Gene Therapy: A Cure for Canine Diabetes

Beagle dog, from Wikipedia commons

Treatment of Diabetes and Long-term Survival Following Insulin and Glucokinase Gene Therapy

David Callejas, Christopher J. Mann, Eduard Ayuso, Ricardo Lage, Iris Grifoll, Carles Roca, Anna Andaluz, Rafael Ruiz-de Gopegui, Joel Montané, Sergio Muñoz, Tura Ferre, Virginia Haurigot, Shangzhen Zhou, Jesús Ruberte, Federico Mingozzi, Katherine A. High, Felix Garcia, and Fatima Bosch

Diabetes (Published online before print) February 1, 2013, doi:10.2337/db12-1113

In both man and dogs, diabetes is a chronic disease for which there is no cure. Almost all diabetic dogs need insulin replacement therapy to survive, but glycemic control is never perfect (1-3). Therefore, long-term complications of diabetes (e.g., cataracts) disease are frequently observed (4-6). In addition, burden of our traditional treatment methods takes a toll on the owners of diabetic pets and results in a proportion of pets being euthanized instead of being treated (7). If pet owners do decide to treat their diabetic pets, they commonly report a significant treatment burden, with a number of negative effects on various aspects of the pet’s and owner’s lifestyle (7,8).

Gene therapy for diabetes
Obviously, a less arduous treatment option for controlling diabetes mellitus in dogs would be welcomed.  Gene therapy has the potential to provide this alternative method of treatment for animals as well as for humans. Simplistically, in gene therapy, particular strings of DNA are "inserted" into cells such that the machinery of the cell takes the DNA, transcribes it into RNA, and then makes the protein that is encoded in the new DNA. This technique allows us to force cells to make proteins that they might not otherwise make, and in the last decade it has started to see success in small trials to treat a number of diseases.

As it turns out, however, designing and targeting a string of DNA at a cell is not easy (8,9). Currently, most investigators use what is called viral vectors to get the DNA inside cells— basically, this involves taking a virus that contains the DNA we want to insert, and infect cells with the virus. The virus enters the cell, where it can integrate its DNA segment into the chromosomes of the host cell (8).

For diabetes, experimental gene therapy has generally involved injection of protein-coding genes into skeletal muscle or the liver. This exploits these tissues' intrinsic ability to read such genes and subsequently constitutively produce the corresponding protein, which is then secreted into the general circulation by virtue of the extensive vascularization of peripheral muscles (10-14).

Study reporting successful gene therapy for canine diabetes
To date, most gene therapy has involved studies in diabetic mice. As reported by Callejas et al. in the  journal Diabetes (15), these researchers from the Center of Animal Biotechnology and Gene Therapy, Universitat Autonoma de Barcelona, Spain, used gene therapy to treat and "cure" 5 Beagle dogs with insulin-deficient diabetes.

This same research group had already tested this type of therapy on mice (10), but the excellent results obtained for the first time with large animals lays the foundation for the clinical translation of this gene therapy approach to veterinary medicine and eventually for diabetic human patients.

The diabetic state in these dogs had been induced by injection of a mixture of streptozotocin and alloxan, both of which are toxic to beta cells (16). After becoming diabetic, the dogs were treated with one of the following:

1. Insulin glargine, administered twice daily
2. Intramuscular injection of the glucokinase gene alone 
3. Intramuscular injection of the insulin gene alone
4. Intramuscular injection of both the insulin and glucokinase genes. 

Not surprisingly, without treatment, the dogs were markedly hyperglycemic. The dogs on twice daily insulin were better controlled but remained hyperglycemic.

To test both the insulin and glucose kinase vectors together, the researchers injected 5 dogs with one or two doses of the viral vectors. These dogs quickly regained normoglycemia, and even the higher doses were tolerated well; none of the dog had any side effects. Unlike the dogs given insulin alone, the dogs given both genes together responded much better to an oral glucose tolerance test, spiking slightly up to between 200 and 300 mg/dL, but stayed well below the levels seen with the diabetic dogs.

