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

LNCaP; delivery of this recombinant adenovirus resulted
in cytotoxicity to the DU145 and LNCaP, but not PC-3,
cell lines and reduced the clonogenic capacity of DU145
cells cultured alone or mixed with various ratios of
irradiated human bone marrow. This finding led to a
strategy for effectively reducing DU145 and erbB-2positive
primary prostate tumor contamination in bone
marrow cultures (Kim et al, 1997). Delivery of an antierbB-2
single chain (sFv) antibody gene for previously
treated ovarian and extraovarian cancer patients is in
clinical trials using adenoviral gene delivery (protocol
#133).
A luciferase expression vector (pRSVLuc) noncovalently
linked to a humanized HER2 antibody
(rhuMAbHER2) covalently modified with poly-L-lysine
bridges was able to direct gene transfer to HER2
expressing cells in vitro (Foster and Kern, 1997).
A targeting gene therapy approach for hematopoietic
stem/progenitor cells has been directed to cell lines
expressing the c-kit receptor; plasmid DNA containing a
luciferase reporter gene was condensed with polylysine
covalently linked to streptavidin (which binds biotinylated
ligand) and with polylysine covalently linked to
adenovirus (to achieve endosomal lysis) with the final
addition of biotinylated steel factor; omission of the
adenovirus endosomalytic agent from the vector resulted
in the loss of gene expression (Schwarzenberger et al,
1996).
Systemic administration of a c-fos antisense, regulated
by mouse mammary tumor virus (MMTV) control
elements in a retroviral vector, showed expression only in
breast epithelium although the vector could be detected in
several tissues thus supporting targeting to MMTVregulated
tissues (Arteaga and Holt, 1996).
Liposomes coated with polyethyleneglycol (PEG) can
be efficiently targeted to tumor cells that express folate
receptors (KB cells) via conjugation of folate to a PEG
spacer of 25 nm in length; shorter PEG spacers were not
efficient in mediating binding of the liposomes to KB cells
(Lee and Low, 1995).
Neri and coworkers (1997) were able to target an
angiogenesis-associated oncofetal fibronectin (B-FN)
isoform by affinity-matured recombinant antibody
fragments. B-FN is present in vessels of neoplastic tissues
during angiogenesis but is absent from mature vessels and
could provide a target for diagnostic imaging and therapy
of cancer. Phage display libraries were screened to isolate
human antibody fragments able to recognize this isoform
across species; imaging of F9 murine teratocarcinomas
grafted in nude mice is shown on Figures 8 and 9.
H. Targeted gene delivery with peptidedisplaying
phages
Boulikas: An overview on gene therapy
26
Development of methods to display and select
collections of peptides specific for binding a target provide
valuable tools to identification of peptide drugs; peptides
could be selected for binding biological targets including
cell surface receptor molecules, DNA, antibodies, or
whole cells. The technique of peptide-displaying phages
has been developed for targeted gene delivery. Selection
of cell surface-binding peptides, ideally specific for each
type of cell in the human body, will be used for
incorporation into gene delivery vehicles to achieve the
long-searched tissue specificity of the vector (reviewed by
Russell, 1996).
Development of the random peptide library as a source
of specific protein binding molecules (Devlin et al, 1990)
and exposure of random peptides on the surface of phages
(Cwirla et al, 1990) has been the catalyst for progress in
this promising field. Libraries of random 8 to 12 amino
acid peptides expressed on the N-terminus of the pIII
protein of the fd phage or on the N-terminus of the pVIII
major coat protein of the same phage have been selected
that bind the extracellular domain of human IL-1 receptor;
screening was against immobilized IL-1 receptor
extracellular domain. Two families of peptides could act
as antagonists blocking triggering of the IL-1 signaling
pathway; because IL-1 levels become elevated in
autoimmune and inflammatory disorders, these peptide
antagonists of IL-1 receptor could provide novel drugs for
these diseases (Yanofsky et al, 1996).
Phages displaying known integrin-binding peptides
have been shown to bind and enter mammalian cells (Hart
et al, 1994). A peptide antagonist to thrombin receptor has
been identified using phage display (Doorbar and Winter,
1994). Production of cell-targeting ligands has been
achieved by cell-binding peptides specific for different
cell types in culture; these peptides are selected through
six rounds of binding (and amplification of phage clones)
to a particular cell type from random peptide-presenting
phage libraries; the selected peptides are apparently
recognizing specific surface receptor molecules. For
example, the 20mer peptide
KTLTLEAALRNAWLREVGLK has been selected for its
high affinity for PEA10 mouse fibroblast cells binding
1000 more efficiently to the cells than random peptides
(Barry et al, 1996).
IX. Gene delivery with polymers,
peptides and other means
A. Delivery of transferrin-polylysine-DNA
complexes
A number of polymers have been tested and shown to
enhance significantly the transfection efficiency of
plasmids but also of viruses; the enhancement in
transfection results from a facilitation in the interaction of