Gene Therapy to Cure Type 1 Diabetes
The goal of this research project is to develop an innovative yet simple therapy to treat Type 1 diabetes mellitus (T1DM) by engineering a lentiviral vector (LV) containing a novel transgene for glucose- dependent insulin secretion from the liver.non-traumatic amputations. About 50,000 WI residents and 1.5 million Americans are afflicted with T1DM (1-3). Experts agree that a treatment which would precisely control blood glucose, will revolutionize T1DM management by preventing secondary complications. We have spent the past 20 years incrementally reaching our goal of more accurately controlling glucose levels by developing a novel insulin gene construct, called TA1m (Diatagene, patented by WARF: # US7425443 B2; second patent by WARF, pending). We have demonstrated that a single injection of TA1m minicircle DNA provided accurate glucose control in the rat model of T1DM for one month and fully normalized growth rate [4]. More importantly, response of TA1m treated diabetic rats to a glucose overload was similar to the normal animals (Fig. 1). To our knowledge, this is the first demonstration of a near-perfect glucose-regulated insulin production from native non- beta cells.

Our goal is to provide long-term benefit by utilizing inherent longevity of lentiviral-based gene therapy. Current treatment protocols do not adequately control blood glucose levels resulting in retinopathy, renal failure, cardiovascular complications and To this end, we have engineered TA1 into a 3rd generation complementing plasmids encoding viral structural genes for packaging was used to produces a safe replication-incompetent lentivirus. The vector lacks Tat, which can cause unwanted off- target gene transcription in transduced cells (5,6, reviewed in 7). We have recently validated therapeutic efficacy of LV-TA1 in a rat model of T1DM. In our first experiment, due to the use of a larger than optimum dose, as recognized by results, an overcorrection of hyperglycemia and weight gain was noted. However, this overcorrection of hyperglycemia is sustained for over 6 months with no signs of deterioration in therapeutic efficacy (Fig. 2). We expect that an optimized dose of lentivirus will accurately correct hyperglycemia and sustain insulin expression up to one year or longer (8,9). To our knowledge, this is the first demonstration of a long-term glucose-regulated insulin production from native non-ß-cells.

lentiviral vector (LV-TA1) A set of In this application we propose to treat diabetic dogs that like humans, spontaneously develop diabetes due to autoimmunity and are therefore considered the best pre-clinical T1DM animal model (10). We propose to transduce hepatocytes, in vivo, with lentivirus and expect that the treatment will result in long-term glucose-regulated insulin production, causing reversal of T1DM in dogs. To minimize any possible immunological reaction, the current TA1 construct will be altered to contain the dog insulin sequence to produce a lentivirus appropriate for use in dogs (LV-TA1D). Success of this study will have the following benefits: 1) provide valuable pre-clinical data for a human trial, 2) make our research more likely to be funded by external sources, such as the NIH, and 3) has potential to generate revenue from treating pets based on a commitment by a private company to fund and assist with the transitional studies required for application in humans. Our goal of achieving long-term T1DM treatment in dogs will be accomplished through the following two specific aims:

Specific Aim 1: Optimize the dose of therapeutic lentiviral particles, assess safety and long-term glucose control in a dog model of T1DM. Groups of experimental dogs (3-5 Beagles/group) will be rendered diabetic and intravenously injected with several single doses of LV-TA1D (5×108–1011 lentiviral particles/kg). Our results from rat studies will serve as a guide for LV doses to determine the optimal dose to achieve euglycemia in diabetic dogs. Using the optimal dose, we will establish the ability of LV-TA1D treatment to confer long-term euglycemia in the dog model. We already have a University of Wisconsin approved protocol for testing insulin gene-therapy in the dog model of T1DM.

Lentivirus vector is already approved for clinical trials by the FDA, it appears safe and proven beneficial (11); we do not anticipate any major safety or toxicity related issues. Nevertheless, appropriate safety and toxicity studies will be performed. All of the experimental dogs will be closely monitored for their general health and specific diabetes-associated markers, such as blood glucose levels, body weights, plasma levels of glycated hemoglobin, lipid levels, and markers of liver function and damage. Three groups of diabetic Beagles treated with LV-TA1D, one group treated at the optimal dosage, second at half the dosage, and the third at twice the dosage, will be euthanized 3-months post-treatment for toxicology and safety related studies. We will perform histology, locate and quantify insulin producing cells in the liver, ascertain the frequency of LV-TA1D integration in liver and other organs, and evaluate the status of immune response. In consultation with a long-term NIH funded and well-regarded Diabetologist Dr. MacDonald, our collaborating team at the Veterinary Science School will follow these animals for any clinical and metabolic investigations to assure the safety of treatment prior to its use in the clinical trial in Aim 2. We anticipate that safety and efficacy portion of the first aim to be complete in less than 1 year.
To determine long-term efficacy, a group of 5-6 diabetic Beagles treated with the optimum dose of LV- TA1D will be closely monitored. We anticipate determining optimized conditions for a single treatment to normalize diabetic hyperglycemia for one year, perhaps longer, as indicated by our ongoing rat study.If despite fine-tuning the therapeutic dosage of LV-TA1D, some animals encounter hypoglycemia, we will destabilize the insulin mRNA by altering its 3’-UTR to reduce mRNA half-life. Due to faster degradation, large amounts of insulin mRNA produced during hyperglycemia will not be available long after euglycemia is established. Specific Aim 2: Treatment of diabetic pet dogs. After establishing the safety and optimal dose of LV- TA1D for treatment, 25-30 pet dogs with autoimmune diabetes will be recruited at our collaborating Veterinary School, with informed consent from their owners, into our study and treated with LV-TA1D.
The Aim 2 is a “clinical trial” to test the LV-TA1D treatment in acquired autoimmune diabetes in pets. Experimental Beagles used in Aim 1, rendered diabetic experimentally, lack the autoimmunity component of the disease that is, like in humans, characteristic of autoimmune diabetes in dogs. Hence, experts in the field agree that dogs with autoimmunity make for the best preclinical model of human T1DM (10).

