應(yīng)用病毒治療結(jié)直腸癌的研究進(jìn)展*

尹磊　孫燕來　綜述　徐忠法③　審校

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應(yīng)用病毒治療結(jié)直腸癌的研究進(jìn)展*

尹磊①②孫燕來②綜述徐忠法②③審校

摘要基于病毒的抗腫瘤治療是一種新興的生物治療方式，病毒載體感染腫瘤組織，引起溶瘤效應(yīng)，制成疫苗激活體內(nèi)抗腫瘤免疫，搭載基因行癌癥的基因治療。隨著對(duì)病毒的不斷改造，各類病毒治療腫瘤更趨于安全和高效，同時(shí)，病毒載體與現(xiàn)有的抗腫瘤療法合理聯(lián)用，可提高治療效果。因此，基于病毒的抗腫瘤治療將作為極具潛力的方法而逐漸引起人們的重視。本文就近些年各種病毒在治療結(jié)直腸癌中的研究進(jìn)展做一綜述。

關(guān)鍵詞結(jié)直腸腫瘤溶瘤病毒病毒載體基因治療免疫治療

作者單位：①山東省醫(yī)學(xué)科學(xué)院，濟(jì)南大學(xué)醫(yī)學(xué)與生命科學(xué)學(xué)院（濟(jì)南市250022）；②山東省腫瘤醫(yī)院外四科；③山東省醫(yī)學(xué)科學(xué)院附屬醫(yī)院

*本文課題受山東省科技發(fā)展計(jì)劃項(xiàng)目（編號(hào)：2013GSF11834）和濟(jì)南市科技計(jì)劃高校自主創(chuàng)新項(xiàng)目（編號(hào)：201401253）資助

結(jié)直腸癌（colorectal carcinoma，CRC）是世界第三大常見的惡性腫瘤，發(fā)病率呈持續(xù)上升趨勢［1］。在中國，結(jié)直腸癌5年生存率僅32%，排位死因順位已上升到第5位［2］。盡管結(jié)直腸癌的診治水平不斷提高，但由于其早期癥狀隱匿，仍有20%患者確診時(shí)即為轉(zhuǎn)移性結(jié)直腸癌［3］，且即使進(jìn)行根治手術(shù)仍有50%～60%因微小轉(zhuǎn)移灶而發(fā)生轉(zhuǎn)移［4］?；煂?duì)于局部淋巴結(jié)轉(zhuǎn)移者療效有限，而在伴有遠(yuǎn)處轉(zhuǎn)移者中療效尚不肯定［5］。遠(yuǎn)處轉(zhuǎn)移、對(duì)化療耐藥、反復(fù)復(fù)發(fā)使結(jié)直腸癌患者的預(yù)后欠佳。為了提高結(jié)直腸癌的療效及預(yù)后，人們不斷深入研究結(jié)直腸癌發(fā)生及發(fā)展機(jī)制的研究，已取得不少成果。隨著基因工程的發(fā)展和分子生物學(xué)的應(yīng)用，利用病毒治療腫瘤在近20年得到迅速發(fā)展，被基因改造的病毒許多已行體內(nèi)外抗腫瘤療效評(píng)估［6］。

1　基于病毒治療腫瘤的發(fā)展史

利用病毒治療癌癥已有100多年歷史。然而其用來治療腫瘤最初是源于偶然事件，腫瘤患者患有感染性疾病時(shí)，腫瘤出現(xiàn)短暫的臨床緩解。1904年有報(bào)道［7］，1例白血病患者在一次流感病毒感染后病情意外好轉(zhuǎn)。100多年來，病毒一直被作為實(shí)驗(yàn)研究對(duì)象行抗腫瘤治療，但人們對(duì)該領(lǐng)域的興趣一直波動(dòng)不定。直到1991年Martuza等［8］報(bào)道，轉(zhuǎn)基因單純皰疹病毒（herpes simplex virus，HSV）在惡性膠質(zhì)瘤治療中有一定療效，才使基于病毒的抗腫瘤療法日益受人關(guān)注。2005年中國國家食品藥品監(jiān)督管理局首次批準(zhǔn)重組人5型腺病毒（安柯瑞）注射液用于臨床治療［9］。至今，全世界已有200多項(xiàng)病毒抗腫瘤治療的研究專利，且許多已完成臨床Ⅰ/Ⅱ期試驗(yàn)。

