal ganglion cells,RGC)的軸突在鞏膜篩孔處匯聚向后延伸形成。外傷性視神經(jīng)損傷(traumatic optic nerve injury,TONI)常導(dǎo)致嚴(yán)重視力下降或盲,以往多種藥物及手術(shù)治療也難見成效。2001年 Acland等[1]用重組腺相關(guān)病毒(recombination adeno-associated virus,rAAV)載體編碼RPE65可使先天性利伯氏黑蒙病狗恢復(fù)視覺功能,從此激發(fā)人們對(duì)轉(zhuǎn)基因治療視神經(jīng)疾病的極大興趣?;蛑委熗ǔJ峭ㄟ^載體將目的基因即治療基因?qū)氚屑?xì)胞,通過表達(dá)產(chǎn)物從而達(dá)到治療目的。已證實(shí)腺相關(guān)病毒(adeno-associated virus vector,AAV)載體具有轉(zhuǎn)染效率高、免疫原性低、無毒性、長期穩(wěn)定表達(dá)等優(yōu)點(diǎn)[2],是目前最常用的轉(zhuǎn)基因治療載體。TONI基因治療最常用的研究模型是哺乳動(dòng)物視神經(jīng)橫斷或擠壓傷,與臨床的視神經(jīng)外傷相似。本文就近年來關(guān)于視神經(jīng)損傷后微環(huán)境變化及應(yīng)用AAV載體基因治療TONI的研究進(jìn)展進(jìn)行綜述。
1.1 視神經(jīng)損傷后微環(huán)境變化 視神經(jīng)損傷后,神經(jīng)結(jié)構(gòu)和周邊微環(huán)境遭到破壞,細(xì)胞軸突離斷、水腫、死亡,同時(shí)也出現(xiàn)一些不利因素,如自由基形成、RGC 內(nèi)超氧化物增加[3]、過氧化物陰離子增加[4]、谷氨酸鹽的毒性作用、K+門控通道 KV1家族出現(xiàn)[5]、caspase-6 及 caspase-8 上 調(diào)[6]、caspase-2 激活[7]、JNK 信號(hào)通路激活[8]、軸突殘端有毒物質(zhì)滲漏、硫苷脂增加[9]等,這些因素均不利于視神經(jīng)修復(fù)。同時(shí),視神經(jīng)損傷后因自身固有的修復(fù)能力會(huì)產(chǎn)生一些有利視神經(jīng)修復(fù)的因素,如自我吞噬功能上調(diào)以保護(hù)神經(jīng)元存活[10]、再生RGC中上調(diào)的晶狀體蛋白β2通過增強(qiáng)睫狀神經(jīng)營養(yǎng)因子的產(chǎn)生而促進(jìn)軸突再生[11]、睫狀神經(jīng)營養(yǎng)因子在星型膠質(zhì)細(xì)胞的顯著表達(dá)[12]和胸腺素-β4 上調(diào)[13]均增強(qiáng) RGC 存活和促進(jìn)軸突再生等,此外尚有許多不為所知的影響因素有待發(fā)現(xiàn)。
1.2 RGC死亡、凋亡特點(diǎn) 視神經(jīng)損傷后周邊微環(huán)境變得不穩(wěn)定使RGC漸漸死亡。成年哺乳動(dòng)物視神經(jīng)損傷存在延時(shí)現(xiàn)象,即視神經(jīng)損傷后3 d才能發(fā)現(xiàn)RGC死亡,RGC程序性死亡相關(guān)改變可能在傷后6 h已經(jīng)開始[14],RGC死亡高峰在視神經(jīng)損傷后5~9 d,在損傷 2周后有 10%~15%的 RGC存活[15]。凋亡是一種繼發(fā)性死亡形式,RGC凋亡高峰出現(xiàn)在傷后3個(gè)月[16],直至傷后6個(gè)月仍可見RGC凋亡[17]。因此,視神經(jīng)損傷后及時(shí)增強(qiáng)RGC生存能力和促進(jìn)其軸突再生是治療的關(guān)鍵。
