Anti-genes: siRNA, ribozymes and antisense.

Scientists have been working on strategies to selectively turn off specific genes in diseased tissues for the past thirty years. In the 1980's, oligodeoxynucleotides (ODNs) with unique chemistries were tested with model systems both in vitro and in vivo with varying degrees of success. In the 1990's, ribozymes with both antisense and catalytic properties were successfully introduced to the field. Ribozymes were shown to selectively knock down targeted genes in human tumors grown in mice but delivery issues for these therapeutic anti-genes limited their clinical utility. Short interfering RNA (siRNA) is currently the fastest growing sector of this anti-gene field for target validation and therapeutic applications. The siRNA field may have an opportunity to impact the clinic faster than antisense and ribozymes if the scientists can overcome the previous anti-gene limitations. Fortuitously, there have been a several developments involving the expansion of our genomic knowledge coupled with the rapid dissemination of disease genes by the digital revolution. This convergence of the knowledge of the human genome with the speed of digital communication will help facilitate swift changes in the detection and treatment of human illnesses. The anti-gene field is positioned to exploit this timely union of two distinct technologies. Anti-gene molecules have an opportunity to become a successful technology in understanding the human genome, as well as, enabling the development of efficacious gene therapy for human diseases in the near future. This review will characterize the advances in this field and address the challenges to the success of for the anti-gene technology.

Ribozyme against mutant K-ras mRNA suppresses tumor growth of pancreatic cancer.

Point mutations in the K-ras gene are observed at a high incidence in human pancreatic carcinomas. These alterations can be used as potential targets for specific ribozyme-mediated reversal of the malignant phenotype. We designed an anti-K-ras ribozyme against codon 12 of the mutant K-ras gene transcripts (GGT right curved arrow GTT), and generated a recombinant adenovirus to express the ribozyme (rAd/anti-K-ras Rz). We inoculated Capan-1 human pancreatic carcinoma cells in athymic mice, and made Capan-1 tumor xenografts. When the Capan-1 tumors in athymic mice became approximately 100 mm(3), rAd/anti-K-ras Rz was directly injected into the tumor xenografts. Fifteen (68%) of 22 tumors injected with rAd/anti-K-ras Rz showed tumor growth suppression or tumor regression; 6 of 15 tumors were completely regressive, and 1 tumor was recurrent after the tumor regression. By using the recombinant adenovirus in a mice model system, it was possible to accomplish efficient reversion of the malignant phenotype in human pancreatic tumors with K-ras gene mutation.

Reversal of drug resistance using hammerhead ribozymes against multidrug resistance-associated protein and multidrug resistance 1 gene.

We examined the effects of suppressing multidrug resistance-associated protein (MRP) and multidrug resistance 1 (MDR1) gene expression in HCT-8DDP human colon cancer cell lines, which showed both cisplatin and multidrug resistance. Hammerhead ribozymes, designed to cleave MRP mRNA (anti-MRP Rz) and MDR1 mRNA (anti-MDR1 Rz), were transfected into the HCT-8DDP cells. Drug sensitivity was estimated by MTT assay in vitro. The HCT-8DDP/anti-MRP Rz cells were more sensitive to doxorubicin (DOX) and etoposide (VP-16) by 2.5- and 4.1-fold, respectively, compared with HCT-8DDP cells. The HCT-8DDP/anti-MDR Rz cells were more sensitive to DOX and VP-16 by 2.3- and 3.8-fold, respectively. The anti-MRP Rz and anti-MDR1 Rz significantly down-regulated resistance to DOX and VP-16, while anti-MRP Rz and anti-MDR1 Rz did not affect resistance to cisplatin, methotrexate and 5-fluorouracil. The hammerhead ribozyme-mediated specific suppression of MRP or MDR1 was sufficient to reverse multidrug resistance in the human colon cancer cell line.

Phase I study of cisdiamminedichloroplatinum in combination with azidothymidine in the treatment of patients with advanced malignancies.

