John Wiley & Sons - 2004 - Analysis of Genes and Genomes

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In vivo

Ex vivo

Figure 13.7. In vivo and ex vivo human gene therapy. See the text for details

cultured in vitro for extended periods. Therefore, disorders of the blood system may be treated ex vivo through the isolation and culturing of bone marrow stem cells. These cells can be modified in vitro and the resulting transgenic cells can then be returned to the patient. The modified stem cells will then produce the various modified differentiated cells that may cure the disease. In vitro manipulation of the cells permits the use of a wide variety of methods to insert the transgene – e.g. viral infection, injection and other methods (see Chapter 12). Other cells and tissues are less amenable to ex vivo treatment. For example, lung epithelial cells – whose function is severely impaired in cystic fibrosis patients – grow very poorly in culture. Even if they could be cultured, it would not be possible to repopulate an affected lung with transgenic lung epithelial cells. Therefore, diseases such as cystic fibrosis must be treated in vivo so that the cells of the defective lung can be modified. This limits the type of transgene insertion that can take place. Viruses, e.g. adenovirus that naturally infect epithelial cells, are usually used to transport the transgene into the affected cells.

13.5Examples and Potential of Gene Therapy

The history of human gene therapy trails is not a particularly happy one. With one notable exception (see below), the effect of introducing a gene into cells rarely promotes more than a transient relief from the symptoms of the disease being treated. Worse still, there have been highly publicized cases where gene

therapy trial patients have suffered as a consequence of the treatment itself. For example, in 1999 an 18-year-old gene therapy trial volunteer from Philadelphia, Jesse Gelsinger, died following a gene therapy trial (Teichler Zallen, 2000). Gelsinger had an ornithine transcarbamylase (OTC) disorder, a rare genetic defect of the liver that renders the body unable to clear ammonia from the bloodstream. He was treated with an adenoviral vector as a mechanism to insert a healthy copy of the gene into his liver, but the virus itself elicited a massive immune reaction that resulted in his death. Cases such as this graphically illustrate the need for the development of vectors characterized by maximum transfection efficiency and minimal toxicity.

Some gene therapy successes have, however, been noted. Children born with severe combined immune deficiency, X-SCID, have a poor prognosis using traditional medicines. The disease is caused by a mutation on the X chromosome in the gene encoding the gamma chain (γ c) of the interleukin-2 receptor. Mutations in this gene prevent two types of white blood cell, the T-cells and natural killer cells, from developing normally (Sugamura et al., 1996). With little or no defence against infection, sufferers usually die within the first year of life unless a bone marrow donor can be found. Stem cells were collected from the bone marrow of an affected infant and treated with a retrovirus carrying a wild-type copy of the γ c gene (Cavazzana-Calvo et al., 2000). When the transgenic stem cells were returned to the infant they were capable of generating all of the cells required for a fully functional immune system for at least 10 months (Fischer, Hacein-Bey and Cavazzana-Calvo, 2002). Removing the bone marrow cells from the body prior to infection with the retrovirus eliminates the danger of acute reaction to the virus itself, and also ensures that the virus only infects the correct cells. Repopulating the immune system with a relatively small number of transgenic bone marrow cells may also cause problems. The treatment specifically selects for proliferating cells and may therefore increase the risk of bone marrow related cancers. It has been noted that some of patients treated in this way develop leukaemia (one out of 10 patients successfully treated), attributed to a result of the integration of the foreign DNA fragments into the genome at random locations. In this case, the retrovirus inserted the therapeutic gene into the regulatory region of a gene called Lmo2 on chromosome 11 (Gansbacher¨ et al., 2003). The activation of the therapeutic gene appeared to cause the expression of Lmo2 which is an oncogene (Davenport, Neale and Goorha, 2000). Even with these problems, these experiments represent the only example to date where a patient is apparently completely cured using gene therapy.

