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Many outstanding problems have been solved in biology and medicine for which scientists have been awarded prestigious prizes including the Nobel Prize, Lasker Award and Breakthrough Prizes in life sciences. These have been the fruits of years of basic research. From time to time, publications have appeared listing “unsolved” problems in biology. In this article, I ask the question whether it is possible to have such a list, if not a unique one, at least one that is analogous to the Millennium Prize in mathematics. My approach to finding an answer to this question was to gather views of leading biologists. I have also included my own views. Analysis of all the responses received over several years has convinced me that it is difficult, but not impossible, to have such a prize. Biology is complex and very interdisciplinary these days at times involving large numbers of teams, unlike mathematics, where Andrew Wiles spent seven years in complete isolation and secrecy solving Fermat's last theorem. Such an approach is simply not possible in biology. Still I would like to suggest that a similar prize can be established by a panel of distinguished scientists. It would be awarded to those who solved one of the listed problems in biology that warrant a verifiable solution. Despite many different opinions, I found that there is some commonality in the responses I received – I go on to discuss what these are and how they may impact future thinking.

We report the use of plasmid DNA-mediated combination gene therapy for tumor-bearing mice using in vivo electroporation, also called electro-gene therapy (EGT), that resulted in uncomplicated and complete cures in more than 90% of the mice. Subcutaneously inoculated CT26 tumors in syngeneic BALB/c mice were subjected to repeated EGT treatments consisting of intratumoral co-injection of naked plasmids encoding the cytokine interleukin-12 (IL-12) (p35 and p40 subunits) and the suicide gene herpes simplex virus thymidine kinase (HSV-tk), followed by in vivo electroporation. The early anti-tumor effect was always stronger, and the rate of cure, as seen in the long-term follow-up, was always greater in the groups treated with combination EGT than in those treated with IL-12 or HSV-tk EGT alone. Systemic levels of IL-12 and IFN-gamma increased in both combination and IL-12-alone EGT-treated groups. Moreover, combination EGT for established subcutaneous tumors strongly reduced hematogenous lung metastases and increased survival time when live CT26 tumor cells were injected through the tail vein. Limited experiments on C57/B16 mice with murine melanoma also showed very similar trends. These results suggest that this simple and safe method of plasmid-mediated combination EGT may provide a potentially effective gene therapy for cancer.

We report on an antitumor treatment involving electrogene therapy (EGT), a newly developed in vivo gene transfer method using electroporation. We carried out in vivo EGT in a subcutaneous model of CT26 colon carcinoma cells, using plasmid DNAs encoding interleukin 12 (IL-12) subunits. For this purpose, we developed two IL-12 expression systems: a cotransfer system using a plasmid encoding the IL-12 p40 subunit and a plasmid encoding the IL-12 p35 subunit, and a single-vector system using a plasmid expressing a p40-p35 fusion protein. Both transfer systems significantly inhibited the growth of CT26 tumor. Immunohistochemical analysis of IL-12 EGT-treated tumors revealed enhanced infiltration of CD8(+) cells into the tumor tissue, while reverse transcriptase-polymerase chain reaction confirmed the increased expression of interferon gamma within treated tumors. The same IL-12 EGT applied to the nude mouse model was not effective, suggesting the critical role of T cell infiltration in this treatment. The inhibitory effects revealed in experiments in which previously treated mice were rechallenged with a second inoculation of CT26 tumor cells suggested that IL-12 EGT may also establish partial systemic antitumor immunity. The growth of IL-12 EGT-treated Renca tumors, a renal cell carcinoma, was also significantly inhibited. These findings suggest that EGT of the IL-12 gene has the potential to be an effective anticancer gene therapy.

The present study examined the use of electroporation (pulsed electric field application) to temporarily permeabilize vascular cell membranes and thereby deliver drugs directly into cells of the vessel wall to treat focal cardiovascular diseases (e.g., postangioplasty restenosis). A unique intravascular porous balloon electroporation catheter has been designed specifically to enhance drug uptake directly into the arterial wall. The effect of heparin on neointimal growth following balloon-induced injury of the carotid arteries of rats was used as a measure of effective drug delivery. Histomorphometrical analysis of arteries showed that neointimal growth and lumen loss were significantly inhibited by heparin in combination with electroporation, but not by heparin delivered without electroporation. We conclude that the application of electroporation technology may be a viable approach to intramural drug delivery of potential therapeutic compounds for the treatment of neointimal growth and negative remodeling during postangioplasty restenosis.

