Survivin启动子控制自杀基因HSV-TK真核表达载体对肝癌细胞杀伤活性的研究

目的研究自杀基因单纯疱疹病毒胸苷激酶(HSV-TK)对人肝癌细胞的特异性杀伤作用,推进该自杀基因治疗原发性肝癌的可行性,从而为肝癌靶向基因治疗奠定一定的基础。方法将pc DNA3.1-p Survivin-TK质粒转染人肝癌细胞Hep G2中,RT-PCR法检测自杀基因m RNA水平,Western blot法检测自杀基因蛋白量的表达;再次转染上述质粒到肝癌细胞Hep G2中,随着更昔洛韦(GCV)剂量的增加,来监测pc DNA3.1-p Survivin-TK重组载体对细胞的杀伤效果,CCK-8法检测GCV对肝癌细胞的毒性作用,流式细胞术检测细胞凋亡的情况。结果通过RT-PCR法、Western blot法表明了转染该重组质粒的Hep G2细胞可以成功表达TK基因;此外,在外源药物GCV作用下也实现了对肝癌细胞的特异杀伤,CCK-8法表明,随着药物浓度的增高质粒转染组细胞存活率逐渐下降;流式细胞术检测得知,细胞经GCV处理48 h后,转染pc DNA3.1-p Survivin-TK重组质粒的肝癌细胞组凋亡率约为(37.37±4.02)%,而未转染质粒组细胞凋亡率约为(1.00±0.62)%,两组间比较有统计学差异(P<0.01)。结论 HSV-TK/GCV自杀基因的毒性作用随着GCV浓度的增高而上升,并且HSV-TK/GCV系统对人肝癌细胞具有特异性靶向杀伤作用。     

Tat8-TK/GCV suicide gene therapy induces pancreatic tumor regression in vivo

Suicide gene therapy using the herpes simplex virus thymidine kinase (TK) gene in combination with ganciclovir (GCV) has been shown to produce therapeutic, but limited, efficacy because of poor gene transfer efficiency and reduced bystander effect. Here we report that fusion of TK to an eight-amino acid peptide from the basic domain of the human immunodeficiency virus (HIV) Tat protein significantly increases the cytotoxic efficacy of the TK/GCV system in pancreatic cancer cells. We demonstrate that Tat8-TK protein is released from the intracellular compartment of Tat8-TK-expressing cells to the extracellular medium after GCV treatment. Interestingly, we show that this conditioned medium is then able to mediate cytotoxicity of wildtype cultures, suggesting the internalization of the Tat8-TK protein. Moreover, a strong antitumoral effect of Tat8-TK/GCV treatment could be achieved by two different in vivo approaches. Tumors injected with NIH 3T3/Tat8-TK cells attached to microcarriers (MC+Tat8-TK) and treated with GCV led to a 35.6% reduction in the initial tumor volume and to 50% tumor eradication. Furthermore, electrogene transfer of TK or Tat8-TK followed by administration of high doses of GCV led to an overall statistically significant reduction in tumor growth. However, the reduction in initial tumor volume was statistically significant only for the Tat8-TK group (59.5% reduction). Moreover, in this group 50% complete tumor eradication was achieved. When moderate doses of GCV were administered, the overall reduction in tumor growth was statistically significant only in the Tat8-TK group. Therefore, our results suggest that fusion of TK to the Tat8 peptide enhances TK/GCV suicide gene therapy.     

Adenoviral vectors capable of replication improve the efficacy of HSVtk/GCV suicide gene therapy of cancer.