Some of the problems associated with random integration of the transgene during gene therapy may be addressed by utilizing site-specific recombination

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systems. For example, DNA fragments have been constructed such that they contain a therapeutic gene adjacent to the recognition sequence of a site-specific recombinase enzyme. If these DNA fragments are injected into the tail veins of mice together with a DNA fragment encoding the integrase, then site-specific genomic integration of the transgene occurs (Olivares et al., 2002). This could be developed from the mouse model into a human therapy.

Other gene therapy trails are currently ongoing for both genetic and nonheritable diseases.

• Haemophilia B. Sufferers lack the gene for factor IX, a critical agent in the blood clotting process. Parvoviruses have been used to insert the missing gene into skeletal muscle cells (High, 2001). The cells then generate the missing factor, thereby removing the need for daily injections of the protein itself.

•Cancer. Some cancer treatments may be amenable to gene therapy (Wadhwa et al., 2002). Modified viral vectors can be used to prime the immune system to attack cancer cells, while other approaches employ viruses to carry suicide genes into the cancer cells.

•HIV. Specifically engineered HIV may eventually be recruited to help control HIV-1 infection (Statham and Morgan, 1999).

Currently, the promise of gene therapy remains just that. Even single gene defect diseases can manifest themselves as deficiencies in a wide variety of different cell types. Being able to correct the defect in one cell type may not be sufficient to cure the disease fully. However, the development and refinement of transgene delivery systems, combined with advances in our understanding of stem cells may generate many more opportunities in the future where gene therapy may be clinically important.

402 GLOSSARY

Centromere – the point or region on a chromosome to which the spindle attaches during mitosis and meiosis

Chromatid – one of the usually paired and parallel strands of a duplicated chromosome joined by a single centromere

Chromatin – a complex of DNA and proteins in the nucleus of a cell

Chromatin immunoprecipitation (ChIP) – a method for identifying proteins bound to particular sequences of DNA

Chromosome – a discrete unit of the genome that is visible as a morphological entity during cell division. Each chromosome is a single DNA molecule

Chromosome walking – the sequential isolation of clones carrying overlapping DNA sequences that allows the sequencing of large regions of the chromosome from a single starting point

Clone – an organism, cell or molecule produced from a single ancestor Cloning vector – a plasmid or phage that is used to carry inserted foreign DNA

Codon – the triplet of nucleotides that result in the insertion of an amino acid or a termination signal into a polypeptide

Codon usage – the frequency at which amino acid codons are used for the production of proteins

Complementary – the sequences on one strand of a nucleic acid molecule can bind to their complementary partners on another strand. A = T, G = C

Conjugation – the transfer of all or part of a chromosome that occurs during bacterial mating

Conservative replication – a disproved model for DNA synthesis in which the newly synthesized DNA strands bind to each other

Contig – a continuous sequence of DNA produced from a number of smaller, overlapping fragments

Cosmid – a plasmid onto which phage lambda cos sites have been inserted. Consequently, the plasmid DNA can be packaged in vitro into the lambda phage coat Cytological map – a type of chromosome map where genes are located on the basis of

the effect that chromosome mutations have on staining patterns

Cytosine – a pyrimidine base found in DNA and RNA. Cytosine bases pairs with guanine

Denatured – in DNA, the conversion of the double-stranded form to a single-stranded form. In proteins, the conversion from an active to an inactive form

Differential display – a technique to visualize difference in the expression of genes from different sources

Dinucleotide – the joining of two nucleotides through the formation of a phosphodiester linkage

Dispersive replication – a disproved model of DNA synthesis in which a random interspersion of parental and new segments are found in daughter DNA molecules

DNA – deoxyribonucleic acid

DNA ligase – the enzyme that catalyses the formation of a phosphodiester bond between two DNA chains

DNA polymerase – the enzyme that synthesizes new DNA strands from a DNA template DNA topoisomerase – an enzyme that changes the linking number of DNA molecules

GLOSSARY 403

Electrophoresis – the application of an electric current to separate molecules (as proteins and nucleic acids) through a gel