Current methods of local drug delivery frequently fail to achieve a prolonged therapeutically effective tissue drug level without producing vascular trauma. A novel double-balloon catheter system, incorporating electroporation technology, has been designed and tested to deliver heparin into rabbit carotid arteries in an overstretch balloon injury model in vivo. Following arterial injury, fluoresceinated heparin was delivered into the volume between the two inflated balloons, and the artery was subjected to an electrical pulse. Catheter deployment and endovascular electrical pulsing were well-tolerated in all animals (N = 21) without adverse hemodynamic and histological changes. Periodic arterial blood samples revealed no abnormalities in the clotting profile or any gross morphological changes in the blood cells up to 8 hr after treatment. Much stronger heparin fluorescence was detected throughout the vessel layers for at least 12 hr in the pulsed samples compared to the control. Histochemical staining of the tissue showed intracellular localization of heparin. Endovascular electroporation may provide better retention and higher therapeutic efficacy than can be achieved by conventional systemic delivery of heparin at clinically safe concentrations.

This article is a compilation of the author's own observations on how biology is changing and is likely to change in the next decade or two, with views from leading biologists on current and future developments, particularly in terms of solving some of the most fundamental problems. Starting from the message of 'universality' which, in the author's view, is coded into the DNA structure, the possible consequences of the completion of the human genome project, namely the sequencing of all of the 50 000–100 000 human genes, the impact of new technology, especially in the field of medicine, and the relentless march of molecular biology in deciphering the secret of life are described. The role of bacteria in new drug development, routine use of gene therapy to tackle single gene disease, problems of evolution, behaviour, and development, and prediction of three-dimensional protein structure from amino acid sequences are discussed. It is argued that biology and technology have to go hand in hand to main...

Knowledge of the mechanisms of interaction between self-aggregating peptides and nucleic acids or other polyanions is key to the understanding of many aggregation processes underlying several human diseases (e.g. Alzheimer's and Parkinson's diseases). Determining the affinity and kinetic steps of such interactions is challenging due to the competition between hydrophobic self-aggregating forces and electrostatic binding forces. Kahalalide F (KF) is an anticancer hydrophobic peptide which contains a single positive charge that confers strong aggregative properties with polyanions. This makes KF an ideal model to elucidate the mechanisms by which self-aggregation competes with binding to a strongly charged polyelectrolyte such as DNA. We use optical tweezers to apply mechanical forces to single DNA molecules and show that KF and DNA interact in a two-step kinetic process promoted by the electrostatic binding of DNA to the aggregate surface followed by the stabilization of the complex due to hydrophobic interactions. From the measured pulling curves we determine the spectrum of binding affinities, kinetic barriers and lengths of DNA segments sequestered within the KF-DNA complex. We find there is a capture distance beyond which the complex collapses into compact aggregates stabilized by strong hydrophobic forces, and discuss how the bending rigidity of the nucleic acid affects such process. We hypothesize that within an in vivo context, the enhanced electrostatic interaction of KF due to its aggregation might mediate the binding to other polyanions. The proposed methodology should be useful to quantitatively characterize other compounds or proteins in which the formation of aggregates is relevant., Comment: 34 pages, 8 figures, Supplementary Information

In this article, I critically analyze the impact of the new biology and the biological revolution. I argue that indiscriminate use of the words such as 'interdisciplinary,' 'integrative,' and 'revolution' has caused only confusion when applied to biology. The recent debate, especially after the exploding field of systems biology, has brought back the controversy whether molecular biology is reductionist or holistic. I look at the issues involved critically. I discuss the problem of defining the word 'gene' and argue that recent attempts to redefine the central dogma of molecular biology about the information flow from DNA to RNA to protein are not justified. I support my view with comments from the scientist who discovered RNA splicing. Several aspects of evo-devo, a new branch of biology, are discussed. I give examples from this evolution-developmental biology to show how some of Darwin's inspired guesses have had resounding victory when it was found that specific genes during embryonic development of the Galapagos finches decided the size and shape of their beaks. I discuss the recent publications which show that the conditions in the island, such as wet to dry to wet season, can bring about evolutionary changes from year to year. Thus it is essential to monitor both short and long-term evolutionary changes to get the full picture of evolution.

The advances in biological sciences have been phenomenal since the structure of DNA was decoded, especially if one considers the input from physical sciences, not only in terms of analytical tools, but also understanding and solving some of the key problems in biology. In this article, I trace briefly the history of this transition, from physical sciences to biology, and argue that progress in modern biology can be accelerated if there is far more meaningful crosstalk between the biologists and the physical scientists, simply because biology has become far more complex and interdisciplinary, and the need for such crosstalk cannot be overemphasized. Without a concerted effort in this area progress will be hindered, and the two camps will continue to work on their own, using their own specialized language, thus making communication highly ineffective. I support my argument giving a vast array of examples and also quoting leading authorities.