A major obstacle to the success of gene therapy strategies that directly target cancer cells is the poor vector distribution within solid tumors. To address this problem, we developed an E1b 55 kDa attenuated, replication-competent adenovirus (Ad.TKRC) which expresses the herpes simplex-1 thymidine kinase (HSVtk) gene to sensitize tumors to ganciclovir (GCV). Efficacy of this combined strategy was tested in nude mice with subcutaneous human A375 melanoma and ME180 cervical carcinomas. Intratumoral injection of a replication-defective adenoviral vector expressing HSVtk (Ad.TK) followed by GCV treatment resulted in doubling of the survival time of mice bearing A375 tumors and 20% long-term survival of mice with ME180 tumors. Treatment of tumors with Ad.TKRC without GCV resulted in a similar antitumor effect, confirming that the replicating vector has an oncolytic effect. When GCV was initiated 3 days after Ad.TKRC injection, survival of mice with each tumor type was greatly prolonged, with 60% of animals with ME180 tumors surviving for over 160 days. These results confirm that both the oncolysis caused by a replicating virus and suicide/prodrug gene therapy with HSVtk/GCV have potent antitumor effects. When combined, these two approaches are complementary resulting in a significantly improved treatment outcome.     

HSV-TK和IL-12联合基因对宫颈癌的杀伤作用

目的观察单纯疱疹病毒胸苷激酶(HSV-TK)丙氧鸟苷(GCV)自杀基因系统联合IL-12基因对人宫颈癌hela细胞系体外杀伤作用。方法采用脂质体转染法将HSV-TK及IL-12联合基因转染hela细胞,并将其用于体外实验,观察GCV对转染联合基因的Hela的杀伤作用及旁观者效应等。结果加入GCV后体外培养联合基因转染的Hela细胞,24 h后细胞明显发生形态上改变,正常对照组细胞生长旺盛;随着GCV剂量的加大,转染联合基因的Hela细胞的存活率呈逐渐下降的趋势,GCV对转染细胞的杀伤作用较对正常细胞的杀伤作用明显增强。并观察到自杀基因系统具有明显的旁观者效应。结论脂质体可介导HSV-TK及IL-12联合基因转入人宫颈癌hela细胞并获得稳定表达,GCV对HSV-TK及IL-12联合基因转染的hela细胞具有明显的杀伤作用。     

Egrl启动子介导的HSV-TK／GCV联合放疗对卵巢癌细胞的治疗作用

目的探讨Egrl启动子介导的HSV-TK／GCV自杀基因系统与放疗联合治疗卵巢癌的疗效。方法以MTT方法测定细胞增殖抑制率,流式细胞仪分析细胞凋亡和坏死情况,倒置光学显微镜和电子显微镜观察治疗前后细胞形态学变化,并与单纯的辐射治疗比较。结果辐射一基因治疗对卵巢癌H08910细胞有生长抑制和凋亡诱导作用,效果明显优于单纯的放疗,干预60h、72h、96h后,辐射．基因治疗组的细胞增殖抑制率分别达到（56．88±5．35）％、（69．09±3．43）％、（86．91±0．75）％,而单纯辐射组对应的细胞增殖抑制率仅为（21．93±11．37）％、（37．99±7．24）％、（69．55±3．26）％。干预48h后,辐射．基因治疗组的细胞死亡率为26．01％（凋亡率：19．45％;坏死率：6．56％）,明显高于单纯辐射组的12．47％（凋亡率：8．68％;坏死率：3．79％）和阴性对照组的4．41％（凋亡率：2．52％;坏死率：1．89％）。细胞形态学观察结果也显示辐射．基因治疗组细胞呈现了典型的凋亡形态学特征,细胞生长状态远不如单纯辐射组和阴性对照组。结论辐射．基因联合应用具有较好的协同和互补效应,比单一的放疗具有更强的抗肿瘤作用。     

Effective suicide gene therapy in vivo by EBV-based plasmid vector coupled with polyamidoamine dendrimer

