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

I. Introduction
Monumental progress in several fields including DNA
replication, transcription factors and gene expression,
repair, recombination, signal transduction, oncogenes and
tumor suppressor genes, genome mapping and sequencing,
and on the molecular basis of human disease are providing
the foundation of a new era of biomedical research aimed
at introducing therapeutically important genes into somatic
cells of patients. The main targets of gene therapy are to
repair or replace mutated genes, regulate gene expression
and signal transduction, manipulate the immune system, or
target malignant and other cells for destruction (reviewed
by Anderson, 1992; Nowak, 1995; Boulikas, 1996a,b;
Culver, 1996; Ross et al, 1996).
Two main approaches have been pursued for gene
transfer to somatic cells (i) direct gene delivery using
murine retroviruses, adenoviruses, adeno-associated virus,
HSV, EBV, liposomes, polymers, or direct plasmid
injection (gene therapy in vivo); and (ii) ex vivo gene
therapy involving removal of syngeneic cells from a
specific organ or tumor of an individual, genetic
correction of the defect in cell culture (ADA deficiency,
LDL-R for FH) or transfer of a different gene (IL-2 to
tumor infiltrating lymphocytes to potentiate the
cytotoxicity to tumors, cytokine genes to tumor cells from
a patient for cancer immunotherapy, multidrug resistance
gene transfer to render bone marrow cells resistant to
certain antineoplastic drugs), followed by reimplantation
of the cells. The reimplanted cells produce the therapeutic
protein.
Several key factors or steps appear to be involved for
the effective gene transfer to somatic cells in a patient or
animal model: (i) the type of vehicle used for gene
delivery (liposomes, adenoviruses, retroviruses, AAV,
HSV, EBV, polymer, naked plasmid) which will
determine not only the half-life in circulation, the
biodistribution in tissues, and efficacy of delivery but also
the route through the cell membrane and fate of the
transgene in the nucleus; (ii) interaction of the genevehicle
system with components in the serum or body
fluids (plasma proteins, macrophages, immune response
cells); (iii) targeting to the cell type, organ, or tumor, and
binding to the cell surface; (iv) port and mode of entrance
to the cell (poration through the cell membrane, receptormediated
endocytosis), (v) release from cytoplasmic
compartments (endosomes, lysosomes), (vi) transport
across the nuclear envelope (nuclear import); (vii) type
and potency of regulatory elements for driving the
expression of the transferred gene in a particular cell type
including DNA sequences that determine integration
versus maintenance of a plasmid or recombinant
virus/retrovirus as an extrachromosomal element; (viii)
expression (transcription) of the transgene producing
heterogeneous nuclear RNA (HnRNA) which is then (ix)
spliced and processed in the nucleus to mature mRNA and
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is (x) exported to the cytoplasm to be (xi) translated into
protein. Additional steps may include posttranslational
modification of the protein and addition of a signal peptide
(at the gene level) for secretion.
All steps can be experimentally manipulated and
improvements in each one can enormously enhance the
level of expression and therapeutic index of a gene therapy
approach. It has been proposed that the plasmid vector is
unable to translocate to the nucleus unless complexed in
the cytoplasm with nuclear proteins possessing nuclear
localization signals (NLSs). NLSs are short karyophilic
peptides on proteins destined to function in the nucleus
used for binding to specific transporter molecules in the
cytoplasm, mediating their passage through the pore
complexes to the nucleus (see Boulikas, 1998, this
volume). NLS are present on histones, transcription
factors, nuclear enzymes, and a number of other nuclear
proteins; nascent chains of DNA-binding polypeptides
could bind to the supercoiled plasmid in the cytoplasm
mediating its translocation to the nucleus.
During delivery of foreign DNA in vivo vehicles may
be attacked by macrophages, lymphocytes, or other
components of the immune system and the vast majority
will be cleared from blood, intracellular, or other body
fluids before it is given the chance to reach the membrane
of the cell target; the half-life of naked plasmids injected
intravenously into animals is about 5 min (Lew et al,
1995). Cationic lipids, other than being very toxic,
mediate efficient gene delivery passing through biological
membranes; those lipid-DNA complexes surviving the
immediate neutralization by serum proteins in the blood
can reach the lung, heart and other tissues after vein or
artery injection with one heart beat and transform
endothelial vascular cells (reviewed by Boulikas, 1996d).
A variety of viral vectors have been developed to
exploit the characteristic properties of each group to
maintain persistence and viral gene expression in infected
cells. Retroviral vectors and AAV integrate into target
chromosomes and the transgene they carry can be
inactivated from position effects from chromatin
surroundings. Vectors with persistence/integration
functions may not result in high levels of gene delivery in
vivo.
Adenoviruses and retroviruses which are of the most
frequently used vehicles for gene transfer can
accommodate up to 7kb of total foreign DNA into their
genome because of packaging limitations. This precludes
their use for the transfer of large genomic regions.
Transfer of intact yeast artificial chromosome (YAC) into
transgenic mice will enable the analysis of large genes or
multigenic loci such as human β-globin locus (reviewed
by Peterson et al, 1997).
A small portion of plasmid molecules crossing the cell
membrane will escape degradation from nucleases in the
lysosomes and become released to the cytoplasm; even a