AbstractThis project focused on the development of an effective gene replacement therapy for the Duchenne muscular dystrophy (DMD) in its mouse models [(X-linked muscular dystrophy, mdx) and mdx-βgeo mice]. Earlier studies on the DMD replacement therapy (usually using mini-dystrophin) were largely not successful because dystrophin is being recognised as an antigen upon re-expression in dystrophic muscles and initiates the specific immune response. This leads to a short-lived or no expression of mini-dystrophin as was found in both human clinical trials and in animal models. It had been shown that the presence of pre-existing Tcells responding to dystrophin was responsible for this effect and immunosuppressive drug treatments in a canine model of DMD (cxmd) resulted in a stable expression of dystrophin for 2 years.
This study investigated the potential of using immunomodulatory factors such as
IDO (Indoleamine 2,3-dioxygenase) and TDO (tryptophan 2,3-dioxygenase) to
prolong expression of newly synthesised antigenic products in dystrophic mice. IDO and TDO are the rate-limiting enzymes of the tryptophan catabolism pathway, which regulate the production of kynurenines. These enzymes are known to increase the survival of grafts in transplantation by targeting dendritic cells, which play an important role in the T-cell activation. The plasmid with the general CMV promoter was used for expression of these enzymes in cell lines (HEK 293 cells and SC5 dystrophic myoblasts) and in skeletal muscles in vivo. To achieve targeting of the immunomodulatory constructs specifically into dendritic cells, the CD11c minimal promoter has been used. The plasmid driven by the CMV promoter was used for expression of the mini-dystrophin (an intracellular, structural protein) or the E.coli b-galactosidase (cytoplasmic but also secreted, strongly antigenic protein) in cells in vitro and muscles in vivo. Another plasmid construct expressing the minidystrophin gene under the muscle- specific creatine kinase promoter and the myosin light chain 1/3 enhancer combination was also used for studies of the effects of muscle-specific expressions of the transgene.
The cloning resulted in the generation of plasmids with the mini-dystrophin driven by MCK or CMV promoters and IDO transcripts under the control of CMV or CD11c minimal promoter (Chapter 3). The Western blotting analyses confirmed the ability of plasmids to drive specific transgenes’ expression in HEK 293, mouse myoblasts and RAW 264.7 macrophage cell lines in vitro. The RT-PCR analyses confirmed the expression of specific plasmids (Chapter 4).
The single plasmid expression experiment using the mini-dystrophin construct targeted into muscles was analysed by Western blotting, RT-PCR and immunohistochemistry. While RT-PCR confirmed its expression, the Western blotting results were ill-reproducible and immunohistochemistry did not confirm transgene expression. Moreover, there was no significant difference in the expression of mini-dystrophin driven by CMV or the muscle-specific MCK promoter/MLC enhancer combination (Chapter 5). The use of Pluronic SP1017-2
did not yield any significant improvement in the expression profile of plasmids as compared with normal saline. Hence, normal saline was used in subsequent
experiments as a vehicle of choice. Moreover, to support the hypothesis, there was a requirement to analyse the fold increase of the target plasmid expression in the presence of immunomodulatory factors. This could not be achieved using the minidystrophin plasmids due to low expression and lack of reproducibility. Therefore, the expression profile of b-galactosidase used as a model transgene was analysed instead. This protein is immunogenic due to its E.coli origin and is a 120 KDa protein, which is very close to 125 KDa size of the mini-dystrophin. The timeline of b-galactosidase expression was established based on the presence of this protein at 7 and 14 days and its absence 21 days post-injection, as assessed by Western blotting.
The expression profiles of IDO1 driven by CMV or CD11c were analysed and confirmed using RT-PCR; IDO1 did not show detectable expression in Western blotting. (Chapter 5).
The effects of co-injection of β-galactosidase with IDO1 driven by CMV or CD11c were analysed by Western blotting, RT-PCR and qPCR. In the control samples, 25% of muscles expressed b-galactosidase two weeks after the injection. This increased to 42% (5 out of 12 muscles) in samples co-injected with CD11c-driven IDO1 and 69% (11 out of 16 muscles) in samples co-injected with IDO1 driven by the CMV promoter. This confirmed the hypothesis that the presence of IDO1 has a potential to sustain the expression of an immunogenic transgene and indicated that the more widespread rather than targeted expression of IDO1 in antigen-presenting cells was more effective in supporting such an expression (Chapter 5).
The RT-PCR data showed IDO1 expression in most samples, also some that were
not showing β-galactosidase in Western blots, and confirmed the plasmid-driven IDO1 expression. The qPCR data also confirmed significantly increased expression of b-galactosidase and IDO1 in co-injected samples compared to β-galactosidaseonly controls. This further supported the hypothesis that co-expression of immunomodulatory IDO1 increases the transgene expression (Chapter 5).
The X-gal staining identified the expression of b-galactosidase in very few
myofibres, which correlated with the Western blotting data and confirmed the low efficiency of the “naked” plasmid uptake by skeletal muscles. The presence of infiltrating immune cells surrounding these β-galactosidase positive myofibres was probed by immunohistochemical methods (Chapter 6).
The qPCR analyses of a selection of the immune cell markers showed statistically significantly higher expression of CD4, CD8a, FoxP3 and COX2 in co-injected samples while expressions of IL-10 and IL-12 were statistically significantly lower in co-injected muscles.
The levels of antibodies against beta-galactosidase were quantified by ELISA in
control, b-galactosidase-only injected samples and IDO1 co-injected samples. The anti-β-galactosidase antibody levels were significantly lower in co-injected samples compared to the controls (Chapter 6).
These results indicate that co-expression of genes encoding immunomodulatory enzymes of the kynurenine pathway can be a feasible strategy for preventing loss of expression of transgenes targeted into muscles with pre-existing inflammation.
Hypothesis: “Co-expression of immunomodulatory factors (IDO/TDO) with minidystrophin or beta-galactosidase transgenes in skeletal muscles of the mdx mouse prolongs the expression of these transgenes.”
Aims:1-To prolong expression of immunogenic transgenes in dystrophic muscles withpre-existing inflammation.2-To prevent this loss of transgene by exploiting co-expression of genes encoding enzymes controlling kynurenine pathways instead of global immunosuppression.
Objectives:1-To modify and validate expression profile of plasmids expressing target genes(mini-dystrophin and beta-galactosidase) and immunomodulatory genes(IDO1/IDO2/TDO/FoxO3) both in vitro and in vivo.2-To check the effects of immunomodulatory genes on the prolongation of target genes expression in vivo.3-To assess the occurrence of the tolerance induction.
|Date of Award
|Darek Gorecki (Supervisor), Qian An (Supervisor) & Colin Sharpe (Supervisor)