Fusion enzymes containing HSV-1 thymidine kinase mutants and guanylate kinase enhance prodrug sensitivity in vitro and in vivo.
Fusion enzymes containing HSV-1 thymidine kinase mutants and guanylate kinase enhance prodrug sensitivity in vitro and in vivo.
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含有HSV-1胸苷激酶突变体和鸟苷酸激酶的融合酶增强了体外和体内前药敏感性。
DOI：
10.1038/cgt.2009.60
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发表时间：
2010-02
影响因子：
6.4
通讯作者：
中科院分区：
文献类型：
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作者：
单纯疱疹病毒胸苷激酶(HSVtk)联合更昔洛韦(GCV)是目前在肿瘤基因治疗中应用最广泛的自杀基因/前药系统。这种疗法的一个主要局限性是HSVtk对其活性抗代谢产物激活GCV的效率低下。我们之前描述了两种克服这一局限性的策略：1)构建具有改善GCV激活潜力的HSVtk突变体；2)构建编码HSVtk和小鼠鸟苷酸激酶(MGMK)的融合蛋白，MGMK是GCV激活途径中的第二种酶。作为进一步增强GCV活性的手段，构建了两个含有HSVtk突变体(突变体30和SR39)的MGMK/HSVtk载体，并在体内外评价了它们对肿瘤和旁观者的杀伤作用。与HSVtk、突变体30或MGMK/HSVtk相比，融合突变体MGMK/30的IC50值分别降低了约12、500倍、100倍和125倍。体外旁观者分析显示，5%的MGMK/30表达细胞足以诱导75%的肿瘤细胞杀伤。在移植瘤模型中，当GCV浓度为1 mg/kg时，MGMK/30对肿瘤生长的抑制作用最强，而对野生型HSVtk-、MGMK/HSVtk-或突变型30转基因细胞没有影响。另一种融合结构，MGMK/SR39，与HSVtk或MGMK/HSVtk相比，分别使大鼠C6胶质瘤细胞对GCV的敏感性增加2500倍或25倍。体外分析显示，携带SR39和MGMK/SR39的细胞的IC50值相似，尽管MGMK/SR39似乎引起更强的旁观者杀伤效应，其中1%的MGMK/SR39细胞导致60%的细胞死亡。在异种移植瘤模型中，尽管观察到肿瘤生长受到抑制，但当剂量为1 mg/kg GCV时，携带SR39和MGMK/SR39转基因细胞的小鼠之间的肿瘤体积没有统计学意义。然而，在较低剂量的更昔洛韦(0.1 mg/kg)下，MGMK/SR39似乎比SR39有更强的肿瘤生长抑制作用(P≤0.05)。体内研究表明，即使只有5%的肿瘤细胞被转染，当存在1 mg/kg的GCV时，这两种突变融合蛋白在旁观者杀伤方面也表现出显著的改善。这种具有特殊前药物转化特性的融合突变体将允许使用较低剂量和非骨髓抑制剂量的GCV，同时提高肿瘤杀伤率，因此有望成为翻译基因治疗研究的候选药物。
Herpes Simplex Virus thymidine kinase (HSVTK) with ganciclovir (GCV) is currently the most widely used suicide gene/prodrug system in cancer gene therapy. A major limitation in this therapy is the inefficient activation of GCV by HSVTK to its active antimetabolites. We previously described two strategies to overcome this limitation: 1) generation of HSVTK mutants with improved GCV activation potential and 2) construction of a fusion protein encoding HSVTK and mouse guanylate kinase (MGMK), the second enzyme in the GCV activation pathway. As a means to further enhance GCV activation, two MGMK/HSVTK constructs containing the HSVTK mutants (mutant 30 and SR39) were generated and evaluated for their tumor and bystander killing effects in vitro and in vivo. One fusion mutant, MGMK/30, demonstrates significant reduction in IC50 values of approximately 12,500-fold, 100-fold, and 125-fold compared to HSVTK, mutant 30 or MGMK/HSVTK, respectively. In vitro bystander analyses reveal that 5% of MGMK/30-expressing cells are sufficient to induce 75% of tumor cell killing. In an xenograft tumor model, MGMK/30 displays the greatest inhibition of tumor growth at a GCV concentration (1mg/kg) that has no effect on wild type HSVTK-, MGMK/HSVTK-, or mutant 30-transfected cells. Another fusion construct, MGMK/SR39, sensitizes rat C6 glioma cells to GCV by 2,500-fold or 25-fold compared to HSVTK or MGMK/HSVTK, respectively. In vitro analyses show similar IC50 values between cells harboring SR39 and MGMK/SR39, although MGMK/SR39 appears to elicit stronger bystander killing effects where 1% of MGMK/SR39-transfected cells result in 60% cell death. In a xenograft tumor model, despite observable tumor growth inhibition, no statistical significance in tumor volume was detected between mice harboring SR39- and MGMK/SR39-transfected cells when dosed with 1mg/kg GCV. However, at a lower dose of GCV (0.1mg/kg), MGMK/SR39 appears to have slightly greater tumor growth inhibition properties compared to SR39 (P≤0.05). In vivo studies indicate that both mutant fusion proteins display substantial improvements in bystander killing in the presence of 1mg/kg GCV, even when only 5% of the tumor cells are transfected. Such fusion mutants with exceptional prodrug converting properties will allow administration of lower and non-myelosuppressive doses of GCV concomitant with improved tumor killing and as such are promising candidates for translational gene therapy studies.
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DOI：
10.1073/pnas.80.13.4139
发表时间：
1983-01-01
期刊：
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA-BIOLOGICAL SCIENCES
影响因子：
--
作者：
FIELD, AK;DAVIES, ME;TOLMAN, RL
通讯作者：
TOLMAN, RL
影响因子：
158.5
作者：
KALDOR, JM;DAY, NE;STOVALL, M
通讯作者：
STOVALL, M
影响因子：
56.9
作者：
CULVER, KW;RAM, Z;BLAESE, RM
通讯作者：
BLAESE, RM
影响因子：
12.4
作者：
Aky端rek, LM;Nallamshetty, S;Nabel, EG
通讯作者：
Nabel, EG
影响因子：
168.9
作者：
Relling, MV;Rubnitz, JE;Pui, CH
通讯作者：
Pui, CH