Gene Therapy Approaches to Diabetes

Gene Therapy Approaches for Diabetes and Its Complications

Summary and Recommendations

Savio Woo and Ake Lernmark, Co-Chairs

Summary

Introduction

As the technology for introducing new genes into cells has been improving, the disease targets for gene therapy have expanded beyond traditional genetic diseases to chronic diseases such as diabetes. This workshop assessed the current understanding of the pathogenesis of both type 1 and type 2 diabetes and its complications and identified strategies for intervening in the induction and progression of diabetes using gene therapy. Investigators described their results using gene therapy approaches to treat diabetes in both animal models and patients. The workshop started with an introductory lecture on Diabetes – Natural History and Therapeutic Intervention by Robert Sherwin, Yale University. Mark Kay, Stanford University, gave a primer on Vector Development for Gene Therapy and Dale Greiner, University of Massachusetts, described the utility of animal models for type 1 diabetes for these studies. The topics for the scientific sessions were divided into four basic sessions: Gene Therapy Approaches for Expressing Insulin; Gene Therapy Approaches for Immunomodulation; Gene Therapy Approaches for Cell-based Therapies; and Gene Therapy Approaches for Complications. As new genes involved in 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 presented their data and discuss strategies for treating diabetes. The outcome of this Workshop will include the identification of the most promising approaches for gene therapy for diabetes as well as identification of areas of gene therapy technology that need further improvements 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 data on glucose stimulated insulin expression from the glucose-6-phosphotase promoter in hepatoma cells. This promoter has the properties of being stimulated by glucose and inhibited by insulin. Mark Magnuson, Vanderbilt University Medical Center, described alterations in glucose metabolism when either the glucokinase or the phosphoenolpyruvate carboxykinase genes were knocked out in mice. Gene therapy could be used to alter glucose metabolism by regulating enzymes in the glycolytic pathway. Using a different strategy to regulate insulin secretion, William Osborne, University of Washington, placed the furin protease under control of a glucose stimulated TGF-alpha promoter. Following glucose injection, the increase levels of furin processes proinsulin to insulin showing significant decreases in glucose. James Wilson, University of Pennsylvania, has found that the protease, PC3, is more efficient than furin at processing insulin. They have investigated regulated gene expression using a promoter that can be induced by an oral agent. Although the change in expression may be too slow to appropriately regulate glucose levels, this could be an extremely useful way to regulate gene expression for safety. Victor Rivera, ARIAD Gene Therapeutics, Inc., has developed a technology to regulate secretion of proteins from the endoplasmic reticulum in response to an oral agent. This technology has the advantage of being more rapid than regulating transcription. In general, these approaches appear to show some promise to an extremely difficult problem of rapidly regulating insulin secretion. Most participants felt that the ability to regulate insulin expression levels was a necessary safety factor before studies could proceed to clinical trials.

Gene Therapy Approaches for Immunomodulation

The session was chaired by Ake Lernmark, University of Washington, who outlined potential targets for gene therapy. These included the expression of GAD65 or IA-2 to tolerize patients to prevent beta-cell destruction and the expression of IL4 to reduce inflammation. Alternative approaches include targeting beta-cells to induce regeneration or to protect against immunodestruction. Gary Fathman, Stanford University, presented his work showing that CD4+ T-cells can transfer diabetes to NOD mice. By using a retrovirus to mark activated T-cells that are stimulated to divide, he can characterize the population of activated T-cells involved in the development of diabetes. These vectors can also be used to introduce cytokine genes into this subset of T cells. Dr. Yoon, University of Calgary, presented data showing that immunization of young mice with a vaccina virus expressing GAD reduced the number of NOD-mice that developed diabetes. Also the expression of TGF-beta reduced the development of diabetes. This would appear to be an important avenue 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 Medical Center, who presented two strategies for treating diabetes. One is the development of encapsulated cell lines that are engineered to secret insulin. The other is to increase gluconeogenesis in the liver by delivering protein phosphotase-1 using gene therapy. Myra Lipes, Harvard Medical School, presented studies using intermediate lobe pituitary cells to express insulin. These cells are not subject to immunodestruction probably because they don’t express MHC antigens. These cells are being engineered to include glucose sensing enzymes to improve glucose responsiveness. Shimon Efrat, Tel Aviv University, presented data on beta-cell lines that are transformed with a temperature sensitive oncogene. He proposed the use of viral genes to elude the immune system. These approaches hold 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 data that demonstrated transduction of nerves by herpes virus expressing NGF. This approach is being investigated in nerves innervating the bladder for treatment of cystopathy and for peripheral neuropathy. Ron Crystal, Cornell Medical Center, summarized his clinical trial using the growth factor, VEGF, in patients with coronary artery disease, 60% have diabetes as 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 the delivery of kallikrein by gene therapy reduces hypertension and kidney disease. David Margolis, University of Pennsylvania, presented data that the delivery of the growth factor, PDGF, by adenovirus into a wound improved healing. This approach is awaiting regulatory approval to begin a clinical trial. These talks illustrated several gene therapy approaches for the treatment of some of the many complications resulting from diabetes.