This study demonstrates in vivo effectiveness of a nonviral vector system, Epstein-Barr virus (EBV)-based plasmid vector coupled with polyamidoamine (PAMAM) dendrimer (EBV/polyplex), in suicide gene therapy of cancer. The EBV-based vector is a plasmid vector containing EBV nuclear antigen 1 (EBNA1) gene and oriP from EBV genome. HSV-1 tk gene was transferred into Ewing's sarcoma cell lines, A4573 and KP-EWS-YI, by using an EBV-based plasmid vector, pSES.Tk, or a conventional plasmid vector, pS.Tk. Cells transfected with pSES.Tk/dendrimer showed approximately 100 times lower ID50 to ganciclovir (GCV) compared with those transfected with pS. Tk/dendrimer. Intratumoral injection of pSES.Tk/dendrimer but not pS. Tk/dendrimer drastically suppressed the growth of tumors which had generated from A4573 or Huh7 hepatocellular carcinoma (HCC) cells inoculated into severe combined immunodeficiency (SCID) mice. The treatment with pSES.Tk/dendrimer also resulted in significant prolongation of survival of the mice implanted with A4573. These results suggest that the EBV/polyplex system could be useful for in vivo suicide gene therapy of cancer. Gene Therapy (2000) 7, 53-60.     

Efficient suicide gene therapy of transduced and distant untransduced ovary tumors is correlated with significant increase of intratumoral T and NK cells

Gene therapy using herpes simplex type 1 thymidine kinase gene (HSV1-TK) transfer followed by ganciclovir (GCV) treatment has revealed an important intratumoral and regional bystander effect that is at least partly immune-mediated. The aim of this work was to study the modifications of T lymphocyte subpopulations in a model of distant bystander effect occurring between ovary tumors. Bilateral ovarian tumors were generated in 21 WKY rats by injection in the ovarian pouch of either parental or HSV1-TK-expressing DWA-OC-1 ovarian cancer cells. After 14 days, rats were treated for two weeks with GCV (75 mg/kg x 2/d) or saline. All rats were killed at day 29 for pathological examination. The tumor-infiltrating mononuclear cells were analyzed by semi-quantitative immunohistochemistry. As compared to rats receiving saline, GCV-treated animals exhibited a complete disappearance of the HSV1-TK+ tumors with residual fibrotic scars (ovary weights: 0.46 +/- 0.4 g vs 10.11 +/- 1.5 g, P < 0.001). Interestingly, the contralateral HSV1-TK negative tumor showed a significant regression (12.39 +/- 1.93 g vs 22.24 +/- 237 g, P < 0.014). Furthermore, a lower incidence of tumoral ascitis was found in the GCV-receiving group (20% vs 90% P < 0.02). Within both TK- and TK+ tumors, there was a significant increase of CD4+, CD8+ and NK cells in the GCV-treated group compared to the saline-treated group. This study thus indicates that a distant bystander effect not only acts between close tumors within a given organ such as the liver, but also between more distant tumors in the peritoneal cavity. This effect is associated with significant infiltration of the tumor by immune system cells, supporting the notion that the distant bystander effect is immune-mediated.     

Utility of TK/GCV in the context of highly effective oncolysis mediated by a serotype 3 receptor targeted oncolytic adenovirus

Arming oncolytic adenoviruses with therapeutic transgenes and enhancing transduction of tumor cells are useful strategies for eradication of advanced tumor masses. Herpes simplex virus thymidine kinase (TK) together with ganciclovir (GCV) has been promising when coupled with viruses featuring low oncolytic potential, but their utility is unknown in the context of highly effective infectivity-enhanced viruses. We constructed Ad5/3-Delta24-TK-GFP, a serotype 3 receptor-targeted, Rb/p16 pathway-selective oncolytic adenovirus, where a fusion gene encoding TK and green fluorescent protein (GFP) was inserted into 6.7K/gp19K-deleted E3 region. Ad5/3-Delta24-TK-GFP killed ovarian cancer cells effectively, which correlated with GFP expression. Delivery of GCV immediately after infection abrogated viral replication, which might have utility as a safety switch. Due to the bystander effect, killing of some cell lines in vitro was enhanced by GCV regardless of timing. In murine models of metastatic ovarian cancer, Ad5/3-Delta24-TK-GFP improved antitumor efficacy over the respective replication-deficient virus with GCV. However, GCV did not further enhance efficacy of Ad5/3-Delta24-TK-GFP in vivo. Simultaneous detection of tumor load and virus replication with bioluminescence and fluorescence imaging provided insight into the in vivo kinetics of oncolysis. In summary, TK/GCV may not add antitumor activity in the context of highly potent oncolysis.     