2　溶瘤病毒直接破壞腫瘤細(xì)胞

溶瘤病毒（oncolytic virus，OV）治療結(jié)直腸癌是基于病毒選擇性地在腫瘤細(xì)胞內(nèi)大量復(fù)制最終破壞腫瘤細(xì)胞，釋放子代，感染周邊腫瘤細(xì)胞，在瘤體內(nèi)廣泛播散，而對(duì)正常組織細(xì)胞無殺傷作用［10］。腫瘤細(xì)胞裂解后，腫瘤釋放原始抗原和熱休克蛋白，導(dǎo)致強(qiáng)烈的機(jī)體抗腫瘤免疫效應(yīng)。另外，有些溶瘤病毒還能產(chǎn)生凋亡蛋白，有些可通過表達(dá)病毒蛋白或非特異性炎癥反應(yīng)來增強(qiáng)抗腫瘤免疫。NV1020是減毒的單純皰疹病毒，刪除了UL/L交界的15 kb區(qū)域和胸苷激酶基因及其啟動(dòng)子UL24，另外還插入了外源性TK基因，該基因受ICP4啟動(dòng)子的控制［11］。NV1020 Ⅰ/Ⅱ期臨床試驗(yàn)［12］以轉(zhuǎn)移性結(jié)直腸癌患者為研究對(duì)象，Ⅰ期13例患者通過肝動(dòng)脈注射NV1020，患者出現(xiàn)短暫的發(fā)熱而無其他不適，認(rèn)為NV1020是安全的。Ⅱ期入組多線治療后病情仍進(jìn)展的患者22例，注射NV1020后，50%患者病情穩(wěn)定，繼續(xù)化療后整體腫瘤控制率為68%（其中PR 1例，SD 14例），中位生存期平均延長6.4個(gè)月。因此NV1020在結(jié)直腸癌肝轉(zhuǎn)移患者中具有良好療效且不良反應(yīng)小，另外，其可能通過特殊的、系統(tǒng)的溶瘤免疫應(yīng)答逆轉(zhuǎn)腫瘤的化療耐藥性，增加放療敏感性［13］。目前，一項(xiàng)NV1020聯(lián)合細(xì)胞毒藥物或靶向藥物殺傷腫瘤細(xì)胞的大型隨機(jī)Ⅱ/Ⅲ期臨床試驗(yàn)正在開展。

胃腸癌腹膜轉(zhuǎn)移是其常見轉(zhuǎn)移形式，約超過一半患者術(shù)后復(fù)發(fā)病例為腹膜轉(zhuǎn)移，患者預(yù)后不理想［14］。Eveno等［15］采用改造的溶瘤痘苗病毒GLV-1h153表達(dá)人鈉碘轉(zhuǎn)運(yùn)體基因（hNIS）行體外實(shí)驗(yàn)，該病毒能感染復(fù)制并溶解人結(jié)直腸腺癌細(xì)胞。在小鼠結(jié)直腸癌腹膜轉(zhuǎn)移模型上腹腔注射GLV-1h153后，腫瘤顯著減小。另外，GLV-1h153感染的組織還可經(jīng)131I SPECT掃描和熒光光學(xué)成像顯示。因此，GLV-1h153不僅可用于治療，還可用于腹膜轉(zhuǎn)移病灶的診斷和評(píng)估。

對(duì)于溶瘤病毒，缺點(diǎn)是大部分需要瘤內(nèi)局部注射，但并非所有的溶瘤病毒均如此，這種區(qū)別的原因尚不清楚。Adair等［16］選取結(jié)直腸癌肝轉(zhuǎn)移患者為試驗(yàn)對(duì)象，在轉(zhuǎn)移病灶切除前先行呼腸孤病毒靜脈注射1個(gè)周期。雖然患者在治療前體內(nèi)已有抗病毒抗體，但是血細(xì)胞中仍能檢測出具有細(xì)胞毒性的呼腸孤病毒，而血漿中卻未檢測出，這提示細(xì)胞轉(zhuǎn)運(yùn)可保護(hù)病毒免受抗體破壞，使其進(jìn)入腫瘤組織，發(fā)揮抗腫瘤效應(yīng)。在轉(zhuǎn)移病灶手術(shù)標(biāo)本中，病毒蛋白在瘤細(xì)胞內(nèi)表達(dá)而在正常肝組織中未表達(dá)。提示呼腸孤病毒可能被免疫細(xì)胞攜帶至腫瘤組織并只在瘤細(xì)胞內(nèi)復(fù)制。另外，細(xì)胞載體也可保護(hù)病毒免遭體內(nèi)抗體的中和，這種載體也較便捷［17］：Mader等［18］用自體脂肪間充質(zhì)干細(xì)胞作為麻疹病毒的載體，已被美國食品藥品監(jiān)督管理局批準(zhǔn)用于卵巢癌的臨床研究。