2.1 AAV載體生物學(xué)特點(diǎn) 野生型 AAV屬于微小病毒科,是依賴病毒類的一種。AAV為無包膜單鏈DNA,外有衣殼蛋白包裹,是動(dòng)物病毒中最小的病毒[18]。AAV載體基因組長約4.6 kb,包括2個(gè)開放閱讀框—rep和cap,兩端各有一個(gè)末端反向重復(fù)序列(inverted terminal repeat,ITR),在保留 ITR 的前提下,AAV載體的rep及cap基因由外源性基因表達(dá)框替代,經(jīng)修飾后形成 rAAV載體。自身互補(bǔ)型rAAV 載體(self-complementary rAAV,ScrAAV)也是在AAV載體的基礎(chǔ)上改良而成,rAAV載體和ScrAAV載體一個(gè)共同點(diǎn)是兩者都含雙鏈基因組。
2.2 AAV載體的血清型及其在眼部轉(zhuǎn)染特點(diǎn) 目前,來自人的 AAV載體血清型有 12種[19],包括AAV1-AAV9、avianAAV、bovineAAV 和 canineAAV等,不同血清型AAV載體對(duì)不同細(xì)胞或組織的轉(zhuǎn)染效率不同。其中,AAV2和AAV5載體最常用于治療眼部疾病,AAV2載體具有親神經(jīng)性、優(yōu)先轉(zhuǎn)染神經(jīng)元、能有效轉(zhuǎn)染RGC[20]、表達(dá)持久等優(yōu)點(diǎn)。而 AAV5載體主要轉(zhuǎn)染光感受器和視網(wǎng)膜色素上皮細(xì)胞。其中rAAV2的雜合載體 rAAV2/2和rAAV2/6轉(zhuǎn)染RGC的效率最高[21]。但 AAV載體也存在不足,如攜帶基因小于5 kb、滴度低和表達(dá)“滯后性”,即被轉(zhuǎn)染細(xì)胞重組AAV-DNA轉(zhuǎn)變?yōu)檗D(zhuǎn)錄活躍的雙鏈模式需要一定時(shí)間。改良型ScAAV載體含有雙鏈基因組,具有更快啟動(dòng)轉(zhuǎn)基因表達(dá)和更高轉(zhuǎn)染效率等特點(diǎn)[22-23],這或許會(huì)彌補(bǔ)AAV載體表達(dá)“滯后性”這一不足。
3.1 AAV載體轉(zhuǎn)導(dǎo)營養(yǎng)因子對(duì)RGC的保護(hù)作用
目前用于治療視神經(jīng)損傷的營養(yǎng)因子主要有睫狀神經(jīng)營養(yǎng)因子(ciliary neurotrophic factor,CNTF)、腦源性神經(jīng)營養(yǎng)因子(brain-derived neurotrophic factor,BDNF)、血管內(nèi)皮生長因子(vascular endothelial growth factor,VEGF)、血小板源性生長因子 CC(platelet-derived growth factor CC,PDGF-CC)等。實(shí)驗(yàn)證實(shí),玻璃體注射BDNF和VEGF能增強(qiáng)RGC存活,而對(duì)軸突再生無明顯促進(jìn)作用[24-25]。D’Onofrio等[25]向成年大鼠玻璃體內(nèi)注射 AAV-VEGF-ZFP(鋅指蛋白),可使RGC的存活數(shù)量提高2倍,ZFP的作用在于上調(diào) VEGF-A,進(jìn)一步促進(jìn) RGC存活。相似地,PDGF-CC也主要體現(xiàn)在增強(qiáng)RGC存活方面[26],PDGF-CC通過調(diào)節(jié)糖原合成酶激酶-3β的磷酸化和表達(dá)對(duì)RGC起保護(hù)作用,同時(shí)也調(diào)節(jié)一些凋亡相關(guān)和神經(jīng)保護(hù)基因的表達(dá)。Hellstr?m等[27]將環(huán)腺苷酸類似物(CTP-cAMP)聯(lián)合 rAAV2-CNTF-GFP注射到成年大鼠玻璃體內(nèi),證實(shí)CNTF可以增強(qiáng)RGC存活和促進(jìn)軸突再生,但在此基礎(chǔ)上,CTP-cAMP并沒有進(jìn)一步的促進(jìn)作用。