Study On An Target Adenovirus For Delivering The Suicide Genes For Cancer Gene Therapy And The Combination Efficacy Of Methylselenol Prodrug And MDV3100for Treatment Of Castration-Resistant Prostate Cancer

1.An Target Adenovirus for Delivering the Suicide Genes for Cancer GeneTherapySuicide gene therapy, or gene-directed enzyme prodrug therapy, is becomingincreasingly popular for cancer treatments. This approach is based on the introductionof viral or bacterial genes into target cells to express an enzyme to catalyze anon-toxic prodrug into a highly toxic metabolite that would subsequently cause tumorcell death. The present study is focused on developing an improved suicide genetherapy strategy for the treatment of human cancer.One of the most important factors affecting the efficiency of suicide genetherapy is the choice of a delivery vector to effectively transfer the therapeutic genesinto the target cells. Replication-defective adenovirus serotype5(Ad5) has become apromising transfer vehicle due to its well-characterized biology, large transgenecapacity, and gene transfer to a wide variety of cell types, both dividing andnon-dividing. Moreover, because of its safety and easy operation, Ad5vector hasbecome the most widely-used viral vector for gene therapy, especially for cancer genetherapy.Although Ad5vectors have exhibited excellent gene transfer activity, relativeresistance of target tissues to Ad5infection has been noted in several reported humanclinical trials based on gene delivery. Deficiency of the coxsackie-adenovirus receptor(CAR) is considered to be the biological basis of this phenomenon, because CAR hasbeen shown to be a docking site for Ad and act as a key receptor for the enhancementof the virus-to-host affinity and the initiation of the virus internalization to host cells. Significant variability in CAR expression between normal tissue and human cancercells has been reported. A major hurdle for Ad5-mediated cancer gene therapy is thata significant fraction of cancer cells lack the CAR receptor. The use of geneticallymodified Ad5to enhance viral tropism and entry is one approach that can overcomethe problem of CAR deficiency in the cancer cells while potentially enhancing thetherapeutic efficacy of suicide gene therapy.The HI-loop is a region on the outer surface of the knob of Ad5. However, thecomplexity of inserting foreign gene corresponding to the motif of interest into theHI-loop hampers the development of targeted vectors. In the present study, wedeveloped a simplified system, using shuttle plasmids and through in vitro ligation, torapidly and efficiently insert a gene of interest in the HI-loop. At the same time, theinclusion of the firefly luciferase reporter gene in the construct allows easymonitoring of gene transfer efficiency. Using this approach, we constructed afiber-mutant Ad5vector containing an Arg-Gly-Asp (RGD) peptide in the HI-loop ofthe fiber knob domain. The incorporation of the RGD peptide has been shown toincrease gene transfer to various types of tumors lacking sufficient CAR expression.The most commonly-used therapeutic suicide gene strategy is based on theherpes simplex virus thymidine kinase gene (HSV-TK) followed by ganciclovir(GCV) administration. The Escherichia coli nitroreductase (Coli.NTR)/CB1954system is another suicide gene therapy approach. However, it also has a strongbystander effect as a result of the generation of a potent DNA cross-linking agent thatinduces apoptosis in both dividing and non-dividing cells. To overcome theshortcomings of using a single suicide gene, we used the combination of two suicidegene systems in one vector in this study to enhance targeted tumor cell killing effects.We have also used the tumor-specific human telomerase reverse transcriptase (hTERT)promoter to improve the specificity of suicide gene expression in cancer cells.Ultimately, this strategy may reduce the amount of prodrugs required for therapy, andimprove anti-tumor efficacy.Compared with the unmodified Ad-Luc, the infectivity of the fiber-modifiedAd-RGD-Luc on all cell models was significantly increased in vitro, and the mostdramatic increase was observed in ZR-75-30cells, which are deficient for CAR but express the high level of αvβ3integrin. After we replace the luciferase gene withdouble suicide genes under the control of hTERT promoter, the greater enhancementof cytotoxicity by Ad-RGD-hT-TK/NTR/GCV+CB1954versus Ad-hT-TK/NTR/GCV+CB1954against ZR-75-30and KB cells as compared to MCF7and Tca-8113cells was likely due to the fact that ZR-75-30and KB cells express higher levels ofintegrin than the other two cell types. These results indicated that the RGD peptide inthe HI-loop and the use of the hTERT promoter to drive the expression of the suicidegenes could improve the sensitivity and specificity of cancer cells to GCV andCB1954.To confirm our in vitro data, we compared the in vivo efficacy of the Ad-RGD-hT-TK/NTR/GCV+CB1954system versus the Ad-hT-TK/NTR/GCV+CB1954system against the growth of ZR-75-30xenograft tumors. The average tumor volumeof the group that received conventional Ad5with the double suicide genes(Ad-hT-TK/NTR/GCV+CB1954) was21.8%of that of the control group(PBS/physiological saline) on Day30. The tumor volume of the fiber-modified Ad5vector group (Ad-RGD-hT-TK/NTR/GCV+CB1954) was10.3%of the control group and47.2%of the conventional Ad5group on Day30(P <0.05). The survival times of the fiber-modified Ad5vectorgroup was197.2%of the control and134.6%of the conventional Ad5groups. Thesedata suggest that the Ad-RGD-hT-TK/NTR/GCV+CB1954treatment significantlyenhanced anti-tumor effects in vivo, reducing the tumor volume to nearly half the sizeof that of the Ad-hT-TK/NTR/GCV+CB1954group. While both the tumor volumeand survival time of the Ad-Blank/GCV+CB1954group were slightly lower than thatof the PBS group, there were no statistically significant differences (P>0.05),indicating that the prodrugs (GCV and CB1954) did not affect tumor growth in theabsence of suicide genes. Histological staining of the sections of the hepatic cordcollected at Day15after gene transfer showed that the cells in all groups werearranged neatly in a radiating pattern around the vein with no apparent hepatotoxicityor hepatic necrosis.In conclusion, this new vector system provides a promising gene therapyapproach for treating CAR-deficient cancers. 