Pharmacologically Regulated Regeneration of Functional Human Pancreatic IsletsIntroductionResultsEx Vivo Expansion of Human Islets with Two Chemically Inducible Growth SwitchesEngineered Human Islets Conserve Functionality upon Induced ProliferationIn Vivo Administration of the CID to Transplanted Diabetic Mice Leads to Normalized Glucose Levels over TimeDiscussionMaterials and MethodsIsolation and culture of human isletsTransplantation of human islets in miceVector production and transductionHistology and immunocytochemistryPerifusion and dosage of human insulinAcknowledgmentsReferencesPharmacologically Regulated Regeneration ofFunctional Human Pancreatic IsletsGary P. Kobinger,1,2Shaoping Deng,3Jean-Pierre Louboutin,1,2Marko Vatamaniuk,4Victor M. Rivera,5Moh-Moh Lian,3James F. Markmann,3Tim Clackson,5Steven E. Raper,3Franz Matschinsky,4James M. Wilson,1,2,*1Gene Therapy Program, Division of Medical Genetics, Department of Medicine,3Department of Surgery, and4The Diabetes Center,University of Pennsylvania Health System, Philadelphia, PA 19104, USA2The Wistar, Philadelphia, PA 19104, USA5ARIAD Pharmaceuticals, Cambridge, MA, USA*To whom correspondence and reprint requests should be addressed at 204 Wistar Institute, 3601 Spruce Street, Philadelphia,PA 19104–4268, USA. Fax: (215) 898 6588. E-mail: [email protected] online 19 October 2004Transplantation of allogeneic islets can correct the metabolic abnormalities of Type I diabetes.Limited availability of donor pancreas tissues restricts the application of this therapeutic modality toa subset of eligible recipients. In an attempt to expand the utility of available donor human pancreastissue, we developed a method to stimulate the proliferation of insulin-secreting B-cells withinhuman islets. A lentiviral vector was used to introduce into human islets chimeric signaling receptorsthat are activated to stimulate cell proliferation through interactions with a small-molecule drugcalled a chemical inducer of dimerization (CID). In vitro exposure of vector-transduced human isletsto the CID expanded the number of cells and increased regulated insulin secretion. Transplantationof the regenerated islets into diabetic immunodeficient mice, followed by in vivo administration ofthe CID, corrected hyperglycemia. This strategy has the potential to reduce the quantity of humanislets required for treatment of patients with Type I diabetes.INTRODUCTIONAutoimmune destruction of islets in the pancreas leads tothe development of insulin-dependent diabetes mellitus(Type I diabetes). Replacement of insulin-producingtissue by transplantation of islets, together with immunesuppression, has corrected hyperglycemia in a number ofpatients. However, the limited availability of donor tissueand the nonproliferative status of islets ex vivo limit thisapproach, since pancreatic islets from at least two donorsare typically necessary for each recipient [1]. A number ofstrategies are being pursued to increase the availability ofallogeneic islets for transplantation, such as including theuse of stem cells and inducing proliferation of islets [2,3].A proliferation switch comprising a drug-inducible chi-meric receptor has been used to induce reversibly theproliferation of lineages of the hematopoietic system[4,5]. The proliferation switch consists of a growth factorreceptor signaling domain fused to binding domains for achemical inducer of dimerization (CID). The CID used inthis study is AP20187, a bivalent cell-permeant com-pound that binds simultaneously to two FKBP12 proteindomains [6]. Addition of AP20187 to engineered cellsinduces dimerization of the FKBP domains fused togrowth factor receptor signaling domains, which can inturn trigger cell division through signal-dependent kinaseactivity [7,8].Incorporation of the proliferation switch in target cellssuch as islets can be performed with gene transfervehicles. A number of gene transfer strategies have beenevaluated for transduction of islets ex vivo [9]. Lentiviralvectors were reported to transduce islets stably with goodefficiency [10–12]. We recently showed that HIV-basedvectors pseudotyped with the envelope protein from thelymphocytic choriomeningitis virus (LCMV) are less toxicand transduce human islets more efficiently than thecommonly used vesicular stomatitis virus G protein (VSV-G)-pseudotyped HIV vector [13]. These data also showedthat LCMV-pseudotyped HIV vector transduces insulin-and glucagon-positive cells (h-and a-cells) with similarefficiencies.The present study explores the possibility of usinggrowth switches for inducing proliferation of humanpancreatic islets ex vivo and in vivo. Results show thatengineered islets with a lentiviral vector encoding agrowth switch could proliferate ex vivo in response toARTICLEdoi:10.1016/j.ymthe.2004.09.010MOLECULAR THERAPY Vol. 11, No. 1, January 2005105Copyright C The American Society of Gene Therapy1525-0016/$30.00pharmacologic stimulation. Analysis of expanded isletsrevealed that insulin secretion was not compromisedupon glucose stimulation, indicating that function waspreserved. Finally, transplantation of expanded modi-fied islets in immunocompromised diabetic mice com-bined with in vivo administration of the CID resultedin progressive normalization of glucose levels overtime.RESULTSEx Vivo Expansion of Human Islets with TwoChemically Inducible Growth SwitchesWe cloned two candidate cell growth switches into HIV-based vectors and concentrated LCMV-pseudotypedparticles were produced. The candidate receptors werederived from the erythropoietin receptor (EpoR) and thetype I fibroblast growth factor receptor (FgfR). Thesereceptors are members of the receptor tyrosine kinasefamily and signal the cell to divide upon dimerization[14,15]. EpoR is expressed by human islets and wasshown to protect islets in culture from cytokine-inducedapoptosis when activated [16]. Inhibition of FgfR activ-ity in mouse pancreatic islets leads to diabetes andregression in the number of h-cells in vivo [17]. Bothreceptors have the capacity to signal through the JAK/STAT pathway, which is functional in pancreatic h-cells[15,18,19].We evaluated both growth switches (FgfR-GS andEpoR-GS) in vitro following transduction of intacthuman islets with an LCMV-pseudotyped HIV vectorexpressing FgfR-GS or EpoR-GS (LCMV-HIV-FgfR-GS orEpoR-GS, respectively). We evaluated transduction effi-ciency of LCMV-HIV-FgfR-GS or EpoR-GS in humanislets by immunodetection of FKBP12, a