StemCell Therapy

The general approach of gene therapy is nothing more than an extension of the technique for clone selection by functional complementation (Chapter 12). The functions absent in the recipient as a result of a defective gene are introduced on a vector that inserts into one of the recipients chromosomes and thereby generates a transgenic animal that has been genetically cured. The technique is of great potential in humans because it offers the hope of correcting hereditary diseases. However, gene therapy is also being applied to mammals other than humans.

The first example of gene therapy in a mammal was the correction of a growth-hormone deficiency in mice. The recessive mutation little (lit) results in dwarf mice. Even though a mouses growth-hormone gene is present and apparently normal, no mRNA for this gene is produced. The initial step in correcting this deficiency was to inject homozygous lit/lit eggs with about 5000 copies of a 5-kb linear DNA fragment that contained the rat growth-hormone structural gene (RGH) fused to a regulatorpromoter sequence from a mouse metallothionein gene (MP). The normal job of metallothionein is to detoxify heavy metals, so the regulatory sequence is responsive to the presence of heavy metals in the animal. The eggs were then implanted into pseudopregnant mice, and the baby mice were raised. About 1 percent of these babies turned out to be transgenic, showing increased size when heavy metals were administered in the course of development. A representative transgenic mouse was then crossed with a homozygous lit/lit female. The ensuing pedigree is shown in . We can see in that mice two to three times the weight of their lit/lit relatives are produced in subsequent generations, with the rat growth-hormone transgene acting as a dominant allele, always heterozygous in this pedigree. The rat growth-hormone transgene also makes lit+ mice bigger ().

The rat growth-hormone gene (RGH), under the control of a mouse promoter region that is responsive to heavy metals, is inserted into a plasmid and used to produce a transgenic mouse. RGH compensates for the inherent dwarfism (lit/lit) in the mouse. RGH (more...)

Transgenic mouse. The mice are siblings, but the mouse on the left was derived from an egg transformed by injection with a new gene composed of the mouse metallothionein promoter fused to the rat growth-hormone structural gene. (This mouse weighs 44g, (more...)

The site of insertion of the introduced DNA in mammals is highly variable, and the DNA is generally not found at the homologous locus. Hence, gene therapy most often provides not a genuine correction of the original problem but a masking of it.

Similar technology has been used to develop transgenic fast-growing strains of Pacific salmon, with spectacular results. A plasmid containing a growth-hormone gene placed next to a metallothionein promoter (all derived from salmon) was microinjected into salmon eggs. A small proportion of the resulting fish proved to be transgenic, testing positive when their DNA was probed with the plasmid construct. These fish were on average 11-fold heavier than the nontransgenic controls (). Progeny inherited the transgene in the same manner as the mice in the earlier example.

Effect of introducing a hormone transgene complex with a strong promoter into Pacific salmon. All salmon shown are the same age. (R. H. Devlin, T. Y. Yesaki, C. A. Biagi, E. M. Donaldson, P. Swanson, and W.-K. Chan, Extraordinary Growth, (more...)

Perhaps the most exciting and controversial application of transgenic technology is in human gene therapy, the treatment and alleviation of human genetic disease by adding exogenous wild-type genes to correct the defective function of mutations. We have seen that the first case of gene therapy in mammals was to cure a genotypically dwarf fertilized mouse egg by injecting the appropriate wild-type allele for normal growth. This technique () has little application in humans, because it is currently impossible to diagnose whether a fertilized egg cell carries a defective genotype without destroying the cell. (However, in an early embryo containing only a few cells, one cell can be removed and analyzed with no ill effects on the remainder.)

Two basic types of gene therapy can be applied to humans, germ line and somatic. The goal of germ-line gene therapy () is the more ambitious: to introduce transgenic cells into the germ line as well as into the somatic cell population. Not only should this therapy achieve a cure of the person treated, but some gametes could also carry the corrected genotype. We have seen that such germinal therapy has been achieved by injecting mice eggs. However, the protocol that is relevant for application to humans is the removal of an early embryo (blastocyst) with a defective genotype from a pregnant mouse and injection with transgenic cells containing the wild-type allele. These cells become part of many tissues of the body, often including the germ line, which will give rise to the gonads. Then the gene can be passed on to some or all progeny, depending on the size of the clone of transgenic cells that lodges in the germinal area. However, no human germ-line gene therapy has been performed to date.