HSV-tk/GCV自杀基因疗法及其影响树突状细胞功能的实验研究

目的 研究单纯疱疹病毒－胸苷激酶／更昔洛韦（ＨＳＶ－ｔｋ／ＧＣＶ）自杀基因系统的特性,探讨转ＨＳＶ－ｔｋ基因后,肿瘤细胞被更昔洛韦（Ｇａｎｃｉｃｌｏｖｉｒ,ＧＣＶ）杀伤时的凋亡现象及对树突状细胞功能的影响。方法 以逆转录病毒法将ＨＳＶ－ｔｋ基因转人乳腺癌细胞株ＭＣＦ－７中,以Ｓｏｕｔｈｅｒｎ ｂｌｏｔ法对肿瘤细胞基因组ＤＮＡ中ｔｋ基因的整合进行鉴定,以流式细胞仪及电镜观察ＧＣＶ杀伤ＭＣＦ－７／ｔｋ细胞时的凋亡现象,由脐血ＣＤ３４＾＋细胞诱生树突状细胞,以＾３Ｈ－ＴｄＲ掺入法检测树突状细胞刺激同种异体Ｔ淋巴细胞增殖的能力。结果 以逆转录病毒法成功地将ＨＳＶ－ｔｋ基因转入ＭＣＦ－７细胞中,转染ｔｋ基因的ＭＣＦ－７细胞能被ＧＣＶ有效杀伤并存在凋亡现象,凋亡率可达３１．３％;由脐血ＣＤ３４＾＋细胞在ＧＭ－ＣＳＦ、ＴＮＦ     

CD/TK双自杀基因系统对Balb/c小鼠大肠癌的杀伤作用

目的:探讨腺病毒介导的以血管内皮生长因子(VEGF)启动的CD/TK 双自杀基因对Balb/c小鼠大肠癌的杀伤作用.方法:将Ad-VECF-CD/TK在293细胞中包装、扩增、纯化、鉴定,用CT26细胞通过皮下注射法建立荷大肠癌Balb/c小鼠模型,随机选取30只荷瘤小鼠分为治疗组和时照组,每组15只小鼠.治疗组小鼠使用重组腺病毒和5.FC、GCV两种前药治疗,对照组注射生理盐水.记录两组小鼠不同时间点肿瘤生长体积,计算抑瘤率,3周后处死小鼠,取瘤,切片行TUNEL原位凋亡检测.结果:治疗组与对照组相比,治疗前肿瘤体积差异无统计学意义,第7、14、21天肿瘤体积差异有统计学意义(P<0.05),治疗组肿瘤体积较对照组明显变小,生长受抑制,第7、14、21天的抑瘤率分别为69.5%、75.1%、84.3%.TUNEL原位凋亡检测显示治疗组肿瘤细胞凋亡率较对照组明显增高,分别为(43.2±6.67)%和(2.4±0.91)%.结论:VEGF启动的CD/TK双自杀基因系统对Balb/c小鼠大肠癌具有明显的杀伤作用.     

Plasma Ip(a) concentration is inversely correlated with the ratio of Kringle IV/Kringle V encoding domains in the apo(a) gene.

Plasma Lp(a) levels correlate with atherosclerosis susceptibility. This lipoprotein consists of an LDL-like particle attached to a large glycoprotein called apo(a). Apo(a) is a complex glycoprotein containing multiple Kringle domains, found to be highly homologous to plasminogen Kringle IV, and a single Kringle domain homologous to plasminogen Kringle V. Lp(a) levels appear to be inversely correlated with apo(a) size in a given individual. In this study, we have used probes specific to the Kringles IV and V domains of apo(a) cDNA in quantitative Southern blotting analysis. By this method, we have determined the ratio of Kringle IV/Kringle V encoding domains in the apo(a) gene of 53 unrelated individuals with different plasma concentrations of Lp(a). This ratio was found to be inversely correlated with log Lp(a) levels (r = -0.90, P less than 0.0001) and directly correlated with apo(a) apparent molecular weight (Mr) (r = 0.79, P less than 0.0001). In summary, by showing that Lp(a) concentrations and apo(a) apparent size are highly correlated with the ratio of Kringle IV/Kringle V encoding domains in the apo(a) gene, we provide a DNA marker for this atherosclerosis risk factor as well as an important insight into the genetic mechanism regulating Lp(a) levels.     

