β-Cell survival factors

Prevention of β-cell destruction could be achieved by genetic manipulation of β-cells so that they produce a β-cell protection and/or survival factor. Such an approach would, in general, leave the immune system unaffected as the transgene production is localized to the islets. Targeting of a survival factor to the β-cells could be applied to individuals in whom autoimmune destruction of β-cells has begun, but not reached the end stage. Candidate transgenes currently under investigation include those encoding antioxidant enzymes: glutathione peroxidase, mitochondrial manganese superoxide dismutase (MnSOD), catalase, cytosolic copper-zinc superoxide dismutase, anti -apoptotic proteins (members of the heat shock proteins and Bcl-2 families) and modulators of cytokine signaling pathways (modulation of nuclear factor κB, NFκB), and suppressors of cytokine signaling. Alternative approaches for cytoprotection of β-cells have been envisaged, such as immune modulators; for example, an interleukin-1 (IL-1) receptor antagonist, hepatocyte growth factor, transforming growth factor β (TGF-β), adenoviral E3 and calcitonin gene-related peptide, inhibitors of Fas ligand signaling, and antistress factors such as thioredoxin. These factors have been addressed experimentally and could possibly, when expressed by β-cells in patients with diabetes, promote β -cell survival.

Genetic modulation of the immune system

In individuals with a high risk of developing T1DM, as indicated by genetic and humoral markers, but who have not yet entered the phase of autoimmune β-cell destruction, it might be possible to prevent the progression of the disease by DNA vaccination. In mice, it has already been observed that DNA vaccination with a glutamic acid decarboxylase 65 (GAD65) gene construct generates a protective humoral immune response and significantly delays the onset of diabetes [5]. Other studies of DNA vaccination in non-obese diabetic (NOD) mice were performed with a preproinsulin/glutamic acid decarboxylase 65 (Ins-GAD) fusion construct as the target antigen to introduce a larger number of autoantigenic target epitopes. DNA co-vaccination with Ins-GAD and B7-1wa (a membrane-bound molecule that can engage cytotoxic T-lymphocyte associated antigen 4 [CTLA-4] and promote negative signaling) generated protective regulatory T-cells and ameliorated the disease [6]. Both insulin and GAD65 may be key autoantigens in T1DM, and if the DNA vaccination approach leads to tolerance, β-cell destruction might be avoided.

A better understanding of the cellular sources for the expansion and turnover of β-cells seen in postnatal life could make way for a possible gene therapy approach leading to in situ regeneration of β-cells in patients with diabetes. Indirect evidence has suggested that postnatal β-cells derive from adult stem cells, proposed to reside in the pancreatic ducts, bone marrow, spleen or within islets. Lineage tracing experiments, however, demonstrated that the vast majority of adult β-cells derive from preexisting β-cells, suggesting that terminally differentiated β-cells retain a significant proliferative capacity and thus could represent an attractive target for expansion using gene therapy [10]. For example, mice over-expressing gastrin and TGF-α display a significantly increased islet cell mass. In addition, the cyclin-dependent kinase 4 (CDK4) and cyclins D1/D2 emerge as key determinants of β-cell proliferation and β-cell mass. Indeed, mice lacking CDK4 display a selective loss of β-cell function and over-expression of CDK4 results in preferential hyperplasia of the β-cells; however, it is likely that all approaches aiming at regenerating β-cells in patients with T1DM must be combined with a strategy to prevent autoimmune destruction of the newly formed β-cells.

With the cloning of the insulin gene in the late 1970s, it was proposed that using a suitable promoter and insulin gene construct, non-insulin-producing cells could be made into insulin-producing substitute cells, and that such cells could restore insulin production in T1DM and some patients with T2DM. The original experiments were carried out on cell lines and fibroblasts, but since then hepatocytes, myocytes, pituitary and exocrine cells have also been made into insulin-producing substitute cells. Proinsulin is only converted into insulin by the prohormone convertases PC2 and PC3, which are not expressed in most substitute cells. This limitation has been overcome by mutating the insulin gene so that a furin cleavable site is generated (INS-FUR). The protease furin, unlike PC2 and PC3, is expressed in most cells. Although processing of proinsulin to insulin can be achieved in substitute cells, none of these cells would be able to respond to insulin secretagogues with a physiologic secretion of insulin. Recent studies showed that both lentivirus [11] and adeno-associated virus vectors [12] were able to deliver INS-FUR cDNA efficiently to hepatocytes in vitro and in vivo. resulting in long-term correction of diabetes in rats.

Given its role in β-cell maintenance, the transcription factor Pdx-1 was investigated for its ability to induce ectopic insulin production in non β-cells in vivo.Ferber and colleagues have used adenoviral-mediated Pdx-1 overexpression for ex vivo transduction of adult human hepatocytes for cell-based therapy. In addition to glucagons, insulin and somatostatin, exogenous expression of Pdx-1 induced transcription of several β-cell products, including glucose transporter 2 and glucokinase, endogenous Pdx-1 and multiple downstream pro-endocrine developmental factors [13].

In a recent study Zhou et al.describe a way to reprogram pancreatic exocrine cells into insulin producing β-cells [14]. By using adenoviruses, they introduced combinations of nine different genes, previously identified to be essential to the embryonic development of β-cells, into the pancreas of live mice. They found that the transfer of three transcription factors (Ngn3, Pdx1 and Mafa) induces trans-differentiation of up to 20% of the successfully manipulated exocrine cells into β-like cells. By lineage tracing experiments, the authors proved that the trans-differentiated cells were, indeed, exocrine cells. These reprogrammed β-cells had similar morphology, protein expression and the capacity to secrete insulin as their “natural” counterparts. Moreover, they were able to improve hyperglycemia in mice with induced diabetes. This study elegantly shows that adult cells can be directly converted into another type of adult cell, which opens the possibility of directly converting cells in vivo for repair and regeneration as a therapeutic tool for diabetes and beyond [14].

This topic is discussed in detail in the first part of this chapter.