by Hanna Kurlanda-Witek
Nobel Prize in Physiology or Medicine in 2007 together with Mario R. Capecchi and Martin J. Evans for “their discoveries of principles for introducing specific gene modifications in mice by the use of embryonic stem cells”.
“All my science has been invention, if you like, not just discovery,” Oliver Smithies explained during an interview with Nathaniel Comfort in 2005. “An inventor” was what Smithies wanted to become as a boy, having not known the profession of scientist. He was born in Halifax, England, in 1925. Smithies recalled having learned practical, manual skills from his father and writing skills from his mother, who taught English at a nearby technical college. The young Smithies was afflicted with a heart murmur, which prevented him from playing sports, but gave him plenty of time to read comic books and try to make devices, such as a radio-controlled boat and string communications system. He attended Heath Grammar School in Halifax, where he received a very good education and excelled in mathematics, physics and chemistry. Smithies obtained a scholarship to attend Balliol College at Oxford University, his acceptance telegram stating that his “mathematics (was) very promising for a person so young.” Upon overcoming his initial homesickness, Smithies greatly enjoyed his studies in physiology, anatomy and organic chemistry in preparation for medical school, yet after receiving a Bachelor of Arts degree in Animal Physiology in 1946, he decided to pursue research in what was then known as physical biochemistry (or molecular biochemistry today). This was in part influenced by Smithies’ mentor and tutor Professor Alexander (Sandy) Ogston, and after obtaining a second Bachelor’s degree, in chemistry, Smithies joined Ogston at his lab in the Department of Biochemistry, where he completed a PhD in two years.
Smithies later referred to his graduate work as “rather an undistinguished PhD”. He studied the osmotic pressures of mixtures of protein and spent most of two years devising a very precise osmometer. Despite his success, the ensuing paper describing the method was never cited by anyone. Smithies himself told a laughing audience during the 65th Lindau Meeting in 2015, “and nobody ever used the method! And I never used the method. (...) (but) I enjoyed doing it. And I learned to do good science.”
Ogston advised Smithies to continue his research career in the United States, but Smithies was reluctant to move there. He was eventually persuaded by an American friend, and joined Professor J.W. Williams’s group as a post-doc at the University of Wisconsin, studying protein purity. The methods of measuring protein purity that Smithies created were published but also never cited by anyone; yet, Smithies credited this period for teaching him meticulous physical chemistry. By this time, Smithies was engaged to Lois Kitze, an American virologist, and was thus unwilling to return to England after his visa had expired. Fortunately, he was offered a job at the Connaught Medical Research Laboratories at the University of Toronto by Dr. David A. Scott, who welcomed Smithies to work on anything he liked “as long as it has something to do with insulin,” which had been famously extracted for treatment of diabetes at the university 30 years earlier. Smithies set to work looking for a precursor to insulin and tried to purify mixtures of proteins using filter-paper electrophoresis. In this technique, a current passed through a small amount of protein mixture, which was dabbed onto a filter paper in solution, and separated into components, which could then be stained for identification. Insulin simply wouldn’t separate as it stuck to the paper, and Smithies tried to use different chemicals to stop this from happening. Starch grains in electrolyte were already being used as an electrophoretic medium, but the method was difficult and particularly time-consuming. In order to speed up the process, Smithies reminded himself of his childhood days when his mother did the laundry. The starch mixture used to stiffen shirt collars would set into a gel if left for long enough. Smithies thought that this starch gel could serve as medium and ran some experiments out of curiosity. The method worked very well, and a graduate student named Gordon Dixon used it to separate cabbage enzymes. Their paper, published in Nature in 1955, was the first to mention starch-gel electrophoresis, and the invention eventually won Smithies the Gairdner Foundation International Award. Polyacrylamide and agarose gels widely used today are improvements of Smithies’ method.