3　基于病毒載體的基因治療

溶瘤病毒具有無限復(fù)制性和靶向性，可作為基因載體，攜帶外源性抗癌基因，使其在發(fā)揮溶瘤功能的同時(shí)，還可大量表達(dá)抗腫瘤基因，產(chǎn)生雙重抗癌效應(yīng)。目前，越來越多攜帶治療基因的溶瘤病毒被用于抗癌研究，并取得了可喜成果［19］。

3.1癌基因和抑癌基因治療

結(jié)直腸癌的發(fā)生是多基因致病的過程，通過轉(zhuǎn)基因的高表達(dá)阻斷腫瘤惡性轉(zhuǎn)化，是控制腫瘤的有效手段之一。FHL2（four and a half LIM domain 2）是結(jié)直腸癌的一種致癌基因［20］。Wu等［20］以腺病毒為載體，構(gòu)建了可下調(diào)FHL2蛋白表達(dá)的rAAV-shR?NA-FHL2病毒，將其轉(zhuǎn)染LOVO細(xì)胞株，行周期檢測顯示細(xì)胞停滯在G0/G1期，細(xì)胞生長受到抑制。ST13是一種結(jié)直腸癌特異性抑制基因，腺病毒SG500能通過hTERT、HRE啟動(dòng)子分別驅(qū)動(dòng)E1A和E1B進(jìn)行雙重調(diào)節(jié)，Yu等［21］將ST13插入腺病毒SG500中構(gòu)建結(jié)直腸癌特異性腺病毒SG500-ST13，比SG500特異性更高，抗腫瘤效果更強(qiáng)。

3.2免疫基因治療

免疫基因治療的主要策略是把免疫相關(guān)基因或細(xì)胞因子基因?qū)肴梭w，增強(qiáng)腫瘤的免疫原性及機(jī)體對(duì)腫瘤抗原的識(shí)別和遞呈能力，提高免疫效應(yīng)細(xì)胞的抗腫瘤免疫功能。Zhao等［22］使用腺病毒為載體構(gòu)建表達(dá)趨化因子CCL21和細(xì)胞因子IL15基因的病毒Ad-CCL21-IL-15，在結(jié)直腸癌荷瘤小鼠行瘤內(nèi)注射病毒后能顯著抑制腫瘤生長。另外，經(jīng)Ad-CCL21-IL-15治療后小鼠體內(nèi)能產(chǎn)生腫瘤特異性細(xì)胞毒T淋巴細(xì)胞免疫反應(yīng)，IFN-γ表達(dá)水平比對(duì)照組高。

