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.

Glycogen storage disease type II (GSDII) is a lysosomal storage disease caused by a deficiency in acid alpha-glucosidase (GAA), and leads to cardiorespiratory failure by the age of 2 years. In this study, we investigate the impact of anti-GAA antibody formation on cross-correction of the heart, diaphragm, and hind-limb muscles from liver-directed delivery of recombinant adeno-associated virus (rAAV)5- and rAAV8-GAA vectors. GAA(-/-) mice receiving 1 x 10(12) vector genomes of rAAV5- or rAAV8-DHBV-hGAA were analyzed for anti-GAA antibody response, GAA levels, glycogen reduction, and contractile function. We demonstrate that restoration of GAA to the affected muscles is dependent on the presence or absence of the antibody response. Immune-tolerant mice had significantly increased enzyme levels in the heart and skeletal muscles, whereas immune-responsive mice had background levels of GAA in all tissues except the diaphragm. The increased levels of activity in immune-tolerant mice correlated with reduced glycogen in the heart and diaphragm and, overall, contractile function of the soleus muscle was significantly improved. These findings highlight the importance of the immune response to rAAV-encoded GAA in correcting GSDII and provide additional understanding of the approach to treatment of GSDII.

Glycogen storage disease type II (GSD II) is a lysosomal storage disease resulting from complete deficiency of acid alpha-glucosidase (GAA). GAA deficiency causes glycogen to accumulate in lysosomes, leading to muscle dysfunction. Respiratory muscle dysfunction is a particular concern because respiratory insufficiency is a major cause of morbitity and mortality in GSD II. Thus, our first purpose was to characterize breathing in a Gaa-/- mouse model of GSD II. Using barometric plethysmography, minute ventilation (MV; mL/min), frequency (F; breaths/min) and tidal volume (TV; mL/min) were measured in 2 year old (n=6/group) control and Gaa-/- mice. Data were collected during quiet breathing (21% O2, balanced N2). MV (control vs. Gaa-/-; 75 5 vs. 32 3), F (223 10 vs. 138 14), and TV (0.35 0.02 vs. 0.24 0.01) were attenuated in Gaa-/- ice compared to controls (MEANSEM). Further, the ratio of minute ventilation/CO2 (VE/VCO2) was 79.6 % of controls in the Gaa-/- mice. Our second purpose was to evaluate the role of gene therapy on the pattern of breathing in Gaa-/- mice. Plethysmography was performed on a group of 2 year old Gaa-/- mice that were injected i.m. into the quadriceps with AAV1-CMV-GAA at 3 months of age. VE/VCO2 was 84.1 % of controls in the treated group, suggesting improved ventilation following AAV1-CMV-GAA administration. Furthermore, mice from the treated group that were sacrificed at 6 months of age had diaphragm GAA enzyme activity levels similar to Gaa-/- mice (control: 4.95 nmol/hr/mg, Gaa-/-: 0.17, Gaa-/- treated: 0.15). These data suggest that the functional improvement identified in the treated group may be due to central nervous system correction, since GAA enzyme levels were not corrected. To investigate this hypothesis, we injected AAV1-CMV-LacZ into the quadriceps of control mice. Four weeks post-injection, LacZ was detected in the spinal cord via PCR. Thus. The therapeutic efficacy may reflect a spinal mechanism of action. This hypothesis is supported by the observation that glycogen (mg/mL/gww; MEANSD) is elevated in the spinal cord of 6 mo old Gaa-/- (13.0 6.5) compared to wild-type (2.8 0.3) mice. Although GSD II is typically viewed as a degenerative disease in the peripheral musculature, CNS abnormalities may play an important role in the pathogenesis of muscle weakness and respiratory insufficiency.