Electroporation – a physical way to introducing DNA into cells using an electric current Epitope – the molecular region on the surface of an antigen capable of eliciting an

immune response

Euchromatic DNA – the gene-rich areas (including both exons and introns) of a genome Exon – a segment of a gene that is represented in the mature mRNA

Expressed sequence tag (EST) – small pieces of cDNA sequence generated by sequencing either one or both ends of an expressed gene

Expression vector – a cloning vector designed so that the genes cloned into it may be transcribed and translated

F episome – a large extrachromosomal circular double-stranded DNA molecule that carries bacterial fertility genes

FISH – fluorescent in situ hybridization

Frame-shift mutation – occurs when the coding sequence of a gene contains a deletion or insertion of bases that are not in multiples of three. This changes the reading frame in which translation occurs

Functional complementation – the identification of genes from one organism by their ability to counteract the defect caused by the lack of a gene in another organism

Gene – a discrete unit of genetic information that is required for the production of a polypeptide. It includes the coding sequence, the promoter and terminator, and introns

Gene knockout – the removal of a gene from the genome

Gene knock-down – the use of silencing techniques to reduce or eliminate the expression of a particular gene

Genetic engineering – the deliberate modification of the characters of an organism by the manipulation of DNA and the transformation of certain genes

Genetic marker – any DNA sequence that can be used to identify a gene or phenotypic trait associated with it

Genetic switch – the control of transcription in response to particular signals Genome – the genetic make-up of an organism

Genomic library – a collection of DNA fragments, derived from the genome of an organism, cloned into a vector

Guanine – a purine base found in DNA and RNA. Guanine base pairs with cytosine HAC – human artificial chromosome

Helper phage – provides certain functions, that are absent from a defective phage, to allow complete phage replication during a mixed infection

Hemimethylated DNA – DNA that contains methylated bases on one strand only Heterochromatic DNA – regions of the chromosome that are highly condensed and not

transcribed

Heterozygous – an individual with different alleles at a particular genomic locus Histone – a DNA binding protein that forms part of the nucleosome

Holliday junction – a central intermediate formed during recombination

Homologous recombination – DNA crossovers that occur between two homologous DNA molecules

404 GLOSSARY

Homology – a similarity often attributable to common origin

Homozygous – an individual with the same allele at a particular genomic locus Hybridization – the pairing of complementary nucleic acids (DNA – DNA or DNA –

RNA)

Hyperchromic effect – the increase in optical density that occurs when DNA become single stranded

Insertional inactivation – the destruction of the function of a gene by cloning a DNA sequence into it

Intron – a segment of DNA that is transcribed, but is removed from the transcript during splicing

Isopycnic centrifugation – the separation of the components of a mixture on the basis of differences in density

Isoschizomer – different restriction enzymes that recognize the same target DNA sequence

Karyotype – the chromosome content of an organism

Kozak sequence – the consensus sequence for the initiation of translation. A binding site for the small subunit of the ribosome

lac operon – the genes required in bacteria for the metabolism of lactose

Lagging strand – short DNA fragments are replicated discontinuously in a 5 – 3 direction and later covalently joined

Lariat – an intermediate of RNA splicing

Leading strand – the strand of DNA that is synthesized continuously during replication in a 5 – 3 direction

Melting temperature (Tm) – the temperature at the mid-point of DNA denaturation Methylated DNA – the modification of DNA bases by the addition of methyl (CH3)

groups

Microarray – sets of miniaturized reaction areas that may also be used to test the binding of DNA fragments

Microsatellites – highly polymorphic DNA markers comprised of a variable number of tandem repeats

Mini-prep – the purification of extra-chromosomal DNA (usually a plasmid) from a small culture (e.g. 1.5 mL) of bacterial cells

Monocistronic – an RNA molecule that codes for one protein

Monoclonal antibody – a highly specific protein that can bind to a single epitope within an antigen

