How to breed successfully, how to avoid disease, and how to live to a decent age are questions that have perplexed our ancestors throughout recorded time. As humans explored new lifestyles and habitats, each new challenge – whether it was agriculture, urban living, colonisation of new territories, domestication of animals, or industrialisation – could have notable rewards but was usually fraught with unpredictable physiological penalties. The history of our species is marked by technical solutions that have made these problems of human biology bearable. Some originated in common sense – the piped water and closed sewers of the nineteenth-century metropolis – and others in the application of science, but even these could sometimes produce less-than-satisfactory outcomes and, in some instances, disaster.

The idea of this book is to examine some of these adventures through the lens of twentieth-century biomedicine and to identify the risks and the rewards involved in each. During the first decade of the last century, crucial developments were afoot; philanthropists and eventually governments were recognising the importance of biomedical science and beginning to devise a financial infrastructure that could support its progress. This process gained an irresistible momentum after the Second World War, when the American government undertook unprecedented investment in the life sciences. Within three decades, profound insights into inheritance and cell biology gave us the powerful tools needed to establish a gene technology industry.

The Surgical Tradition

The diary of Samuel Pepys describes how, in 1658, after several years of excruciating discomfort, he bravely decides that his bladder stone must be removed. Completely aware of the hideous unpleasantness of the operation, he sees the risks as preferable to the misery the condition will bring to the rest of his life. A few skilled surgeons existed in London at the time who evidently specialised in this operation with reasonable success.

Should We Worry about Unknown Infectious Enemies?

Industrial countries with well-developed sanitation and public health systems are no longer at risk from the old villains such as plague, cholera, and typhus, but new infectious diseases can still surprise us.

DNA Transmits Genetic Information

Once scientists started to understand how the characteristics of animals and plants were inherited, they inevitably wondered what part of a cell carried genetic information. The answer to this profoundly important question came from an unexpected quarter – a lowly pneumonia-causing microbe called Pneumococcus – and the penetrating insight of a clinical pathologist, Fred Griffith, in London in 1929. In order to understand his esoteric discovery, one needs to know that disease-causing Pneumococci, when cultured in a bacteriologist's Petri dish, grow as smooth slimy-looking colonies; colonies of harmless strains have a rough appearance. Griffith's interest was aroused by the curious outcome of an experiment. Mice had been injected with the “rough” harmless variety of Pneumococcus together with sterilised cells of the “smooth,” disease-causing version of the same bug.

The Power of Chemistry

Experimentation with the physiological effects of natural products must be as old as human civilisation. Alcohol, narcotics, mind-affecting drugs, poisons, and many other natural products with pharmacological effects were widely known in ancient times. More puzzling, though, is why the ancients thought these products might cure human sickness. The ancient Egyptian Ebers' papyrus lists many medicines; whether they were efficacious we shall never know, but the betting must surely be not. One offering contains human excrement! The puritanical Hebrews, who would have known the Egyptian predilection for medicine, seemed to reject this kind of hocus-pocus along with alcohol.

In the developed world, human life expectancy has more than doubled in the last two centuries and is still increasing. The trend may not be sustainable, but life expectancy is evidently continually improving. Our bodies intrinsically lack the resources to provide foolproof maintenance long after our reproductive years, but our individual capacities are notably variable because of the heterogeneity of our genetic legacies and our life experiences. Even before birth, nutritional stress, toxic substances, and viral infections can have effects on the development of the foetus that will shorten an adult life span. After birth, accumulation of somatic mutations – sometimes originating in poor nutrition or chronic inflammation – creates a risk of cancer and other diseases of late middle age and may advance the ageing process. However, although our bodies may be unable to repair all biochemical damage, we can, in principle, reduce some of the risk to health and life expectancy it imposes.

New health challenges originating in life experience still emerge. Breast cancer, allergic reactions, and obesity are three very different kinds of health threat that are becoming increasingly common in the developed world. Breast cancer was well known before the twentieth century, but the lifetime risk of developing such a malignancy in Britain and the United States (and probably in many other countries) has now reached one in eight.

The Perfect Nursery?

Imagine the reproduction of fish and frogs. Eggs are laid in thousands; the likelihood of fertilisation is low; the progeny are easily eaten, infected, or poisoned. However, if more than a few are fertilised and survive, the species prospers. Reproductive duties complete, the parents of these lucky few abandon their progeny to the lonely battle for survival. Mammals avoid this wasteful lottery. Eggs are fertilised with more certainty, and the resulting embryo is raised in an extraordinary high-grade “nursery,” in relative safety.

The Architecture of Cells

Just as powerful telescopes revolutionised our perception of our place in the solar system, so advances in microscopy revolutionised perceptions of our material character. Early nineteenth-century microscopists realised that all living things are made up of cells and that only division of preexisting cells could generate more cells. The immensely influential Prussian biologist Rudolph Virchow was one of the first to absorb this lesson and to see that more was to be gained in studying pathology by looking at cells than by looking at the gross anatomy of cadavers.

Historic Epidemics

Nobody knows exactly when infectious disease became a great threat to human existence, but the first settled communities were probably much more vulnerable to devastating epidemics than their nomadic ancestors. Demographers believe that although the birth rate was high, these communities grew slowly and spasmodically because of periodic outbreaks of disease. Without efficient sanitation, sound hygienic practices, or immunity to these diseases, human societies were always at the mercy of microbes, a susceptibility greatly increased by malnutrition. The Old Testament is, again, an indication of the perceptions of an ancient people (see Fig. 9.1).