Top of pageAbstract Glycogen storage disease type II (GSDII), is a lysosomal storage disease caused by a partial to complete deficiency in the lysosomal hydrolase, acid |[alpha]|-glucosidase (GAA). From gestation, affected individuals store glycogen in their lysosomal compartments, resulting in severe hypertrophic cardiomyopathy and respiratory insufficiency. Previous studies with intrahepatic and intramuscular delivery of recombinant adeno-associated viral (rAAV) vectors to GSDII mice resulted in a variable anti-GAA immune response and insufficient levels of circulating protein in immune-responsive mice. In this study, we evaluated the potential for intravenously delivered rAAV vectors to correct the biochemical, histological and functional phenotype in GSDII mice over a sustained period of time. Neonatal GSDII mice were intravenously administered 5 |[times]| 1010 particles of rAAV serotype 1 vector encoding human GAA under control of the CMV promoter and analyzed at 2.5, 6 and 11 months post-injection (PI). GAA levels in the heart and diaphragm peaked at 6 months PI with averages of 42 |[plusmn]| 23-fold and 1.4 |[plusmn]| 0.6-fold normal respectively. At 11 months PI, cardiac levels dropped but remained therapeutic at 6.4 |[plusmn]| 1.9-fold normal and these levels were concomitant with glycogen clearance in the heart. Additionally, at 11 months, PI, contractile function of hind-limb muscles was significantly improved and the observed mobility of the treated animals was improved compared to age-matched mice. Six of the 26 treated mice formed anti-GAA antibodies between 4 and 10 weeks PI that quickly dropped to background levels by 15 weeks PI. Based on historical data, the therapeutic target for GAA restoration is 20-30% of normal. In this study, we achieved long-term therapeutic levels of correction from a single intravenous delivery in the two most critically affected muscles, the heart and diaphragm, with functional correction in the skeletal muscle. These results demonstrate the utility of intravenously administered rAAV1 vectors for both skeletal and cardiac muscle gene transfer in a model of fatal cardiomyopathy.

Several human genetic diseases that affect striated muscle have been modeled by creating knockout mouse strains. However, many of these are perinatal lethal mutations that result in death from respiratory distress within hours after birth. As the diaphragm muscle does not contract until birth, the sudden increase in diaphragm activity creates permanent injury to the muscle causing it to fail to meet respiratory demands. Therefore, the impact of these mutations remains hidden throughout embryonic development and early death prevents investigators from performing detailed studies of other striated muscle groups past the neonatal stage. Glycogen storage disease type II (GSDII), caused by a deficiency in acid α-glucosidase (GAA), leads to lysosomal accumulation of glycogen in all cell types and abnormal myofibrillogenesis in striated muscle. Contractile function of the diaphragm muscle is severely affected in both infantile-onset and late-onset individuals, with death often resulting from respiratory failure. The knockout mouse model of GSDII survives well into adulthood despite the gradual weakening of all striated muscle groups. Using this model, we investigated the delivery of recombinant adeno-associated virus (rAAV) vectors encoding the human GAA cDNA to the developing embryo. Results indicate specific high-level transduction of diaphragm tissue, leading to activity levels up to 10-fold higher than normal and restoration of normal contractile function. Up to an estimated 50 vector copies per diploid genome were quantified in treated diaphragms. Histological glycogen staining of treated diaphragms revealed prevention of lysosomal glycogen accumulation in almost all fibers when compared with untreated controls. This method could be employed with disease models where specific rescue of the diaphragm would allow for increased survival and thus further investigation into the impact of the gene deletion on other striated muscle groups.