Morula – a globular solid mass of cells formed by the cleavage of a zygote that precedes the blastula

mRNA – messenger RNA

Northern blot – a technique for the separation of RNA molecules through agarose gels followed by detection of specific RNAs through hybridization with singlestranded DNA

Nucleoside – a purine or pyrimidine base combined with either deoxyribose or ribose found in DNA or RNA

Nucleosome – the basic structural unit of chromatin

Nucleotide – a purine or pyrimidine base combined with either deoxyribose or ribose and a phosphate group found in DNA or RNA

Oligonucleotide – a short molecule of single-stranded DNA, usually synthesized chemically

Oocyte – an egg before maturation

Opine – a product produced by the condensation product of an amino acid with either a keto-acid or a sugar

Origin of replication – the nucleotide sequence at which DNA synthesis (replication) is initiated

PAC – P1 derived artificial chromosome. A vector, based on the bacteriophage P1 genome, used to clone large DNA fragments in E. coli

Palindromic – a sequence of DNA that read on one strand in a 5 – 3 direction is the same as than on the other strand in a 5 – 3 direction

PCR – polymerase chain reaction – cycles of DNA denaturation, primer annealing and extension with DNA polymerase lead to a amplification of the target DNA sequence

PFGE – Pulsed field gel electrophoresis Phage – bacteriophage – a bacterial virus

Phage display – a technique that fuses peptides to capsid proteins on the surface of phages. Libraries of phage displayed peptides may be screened for binding to specific ligands

Phenotype – the functional and structural characteristics of an organism as determined by the interaction of the genotype with the environment

Pilus – a filament-like projection from the surface of a bacterial cell

Phosphodiester bond – a covalent bond between the 5 phosphate group on one nucleotide and the 3 hydroxyl group on an adjacent nucleotide

Plasmid – an autonomous self-replicating extra-chromosomal closed-circular DNA Polycistronic – an mRNA molecule that contains more than one coding region Polyclonal antibody – a mixture of many antibodies, each raised against the same

antigen

Polynucleotide – long chains of nucleotides linked together by phosphodiester bonds

Polypeptide – a chain of amino acids connected by peptide linkages

Polysome – multiple ribosomes actively translating a single mRNA molecule into polypeptides

Primer – a short DNA or RNA sequence that is paired with one strand of DNA and provides a free 3 hydroxyl group at which DNA replication can initiate

Promoter – a DNA sequence which serves as the binding site for transcription factors and RNA polymerase during the initiation of transcription

Pronucleus – the haploid nucleus of an egg or sperm cell prior to fertilization, and immediately after fertilization before the sperm and egg nuclei have fused into a single diploid nucleus

Propeller twist – the rotation of individual DNA base pairs within the double helix Purine – a nitrogen containing, double-ring basic compound that occurs in nucleic

acids. The purines in DNA and RNA are adenine and guanine

406 GLOSSARY

Pyrimidine – a nitrogen containing, single-ring basic compound that occurs in nucleic acids. The pyrimidines in DNA are cytosine and thymine, and those in RNA are cytosine and uracil

Quiescence – a dormant phase of cell growth. All but the most basic functions of a cell or group of cells have stopped, usually in response to an unfavourable environment. The cell remains dormant until its surroundings are more favourable

Replicon – part of the genome in which replication is initiated. Contains origins of replication

Restriction enzyme – (or restriction endonuclease) recognizes short, often palindromic, DNA sequences (recognition sites) and cleaves the DNA

Restriction map – a linear map of the restriction enzyme recognition sites with a DNA molecule

Reverse transcriptase – the enzyme that synthesizes DNA from RNA templates Reverse transcription – the synthesis of DNA from RNA

RFLP – restriction fragment length polymorphism – changes in DNA sequence that result in altered lengths of DNA when cleaved with a restriction enzyme

Ribosome – RNA-rich cytoplasmic granules that are sites of protein synthesis RNA – ribonucleic acid

