History of genetics

The history of genetics is generally held to have started with the work of an Augustinian monk, Gregor Mendel. His work on pea plants, published in 1866, described what came to be known as Mendelian inheritance. In the centuries before—and for several decades after—Mendel's work, a wide variety of theories of heredity proliferated (see below). 1900 marked the "rediscovery of Mendel" by Hugo de Vries, Carl Correns and Erich von Tschermak, and by 1915 the basic principles of Mendelian genetics had been applied to a wide variety of organisms—most notably the fruit fly Drosophila melanogaster. Led by Thomas Hunt Morgan and his fellow "drosophilists", geneticists developed the Mendelian-chromosome theory of heredity, which was widely accepted by 1925. Alongside experimental work, mathematicians developed the statistical framework of population genetics, bring genetical explanations into the study of evolution.

With the basic patterns of genetic inheritance established, many biologists turned to investigations of the physical nature of the gene. In the 1940s and early 1950s, experiments pointed to DNA as the portion of chromosomes (and perhaps other nucleoproteins) that held genes. A focus on new model organisms such as viruses and bacteria, along with the discovery of the double helical structure of DNA in 1953, marked the transition to the era of molecular genetics. In the following years, chemists developed techniques for sequencing both nucleic acids and proteins, while others worked out the relationship between the two forms of biological molecules: the genetic code. The regulation of gene expression became a central issue in the 1960s; by the 1970s gene expression could be controlled and manipulated through genetic engineering. In the last decades of the 20th century, many biologists focused on large-scale genetics projects, sequencing entire genomes.

Pre-Mendelian ideas on heredity

Ancient theories

The most influential early theories of heredity were that of Hippocrates and Aristotle. Hippocrates' theory (possibly based on the teachings of Anaxagoras) was similar to Darwin's later ideas on pangenesis, involving heredity material that collects from throughout the body. Aristotle suggested instead that the (nonphysical) form-giving principle of an organism was transmitted through semen (which he considered to be a purified form of blood) and the mother's menstrual blood, which interacted in the womb to direct an organism's early development. For both Hippocrates and Aristotle—and nearly all Western scholars through to the late 19th century—the inheritance of acquired characters was a supposedly well-established fact that any adequate theory of heredity had to explain. At the same time, individual species were taken to have a fixed essence; such inherited changes were merely superficial.

In the 9th century CE, the Afro-Arab writer Al-Jahiz considered the effects of the environment on the likelihood of an animal to survive, and first described the struggle for existence. His ideas on the struggle for existence in the Book of Animals have been summarized as follows:

"Animals engage in a struggle for existence; for resources, to avoid being eaten and to breed. Environmental factors influence organisms to develop new characteristics to ensure survival, thus transforming into new species. Animals that survive to breed can pass on their successful characteristics to offspring."

In 1000 CE, the Arab physician, Abu al-Qasim al-Zahrawi (known as Albucasis in the West), wrote the first clear description of haemophilia, a hereditary genetic disorder, in his Al-Tasrif. In this work, he wrote of an Andalusian family whose males died of bleeding after minor injuries.

Plant systematics and hybridization

In the 18th century, with increased knowledge of plant and animal diversity and the accompanying increased focus on taxonomy, new ideas about heredity began to appear. Linnaeus and others (among them Joseph Gottlieb Kölreuter, Carl Friedrich von Gärtner, and Charles Naudin) conducted extensive experiments with hybridization, especially species hybrids. Species hybridizers described a wide variety of inheritance phenomena, include hybrid sterility and the high variability of back-crosses.

Plant breeders were also developing an array of stable varieties in many important plant species. In the early 19th century, Augustin Sageret established the concept of dominance, recognizing that when some plant varieties are crossed, certain characters (present in one parent) usually appear in the offspring; he also found that some ancestral characters found in neither parent may appear in offspring. However, plant breeders made little attempt to establish a theoretical foundation for their work or to share their knowledge with current work of physiology.

Mendel

In breeding experiments between 1856 and 1865, Gregor Mendel first traced inheritance patterns of certain traits in pea plants and showed that they obeyed simple statistical rules. Although not all features show these patterns of Mendelian inheritance, his work acted as a proof that application of statistics to inheritance could be highly useful. Since that time many more complex forms of inheritance have been demonstrated.

From his statistical analysis Mendel defined a concept that he described as an allele, which was the fundamental unit of heredity. The term allele as Mendel used it is nearly synonymous with the term gene, and now means a specific variant of a particular gene.

Mendel's work was published in 1866 as "Versuche über Pflanzen-Hybriden" (Experiments on Plant Hybridization) in the Verhandlungen des Naturforschenden Vereins zu Brünn (Proceedings of the Natural History Society of Brünn), following two lectures he gave on the work in early 1865.

Post-Mendel, pre-re-discovery

Mendel's work was published in a relatively obscure scientific journal, and it was not given any attention in the scientific community. Instead, discussions about modes of heredity were galvanized by Darwin's theory of evolution by natural selection, in which mechanisms of non-Lamarckian heredity seemed to be required. Darwin's own theory of heredity, pangenesis, did not meet with any large degree of acceptance. A more mathematical version of pangenesis, one which dropped much of Darwin's Lamarckian holdovers, was developed as the "biometrical" school of heredity by Darwin's cousin, Francis Galton. Under Galton and his successor Karl Pearson, the biometrical school attempted to build statistical models for heredity and evolution, with some limited but real success, though the exact methods of heredity were unknown and largely unquestioned.

Classical genetics

The significance of Mendel's work was not understood until early in the twentieth century, after his death, when his research was re-discovered by other scientists working on similar problems. Hugo de Vries, Carl Correns and Erich von Tschermak.

There was then a feud between Bateson and Pearson over the hereditary mechanism. Fisher solved this in The Correlation Between Relatives on the Supposition of Mendelian Inheritance

1865 Gregor Mendel's paper, Experiments on Plant Hybridization

1869 Friedrich Miescher discovers a weak acid in the nuclei of white blood cells that today we call DNA

1880-1890 Walther Flemming, Eduard Strasburger, and Edouard van Beneden elucidate chromosome distribution during cell division

1889 Hugo de Vries postulates that "inheritance of specific traits in organisms comes in particles", naming such particles "(pan)genes"

1903 Walter Sutton hypothesizes that chromosomes, which segregate in a Mendelian fashion, are hereditary units

1905 William Bateson coins the term "genetics" in a letter to Adam Sedgwick and at a meeting in 1906

1908 Hardy-Weinberg law derived.

1910 Thomas Hunt Morgan shows that genes reside on chromosomes

1913 Alfred Sturtevant makes the first genetic map of a chromosome

1913 Gene maps show chromosomes containing linear arranged genes

1918 Ronald Fisher publishes "The Correlation Between Relatives on the Supposition of Mendelian Inheritance" the modern synthesis of genetics and evolutionary biology starts. .

1928 Frederick Griffith discovers that hereditary material from dead bacteria can be incorporated into live bacteria

1931 Crossing over is identified as the cause of recombination

1933 Jean Brachet is able to show that DNA is found in chromosomes and that RNA is present in the cytoplasm of all cells.

1941 Edward Lawrie Tatum and George Wells Beadle show that genes code for proteins; see the original central dogma of genetics

The DNA era

James Watson and colleagues discovered the structure of DNA

1944 Oswald Theodore Avery, Colin McLeod and Maclyn McCarty isolate DNA as the genetic material (at that time called transforming principle).

1950 Erwin Chargaff shows that the four nucleotides are not present in nucleic acids in stable proportions, but that some general rules appear to hold (e.g., that the amount of adenine, A, tends to be equal to that of thymine, T). Barbara McClintock discovers transposons in maize.

1952 The Hershey-Chase experiment proves the genetic information of phages (and all other organisms) to be DNA.

1953 DNA structure is resolved to be a double helix by James D. Watson and Francis Crick1956 Joe Hin Tjio and Albert Levan established the correct chromosome number in humans to be 46.

1958 The Meselson-Stahl experiment demonstrates that DNA is semiconservatively replicated.

1961-1967 Combined efforts of scientists "crack" the genetic code, including Marshall Nirenberg, Har Gobind Khorana, Sydney Brenner & Francis Crick1964 Howard Temin showed using RNA viruses that the direction of DNA to RNA transcription can be reversed.

1970 Restriction enzymes were discovered in studies of a bacterium, Haemophilus influenzae, enabling scientists to cut and paste DNA.

The genomics era

1972, Walter Fiers and his team at the Laboratory of Molecular Biology of the University of Ghent (Ghent, Belgium) were the first to determine the sequence of a gene: the gene for bacteriophage MS2 coat protein.

1976, Walter Fiers and his team determine the complete nucleotide-sequence of bacteriophage MS2-RNA

1977 DNA is sequenced for the first time by Fred Sanger, Walter Gilbert, and Allan Maxam working independently. Sanger's lab sequence the entire genome of bacteriophage Φ-X174.