The Burden of Genetic Disease

There can be few parental heartaches greater than the implacable advance of a genetic disease slowly crushing a child's unique personality.

Leaving Eden

For more than a hundred millennia our species has roamed the earth multiplying slowly, subsisting on animal and plant food, constantly adapting to changes in climate and to potential prey. We know little of their lives except that the Cro-Magnon people, the immediate ancestors of Europeans, hunted big game with what seems a mystical enthusiasm and buried their dead with some reverence. By the end of the last Ice Age, the herds of deer had dwindled away and human populations were reduced at one point to perhaps just 10,000 pairs, while our Neanderthal cousins became extinct. When the ice receded, the survivors' frugal existence, dependent on small animals and plant foods, began to improve as they learnt to grow plants and to manage domesticated animals.

Twentieth-Century Nightmare

In the generation that reached middle age in the early twentieth century, the family was frequently faced with cancer for the first time in its collective memory. No remedies or palliative care could ease a terrible illness that often struck when its victims were still in their prime.

Introduction

Novel tools for evaluating gene function in vivo such as ribozymes and RNA interference (RNAi) are emerging as the most highly effective strategies (Sioud, 2001; Sioud, 2004; Hannon, 2002). RNAi is sequence-specific posttranscriptional gene silencing, which is triggered by double stranded RNA (dsRNA). This evolutionally conserved gene-silencing pathway triggered by dsRNAs was first described in the nematode worm Caenorhabditis elegans (Fire et al., 1998). This process has been linked to many previously described phenomena such as post-transcriptional gene silencing (PTGS) in plants (Jorgensen, 1990). The difficulty of using RNAi in somatic mammalian cells was overcome when Tuschl and colleagues discovered that siRNAs (21 nt), normally generated from long dsRNA during RNAi, could be used to inhibit specific gene targets (Elbashir et al., 2001). Currently, there is a great interest in the use of small interfering RNA as a research tool to study gene function and drug target validation (Dykxhoorn et al., 2003; Sørensen et al., 2003).

The therapeutic application of siRNAs, however, is largely dependent on the development of a delivery vehicle that can efficiently deliver the siRNAs to target cells. In addition, such delivery vehicles should be administered efficiently, safely and repeatedly. Cationic liposomes represent one of the few examples that can meet these requirements (Templeton, 2002). These agents are composed of positively charged lipid bilayers, and can be complexed to negatively charged siRNA duplexes. The routes of delivery include direct injection (e.g. intratumoral), intravenous, intraperitoneal, intraarterial, intracranial, and others.

Introduction

A major challenge in the post genomic era of biology is to decipher the molecular function of over 30,000 genes. The gene knock-out by homologous recombination has proven to be very useful but is laborious and expensive. RNA interference has recently emerged as a novel pathway that allows modulation of gene expression. The basic biology of RNAi is described in the next section. Briefly, long dsRNA molecules are processed by the endonuclease Dicer into short 21–23 nucleotide small interfering RNAs (siRNAs), which are then incorporated into RISC (RNA-induced silencing complex), a multi-component nuclease complex that selects and degrades mRNAs that are homolgous to the dsRNA initially delivered (Fjose et al., 2001; Hannon, 2002). In mammalian systems, synthetic siRNA's can be delivered exogenously (Elbashir et al., 2001) or can be expressed endogenously from RNA Pol III promoters, resulting in a powerful tool for achieving specific downregulation of target mRNA's (Miyagashi and Taira, 2002; Paul et al., 2002; Oliviera and Goodell, 2003). The delivery of synthetic siRNAs to cells in culture is hampered by limitations in transfection efficiency for many cell types and the transient nature of the silencing effect. In vivo, delivering siRNAs to target cells is difficult due to lack of stability of siRNA and low uptake efficiency in the absence of transfection agents (Isacson et al., 2003). Thus in order to apply this potent technique to both basic biological questions and therapeutic strategies, efficient siRNA delivery methods must be developed.

Introduction

In eukaryotic organisms, RNA interference (RNAi) is the sequence-specific gene silencing that is induced by double-stranded RNA (dsRNA) homologous to the silenced gene. In the cytoplasm of mammalian cells, long dsRNAs (>30 nt) can activate the potent interferon and a protein kinase-mediated pathway, which lead to non-sequence-specific effects that can include apoptosis (Kumar and Carmichael, 1998). Elbashir and coworkers (2001a) made the important discovery that small interfering RNAs (siRNAs) of about 21 nt specifically inhibit gene expression, because siRNAs are too short to activate the interferon or protein kinase pathway. The silencing by synthetic siRNAs is transient. This limitation can be overcome by stably expressed short hairpin RNAs (shRNAs), which are processed by Dicer into siRNAs (Paddison et al., 2002; Brummelkamp et al., 2002). However, it was recently reported that shRNA vectors can induce an interferon response (Bridge et al., 2003).

Because target recognition presumably depends on Watson-Crick base pairing, the RNAi machinery is widely believed to be exquisitely specific. As a reverse genetic tool, RNAi has set the standard in high-throughput functional genomics (Barstead, 2001; Tuschl, 2003). RNAi has also become an important tool in the identification and validation of drug targets in preclinical therapeutic development (Thompson, 2002; Appasani, 2003). Furthermore, RNAi-based human therapeutics are under development.

Initial empirical rules have been established by the Tuschl lab for the design of siRNAs. However, large variation in the potency of siRNAs is commonly observed, and often only a small proportion of the tested siRNAs are effective.