RNA interference (RNAi) – the process in which the introduction of double-stranded RNA into a cell inhibits the expression of genes

RNA polymerase – the enzyme that synthesizes RNA from a DNA template

RT-PCR – a method for the amplification of a specific mRNA. Reverse transcriptase is used to form a cDNA which is then amplified using PCR

Semi–conservative replication – the separation of the strands of DNA during replication with each acting as a template for the synthesis of a new complementary DNA strand Shine-Dalgarno sequence – an mRNA sequence that precedes the translation initiation

codon and is complementary to a ribosomal RNA

Signal sequence – a short amino acid sequence that determines the localization of a protein within the cell

Silent mutation – a mutation with DNA that does not cause an alternation to the encoded amino acid sequence

Single-nucleotide polymorphisms (SNPs) – single-base-pair variations scattered within the genetic code of the individuals within a population

siRNA – small interfering RNAs. Synthetic short double-stranded RNA molecules that inhibit gene expression

Site-specific recombination – occurs between two specific, but not necessarily homologous, DNA sequences

Southern blot – a technique for the separation of DNA molecules through agarose gels followed by detection of specific DNAs after denaturation through hybridization with single-stranded DNA

Spliceosome – a complex consisting of RNA and small nuclear ribonucleoproteins (snRNPs). The spliceosome splices RNA transcripts by excising introns and ligating the ends of exons

Splicing – the removal of introns and joining of exons to form a mature mRNA

GLOSSARY 407

Stem cell – a cell from the embryo, foetus or adult that has capability to reproduce itself. It can give rise to specialized cells that make up the tissues and organs of the body

Supercoiling – the way in which closed-circular DNA crosses over its own axis in three-dimensional space

T-DNA – the portion of the Agrobacterium tumefaciens Ti plasmid that is inserted into the genome of the host plant cell

TA cloning – a method for cloning PCR products that relies on the addition of template independent A residues to PCR products by Taq DNA polymerase

Telomere – the repetitive DNA sequences at the end of a chromosome

Terminal transferase – an enzyme that catalyses the addition of nucleotides to the 3 -terminus of DNA

Terminator – the sequence of DNA that causes RNA polymerase to stop transcription Terminator technology – plants are engineered so when crops are harvested, all new

seeds produced are sterile

Thymine – a pyrimidine base found in DNA. Thymine base pairs with adenine Transcription – the synthesis of RNA from a DNA template

Transfection – the addition of DNA to eukaryotic cells

Transformation – the addition of DNA to bacteria. In eukaryotes, this term also refers to the state of cells that undergo tumour-like growth

Transforming principle – the factor identified by Griffith as being able to convert one bacterial type to another

Transgenic animals – created by inserting DNA sequences into the germ line through addition to the egg

Transition mutation – a mutation in DNA in which one purine – pyrimidine base pair is changed to a different purine – pyrimidine base pair

Translation – the synthesis of protein using an mRNA template

Transversion mutation – a mutation in DNA in which a purine – pyrimidine base pair is changed to a pyrimidine – purine base pair

Trinucleotide – the joining of three nucleotides through phosphodiester linkages Two-hybrid screen – a technique to identify proteins that are able to interact with

each other

Uracil – a pyrimidine base usually only found in RNA. Uracil bases pairs with adenine Variable number tandem repeats (VNTRs) – different numbers of tandemly repeated

DNA sequences at a given locus.

Vector – a DNA molecule that possesses the ability to self-replicate. Used to introduce foreign DNA into host cells, where it is replicated autonomously in large quantities Western blot – the detection of specific proteins following electrophoresis using anti-

bodies

Xenobiotic – a compound with a chemical structure that is foreign to an organism Xenotransplantation – the surgical transplantation of tissue or organs from an individ-

ual of one species into an individual of another species YAC – yeast artificial chromosome

Zygote – a cell formed by the union of two gametes (egg and sperm)