1983 Kary Banks Mullis discovers the polymerase chain reaction enabling the easy amplification of DNA.

1989 The human gene that encodes the CFTR protein was sequenced by Francis Collins and Lap-Chee Tsui. Defects in this gene cause cystic fibrosis.

1995 The genome of Haemophilus influenzae is the first genome of a free living organism to be sequenced.

1996 Saccharomyces cerevisiae is the first eukaryote genome sequence to be released1998 The first genome sequence for a multicellular eukaryote, Caenorhabditis elegans, is released

2001 First draft sequences of the human genome are released simultaneously by the Human Genome Project and Celera Genomics.

2003 (14 April) Successful completion of Human Genome Project with 99% of the genome sequenced to a 99.99% accuracy.

Quantitative genetics

Quantitative genetics is the study of continuous traits (such as height or weight) and its underlying mechanisms. It is effectively an extension of simple Mendelian inheritance in that the combined effect of the many underlying genes results in a continuous distribution of phenotypic values.

History

The field was founded, in evolutionary terms, by the originators of the modern synthesis, R.A. Fisher, Sewall Wright and J. B. S. Haldane, and aimed to predict the response to selection given data on the phenotype and relationships of individuals.

Analysis of Quantitative trait loci, or QTL, is a more recent addition to the study of quantitative genetics. A QTL is a region in the genome that affects the trait or traits of interest. Quantitative trait loci approaches require accurate phenotypic, pedigree and genotypic data from a large number of individuals.

Traits

Quantitative genetics is not limited to continuous traits, but to all traits that are determined by many genes. This includes:

• Continuous traits are quantitative traits with a continuous phenotypic range. They are often polygenic, and may also be influenced significantly by environmental effects.
• Meristic traits or other ordinal numbers are expressed in whole numbers, such as number of offspring, or number of bristles on a fruit fly. These traits can be either treated as approximately continuous traits or as threshold traits.
• Some qualitative traits can be treated as if they have an underlying quantitative basis, expressed as a threshold trait (or multiple thresholds). Some human diseases (such as, schizophrenia) have been studied in this manner.

Basic principles

The phenotypic value (P) of an individual is the combined effect of the genotypic value (G) and the environmental deviation (E):

P = G + E

The genotypic value is the combined effect of all the genetic effects, including nuclear genes, mitochondrial genes and interactions between the genes. It is therefore often subdivided in an additive (A) and a dominance component (D). The additive effect described the cumulative effect of the individual genes, while the dominance effect is the result of interactions between those genes. The environmental deviation can be subdivided in a pure environmental component (E) and an interaction factor (I) describing the interaction between genes and the environment. This can be described as:

P = A + D + E + I

The contribution of those components cannot be determined in a single individual, but they can be estimated for whole populations by estimating the variances for those components, denoted as:

V_P = V_A + V_D + V_E + V_I

The heritability of a trait is the proportion of the total (i.e. phenotypic) variation (VP) that is explained by the genetic variation. This is the total genetic variation (VG) in broad sense heritabilities (H2), while only the additive genetic variation (VA) is used for narrow sense heritabilities (h2), often simply called heritability. The latter gives an indication how a trait will respond to natural or artificial selection.

Resemblance between relatives

Central in estimating the variances for the various components is the principle of relatedness. A child has a father and a mother. Consequently, the child and father share 50% of their alleles, as do the child and the mother. However, the mother and father normally do not share genes as a result of shared ancestors. Similarly, two full siblings share also on average 50% of the alleles with each other, while half sibs share only 25% of their alleles. This variation in relatedness can be used to estimate which proportion of the total phenotypic variance (V_P) is explained by the above-mentioned components.

Correlated traits

Although some genes have only an effect on a single trait, many genes have an effect on various traits. Because of this, a change in a single gene will have an effect on all those traits. This is calculated using covariances, and the phenotypic covariance (CovP) between two traits can be partitioned in the same way as the variances described above. The genetic correlation is calculated by dividing the covariance between the additive genetic effects of two traits by the square root of the product of the variances for the additive genetic effects of the two traits:

Psychiatric genetics

Psychiatric genetics, a subfield of behavioral neurogenetics, studies the role of genetics in psychological conditions such as alcoholism, schizophrenia, bipolar disorder, and autism. The basic principle behind psychiatric genetics is that genetic polymorphisms, as indicated by linkage to e.g. a single nucleotide polymorphism (SNP), are part of the etiology of psychiatric disorders.

The goal of psychiatric genetics is to better understand the etiology of psychiatric disorders, to use that knowledge to improve treatment methods, and possibly also to develop personalized treatments based on genetic profiles. In other words, the goal is to transform parts of psychiatry into a neuroscience-based discipline.

Linkage, association, and microarray studies generate raw material for findings in psychiatric genetics. Copy number variants have also been associated with psychiatric conditions.

Most psychiatric disorders are highly heritable; the estimated heritability for bipolar disorder, schizophrenia, and autism (80% or higher) is much higher than that of diseases like breast cancer and Parkinson disease. However, linkage analysis and genome-wide association studies have found few reproducible risk factors.

Several genetic risk factors have been found with the endophenotypes of psychiatric disorders, rather than with the diagnoses themselves. That is, the risk factors are associated with particular symptoms, not with the overall diagnosis.

Population genetics

Population genetics is the study of the allele frequency distribution and change under the influence of the four evolutionary forces: natural selection, genetic drift, mutation and gene flow. It also takes account of population subdivision and population structure in space. As such, it attempts to explain such phenomena as adaptation and speciation. Population genetics was a vital ingredient in the modern evolutionary synthesis, its primary founders were Sewall Wright, J. B. S. Haldane and R. A. Fisher, who also laid the foundations for the related discipline of quantitative genetics.

Scope and theoretical considerations

Perhaps the most significant "formal" achievement of the modern evolutionary synthesis has been the framework of mathematical population genetics. Indeed some authors (Beatty 1986) would argue that it does define the core of the modern synthesis.

Lewontin (1974) outlined the theoretical task for population genetics. He imagined two spaces: a "genotypic space" and a "phenotypic space". The challenge of a complete theory of population genetics is to provide a set of laws that predictably map a population of genotypes (G1) to a phenotype space (P1), where selection takes place, and another set of laws that map the resulting population (P2) back to genotype space (G2) where Mendelian genetics can predict the next generation of genotypes, thus completing the cycle. Even leaving aside for the moment the non-Mendelian aspects revealed by molecular genetics, this is clearly a gargantuan task. Visualizing this transformation schematically:

(adapted from Lewontin 1974, p. 12). XD

T₁ represents the genetic and epigenetic laws, the aspects of functional biology, or development, that transform a genotype into phenotype. We will refer to this as the "genotype-phenotype map". T2 is the transformation due to natural selection, T3 are epigenetic relations that predict genotypes based on the selected phenotypes and finally T4 the rules of Mendelian genetics.

In practice, there are two bodies of evolutionary theory that exist in parallel, traditional population genetics operating in the genotype space and the biometric theory used in plant and animal breeding, operating in phenotype space. The missing part is the mapping between the genotype and phenotype space. This leads to a "sleight of hand" (as Lewontin terms it) whereby variables in the equations of one domain, are considered parameters or constants, where, in a full-treatment they would be transformed themselves by the evolutionary process and are in reality functions of the state variables in the other domain. The "sleight of hand" is assuming that we know this mapping. Proceeding as if we do understand it is enough to analyze many cases of interest. For example, if the phenotype is almost one-to-one with genotype (sickle-cell disease) or the time-scale is sufficiently short, the "constants" can be treated as such; however, there are many situations where it is inaccurate.

Population geneticists

The three founders of population genetics were the Britons R.A. Fisher and J.B.S. Haldane and the American Sewall Wright. Fisher and Wright had some fundamental disagreements and a controversy about the relative roles of selection and drift continued for much of the century between the Americans and the British. The Frenchman Gustave Malécot was also important early in the development of the discipline. John Maynard Smith was Haldane's pupil, whilst W.D. Hamilton was heavily influenced by the writings of Fisher. The American George R. Price worked with both Hamilton and Maynard Smith. On the American side, Richard Lewontin and the Japanese Motoo Kimura were heavily influenced by Wright.

Gene therapy

Gene therapy is the insertion of genes into an individual's cells and tissues to treat a disease, and hereditary diseases in which a defective mutant allele is replaced with a functional one. Although the technology is still in its infancy, it has been used with some success. Antisense therapy is not strictly a form of gene therapy, but is a genetically-mediated therapy and is often considered together with other methods.

Gene therapy using an Adenovirus vector. A new gene is inserted into an adenovirus vector, which is used to introduce the modified DNA into a human cell. If the treatment is successful, the new gene will make a functional protein.