Top of pageAbstract Pompe disease is a lysosomal storage disease caused by a deficiency of the lysosomal enzyme acid α-glucosidase (GAA). The disorder causes cardiac and skeletal myopathy in infants, is fatal within two years of life, and protein therapeutic trials are currently underway. The ultimate goal of viral vector-mediated correction includes secretion/re-uptake of recombinant hGAA via the mannose 6-phosphate receptor (M6PR) pathway. Previous experience with rAAV serotype 2 vectors demonstrated moderate levels of transgene expression in the liver. We anticipate that significant hepatic overexpression of human GAA will be necessary for systemic correction of Pompe disease. Therefore, we tested the ability of rAAV serotype 5 and 8 (rAAV5 and rAAV8) vectors to direct GAA overexpression and secretion from the liver. We cross-packaged rAAV2-ITR containing genomes carrying the hGAA cDNA under the control of the duck hepatitis B viral (DHBV) promoter into AAV5 and AAV8 capsids, respectively. In a pilot study using the rAAV5 vector in Gaa-/- mice, we delivered 1012 vector genomes (vg) intraportally, which resulted in 4- to 16-fold overexpression of GAA; inhibitory anti-GAA antibody formation; no detectable enzyme activity in the heart or skeletal muscle; and significant levels of enzyme activity in the diaphragm that was dependent on both antibody titer and hepatic expression levels. In an effort to modulate this observed immune response, we developed a neonatal model of tolerization using subcutaneous, low dose delivery of recombinant hGAA to 1-day-old Gaa-/- mice. At 8 weeks post-tolerization, we intraportally delivered 1012 vg of rAAV5 and rAAV8 respectively. Hepatic expression of hGAA was 1.7-fold greater in rAAV8-treated mice compared to rAAV5 (26 ± 4.8-fold wild-type v. 15 ± 1.6-fold wild-type). Some mice from both groups exhibited antibody formation despite neonatal tolerization, and rAAV8 mice, as a group, had higher antibody titers (43-fold untreated v. 7-fold for rAAV5). Specific subjects in both rAAV5- and rAAV8-treated groups maintained tolerance, and in those subjects, significantly higher GAA activities were observed in the heart, diaphragm and skeletal muscle tissues, compared to non-tolerant, treated mice. Specifically, tolerant rAAV8-treated mice had hepatic GAA activities that were 34-fold wild-type, while tolerant rAAV5 mice had 10-fold hepatic overexpression. For both rAAV5 and rAAV8 GAA-tolerant mice, we observed diaphragmatic enzyme activities up to 3-fold of wild-type and cardiac activities reached 50% of wild-type, with concomitant histological reduction of stored glycogen. In summary, we have established that superphysiologic levels of liver GAA expression can be directed by both rAAV5 and rAAV8. These levels are sufficient to mediate cross-correction of target tissues, but can be adversely modulated by a humoral response. Clinical therapeutic strategies may require a combination of high levels of liver GAA expression and immunomodulation.

Genetically modified mice are important models for evaluation of potential gene therapies for human diseases. However, their small size often precludes the use of clinically feasible methods for vector delivery, therefore, alternative methods must be used. We have developed a gel-based method for delivery of recombinant adeno-associated virus vectors to the mouse diaphragm, an important target organ for many myopathic diseases. We hypothesized that delivery of vectors in a viscous solution would increase transduction by providing a longer exposure time to target cells. We demonstrate that gel-mediated delivery of rAAV serotypes 1, 2, and 5 results in higher transduction efficiencies than free vectors alone when administered in vivo to mouse diaphragms. We further establish greater tropism of rAAV1 vectors for the diaphragm compared to serotypes 2 and 5. This report describes a novel method for efficient delivery of rAAV vectors to the mouse diaphragm and is the first demonstration of gene transfer to the diaphragm using recombinant adeno-associated virus vectors.

Recombinant adeno-associated viral (rAAV) vectors based on serotype 2 are currently being evaluated most extensively in animals and human clinical trials. rAAV vectors constructed from other AAV serotypes (serotypes 1, 3, 4, 5, and 6) can transduce certain tissues more efficiently and with different specificity than rAAV2 vectors in animal models. Here, we describe reagents and methods for the production and purification of AAV2 inverted terminal repeat-containing vectors pseudotyped with AAV1 or AAV5 capsids. To facilitate pseudotyping, AAV2rep/AAV1cap and AAV2rep/AAV5cap helper plasmids were constructed in an adenoviral plasmid backbone. The resultant plasmids, pXYZ1 and pXYZ5, were used to produce rAAV1 and rAAV5 vectors, respectively, by transient transfection. Since neither AAV5 nor AAV1 binds to the heparin affinity chromatography resin used to purify rAAV2 vectors, purification protocols were developed based on anion-exchange chromatography. The purified vector stocks are 99% pure with titers of 1×1012 to 1×1013vector genomes/ml.