3.3自殺基因治療

自殺基因治療即將某些病毒或細(xì)菌所特有的前體藥物轉(zhuǎn)換酶基因?qū)肽[瘤細(xì)胞，該基因能編碼特殊的酶，使無毒的藥物前體在腫瘤細(xì)胞內(nèi)轉(zhuǎn)換為細(xì)胞毒性產(chǎn)物，從而達(dá)到殺滅腫瘤細(xì)胞的目的。目前研究較多的有單純皰疹病毒胸苷激酶/更昔洛韋、胞嘧啶脫氨基酶/5-氟胞嘧啶（5-FC）、硝基還原酶/硝苯亞胺。Yamada等［23］通過表達(dá)胞嘧啶脫氨基酶的溶瘤單純皰疹病毒闡述了病毒的復(fù)制、基因的表達(dá)和前體藥物的活化之間的關(guān)系。該研究在小鼠結(jié)直腸癌模型上進(jìn)行實(shí)驗(yàn)發(fā)現(xiàn)，早期加入5-FC相對(duì)于晚期加入有更大的細(xì)胞毒性；病毒瘤內(nèi)注射后第6天轉(zhuǎn)換5-FC成5-氟尿嘧啶（5-FU）的效率最高，治療效果最強(qiáng)；此外，瘤內(nèi)5-FU產(chǎn)物的形成未引起小鼠整體水平的5-FU升高。該研究為臨床應(yīng)用奠定了基礎(chǔ)。許多實(shí)驗(yàn)表明單自殺基因抑瘤效果不及雙自殺基因。Boulaiz等［24］利用逆轉(zhuǎn)錄病毒為載體攜帶凋亡素基因（apoptin）和鳥苷酸交換因子（GEF）轉(zhuǎn)染DLD-1人結(jié)直腸癌細(xì)胞株，結(jié)果顯示同時(shí)表達(dá)apoptin和GEF兩種基因的比只表達(dá)其中一種基因的病毒載體更能促進(jìn)細(xì)胞凋亡和抑制細(xì)胞活性。

3.4抗腫瘤血管的基因治療

腫瘤的生長和轉(zhuǎn)移有賴于血管的形成，血管內(nèi)皮生長因子（VEGF）促進(jìn)腫瘤細(xì)胞遷移，抑制瘤細(xì)胞凋亡和誘導(dǎo)腫瘤新生血管。Qiu等［25］采用慢病毒構(gòu)建VEGFA靶向RNAi病毒載體感染RKO大腸癌細(xì)胞，實(shí)驗(yàn)結(jié)果表明抑制VEGFA能顯著降低RKO大腸癌的細(xì)胞增殖、侵襲轉(zhuǎn)移和腫瘤生長，其機(jī)制可能是抑制VEGFA可導(dǎo)致MEK/ERK-Smac/Diablo信號(hào)通路表達(dá)受阻。VB-111由非復(fù)制型腺病毒載體和修飾的小鼠前內(nèi)皮素前體基因的啟動(dòng)子組成，其啟動(dòng)子通過在血管內(nèi)皮細(xì)胞表達(dá)Fas的嵌合體基因?qū)е录?xì)胞凋亡。Brenner等［26］用VB-111在33例晚期實(shí)體瘤患者中（包括晚期結(jié)直腸癌）行劑量遞增性研究發(fā)現(xiàn)，病毒劑量>3×1011VPs時(shí)患者出現(xiàn)自限性發(fā)熱；治療28天后療效評(píng)價(jià)顯示53%患者病情穩(wěn)定；治療后轉(zhuǎn)移病灶中能檢測到轉(zhuǎn)基因的表達(dá)，而在血液中轉(zhuǎn)基因的表達(dá)陰性。

4　基于病毒載體的腫瘤疫苗治療

腫瘤疫苗是直接應(yīng)用腫瘤抗原進(jìn)行主動(dòng)免疫治療的一種產(chǎn)物，可增強(qiáng)和調(diào)節(jié)宿主免疫系統(tǒng)，誘導(dǎo)產(chǎn)生特異性抗腫瘤反應(yīng)［27］。早在1990年人們已開發(fā)出聯(lián)合痘病毒載體表達(dá)CEA的結(jié)直腸癌疫苗。研究表明，病毒載體比傳統(tǒng)佐劑有更強(qiáng)的免疫原性，其感染腫瘤組織引起炎癥反應(yīng)使得其成為強(qiáng)大的抗腫瘤免疫誘導(dǎo)劑［28-29］。大部分腫瘤疫苗能激活特異性T淋巴細(xì)胞，記憶T淋巴細(xì)胞是結(jié)直腸癌的重要預(yù)后因素［30］。另外，對(duì)于單純皰疹病毒（herpes simplex vi?rus，HSV）載體，機(jī)體適應(yīng)的抗病毒免疫可以提高抗腫瘤免疫［31］。

