Introduction: Biotechnology — An Ever Expanding Toolbox for Medicine

LARA V. MARKS

University College London, UK Email: l.marks@ucl.ac.uk

1.1 INTRODUCTION

Biotechnology is intrinsic to medicine. Everywhere you look today, from medical research conducted in the laboratory through to the diagnosis and clinical treatment of a patient, biotechnology is pivotal to that process. Despite its importance, few non-scientists understand what biotechnology is, where it has come from or the many different functions it serves in everyday healthcare.

The term biotechnology was first coined in 1919 by Károly Ereky, a Hungarian agricultural engineer and economist. At its most basic level biotechnology refers to the controlled and deliberate manipulation of organisms and living cells to create products for the benefit of humans. In one form or another, humans have deployed biotechnology for thousands of years. Since prehistoric times, for example, they have used yeast to get bread dough to rise and to produce alcoholic drinks. Bacteria have also been added to milk for generations to make cheese and yoghurt. Animals and plants have also been selectively bred over many centuries to generate stronger and more productive offspring for multiple purposes. In more recent years, increasing knowledge about how to manipulate and control the functions of various cells and organisms, including their genes, has given birth to a burgeoning number of products and technologies for combating human disease.

Biotechnology is currently one of the hottest growth areas in medicine. Between 2001 and 2012 investment in medical biotechnology research rose globally from £6.7bn to £66bn. Such work is helping to determine the molecular causes of disease, to generate more accurate and faster diagnostic platforms and to develop drugs that are more precise in their target and personalised for individual patients.

Just how important biotechnology has become can be seen from the fact that by 2013 seven out of ten of the best selling drugs were biological products. Also known as biologics, such drugs replicate natural substances in our body, including enzymes, antibodies and hormones. They are made from a variety of natural resources — human, animal, and microorganism — and are usually manufactured using biotechnology techniques. Living entities, such as cells and tissues, also comprise biological products. Analysis in 2013 predicted that by 2018 biological drugs would account for a quarter of all drug spending worldwide and for more than 50 percent of the top selling drugs in the world. Such therapeutics include those that are manufactured in either animal cells or bacteria and make use of the body's natural immune system to fight disease.

1.2 MOLECULAR DISEASE AND DNA

Much of the application of biotechnology in medicine is directed towards addressing structural changes at the molecular level that cause disease. This rests on the premise that an illness can be driven by an abnormality or deficiency of a particular molecule. Such thinking can be traced back to the work of Linus Pauling and colleagues at the California Institute of Technology in the late 1940s. Importantly, they demonstrated that sickle-cell anaemia, an inherited blood disorder, was linked to an abnormal haemoglobin, the protein responsible for delivering oxygen to cells in the body. They proposed the hypothesis that a mistake in the protein was caused by a defective gene. Pauling would go on to win the Nobel Prize in Chemistry for this work in 1954. A gene is a distinct stretch of DNA (deoxyribonucleic acid) that carries the instructions needed to create proteins, specific molecules that are essential to the functioning of the body. Proteins not only do most of the work in cells they are also vital to the structure, function and regulation of the body's tissues and organs.

The concept that DNA could play a role in the disease process was highly novel for the time. DNA had been first discovered in the late nineteenth century, but remained little studied for many decades. In part this was due to the belief that DNA was an inert substance incapable of carrying genetic information because of its simple structure. Instead, proteins, which had a more complex structure, were assumed to act as genetic material. Attitudes to DNA began to shift as a result of some experiments by the physician and molecular biologist Oswald Avery and colleagues at the Rockefeller Institute in New York. In 1944 Avery showed that DNA could transform non-infectious bacteria associated with pneumonia into dangerous virulent forms. Avery's work ignited a new interest in DNA. It would take time, however, for scientists to agree that it was DNA, not proteins, that carried genetic information. Consensus finally emerged after experiments conducted by the geneticists Alfred Hershey and Martha Chase at Cold Spring Harbor in 1952.

By the 1950s a number of researchers had begun to investigate the structure of DNA in the hope that this would reveal how the molecule worked. The structure of DNA was finally cracked in 1953 as a result of the culmination of efforts by the biophysicists Rosalind Franklin, Maurice Wilkins and Ray Gosling, based at King's College London, and Francis Crick and James Watson based in the Cavendish Laboratory, Cambridge University. Their work showed DNA to be a long molecule made up of two strands coiled around each other in a spiral configuration called a double helix. Each strand was composed of four complementary nucleotides, chemical sub-units: adenine (A), cytosine (C), guanine (G) and thymine (T). The two strands were oriented in opposite directions so that adenines always joined thymines (A T) and cytosines were linked with guanines (C G). This structure helped each strand to reconstruct the other and facilitate the passing on of hereditary information.

