Long-term efficacy following readministration of an adeno-associated virus vector in dogs with glycogen storage disease type Ia

Abstract

Glycogen storage disease type Ia (GSD-Ia) is the inherited deficiency of glucose-6-phosphatase (G6Pase), primarily found in liver and kidney, which causes life-threatening hypoglycemia. Dogs with GSD-Ia were treated with double-stranded adeno-associated virus (AAV) vectors encoding human G6Pase. Administration of an AAV9 pseudotyped (AAV2/9) vector to seven consecutive GSD-Ia neonates prevented hypoglycemia during fasting for up to 8 hr; however, efficacy eventually waned between 2 and 30 months of age, and readministration of a new pseudotype was eventually required to maintain control of hypoglycemia. Three of these dogs succumbed to acute hypoglycemia between 7 and 9 weeks of age; however, this demise could have been prevented by earlier readministration an AAV vector, as demonstrated by successful prevention of mortality of three dogs treated earlier in life. Over the course of this study, six out of nine dogs survived after readministration of an AAV vector. Of these, each dog required readministration on average every 9 months. However, two were not retreated until >34 months of age, while one with preexisting antibodies was re-treated three times in 10 months. Glycogen content was normalized in the liver following vector administration, and G6Pase activity was increased in the liver of vector-treated dogs in comparison with GSD-Ia dogs that received only with dietary treatment. G6Pase activity reached approximately 40% of normal in two female dogs following AAV2/9 vector administration. Elevated aspartate transaminase in absence of inflammation indicated that hepatocellular turnover in the liver might drive the loss of vector genomes. Survival was prolonged for up to 60 months in dogs treated by readministration, and all dogs treated by readministration continue to thrive despite the demonstrated risk for recurrent hypoglycemia and mortality from waning efficacy of the AAV2/9 vector. These preclinical data support the further translation of AAV vector-mediated gene therapy in GSD-Ia.

FIG. 1.

Prevention of hypoglycemia following neonatal AAV2/9-G6Pase administration. Blood glucose was determined every 2 hr following fasting of duration indicated. (A) Dog W was treated with AAV2/9-G6Pase at 2 days of age (2×10¹³ vp/kg), and subsequently fasted for 8 hr at the indicated ages. (B) Puppies Du, F, and Ro were treated with AAV2/9-G6Pase at 2 days of age (4×10¹³ vp/kg), and subsequently fasted for 8 hr at 2–3 weeks of age. These three puppies did not survive past 3 months of age. (C) Puppies De, H, and T were treated with AAV2/9-G6Pase at 2 days of age (4×10¹³ vp/kg), and subsequently fasted for 8 hr at 2–3 weeks of age. These three puppies were re-treated with AAV2/7- and AAV2/8-G6Pase as a group to prevent mortality (Table 1). Glucose curves for normal puppies (n=10) and GSD-Ia puppies treated with dietary therapy alone (n=4) are shown for comparison. AAV, adeno-associated virus; AAV2, adeno-associated virus serotype 2; G6Pase, glucose-6-phosphatase; GSD-Ia, glycogen storage disease type Ia; vp, vector particles. Color images available online at www.liebertonline.com/hum

FIG. 2.

Sustained efficacy following readministration of AAV-G6Pase. Puppies De, H, and T were treated with AAV2/9-G6Pase at 2 days of age (4×10¹³ vp/kg), and subsequently re-treated with AAV2/7- and AAV2/8-G6Pase as a group to prevent mortality. Blood glucose following 4 and 6 hr of fasting at the indicated ages. Fasting was performed 2 weeks following vector administration, and every 2–3 months subsequently. (A) Dog De. (B) Dog H. (C) Dog T. Dog T had recurrent hypoglycemia and anorexia at 5 months of age that precipitated re-treatment with AAV2/1-G6Pase (Table 1). Color images available online at www.liebertonline.com/hum

FIG. 3.