Background

On September 14, 1990 at the U.S. National Institutes of Health W. French Anderson, M.D., and his colleagues R. Michael Blaese, M.D., C. Bouzaid, M.D., and Kenneth Culver, M.D., performed the first approved gene therapy procedure on four-year old Ashanthi DeSilva. Born with a rare genetic disease called severe combined immunodeficiency (SCID), she lacked a healthy immune system, and was vulnerable to every passing germ or infection. Children with this illness usually develop overwhelming infections and rarely survive to adulthood; a common childhood illness like chickenpox is life-threatening. Ashanthi led a cloistered existence -- avoiding contact with people outside her family, remaining in the sterile environment of her home, and battling frequent illnesses with massive amounts of antibiotics.

In Ashanthi's gene therapy procedure, doctors removed white blood cells from the child's body, let the cells grow in the lab, inserted the missing gene into the cells, and then infused the genetically modified blood cells back into the patient's bloodstream. Laboratory tests have shown that the therapy strengthened Ashanthi's immune system by 40%; she no longer has recurrent colds, she has been allowed to attend school, and she was immunized against whooping cough. This procedure was not a cure; the white blood cells treated genetically only work for a few months, after which the process must be repeated (VII, Thompson [First] 1993). As of early 2007, she was still in good health, and she was attending college. However, there is no consensus on what portion of her improvement should be attributed to gene therapy versus other treatments. Some would state that the case is of great importance despite its indefinite results, if only because it demonstrated that gene therapy could be practically attempted without adverse consequences.

Although this simplified explanation of a gene therapy procedure sounds like a happy ending, it is little more than an optimistic first chapter in a long story; the road to the first approved gene therapy procedure was rocky and fraught with controversy. The biology of human gene therapy is very complex, and there are many techniques that still need to be developed and diseases that need to be understood more fully before gene therapy can be used appropriately. The public policy debate surrounding the possible use of genetically engineered material in human subjects has been equally complex. Major participants in the debate have come from the fields of biology, government, law, medicine, philosophy, politics, and religion, each bringing different views to the discussion.

Scientists took the logical step of trying to introduce genes straight into human cells, focusing on diseases caused by single-gene defects, such as cystic fibrosis, hemophilia, muscular dystrophy and sickle cell anemia. However, this has been much harder than modifying simple bacteria, primarily because of the problems involved in carrying large sections of DNA and delivering them to the correct site on the comparatively large human genome.

Basic process

In most gene therapy studies, a "correct copy" or "wild type" gene is provided or inserted into the genome. Generally, it is not an exact replacement of the "abnormal," disease-causing gene, but rather extra, correct copies of genes are provided to complement the loss of function. A carrier called a vector must be used to deliver the therapeutic gene to the patient's target cells. Currently, the most common type of vectors are viruses that have been genetically altered to carry normal human DNA. Viruses have evolved a way of encapsulating and delivering their genes to human cells in a pathogenic manner. Scientists have tried to harness this ability by manipulating the viral genome to remove disease-causing genes and insert therapeutic ones.

Target cells such as the patient's liver or lung cells are infected with the vector. The vector then unloads its genetic material containing the therapeutic human gene into the target cell. The generation of a functional protein product from the therapeutic gene restores the target cell to a normal cell.

Types of gene therapy

Gene therapy may be classified into the following types:

Germ line gene therapy

In the case of germ line gene therapy, germ cells, i.e., sperm or eggs, are modified by the introduction of functional genes, which are ordinarily integrated into their genomes. Therefore, the change due to therapy would be heritable and would be passed on to later generations. This new approach, theoretically, should be highly effective in counteracting genetic disorders. However, this option is prohibited for application in human beings, at least for the present, for a variety of technical and ethical reasons.

Somatic gene therapy

In the case of somatic gene therapy, theraputic genes are transferred into the somatic cells of a patient. Any modifications and effects will be restricted to the individual patient only, and will not be inherited by the patient's offspring.

Broad methods

There are a variety of different methods to replace or repair the genes targeted in gene therapy.

• A normal gene may be inserted into a nonspecific location within the genome to replace a nonfunctional gene. This approach is most common.
• An abnormal gene could be swapped for a normal gene through homologous recombination.
• The abnormal gene could be repaired through selective reverse mutation, which returns the gene to its normal function.
• The regulation (the degree to which a gene is turned on or off) of a particular gene could be altered.

Vectors in gene therapy

Viruses

All viruses bind to their hosts and introduce their genetic material into the host cell as part of their replication cycle. This genetic material contains basic 'instructions' of how to produce more copies of these viruses, hijacking the body's normal production machinery to serve the needs of the virus. The host cell will carry out these instructions and produce additional copies of the virus, leading to more and more cells becoming infected. Some types of viruses physically insert their genes into the host's genome (a defining feature of retroviruses, the family of viruses that includes HIV, is that the virus will introduce the enzyme reverse transcriptase into the host and thus use its RNA as the "instructions"). This incorporates the genes of that virus among the genes of the host cell for the life span of that cell.

Doctors and molecular biologists realized that viruses like this could be used as vehicles to carry 'good' genes into a human cell. First, a scientist would remove the genes in the virus that cause disease. Then they would replace those genes with genes encoding the desired effect (for instance, insulin production in the case of diabetics). This procedure must be done in such a way that the genes which allow the virus to insert its genome into its host's genome are left intact. This can be confusing, and requires significant research and understanding of the virus' genes in order to know the function of each. An example: A virus is found which replicates by inserting its genes into the host cell's genome. This virus has two genes- A and B. Gene A encodes a protein which allows this virus to insert itself into the host's genome. Gene B causes the disease this virus is associated with. Gene C is the "normal" or "desirable" gene we want in the place of gene B. Thus, by re-engineering the virus so that gene B is replaced by gene C, while allowing gene A to properly function, this virus could introduce the required gene - gene C into the host cell's genome without causing any disease.

All this is clearly an oversimplification, and numerous problems exist that prevent gene therapy using viral vectors, such as: trouble preventing undesired effects, ensuring the virus will infect the correct target cell in the body, and ensuring that the inserted gene doesn't disrupt any vital genes already in the genome. However, this basic mode of gene introduction currently shows much promise and doctors and scientists are working hard to fix any potential problems that could exist.

Retroviruses

The genetic material in retroviruses is in the form of RNA molecules, while the genetic material of their hosts is in the form of DNA. When a retrovirus infects a host cell, it will introduce its RNA together with some enzymes, namely reverse transcriptase and integrase, into the cell. This RNA molecule from the retrovirus must produce a DNA copy from its RNA molecule before it can be integrated into the genetic material of the host cell. The process of producing a DNA copy from an RNA molecule is termed reverse transcription. It is carried out by one of the enzymes carried in the virus, called reverse transcriptase. After this DNA copy is produced and is free in the nucleus of the host cell, it must be incorporated into the genome of the host cell. That is, it must be inserted into the large DNA molecules in the cell (the chromosomes). This process is done by another enzyme carried in the virus called integrase.

Now that the genetic material of the virus is incorporated and has become part of the genetic material of the host cell, it can be said that the host cell is now modified to contain a new gene. If this host cell divides later, its descendants will all contain the new genes. Sometimes the genes of the retrovirus do not express their information immediately.

One of the problems of gene therapy using retroviruses is that the integrase enzyme can insert the genetic material of the virus in any arbitrary position in the genome of the host- it randomly shoves the genetic material into a chromosome. If genetic material happens to be inserted in the middle of one of the original genes of the host cell, this gene will be disrupted (insertional mutagenesis). If the gene happens to be one regulating cell division, uncontrolled cell division (i.e., cancer) can occur. This problem has recently begun to be addressed by utilizing zinc finger nucleases or by including certain sequences such as the beta-globin locus control region to direct the site of integration to specific chromosomal sites.

Gene therapy trials using retroviral vectors to treat X-linked severe combined immunodeficiency (X-SCID) represent the most successful application of gene therapy to date. More than twenty patients have been treated in France and Britain, with a high rate of immune system reconstitution observed. Similar trials were halted or restricted in the USA when leukemia was reported in patients treated in the French X-SCID gene therapy trial. To date, four children in the French trial and one in the British trial have developed leukemia as a result of insertional mutagenesis by the retroviral vector. All but one of these children responded well to conventional anti-leukemia treatment. Gene therapy trials to treat SCID due to deficiency of the Adenosine Deaminase (ADA) enzyme continue with relative success in the USA, Britain, Italy and Japan.

Adenoviruses

Adenoviruses are viruses that carry their genetic material in the form of double-stranded DNA. They cause respiratory, intestinal, and eye infections in humans (especially the common cold). When these viruses infect a host cell, they introduce their DNA molecule into the host. The genetic material of the adenoviruses is not incorporated (transient) into the host cell's genetic material. The DNA molecule is left free in the nucleus of the host cell, and the instructions in this extra DNA molecule are transcribed just like any other gene. The only difference is that these extra genes are not replicated when the cell is about to undergo cell division so the descendants of that cell will not have the extra gene. As a result, treatment with the adenovirus will require readministration in a growing cell population although the absence of integration into the host cell's genome should prevent the type of cancer seen in the SCID trials. This vector system has shown real promise in treating cancer and indeed the first gene therapy product to be licensed to treat cancer is an adenovirus.