但后來 Hellstr?m等[28]行鼠玻璃體內(nèi)注射 CTP-cAMP和 rAAV2-CNTF-GFP、rCNTF治療,結(jié)果發(fā)現(xiàn)約1/3RGC存活,其中27%的RGC軸突再生。這是一種基因聯(lián)合藥物治療方法,實(shí)驗(yàn)注藥時(shí)間幾乎與視神經(jīng)破壞手術(shù)同時(shí)進(jìn)行,并提出rCNTF對(duì)于急性視神經(jīng)損傷有潛在的治療意義。以上實(shí)驗(yàn)證明,CNTF能增強(qiáng)RGC存活和促進(jìn)軸突再生,正常情況下,CNTF主要存在于星型膠質(zhì)細(xì)胞內(nèi),神經(jīng)受損后,CNTF在Müller細(xì)胞也有表達(dá)。
3.2 敲除 SOCS3、PTEN和 ephrinB3基因?qū)σ暽窠?jīng)修復(fù)的促進(jìn)作用 機(jī)體組織破壞后,通常進(jìn)入積極的修復(fù)狀態(tài),但是以往一直認(rèn)為視神經(jīng)損傷后不可修復(fù),損傷的神經(jīng)元內(nèi)是否存在抑制修復(fù)的基因?Smith等[29]把 AAV-cre(重組酶)注射到成年小鼠玻璃體內(nèi),即利用AAV載體表達(dá)cre以敲除SOCS3基因,與沒有敲除 SOCS3組相比,AAV-cre組 RGC表現(xiàn)出顯著的再生能力,再向AAV-cre組玻璃體內(nèi)注入CNTF,能進(jìn)一步加強(qiáng) RGC軸突再生。后來,Hellstr?m 等[27]從正面證實(shí) SOCS3對(duì)視神經(jīng)修復(fù)的抑制作用,他們把rAAV2-SOCS3-GFP注射到成年大鼠玻璃體內(nèi),使 RGC過度表達(dá) SOCS3,結(jié)果導(dǎo)致RGC軸突幾乎完全不能再生,同時(shí)也提出AAV2轉(zhuǎn)導(dǎo)CNTF表達(dá)比玻璃體內(nèi)直接注射rCNTF治療更有效。
視神經(jīng)損傷的修復(fù)不完全等同于RGC軸突的再生,還包括再生軸突能否進(jìn)入大腦,經(jīng)過視交叉,到達(dá)相應(yīng)靶區(qū),并恢復(fù)視功能。2010年 Kurimoto等[30]提出敲除鼠 RGC內(nèi) PETN基因有利于細(xì)胞軸突再生,時(shí)隔2 a,他們?cè)俅芜M(jìn)行了相似的實(shí)驗(yàn)[31],他們向成年小鼠眼內(nèi)注射AAV2-cre,以敲除PETN,2周后建立視神經(jīng)橫切外傷模型,同時(shí)眼內(nèi)注射酵母多糖(Zymosan)及 CPT-cAMP,以霍亂毒素 B(CTB)示蹤再生軸突,實(shí)驗(yàn)反復(fù)向玻璃體內(nèi)注射Zymosan及CPT-cAMP,10周后發(fā)現(xiàn)36%RGC存活,12周后大量視神經(jīng)全長呈CTB陽性,即視神經(jīng)全長再生,大部分再生軸突到達(dá)視交叉上核,而僅注射AAV2-GFP、Zymosan及 CPT-cAMP組10周 RGC存活率為16%,視神經(jīng)CTB陽性很少。該實(shí)驗(yàn)證明了視神經(jīng)損傷后視覺中央回路重建及部分視力恢復(fù)的可行性,實(shí)驗(yàn)中Zymosan主要作用是造成輕微持續(xù)的眼內(nèi)炎癥反應(yīng),并促使一些炎性細(xì)胞如巨噬細(xì)胞分泌生長因子和癌調(diào)蛋白,CPT-cAMP與酵母多糖共同促進(jìn)癌調(diào)蛋白與RGC捆綁以提高細(xì)胞再生[30],這種輕微足量的連續(xù)炎癥刺激可以使軸突細(xì)胞再生并到達(dá)視神經(jīng)的全長。對(duì)于這一類似問題,目前尚有爭議,有研究者[32]認(rèn)為癌調(diào)蛋白水平在玻璃體內(nèi)注射Zymosan后并沒有明顯增加,并推斷巨噬細(xì)胞源性癌調(diào)蛋白不可能是介導(dǎo)由眼內(nèi)炎癥引起這種有益作用的主要因素,而一定是另一機(jī)制所啟動(dòng),同時(shí)也提出,癌調(diào)蛋白的作用與細(xì)胞內(nèi)cAMP水平上升密切相關(guān)。