PURPOSE: Azidothymidine (AZT, zidovudine) has been shown to reverse cisplatin resistance in cell culture. This phase I study was performed to determine the maximally tolerated dose (MTD) and dose-limiting toxicities of AZT when administered by continuous intravenous infusion in combination with cisplatin (CDDP), and to evaluate the pharmacokinetics of AZT in this setting. PATIENTS AND METHODS: Entered in the study were 61 patients with advanced, histologically confirmed malignancies which were unresponsive to or for which no "standard" chemotherapeutic regimen existed. AZT was administered as a 72-h infusion on days 1-3 and 14-16 of a 28-day cycle at dose levels from 400 through 14,364 mg/m(2) per day. CDDP at dose levels of 30, 45, or 60 mg/m(2) was administered at hour 36 of each AZT infusion. The plasma pharmacokinetics of AZT were determined in patients treated at representative dose levels. RESULTS: Of the 61 patients who completed 125 courses of therapy, 21 had stable disease for a median of four cycles (range two to eight), 33 progressed on therapy, and 7 were not assessable for response. The major observed toxicity was myelosuppression. The MTD of AZT was 8135 mg/m(2) per day when administered on this schedule. Escalation of CDDP did not result in additive toxicity. The mean steady-state level of AZT at the MTD was 44 microM (range 35-51 microM). CONCLUSIONS: Steady-state concentrations of AZT increased with dose. The plasma levels achieved at the MTD exceeded those required for drug resistance reversal in vitro. The administration of CDDP had no effect on AZT steady-state levels. The dose-limiting toxicity of this drug combination is myelosuppression. AZT may be useful in further studies utilizing combination therapy to achieve increased chemotherapy effectiveness.

Cancer gene therapy: challenges and opportunities.

Understanding the molecular basis of human disease has been the corner-stone of rationally designed molecular therapies. Medicine has a long history of treating patients with cell therapies (i.e., blood transfusions) and protein therapies (i.e., growth factors and cytokines). Gene therapies are the newest therapeutic strategy for treating human diseases. Where will gene therapy be in five years after the euphoria and frustrations of the last 14 years? This is a complex question, but the primary challenge for gene therapy will be to successfully deliver an efficacious dose of a therapeutic gene to the defective tissue. Will the delivery systems return to the early clinical trials of ex vivo gene therapy or will there still be a high demand for systemic therapy? Will systemic therapy continue to depend on viral vectors, or will non-viral and nano-particles become the new mode for gene delivery? The future success of gene therapy will be built on achievements in other fields, such as medical devices, cell therapy, protein therapy and nano-particle technology. This review describes the advances being made in the gene therapy field, as well as addressing the challenges of the near future for cancer gene therapy.

Construction and transfection of a ribozyme targeting human caspase-3.

Caspase-3 is a key executioner cysteine protease involved in programmed cell death or apoptosis. A ribozyme to human caspase-3 was designed, tested by in vitro cleavage, and transfected into a drug-resistant variant (DLKP-A5F) of a human lung carcinoma cell line (DLKP). By both stable and transient transfection, this ribozyme was shown to be effective at down-regulating human caspase-3 mRNA and protein levels.

Commercializing medical technology.

As medicine moves into the 21st century, life saving therapies will move from inception into medical products faster if there is a better synergy between science and business. Medicine appears to have 50-year innovative cycles of education and scientific discoveries. In the 1880's, the chemical industry in Germany was faced with the dilemma of modernization to exploit the new scientific discoveries. The solution was the spawning of novel technical colleges for training in these new chemical industries. The impact of those new employees and their groundbreaking compounds had a profound influence on medicine and medical education in Germany between 1880 and 1930. Germany dominated international science during this period and was a training center for scientists worldwide. This model of synergy between education and business was envied and admired in Europe, Asia and America. British science soon after evolved to dominate the field of science during the prewar and post World War (1930's-1970's) because the German scientists fled Hitler's government. These expatriated scientists had a profound influence on the teaching and training of British scientists, which lead to advances in medicine such as antibiotics. After the Second World War, the US government wisely funded the development of the medical infrastructure that we see today. British and German scientists in medicine moved to America because of this bountiful funding for their research. These expatriated scientists helped drive these medical advances into commercialized products by the 1980's. America has been the center of medical education and advances of biotechnology but will it continue? International scientists trained in America have started to return to Europe and Asia. These American-trained scientists and their governments are very aware of the commercial potential of biotechnology. Those governments are now more prepared to play an active role this new science. Germany, Ireland, Britain, Singapore, Taiwan and Israel are such examples of this government support for biotechnology in the 21st century. Will the US continue to maintain its domination of biotechnology in this century? Will the US education system adjust to the new dynamic of synergistic relationships between the education system, industry and government? This article will try to address these questions but also will help the reader understand who will emerge by 2015 as the leader in science and education.