Evaluation of miR-122-regulated suicide gene therapy for hepatocellular carcinoma in an orthotopic mouse model

Objective： Intratumoral administration of adenoviral vector encoding herpes simplex virus （HSV） thymidine kinase （TK） gene （Ad-TK） followed by systemic ganciclovir （GCV） is an effective approach in treating experimental hepatocellular carcinoma （HCC）. However, hepatotoxicity due to unwanted vector spread and suicide gene expression limited the application of this therapy, miR-122 is an abundant, liver-specific microRNA whose expression is decreased in human primary HCC and HCC-derived cell lines. These different expression profiles provide an opportunity to induce tumor-specific gene expression by miR-122 regulation. Methods： By inserting miR-122 target sequences （miR-122T） in the 3＇ untranslated region （UTR） ofTK gene, we constructed adenovirus （Ad） vectors expressing miR-122-regulated TK （Ad-TK-122T） and report genes. After intratumoral administration of Ad vectors into an orthotopic miR-122-deficient HCC mouse model, we observed the miR-122-regulated transgene expression and assessed the antitumor activity and safety of Ad-TK-122T. Results： Insertion of miR-122T specifically down-regulated transgene expression in vitro and selectively protected the miR-122-positive cells from killing by TK/GCV treatment. Insertion of miR-122T led to significant reduction of tansgene expression in the liver without inhibition of its expression in tumors in vivo, resulting in an 11-fold improvement of tumor-specific transgene expression. Intratumoral injection of Ad vectors mediated TK/GCV system led to a vector dosage-dependent regression of tumor. The insertion of miR-122T does not influence the antitumor effects of suicide gene therapy. Whereas mice administrated with Ad-TK showed severe lethal hepatotoxicity at the effective therapeutic dose, no liver damage was found in Ad-TK-122T group. Conclusions： miR-122-regulated TK expression achieved effective anti-tumor effects and increased the safety of intratumoral delivery of adenovirus-mediated TK/GCV gene therapy for miR-122-deficient HCC.

ENHANCED ANTITUMOR EFFECTS OF SUICIDE GENETHERAPY BY SIMULTANEOUS TRANSFER OF GM CSF GENE IN LEUKEMIABEARING MICE

In the present report, antitumor effect of combined transfer of suicide gene and cytokine gene was studied. Adenovirus engineered to express E. Coli. Cytosine deaminase (AdCD) and/or adenovirus engineered to express murine granulocytemacrophage colonystimulating factor (AdGMCSF) were used for the treatment of leukemiabearing mice. The mice were inoculated s.c. with FBL3 erythroleukemia cells and 3 days later received intratumoral injection of AdCD in the presence or absence of AdGMCSF followed by intraperitoneal 5fluorocytosine (5FC) treatment. The results demonstrated that mice received combined therapy of AdCD/5FC and AdGMCSF developed tumors most slowly and survived much longer when compared with mice treated with AdCD/5FC alone, AdGMCSF alone, AdlacZ/5FC or PBS. Combined transfer of CD gene and GMCSF gene achieved higher specific CTL activity than control therapies. Pathological examination illustrated that the tumor mass showed obvious necrosis and inflammatory cell infiltration in mice after combined therapy. The results demonstrated that combined transfer of suicide gene and cytokine gene could synergistically inhibit the growth of leukemia in mice and induce antitumor immunity of the host. The combination therapy might be a potential approach for cancer gene therapy.

Bifunctional chimeric SuperCD suicide gene -YCD: YUPRT fusion is highly effective in a rat hepatoma model

AIM： To investigate the effects of catalytically superior gene-directed enzyme prodrug therapy systems on a rat hepatoma model. METHODS： To increase hepatoma cell chemosensitivity for the prodrug 5-fluorocytosine （5-FC）, we generated a chimeric bifunctional SuperCD suicide gene, a fusion of the yeast cytosine deaminase （YCD） and the yeast uracil phosphoribosyltransferase （YUPRT） gene. RESULTS： In vitro stably transduced Morris rat hepatoma cells （MH） expressing the bifunctional SuperCD suicide gene （MH SuperCD） showed a clearly marked enhancement in cell killing when incubated with 5-FC as compared with MH cells stably expressing YCD solely （MH YCD） or the cytosine deaminase gene of bacterial origin （MH BCD）, respectively. In vivo, MH SuperCD tumors implanted both subcutaneously as well as orthotopically into the livers of syngeneic ACI rats demonstrated significant tumor regressions （P〈0.01） under both high dose as well as low dose systemic 5-FC application, whereas MH tumors without transgene expression （MH naive） showed rapid progression. For the first time, an order of in vivo suicide gene effectiveness （SuperCD〉〉 YCD〉〉BCD〉〉〉negative control） was defined as a result of a direct in vivo comparison of all three suicide genes. CONCLUSION： Bifunctional SuperCD suicide gene expression is highly effective in a rat hepatoma model, thereby significantly improving both the therapeutic index and the efficacy of hepatocellular carcinoma killing by fluorocytosine.