cAMP是調(diào)節(jié)神經(jīng)元存活和神經(jīng)軸突再生至關(guān)重要的第二信使,輔助促進(jìn)軸突再生,CPT-cAMP的濃度也是實(shí)驗(yàn)成功的要素之一。此外,Duffy等[33]發(fā)現(xiàn),敲除小鼠 ephrinB3基因,可在視神經(jīng)損傷點(diǎn)外1000 μm發(fā)現(xiàn)再生軸突,明顯促進(jìn)軸突再生,并認(rèn)為ephrinB3是軸突生長重要的鞘磷脂相關(guān)的生理抑制因子。
本文綜述了神經(jīng)損傷后周圍微環(huán)境的改變以及利用AAV載體基因治療TONI的相關(guān)進(jìn)展,表明視神經(jīng)損傷后微環(huán)境變化的復(fù)雜性,顯示AAV載體在視神經(jīng)損傷基因治療中強(qiáng)大的應(yīng)用潛能,同時(shí)也啟示我們視神經(jīng)損傷基因治療會(huì)是一種更有效的治療方法。目前AAV載體攜帶基因容量小等問題尚未得到有效解決,而且基因治療過程復(fù)雜,涉及從胞外到胞核及轉(zhuǎn)染表達(dá)等一系列步驟,其間可能發(fā)生AAV顆粒降解,或因缺乏對(duì)靶細(xì)胞的專一性而轉(zhuǎn)染其他組織,或不能在特定組織大量有效的轉(zhuǎn)染等,都是有待解決的問題。另外,目前多數(shù)實(shí)驗(yàn)采用成年鼠視神經(jīng)外傷為模型,然而有研究表明:鼠RGC固有生存能力受其年齡影響[34],外傷后RGC死亡和再生能力受到 RGC發(fā)育形成時(shí)間先后的影響[35],最后,在正常視網(wǎng)膜和病變視網(wǎng)膜中,AAV載體介導(dǎo)轉(zhuǎn)染能力也是不同的[36]??傊?,影響基因治療療效的因素遠(yuǎn)不止這些,相信隨著分子生物學(xué)、細(xì)胞生物學(xué)的發(fā)展,神經(jīng)細(xì)胞死亡、軸突再生機(jī)制的進(jìn)一步闡明,會(huì)有更佳的改良型AAV載體應(yīng)用于臨床。
1 Acland GM,Aguirre GD,Ray J,Zhang Q,Aleman TS,Cideciyan AV,et al.Gene therapy restores vision in a canine model of childhood bl indnes[J].Nat Genet,2001,28(1):92-95.
2 Stieger K,Schroeder J,Provost N,Mendes-Madeira A,Belbellaa B,Le Meur G,et al.Detection of intact rAAV particles up to 6 years after successful gene transfer in the retina of dogs and primates[J].Mol T-her,2009,17(3):516-523.
3 Kanamori A,Catrinescu MM,Kanamori N,Mears KA,Beaubien R,Levin LA.Superoxide is an associated signal for apoptosis in axonal injury[J].Brain,2010,133(9):2612-2625.
4 Catrinescu MM,Chan W,Mahammed A,Gross Z,Levin LA.Superoxide signaling and cell death in retinal ganglion cell axotomy:Effects of metallocorroles[J].Exp Eye Res,2012,97(1):31-35.