Survival following initial AAV2/9 vector administration. Survival following neonatal administration of AAV2/9-G6Pase. Proportion surviving by age shown (n=7), in comparison with puppies historically treated with dietary therapy alone (n=8; Koeberl et al., 2009). The fraction surviving is indicated for major steps in each survival curve, for example 1/8 diet-treated puppies at 2 months of age.

FIG. 4.

Evaluation of efficacy in adult dogs following AAV-G6Pase administration. Blood glucose during prolonged fasting for the indicated duration in older dogs (Table 1). (A) Fasting for 6 and 8 hr for Dog R at older ages. Dog R was treated by readministration of AAV-G6Pase in response to recurrent symptoms. (B) Fasting for 6 and 8 hr for Dog L at older ages. Dog L was treated by readministration of AAV-G6Pase to prevent recurrence of symptoms. (C) Fasting for 6 and 8 hr for Dog W at older ages. Dog W was treated by readministration of AAV-G6Pase to in response to life-threatening hypoglycemia, anorexia, and pancreatitis (Table 1). Color images available online at www.liebertonline.com/hum

FIG. 5.

Anti-AAV antibody formation prior to and following AAV-G6Pase administration. Plasma was retrospectively analyzed from samples drawn during fasting for dogs with GSD-Ia or drawn following birth of litters from carrier females. Samples for dogs treated with AAV-G6Pase were obtained 2 months following vector administration, with the exception of Dog Du, which was obtained at 0.75 months. Dogs were treated initially with AAV2/8 or AAV2/9 as indicated. The elevation of absorbance >0.2 indicated detectable anti-capsid IgG antibodies. Absorbance for titer of 1:20 shown, except where otherwise indicated. Samples that were unavailable are indicated (#). (A) Anti-AAV9 in vector-treated dogs. (B) Anti-AAV8 in vector-treated dogs. (C) Anti-AAV7 in vector treated dogs. (D) Titering of anti-capsid IgG for Dog T at 2 days of age, and at 1 month (anti-AAV7) to 2 months (anti-AAV8 and anti-AAV9) of age. (E) Anti-AAV9 in carrier females. Dog A was not exposed to puppies treated with AAV2/9-G6Pase, unlike the other carrier females.

FIG. 6.

Biochemical correction and vector genome quantification for liver following initial AAV-G6Pase administration and following readministration. G6Pase activity and glycogen content in liver following AAV vector administration or dietary therapy alone for dogs with GSD-Ia (Affected+dietary therapy), and for normal dogs. Mean±standard deviation shown. (A) Liver G6Pase analysis 2 months following AAV2/9-G6Pase administration (Affected+AAV2/9), following a second AAV vector administration (Affected+2nd AAV vector), and following administration of >2 vectors. (B) Liver glycogen content analysis for samples from (A). (C) G6Pase analysis for serial liver biopsies from individual dogs with GSD-Ia following the indicated number of vector treatments (see Table 1 for additional details). GSD-Ia dogs treated with dietary therapy (Aff.). (D) Liver glycogen content for samples from (C). (E) Vector quantification for postmortem tissue samples. Limit of detection was 0.1.

FIG. 7.

Histology of GSD-Ia following AAV-G6Pase administration. Liver biopsies obtained at the indicated ages. Hematoxylin and eosin staining shown. Original magnification, ×400, and scale bar indicates 50 μm. (A) Dog Ro at 6 weeks of age, following AAV2/9 vector administration at 2 days of age. (B) Dog T at 6 months of age, 6 weeks following AAV2/1 vector administration. (C) Dog W at 16 months of age, 1 month following AAV2/8 vector administration. (D) Dog R at 46 months of age, 3 months following AAV2/7 vector administration. (E) GSD-Ia dog following dietary therapy alone at 2 years of age. (F) Normal dog liver. Color images available online at www.liebertonline.com/hum

FIG. 8.

Monitoring for liver damage or renal failure following AAV-G6Pase administration. (A) Serum aspartate aminotransferase was quantified during episodes of recurrent hypoglycemia in dogs with GSD-Ia at the indicated ages. (B) Serum creatinine. Normal ranges indicated (dashed boxes).