Synergy of adoptive T-cell therapy and intratumoral suicide gene therapy is mediated by host NK cells

In situ tumor cell killing by the herpes simplex virus thymidine kinase (HSVtk) gene can effectively prime antitumor T-cell responses, at least in part through local induction of a pro-inflammatory environment. Therefore, we reasoned that tumor-associated HSVtk expression would significantly enhance the efficacy of adoptive T-cell transfer (ACT) of (tumor) antigen-specific T cells into tumor-bearing hosts. When B16ovaHSVtk tumors were treated with ganciclovir (GCV), along with suboptimal numbers of activated OT-1T cells, complete tumor regressions were observed where GCV, or ACT, alone was completely ineffective. To our surprise, analysis of regressing tumors showed no increases in intratumoral OT-1T cell trafficking. However, the intratumoral percentages of both OT-1 and endogenous natural killer (NK) cells were substantially increased over controls. Depletion of endogenous NK cells abrogated the efficacy of the combination therapy and reduced the percentages of interferon-gamma(IFNgamma)-secreting OT-1T cells in mice that received combined therapy to levels similar to those of control mice. These data suggest that even relatively low levels of gene transfer of suicide genes into tumors may have therapeutic value as an adjuvant for other T-cell therapies, by providing immunological signals that support T-cell activation and expansion in vivo.     

ERK-dependent suicide gene therapy for selective targeting of RTK/RAS-driven cancers

1. Download : Download high-res image (180KB)
2. Download : Download full-size image

     

Double suicide gene therapy augments the antitumor activity of a replication-competent lytic adenovirus through enhanced cytotoxicity and radiosensitization

Replication-competent adenoviruses may provide a highly efficient means of delivering therapeutic genes to tumors. Previously, we evaluated in vitro a replication-competent adenovirus (Ad5-CD/TKrep) containing a cytosine deaminase (CD)/herpes simplex type 1 thymidine kinase (HSV-1 TK) fusion gene that allows lytic viral therapy to be combined with double suicide gene therapy. Both the CD/5-FC and HSV-1 TK/GCV enzyme/prodrug systems enhanced the tumor cell-specific cytopathic effects of the Ad5-CD/TKrep virus in vitro and sensitized cells to radiation. To extend these in vitro findings in vivo, we evaluated the antitumor activity of the Ad5-CD/TKrep virus in combination with double prodrug therapy and radiation therapy. The Ad5-CD/TKrep virus independently demonstrated significant antitumor activity against C33A cervical carcinoma xenografts. Therapeutic outcome was dramatically improved with systemic administration of double, but not single, prodrug (5-FC + GCV) therapy. When used in a neoadjuvant setting, Ad5-CD/TKrep-mediated double suicide gene therapy dramatically potentiated the effectiveness of radiation therapy. The trimodal approach of Ad5-CD/TKrep viral, double suicide gene, and radiotherapies produced significant tumor regression and ultimately 100% tumor cure. The results demonstrate the high therapeutic potential of the trimodal approach and provide a solid foundation for future clinical trials.     

Integration of active human beta-galactosidase gene (100 kb) into genome using HSV/AAV amplicon vector

