Currently, the canine model of GSDIa is being studied primarily as part of early stage preclinical trials of recombinant adeno-associated virus (AAV) vector-based therapies. These types of therapy offer great promise for the treatment of GSDIa since they target the underlying problem rather than just palliating some of the clinical effects of G6Pase deficiency.
With the ability to generate large numbers of affected animals quickly, the murine model of GSDIa has provided a useful platform to prove the concept of gene therapy for correction of GSDIa and to compare the efficacy of various types of vectors [33
]. Gene therapy has mediated prolonged survival, sustained correction of glucose homeostasis and normalization of triglycerides, cholesterol, and uric acid in mice using a variety of different vectors, although substantial variation in efficacy and specific effects of the different vectors has been identified [33
]. The lack of ability to address all of the clinical manifestations (e.g., lactic acidosis) of GSDIa in this model and the lack of ability to address long-term safety and efficacy due to the short overall lifespan of the mice demonstrate the need for additional studies in a different model.
The first reported results of gene therapy in the canine model utilized an AAV vector administered between 3-4 days after birth [32
]. Puppies that received the vector demonstrated increased G6Pase activity in the liver and decreased glycogen compared to untreated, affected dogs. Some dogs also had improvement in blood glucose, triglyceride, cholesterol, and lactate levels after a three-hour fast compared to untreated, affected dogs at a least one time point after vector administration. When compared to carriers or wild-type dogs; however, these treated dogs demonstrated only partial improvements in biomarker levels and all succumbed to a GSDIa-related medical crisis between 20–86 days of age.
Later reports indicated that gene therapy utilizing an AAV2/8 vector significantly improved survival in three dogs compared to untreated controls and the dogs from the previous report by Beaty et al. [32
]. All three dogs survived passing 11 months of age. In addition to improved survival, treated dogs were able to maintain normal blood glucose levels during a fasting period of at least 2 hours (longer times are not reported) starting after 1–6 month (s) of age. While treated dogs still required frequent feeding (q6–10
hr) compared to normal dogs, they did not require additional carbohydrate supplementation. Glycogen and fat storage in the liver was reduced, and G6Pase activity was increased compared to values from untreated dogs and were comparable to levels reported in carrier dogs which showed no overt clinical signs of GSDIa. While lactate levels after a 2-hour fast were significantly improved in the treated dogs compared to untreated controls, they remained well above normal laboratory standards for dogs (>2.1
Several dogs from a colony at the University of Florida have also been treated with gene therapy utilizing AAV vectors. The first dog was treated with an AAV2/8 vector on postnatal day 1 [34
]. Two dogs from each of two subsequent litters have received gene therapy with AAV vectors at postnatal days 1 or 2. Four of five treated dogs demonstrated an ability to maintain normal glucose and lactate levels during a two-hour fast at one month after treatment. The other dog did not receive the entire IV dose of vector due to subcutaneous extravasation and remained unable to tolerate a 2-hour fast.
While the dogs clinically improved after the initial dose of gene therapy, this level of correction was not sustained in any of these dogs. By two months of age, fasting glucose concentrations fell to levels similar to those of an untreated puppy and elevated lactate concentrations were consistently seen with fasting. In addition, growth curves for these dogs remained more similar to untreated controls than to wild-type puppies during the first few months of life. Similar results are reported in mice, in which intravenous delivery of AAV vector to deliver the gene of interest to neonates did not result in sustained correction.
Due to lack of sustained correction, the first dog was treated again at 20 weeks of age with a second dose of gene therapy delivered by an AAV2/1 vector injected into the portal vein [34
]. This resulted in a sustained partial correction of the G6Pase deficiency and a dramatic improvement in the metabolic status of the dog. One month after treatment with the AAV2/1 vector, dextrose supplementation was discontinued and this dog continues to do well clinically three years later receiving a mixture of canned and dry preparations of a commercially available dog food supplemented with UCCS offered every 4–6 hours. Serial fasting studies show the best response at two months after treatment, when fasting blood glucose and lactate levels were normal for greater than 5 hours and still close to normal at 9 hours into the fast. The response was somewhat diminished at 15 months after injection, but in contrast to dogs described in prior reports, this dog has consistently maintained normal blood glucose and lactate levels throughout fasting periods of 2–4 hours. Liver biopsies collected at 30 months had greater G6Pas activity and much less glycogen and fat accumulation than biopsies from an untreated control; however, they were still abnormal when compared to wild-type controls.
The second and third dogs were doing well clinically until developing signs of acute respiratory distress at 74 days of age. Dyspnea and hypoxemia developed rapidly, and the dogs died from this complication despite aggressive critical care. Necropsy and infectious disease testing revealed severe viral pneumonia caused by Canine adenovirus-2 (which is distinct from, and unrelated to, the adeno-associated virus used as a vector). The death of these dogs was not deemed to be related to the gene therapy.
Two other dogs from our colony have recently been treated and have shown results similar to the first dog. They received their second doses of AAV vector at approximately 10 weeks of age. Both dogs are clinically healthy with no glucose supplementation at this time. However, these dogs are currently only five months old, so it is too early to determine if there will be a sustained partial correction of their condition.
Overall, the collective work of the two centers currently using the canine model of GSDIa in preclinical trials of AAV vector-mediated gene therapy have demonstrated significant improvements in biomarker levels and histopathologic abnormalities, and dramatically improved survival times for dogs treated with gene therapy compared to controls [33