Once the starch-gel method revealed that there were more proteins present in plasma than the established five, Smithies abandoned his studies on insulin and continued to analyse protein in blood plasma. The differences in proteins from the blood of different persons led to work with Norma Ford Walker, a geneticist at the Hospital for Sick Children in Toronto. They jointly published a paper on the inherited differences of the protein haptoglobin, a binding protein in haemoglobin, also in Nature, in 1956, and this research forged Smithies’ lasting interest in molecular genetics.
Smithies returned to the University of Wisconsin in 1960 and joined the Department of Genetics. He taught molecular genetics and further studied the differences in the alleles, or gene variants, of haptoglobin, still collaborating with George Connell and Gordon Dixon from the University of Toronto. By 1961, Smithies and his co-workers deducted that one of the haptoglobin genes that was larger in structure, was the result of a recombination between the other two genes. This was the first detection of non-homologous recombination, a recombination between genes in different parts of the DNA sequence, which could be called a rare and random cross-over event. The non-homologous recombination in turn leads to predictable homologous recombination, or crossing over of similar or identical DNA strands. In terms of haptoglobin, Smithies and his team discovered that the larger gene resulting from non-homologous recombination would independently produce a fourth variant of the gene, as a consequence of homologous recombination, in various populations around the world.
In the next 16 years at the University of Wisconsin, Smithies became dedicated to teaching, but would never abandon the lab bench for long, conducting experiments on genetic variants of blood proteins, such as individual haemoglobin genes, and remaining at the forefront of the advancement of molecular genetics. During a sabbatical in 1978, Smithies spent some time in Fred Blattner’s lab, which was one floor below his own lab, learning to clone DNA and work with bacteriophages. Smithies and his co-workers were one of the first scientists to isolate individual genes from DNA. At this point, Smithies envisioned that it could be possible to substitute a faulty piece of DNA with a correct form of the DNA, particularly in diseases which are caused by the mutation of a single gene, such as sickle cell anaemia. The publication of two important papers prompted Smithies to plan an experiment on how to use homologous recombination to correct a faulty gene, the blueprint for gene targeting. The first paper, by Terry Orr-Weaver et al. (1981) demonstrated that plasmids could be inserted in the yeast genome due to homologous recombination. A double strand break was created in a gene, and a particular sequence of DNA could be implanted in the resulting gap. The second paper, by Mitchell Goldfarb et al. (1982), reported the study of a gene rescue technique to isolate a gene from human bladder carcinoma cells. The basis of gene rescue is cutting out a DNA fragment using the enzyme endonuclease, after which the DNA fragment is joined onto a particular marker sequence. After the gene transfer experiments, the gene is isolated based on its link with the marker sequence. Smithies taught the outcomes of these papers to his students, and at the same time wondered how to modify Goldfarb’s technique to make an assay for gene placement. Within ten days, he had outlined a method for his experiment.
Smithies and his team set to work making a large targeting construct, a cosmid, a DNA sequence used as a cloning vector, which contains the cos gene from a bacteriophage. The cosmid, named Cosos 17, took seven months to make. Smithies also made a tester plasmid, a precursor of Cosos 17. The first small-scale experiments using this precursor demonstrated that homologous recombination had taken place and the bacteriophage gene rescue assay had worked; however, the large-scale experiment with Cosos 17 and human bladder carcinoma cells failed to produce any positive results. After nearly a year of discouraging results, the group decided to use mouse-human hybrid erythroleukemia cells, which expressed human beta-globin, instead of the human bladder carcinoma cells, which did not express beta-globin. As the erythroleukemia cells grew in suspension, electroporation, or the process of applying electricity to cells in order to facilitate DNA insertion through cell membranes, was required for the experiment to work. This was a new technique, and hence electroporators weren’t yet commercially available. Ever the inventor, Smithies designed and built his own electroporator. Subsequent experimental work showed that the electroporator succeeded in DNA incorporation in about 80 percent of the cells.