Adeno-associated viruses

Adeno-associated viruses, from the parvovirus family, are small viruses with a genome of single stranded DNA. The wild type AAV can insert genetic material at a specific site on chromosome 19 with near 100% certainty. But the recombinant AAV, which does not contain any viral genes and only the therapeutic gene, does not integrate into the genome. Instead the recombinant viral genome fuses at its ends via the ITR (inverted terminal repeats) recombination to form circular, episomal forms which are predicted to be the primary cause of the long term gene expression. There are a few disadvantages to using AAV, including the small amount of DNA it can carry (low capacity) and the difficulty in producing it. This type of virus is being used, however, because it is non-pathogenic (most people carry this harmless virus). In contrast to adenoviruses, most people treated with AAV will not build an immune response to remove the virus and the cells that have been successfully treated with it. Several trials with AAV are on-going or in preparation, mainly trying to treat muscle and eye diseases; the two tissues where the virus seems particularly useful. However, clinical trials have also been initiated where AAV vectors are used to deliver genes to the brain. This is possible because AAV viruses can infect non-dividing (quiescent) cells, such as neurons in which their genomes are expressed for a long time.

Envelope protein pseudotyping of viral vectors

The viral vectors described above have natural host cell populations that they infect most efficiently. Retroviruses have limited natural host cell ranges, and although adenovirus and adeno-associated virus are able to infect a relatively broader range of cells efficiently, some cell types are refractory to infection by these viruses as well. Attachment to and entry into a susceptible cell is mediated by the protein envelope on the surface of a virus. Retroviruses and adeno-associated viruses have a single protein coating their membrane, while adenoviruses are coated with both an envelope protein and fibers that extend away from the surface of the virus. The envelope proteins on each of these viruses bind to cell-surface molecules such as heparin sulfate, which localizes them upon the surface of the potential host, as well as with the specific protein receptor that either induces entry-promoting structural changes in the viral protein, or localizes the virus in endosomes wherein acidification of the lumen induces this refolding of the viral coat. In either case, entry into potential host cells requires a favorable interaction between a protein on the surface of the virus and a protein on the surface of the cell. For the purposes of gene therapy, one might either want to limit or expand the range of cells susceptible to transduction by a gene therapy vector. To this end, many vectors have been developed in which the endogenous viral envelope proteins have been replaced by either envelope proteins from other viruses, or by chimeric proteins. Such chimera would consist of those parts of the viral protein necessary for incorporation into the virion as well as sequences meant to interact with specific host cell proteins. Viruses in which the envelope proteins have been replaced as described are referred to as pseudotyped viruses. For example, the most popular retroviral vector for use in gene therapy trials has been the lentivirus Simian immunodeficiency virus coated with the envelope proteins, G-protein, from Vesicular stomatitis virus. This vector is referred to as VSV G-pseudotyped lentivirus, and infects an almost universal set of cells. This tropism is characteristic of the VSV G-protein with which this vector is coated. Many attempts have been made to limit the tropism of viral vectors to one or a few host cell populations. This advance would allow for the systemic administration of a relatively small amount of vector. The potential for off-target cell modification would be limited, and many concerns from the medical community would be alleviated. Most attempts to limit tropism have used chimeric envelope proteins bearing antibody fragments. These vectors show great promise for the development of "magic bullet" gene therapies.

Non-viral methods

Non-viral methods present certain advantages over viral methods, with simple large scale production and low host immunogenicity being just two. Previously, low levels of transfection and expression of the gene held non-viral methods at a disadvantage; however, recent advances in vector technology have yielded molecules and techniques with transfection efficiencies similar to those of viruses.

Naked DNA

This is the simplest method of non-viral transfection. Clinical trials carried out of intramuscular injection of a naked DNA plasmid have occurred with some success; however, the expression has been very low in comparison to other methods of transfection. In addition to trials with plasmids, there have been trials with naked PCR product, which have had similar or greater success. This success, however, does not compare to that of the other methods, leading to research into more efficient methods for delivery of the naked DNA such as electroporation, sonoporation, and the use of a "gene gun", which shoots DNA coated gold particles into the cell using high pressure gas.

Oligonucleotides

The use of synthetic oligonucleotides in gene therapy is to inactivate the genes involved in the disease process. There are several methods by which this is achieved. One strategy uses antisense specific to the target gene to disrupt the transcription of the faulty gene. Another uses small molecules of RNA called siRNA to signal the cell to cleave specific unique sequences in the mRNA transcript of the faulty gene, disrupting translation of the faulty mRNA, and therefore expression of the gene. A further strategy uses double stranded oligodeoxynucleotides as a decoy for the transcription factors that are required to activate the transcription of the target gene. The transcription factors bind to the decoys instead of the promoter of the faulty gene, which reduces the transcription of the target gene, lowering expression. Additionally, single stranded DNA oligonucleotides have been used to direct a single base change within a mutant gene. The oligonucleotide is designed to anneal with complementarity to the target gene with the exception of a central base, the target base, which serves as the template base for repair. This technique is referred to as oligonucleotide mediated gene repair, targeted gene repair, or targeted nucleotide alteration.

Lipoplexes and polyplexes

To improve the delivery of the new DNA into the cell, the DNA must be protected from damage and its entry into the cell must be facilitated. To this end new molecules, lipoplexes and polyplexes, have been created that have the ability to protect the DNA from undesirable degradation during the transfection process.

Plasmid DNA can be covered with lipids in an organized structure like a micelle or a liposome. When the organized structure is complexed with DNA it is called a lipoplex. There are three types of lipids, anionic (negatively charged), neutral, or cationic (positively charged). Initially, anionic and neutral lipids were used for the construction of lipoplexes for synthetic vectors. However, in spite of the facts that there is little toxicity associated with them, that they are compatible with body fluids and that there was a possibility of adapting them to be tissue specific; they are complicated and time consuming to produce so attention was turned to the cationic versions.

Cationic lipids, due to their positive charge, were first used to condense negatively charged DNA molecules so as to facilitate the encapsulation of DNA into liposomes. Later it was found that the use of cationic lipids significantly enhanced the stability of lipoplexes. Also as a result of their charge, cationic liposomes interact with the cell membrane, endocytosis was widely believed as the major route by which cells uptake lipoplexes. Endosomes are formed as the results of endocytosis, however, if genes can not be released into cytoplasm by breaking the membrane of endosome, they will be sent to lysosomes where all DNA will be destroyed before they could achieve their functions. It was also found that although cationic lipids themselves could condense and encapsulate DNA into liposomes, the transfection efficiency is very low due to the lack of ability in terms of “endosomal escaping”. However, when helper lipids (usually electroneutral lipids, such as DOPE) were added to form lipoplexes, much higher transfection efficiency was observed. Later on, it was figured out that certain lipids have the ability to destabilize endosomal membranes so as to facilitate the escape of DNA from endosome, therefore those lipids are called fusogenic lipids. Although cationic liposomes have been widely used as an alternative for gene delivery vectors, a dose dependent toxicity of cationic lipids were also observed which could limit their therapeutic usages.

The most common use of lipoplexes has been in gene transfer into cancer cells, where the supplied genes have activated tumor suppressor control genes in the cell and decrease the activity of oncogenes. Recent studies have shown lipoplexes to be useful in transfecting respiratory epithelial cells, so they may be used for treatment of genetic respiratory diseases such as cystic fibrosis.

Complexes of polymers with DNA are called polyplexes. Most polyplexes consist of cationic polymers and their production is regulated by ionic interactions. One large difference between the methods of action of polyplexes and lipoplexes is that polyplexes cannot release their DNA load into the cytoplasm, so to this end, co-transfection with endosome-lytic agents (to lyse the endosome that is made during endocytosis, the process by which the polyplex enters the cell) such as inactivated adenovirus must occur. However, this isn't always the case, polymers such as polyethylenimine have their own method of endosome disruption as does chitosan and trimethylchitosan.

Hybrid methods

Due to every method of gene transfer having shortcomings, there have been some hybrid methods developed that combine two or more techniques. Virosomes are one example; they combine liposomes with an inactivated HIV or influenza virus. This has been shown to have more efficient gene transfer in respiratory epithelial cells than either viral or liposomal methods alone. Other methods involve mixing other viral vectors with cationic lipids or hybridising viruses.

Dendrimers

A dendrimer is a highly branched macromolecule with a spherical shape. The surface of the particle may be functionalized in many ways and many of the properties of the resulting construct are determined by its surface.

In particular it is possible to construct a cationic dendrimer, i.e. one with a positive surface charge. When in the presence of genetic material such as DNA or RNA, charge complimentarity leads to a temporary association of the nucleic acid with the cationic dendrimer. On reaching its destination the dendrimer-nucleic acid complex is then taken into the cell via endocytosis.