4.1針對(duì)腫瘤特異性抗原的腫瘤疫苗

這類疫苗可以打破機(jī)體對(duì)自身抗原的耐受性。MUC1是人們發(fā)現(xiàn)的首個(gè)結(jié)直腸癌抗原［32］，但許多臨床研究顯示機(jī)體對(duì)MUC1有較強(qiáng)的免疫耐受性，不能誘導(dǎo)出MUC1特異性免疫應(yīng)答［33］。但近期研究顯示處于癌前病變時(shí)的腫瘤微環(huán)境對(duì)MUC1特異性免疫具有塑造作用。Takashi等［34］以痘病毒為載體構(gòu)建攜帶MUC1的結(jié)直腸癌疫苗，將其注入39例晚期結(jié)腸腺癌患者中，結(jié)果發(fā)現(xiàn)其中43.6%患者能誘導(dǎo)出MUC1相關(guān)免疫反應(yīng)，而另外56.4%患者未能產(chǎn)生免疫應(yīng)答，這是因?yàn)樵诮臃N疫苗前，其體內(nèi)已有高水平的髓源性抑制細(xì)胞（MDSC），導(dǎo)致其免疫功能被抑制。這提示在癌前病變階段者或免疫功能抑制者中預(yù)防性接種MUC1疫苗能避免癌前病變發(fā)展為癌癥。此外，還有多種腫瘤相關(guān)抗原的病毒疫苗被用于結(jié)直腸癌靶向治療的研究中，如p53［35］、KSA［36］、5T4［37］等。

4.2自體腫瘤疫苗

自體腫瘤細(xì)胞疫苗是使用患者自身的腫瘤細(xì)胞，在體外行放射處理后偶聯(lián)免疫佐劑，再接種到患者自身。Onco-VAX是種以卡介苗為佐劑的自體腫瘤細(xì)胞疫苗，Ⅲ期臨床試驗(yàn)顯示，Ⅰ～Ⅳ期結(jié)直腸癌患者腫瘤復(fù)發(fā)率減低，生存期延長［38］。ATV-NDV是利用新城疫病毒（newcastle disease virus）感染從患者腫瘤組織分離的細(xì)胞而制成的疫苗。Schulze等［39］將入組的結(jié)直腸癌肝轉(zhuǎn)移患者隨機(jī)分為對(duì)照組和接種疫苗組。肝轉(zhuǎn)移病灶完整切除后，疫苗組的患者接受6次ATV-NDV治療，結(jié)果顯示接種疫苗的患者無轉(zhuǎn)移生存期和總生存期顯著延長。一項(xiàng)關(guān)于Ⅱ期或Ⅲ期結(jié)直腸癌術(shù)后殘留微小瘤灶行主動(dòng)特異性免疫療法（ASI）的Meta分析顯示，ASI能顯著提高無進(jìn)展生存期（HR=0.76，P=0.03）和總生存期（HR=0.76，P= 0.007）。所有亞組患者對(duì)疫苗的副反應(yīng)率為1.68%，且無顯著的不良事件報(bào)道［40］。

4.3添加共刺激因子的腫瘤疫苗

痘病毒屬轉(zhuǎn)染效率高，無插入突變風(fēng)險(xiǎn)，能產(chǎn)生強(qiáng)大的免疫刺激作用［41］。結(jié)直腸癌患者接種痘病毒疫苗rV-CEA行主動(dòng)免疫，雖然觀察到特異性的細(xì)胞毒T淋巴細(xì)胞反應(yīng)，但客觀反應(yīng)率卻是令人失望的［42］。隨后，痘病毒載體表達(dá)CEA和共刺激因子B7.1的腫瘤疫苗ALVAC-CEA/B7.1被研發(fā)［43］。Ⅰ期臨床試驗(yàn)發(fā)現(xiàn)晚期腫瘤患者（包括結(jié)直腸癌患者）體內(nèi)能產(chǎn)生CEA特異性T淋巴細(xì)胞免疫，40%患者病情穩(wěn)定至少達(dá)4個(gè)月［44］。Ⅱ期臨床試驗(yàn)應(yīng)用ALVAC-CEA/B7.1聯(lián)合化療治療結(jié)直腸癌，結(jié)果顯示化療藥物并不影響ALVAC-CEA/B7.1誘導(dǎo)抗腫瘤免疫，有40%患者能達(dá)到客觀反應(yīng)，為腫瘤疫苗聯(lián)合化療的臨床應(yīng)用提供了支持［45］。還有臨床實(shí)驗(yàn)顯示，表達(dá)CEA和ICAM-1 或LFA-3等共刺激因子的牛痘病毒和禽類痘病毒有較好的臨床療效［46］。目前，基于病毒載體的腫瘤疫苗能直接產(chǎn)生免疫反應(yīng)，而無需感染腫瘤組織，有望成為新的腫瘤治療方法。然而，基于病毒的腫瘤疫苗需要依賴完善的機(jī)體免疫，真正的腫瘤抗原特異性免疫應(yīng)答還很低，這可能更適合微小轉(zhuǎn)移灶的治療。