Antitumor activity of mutant bacterial cytosine deaminase gene for colon cancer

AIM： To evaluate bacterial cytosine deaminase （bCD） mutant D314A and 5-fluorocytosine （5-FC） for treatment of colon cancer in a mouse model. METHODS： Recombinant lentivirus vectors that contained wild-type bCD gene （bCDwt）, and bCD mutant D314A gene （bCD-D314A） with green fluorescence protein gene were constructed and used to infect hu- man colon carcinoma LoVo cells, to generate stable transfected cells, LoVo/null, LoVo/bCDwt or LoVo/bCD- D314A. These were injected subcutaneously into Balb/c nude mice to establish xenograft models. Two weeks post-LoVo cell inoculation, PBS or 5-FC （500 mg/kg） was administered by intraperitoneal （i.p.） injection once daily for 14 d. On the day after LoVo cell injection, mice were monitored daily for tumor volume and survival. RESULTS： Sequence analyses confirmed the construction of recombinant lentiviral plasmids that contained bCDwt or bCD-D314A. The lentiviral vector had high elficacy for gene delivery, and RT-PCR showed that bCDwt or bCD-D314A gene was transferred to LoVo cells. Among these treatment groups, gene delivery or 5-FC administration alone had no effect on tumor growth. However, bCDwt/5-FC or bCD-D314A/5-FC treatment inhibited tumor growth and prolonged survival of mice significantly （P 〈 0.05）. Importantly, the tumor volume in the bCD-D314A/5-FC-treated group was lower than that in the bCDwt/5-FC group （P 〈 0.05）, and bCD- D314A plus 5-FC significantly prolonged survival of mice in comparison with bCDwt plus 5-FC （P 〈 0.05）. CONCLUSION： The bCD mutant D314A enhanced significantly antitumor activity in human colon cancer xenograft models, which provides a promising approach for human colon carcinoma therapy.

Telomerase activity: An attractive target for cancer therapeutics

Telomeres are non-coding tandem repeats of 1000-2000 TTAGGG nucleotide DNA sequences on the 3’ termini of human chromosomes where they serve as protective “caps” from degradation and loss of genes. The “cap” at the end of chromosome required to protect its integ-rity is a 150-200 nucleotide-long single stranded G-rich 3’ overhang that forms two higher order structures, a T-loop with Sheltering complex, or a G-quadruplex com-plex. Telomerase is a human ribonucleoprotein reverse transcriptase that continually added single stranded TTAGGG DNA sequences onto the single strand 3’ of telomere in the 5’ to 3’ direction. Telomerase activity is detected in male germ line cells, proliferative cells of renewal tissues, some adult pluripotent stem cells, embryonic cells, but in most somatic cells is not de-tected. Re-expression or up-regulation of telomerase in tumours cells is considered as a critical step in cell tumorigenesis and telomerase is widely considered as a tumour marker and a target for anticancer drugs. Dif-ferent approaches have been used in anticancer thera-peutics targeting telomerase. Telomerase inhibitors can block directly Human TElomerase Reverse Transcrip-tase （hTERT） or Human TElomerase RNA telomerase subunits activity, or G-quadruplex and Sheltering complex components, shortening telomeres and inhibiting cell proliferation. Telomerase can become an immune target and GV1001, Vx-001, I540 are the most wide-spread vaccines used with encouraging results. Another method is to use hTERT promoter to drive suicide gene expression or to control a lytic virus replication. Recently telomerase activity was used to activate pro-drugs such as Acycloguanosyl 5’-thymidyltriphosphate, a synthetic ACV-derived molecule when it is activated by telomer-ase it does not require any virus or host active immune response to induce suicide gene therapy. Advantage of all these therapies is that target only neoplastic cells without any effects in normal cells, avoiding toxicity and adverse effects of the current chemotherapy. However, as not all the approaches are equally effcient, further studies will be necessary.

Non-Viral Deoxyribonucleoside Kinases——Diversity and Practical Use

Deoxyribonucleoside kinases （dNKs） phosphorylate deoxyribonucleosides to their corresponding monophosphate compounds, dNks also phosphorylate deoxyribonucleoside analogues that are used in the treatment of cancer or viral infections. The study of the mammalian dNKs has therefore always been of great medical interest. However, during the last 20 years, research on dNKs has gone into non- mammalian organisms. In this review, we focus on non-viral dNKs, in particular their diversity and their practical applications. The diversity of this enzyme family in different organisms has proven to be valuable in studying the evolution of enzymes. Some of these newly discovered enzymes have been useful in numerous practical applications in medicine and biotechnology, and have contributed to our understanding of the structural basis of nucleoside and nucleoside analogue activation.