The dogs gained weight, had lower serum fructosamine levels, and experienced long-term remission of the diabetes without secondary complications. Therapy that included only the insulin gene or the glucose kinase gene but not both did not result in the same level of success.

Click here to see a video of the treated dogs in diabetic remission.

My Bottom Line:

Since the 1922 breakthrough discovery of Banting and Best (17), who corrected hyperglycemia in dogs using pancreatic extracts, exogenous insulin administration has been the mainstay of diabetes therapy (1-3). Alternative therapies have been studied, but thus far only a handful of approaches, mainly involving allo- or xeno-transplantation of pancreatic islets, have reached clinical application (18).

The authors of this landmark study (15) took a different approach: instead of trying to transplant or manufacture beta cells that respond to glucose and produce insulin, they used dual gene therapy to develop a “glucose sensor” in skeletal muscle that permitted long-term, normoglycemia in this canine model of diabetes.

The gene therapy used in these dogs consisted of a single session of intramuscular injections to introduce gene therapy vectors, with two ultimate objectives: to express the insulin gene, on the one hand, and that of glucokinase, on the other. Glucokinase is an enzyme that regulates the uptake of glucose from the blood (19). When both insulin and glucokinase genes act simultaneously, they function together as a glucose sensor to automatically regulate the uptake of glucose from the blood, thus reducing hyperglycemia (20).

The results of this study look very promising indeed. The dogs in the study, once treated, experienced long-term normoglycemia, both in the fasting or fed state, with no need for exogenous insulin therapy. There were no episodes of hypoglycemia, even after the dogs were exercised. In addition, all dogs regained lost body weight and developed no secondary complications 4 years after treatment.

Hopefully, this study will provide the basis for the initiation of clinical studies in dogs (and cats?) with naturally-occurring diabetes. Such veterinary clinical trials should also help investigators in preparing for the use of this approach in humans patients with diabetes.  

References:

1. Davison LJ, Herrtage ME, Catchpole B. Study of 253 dogs in the United Kingdom with diabetes mellitus. Vet Rec 2005;156:467-471.
2. Nelson RW. Canine diabetes mellitus In: Ettinger SJ, Feldman EC, eds. Textbook of Veterinary Internal Medicine: Diseases of the Dog and Cat. Seventh ed. St. Louis: Saunders Elsevier, 2010;1449-1474.
3. Davison LJ. Canine diabetes mellitus In: Mooney CT, Peterson ME, eds. BSAVA Manual of Canine and Feline Endocrinology. Fourth ed. Quedgeley, Gloucester: British Small Animal Veterinary Association, 2012;116-132.
4. Munana KR. Long-term complications of diabetes mellitus, Part I: Retinopathy, nephropathy, neuropathy. Vet Clin North Am Small Anim Pract 1995;25:715-730. 
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14. Ren B, O'Brien BA, Byrne MR, et al. Long-term reversal of diabetes in non-obese diabetic mice by liver-directed gene therapy. J Gene Med 2013;15:28-41. 
15. Callejas D, Mann CJ, Ayuso E, et al. Treatment of diabetes and long-term survival following insulin and glucokinase gene therapy. Diabetes 2013 (in press). 
16. Anderson HR, Stitt AW, Gardiner TA, et al. Induction of alloxan/streptozotocin diabetes in dogs: a revised experimental technique. Lab Anim 1993;27:281-285. 
17. Banting FG, Best CH, Collip JB, et al. Pancreatic extracts in the treatment of diabetes mellitus. Can Med Assoc J 1922;12:141-146. 
18. Robertson RP. Islet transplantation a decade later and strategies for filling a half-full glass. Diabetes 2010;59:1285-1291.  
19. Printz RL, Magnuson MA, Granner DK. Mammalian glucokinase. Annu Rev Nutr 1993;13:463-496. 
20. Otaegui PJ, Ferre T, Pujol A, et al. Expression of glucokinase in skeletal muscle: a new approach to counteract diabetic hyperglycemia. Hum Gene Ther 2000;11:1543-1552. 

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