5　展望

結(jié)直腸癌基于病毒的療法有著廣闊的前景，但也存在著很多障礙。一方面，腫瘤是復(fù)雜、異質(zhì)性的，腫瘤干細(xì)胞會(huì)休眠，因此溶瘤病毒難以達(dá)到理想的治療效果；另一方面，病毒本身具有抗原性，能激活機(jī)體抗病毒免疫，病毒的進(jìn)化變異會(huì)造成新的病原體感染。但隨著對(duì)病毒基因和結(jié)直腸癌相關(guān)機(jī)制的了解，基于病毒治療結(jié)直腸癌的策略也在不斷完善。Pan等［47］利用髓源抑制細(xì)胞運(yùn)載溶瘤水皰性口炎病毒，行靜脈注射治療結(jié)直腸癌轉(zhuǎn)移小鼠后，小鼠生存期顯著延長且無明顯毒性。乏氧會(huì)造成腫瘤對(duì)放化療產(chǎn)生耐受［48］。Cherry等［49］針對(duì)腫瘤微環(huán)境中乏氧誘導(dǎo)因子（HIF）通路的激活構(gòu)建新的溶瘤腺病毒HIF-Ad和HIF-Ad-IL4，實(shí)驗(yàn)結(jié)果顯示其均僅在乏氧條件下復(fù)制、表達(dá)E1A基因，抗腫瘤效果顯著。另外，病毒在聯(lián)合化療、放療和靶向藥物治療腫瘤時(shí)，顯示無交叉耐受性，抗腫瘤效應(yīng)可達(dá)到疊加或協(xié)同［50-52］。

總之，基于病毒的抗腫瘤治療可能將代表一種新的選擇應(yīng)用于臨床，不同于傳統(tǒng)的腫瘤治療方法，病毒的復(fù)制和腫瘤的發(fā)展之間存在許多相關(guān)聯(lián)的治療路徑，未來病毒治療癌癥將個(gè)體化到每位患者和每種腫瘤，而這種靶向治療也減少病毒突變體的產(chǎn)生。

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（2015-12-02收稿）

（2016-01-05修回）

（編輯：邢穎校對(duì)：楊紅欣）

尹磊專業(yè)方向?yàn)槲改c道腫瘤的外科治療。E-mail：15056997829@163.com

Advances in virus-based therapies for colorectal cancer

Lei YIN1,2, Yanlai SUN2, Zhongfa XU2,3
Correspondence to: Zhongfa XU; E-mail: xzf2216@126.com
1School of Medicine and Life Sciences, Jinan University and Shandong Academy of Medical Sciences, Ji'nan 250022, China;2The Fourth Surgical Department, Shandong Tumor Hospital, Ji'nan 250117, China;3The Affiliated Hospital of Shandong Academy of Medical Sciences, Ji'nan 250031, China
This study was supported by the Science & Technology Development Projects of Shandong Province (No. 2013GSF11834) and the Jinan Science & Technology Plan of Independent Innovation Projects in Colleges and Universities (No. 201401253)

AbstractVirus-based anti-tumor therapies are novel biological treatments. Viral vectors can infect tumors to kill cancers directly (oncolysis), act as cancer vaccines to activate the immune system, and deliver genes with anti-tumor activity to the cancer cells. Genetic engineering has been applied to viruses to achieve more specific and efficient cancer treatment. Simultaneously, a reasonable combination of viral vectors and existing anti-tumor therapy can improve the therapeutic effect. Consequently, virus-based therapy is expected to serve as an effective anti-tumor strategy. We reviewed recent studies on the anti-tumor viral therapy of colorectal carcinoma.

Keywords:colorectal neoplasm, oncolytic virus, viral vector, genetic therapy, immunotherapy

作者簡介

通信作者：徐忠法xzf2216@126.com

doi:10.3969/j.issn.1000-8179.2016.03.301