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

smaller portion of these molecules will enter nuclei;
finally, after successfully reaching the nucleus, plasmids
with therapeutic genes are usually degraded by nuclear
enzymes and transgene expression is permanently lost
after about 2-7 days from animal tissues following
successful gene delivery. During the peak of transgene
expression (usually 7-48 h from injection) the transgene
transcript can follow the normal fate of other nuclear
transcripts when proper polyadenylation signals are
provided; its processed mRNA will be exported to the
cytoplasm and translated into the therapeutic protein.
The choice of the appropriate delivery system for
successful somatic gene transfer demands understanding
of the drawbacks and advantages of each delivery system,
such as limitations in the total length of the DNA that can
be introduced, including the cDNA of the therapeutically
important gene and control elements. Understanding the
pathophysiology of the disease and the cell targets can
give clues on the way of introducing the gene (i.v., i.p.,
intratumoral, s.c. injection) or direct the gene therapist to
designing methods to target and secrete a therapeutic
protein from a tissue which is not the normal site of
production of a therapeutic protein. The type of control
elements required for the anticipated tissue-specific
expression of the construct, the presence of viral or other
origins of replication as well as of the cDNA encoding the
viral replication initiator protein for an episomal
replication of the transgene, sequences that prompt
integration and others that insulate the gene from the
chromatin surroundings at the integration site, are also
important for successful gene transfer.
Cancer gene therapy and immunotherapy has been the
first priority of human gene therapy protocols. New gene
targets are being defined and new clinical protocols are
being proposed and approved. Effective eradication of a
great variety of tumors with drugs which inhibit
angiogenesis has been extraordinarily successful on
animal models and the method moves fast to clinical trials;
transfer of anti-angiogenesis genes will be the next step. A
number of anticancer genes are being tested in preclinical
or clinical cancer trials including p53, RB, BRCA1, E1A,
bcl-2, MDR-1, HER2, p21, p16, bax, bcl-xs, E2F,
antisense IGF-I, antisense c-fos, antisense c-myc,
antisense K-ras and the cytokine genes GM-CSF, IL-12,
IL-2, IL-4, IL-7, IFN-γ, and TNF-α. A promising
approach is transfer of the herpes simplex virus thymidine
kinase (HSV-tk) gene (suicide gene) and systemic
treatment with the prodrug ganciclovir which is converted
by HSV-tk into a toxic drug killing dividing cells.
Theoretically, expression of therapeutic genes
preferentially in cancer cells could be achieved by
regulatory elements from tumor-specific genes such as
carcinoembryonic antigen.
The first gene therapy products are expected to receive
FDA approval by the year 2000; the market for gene
Boulikas: An overview on gene therapy
4
therapy products is expected to exceed $45 billion by
2010.
This article reviews the molecular mechanisms and
recent developments for the gene therapy of cancer, HIV,
ADA deficiency, Parkinson's disease, lysosomal storage
disease, hemophilia A and B, α1-antitrypsin deficiency,
cystic fibrosis, rheumatoid arthritis, hypertension, familial
hypercholesterolemia, atherosclerosis/restenosis, wound
healing, and obesity including the treatment of cancer and
heart diseases with angiogenesis inhibitors and gene
transfer to the arterial wall. It is my intention to give a
general overview rather to exhaust the field.
DIVISION ONE: GENE DELIVERY
SYSTEMS AND GENE EXPRESSION
II. Gene delivery using retroviruses
A. Recombinant murine retroviruses
The recombinant Moloney murine leukemia virus
(Mo-MLV or MLV) has been extensively used for gene
transfer. Retroviral vectors derived from Mo-MLV
promote the efficient transfer of genes into a variety of cell
types from many animal species; up to 8 kb of foreign
DNA can be packaged in a retroviral vector. Recombinant
retroviruses have been the most frequently used and
promising vehicles for the delivery of therapeutic genes in
human gene therapy protocols (Appendix 1). Retroviral
vectors cause no detectable harm as they enter their target
cells; the retroviral nucleic acid becomes integrated into
chromosomal DNA, ensuring its long-term persistence and
stable transmission to all future progeny of the transduced
cell.
The life cycle of the retrovirus is well understood and
can be effectively manipulated to generate vectors that can
be efficiently and safely packaged. An important
contribution to their utility has been the development of
retrovirus packaging cells, which allow the production of
retroviral vectors in the absence of replication-competent
virus.
Recombinant retroviruses stably integrate into the
DNA of actively dividing cells, requiring host DNA
synthesis for this process (Miller et al, 1990). Although
this is a disadvantage for targeting cells at G0, such as the
totipotent bone marrow stem cells, it is a great advantage
for targeting tumor cells in an organ without affecting the
normal cells in the surroundings. This approach has been
used to kill gliomas in rat brain tumors by injection of
murine fibroblasts stably transduced with a retroviral
vector expressing the HSV-tk gene (Culver et al, 1992; see
below).
B. Retrovirus packaging cell lines