We have seen that most transforming fragments will insert ectopically throughout the genome. This is a disadvantage in human gene therapy not only because of the possibility of the ectopic insert causing gene disruption, but also because, even if the disease phenotype is reversed, the defective allele is still present and can segregate away from the transgene in future generations. Therefore, for effective germinal gene therapy, an efficient targeted gene replacement will be necessary, in which case the wild-type transgene replaces the resident defective copy by a double crossover.

Somatic gene therapy () focuses only on the body (soma). The approach is to attempt to correct a disease phenotype by treating some somatic cells in the affected person. At present, it is not possible to render an entire body transgenic, so the method addresses diseases whose phenotype is caused by genes that are expressed predominantly in one tissue. In such cases, it is likely that not all the cells of that tissue need to become transgenic; a percentage of cells being transgenic can ameliorate the overall disease symptoms. The method proceeds by removing some cells from a patient with the defective genotype and making these cells transgenic through the introduction of copies of the cloned wild-type gene. The transgenic cells are then reintroduced into the patients body, where they provide normal gene function.

Currently, there are two ways of getting the transgene into the defective somatic cells. Both methods use viruses. The older method uses a disarmed retrovirus with the transgene spliced into its genome, replacing most of the viral genes. The natural cycle of retroviruses includes the integration of the viral genome at some location in one of the host cells chromosomes. The recombinant retrovirus will carry the transgene along with it into the chromosome. This type of vector poses a potential problem, because the integrating virus can act as an insertional mutagen and inactivate some unknown resident gene, causing a mutation. Another problem with this type of vector is that a retrovirus attacks only proliferating cells such as blood cells. This procedure has been used for somatic gene therapy of severe combined immunodeficiency disease (SCID), otherwise known as bubble-boy disease. This disease is caused by a mutation in the gene encoding the blood enzyme adenosine deaminase (ADA). In an attempt at gene therapy, blood stem cells are removed from the bone marrow, the transgene is added, and the transgenic cells are reintroduced into the blood system. Prognosis for such patients is currently good.

Even solid tissues seem to be accessible to somatic gene therapy. In a dramatic case, gene therapy was administered to a patient homozygous for a recessive mutant allele of the LDLR gene for low-density-lipoprotein receptor (genotype LDLR/LDLR). This mutant allele increases the risk of atherosclerosis and coronary disease. The receptor protein is made in liver cells, so 15 percent of the patients liver was removed, and the liver cells were dissociated and treated with retrovirus carrying the LDLR+ allele. Transgenic cells were reintroduced back into the body by injection into the portal venous system, which takes blood from the intestine to the liver. The transgenic cells took up residence in the liver. The latest reports are that the procedure seems to be working and the patients lipid profile has improved.

The other vector used in human gene therapy is the adenovirus. This virus normally attacks respiratory epithelia, injecting its genome into the epithelial cells. The viral genome does not integrate into a chromosome but persists extrachromosomally in the cells, which eliminates the problem of insertional mutagenesis by the vector. Another advantage of the adenovirus as a vector is that it attacks nondividing cells, making most tissues susceptible in principle. Inasmuch as cystic fibrosis is a disease of the respiratory epithelium, adenovirus is an appropriate choice of vector for treating this disease, and gene therapy for cystic fibrosis is currently being attempted with the use of this vector. Viruses bearing the wild-type cystic fibrosis allele are introduced through the nose as a spray. It is also possible to use the adenovirus to attack cells of the nervous system, muscle, and liver.

A promising type of construct that should find use in gene therapy is the human artificial chromosome (HAC). HACs contain essentially the same components as YACs. They were made by mixing human telomeric DNA, genomic DNA, and arrays of repetitive -satellite DNA (thought to have centrometric activity). To this unjoined mixture was added lipofectin, a substance needed for passage through the membrane, and the complete mixture was added to cultured cells. Some cells were observed to contain small new chromosomes that seemed to have assembled de novo inside the cell from the added components (). When the technology has been perfected, these HACs should be potent vectors capable of transferring large amounts of human DNA into cells in a stable replicating form.

An artificial human chromosome (at arrow). (John J. Harrington, G. Van Bokkelen, R. W. Mays, K. Gustashaw, and H. F. Willard, Formation of de Novo Centromeres and Construction of First-Generation Human Artificial Microchromosomes, Nature (more...)

Gene therapy introduces transgenic cells either into somatic tissue to correct defective function (somatic therapy) or into the germ line for transmission to descendants (germ-line therapy).

Original post:
Gene therapy - An Introduction to Genetic Analysis - NCBI ...

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