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

AAV might be better suited. Administration of single
doses of AAV-CFTR vector to 10 rhesus macaques by
fiberoptic bronchoscopy to the right lower lobe of lungs
showed that transfer of the CFTR gene occurred in
bronchial epithelial cells of each animal by in situ DNA
PCR; vector mRNA was detectable for 180 days after
administration as detected by RT-PCR and there was no
evidence of inflammation (Conrad et al, 1996).
Transfer of CFTR gene was also achieved using
retroviral vectors; sodium butyrate treatment of murine
retrovirus packaging cells producing the vector increased
the production of the retrovirus vector between 40- and
1,000-fold (Olsen and Sechelski, 1995).
Yoshimura and coworkers (1992) have transduced
airway epithelial cells in mice by intratracheal instillation
of a plasmid carrying the CFTR gene under control of the
Rous sarcoma virus promoter with cationic liposomes. Use
of liposomes have successfully transferred the CFTR gene
to epithelia and to alveoli deep in the lung leading to
correction of the ion conductance defects found in the
trachea of transgenic mice (Hyde et al, 1993).
Complexes of cationic polymers and cationic lipids
with adenovirus enhanced gene transfer to the nasal
epithelium of cystic fibrosis mice in vivo (Fasbender et al,
1997). The novel cationic lipid EDMPC (1,2dimyristoylsn-glycero-3-ethylphosphocholine,
chloride
salt) mediated efficient intralobar DNA delivery of CFTR
to rodents; there was no correlation between DNA-
EDMPC formulations that delivered DNA most efficiently
in vitro and those that worked best in vivo (Gorman et al,
1997). The structures of several novel cationic lipids that
were effective for CFTR gene delivery to the lungs of
mice were investigated; an amphiphile (lipid # 67)
consisting of a cholesterol anchor linked to a spermine
head-group in a "T-shape" configuration was shown to
sustain a 1,000-fold increase in expression above that
obtained in animals instilled with naked pDNA alone and
was greater than 100-fold more active than other cationic
lipids and comparable to that of adenoviral vectors (Lee et
al, 1996).
C. Clinical trials on cystic fibrosis patients
A significant number of clinical trials on CF have
received RAC approval (Appendix 1 and Table 4 in
Martin and Boulikas, following article). The clinical
protocols approved use adenoviral delivery of CFTR
(#118-123, 125, 128, 129) or cationic lipids (#193, 203,
212, and 214). The only two protocols using AAV in
clinical trials are for CF (#165, 166) whereas no retroviral
protocol has been approved for CF as of December 1997.
Results of clinical trials have been reported and more
will become available in the near future. A single dose of
400 µg pCMV-CFTR in complex with 2.4 mg DOTAP,
administered to the nasal epithelium of eight CF patients
Boulikas: An overview on gene therapy
112
resulted in partial, sustained correction of CFTR-related
functional changes toward normal values in two treated
patients; transgene DNA was detected in seven of eight
treated patients for up to 28 days after treatment; vector
derived CFTR mRNA was detected in two of the seven
patients at 3 and 7 days from treatment using PCR
(Porteous et al, 1997). Complexes of plasmids with
DOTAP liposomes rendered their DNA resistant to
DNaseI something relevant to clinical trials for gene
therapy of CF, in which patients are normally removed
from treatment with DNase before receiving
administration of DNA (Crook et al, 1997).
A formulation of plasmid encoding CFTR (pCF1-
CFTR) was at least as effective as complexes of DNA
with lipid in partially correcting the Cl- transport defect in
CF patients by administering complexes of DNA-lipid to
one nostril and DNA alone to the other nostril in a
randomized, double-blind study (Zabner et al, 1997).
The safety of the nebulised cationic lipid formulation
(GL-67/DOPE/DMPE-PEG5000) to be used for the
transfer of CFTR to CF patients was first tested on 15
healthy volunteers in the absence of plasmid DNA; no
adverse clinical events were seen (Chadwick et al, 1997).
XXXIV. Rheumatoid arthritis (RA)
A. Molecular mechanisms for development
of RA
RA is a systemic autoimmune disease caused by
genetic and environmental factors; chronic inflammation
and hyperactivation of synovial cells in the joints is the
salient feature of RA resulting in the thickening
(hyperplasia) of the synovial membrane lining the interior
surface of the joint capsule. The mechanism involves
synovial cell proliferation, infiltration by leukocytes, and
excessive extracellular cell matrix deposition. The
activated synovial cells in the hypertrophied synovium
produce inflammatory cytokines and degradative enzymes
that invade and erode the articular cartilage leading to
partial or complete destruction of cartilage and bones. The
inflamed synovium in RA is infiltrated by lymphocytes
and monocytes, a process mediated by the enhanced
binding of the very late antigen-4 (VLA-4) to vascular cell
adhesion molecule-1 (VCAM-1) (Chen et al, 1995).
Several well-characterized murine models of arthritis
closely resemble RA immunologically, genetically, and
histopathologically and have been developed to study RA.
Collagen-induced arthritis in DBA/1 mice is one model of
RA; the animals exhibit marked synovitis and erosions.
The disease can be adoptively transferred to SCID mice
using arthritogenic splenocytes from DBA/1 mice injected
with bovine collagen type II (Chernajovsky et al, 1997).
Bacterial cell wall-induced arthritis in rats is another
model (Makarov et al, 1996).