Gene Therapy for Type I Diabetes
Our
long-term objective is to genetically engineer ex vivo pancreatic islets
to enhance cell survival and function following transplantation as therapy for
type I diabetes mellitus. Oxygen free radicals produced endogenously and via
the action of pro-inflammatory cytokines play a major role in b cell destruction in type I diabetes as well
as following islet cell transplantation, as depicted in the model below.
To restore the
cellular redox state, cells and tissues respond by inducing cytoprotective,
antioxidant and anti-inflammatory enzymes such as heme oxygenase-1 (HO-1) and
manganese superoxide dismutase (MnSOD). Induction of these enzymes has been
shown to play an important cytoprotective role in pancreatic islets exposed to
the inflammatory effects of cytokines or oxygen-derived free radicals.
To
improve the inherent resistance of pancreatic islets to pro-inflammatory mediators
and enhance islet cell survival following transplantation, we are over-expressing
these genes either alone or in combination in an in vitro model of
cytokine-induced islet cell injury. HO-1 engineered AAV vector plasmids are
depicted with a constitutive promoter, CMV enhancer/β-actin promoter
(CBAp) as well as an inducible MnSOD enhancer element coupled to the minimal
viral thymidine kinase promoter (TKp).
The inherent antioxidant qualities of both HO-1 and
MnSOD along with recent evidence proposing that the protective effects of
HO-1-derived carbon monoxide (CO) may be a consequence of MnSOD activation has
prompted us to propose co-expression of these proteins in a bicistronic AAV
vector system. Based on the simplified
model depicted above we have rationalized that: 1) HO-1 will break the heme
group of cytochrome c blocking downstream activation of the caspase pathway and
cell death; 2) MnSOD will dismutate electron transport-derived superoxide
radical preventing alterations of mitochondrial membrane potential, subsequent
cytochrome c release and peroxynitrite formation; and 3) the elevated HO-1
activity will release CO which could further induce endogenous MnSOD synthesis,
creating a cycle of cytoprotective activity.
The
ex vivo engineered islets will subsequently be evaluated in vivo
via transplantation in animal models of diabetes.
Below are
examples of mouse and human islets transduced with a bicistronic rAAV vector
containing the red fluorescence protein (RFP) and the green fluorescence
protein (GFP) to illustrate the efficacy of our studies.
The photo below
illustrates transduction of isolated islets with a rAAV/GFP vector which were
subsequently transplanted under the kidney capsule and evaluated for GFP
expression one month after the transplant.
