(500c) Development of a Pancreatic Substitute Based On Genetically Engineered Intestinal Endocrine Cells

Introduction: A tissue engineered pancreatic
substitute constitutes a promising cell-based insulin therapy for achieving
tight glycemic regulation in diabetes patients. In
particular, non-beta cells genetically engineered to secrete insulin can
address the major issues of immune rejection and donor cell availability
through their potential autologous nature. The
objective of this study was to generate a recombinant system of luminescent and
insulin-secreting intestinal L-cells that allows treatment of diabetic mice and
direct monitoring of the graft over time. Recombinant insulin-secreting GLUTag-INS cells were previously generated by stably transfecting murine intestinal
endocrine GLUTag cells with human B10 insulin [1]. Although insulin secretion was
achieved, in vivo therapeutically relevant levels were not obtained [2]. Further genetic engineering
via transduction with a lentivirus containing the
wild-type human insulin gene followed by a GFP reporter gene, generated cell
line GLUTag-eINS with enhanced insulin secretion and
promise for therapeutic application. As these cells constitute allogeneic therapy in diabetic mice, we are pursuing cell
microencapsulation in alginate material as a protective barrier against in
vivo immune rejection. In addition, we are investigating luciferase incorporation for non-invasive monitoring via
bioluminescence of in vivo graft survival.

Materials
and Methods:
Sequential lentiviral transductions were performed on
GLUTag-INS cells: the first contained the wild-type
insulin gene followed by a GFP reporter gene, and the second contained the luciferase reporter gene. Fluorescence-activated sorting
was employed to enrich the transduced population to
98% fluorescence; bioluminescence images and signals were captured with the
IVIS Lumina (Xenogen). Insulin concentrations in
secretion tests were measured by radioimmunoassay (Millipore, MA). Cell
microencapsulation was performed in 3.3 wt% alginate (FMC Biopolymer, Norway)
cross-linked with barium using an electrostatic droplet generator [3].

Results
and Discussion: The
first lentivirus transduction generated the enhanced
cell line GLUTag-eINS and significantly improved
insulin secretion from GLUTag-INS, making them
promising for therapeutic application. The second lentivirus
transduction generated the cell line GLUTag-eINS-fluc
with no negative effects on insulin secretion (Figure 1). Strong
bioluminescence signal of 1.5-1.8x10⁸ photons/(s∙cm²∙sr)
was obtained from GLUTag-eINS-fluc monolayers in 12-well plates and was stable upon expansion
and passaging.

Insulin
secretion from GLUTag-eINS cells in barium alginate
microcapsules was comparable to that from cell monolayers;
a protective barrier can therefore be provided without impeding insulin
secretion.  Bioluminescence signal from microencapsulated GLUTag-eINS-fluc cells was also similar to monolayers, thus offering promise for in vivo
monitoring capabilities. In vivo experiments with microencapsulated GLUTag-eINS-fluc cells are currently in progress in normal
mice at subtherapeutic levels as a first step in
characterizing this new system toward in vivo efficacy studies.

Conclusion: A system of recombinant
luminescent and insulin-secreting intestinal L-cells can potentially serve as a
pancreatic substitute for the treatment of diabetic animal models, as well as
offer the ability for direct monitoring of graft survival
over time.

References:

1.           
Bara, H. and A. Sambanis, Insulin-secreting L-cells for the treatment of
insulin-dependent diabetes. Biochem Biophys Res Commun,
2008. 371(1): p. 39-43.

2.           
Bara, H., P.M. Thule, and A. Sambanis, A
cell-based approach for diabetes treatment using engineered non-beta cells.
J Diabetes Sci Technol,
2009. 3(3): p. 555-61.

3.           
Goh, F. and A. Sambanis, In vivo noninvasive monitoring of dissolved oxygen
concentration within an implanted tissue-engineered pancreatic construct. Tissue Eng Part C Methods, 2011. 17(9): p. 887-94.