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152 Part IV: Molecular and Cellular Hematology Chapter 10: Genetic Principles and Molecular Biology 153

INTERFERENCE WITH GENE EXPRESSION levels of human hemoglobin S to have some potential as an animal
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Antisense RNA and DNA model of sickle disease (Chaps. 48 and 49). Another valuable tech-
nique for the study of gene function is targeted disruption (“knocking
It is possible to interdict the expression of a gene at several different levels. out”) of genes. In this technique, a DNA construct that contains regions
The translation of mRNA can be inhibited and the mRNA degraded by homologous to the gene being targeted and selectable markers is trans-
antisense RNA or DNA, molecules that have a sequence complemen- fected into an embryonic mouse stem cell. Once a cell in which recom-
tary to the mRNA that is to be inactivated. When such oligonucleotides bination has occurred within a gene is found, it can be implanted into a
are present, they inhibit gene expression through a variety of mecha- blastocyst, with the hope that some of the progeny of the implanted cell
nisms. For example, they form a double strand with the RNA, just as will become germ cells. If this does occur, the knockout can be prop-
two complementary strands of DNA will hybridize to form the normal agated and homozygous animals bred. The value of the technique is
double-stranded form of DNA. Because the double-stranded form can- often limited by the fact that the knockout may be lethal (e.g., G6PD75
not be translated and is probably degraded rapidly, the production of its deficiency and Gaucher disease) or may not have any abnormal pheno-
protein product is inhibited specifically. In experimental systems, anti- type. But in some diseases, such as hemochromatosis, knockout mod-
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sense DNA or stable DNA analogues, such as the methylphosphonates, els of various forms of the disease are valuable resources. In situations
can be transfected directly into cells or the RNA can be made off of a in which a knockout proves to be lethal, or where it would be useful
plasmid with the appropriate DNA template and a promoter. Originally to limit the deficiency to a single-organ system, the Cre/LoxP site-spe-
this approach was used, for example, to suppress lymphoma growth cific recombination system has proven to be very useful. The LoxP
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with DNA oligonucleotides antisense to introns of the oncogene c-myc, sequence, a 13-bp inverted repeat, is inserted so that it flanks the gene
to suppress marrow cells from patients with chronic myelogenous leu- that is to be removed. Site specific recombination is catalyzed by the P-1
kemia by antisense DNA directed at the BCR-ABL junction, or to sup- bacteriophage Cre recombinase, excising the intervening DNA targeted
press BCL-2–positive lymphoma cells in culture by BCL-2 antisense. by the LoxP sequence and ligating the remaining 5′ and 3′ DNA. Tissue-
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Because antisense RNA can be produced in vivo by transcribing the specific excision can be achieved by inserting the Cre-recombinase
complementary strand of a gene, it may represent a natural regulatory downstream from a tissue-specific promoter. Random mutagenesis with
mechanism.
agents such as N-ethyl-N-nitrosourea (ENU) can identify functions of
genes whose role in a metabolic pathway was unsuspected. For exam-
Interfering RNA ple, a mutation in a membrane serine protease of unknown function
RNA plays a much broader role in the physiologic regulation of genes revealed that it was a negative regulator of hepcidin, and subsequent
than merely the formation of antisense mRNAs. siRNAs (small inter- investigations revealed that mutations of this gene caused hereditary
fering ribonucleic acids) and the closely related miRNAs (microribo- iron deficiency in humans. 60
nucleic acids) represent a mechanism for silencing of genes, through a
process known as RNA interference (RNAi 52,53 ). In the case of siRNA,
double-stranded RNA is cleaved by the “dicer” enzyme into approxi- GENE THERAPY
mately 22 bp segments that trigger the destruction through the RNA-
induced silencing complex (RISC) of the homologous targeted mRNA. In somatic cell gene therapy, the DNA of a specific set of a patient’s
Although siRNAs tend to operate through RISC and slice the targeted somatic cells is altered. It is also possible to carry out germline therapy,
mRNA, the miRNAs that represent endogenous duplexes can decrease which affects all cells, including reproductive cells, but for technical and
the amount of target mRNA(s) or can also posttranscriptionally regu- ethical reasons this is not being pursued in humans. Most commonly,
late gene expression by complexing with the same RISC and interfering somatic cell therapy is used for conditions in which a mutation has
with the targeted mRNA translation. miRNAs may play an important caused the absence of a gene product in a cell (e.g., adenosine deaminase
role in hematopoietic differentiation and seem to be widely used as a in T cells, which leads to an autosomal recessive form of severe com-
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gene regulatory and antiviral measure. The use of siRNA has become bined immunodeficiency [SCID]). A vector is used to carry a normal
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very useful to molecular biologists as a powerful method for the down- copy of the mutated gene into the patient’s cells. These vectors are usually
regulation of genes in experimental systems. viruses, such as retroviruses or adenoviruses, which have been geneti-
cally modified so that they contain the normal human gene and cannot
Ribozymes make copies of themselves (otherwise they could cause a viral infection).
Cleaving RNA at defined sequences, much as restriction endonucleases Once inside the patient’s cells, the normal human gene begins to encode
cleave DNA, is one of the known enzymatic functions of RNA, and this the missing gene product. For diseases caused by a gain of function, tech-
function provides a means by which the expression of a gene can be niques such as antisense DNA or RNA and RNAi (discussed in the sec-
interdicted in experimental systems. This ribozyme approach has been tion on Interference with Gene Expression.) are being used.
used, for example, in preventing replication of the HIV-1 virus and by Gene therapy has faced a number of technical hurdles, including
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cleaving BCR-ABL fusion transcript with a view to developing a treat- immune responses against the vector, limited efficiency of antisense
ment for chronic myelogenous leukemia. 56 and RNAi approaches, and difficulties in producing sufficient quanti-
ties of a desired gene product. In one case, an immune response against
Transgenic and Knockout Animal Models an adenoviral vector proved fatal, and several cases of leukemia have
The insertion of DNA fragments into the nucleus of a fertilized ovum resulted from the insertion of a modified retrovirus near an oncogene.
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provides a means for altering the genetic constitution of animals. Ani- Nevertheless, gene therapy has now been successful in treating a
mals that have been engineered in this manner are referred to as trans- number of inherited conditions, including X-linked and ADA SCID,
genic. The use of promoters that are inducible or tissue specific permits β-thalassemia, hemophilia B, and X-linked adrenoleukodystrophy. 63–66
studies of the effect of a gene product that might be lethal if expressed In addition to the treatment of hereditary diseases, gene therapy is being
in all tissues or at all times during embryogenesis. Transgenic mice that used to alter tumor cells in the treatment of various types of cancer. It is
carry the human sickle β-globin gene have been produced and when hoped that further research will lead to safe, efficient and cost-effective
superimposed on a murine thalassemic genotype produce high enough treatment of many human diseases through gene therapy.

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