Vectors based on herpes simplex virus type-1 (HSV-1) permit delivery of transgenes of up to 150 kb, while the inverted terminal repeats and Rep of the adeno-associated virus (AAV) can confer site-specific integration into the AAVS1 site, which allows sustained expression of a transgene. In this study, combination of the viral elements in HSV/AAV hybrid vectors has been applied for the infectious transfer of the human lysosomal beta-galactosidase (BGAL) gene of 100 kb. Temporary expression and functional activity of beta-galactosidase (beta-gal) could be detected in human beta-gal-deficient patient and glioblastoma (Gli36) cells upon infection with the basic BGAL amplicon vector. Sustained expression of beta-gal was achieved in Gli36 cells infected with rep-plus, but not rep-minus, HSV/AAV hybrid vectors. None of five clones isolated after rep-minus hybrid vector infection showed elevated beta-gal activity or site-specific integration. In contrast, 80% of the rep-plus clones possessed beta-gal activity at least twofold greater than normal levels for up to 4 months of continuous growth, and 33% of the clones exhibited AAVS1-specific integration of the ITR-flanked transgene. One of the rep-plus clones displayed integration of the ITR cassette only at the AAVS1 site, with no sequences outside the cassette detectable and beta-gal activity fourfold above normal levels. These data demonstrate AAVS1-specific integration of an entire genomic locus and expression of the transgene from the endogenous promoter mediated by an HSV/AAV hybrid vector.     

HSV trafficking and development of gene therapy vectors with applications in the nervous system

Herpes simplex virus type 1 (HSV-1) is a neurotropic double-stranded DNA virus that causes cold sores, keratitis, and rarely encephalitis in humans. Nonpathogenic HSV-1 gene transfer vectors have been generated by elimination of viral functions necessary for replication. The life cycle of the native virus includes replication in epithelial cells at the site of initial inoculation followed by retrograde axonal transport to the nuclei of sensory neurons innervating the area of cutaneous primary infection. In this review, we summarize the current understanding of the molecular basis for HSV cell entry, nuclear transport of the genome, virion egress following replication, and retrograde and anterograde axonal transport in neurons. We discuss how each of these properties has been exploited or modified to allow the generation of gene transfer vectors with particular utility for neurological applications. Recent advances in engineering virus entry have provided proof of principle that vector targeting is possible. Furthermore, significant and potentially therapeutic modifications to the pathological responses to various noxious insults have been demonstrated in models of peripheral nerve disease. These applications exploit the natural axonal transport mechanism of HSV, allowing transgene expression in the cell nucleus within the inaccessible trigeminal ganglion or dorsal root ganglion, following the noninvasive procedure of subcutaneous vector inoculation. These findings demonstrate the importance of understanding basic virology in the design of vector systems and the powerful approach of exploiting favorable properties of the parent virus in the generation of gene transfer vectors.     

Neuronal precursor-restricted transduction via in utero CNS gene delivery of a novel bipartite HSV amplicon/transposase hybrid vector.

The ability to modify genetically in utero the precursors of neuronal lineage contributing to multiple postmitotic cell types in the adult central nervous system would provide a means to evaluate strategies to ameliorate conditions affecting cellular patterning, metabolism, or survival. The herpes simplex virus (HSV)-derived amplicon, a vector devoid of viral genes and with the largest known payload capacity, normally exists episomally within nuclei of transduced cells, thus precluding conveyance during mitosis. Herein, we modify the Tc1-like Sleeping Beauty (SB) transposon system to create an integrating amplicon vector platform wherein provision of transposase in trans effectively catalyzes integration of a transgenomic segment. Cotransduction with a Rous sarcoma virus promoter-driven beta-galactosidase-neomycin (betageo) fusion flanked by SB terminal repeats (HSVT-betageo) and a second expressing the SB transposase gene under HSV immediate-early 4/5 gene promoter control (HSVsb) resulted in integration and extension of expression duration. Most notably, in utero intraventricular application led to extensive transgene expression within neuronal precursors and their derivatives without attendant adverse consequences, suggesting this new platform could be used to evaluate prenatally the function of gene products in neuronal lineages and evaluate therapeutic strategies for correction of genetic abnormalities affecting the developing CNS.     

