Strategies to Improve Islet Graft Function (a collaboration with Dr. J Joseph– Waterloo)

 

Background

Islet Graft Gene Transfer: The manipulation of gene expression prior to islet engraftment is a promising approach for the induction of immune tolerance [1,2] and may also benefit graft function. Most approaches to pre-transplantation gene manipulation have been to direct gene expression vectors to islets following isolation from the pancreas using one of several viral-based vectors. These approaches suffer from two main drawbacks: 1) penetration of gene expression to the islet core is often poor, limiting gene transduction [3]; and 2) viral infection alone may compromise islet function and increase immunogenicity [4,5]. The issue of islet penetration had been addressed in the past by direct infusion of adenovirus into the pancreatic circulation where entry into the islet core is facilitated by perfusion through the islet microcirculation prior to islet isolation. This has allowed gene expression throughout the islet [3,6]. Promising technological advances may be able to address the second issue, as non-viral methods of gene delivery are developed. In one recent study [7] a human insulin gene was delivered to the islets of live rats, resulting in the production of human insulin peptide and a reduction in blood glucose. This gene delivery was achieved with a technique termed ‘Ultrasound Targeted Microbubble Destruction’ (UTMD).

 

What is UTMD?:  In this technique, naked DNA is loaded into phospholipid-stabilized perfluoropropane gas microbubles suspended in a salt solution  [8]. These are injected into the circulation and then destroyed within the microvasculature of the target tissue by standard ultrasound using what is known as the ‘ultraharmonic’ mode. This results in the release of DNA-lipid particles that are taken up by surrounding cells, which then express the gene of interest. This methodology has been used in the past to transduce vascular cells in rat myocardium [9–11] and pancreas [7,12]. The specific delivery of genes to islets is achieved two ways: first, the ultrasound is directed specifically to the pancreas (targeting is verified by the accompanied ultrasound imaging); and secondly, the genes of interest are placed under the control of the insulin promoter such that they are only expressed in islet b-cells. Of importance to the present application, we propose that UTMB can be performed immediately following pancreas procurement (rather than in live animals) prior to isolation of islets for transplantation, thus allowing efficient delivery of genes to the islet core using a non-viral method.

 

Enhancing islet function by augmenting electrical responses: A key objective of this project is to utilize the UTMD approach to introduce gene ‘X’ into islets to augment insulin secretion. We believe that a gene that enhances the islet electrical responses provides an ideal proof of principle approach since the effects on insulin secretion are well known and straightforward to analyze.

             Much like the electrical impulses that control the contraction of heart muscle or the transmission of information along nerves, the secretion of insulin from pancreatic islets depends critically on electrical activity [13,14]. This so called ‘excitability’ is controlled by numerous ion channels (Fig. 1), the most well-studied being the ATP-sensitive K⁺ (K_ATP) channel [15] which is the target of the sulfonylurea drugs used to enhance insulin secretion in type 2 diabetes. As shown in Figure 1 however, the K_ATP channel is not the only channel controlling b-cell electrical responses. Notably, our previous work [16,17] has identified another K⁺ channel (a voltage-dependent K⁺ channel- Kv2.1) as an important regulator of b-cell electrical activity. It is well-known that inhibition of this ion channel increases glucose-stimulated insulin secretion [17–19].

             Kv2.1 is an attractive pharmacological target for improving islet function, and several approaches have been investigated to inhibit this channel as a means of improving islet function [16,17]. The most interesting in the present context is inhibition with a peptide neurotoxin called hanatoxin (HaTX) [20,21] which was initially isolated from tarantula venom [22]. We propose to induce islet cells to produce endogenous HaTX, which is known to enhance insulin secretion, through UTMD as a means to improve islet graft insulin responses and transplantation. This approach has several benefits: the peptide can be produced in islet cells by introducing the HaTX gene; HaTX has straightforward and well known effects on b-cell ion channels and insulin secretion which will allow us to easily determine the effectiveness of the UTMD treatment; and finally HaTX is expected to be secreted (through the regulated secretory pathway) to interact not only with the cells that received the gene, but also with their neighbors (the result being that we will not need to genetically modify every b-cell to obtain a beneficial effect).

 

Significance to Type 1 Diabetes

Islet transplantation as a treatment for Type 1 Diabetes is presently limited by both islet supply and degradation of graft function over time [23–25]. Improvement of islet graft function can be expected to improve the outcome of marginal mass transplants and perhaps lower the islet mass required for effective glucose-lowering. Improving islet graft function is therefore seen as an important step towards improving transplantation outcomes and addressing issues of limited tissue supply.

Literature Cited

 

      1.     Jun HS, Yoon JW (2005) Approaches for the cure of type 1 diabetes by cellular and gene therapy. Curr Gene Ther 5: 249-262.

      2.     Van Linthout S, Madeddu P (2005) Ex vivo gene transfer for improvement of transplanted pancreatic islet viability and function. Curr Pharm Des 11: 2927-2940.

      3.     Barbu AR, Bodin B, Welsh M, Jansson L, Welsh N (2006) A perfusion protocol for highly efficient transduction of intact pancreatic islets of Langerhans. Diabetologia 49: 2388-2391.

      4.     Sigalla J, David A, Anegon I, Fiche M, Huvelin JM, Boeffard F, Cassard A, Soulillou JP, Le Mauff B (1997) Adenovirus-mediated gene transfer into isolated mouse adult pancreatic islets: normal beta-cell function despite induction of an anti-adenovirus immune response. Hum Gene Ther 8: 1625-1634.

      5.     Zhang N, Schroppel B, Chen D, Fu S, Hudkins KL, Zhang H, Murphy BM, Sung RS, Bromberg JS (2003) Adenovirus transduction induces expression of multiple chemokines and chemokine receptors in murine beta cells and pancreatic islets. Am J Transplant 3: 1230-1241.

