Introduction
As the technology for introducing new genes into cells has been improving, the diseasetargets for gene therapy have expanded beyond traditional genetic diseases to chronicdiseases such as diabetes. This workshop assessed the current understanding of thepathogenesis of both type 1 and type 2 diabetes and its complications and identifiedstrategies for intervening in the induction and progression of diabetes using genetherapy. Investigators described their results using gene therapy approaches to treatdiabetes in both animal models and patients. The workshop started with an introductorylecture on Diabetes – Natural History and Therapeutic Intervention by Robert Sherwin,Yale University. Mark Kay, Stanford University, gave a primer on Vector Development forGene Therapy and Dale Greiner, University of Massachusetts, described the utility ofanimal models for type 1 diabetes for these studies. The topics for the scientificsessions were divided into four basic sessions: Gene Therapy Approaches for ExpressingInsulin; Gene Therapy Approaches for Immunomodulation; Gene Therapy Approaches forCell-based Therapies; and Gene Therapy Approaches for Complications. As new genes involvedin type 2 diabetes are discovered, additional targets for gene therapy will be identified.Experts in both the pathogenesis and treatment of diabetes and gene therapy presentedtheir data and discuss strategies for treating diabetes. The outcome of this Workshop willinclude the identification of the most promising approaches for gene therapy for diabetesas well as identification of areas of gene therapy technology that need furtherimprovements in order to achieve successful gene therapy for diabetes.
Gene Therapy Approaches for Expressing Insulin
The session was chaired by Savio Woo, Mt. Sinai School of Medicine, who presented dataon glucose stimulated insulin expression from the glucose-6-phosphotase promoter inhepatoma cells. This promoter has the properties of being stimulated by glucose andinhibited by insulin. Mark Magnuson, Vanderbilt University Medical Center, describedalterations in glucose metabolism when either the glucokinase or the phosphoenolpyruvatecarboxykinase genes were knocked out in mice. Gene therapy could be used to alter glucosemetabolism by regulating enzymes in the glycolytic pathway. Using a different strategy toregulate insulin secretion, William Osborne, University of Washington, placed the furinprotease under control of a glucose stimulated TGF-alpha promoter. Following glucoseinjection, the increase levels of furin processes proinsulin to insulin showingsignificant decreases in glucose. James Wilson, University of Pennsylvania, has found thatthe protease, PC3, is more efficient than furin at processing insulin. They haveinvestigated regulated gene expression using a promoter that can be induced by an oralagent. Although the change in expression may be too slow to appropriately regulate glucoselevels, this could be an extremely useful way to regulate gene expression for safety.Victor Rivera, ARIAD Gene Therapeutics, Inc., has developed a technology to regulatesecretion of proteins from the endoplasmic reticulum in response to an oral agent. Thistechnology has the advantage of being more rapid than regulating transcription. Ingeneral, these approaches appear to show some promise to an extremely difficult problem ofrapidly regulating insulin secretion. Most participants felt that the ability to regulateinsulin expression levels was a necessary safety factor before studies could proceed toclinical trials.
Gene Therapy Approaches for Immunomodulation
The session was chaired by Ake Lernmark, University of Washington, who outlinedpotential targets for gene therapy. These included the expression of GAD65 or IA-2 totolerize patients to prevent beta-cell destruction and the expression of IL4 to reduceinflammation. Alternative approaches include targeting beta-cells to induce regenerationor to protect against immunodestruction. Gary Fathman, Stanford University, presented hiswork showing that CD4+ T-cells can transfer diabetes to NOD mice. By using a retrovirus tomark activated T-cells that are stimulated to divide, he can characterize the populationof activated T-cells involved in the development of diabetes. These vectors can also beused to introduce cytokine genes into this subset of T cells. Dr. Yoon, University ofCalgary, presented data showing that immunization of young mice with a vaccina virusexpressing GAD reduced the number of NOD-mice that developed diabetes. Also the expressionof TGF-beta reduced the development of diabetes. This would appear to be an importantavenue of research for potential treatments of type 1 diabetes.
Gene Therapy Approaches for Cell-based Therapies
The session was chaired by Chris Newgard, University of Texas Southwestern MedicalCenter, who presented two strategies for treating diabetes. One is the development ofencapsulated cell lines that are engineered to secret insulin. The other is to increasegluconeogenesis in the liver by delivering protein phosphotase-1 using gene therapy. MyraLipes, Harvard Medical School, presented studies using intermediate lobe pituitary cellsto express insulin. These cells are not subject to immunodestruction probably because theydon’t express MHC antigens. These cells are being engineered to include glucosesensing enzymes to improve glucose responsiveness. Shimon Efrat, Tel Aviv University,presented data on beta-cell lines that are transformed with a temperature sensitiveoncogene. He proposed the use of viral genes to elude the immune system. These approacheshold great promise for the treatment of both type 1 and type 2 diabetes.
Gene Therapy Approaches for Complications
One of the most promising areas for gene therapy is the treatment of complications.This session was chaired by Joe Glorioso, University of Pittsburgh, who presented datathat demonstrated transduction of nerves by herpes virus expressing NGF. This approach isbeing investigated in nerves innervating the bladder for treatment of cystopathy and forperipheral neuropathy. Ron Crystal, Cornell Medical Center, summarized his clinical trialusing the growth factor, VEGF, in patients with coronary artery disease, 60% have diabetesas a co-morbid condition. So far 26 patients have received this experimental treatment.Dr. Chao, University of South Carolina, showed data in several rat models showing that thedelivery of kallikrein by gene therapy reduces hypertension and kidney disease. DavidMargolis, University of Pennsylvania, presented data that the delivery of the growthfactor, PDGF, by adenovirus into a wound improved healing. This approach is awaitingregulatory approval to begin a clinical trial. These talks illustrated several genetherapy approaches for the treatment of some of the many complications resulting fromdiabetes.