AAV9/MFSD8 gene therapy is effective in preclinical models of neuronal ceroid lipofuscinosis type 7 disease

Neuronal ceroid lipofuscinosis type 7 (CLN7) disease is a lysosomal storage disease caused by mutations in the facilitator superfamily domain containing 8 (MFSD8) gene, which encodes a membrane-bound lysosomal protein, MFSD8. To test the effectiveness and safety of adeno-associated viral (AAV) gene therapy, an in vitro study demonstrated that AAV2/MFSD8 dose dependently rescued lysosomal function in fibroblasts from a CLN7 patient. An in vivo efficacy study using intrathecal administration of AAV9/MFSD8 to Mfsd8^{– /–} mice at P7–P10 or P120 with high or low dose led to clear age- and dose-dependent effects. A high dose of AAV9/MFSD8 at P7–P10 resulted in widespread MFSD8 mRNA expression, tendency of amelioration of subunit c of mitochondrial ATP synthase accumulation and glial fibrillary acidic protein immunoreactivity, normalization of impaired behaviors, doubled median life span, and extended normal body weight gain. In vivo safety studies in rodents concluded that intrathecal administration of AAV9/MFSD8 was safe and well tolerated. In summary, these results demonstrated that the AAV9/MFSD8 vector is both effective and safe in preclinical models.     

Systemic administration of a matrix-targeted retroviral vector is efficacious for cancer gene therapy in mice

Targeting cytocidal vectors to tumors and associated vasculature in vivo is a long-standing goal of human gene therapy. In the present study, we demonstrated that intravenous infusion of a matrix (i.e., collagen)-targeted retroviral vector provided efficacious gene delivery of a cytocidal mutant cyclin G1 construct (designated Mx-dnG1) in human cancer xenografts in nude mice. A nontargeted CAE-dnG1 vector (p = 0.014), a control matrix-targeted vector bearing a marker gene (Mx-nBg; p = 0.004), and PBS served as controls (p = 0.001). Enhanced vector penetration and transduction of tumor nodules (35.7 +/- 1.4%, mean +/- SD) correlated with therapeutic efficacy without associated toxicity. Kaplan-Meier survival studies were conducted in mice treated with PBS placebo, the nontargeted CAE-dnG1 vector, and the matrix-targeted Mx-dnG1 vector. Using the Tarone log-rank test, the overall p value for comparing all three groups simultaneously was 0.003, with a trend that was significant to a level of 0.004, indicating that the probability of long-term control of tumor growth was significantly greater with the matrix-targeted Mx-dnG1 vector than with the nontargeted CAE-dnG1 vector or PBS placebo. The present study demonstrates that a matrix-targeted retroviral vector deployed by peripheral vein injection (1) accumulated in angiogenic tumor vasculature within 1 hr, (2) transduced tumor cells with high-level efficiency, and (3) enhanced therapeutic gene delivery and long-term efficacy without eliciting appreciable toxicity.     

Intrinsic bioactivity of thyrotropin in human serum is inversely correlated with thyroid hormone concentrations. Application of a new bioassay using the FRTL-5 rat thyroid cell strain.

We have developed a new bioassay for thyrotropin (TSH) in human serum to evaluate bioactivity in normal individuals and patients with different degrees of primary hypothyroidism. Unpurified TSH in serum showed no stimulation of cyclic AMP production in cultured FRTL-5 rat thyroid cells, but after immunopurification showed potent stimulatory activity. Immunoaffinity purification permitted up to 400-fold concentration of serum TSH, allowing bioactivity measurements even in certain normal sera. The limit of detection in the FRTL-5 bioassay was 10 microU of human TSH per 0.5 ml incubate, and half-maximal responses for standard human TSH was 102 +/- 26 (+/- SE) microU/0.5 ml. Immunoaffinity-purified serum TSH varied in bioactivity-to-immunoactivity (B/I) ratios from less than 0.25 to 1.21 among four euthyroid subjects and eight primary hypothyroid patients. An inverse correlation was found between B/I ratios of immunopurified basal TSH and the serum-free T4 (r = -0.7237, P less than 0.01), T4 (r = -0.6650, P less than 0.05), and T3 (r = -0.6382, P less than 0.05). B/I ratios of immunopurified TSH from three hypothyroid patients before and after acute stimulation by thyrotropin-releasing hormone showed no significant change, despite major changes in serum TSH. In summary, the present study shows an inverse relationship between the metabolic status of an individual and the intrinsic bioactivity of TSH.