5 Koeberle PD,Wang Y,Schlichter LC.Kv1.1 and Kv1.3 channels contribute to the degeneration of retinal ganglion cells after optic nerve transection in vivo[J].Cell Death Differ,2010,17(1):134-144.
6 Monnier PP,D’Onofrio PM,Magharious M,Hollander AC,Tassew N,Szydlowska K,et al.Involvement of caspase-6 and caspase-8 in neuronal apoptosis and the regenerative failure of injured retinal ganglion cells[J].J Neurosci,2011,31(29):10494-10505.
7 Ahmed Z,Kalinski H,Berry M,Almasieh M,Ashush H,Slager N,et al.Ocular neuroprotection by siRNA targeting caspase-2[J].Cell Death Dis,2011,16(2):e173.
8 Fernandes KA,Harder JM,F(xiàn)ornarola LB,F(xiàn)reeman RS,Clark AF,Pang IH,et al.JNK2 and JNK3 are major regulators of axonal injury-induced retinal ganglion cell death[J].Neurobiol Dis,2012,46(2):393-401.
9 Winzeler AM,Mandemakers WJ,Sun MZ,Stafford M,Phillips CT,Barres BA.The lipid sulfatide is a novel myelin-associated inhibitor of CNS axon outgrowth[J].J Neurosci,2011,31(17):6481-6492.
10 Rodríguez-Muela N,Boya P.Axonal damage,autophagy and neuronal survival[J].Autophagy,2012,8(2):286-288.
11 Thanos S,B?hm MR,Schallenberg M,Oellers P.Traumatology of the optic nerve and contribution of crystallins to axonal regeneration[J].Cell Tissue Res,2012,349(1):49-69.
12 Leibinger M,Müller A,Andreadaki A,Hauk TG,Kirsch M,F(xiàn)ischer D.Neuroprotective and axon growth-promoting effects following inflammatory stimulation on mature retinal ganglion cells in mice depend on ciliary neurotrophic factor and leukemia inhibitory factor[J].J Neurosci,2009,29(45):14334-14341.
13 Magharious M,D’Onofrio PM,Hollander A,Zhu P,Chen J,Koeberle PD.Quantitative iTRAQ analysis of retinal ganglion cell degeneration after optic nerve crush[J].J Proteome Res,2011,10(8):3344-3362.
14 Lukas TJ,Wang AL,Yuan M,Neufeld AH.Early cellular signaling responses to axonal injury[J].Cell Commun Signal,2009,13:5.
15 Nadal-Nicolás FM,Jiménez-López M,Sobrado-Calvo P,Nieto-López L,Cánovas-Martínez I,Salinas-Navarro M,et al.Brn3a as a marker of retinal ganglion cells:Qualitative and quantitative time course studies in naive and optic nerve injured retinas[J].Invest Ophthalmol Vis Sci,2009,50(8):3860-3868.
16 Levkovitch-Verbin H,Dardik R,Vander S,Melamed S.Mechanism of retinal ganglion cells death in secondary degeneration of the optic nerve[J].Exp Eye Res,2010,91(2):127-134.
17 Vander S,Levkovitch-Verbin H.Regulation of cell death and survival pathways in secondary degeneration of the optic nerve-a long-term study[J].Curr Eye Res,2012,37(8):740-748.
18 Goncalves MA.Adeno-associated virus:from defective virus to effective vector[J].Virol J,2005,6(2):43-59.
19 Daya S,Berns KI.Gene therapy using adeno-associated virus vectors[J].Clin Microbiol Rev,2008,21(4):583-593.
20 Kolstad KD,Dalkara D,Guerin K,Visel M,Hoffmann N,Schaffer DV,et al.Changes in adeno-associated virus-mediated gene delivery in retinal degeneration.changes in adeno-associated virus-mediated gene delivery in retinal degeneration[J].Hum Gene Ther,2010,21(5):571-578.
21 Hellstr?m M,Ruitenberg MJ,Pollett MA,Ehlert EM,Twisk J,Verhaagen J,et al.Cellular tropism and transduction properties of seven adeno-associated viral vector serotypes in adult retina after intravitreal injection[J].Gene Ther,2009,16(4):521-532.