In recent years the benchmark for transfection agents has been cationic lipids. Limitations of these competing reagents have been reported to include: the lack of ability to transfect a number of cell types, the lack of robust active targeting capabilities, incompatibility with animal models, and toxicity. Dendrimers offer robust covalent construction and extreme control over molecule structure, and therefore size. Together these give compelling advantages compared to existing approaches.

Producing dendrimers has historically been a slow and expensive process consisting of numerous slow reactions, an obstacle that severely curtailed their commercial development. The Michigan based company Dendritic Nanotechnologies discovered a method to produce dendrimers using kinetically driven chemistry, a process that not only reduced cost by a magnitude of three, but also cut reaction time from over a month to several days. These new "Priostar" dendrimers can be specifically constructed to carry a DNA or RNA payload that transfects cells at a high efficiency with little or no toxicity.

Major developments in gene therapy

2002 and earlier

New gene therapy approach repairs errors in messenger RNA derived from defective genes. This technique has the potential to treat the blood disorder thalassaemia, cystic fibrosis, and some cancers. See Subtle gene therapy tackles blood disorder at NewScientist.com (October 11, 2002).

Researchers at Case Western Reserve University and Copernicus Therapeutics are able to create tiny liposomes 25 nanometers across that can carry therapeutic DNA through pores in the nuclear membrane. See DNA nanoballs boost gene therapy at NewScientist.com (May 12, 2002).

Sickle cell disease is successfully treated in mice. See Murine Gene Therapy Corrects Symptoms of Sickle Cell Disease from March 18, 2002, issue of The Scientist.

The success of a multi-center trial for treating children with SCID (severe combined immune deficiency or "bubble boy" disease) held from 2000 and 2002 was questioned when two of the ten children treated at the trial's Paris center developed a leukemia-like condition. Clinical trials were halted temporarily in 2002, but resumed after regulatory review of the protocol in the United States, the United Kingdom, France, Italy, and Germany. (V. Cavazzana-Calvo, Thrasher and Mavilio 2004; see also 'Miracle' gene therapy trial halted at NewScientist.com, October 3, 2002). NO current resourse aquired

In 1993 Andrew Gobea was born with a rare, normally fatal genetic disease - severe combined immunodeficiency (SCID). Genetic screening before birth showed that he had SCID. Blood was removed from Andrew's placenta and umbilical cord immediately after birth, containing stem cells. The allele that codes for ADA was obtained and was inserted into a retrovirus. Retroviruses and stem cells were mixed, after which they entered and inserted the gene into the stem cells' chromosomes. Stem cells containing the working ADA gene were injected into Andrew's blood system via a vein. For four years T-cells (white blood cells), produced by stem cells, made ADA enzymes using the ADA gene. After four years more treatment was needed.

2003

In 2003 a University of California, Los Angeles research team inserted genes into the brain using liposomes coated in a polymer called polyethylene glycol (PEG). The transfer of genes into the brain is a significant achievement because viral vectors are too big to get across the "blood-brain barrier." This method has potential for treating Parkinson's disease. See Undercover genes slip into the brain at NewScientist.com (March 20, 2003).

RNA interference or gene silencing may be a new way to treat Huntington's. Short pieces of double-stranded RNA (short, interfering RNAs or siRNAs) are used by cells to degrade RNA of a particular sequence. If a siRNA is designed to match the RNA copied from a faulty gene, then the abnormal protein product of that gene will not be produced. See Gene therapy may switch off Huntington's at NewScientist.com (March 13, 2003).

2006

Scientists at the National Institutes of Health (Bethesda, Maryland) have successfully treated metastatic melanoma in two patients using killer T cells genetically retargeted to attack the cancer cells. This study constitutes the first demonstration that gene therapy can be effective in treating cancer. The study results have been published in Science (October 2006).

In May 2006 a team of scientists led by Dr. Luigi Naldini and Dr. Brian Brown from the San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET) in Milan, Italy reported a breakthrough for gene therapy in which they developed a way to prevent the immune system from rejecting a newly delivered gene. Similar to organ transplantation, gene therapy has been plagued by the problem of immune rejection. So far, delivery of the 'normal' gene has been difficult because the immune system recognizes the new gene as foreign and rejects the cells carrying it. To overcome this problem, the HSR-TIGET group utilized a newly uncovered network of genes regulated by molecules known as microRNAs. Dr. Naldini's group reasoned that they could use this natural function of microRNA to selectively turn off the identity of their therapeutic gene in cells of the immune system and prevent the gene from being found and destroyed. The researchers injected mice with the gene containing an immune-cell microRNA target sequence, and spectacularly, the mice did not reject the gene, as previously occurred when vectors without the microRNA target sequence were used. This work will have important implications for the treatment of hemophilia and other genetic diseases by gene therapy.

In March 2006 an international group of scientists announced the successful use of gene therapy to treat two adult patients for a disease affecting myeloid cells. The study, published in Nature Medicine, is believed to be the first to show that gene therapy can cure diseases of the myeloid system.

2007

On 1 May 2007 Moorfields Eye Hospital and University College London's Institute of Ophthalmology announced the world's first gene therapy trial for inherited retinal disease. The first operation was carried out on a 23 year-old British male, Robert Johnson, in early 2007. Leber's congenital amaurosis is an inherited blinding disease caused by mutations in the RPE65 gene. The results of the Moorfields/UCL trial were published in New England Journal of Medicine in April 2008. They researched the safety of the subretinal delivery of recombinant adeno associated virus (AAV) carrying RPE65 gene, and found it yielded positive results, with patients having modest increase in vision, and, perhaps more importantly, no apparent side-effects.

Problems and ethics

For the safety of gene therapy, the Weismann barrier is fundamental in the current thinking. Soma-to-germline feedback should therefore be impossible. However, there are indications that the Weissman barrier can be breached. One way it might possibly be breached is if the treatment were somehow misapplied and spread to the testes and therefore would infect the germline against the intentions of the therapy.

Some of the problems of gene therapy include:

• Short-lived nature of gene therapy - Before gene therapy can become a permanent cure for any condition, the therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy.
• Immune response - Anytime a foreign object is introduced into human tissues, the immune system has evolved to attack the invader. The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a possibility. Furthermore, the immune system's enhanced response to invaders it has seen before makes it difficult for gene therapy to be repeated in patients.
• Problems with viral vectors - Viruses, while the carrier of choice in most gene therapy studies, present a variety of potential problems to the patient --toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease.
• Multigene disorders - Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy. Unfortunately, some of the most commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the combined effects of variations in many genes. Multigene or multifactorial disorders such as these would be especially difficult to treat effectively using gene therapy.
• Chance of inducing a tumor (insertional mutagenesis) - If the DNA is integrated in the wrong place in the genome, for example in a tumor suppressor gene, it could induce a tumor. This has occurred in clinical trials for X-linked severe combined immunodeficiency (X-SCID) patients, in which hematopoietic stem cells were transduced with a corrective transgene using a retrovirus, and this led to the development of T cell leukemia in 3 of 20 patients.
• Religious concerns - Religious groups and creationists may consider the alteration of an individual's genes as tampering or corrupting God's work.

Deaths have occurred due to gene therapy, including that of Jesse Gelsinger.

In popular culture

• In the TV series Dark Angel gene therapy is mentioned as one of the practices performed on transgenics and their surrogate mothers at Manticore, and in the episode Prodigy, Dr. Tanaka uses a groundbreaking new form of gene therapy to turn Jude, a premature, vegitative baby of a crack/cocaine addict, into a boy genius.

• Gene therapy is a crucial plot element in the video game Metal Gear Solid, where it has been used to enhance the battle capabilities of enemy soldiers.

• Gene therapy plays a major role in the sci-fi series Stargate Atlantis, as a certain type of alien technology can only be used if one has a certain gene which is given to the members of the team through gene therapy.

• Gene therapy also plays a major role in the plot of the James Bond movie Die Another Day.

• The Yellow Bastard from Frank Miller's Sin City was also apparently the recipient of gene therapy.

• In the The Dark Knight Strikes Again, Dick Grayson, the first Robin, becomes a victim of extensive gene therapy for years by Lex Luthor to become The Joker.

• Gene therapy plays a recurring role in the present-time sci-fi television program ReGenesis, where it is used to cure various diseases, enhance athletic performance and produce vast profits for bio-tech corporations. (e.g. an undetectable performance-enhancing gene therapy was used by one of the characters on himself, but to avoid copyright infringement, this gene therapy was modified from the tested-to-be-harmless original, which produced a fatal cardiovascular defect)

• Gene therapy is the basis for the plot line of the film I Am Legend.

• Gene therapy is an important plot key in the game Bioshock where the game contents refer to plasmids and [gene] splicers.

• The book Next by Michael Crichton unravels a story in which fictitious biotechnology companies which experiment with gene therapy are involved.

• In the television show Alias, a breakthrough in molecular gene therapy is discovered, whereby a patient's body is reshaped to identically resemble someone else. Protagonist Sydney Bristow's best friend was secretly killed and her "double" resumed her place.