Despite many setbacks and tedious work (leading to graduate students abandoning the project), Smithies and his colleagues managed to unravel problems and adjust the experiment, also by introducing a new targeting construct, which increased the frequency of homologous recombination. Over three years (and seven lab notebooks) after the idea was formulated, the assay for gene targeting proved to have worked; DNA sequences were incorporated into human chromosomal beta-globin as a result of homologous recombination. The paper describing the method was published in Nature in 1985 and stressed that gene targeting was possible, but new, easier methods would have to be developed. Smithies realised that gene therapy, even in single-gene disorders, couldn’t be anticipated in the near future.
At around this time, Smithies became familiar with the work of Martin Evans from the University of Cambridge, who in 1981 famously isolated embryonic stem cells from a mouse and then grew them in cell cultures, which were reintroduced into another mouse. The genetic modification could then be inherited by the mouse’s offspring. Smithies realised that the method could be used to generate mouse models, in which genes could be corrected to eradicate human single-gene illnesses. In his 2015 Lindau Lecture, Smithies recalled how Evans himself brought him stem cells in a vial, saying “Here they are Oliver!” encapsulating the importance of scientific collaboration. At the same time, Mario Capecchi at the University of Utah used stem cells in gene targeting, whereby a gene was made defective, or “knocked out”. Both groups worked on the hypoxanthine phosphoribosyl transferase (HPRT) gene, and, unbeknownst to either group, their papers were published within ten days of each other. Many years later, Smithies explained that, while he was primarily interested in correcting genes, the idea behind Capecchi’s experiments was very rational: “In genetics as a whole (…) the ability to knock out a gene is more useful (…) if you’ve got a whole bunch of genes and you want to understand what they’re doing, the simplest thing you can do is to knock them out and see what happens.”
The year 1988 marked a significant change for Smithies, both personally and professionally. In the 1980s, Nobuyo Maeda, a post-doc from Japan, joined Smithies’ lab and their common research interests and love of science naturally drew them together. By that time, Smithies had been divorced from his first wife for several years. Maeda was unable to obtain a faculty position at the University of Wisconsin and had to look elsewhere, with assurance from Smithies that he would follow despite having spent 28 years in Wisconsin. The pair chose the University of North Carolina at Chapel Hill, because of its reputation for excellent research in their field, but also because of good conditions for flying airplanes, a passion of Smithies since the 1970s.
Upon joining the School of Medicine at UNC, Smithies concentrated on both common and rare diseases, constructing mice models for such diseases as cystic fibrosis, hypertension, and Lesch-Nyhan syndrome, among many others. Smithies spent nearly 30 years at UNC, the highlight of which was winning the Nobel Prize in 2007, along with Capecchi and Evans. The Nobel Prize brings fame and recognition, but Smithies continued to do what he loved best: work at the lab bench, often at the weekends as well. He once said, “On Saturday morning you can do an experiment that is crazy. It doesn’t have to be terribly logical, and you don’t have to measure anything, and you can just use any chemical you feel like and do anything, because you shouldn’t be there on Saturday morning anyway.” Smithies died on January 11, 2017 after a short illness. He had been a dedicated researcher for almost 70 years, affirmed not only by his many discoveries, inventions, awards and professional memberships, but also by 150 lab notebooks, examples of which he presented even during his Nobel Lecture. Also, perhaps remarkably from today’s perspective, Smithies very rarely patented his inventions. He worked in many areas of research, from medicine, to biochemistry, microbiology and genetics, ultimately making genetics into the experimental science it is today.
Those who knew Smithies remember him as a warm and joyful person, eager to share his enthusiasm for science. Many of his experiments were motivated by curiosity, but as Smithies himself pointed out during his first meeting in Lindau in 2010, three factors were key in his scientific career: chance, opportunity and planning.
http://gazette.unc.edu/2013/08/06/separate-research-paths-lead-to-a-lifelong-partnership/, Separate Research Paths Lead to a Lifelong Partnership, University Gazette, August 6, 013, the University of North Carolina at Chapel Hill
Interview with Nathaniel Comfort: http://ohhgp.pendari.com/files/980906a2_90b3_46a3_812a_90f4df5713b0.pdf
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