Soon after this breakthrough, in 1955, Fred Sanger, a biochemist at the William Dunn Institute of Biochemistry, Cambridge University, unveiled the molecular composition of the first protein: insulin. This protein, Sanger showed, had a specific sequence of building blocks, known as amino acids. Sanger's finding was quickly seized upon by Crick, who by 1958 had developed a theory that the arrangement of nucleotides in DNA determined the sequence of amino acids in proteins and that this in turn regulated how a protein folded into its final shape. Crick argued that it was this shape that decided each protein's function. He further proposed that an intermediary molecule helped the DNA to specify the sequence of the amino acids in a protein. The key question was how to prove his hypothesis.

Crick recognised that one way to find out would be to investigate sickle-cell anaemia. Pauling and his colleagues had proposed the hypothesis that the difference in haemoglobin found in sickle-cell patients and healthy individuals could be down to a difference in the number of amino acids. How many amino acids were involved, remained unknown. Was it just one amino acid or more? Crick realised this could be resolved with the technique Sanger had developed to work out the composition of amino acids in insulin.

Based on this thinking, Crick launched a collaboration with Sanger and Vernon Ingram, a fellow colleague in the Cavendish laboratory. By 1957, after many hours of painstaking work, Ingram had determined that the difference between normal and sickle-cell haemoglobin was down to the replacement of 'only one of nearly 300 amino acids'. Ingram's finding was a significant breakthrough. Not only did it challenge the scepticism of many scientists that the alteration of just one amino acid could produce a molecule as lethal as sickle-cell haemoglobin, it also marked the first time that anyone had managed to break the genetic code, the process by which cells translate information stored in DNA into proteins. Ultimately the work on sickle-cell anaemia laid the foundation for a whole new approach in medicine, known as molecular medicine. Critically, it ignited a search for other genes or molecules that contributed to disease and ways to harness them for treatment.

1.3 GENETIC ENGINEERING AND ITS CONTROVERSIAL BEGINNING

Unravelling the genetic process behind sickle-cell anaemia was just one investigation among many undertaken in the 1950s in order to understand the relationship between DNA and disease. Elsewhere microbiologists and biologists were examining the role of genetics in drug resistance. They were hunting for the biological mechanism bacteria use to resist viruses and other pathogens and thwart natural anti-microbial substances designed to kill or inhibit their growth. Such work was part of a broader effort to understand the mechanisms underlying the rising resistance of bacteria to antibiotic drugs, widely prescribed for medical treatment from the early 1940s. One of the fruits of such endeavours was the discovery of some biological mechanisms for manipulating and copying DNA.

Plasmids were one of the earliest biological tools scientists unearthed. Discovered in the 1940s, plasmids are small independent self-replicating strands of DNA that naturally exist in most bacteria and some fungi, protozoa, plants and animals. They come in a wide variety of lengths and provide the host organism with the necessary genes for coping with stress-related conditions, such as when encountering substances like antibiotics that impede their growth or threaten their survival. Plasmids have several useful characteristics. Firstly they contain only a small number of genes. Secondly, they snap quickly back into shape when cut open. Because of these features, scientists rapidly explored their use as a vehicle, or vector, for cloning, transferring and manipulating genes within the laboratory.

Soon after finding plasmids, scientists discovered some biochemical enzymes capable of cutting and pasting DNA. One of the first was polymerase, discovered in 1957. All living organisms make polymerase. It helps replicate a cell's DNA. Another important group of enzymes were restriction enzymes. This is a group of enzymes which bacteria use to cleave and destroy the DNA of invading viruses. Restriction enzymes were suggested to exist as early as 1952, but the first one was only isolated and characterised in the late 1960s. Often described as 'molecular scissors', restriction enzymes provided the means to cut DNA very precisely for the first time within the laboratory. By 1968 scientists had isolated another type of enzyme, known as ligase, which bacteria use to repair single-strand breaks in DNA. This provided an avenue for joining different DNA fragments together.

The discovery of plasmids and the different biochemical enzymes laid the foundation for the development of genetic engineering. This method involves selecting and cutting out a gene at specific point on a strand of DNA using restriction enzymes, and then inserting it into a plasmid to produce recombinant DNA. The very first piece of recombinant DNA was generated in June 1972 by Janet Mertz, a biochemistry graduate student working with Paul Berg at Stanford University, a subsequent Nobel Prize winner in 1980. This she did as part of a project to understand gene expression in human cells and its misregulation in cancer. Her recombinant DNA contained genes from the simian virus (SV40), a virus that lives in some monkey species, and a bacteriophage, a type of virus that infects bacteria.

