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

When a subfragment of only 513 bp of this
MAR/ORI/ENH was placed at the flanks of the luciferase
gene it was able to sustain episomal replication in human
culture cells (K562 erythroleukemia) for more than 4
months. The actual 3.6 kb ChAT ORI region comprises a
1.2 kb silencer whose presence inhibits the ORI function;
thus, mammalian origins of replication are much more
sophisticated than viral ORIs and contain a number of
control elements, including silencers, for the cell type and
developmental stage-specific regulation. Identification and
elimination of silencers from human ORIs is of
importance in the exploitation of ORI fragments in the
episomal replication of therapeutic genes.
MAR sequences sorted out into MAR/ORI,
MAR/enhancer and MAR/insulators can be used to
promote extrachromosomal replication, to enhance the
transcription of genes or to insulate genes from position
effects from chromatin surroundings after integration. A
number of studies show that MARs act as insulators of
genes shielding them from position effect variegation from
neighboring chromatin domains in transgenic studies; this
shielding results in a 2 to 1000-fold increase in the
expression level of transgenes when MARs are included
on both sides of the foreign gene (see Boulikas 1995b).
Identification of tumor-specific MARs, such as
identification of the MARs of the carcinoembryonic
antigen (CEA) gene, the breast cancer/ovarian cancer
BRCA1 gene, and others can lead to the development of
plasmid vectors able to drive the expression of therapeutic
genes in specific tumor cell types. In the postgenomic era,
identification of a reasonable fraction of regulatory
regions will revolutionarize our approaches to human
disease.
D. What is next on gene therapy?
Theoretically, most human disorders could constitute
targets for gene therapy, aimed at correcting the defect
either by transferring the wild-type gene in all somatic
cells of the body or to those specific cell types responsible
mainly for the synthesis of the particular protein (e.g.
factor IX gene in liver cells of hemophilia B patients).
Nuclear localization signal (NLS) peptides hooked to
triplex-oligonucleotides or to plasmids, or complexation of
plasmids with nuclear proteins possessing multiple NLSs
are expected to increase nuclear localization and enhanced
expression of foreign genes.
A significant number of discoveries in molecular
biology of human diseases have opened doors to the
development of strategies for gene therapy. New genes
whose mutations are responsible for human disease, from
mild to life threatening, are being discovered and the
molecular mechanisms are being unraveled. Many pieces
of the puzzle aimed at elucidating mechanisms leading to
human disease and the genes implicated have been solved
Gene Therapy and Molecular Biology Vol 1, page 129
129
and lie as scattered pieces of knowledge in various
publications, lab notebooks, or patent applications.
Preexisting Biotech Companies redefine their missions
and new Biotech Companies are being founded to explore
new discoveries and develop new drugs; to win the race in
the fight against human disease, especially cancer and
AIDS, we need to gather the right components into a
successful assemble.
Retroviruses, adenoviruses, AAV, HSV, naked
plasmid delivery, and liposomes all have a good share as
delivery vehicles for genes and it seems that they will be
developed independently, each with its own strengths and
limitations for particular gene therapy protocols. For
example, liposomes have a distinct advantage over other
systems for the delivery of oligonucleotides, stealth
liposomes could prove their strength in the systemic
delivery of genes by intravenous injection, retroviruses
and adenoviruses for their high transfection efficiency,
AAV for not stimulating inflammation, HSV as a vehicle
for gene therapy to the nervous system, HIV and HSV
vectors for their high payload capacity. Furthermore,
adenoviruses, AAV, HSV-1, HIV-1 vectors can transduce
nondividing cells (Table 1 on page 29).
A lot has been learned about the involvement of the
tumor suppressor p53 protein in cancer etiology. The
current view is that an initiated tumor cell in the body,
having mutations in one or more oncogenes needs to
acquire loss in function in both alleles of p53 or other
tumor suppressor gene in order to expand into the tumor
cell mass. Expression of the wild-type (non-mutated form)
of p53 arrests the proliferation in tumor cells and induces
apoptosis (suicidal programmed death) by boosting the
expression of the genes of p21, bax, and Gadd45 and by
repressing the bcl-2 gene. Transfer of the p53 gene with
adenovirus or retrovirus after intratumoral injection has
successfully led to eradication of tumors in animal models
and in human patients at advanced stages of non small cell
lung cancer. Intratumoral injection, however, is not
expected to be applicable to metastases very frequently
associated with advanced stages of cancer. Stealth
liposomes might offer a solution to this problem.
Anti-angiogenesis therapy, both drug-mediated and
gene therapy, would bring important ammunition in the
fight against cancer.
Improvements in oligonucleotide delivery in vivo, a
very promising field that is in its infancy at the delivery
level, will advance the field of pharmacogenomics by
providing triplex-forming oligonucleotide drugs to inhibit
the transcription of specific genes or ribozyme drugs to
lower the mRNA level of a specific target protein.
We expect the final victory of the human race on
cancer to be accomplished over the next 10 years. Gene
therapy would, no doubt, have an important role to play. It
is likely that a combination of gene therapy (p53, HSV-tk,
angiostatin) along with the already existing antineoplastic