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

sensitive mutations which allow propagation of the virus
in available packaging cell lines at permissive
temperatures are strategies for improving the therapeutic
efficacy of recombinant adenoviruses and for minimizing
the immune response elicited to the host (Fisher et al,
1996).
E1-defective, recombinant adenoviruses can be
replication-enabled by the codelivery of a plasmid
encoding the deleted E1 functions, a strategy now
designated as “conditional replication-enablement system
for adenovirus” (CRESA); when the original replicationenabling
plasmid was replaced by two separate plasmids
that encoded the necessary E1A and E1B functions the
E1-defective adenovirus could become conditionally
replication-enabled by an RNA transcript encoding the
required E1 functions. The RNA transcript of E1A
enhanced the therapeutic efficacy of the E1-defective
adenovirus: subcutaneous human tumor nodules
containing a fraction of cells cotransduced with the
replication-enabling RNA + DNA and an HSV-tk
adenovirus were reduced to a greater extent than control
nodules generated from the same fraction of cells
cotransduced with the HSV-tk adenovirus and an
irrelevant plasmid (Dion et al, 1996).
A new type of recombinant adenovirus, (called deltarAd),
deprived of all viral open reading frames and
retaining only the essential cis elements (i.e., ITRs and
contiguous packaging sequence), was propagated in 293
cells in the presence of E1-deleted helper virus (Fisher et
al, 1996). This adenovirus was packaged as concatamers
into virions and was used to deliver successfully the CFTR
gene to human airway epithelial cells in culture derived
from a cystic fibrosis patient. The new delivery system
needs improvements in its production and purification to
allow its evaluation and use in vivo.
F. Immune response to adenoviruses
eliminate therapeutic cells
Adenoviruses can achieve high levels of gene transfer
(Haffe et al, 1992; Morsy et al, 1993; Herz and Gerard;
Wilson, 1995; Kozarsky et al, 1996). However, the
duration of transgene expression is limited (i) by clearance
of the infected cells because of the cellular and humoral
immune response (including those mediated by cytotoxic
T lymphocytes) to adenoviral antigens (Yang Y et al,
1994, 1995) and (ii) by loss of adenoviral episomes in
progeny cells (Feng et al, 1997). To circumvent this
problem adenoviral/retroviral chimeric vectors were
constructed where the nonintegrative adenoviral vector
was able to induce target cells to function as transient
retrovirus producer cells and the retroviral particles were
able to transduce neighboring cells; thus the recombinant
adenovirus became integrative via the intermediate
generation of a retroviral producing cell (Feng et al, 1997).
First generation adenovirus-mediated gene transfer of
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CFTR to the mouse lung resulted in the expression of viral
proteins leading to the elimination of the therapeutic cells
expressing CFTR by cellular immune responses and
repopulation of the lung with nontransgene containing
cells; second generation E1-deleted viruses, also crippled
by a temperature sensitive mutation in the E2A gene,
displayed substantially longer recombinant gene
expression and induced a lower inflammatory response
(Yang et al, 1994).
In order to circumvent the elimination of adenovirustransduced
cells by immune responses and for achieving
persistence of transgene expression strategies to reduce the
potential for viral gene expression have been developed;
for example, an E4 modified adenovirus which was
replication defective in cotton rats and displayed a reduced
potential for viral gene expression in vivo was engineered
(Armentano et al, 1997). Vectors containing a wild-type
E4 region, E4 open reading frame 6, or a complete E4
deletion were compared in the lungs of BALB/c mice for
persistence of CFTR or lacZ expression; expression was
transient from the E1a promoter with all vectors but
persisted from the CMV promoter only with a vector
containing a wild-type E4 region; thus, transient
expression from adenoviral vectors may result from the
down-regulation of a promoter and not necessarily from
immune response-related factors (Armentano et al, 1997).
The elimination of therapeutically important cells from
the body after recombinant adenovirus-mediated delivery
seems to be a great limiting factor for the use of
adenoviruses in long-term gene therapy (Dai et al, 1995).
This problem can be partially circumvented by daily
administration of the immunosupressant cyclosporin A
prohibiting the elimination of virally-transduced cell by
activated T lymphocytes (Fang et al, 1995). A different
approach to suppress elimination of therapeuticallytransduced
cells after intra-articular delivery of genes to
treat RA is by pretreatment of the joints with the anti-T
cell receptor monoclonal antibody H57, a treatment which
resulted in a significant reduction in lymphocytic
infiltration and a persistence of transgene expression
(Sawchuk et al, 1996).
The prokaryotic Cre-LoxP recombination system was
adapted to generate recombinant adenoviruses with
extended deletions in the viral genome in order to
minimize expression of immunogenic and/or cytotoxic
viral proteins. An adenovirus was produced with a 25-kb
deletion that lacked E1, E2, E3, and late gene expression;
this vector exhibited viral titers similar to those achieved
with first-generation (E1a-deleted) vectors which was
efficient for gene transfer to cell culture but gene
expression declined to undetectable levels much more
rapidly than that sustained from first-generation vectors.
Vectors deleted only at E1a were sustaining a better
reporter gene expression because of their ability to
replicate (Lieber et al, 1996).