Reverse genetics

Avian Flu vaccine development by Reverse Genetics techniques. Courtesy: National Institute of Allergy and Infectious Diseases

Reverse genetics is an approach to discovering the function of a gene that proceeds in the opposite direction of so called forward genetic screens of classical genetics. Simply put, while forward genetics seeks to find the genetic basis of a phenotype or trait, reverse genetics seeks to find the possible phenotypes that may derive from a specific genetic sequence obtained by DNA sequencing.

Automated DNA sequencing generates large volumes of genomic sequence data relatively rapidly. Many genetic sequences are discovered in advance of other, less easily obtained, biological information. Reverse genetics attempts to connect a given genetic sequence with specific effects on the organism.

Techniques used in reverse genetics

To learn the influence a sequence has on phenotype, or to discover its biological function, researchers can engineer a change or disruption in the DNA. After this change has been made a researcher can look for the effect of such alterations in the whole organism. There are several different methods of reverse genetics that have proved useful:

Random deletions, insertions and point mutations

These are three similar techniques that involve creating large mutagenised populations in a similar way to forward genetic screens. These populations are generated using either chemical (point mutations), gamma radiation (deletions) or DNA insertions (insertional knockouts). These large libraries of mutants can be screened for specific changes at the gene of interest using PCR. For some organisms, such as Drosophila and Arabidopsis there are large online databases that indicate the locations of all the DNA insertions in a particular library.

Directed deletions and point mutations

Site-directed mutagenesis is a sophisticated technique that can either change regulatory regions in the promoter of a gene or make subtle codon changes in the open reading frame to identify important amino residues for protein function.

Alternatively, the technique can be used to create null alleles so that the gene is not functional. For example, deletion of a gene by gene knockout can be done in some organisms, such as yeast, mice and moss. Unique among plants, in Physcomitrella patens, gene knockout via homologous recombination is nearly as efficient as in yeast. In the case of the yeast model system directed deletions have been created in every non-essential gene in the yeast genome. In the case of the plant model system huge mutant libraries have been created based on gene disruption constructs .

In some cases conditional alleles can be used that have normal function until the allele is activated. This is known as gene knocking. This might entail ‘knocking in’ recombinase sites (such as lox or frt sites) that will cause a deletion at the gene of interest when a specific recombinase (such as CRE, FLP) is induced. Cre or Flp recombinases can be induced with chemical treatments, heat shock treatments or be restricted to a specific subset of tissues.

Gene silencing

The discovery of gene silencing using double stranded RNA, also known as RNA interference (RNAi), and the development of gene knockdown using Morpholino oligos have made disrupting gene expression an accessible technique for many more investigators. This method is often referred to as a gene knockdown since the effects of these reagents are generally temporary, in contrast to gene knockouts which are permanent.

RNAi creates a specific knockout effect without actually mutating the DNA of interest. In C. elegans, RNAi has been used to systematically interfere with the expression of most genes in the genome. RNAi acts by directing cellular systems to degrade target messenger RNA (mRNA).

While RNA interference relies on cellular components for efficacy (e.g. the Dicer proteins, the RISC complex) a simple alternative for gene knockdown is Morpholino antisense oligos. Morpholinos bind and block access to the target mRNA without requiring the activity of cellular proteins and without necessarily accelerating mRNA degradation. Morpholinos are effective in systems ranging in complexity from cell-free translation in a test tube to in vivo studies in large animal models.

Interference using transgenes

A molecular genetic approach is the creation of transgenic organisms that overexpress a normal gene of interest. The resulting phenotype may reflect the normal function of the gene.

Alternatively it is possible to overexpress mutant forms of a gene that interfere with the normal (wildtype) genes function. For example, over expression of a mutant gene may result in high levels of a non-functional protein resulting in a dominant negative interaction with the wildtype protein. In this case the mutant version will out compete for the wildtype proteins partners resulting in a mutant phenotype.

Other mutant forms can result in a protein that is abnormally regulated and constitutively active (‘on’ all the time). This might be due to removing a regulatory domain or mutating a specific amino residue that is reversibly modified (by phosphorylation methylation or ubiquitination). Either change is critical for modulating protein function and often result in informative phenotypes.

Molecular genetics

Molecular genetics is the field of biology which studies the structure and function of genes at a molecular level. The field studies how the genes are transferred from generation to generation. Molecular genetics employs the methods of genetics and molecular biology. It is so-called to differentiate it from other sub fields of genetics such as ecological genetics and population genetics. An important area within molecular genetics is the use of molecular information to determine the patterns of descent, and therefore the correct scientific classification of organisms: this is called molecular systematics.

Along with determining the pattern of descendants, molecular genetics helps in understanding genetic mutations that can cause certain types of diseases. Through utilizing the methods of genetics and molecular biology, molecular genetics discovers the reasons why traits are carried on and how and why some may mutate.

Forward genetics

One of the first tools available to molecular geneticists is the forward genetic screen. The aim of this technique is to identify mutations that produce a certain phenotype. A mutagen is very often used to accelerate this process. Once mutants have been isolated, the mutated gene can be molecularly identified.

Reverse genetics

While forward genetic screens are productive, a more straightforward approach would be to determine the phenotype that results from mutating a given gene. This is called reverse genetics. In some organisms, such as yeast and mice, it is possible to induce the deletion of a particular gene, creating a gene knockout. Alternatives include the random induction of DNA deletions and subsequent selection for deletions in a gene of interest, the application of RNA interference and the creation of transgenic organisms that do not express a gene of interest.

Gene Therapy

A mutation in a gene can result in a severe medical condition. A protein encoded by a mutated gene may malfunction and cells that rely on the protein might therefore fail to function properly. This can cause problems for specific tissues or organs, or for the entire body. This might manifest through the course of development (like a cleft palate) or as an abnormal response to stimuli (like a peanut allergy). Conditions related to gene mutations are called genetic disorders. One way to fix such a physiological problem is gene therapy. By adding a corrected copy of the gene, a functional form of the protein can be produced, and affected cells, tissues, and organs may work properly. As opposed to drug-based approaches, gene therapy repairs the underlying genetic defect.

Gene therapy is the process of treating or alleviating diseases by genetically modifying the cells of the affected person, causing the gene to function properly. When a human disease gene has been recognized, molecular genetics tools can be used to explore the process of the gene in both the normal and mutant states. From there, the gene is transferred either in vivo or ex vivo and the body begins to make proteins according to the instructions in the new gene. Gene therapy has to be repeated several times for the infected patient to continually be relieved, however, as repeated cell division and death slowly randomizes the body's ratio of functional-to-mutant genes.

Currently, gene therapy is still being experimented with and products are not approved by the U.S. Food and Drug Administration. There have been several setbacks in the last 15 years that have restricted further developments in gene therapy. As there are unsuccessful attempts, there continue to be a growing number of successful gene therapy transfers which have furthered the research.

Major diseases that can be treated with gene therapy include viral infections, cancers, and inherited disorders, including immune system disorders.

Classical gene therapy

Classical gene therapy is the approach which delivers genes, via a modified virus or "vector" to the appropriate target cells with a goal of attaining optimal expression of the new, introduced gene. Once inside the patient, the expressed genes are intended to produce a product that the patient lacks, kill diseased cells directly by producing a toxin, or activate the immune system to help the killing of diseased cells.

Nonclassical gene therapy

Nonclassical gene therapy inhibits the expression of genes related to pathogenesis, or corrects a genetic defect and restores normal gene expression.

In vivo gene transfer

During In vivo gene transfer, the genes are transferred directly into the tissue of the patient and this can be the only possible option in patients with tissues where individual cells cannot be cultured in vitro in sufficient numbers (e.g. brain cells). Also, in vivo gene transfer is necessary when cultured cells cannot be re-implanted in patients effectively.

Ex vivo gene transfer

During ex vivo gene transfer the cells are cultured outside the body and then the genes are transferred into the cells grown in culture. The cells that have been transformed successfully are expanded by cell culture and then introduced into the patient.

Principles for gene transfer

Classical gene therapies usually require efficient transfer of cloned genes into the disease cells so that the introduced genes are expressed atsufficiently high levels to change the patient's physiology. There are several different physicochemical and biological methods that can be used to transfer genes into human cells. The size of the DNA fragments that can be transferred is very limited, and often the transferred gene is not a conventional gene. Horizontal gene transfer is the transfer of genetic material from one cell to another that is not its offspring. Artificial horizontal gene transfer is a form of genetic engineering.

Techniques in Molecular Genetics

There are three general techniques used for molecular genetics: amplification, separation and detection, and expression. Specifically used for amplification is polymerase chain reaction, which is an “indispensable tool in a great variety of applications”. In the separation and detection technique DNA and mRNA are isolated from their cells. Gene expression in cells or organisms is done in a place or time that is not normal for that specific gene.