A major hurdle in most current gene therapy modalities is the ability to transduce target tissues at very high efficiencies that ultimately lead to therapeutic levels of transgene expression. We have developed a novel method of recombinant adeno-associated virus 2 (rAAV) delivery that results in increased vector transduction efficiencies using microspheres reversibly conjugated to rAAV vectors. We hypothesize that conjugation to microspheres should result in a higher effective concentration of vector as well as longer relative exposure time of vector to target cells as it moves through the tissue vasculature. In vitro experiments demonstrate that the same level of transduction seen with free vector can be achieved using 1% of vector when conjugated to microspheres. In addition, using magnetic microspheres, the region of infection can be targeted. In vivo, we demonstrate that microsphere-mediated delivery of rAAV vector results in higher transduction efficiencies than delivery with free vector alone when administered either intramuscularly or intravenously. Furthermore, we demonstrate targeting of transgene expression to specific tissues by retention of microsphere-bound vector in the capillary bed. These studies demonstrate a novel method to deliver rAAV vectors more effectively that could prove to be a successful alternative mode of virus-mediated human gene therapy.

Pompe disease is a lysosomal storage disease caused by the absence of acid alpha-1,4 glucosidase (GAA). The pathophysiology of Pompe disease includes generalized myopathy of both cardiac and skeletal muscle. We sought to use recombinant adeno-associated virus (rAAV) vectors to deliver functional GAA genes in vitro and in vivo. Myotubes and fibroblasts from Pompe patients were transduced in vitro with rAAV2-GAA. At 14 days postinfection, GAA activities were at least fourfold higher than in their respective untransduced controls, with a 10-fold increase observed in GAA-deficient myotubes. BALB/c and Gaa(-/-) mice were also treated with rAAV vectors. Persistent expression of vector-derived human GAA was observed in BALB/c mice up to 6 months after treatment. In Gaa(-/-) mice, intramuscular and intramyocardial delivery of rAAV2-Gaa (carrying the mouse Gaa cDNA) resulted in near-normal enzyme activities. Skeletal muscle contractility was partially restored in the soleus muscles of treated Gaa(-/-) mice, indicating the potential for vector-mediated restoration of both enzymatic activity and muscle function. Furthermore, intramuscular treatment with a recombinant AAV serotype 1 vector (rAAV1-Gaa) led to nearly eight times normal enzymatic activity in Gaa(-/-) mice, with concomitant glycogen clearance as assessed in vitro and by proton magnetic resonance spectroscopy.

Pompe disease is a lethal cardioskeletal myopathy in infants and results from genetic deficiency of the lysosomal enzyme acid alpha-glucosidase (GAA). Genetic replacement of the cDNA for human GAA (hGAA) is one potential therapeutic approach. Three months after a single intramuscular injection of 10(8) plaque-forming units (PFU) of E1-deleted adenovirus encoding human GAA (Ad-hGAA), the activity in whole muscle lysates of immunodeficient mice is increased to 20 times the native level. Direct transduction of a target muscle, however, may not correct all deficient cells. Therefore, the amount of enzyme that can be transferred to deficient cells from virally transduced cells was studied. Fibroblasts from an affected patient were transduced with AdhGAA, washed, and plated on transwell culture dishes to serve as donors of recombinant enzyme. Deficient fibroblasts were plated as acceptor cells, and were separated from the donor monolayer by a 22-microm pore size filter. Enzymatic and Western analyses demonstrate secretion of the 110-kDa precursor form of hGAA from the donor cells into the culture medium. This recombinant, 110-kDa species reaches the acceptor cells, where it can be taken up by mannose 6-phosphate receptor-mediated endocytosis. It then trafficks to lysosomes, where Western analysis shows proteolytic processing to the 76- and 70-kDa lysosomal forms of the enzyme. Patient fibroblasts receiving recombinant hGAA by this transfer mechanism reach levels of enzyme activity that are comparable to normal human fibroblasts. Skeletal muscle cell cultures from an affected patient were also transduced with Ad-hGAA. Recombinant hGAA is identified in a lysosomal location in these muscle cells by immunocytochemistry, and enzyme activity is transferred to deficient skeletal muscle cells grown in coculture. Transfer of the precursor protein between muscle cells again occurs via mannose 6-phosphate receptors, as evidenced by competitive inhibition with 5 mM mannose 6-phosphate. In vivo studies in GAA-knockout mice demonstrate that hepatic transduction with adenovirus encoding either murine or human GAA can provide a depot of recombinant enzyme that is available to heart and skeletal muscle through this mechanism. Taken together, these data show that the mannose 6-phosphate receptor pathway provides a useful strategy for cell-to-cell distribution of virally derived recombinant GAA.