Cancer of the pancreas is currently the fifth leading cause of cancer related deaths with a five year survival of less than 1% In the United States (1). It is one of the most difficult cancers to treat, since it is hard to detect in the early stages. The patients remain asymptomatic until late in the course of the disease. An excellent review of pancreatic carcinoma has appeared (2). Despite the progress made in our understanding of the biology of this cancer (3), the final outcome for this disease has remained extremely poor. Conventional chemotherapeutic agents have not been very effective for human pancreatic adenocarcinoma (4). Use of intratumoral chemotherapy in combination with monoclonal antibodies have been reported to produce better response rate and also reduced toxicity (5,6). Smith and colleagues (7) have recently shown that an injectable gel with a sustained release profile can inhibit tumor growth in vivo in human pancreatic cancer xenografts. This was demonstrated in nude mice with BxPC-3 xenografts using fluorouracil, cisplatin, and doxorubicin with a consequent size reduction of the tumors between 72% and 79%, compared to the controls at day 28 after the first treatment. Although these figures are impressive, by any standard, no cure was reported.

Hemophilia B is an X-linked genetic disorder that typically results from chronic circulating deficiency of blood coagulation factor IX (FIX) (1). While the occurrence of hemophilia B is significantly less frequent than hemophilia A (factor VIII, deficiency) it has received special attention as a model for gene therapy. This is because hemophilia B is one of the least complicated genetic diseases from the point of view of demonstrating the proof of principle of a gene therapy protocol. Specifically, hemophilia B is a single gene recessive disorder and a wide range of tissues can be targeted for FIX gene delivery and strict regulation of FIX expression is not required. In addition, the 2.8 kb FIX cDNA is much smaller than the 9 kb FVIII cDNA, and FIX expression in transfected mammalian cells has been less problematic than FVIII expression (2). Since clinical severity of bleeding episodes closely corresponds to a patient's FIX activity, achieving even partial restoration of normal FIX levels in the bloodstream can alleviate internal bleeding. Individuals with FIX levels less than 1% of normal experience severe symptomatic episodes but providing roughly 5% of normal levels (i.e., 250 ng/mL plasma) can significantly reduce the frequency and severity of bleeding episodes and reduce long term complications (3). Treatment of hemophilia B primarily relies on intravenous injections of FIX protein purified from pooled human plasma, or very recently, on newly developed recombinant FIX. Treatment is applied typically only when bleeding episodes have occurred or are expected, for example, in case of a trauma or surgery. Although the risk of viral transmission of HIV and hepatitis viruses has been largely eliminated the absolute safety of any product derived from blood cannot be guaranteed. Furthermore, supplies of factor concentrates are limited and costs (especially if prophylactic treatment is being considered) are high. Thus, the application of gene therapy to hemophilia, whereby long-term correction of factor IX deficiency might be achieved, would be extremely useful.

Electroporation, a standard laboratory method of introducing exogenous molecules into cells, has been gaining importance as a very effective non-viral physical technique of gene delivery. In this study, we have used subcutaneous model of the C6 rat glioma cells and established an optimal condition to obtain very high gene expression in tumor tissues using both reporter and functional genes. Tumors grown on the flanks of Wistar rats are exposed and directly injected with plasmid DNA having the constructs of luciferase, green fluorescent protein and, the fragment of the diphtheria toxin, DT-A. The tumors are then subjected to square wave pulses from an electroporator. Gene expression is found to be several orders of magnitude higher when the tumors are pulsed with the optimized electrical parameters compared to the controls. For luciferase, the enhancement is approximately 135-fold, for the green fluorescent protein, gene expression is seen over a wide area within the sections examined, as contrast to a few punctate dots in the control specimens, and finally, DT-A shows massive death in the tumor tissue. A special circular array of six needles through which pulses are delivered with rotating electric field is found to be highly efficient in transferring genes inside the tumor. Direct injection of plasmid DNA followed by electroporation allows very high in vivo gene transfer and its subsequent expression into tumor tissues. This method may be applicable to any solid tumor.

We report successful electro-gene therapy (EGT) by using plasmid DNA for tumor-bearing mice. Subcutaneously inoculated CT26 tumor was subjected to EGT, which consists of intratumoral injection of a naked plasmid encoding a marker gene or a therapeutic gene, followed by in vivo electroporation (EP). When this treatment modality is carried out with the plasmid DNA for the green fluorescent protein gene, followed by in vivo EP with the optimized pulse parameters, numerous intensely bright green fluorescent signals appeared within the tumor. EGT, by using the “A” fragment of the diphtheria toxin gene significantly inhibited the growth of tumors, by about 30%, on the flank of mice. With the herpes simplex virus thymidine kinase gene, followed by systemic injection of ganciclovir, EGT was far more effective in retarding tumor growth, varying between 50% and 90%, compared with the other controls. Based on these results, it appears that EGT can be used successfully for treating murine solid tumors.