Recommendations
Gene Therapy Approaches to Ectopic Insulin Expression
The expression of insulin at sites where the hormone is not normally produced, showspromise in terms of the use of both in vivo and ex vivo gene therapy. Progress has beenmade to better understand the way proinsulin is processed in non-beta cells followingtransfection of the pre-proinsulin gene. Further studies are needed to expand the use ofdifferent secretory cells to be the host of the pre-proinsulin gene for processing andproper secretion.
Other avenues of research include the use of multicistronic vectors that provideglucose sensitivity either at the level of insulin gene expression or the expression ofconverting factors. Certainly improved technical approaches will be needed and researchsupport to improve the basic science of gene therapy is strongly recommended. Expandedresearch in ectopic insulin production would be aided by the development of novel vectorsand cell combinations to achieve a difficult but extremely important goal. Diabetic animalmodels are well developed for this purpose and there is a logical series of preclinicaltests that can be carried out and supported to pave the way for safe clinical trials. Inparticular research is needed in the following areas:
To develop non-toxic, persistent and targetable vectors for therapeutic gene transfer in vivo.
To develop safe and effective strategies to achieve vector re-administration over time into animals that manifest autoimmunity.
To achieve glucose responsive insulin gene expression and glucose stimulated insulin secretion.
To explore auxiliary therapeutic genes that are complementary to insulin for improved efficacy.
Gene Therapy of Type 1 Diabetes Pathogenesis
There are a large number of gene therapy approaches that can be explored to interferewith progressive type 1 islet autoimmunity and beta-cell killing. There seems to besufficient understanding of type 1 diabetes immune mediated beta-cell killing to allowstudies of gene therapy approaches that directly interfere with the inflammatory process.It may also be possible to interfere with those processes that are inducing an anti-betacell response. The gene therapy will not be to correct a defective genetic makeup butrather to interfere with the disease pathogenesis.
Fundamental research would focus on induction of immunological tolerance using variousways of providing expression of autoantigens to induce tolerance. DNA-based vaccineapproaches should be explored as a method to induce tolerance. Preclinical work hasalready tested the hypothesis that the genetic manipulation to express autoantigens reducebeta-cell self reactivity. This approach would be applicable to either primary orsecondary prevention.
Gene therapy applied to secondary or tertiary intervention was also presented. Theresearch utilizing T cell homing technologies to deliver immune suppressive cytokines orother factors at the site of the inflammation is a very exciting prospect. This is anincredibly important area of research that may yield therapeutic modalities that wouldinterfere with progressive inflammatory diseases including beta-cell killing in type 1diabetes. It would seem that the antigen or epitope specificity of the immune system withrespect to the use of either antibodies, T cells or both has not been fully explored fordisease protection and inhibition of progressive autoimmunity diseases. The gene therapytechnology would involve both ex vivo and in vivo approaches.
Another area of diabetes pathogenesis research that needs to be expanded and supportedis to manipulate transplanted pancreatic beta-cells or transplanted islets by theautoimmune process, to reduce or prevent destruction, allograft rejection or both. Ongoingresearch at the preclinical level suggests that islets, which are expressing certainligands or immune suppressive cytokines, have an improved survival. This is a novel andhitherto uncharted area of research that deserves support. In particular, research isneeded to:
To investigate strategies to induce tolerance to beta-cell antigens
To investigate strategies for T-cell homing to deliver immunosuppressive agents
To elucidate the role of cytokine expression in suppression of the inflammatory process
To develop vectors that are targeted to the pancreas would be important to deliver genes that interfere with its immunodestruction in vivo
Gene Therapy Approaches for Cell-based Therapies
Novel approaches to create human beta-cells and beta-cells line needs further supportas a alternative to non-beta cells lines with less vigorous ability to process and secreteinsulin. This area of research has made considerable progress and there seems to be acritical mass of investigative power available to justify a specific request forapplications in this area of research. In particular, research is needed to:
To derive ideal human beta-cell lines for allogeneic transplantation into multiple patients.
To optimally construct engineered surrogate human beta-cell lines for the same purpose.
To understand mouse and human stem/progenitor cell development into beta-cells.
To develop optimal microporous encapsulating devices with sufficient capacity that are long lasting and retrievable.
Gene Therapy Approaches for Diabetic Complications
Although the conference did not specifically review the wide spectrum of diabetescomplications, it was clear that the selected areas of investigation utilizing genetherapy approaches in macrovascular disease, neuropathy and microvascular disease werevery promising. Some of these studies have already progressed to phase I clinical trials.Since glucose management remains a difficult problem, complications from diabetes continueto be a high priority area for support. Research that deserves further supportinclude:
To develop strategies for prevention and/or delay the onset of complications such as the targeted use of growth factors.
To develop methodologies to effectively treat various end-stage diseases and validate them in relevant animal models, followed by carefully designed clinical translational studies.
Summary
Taken together, this conference has clearly demonstrated that there is considerableneed for support to explore the use of gene therapy for diabetes. The conference alsodemonstrated that considerable preliminary results have been obtained to demonstrateprogress in each one of the four areas indicated above. It is therefore stronglyrecommended that NIH provide targeted research support to each of the four areas sinceconsiderable progress can be expected in each one the them. All four areas of research arein high priority in terms of prediction, prevention and cure, as well therapy for patientswith diabetes.
From the NIDDK
Undated webpage
http://www.niddk.nih.gov/fund/reports/gene_therapy_summ.htm