      6.     Wang MY, Shimabukuro M, Lee Y, Trinh KY, Chen JL, Newgard CB, Unger RH (1999) Adenovirus-mediated overexpression of uncoupling protein-2 in pancreatic islets of Zucker diabetic rats increases oxidative activity and improves beta-cell function. Diabetes 48: 1020-1025.

      7.     Chen S, Ding JH, Bekeredjian R, Yang BZ, Shohet RV, Johnston SA, Hohmeier HE, Newgard CB, Grayburn PA (2006) Efficient gene delivery to pancreatic islets with ultrasonic microbubble destruction technology. Proc Natl Acad Sci U S A 103: 8469-8474.

      8.     Shohet RV, Chen S, Zhou YT, Wang Z, Meidell RS, Unger RH, Grayburn PA (2000) Echocardiographic destruction of albumin microbubbles directs gene delivery to the myocardium. Circulation 101: 2554-2556.

      9.     Bekeredjian R, Chen S, Frenkel PA, Grayburn PA, Shohet RV (2003) Ultrasound-targeted microbubble destruction can repeatedly direct highly specific plasmid expression to the heart. Circulation 108: 1022-1026.

    10.     Bekeredjian R, Chen S, Pan W, Grayburn PA, Shohet RV (2004) Effects of ultrasound-targeted microbubble destruction on cardiac gene expression. Ultrasound Med Biol 30: 539-543.

    11.     Chen S, Shohet RV, Bekeredjian R, Frenkel P, Grayburn PA (2003) Optimization of ultrasound parameters for cardiac gene delivery of adenoviral or plasmid deoxyribonucleic acid by ultrasound-targeted microbubble destruction. J Am Coll Cardiol 42: 301-308.

    12.     Chen S, Ding J, Yu C, Yang B, Wood DR, Grayburn PA (2007) Reversal of streptozotocin-induced diabetes in rats by gene therapy with betacellulin and pancreatic duodenal homeobox-1. Gene Ther 14: 1102-1110.

    13.     Dean PM, Matthews EK (1968) Electrical activity in pancreatic islet cells. Nature 219: 389-390.

    14.     Dean PM, Matthews EK (1970) Glucose-induced electrical activity in pancreatic islet cells. J Physiol 210: 255-264.

    15.     Ashcroft FM, Rorsman P (1989) Electrophysiology of the pancreatic beta-cell. Prog Biophys Mol Biol 54: 87-143.

    16.     Macdonald PE, Sewing S, Wang J, Joseph JW, Smukler SR, Sakellaropoulos G, Wang J, Saleh MC, Chan CB, Tsushima RG, Salapatek AM, Wheeler MB (2002) Inhibition of Kv2.1 voltage-dependent K⁺ channels in pancreatic b-cells enhances glucose-dependent insulin secretion. J Biol Chem 277: 44938-44945.

    17.     Macdonald PE, Ha XF, Wang J, Smukler SR, Sun AM, Gaisano HY, Salapatek AM, Backx PH, Wheeler MB (2001) Members of the Kv1 and Kv2 voltage-dependent K⁺ channel families regulate insulin secretion. Mol Endocrinol 15: 1423-1435.

    18.     Philipson LH, Rosenberg MP, Kuznetsov A, Lancaster ME, Worley JF, III, Roe MW, Dukes ID (1994) Delayed rectifier K⁺ channel overexpression in transgenic islets and b-cells associated with impaired glucose responsiveness. J Biol Chem 269: 27787-27790.

    19.     Herrington J, Sanchez M, Wunderler D, Yan L, Bugianesi RM, Dick IE, Clark SA, Brochu RM, Priest BT, Kohler MG, McManus OB (2005) Biophysical and pharmacological properties of the voltage-gated potassium current of human pancreatic b-cells. J Physiol

    20.     Tamarina NA, Kuznetsov A, Fridlyand LE, Philipson LH (2005) Delayed-rectifier (KV2.1) regulation of pancreatic b-cell calcium responses to glucose: inhibitor specificity and modeling. Am J Physiol Endocrinol Metab 289: E578-E585.

    21.     Herrington J (2007) Gating modifier peptides as probes of pancreatic beta-cell physiology. Toxicon 49: 231-238.

    22.     Swartz KJ, MacKinnon R (1995) An inhibitor of the Kv2.1 potassium channel isolated from the venom of a Chilean tarantula. Neuron 15: 941-949.

    23.     Emamaullee JA, Shapiro AM (2007) Factors influencing the loss of beta-cell mass in islet transplantation. Cell Transplant 16: 1-8.

    24.     Truong W, Lakey JR, Ryan EA, Shapiro AM (2005) Clinical islet transplantation at the University of Alberta--the Edmonton experience. Clin Transpl 153-172.

    25.     Witkowski P, Zakai SB, Rana A, Sledzinski Z, Hardy MA (2006) Pancreatic islet transplantation, what has been achieved since Edmonton break-through. Ann Transplant 11: 5-13.

    26.     Macdonald PE, Salapatek AM, Wheeler MB (2003) Temperature and redox state dependence of native Kv2.1 currents in rat pancreatic b-cells. J Physiol 546: 647-653.

    27.     Jacobson DA, Lopez J, Eames S, Mendez F, Kuznetsov A, Philipson LH (2007) Islet electrical activity in mice that do not express the delayed rectifier potassium channel Kv2.1. Diabetes 56: 0011-OR.

 

BACKGROUND

PROJECTS

TECHNIQUES

THE LAB

THE PEOPLE

The MacDonald Islet Biology Lab

bcell.org

bcell.org

HOME