Recommendations

Gene Therapy Approaches to Ectopic Insulin Expression

The expression of insulin at sites where the hormone is not normally produced, shows promise in terms of the use of both in vivo and ex vivo gene therapy. Progress has been made to better understand the way proinsulin is processed in non-beta cells following transfection of the pre-proinsulin gene. Further studies are needed to expand the use of different secretory cells to be the host of the pre-proinsulin gene for processing and proper secretion.

Other avenues of research include the use of multicistronic vectors that provide glucose sensitivity either at the level of insulin gene expression or the expression of converting factors. Certainly improved technical approaches will be needed and research support to improve the basic science of gene therapy is strongly recommended. Expanded research in ectopic insulin production would be aided by the development of novel vectors and cell combinations to achieve a difficult but extremely important goal. Diabetic animal models are well developed for this purpose and there is a logical series of preclinical tests that can be carried out and supported to pave the way for safe clinical trials. In particular 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 interfere with progressive type 1 islet autoimmunity and beta-cell killing. There seems to be sufficient understanding of type 1 diabetes immune mediated beta-cell killing to allow studies 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-beta cell response. The gene therapy will not be to correct a defective genetic makeup but rather to interfere with the disease pathogenesis.

Fundamental research would focus on induction of immunological tolerance using various ways of providing expression of autoantigens to induce tolerance. DNA-based vaccine approaches should be explored as a method to induce tolerance. Preclinical work has already tested the hypothesis that the genetic manipulation to express autoantigens reduce beta-cell self reactivity. This approach would be applicable to either primary or secondary prevention.

Gene therapy applied to secondary or tertiary intervention was also presented. The research utilizing T cell homing technologies to deliver immune suppressive cytokines or other factors at the site of the inflammation is a very exciting prospect. This is an incredibly important area of research that may yield therapeutic modalities that would interfere with progressive inflammatory diseases including beta-cell killing in type 1 diabetes. It would seem that the antigen or epitope specificity of the immune system with respect to the use of either antibodies, T cells or both has not been fully explored for disease protection and inhibition of progressive autoimmunity diseases. The gene therapy technology would involve both ex vivo and in vivo approaches.

Another area of diabetes pathogenesis research that needs to be expanded and supported is to manipulate transplanted pancreatic beta-cells or transplanted islets by the autoimmune process, to reduce or prevent destruction, allograft rejection or both. Ongoing research at the preclinical level suggests that islets, which are expressing certain ligands or immune suppressive cytokines, have an improved survival. This is a novel and hitherto uncharted area of research that deserves support. In particular, research is needed 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 support as a alternative to non-beta cells lines with less vigorous ability to process and secrete insulin. This area of research has made considerable progress and there seems to be a critical mass of investigative power available to justify a specific request for applications 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 diabetes complications, it was clear that the selected areas of investigation utilizing gene therapy approaches in macrovascular disease, neuropathy and microvascular disease were very promising. Some of these studies have already progressed to phase I clinical trials. Since glucose management remains a difficult problem, complications from diabetes continue to be a high priority area for support. Research that deserves further support include:

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 considerable need for support to explore the use of gene therapy for diabetes. The conference also demonstrated that considerable preliminary results have been obtained to demonstrate progress in each one of the four areas indicated above. It is therefore strongly recommended that NIH provide targeted research support to each of the four areas since considerable progress can be expected in each one the them. All four areas of research are in high priority in terms of prediction, prevention and cure, as well therapy for patients with diabetes.