01. Gene therapy Boulikas.pdf - Gene therapy & Molecular Biology

XLIII. Prospects
A. Gene discovery: novel horizons in gene
therapy
By the year 2005 the human genome project will be
completed and by the end of 1998 the entire cDNA
repertoire of the 125,000 human genes will be sequenced
completely (Incyte Pharmaceuticals, Palo Alto, CA).
Every single open reading frame of the human genome
will become known.
Genes implicated in human disease are being identified
by mapping the mutation to a chromosomal locus after
examining the DNA from a number of patients; candidate
genes residing in this locus are then examined in a large
number of patients for inactivating mutations (e.g.
Polymeropoulos et al, 1996, 1997). A number of other
classical techniques are aimed at identifying novel tumor
suppressor genes or genes involved in metastasis, adding
new weapons to the fight against cancer. The elucidation
of many of the pathways implicated in the regulation of
the cell cycle, signaling pathways with cytokines, and
activation and action of transcription factors on the
regulatory regions of genes lead to the discovery of new
drugs interfering with those pathways. The elucidation of
a number of players in apoptosis provides also targets not
only for cancer treatment but for a number of
neurodegenerative diseases. All these studies will
ultimately provide new targets and genes for gene therapy.
B. Mutations in DNA and human disease
A number of human disorders have been linked to
mutations in specific genes that result in loss of function
of a specific protein in all somatic cells of the body. In the
majority of cases known today the mutation is at the
coding region of the gene resulting in one amino acid
substitution at an important domain of the encoded
protein, in amino acid deletions, or in protein truncation.
For example, deletion of three nucleotides resulting in
deletion of a single phenylalanine at the protein level of
the CFTR molecule is responsible for cystic fibrosis
(Riordan et al, 1989); an A to T transversion leading to a
premature stop at amino acid 337 in one allele and a C to
T transition triggering an erroneous splice event and to
frameshift in the other allele are associated with mutations
in the ERCC6 helicase in Cockayne's syndrome (Troelstra
et al, 1992).
Mutations could result in failure of the protein to
interact with DNA (mutated p53), with other regulatory
proteins, or in enzymatic dysfunction of the molecule.
Defects at the nuclear localization signal of a nuclear
protein resulting in its cytoplasmic retention have been
identified in cancer cells (Chen et al, 1995). Mutations in
regulatory regions (promoters, enhancers) of genes, poorly
understood but expected to play an important role in
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human disease, could result in down-regulation of the
gene they dictate; mutation in the DNA-binding or
transactivation domains of transcription factors are
expected to down-regulate the expression of their target
genes. Most important, mutations in genes involved in
DNA repair are expected to have a domino effect on the
appearance of mutations in other regions of the genome,
since it is these genes that are responsible for removal of
premutagenic lesions incurring by a number of xenobiotics
by patrolling the human genome (Boulikas, 1996c).
C. Regulatory regions and the MAR
project
The identification of the regulatory regions from the
human genome should also become a first priority.
Regulatory regions will provide new DNA control
elements for the tissue-specific expression, episomal
replication, insulation, and silencing of genes in gene
therapy protocols but also targets, using small
oligonucleotides (e.g. triplex) to abort transcription of
specific genes. Regulatory regions include enhancers
(ENHs, at least two for each gene), promoters (about
125,000 total, a significant fraction of which might be
known because of their proximity to the 5' end), origins of
replication (ORIs, about 50,000 have been estimated),
silencers, locus control regions (LCRs, perhaps several
thousand), and matrix-attached regions (seem to coincide
with enhancers and ORIs). 445,000 is a modest estimate of
the total number of regulatory regions in the human
genome. Their size ranges from 100-500 bp but much
more for LCRs and some ORIs. Identification of strong
regulatory regions from the human genome is expected to
provide strong promoter and enhancer sequences for the
universal and cell type-specific expression of transgenes in
gene therapy but also strong human ORIs able to sustain
extrachromosomal replication of plasmids loaded with
therapeutic genes in human and animal model tissues.
Transcription factor recognition sequence databases
can be used in conjuction with other software methods
(DNA curvature, inverted repeats, triplex DNA, Z-DNA,
phased nucleosomes) to predict regulatory regions from
the large DNA sequence information arising from the
human genome project (Boulikas, 1995b; Bode et al,
1998, this volume).
A technology developed in our laboratory based on
isolation and cloning of matrix-attached regions, shown to
harbor a large fraction of regulatory regions from the
human and other genomes, is being applied for identifying
human regulatory regions (MAR project). MAR libraries
include tissue-specific and tumor-specific regulatory
regions. One particular MAR clone that has been
extensively characterized (Boulikas et al, in preparation)
represents the ORI, enhancer, and MAR of the human
choline acetyltransferase gene, of crucial importance in
neurological disorders including Alzheimer's disease.