Gel-mediated delivery of AAV1 vectors corrects ventilatory function in Pompe mice with established disease

Abstract

Pompe disease is a muscular dystrophy that results in respiratory insufficiency. We characterized the outcomes of targeted delivery of recombinant adeno-associated virus serotype 1 (rAAV2/1) vector to diaphragms of Pompe mice with varying stages of disease progression. We observed significant improvement in diaphragm contractile strength in mice treated at 3 months of age that is sustained at least for 1 year and enhanced contractile strength in mice treated at 9 and 21 months of age, measured 3 months post-treatment. Ventilatory parameters including tidal volume/inspiratory time ratio, minute ventilation/expired CO2 ratio, and peak inspiratory airflow were significantly improved in mice treated at 3 months and tested at 6 months. Despite early improvement, mice treated at 3 months and tested at 1 year had diminished normoxic ventilation, potentially due to attenuation of correction over time or progressive degeneration of nontargeted accessory tissues. However, for all rAAV2/1-treated mice (treated at 3, 9, and 21 months, assayed 3 months later; treated at 3 months, assayed at 1 year), minute ventilation and peak inspiratory flows were significantly improved during respiratory challenge. These results demonstrate that gel-mediated delivery of rAAV2/1 vectors can significantly augment ventilatory function at initial and late phases of disease in a model of muscular dystrophy.

Figure 1

AAV2/1 can transverse the entire diaphragm thickness. Recombinant AAV2/1 (AAV1) and AAV2 vectors encoding for cytomegalovirus promoter-driven lacZ were applied to the peritoneal side (right side in this figure) of the diaphragm of normal mice using the gel method of delivery. Six weeks postinfection, tissues were harvested and stained for lacZ expression by X-gal staining as described in Methods. AAV1 transgene is observed on both thoracic and peritoneal surfaces. AAV, adeno-associated virus.

Figure 2

Gel-mediated delivery of AAV1-CMV-hGAA to diaphragms of Gaa^−/− mice leads to clearance of accumulated glycogen. (a) Periodic acid-Schiff staining of diaphragms from a 1-year-old wild-type (Gaa^+/+), 1- and 2-year-old Gaa^−/−, 1-year-old Gaa^−/− mice treated with AAV2/1 either at 3 or 9 months of age, and 2-year-old Gaa^−/− mouse treated at 21 months of age. Purple staining is indicative of glycogen. (b) Perchloric acid extracts of diaphragm from untreated Gaa^−/−, Gaa^−/− mice treated at 3 months of age and assayed at 6 months (GEL 6 months) or 1 year of age (GEL 1 year), or C57BL6/129SvJ (WT) were analyzed by ¹H-MRS for the presence of glycogen. AAV, adeno-associated virus; CMV, cytomegalovirus; WT, wild type.

Figure 3

The mouse model of Pompe disease exhibits decreasing diaphragm contractile strength with age. Diaphragm was isolated from 3, 6, 12, and 24 month-old Gaa^−/− mice and contractile strength was assessed by determining force–frequency relationships (n = 3 for each group). GAAKO, Gaa^−/−. m.o., month(s) old; y.o., year(s) old.

Figure 4

Correction of diaphragmatic contractile strength after gel-mediated delivery of AAV1-CMV-hGAA to diaphragm. (a) Three-month-old Gaa^−/− mice were administered AAV1 vector and contractile strength was determined 3 months post-treatment (n = 3 for each group). (b) Three- or 9-month-old Gaa^−/− mice were administered AAV2/1 vector and contractile strength was determined at 1 year of age (n = 3 for each group). (c) 21-month-old Gaa^−/− mice were administered AAV1 vector and contractile strength was determined at 2 years of age (n = 3 for each group). AAV, adeno-associated virus; CMV, cytomegalovirus, m.o., month(s) old; y.o., year(s) old.

Figure 5

Baseline respiration is significantly improved in AAV1-treated mice. Three-month-old Gaa^−/− mice were treated with AAV1-CMV-hGAA via the gel method of delivery to diaphragm. Three months postinfection, respiratory function was assayed by awake, unrestrained, whole-body barometric plethysmography (n = 9). Age-matched untreated Gaa^−/− (GAAKO) and wild-type C57BL6/129SvJ (B6129) mice were assayed as well, (n = 10 for each group). Baseline function was measured for 1 hour under normoxic conditions. V_E/V_CO2 represents ventilatory efficiency (the ratio of minute ventilation to expired CO₂); V_T/T_I represents the mean inspiratory flow (ratio of tidal volume to inspiratory time).

Figure 6

Ventilatory function is significantly improved in rAAV2/1-treated mice. Ventilatory function was assayed by awake, unrestrained, whole-body barometric plethysmography. (a) Graphs show the minute ventilation response to hypercapnia over the 10-minute period of time in untreated Gaa^−/− (n = 10), wild-type (n = 10), and rAAV2/1-treated Gaa^−/− mice (n = 9 for mice treated at 3 months and assayed at 6 months; n = 4 for mice treated at 3 months and assayed at 1 year; n = 9 for mice treated at 9 months; n = 7 for mice treated at 21 months). (b) Graphs show the peak inspiratory flow response to hypercapnia over the 10-minute period of time in the same animals represented in a. m.o., month(s) old; y.o., year(s) old.

Figure 7

Inspiratory phrenic nerve bursting in an rAAV2/1 treated mouse. Efferent phrenic nerve electrical activity was recorded in anesthetized mice (n = 1, each untreated and rAAV2/1-treated at 21 months and assayed at 2 years of age) using standard neurophysiology methods.²⁵ The left and middle panels show inspiratory phrenic bursting during normoxic conditions in untreated and rAAV2/1 treated mice, respectively. In these panels, the bottom trace is the unprocessed phrenic neurogram, and the top trace is the “integrated” (∫) phrenic signal. The right panel provides an overlay plot comparing a single integrated inspiratory phrenic burst in an untreated versus rAAV2/1 treated mouse. The rAAV2/1 treated mouse had considerably more robust phrenic bursting compared to the untreated mouse. AAV, adeno-associated virus.