Amplification

There are other methods for amplification besides polymerase chain reaction. Cloning DNA in bacteria is also a way to amplify DNA in genes.

Polymerase chain reaction

The main materials used in polymerase chain reaction are DNA nucleotides, template DNA, primers and Taq polymerase. DNA nucleotides are the base for the new DNA, the template DNA is the specific sequence being amplified, primers are complementary nucleotides that can go on either side of the template DNA, and Taq polymerase is a heat stable enzyme that jump-starts the production of new DNA. This technique does not need to use living bacteria or cells, all that is needed is the base sequence of the DNA needing amplification.

Cloning DNA in Bacteria

The word cloning for this type of amplification entails making multiple identical copies of a sequence of DNA. The target DNA sequence is then inserted into a [cloning vector]. Because this vector originates from a self-replicating virus, plasmid, or higher organism cell when the appropriate size DNA is inserted the “target and vector DNA fragments are then ligated”^[3] and create a recombinant DNA molecule. The recombinant DNA molecules are then put into a bacteria strain (usually E. coli) which produces several identical copies by transformation. Transformation is the DNA uptake mechanism possessed by bacteria. However, only one recombinant DNA molecule can be cloned within a single bacteria cell, so each clone is of just one DNA insert.

Separation and Detection

In separation and detection DNA and mRNA are isolated from cells (the separation) and then detected simply by the isolation. Cell cultures are also grown to provide a constant supply of cells ready for isolation.

Cell Cultures

A cell culture for molecular genetics is a culture that is grown in artificial conditions. Some cell types grow well in cultures such a skin cells, but other cells are not as productive in cultures. There are different techniques for each type of cell, some only recently being found to foster growth in stem and nerve cells. Cultures for molecular genetics are frozen in order to preserve all copies of the gene specimen and thawed only when needed. This allows for a steady supply of cells.

DNA Isolation

DNA isolation extracts DNA from a cell in a pure form. First, the DNA is separated from cellular components such as proteins, RNA, and lipids. This is done by placing the chosen cells in a tube with a solution that mechanically, chemically, breaks the cells open. This solution contains enzymes, chemicals, and salts that breaks down the cells except for the DNA. It contains enzymes to dissolve proteins, chemicals to destroy all RNA present, and salts to help pull DNA out of the solution.

Next, the DNA is separated from the solution by being spun in a centrifuge, which allows the DNA to collect in the bottom of the tube. After this cycle in the centrifuge the solution is poured off and the DNA is resuspended in a second solution that makes the DNA easy to work with in the future.

This results in a concentrated DNA sample that contains thousands of copies of each gene. For large scale projects such as sequencing the human genome, all this work is done by robots.

mRNA Isolation

Expressed DNA that codes for the synthesis of a protein is the final goal for scientists and this expressed DNA is obtained by isolation mRNA (Messenger RNA). First, laboratories use a normal cellular modification of mRNA that adds up to 200 adenine nucleotides to the end of the molecule (poly(A) tail). Once this has been added, the cell is ruptured and its cell contents are exposed to synthetic beads that are coated with thymine string nucleotides. Because Adenine and Thymine pair together in DNA, the poly(A) tail and synthetic beads are attracted to one another, and once they bind in this process the cell components can be washed away without removing the mRNA. Once the mRNA has been isolated, reverse transcriptase is employed to convert it to single-stranded DNA, from which a stable double-stranded DNA is produced using DNA polymerase. Complementary DNA (cDNA) is much more stable than mRNA and so, once the double-stranded DNA has been produced it represents the expressed DNA sequence scientists look for.

The Molecular Genetics Project

The Human Genome Project is a molecular genetics project that began in the 1980s and was projected to take fifteen years to complete. However, because of technological advances the progress of the project was advanced and the project finished in 2003, taking only thirteen years. The project was started by the U.S. Department of Energy and the National Institutes of Health in an effort to reach six set goals. These goals included:

1. identifying 20,000 to 25,000 genes in human DNA (although initial estimate were approximately 100,000 genes),
2. determining sequences of chemical based pairs in human DNA,
3. storing all found information into databases,
4. improving the tools used for data analysis,
5. transferring technologies to private sectors, and
6. addressing the ethical, legal, and social issues (ELSI) that may arise from the projects.

The project was worked on by eighteen different countries including the United States, Japan, France, Germany, and the United Kingdom. The collaborative effort resulted in the discovery of the many benefits of molecular genetics. Discoveries such as molecular medicine, new energy sources and environmental applications, DNA forensics, and livestock breeding, are only a few of the benefits that molecular genetics can provide.

Microbial genetics

Microbial Genetics is a subject area within biotechnology and genetic engineering. It studies the genetics of very small (micro) organisms. This involves the study of the genotype of microbial species and also the expression system in the form of phenotypes.

Heritability

In genetics, Heritability is the proportion of phenotypic variation in a population that is attributable to genetic variation among individuals. Variation among individuals may be due to genetic and/or environmental factors. Heritability analyses estimate the relative contributions of differences in genetic and non-genetic factors to the total phenotypic variance in a population.

Definition

Figure 1. Relationship of phenotypic values to additive and dominance effects using a completely dominant locus.

Consider a statistical model for describing some particular phenotype:

Phenotype (P) = Genotype (G) + Environment (E).

Considering variances (Var), this becomes:

Var(P) = Var(G) + Var(E) + 2 Cov(G,E).

In planned experiments, we can often take Cov(G,E) = 0. Heritability is then defined as:

.

The parameter H² is the broad-sense heritability and reflects all possible genetic contributions to a population's phenotypic variance. Included are effects due to allelic variation (additive variance), dominance variation or which act epistatically (multi-genic interactions), as well as maternal and paternal effects, where individuals are directly affected of their parents' phenotype (such as with milk production in mammals).

These additional terms can be included in genetic models. For example, the simplest genetic model involves a single locus with two alleles that effect some quantitative phenotype, as shown by + in Figure 1. We can calculate the linear regression of phenotype on the number of B alleles (0, 1, or 2), which is shown as the Linear Effect line. For any genotype, B_iB_j, the expected phenotype can then be written as the sum of the overall mean, a linear effect, and a dominance deviation:

P_ij = μ + α_i + α_j + dij = Population mean + Additive Effect (a_ij = α_i + α_j) + Dominance Deviation (d_ij).

The additive genetic variance is the weighted average of the squares of the additive effects:

where f(bb)a_bb + f(Bb)a_Bb + f(BB)a_BB = 0.

There is a similar relationship for variance of dominance deviations:

where f(bb)d_bb + f(Bb)d_Bb + f(BB)d_BB = 0.

Narrow-sense heritability is defined as

and quantifies only the portion of the phenotypic variation that is additive (allelic) by nature (note upper case H² for broad sense, lower case h² for narrow sense). When interested in improving livestock via artificial selection, for example, knowing the narrow-sense heritability of the trait of interest will allow predicting how much the mean of the trait will increase in the next generation as a function of how much the mean of the selected parents differs from the mean of the population from which the selected parents were chosen. The observed response to selection leads to an estimate of the narrow-sense heritability (called realized heritability).

Estimating heritability

Estimating heritability is not a simple process, since only P can be observed or measured directly. Measuring the genetic and environmental variance requires various sophisticated statistical methods. These methods give better estimates when using data from closely related individuals - such as brothers, sisters, parents and offspring, rather than from more distantly related ones. The standard error for heritability estimates are generally very poor unless the dataset is large.

Figure 2. Heritability for nine psychological traits as estimated from twin studies. All sources are twins raised together (sample size shown inside bars). MZ: Monozygotic twins, DZ: Dizygotic twins

In non-human populations it is often possible to collect information in a controlled way. For example, among farm animals it is easy to arrange for a bull to produce offspring from a large number of cows. Due to ethical concerns, such a degree of experimental control is impossible when gathering human data. As a result, studies of human heritability often contrast identical twins who have been separated early in life and raised in different environments (see for example Fig. 2). Such individuals have identical genotypes and can be used to separate the effects of genotype and environment. Twin studies entail problems of its own, such as: independently raised twins shared a common prenatal environment; they may have undergone intrauterine competition; the mother may be more physically stressed (less nutrients); and twins reared apart are difficult to find, and may reflect certain types of environments.

Heritability estimates are always relative to the genetic and environmental factors in the population, and are not absolute measurements of the contribution of genetic and environmental factors to a phenotype. Heritability estimates reflect the amount of variation in genotypic effects compared to variation in environmental effects. Heritability can be made larger by diversifying the genetic background, e.g., by using only very outbred individuals (which increases the Variance(G)) and/or by minimizing environmental effects (which decreases the Variance(E)). Smaller heritability, on the other hand, can be generated by using inbred individuals (which decreases the Variance(G)) or individuals reared in very diverse environments (which increases the Variance(E)). Due to such effects, different populations of a species might have different heritabilities even for the same trait.

In observational studies G and E may be correlated, giving rise to gene environment correlation. Depending on the methods used to estimate heritability, correlations between genetic factors and shared or non-shared environments may or may not be included in the total heritability estimate.

