Development of genetically engineered human intestinal cells for regulated insulin secretion using rAAV-mediated gene transfer

Tang, Shiue–Cheng; Sambanis, Athanassios

doi:10.1016/s0006-291x(03)00399-1

Cited by 31 publications

(31 citation statements)

References 28 publications

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“…In previous studies in which an insulin-EGFP fusion protein was transiently expressed in human L-cells, engineered insulin-EGFP and endogenous GLP-1 co-localized in secretory granules [14]. The similarity in the secretion of insulin and GLP-1 from GLUTag-INS cells suggests that colocalization of insulin and GLP-1 occurs in GLUTag-INS cells as well.…”

Section: Induced Secretion Of Insulin and Glp-1 From Glutag-ins Cellsmentioning

confidence: 93%

“…With approximately 2.4 million cells per well, this level of insulin expression is on par with values reported for other insulinsecreting non-β-cells, but about 5-fold lower than that reported for β-cell lines. The basal rates of insulin secretion for engineered AtT20 cells [32], engineered NCI-H716 cells [14], and GLUTag-INS cells are 60, 79, 86 fmole/(10 6 cells·hr) respectively, while the basal rate of secretion for βTC3 cells [29] is 384 fmole/(10 6 cells·hr). For GLP-1, the basal secretion rate was 1301.5 ± 245.1 fmole/(well·2hr).…”

Section: Induced Secretion Of Insulin and Glp-1 From Glutag-ins Cellsmentioning

confidence: 99%

“…Previously, our lab engineered a human L-cell line to release insulin in response to nutrient administration [14] and showed preferential transduction by adeno-associated virus for L-cells over enterocytes in a co-culture model [15]. In advancing L-cell mediated insulin therapy to a tissue-engineered treatment for an adult mouse model for IDD, the murine L-cell line, GLUTag, was selected for this study, as it would represent a closer allograft model for mice and glucagonlike peptide (GLP-1) secretion from this cell line has been found similar to those of primary cell cultures and in vivo [16].…”

Section: Introductionmentioning

confidence: 99%

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Insulin-secreting L-cells for the treatment of insulin-dependent diabetes

Bara

Sambanis

2008

Biochemical and Biophysical Research Communications

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Cell-based treatments for insulin-dependent diabetes (IDD) may provide more physiologic regulation of blood glucose levels than daily insulin injections, thereby reducing the occurrence of secondary complications associated with diabetes. An autologous cell source is especially attractive for regulatory and ethical reasons in addition to eliminating the need for immunosuppression. This study uses non-β-cells, genetically modified for physiologic insulin secretion. Enteroendocrine L-cells, exhibit regulated secretion in response to physiologic stimuli and their endogenous products are fully compatible with prandial metabolism. Murine GLUTag L-cells were transfected with a plasmid coexpressing human insulin and neomycin resistance and the stable cell line, GLUTag-INS, was established. Secretion properties of GLUTag-INS cells were investigated in vitro through induced secretion tests using meat hydrolysate or 3-isobutyl-1-methylxanthine and forskolin as secretagogues. GLUTag-INS cells rapidly co-secreted recombinant insulin and endogenous glucagon-like peptide in response to metabolic cues from the surrounding medium and demonstrated efficient processing of proinsulin to insulin.

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Section: Induced Secretion Of Insulin and Glp-1 From Glutag-ins Cellsmentioning

confidence: 93%

Section: Induced Secretion Of Insulin and Glp-1 From Glutag-ins Cellsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Insulin-secreting L-cells for the treatment of insulin-dependent diabetes

Bara

Sambanis

2008

Biochemical and Biophysical Research Communications

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“…No significant change was found in the amount of vector associated with Caco-2 cells by the Cyto-D treatment. A luciferase assay (Promega) was used to quantify rAAV2-luciferase-enhanced green fluorescent protein (EGFP) (Tang & Sambanis, 2003) transduction of polarized intestinal epithelial cells in culture. Treatment with 1 and 10 mg Cyto-D ml 21 resulted in 8.0(±1.0)-fold and 6.6(±3.0)-fold inductions of apical rAAV2 delivery, respectively (Fig.…”

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confidence: 99%

Transient cytochalasin-D treatment induces apically administered rAAV2 across tight junctions for transduction of enterocytes

Sibley

Tang

2008

Journal of General Virology

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Enteropathogens are known to disrupt apical actin filaments and/or tight-junction barriers of intestinal epithelial cells to promote infection. In this study, we show that a controlled, cytochalasin-D (Cyto-D)-mediated disruption of actin filaments and tight junctions enhanced the apical delivery of the gene-therapy vector recombinant adeno-associated virus serotype 2 (rAAV2). This increase in transduction efficiency can be attributed to the enhanced delivery of rAAV2 across the Cyto-D disrupted tight junctions, allowing basolateral entry of rAAV2. Previously, we have shown that MG101 and doxorubicin are capable of overcoming proteasome-mediated transduction barriers of rAAV2 in enterocytes. In this study, when Cyto-D was combined with MG101 and doxorubicin in apical delivery of rAAV2 to transduce the differentiated Caco-2 enterocytes, a synergistic >2300-fold increase in transgene expression was achieved. We conclude that Cyto-D is capable of permeating the polarized enterocytes for rAAV2 transduction, which may potentially be a useful device to facilitate intestinal gene transfer via the gut lumen.

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“…These features were exploited in a number of studies aimed at restoring insulin production, [2][3][4] at facilitating islet transplantation 5,6 and at inducing immunoregulation in prediabetic NOD mice. 7,8 Perhaps the most crucial determinant of high-level transgene expression from these vectors was the serotype.…”

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confidence: 99%