2. Combination Efficacy of Methylselenol Prodrug and MDV3100for Treatmentof Castration-Resistant Prostate CancerRelapse with CRPC after androgen deprivation therapy constitutes a major causeof prostate cancer mortality. There is now compelling evidence supporting resurgentAR signaling as a key driver of castration-resistant progression. This has led to thedevelopment of next-generation AR-targeting therapies that have recently receivedFDA approval for the treatment of metastatic CRPC, such as the potent AR antagonistMDV3100.MDV3100has5-8fold higher affinity to AR as compared to bicalutamide, theanti-androgen currently used in clinics. MDV3100prolonged overall survival ofmetastatic CRPC patients who progressed following chemotherapy in a Phase III trial.However, many of the patients presented with therapy-resistant disease, and the vastmajority who initially responded acquired resistance over time. The median survivalwas improved by only4.8months over placebo. Thus, developing more effectivemodality to improve the therapeutic outcome of MDV3100is urgently needed.A large number of AR-Vs that are devoid of the functional AR ligand-bindingdomain (LBD) as a result of alternative splicing have been recently identified. Themajority of the AR-Vs identified to date display constitutive activity. AR-Vs aredetected in most CRPC specimens, and the levels are significantly upregulatedcompared to hormone-na ve cancers. In fact, the AR-V proteins are expressed at alevel comparable to that of AR-FL, or even represent the predominant form of AR, ina significant portion of metastatic CRPC tissues.Based on its AR-LBD-targeting nature, intuitively, MDV3100may not beeffective against AR-Vs. Although a study showed that ectopic expression of AR-V7in LNCaP xenograft tumors does not alter the ability of MDV3100to inhibit thegrowth of the tumors, this effect might be model specific. It was reported recently that,in CRPC cells expressing endogenous AR-FL and AR-Vs, AR-Vs drive resistance toMDV3100by functioning as independent drivers of the AR transcriptional program.Thus, combining MDV3100with an AR-V-targeting agent may represent a viableapproach to overcome resistance to MDV3100. Methylselenol is considered the active metabolite for the anticancer effect ofmethyl-selenium compounds. Previously, we showed that two methylselenol prodrugs,methylseleninic acid (MSA) and methylselenocysteine (MSC), are effective indownregulating AR-FL mRNA and protein. A marked downregulation of AR-FLmRNA could be detected as early as3hr post-treatment, while AR mRNA stabilitywas not decreased within this duration, indicating that repressed transcription of theAR gene may contribute to the downregulation. Since AR-Vs are generated byalternative splicing and utilize the same promoter as AR-FL, repressed transcriptionof the AR gene is also expected to lead to a reduction in AR-Vs. Since themetabolism of MSC to methylselenol requires the activity of β-lyase, which isexpressed in the liver and kidney, but not the prostate, MSC was used only in theanimal experiments. Accordingly, in the present study, we tested the hypothesis thatmethylselenol prodrug downregulates AR-Vs and improves the therapeutic efficacyof MDV3100.The present study demonstrates a clear example of a rationally designed andpotentially effective combination modality for improving MDV3100efficacy againstCRPC. We reported previously that MSA inhibits the expression and activity ofAR-FL. Here, we showed that the inhibitory effect, which is independent of androgen,extends to AR-Vs, and could be attributable to MSA suppression of the transcriptionof the AR gene. Combined treatment of cultured CRPC cells with MSA andMDV3100more significantly suppressed AR signaling and synergistically inhibitedcell growth. The combinatorial efficacy was observed in not only AR-V-expressingcells but also cells expressing predominantly AR-FL, likely owing to the ability of thetwo drugs to block the AR signaling cascade at distinct steps. Ectopic expression ofAR-FL or AR-V7attenuated the combinatorial efficacy, indicating thatdownregulating AR-FL and AR-V7is importantly involved in mediating thecombinatorial efficacy.Surprisingly, although MSA was able to downregulate AR-FL and AR-Vs in vivoand inhibited the growth of22Rv1tumors, it failed to enhance the antitumor effectwhen combined with MDV3100. In contrast, MSC, another methylselenol prodrug,significantly improved the antitumor efficacy of MDV3100. The lack of in vivo combinatorial efficacy between MSA and MDV3100may be a result of conjugationbetween MSA with MDV3100, and the conjugation may be an issue particular to thetreatment protocol used in our in vivo experiment because the drugs were mixedtogether at very high concentrations before administered to mice. To solve thisproblem, we will explore the possibility of sequential treatment with MSA andMDV3100rather than using a cocktail. Nonetheless, the findings from this studysupport the potential of using methylselenol prodrug to improve MDV3100efficacyIn conclusion，our present study demonstrates that methylselenol prodrug, whicheffectively downregulates AR-Vs, can significantly potentiate MDV3100efficacy,and more importantly, overexpression of AR-V can attenuate the potentiating effect.Our finding therefore further substantiates the contribution of AR-Vs to MDV3100resistance, and provides a foundation for the development of a more effectivecombinatorial modality for CRPC.