| dc.description.abstract | Rheumatoid arthritis (RA) is an autoimmune chronic inflammatory disease, of
unknown aetiology. Several disease-modulating approaches have been developed in
the past years, however these are expensive, usually accompanied by unwanted
side-effects and 30% of the patients fail to respond. The transcription factor NF-kB is
a key factor in the development and perpetuation of the disease, as it regulates a
number of inflammatory genes. The activity of certain signalling pathways can be
modulated by delivering into cells inhibitors coupled to Protein Transduction Domains
(PTDs). The aim of this study was to develop a secretable PTD-fusion NF-κΒ inhibitor
that is produced and secreted by genetically engineered mammalian cells in sufficient
amounts to subsequently transduce and regulate NF-κΒ activity in neighbouring cells.
Such methodology could be useful for the management of RA by transplantation of
engineered cells or directly using gene delivery into the synovial joints.
In this study, PTD-fusion proteins were fused to the IL-2 secretion signal and
their ability to be secreted from mammalian cells was explored. Secretable forms of
TAT-IgG2A and TAT-eGFP were generated as control PTD-fusion proteins, and the
TAT-srIκΒα (super repressor IκBα, a NF-κΒ inhibitor) was generated as an NF-κΒ
PTD-fusion inhibitor. Western blotting analysis of supernatants from transiently
transfected 293T cells revealed that TAT-IgG2A, TAT-eGFP and TAT-srIκΒα are
secreted with variable efficiencies. When concentrated, PTD proteins were able to
transduce mammalian cells, as demonstrated with Jurkat cells by confocal
microscopy and western blotting analysis.
The TAT PTD domain was replaced to a more stable, furin cleavage-resistant
and less positively charged PTD domain, the TAT3 PTD domain, to ensure that PTDfusion
proteins will be secreted more efficiently. This change of the PTD domain did
not increase secretion levels of the srΙκBα. Subsequently, the Latent Associated
Peptide (LAP) of TGFβ, was fused to the TAT3-srIκΒα inhibitor, via a matrix
metalloproteinase (MMP) cleavage linker. This LAP-MMP-PTD-fusion NF-κΒ inhibitor
was again poorly secreted. In turn, the srIκΒα inhibitor was replaced with a small
synthetic NF-κΒ inhibitor, termed Nemo Binding Domain (NBD), in the form of LAP
MMP-TAT3-NBD NF-κΒ inhibitor. Western blotting analysis of supernatants from
transiently transfected 293T cells revealed that the LAP-MMP-TAT3-NBD was
efficiently secreted.
The ability of LAP-MMP-TAT3-NBD to inhibit NF-κΒ was tested in vitro with the
use of a cell-assay based on HeLa cells that are permanently transfected with the
luciferase gene driven by an NF-κB regulated promoter. In this assay, HeLa cells that
were treated with the secreted LAP-MMP-TAT3-NBD, showed reduced levels of
luciferase activity after IL-1β stimulation. Subsequently, using a replication-deficient
lentiviral vector, genetically engeneered DBA/1 fibroblasts (DTF) able to produce the
secreted LAP-MMP-TAT3-NBD were generated. The NF-κB inhibitory properties of
the secreted LAP-MMP-TAT3-NBD were tested in vivo in the Carrageenan-induced
paw oedema, Antigen Induced Arthritis and Air-Pouch acute inflammation models.
Paws of mice that were treated with engineered cells or lentivirus encoding LAPMMP-
TAT3-NBD demonstrated milder paw swelling, suggesting that LAP-MMPTAT3-
NBD had a protective role in the induction of inflammation. However, the LAPMMP-
TAT3-NBD did not demonstrate anti-inflammatory effects in the Air-Pouch
model.
In this study, I present a method to design PTD-fusion proteins that can be
efficiently secreted from mammalian cells and I demonstrate a novel gene therapy
approach for the local delivery of a therapeutic agent. | en_US |