The incidence of T1DM in humans has now reached epidemic proportions and is predicted to rise. More than 20 years ago we initiated research in our laboratory to find a simple, affordable and non-invasive method to treat T1DM which would be available for all T1DM patients. The creation of an insulin gene with glucose-sensitive-response element in our laboratory brought us closer to this goal; the proof-of- principle has been achieved and published (3) and it makes for a compelling case for further investigation.

All experts agree that for any T1DM therapy to be meaningful, it must be evaluated in an autoimmune large animal model (10). It is estimated that about 1% of all pet dogs will develop autoimmune diabetes, which is identical to human T1DM in its clinical, metabolic and autoimmune aspects. Therefore, in collaboration with the School of Veterinary Medicine (University of Wisconsin), we propose to conduct a clinical trial using Gene Therapy to free the animals from insulin injections. Success of this project depends on extensive collaboration with Dr.Viviano, who will be the Veterinarian in-charge of recruitment and all clinical aspects of study. Dr. Michael MacDonald will lend the benefit his extensive expertise. Our team approach supplies the synergy required to move this novel T1DM therapeutic approach forward to application in man. At present, the clinic follows 24 chronic T1DM dogs. Veterinarians from all areas of Wisconsin have expressed sincere interest in referring more patients to reach the projected number of 25-30 dogs for enrollment in the trial.

In summary, we propose a novel T1DM treatment using an ideal pre-clinical T1DM model that can only come to fruition through a major collaborative effort between our Laboratory in the Dept. of Surgery, the Veterinary School, U.W. Madison, and the expertise of Dr. MacDonald (Pediatrics, UW Madison).Successful completion of the overall project is likely to yield a therapy for human T1DM patients with glucose control far superior to other available treatments.This innovative treatment would result in the attracting pivotal trials to the University of Wisconsin with a potential for developing a new therapeutic for T1DM.

Bibliography
1. Vehik, K., Ajami, N. J., Hadley, D., Petrosino, J. F., and Burkhardt, B. R. (2013) The changing landscape of type 1 diabetes: recent developments and future frontiers, Current diabetes reports 13, 642-650.
2. Centers for Disease Control and Prevention. Diabetes Data & Trends. Atlanta, GA: Available at: http://www.cdc.gov/diabetes/statistics/prevalence_national.htm
3 Alam, T., Wai, P., Held, D., Vakili, S. T., Forsberg, E., and Sollinger, H. (2013) Correction of Diabetic Hyperglycemia and Amelioration of Metabolic Anomalies by Minicircle DNA Mediated Glucose-Dependent Hepatic Insulin Production, PLoS One 8, e67515.
4. Review
4. American Gene Technologies International, Inc. http://americangene.com/
5. Demarchi, F., Gutierrez, M. I., and Giacca, M. (1999) Human immunodeficiency virus type 1 tat
protein activates transcription factor NF-kappaB through the cellular interferon-inducible, double-
stranded RNA-dependent protein kinase, PKR, Journal of virology 73, 7080-7086.
6. Maggirwar, S. B., Tong, N., Ramirez, S., Gelbard, H. A., and Dewhurst, S. (1999) HIV-1 Tat-
mediated activation of glycogen synthase kinase-3beta contributes to Tat-mediated neurotoxicity,
Journal of neurochemistry 73, 578-586.
7. Papayannakos, C., and Daniel, R. (2013) Understanding lentiviral vector chromatin targeting:
working to reduce insertional mutagenic potential for gene therapy, Gene Therapy 20, 581-588.
8. Elsner, M., Terbish, T., Jorns, A., Naujok, O., Wedekind, D., Hedrich, H. J., and Lenzen, S.
(2012) Reversal of diabetes through gene therapy of diabetic rats by hepatic insulin expression via lentiviral transduction, Molecular therapy : the journal of the American Society of Gene Therapy 20, 918-926.
9. Ren, B., O’Brien, B. A., Byrne, M. R., Ch’ng, E., Gatt, P. N., Swan, M. A., Nassif, N. T., Wei, M. Q., Gijsbers, R., Debyser, Z., and Simpson, A. M. (2013) Long-term reversal of diabetes in non- obese diabetic mice by liver-directed gene therapy, The journal of gene medicine 15, 28-41.
10. Sakata, N., Yoshimatsu, G., Tsuchiya, H., Egawa, S., Unno, M. (2012) Animal models of diabetes mellitus for islet transplantation, Experimental Diabetes Research 2012, 256707, 11 pages.
11. Biffi, A., Montini, E. Lorioli, L. et al. (2013) Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy, Science 341, 1233158-1.
12. Wisconsin Diabetes Prevention and Control Program, Division of Public Health, Department of Health Services. Wisconsin Diabetes Surveillance Report 2012. Available at: http://www.dhs.wisconsin.gov/diabetes