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Chapter 1 Anatomy and Physiology of the Gene 15

stem cells and for performing gene transfer into those cells has
advanced rapidly, and clinical trials have begun to test the applicabil-
ity of these techniques. Despite the fact that gene therapy has pro-
Embryonic stem cell gressed to the enrollment of patients in clinical protocols, major
technical problems still need to be solved. Presently, there are only
few (but increasing, such as severe combined immunodeficiency
syndromes, Wiskott–Aldrich disease, and others) proven therapeutic
successes from gene therapy.
Progress in this field continues rapidly and is likely to accelerate
Gene of interest as a consequence of the development of “gene editing” technologies.
Among these, “CRISPR” is the most prominent current example. It
neo R Engineered plasmid is based on the discovery of enzyme systems used by microorganisms
to excise foreign DNA sequences (e.g., integrated viral genomes) from
the host genome. These systems can be adapted to insert, replace, or
delete, in principle, any desired DNA sequence in its naturally occur-
Cells selected for ring position in the host genome. For example, one could excise the
resistance to G418 mutation causing sickle cell anemia and replace it with the normal
DNA sequence in the β-globin gene of a patient’s hematopoietic stem
cells, and then re-introduce them into the patient’s bone marrow
without introducing any foreign DNA. This exciting technology is
rapidly moving toward clinical trials. The scientific basis for gene
therapy and the clinical issues surrounding this approach are discussed
in Chapter 98.

Resistant cells inserted Antisense Therapy
into blastocyst
The recognition that abnormal expression of oncogenes plays a role
in malignancy has stimulated attempts to suppress oncogene expres-
sion to reverse the neoplastic phenotype. One way of blocking mRNA
expression is with antisense oligonucleotides. These are single-
stranded DNA sequences 17 to 20 bases long, having a sequence
complementary to the transcription or translation start of the mRNA.
These relatively small molecules freely enter the cell and complex to
Blastocyst implanted the mRNA by their complementary DNA sequence. This often
into mouse results in a decrease in gene expression. The binding of the oligonucle-
otide may directly block translation and clearly enhances the rate of
mRNA degradation. This technique has been shown to be promising
Fig. 1.9 GENE “KNOCKOUT” BY HOMOLOGOUS RECOMBINA- in suppressing expression of bcr-abl and to suppress cell growth in
TION. A plasmid containing genomic DNA homologous to the gene of chronic myelogenous leukemia. The technique is being tried as a
interest is engineered to contain a selectable marker positioned so as to disrupt therapeutic modality for the purging of tumor cells before autologous
expression of the native gene. The DNA is introduced into embryonic stem transplantation in patients with chronic myelogenous leukemia.
cells, and cells resistant to the selectable marker are isolated and injected into
a mouse blastocyst, which is then implanted into a mouse. Offspring mice
that contain the knockout construct in their germ cells are then propagated, FUTURE DIRECTIONS
yielding mice with heterozygous or homozygous inactivation of the gene of
interest. The elegance of recombinant DNA technology and its successor
technologies of genomics, epigenomics, and proteomics resides in the
capacity they confer on investigators to examine each gene as a dis-
crete physical entity that can be purified, reduced to its basic building
their cells. These heterozygous mice can be further bred to produce blocks for decoding of its primary structure, analyzed for its patterns
mice homozygous for the null allele. Such knockout mice reveal the of expression, and perturbed by alterations in sequence or molecular
function of the targeted gene by the phenotype induced by its environment so that the effects of changes in each region of the gene
absence. Genetically altered mice have been essential for discerning can be assessed. Purified genes can be deliberately modified or
the biologic and pathologic roles of large numbers of genes implicated mutated to create novel genes not available in nature. These provide
in the pathogenesis of human disease. the potential to generate useful new biologic entities, such as modi-
fied live virus or purified peptide vaccines, modified proteins custom-
ized for specific therapeutic purposes, and altered combinations of
DNA-BASED THERAPIES regulatory and structural genes that allow for the assumption of new
functions by specific gene systems.
Gene Therapy Purified genes facilitate the study of gene regulation in many ways.
First, a cloned gene provides characterized DNA probes for molecular
The application of gene therapy to genetic hematologic disorders is hybridization assays. Second, cloned genes provide the homogeneous
an appealing idea. In most cases, this would involve isolating hema- DNA moieties needed to determine the exact nucleotide sequence.
topoietic stem cells from patients with diseases with defined genetic Sequencing techniques have become so reliable and efficient that it
lesions, inserting normal genes into those cells, and reintroducing the is often easier to clone the gene encoding a protein of interest and
genetically engineered stem cells back into the patient. A few candi- determine its DNA sequence than it is to purify the protein and
date diseases for such therapy include sickle cell disease, thalassemia, determine its amino acid sequence. The DNA sequence predicts
hemophilia, and adenosine deaminase–deficient severe combined exactly the amino acid sequence of its protein product. By comparing
immunodeficiency. The technology for separating hematopoietic normal sequences with the sequences of alleles cloned from patients