22 Natkunarajah M,Trittibach P,McIntosh J,Duran Y,Barker SE,Smith AJ,et al.Assessment of ocular transduction using single-stranded and self-complementary recombinant adeno-associated virus serotype 2/8[J].Gene Ther,2008,15(6):463-467.
23 Kong F,Li W,Li X,Zheng Q,Dai X,Zhou X,et al.Self-complementary AAV5 vector facilitates quicker transgene expression in photoreceptor and retinal pigment epithelial cells of normal mouse[J].Exp Eye Res,2010,90(5):546-554.
24 Hellstr?m M,Harvey AR.Retinal ganglion cell gene therapy and visual system repair[J].Curr Gene Ther,2011,11(2):116-131.
25 D’Onofrio PM,Thayapararajah M,Lysko MD,Magharious M,Spratt SK,Lee G,et al.Gene therapy for traumatic central nervous system injury and stroke using an engineered zinc finger protein that upregulatesVEGF-A[J].J Neurotrauma,2011,28(9):1863-1879.
26 Tang Z,Arjunan P,Lee C,Li Y,Kumar A,Hou X,et al.Survival effect of PDGF-CC rescues neurons from apoptosis in both brain and retina by regulating GSK3beta phosphorylation[J].J Exp Med,2010,207(4):867-880.
27 Hellstr?m M,Muhling J,Ehlert EM,Verhaagen J,Pollett MA,Hu Y,et al.Negative impact of rAAV2 mediated expression of SOCS3 on the regeneration of adult retinal ganglion cell axons[J].Mol Cell Neurosci,2011,46(2):507-515.
28 Hellstr?m M,Pollett MA,Harvey AR.Post-injury delivery of rAAV2-CNTF combined with short-term pharmacotherapy is neuroprotective and promotes extensive axonal regeneration after optic nerve trauma[J].J Neurotrauma,2011,28(12):2475-2483.
29 Smith PD,Sun F,Park KK,Cai B,Wang C,Kuwako K,et al.SOCS3 deletion promotes optic nerve regeneration in vivo[J].Neuron,2009,64(5):617-623.
30 Kurimoto T,Yin Y,Omura K,Gilbert HY,Kim D,Cen LP,et al.Longdistance axon regeneration in the mature optic nerve:contributions of oncomodulin,cAMP,and pten gene deletion[J].J Neurosci,2010,30(46):15654-15663.
31 de Lima S,Koriyama Y,Kurimoto T,Oliveira JT,Yin Y,Li Y,et al.Full-length axon regeneration in the adult mouse optic nerve and partial recovery of simple visual behaviors[J].Proc Natl Acad Sci U S A,2012,109(23):9149-9154.
32 Hauk TG,Müller A,Lee J,Schwendener R,F(xiàn)ischer D.Neuroprotective and axon growth promoting effects of intraocular inflammation do not depend on oncomodulin or the presence of large numbers of activated macrophages[J].Exp Neurol,2008,209(2):469-482.
33 Duffy P,Wang X,Siegel CS,Tu N,Henkemeyer M,Cafferty WB,et al.Myelin-derived ephrinB3 restricts axonal regeneration and recovery after adult CNS injury[J].Proc Natl Acad Sci U S A,2012,109(13):5063-5068.
34 Luo JM,Geng YQ,Zhi Y,Zhang MZ,van Rooijen N,Cui Q.Increased intrinsic neuronal vulnerability and decreased beneficial reaction of macrophages on axonal regeneration in aged rats[J].Neurobiol Aging,2010,31(6):1003-1009.
35 Dallimore EJ,Park KK,Pollett MA,Taylor JS,Harvey AR.The life,death and regenerative ability of immature and mature rat retinal ganglion cells are influenced by their birthdate[J].Exp Neurol,2010,225(2):353-365.
36 Kolstad KD,Dalkara D,Guerin K,Visel M,Hoffmann N,Schaffer DV,et al.Changes in adeno-associated virus-mediated gene delivery in retinal degeneration[J].Hum Gene Ther,2010,21(5):571-578.