Despite her achievement, Mertz was prevented from cloning the DNA. This involved inserting the recombinant DNA into bacteria for replication by its cell machinery. Mertz was unable to take the next step because of a controversy that broke out following her attendance at a workshop being run by Robert Pollack at Cold Spring Harbor Laboratory in June 1971. Pollack was alarmed to hear during the workshop that she was proposing to insert genes from SV40, into Escherichia coli (E. coli), bacteria that live in the guts of humans and other animals. SV40 is a largely harmless virus. While not known to cause any diseases in humans, SV40 had been shown within the laboratory to be capable of inducing the formation of tumours in rodents and human cells cultivated in culture. Pollack was particularly worried that some bacteria with the SV40 genes could escape from the laboratory, thereby infecting people and other mammals and possibly giving them cancer.

Mertz proposed the risk could be minimised by using an E.coli strain unable to survive outside of the laboratory. But Pollack continued to raise concerns. This persuaded Berg to self-impose a moratorium against anyone performing genetic engineering experiments in his laboratory that introduced SV40 genes into E. coli until the potential safety concerns had been addressed. In the end, the first cloning of recombinant DNA was carried out in June 1973 by Stanley Cohen and Herbert Boyer, based respectively at Stanford University and the University of California in San Francisco. They achieved this on the back of the methods originally outlined by Mertz.

News of Boyer and Cohen's experiment immediately ignited a fierce public debate about the safety of genetic engineering. So great was the furore that in 1974 a group of American scientists agreed to self-impose a voluntary moratorium on experiments involving genetic engineering. This was lifted a year later following the introduction of strict guidelines drawn up by an international conference organised by Berg in Asilomar, California. The guidelines required laboratories to install tight security facilities to contain any experiments with recombinant DNA. The Asilomar conference is now held up as a model of self-regulation by scientists. Indeed, it was recently used as a framework for experiments with CRISPR, a new form of gene editing described below. Significantly, Paul Berg played an important role in debating the guidelines for CRISPR.

Despite the initial controversy, genetic engineering soon grabbed the attention of venture capitalists, who swiftly began partnering with academic scientists to set up biotechnology companies to exploit the new technology. Genentech was the first company in the field, founded in 1976 in San Francisco. The new bioentrepeneurs envisaged inserting human genes into bacteria to encourage the production of unlimited quantities of heretofore scarce therapeutic proteins. Their vision was to spawn an entire new industry. The first two successful products prepared with genetic engineering were insulin, approved by the US Food and Drug Administration (FDA) in 1982, for the treatment of diabetes, and human growth hormone, approved by the FDA in 1985, to treat children with severely restricted growth. Since then genetic engineering has been used to generate medical drugs for a range of other diseases, including cancer, immune deficiency, HIV and heart attacks. As Chapter 2 by Alldread and Birch and Chapter 3 by Buckland show, the technique now underpins the production of many different drugs as well as vaccines. Yet, as these two chapters highlight, the adoption of genetic engineering in this sphere was not as easy or straightforward as originally anticipated. Indeed, it involved significant technical and scientific challenges on the manufacturing front.

1.4 MONOCLONAL ANTIBODIES

Within two years of Boyer and Cohen's successful cloning of recombinant DNA, another important tool appeared on the scene — a technique for the laboratory production of monoclonal antibodies (Mabs). These antibodies are derived from the millions of antibodies the immune system makes every day to fend off bacteria, viruses, pollen, fungi, and any other substance that can threaten the body, including toxins and chemicals. The first Mabs were produced in 1975 by Georges Köhler and Cesar Milstein, based at the Laboratory of Molecular Biology, Cambridge, UK. They developed this as part of their search for a research tool to investigate how the immune system produces so many different types of antibodies specifically targeted to the infinite number of foreign substances that invade the body.

While Köhler and Milstein were subsequently awarded the Nobel Prize in 1994, their invention attracted far less public fanfare at the time than the development of recombinant DNA. Over time, however, their innovation was to have an even more far-reaching impact in the medical field. Able to bind to specific markers found on the surface of cells, Mabs provided an important tool for the detection of unknown molecules and established their function for the first time. As Marks points out in Chapter 4, Mabs opened up new pathways for understanding multiple diseases on an unprecedented scale and greatly enhanced the speed and accuracy of diagnostics. In addition they provided new avenues of treatment. Mab drugs are currently used to treat over 50 major diseases. Just how important they have become can be seen from the fact that Mab drugs now comprise six out of ten of the best-selling drugs worldwide and make up a third of all newly introduced medicines. Mabs are also now at the forefront of the development of immunotherapy for cancer, the subject of Chapter 5 by Marks. Hailed as one of the major advances in cancer treatment in recent years, such therapy is designed to induce, enhance or suppress the body's immune system to combat cancer.