Because of the contextual nature of measured heritabilities, paradoxes often arise. For example, the heritability of a trait could be near 100% in one study and close to zero in another. In one study, e.g., a group of unrelated army recruits may be given identical training and nutrition and then their muscular strength may be measured. The variation in strength observed after the (identical) training will translate into a high heritability estimate. In another study, whose purpose might be to assess the efficacy of various workout regimes or nutritional programs, study subjects may be first chosen to match each other as closely as possible in prior physical characteristics before some of them are put onto Program A and others onto Program B, and this will lead to a low heritability estimate.

Heritability estimates are often misinterpreted. Heritability refers to the proportion of variation between individuals in a population that is influenced by genetic factors. Heritability describes the population, not individuals within that population. For example, It is incorrect to say that since the heritability of a personality trait is about .6, that means that 60% of your personality is inherited from your parents and 40% comes from the environment. The heritability estimate changes according to the genetic and environmental variability present in the population. In studies of genetically identical inbred animals, all traits have zero heritability. Heritability estimates can be much higher in outbred (genetically variable) populations under very homogeneous environments.

A highly genetically loaded trait (such as eye color) still assumes environmental input within normal limits (a certain range of temperature, oxygen in the atmosphere, etc.). A more useful distinction than "nature vs. nurture" is "obligate vs. facultative" -- under typical environmental ranges, what traits are more "obligate" (e.g., the nose -- everyone has a nose) or more "facultative" (sensitive to environmental variations, such as specific language learned during infancy). Another useful distinction is between traits that are likely to be adaptations (such as the nose) vs. those that are byproducts of adaptations (such the white color of bones), or are due to random variation (non-adaptive variation in, say, nose shape or size).

Estimation methods

There are essentially two schools of thought regarding estimation of heritability.

One school of thought was developed by Sewall Wright at The University of Chicago, and further popularized by C. C. Li (University of Chicago) and J. L. Lush (Iowa State University). It is based on the analysis of correlations and, by extension, regression. Path Analysis was developed by Sewall Wright as a way of estimating heritability.

The second was originally developed by R. A. Fisher and expanded at The University of Edinburgh, Iowa State University, and North Carolina State University, as well as other schools. It is based on the analysis of variance of breeding studies, using the intraclass correlation of relatives. Various methods of estimating components of variance (and, hence, heritability) from ANOVA are used in these analyses.

Regression/correlation methods of estimation

The first school of estimation uses regression and correlation to estimate heritability.

Selection experiments

Figure 3. Strength of selection (S) and response to selection (R) in an artificial selection experiment, h²=R/S.

Calculating the strength of selection, S (the difference in mean trait between the population as a whole and the selected parents of the next generation, also called the selection differential ^[3]) and response to selection R (the difference in offspring and whole parental generation mean trait) in an artificial selection experiment will allow calculation of realized heritability as the response to selection relative to the strength of selection, h²=R/S as in Fig. 3.

Comparison of close relatives

In the comparison of relatives, we find that in general,

where r can be thought of as the coefficient of relatedness, b is the coefficient of regression and t the coefficient of correlation.

Parent-offspring regression

Figure 4. Sir Francis Galton's (1889) data showing the relationship between offsping height (928 individuals) as a function of mean parent height (205 sets of parents).

Heritability may be estimated by comparing parent and offspring traits (as In Fig. 4). The slope of the line (0.57) approximates the heritability of the trait when offspring values are regressed against the average trait in the parents. If only one parent's value is used then heritability is twice the slope. (note that this is the source of the term "regression", since the offspring values always tend to regress to the mean value for the population, i.e., the slope is always less than one).

Full-sib comparison

Full-sib designs compare phenotypic traits of siblings that share a mother and a father with other sibling groups. The estimate of the sibling phenotypic correlation is an index on familiality which is equal to half the additive genetic variance plus the common environment variance when there is only additive gene action.

Half-sib comparison

Half-sib designs compare phenotypic traits of siblings that share one parent with other sibling groups.

Twin studies

Figure 5. Twin concordances for seven psychological traits (sample size shown inside bars).

Heritability for traits in humans is most frequently estimated by comparing resemblances between twins (Fig. 2 & 5). Identical twins (MZ twins) are twice as genetically similar as fraternal twins (DZ twins) and so heritability is approximately twice the difference in correlation between MZ and DZ twins, h²=2(r(MZ)-r(DZ)). The effect of shared environment, c², contributes to similarity between siblings due to the commonality of the environment they are raised in. Shared environment is approximated by the DZ correlation minus half heritability, which is the degee to which DZ twins share the same genes, c²=DZ-1/2h². Unique environmental variance, e², reflects the degree to which identical twins raised together are dissimilar, e²=1-r(MZ).

The methodology of the classical twin study has been criticized, but these criticisms do not take into account the methodological innovations and refinements described above.

Analysis of variance methods of estimation

The second set of methods of estimation of heritability involves ANOVA and estimation of variance components.

Basic model

We use the basic discussion of Kempthorne (1957 [1969]). Considering only the most basic of genetic models, we can look at the quantitative contribution of a single locus with genotype G_i as

y_i = μ + g_i + e

where

g_i is the effect of genotype G_i

and e is the environmental effect.

Consider an experiment with a group of sires and their progeny from random dams. Since the progeny get half of their genes from the father and half from their (random) mother, the progeny equation is

Intraclass correlations

Consider the experiment above. We have two groups of progeny we can compare. The first is comparing the various progeny for an individual sire (called within sire group). The variance will include terms for genetic variance (since they did not all get the same genotype) and environmental variance. This is thought of as an error term.

The second group of progeny are comparisons of means of half sibs with each other (called among sire group). In addition to the error term as in the within sire groups, we have an addition term due to the differences among different means of half sibs. The intraclass correlation is

,

since environmental effects are independent of each other.

The ANOVA

In an experiment with n sires and r progeny per sire, we can calculate the following ANOVA, using V_g as the genetic variance and V_e as the environmental variance:

Table 1: ANOVA for Sire experiment

Source	d.f.	Mean Square	Expected Mean Square
Among sire groups	n − 1	S
Within sire groups	n(r − 1)	W

The term is the intraclass correlation among half sibs. We can easily calculate . The Expected Mean Square is calculated from the relationship of the individuals (progeny within a sire are all half-sibs, for example), and an understanding of intraclass correlations.

Model with additive and dominance terms

For a model with additive and dominance terms, but not others, the equation for a single locus is

y_ij = μ + α_i + α_j + d_ij + e,

where

α_i is the additive effect of the i^th allele, α_j is the additive effect of the j^th allele, d_ij is the dominance deviation for the ij^th genotype, and e is the environment.

Experiments can be run with a similar setup to the one given in Table 1. Using different relationship groups, we can evaluate different intraclass correlations. Using Va as the additive genetic variance and Vd as the dominance deviation variance, intraclass correlations become linear functions of these parameters. In general,

Intraclass correlation = rV_a + θV_d,

where r and θ are found as

r = P[ alleles drawn at random from the relationship pair are identical by descent], and

θ = P[ genotypes drawn at random from the relationship pair are identical by descent].

Some common relationships and their coefficients are given in Table 2.

Table 2: Coeffients for calculating variance components

Relationship	r	θ
Identical Twins	1	1
Parent-Offspring		0
Half Siblings		0
Full Siblings
First Cousins		0
Double First Cousins

Larger models

When a large, complex pedigree is available for estimating heritability, the most efficient use of the data is in a restricted maximum likelihood (REML) model. The raw data will usually have three or more datapoints for each individual: a code for the sire, a code for the dam and one or several trait values. Different trait values may be for different traits or for different timepoints of measurement. The currently popular methodology relies on high degrees of certainty over the identities of the sire and dam; it is not common to treat the sire identity probabilistically. This is not usually a problem, since the methodology is rarely applied to wild populations (although it has been used for several wild ungulate and bird populations), and sires are invariably known with a very high degree of certainty in breeding programmes. There are also algorithms that account for uncertain paternity.

The pedigrees can be viewed using programs such as Pedigree Viewer , and analysed with programs such as ASReml, VCE , WOMBAT or BLUPF90 family's programs.

Response to Selection

In selective breeding of plants and animals, the expected response to selection can be estimated by the following equation:

R = h²S

In this equation, the Response to Selection (R) is defined as the realized average difference between the parent generation and the next generation. The Selection Differential (S) is defined as the average difference between the parent generation and the selected parents.

For example, imagine that a plant breeder is involved in a selective breeding project with the aim of increasing the number of kernels per ear of corn. For the sake of argument, let us assume that the average ear of corn in the parent generation has 100 kernels. Let us also assume that the selected parents produce corn with an average of 120 kernels per ear. If h² equals 0.5, then the next generation will produce corn with an average of 0.5(120-100) = 10 additional kernels per ear. Therefore, the total number of kernels per ear of corn will equal, on average, 110.