AAV Gene Therapy Utilizing Glycosylation-Independent Lysosomal Targeting Tagged GAA in the Hypoglossal Motor System of Pompe Mice.

Pompe disease is caused by mutations in the gene encoding the lysosomal glycogen-metabolizing enzyme, acid-alpha glucosidase (GAA). Tongue myofibers and hypoglossal motoneurons appear to be particularly susceptible in Pompe disease. Here we used intramuscular delivery of adeno-associated virus serotype 9 (AAV9) for targeted delivery of an enhanced form of GAA to tongue myofibers and motoneurons in 6-month-old Pompe (Gaa -/- ) mice. We hypothesized that addition of a glycosylation-independent lysosomal targeting tag to the protein would result in enhanced expression in tongue (hypoglossal) motoneurons when compared to the untagged GAA. Mice received an injection into the base of the tongue with AAV9 encoding either the tagged or untagged enzyme; tissues were harvested 4 months later. Both AAV9 constructs effectively drove GAA expression in lingual myofibers and hypoglossal motoneurons. However, mice treated with the AAV9 construct encoding the modified GAA enzyme had a >200% increase in the number of GAA-positive motoneurons as compared to the untagged GAA (p < 0.008). Our results confirm that tongue delivery of AAV9-encoding GAA can effectively target tongue myofibers and associated motoneurons in Pompe mice and indicate that the effectiveness of this approach can be improved by addition of the glycosylation-independent lysosomal targeting tag.

Sustained correction of motoneuron histopathology following intramuscular delivery of AAV in pompe mice.

Pompe disease is an autosomal recessive disorder caused by mutations in the acid-α glucosidase (GAA) gene. Lingual dysfunction is prominent but does not respond to conventional enzyme replacement therapy (ERT). Using Pompe (Gaa(-/-)) mice, we tested the hypothesis that intralingual delivery of viral vectors encoding GAA results in GAA expression and glycogen clearance in both tongue myofibers and hypoglossal (XII) motoneurons. An intralingual injection of an adeno-associated virus (AAV) vector encoding GAA (serotypes 1 or 9; 1 × 10(11) vector genomes, CMV promoter) was performed in 2-month-old Gaa(-/-) mice, and tissues were harvested 4 months later. Both serotypes robustly transduced tongue myofibers with histological confirmation of GAA expression (immunochemistry) and glycogen clearance (Period acid-Schiff stain). Both vectors also led to medullary transgene expression. GAA-positive motoneurons did not show the histopathologic features which are typical in Pompe disease and animal models. Intralingual injection with the AAV9 vector resulted in approximately threefold more GAA-positive XII motoneurons (P < 0.02 versus AAV1); the AAV9 group also gained more body weight over the course of the study (P < 0.05 versus AAV1 and sham). We conclude that intralingual injection of AAV1 or AAV9 drives persistent GAA expression in tongue myofibers and motoneurons, but AAV9 may more effectively target motoneurons.

Striatal readministration of rAAV vectors reveals an immune response against AAV2 capsids that can be circumvented.

Recombinant adeno-associated virus (rAAV) expresses no viral genes after transduction. In addition, because the brain is relatively immunoprivileged, intracranial rAAV transduction may be immunologically benign due to a lack of antigen presentation. However, preexposure to AAV allows neutralizing antibodies (nAbs) to block brain transduction and rAAV readministration in the brain leads to an inflammatory response in the second-injection site. In this study, we replicate our striatal rAAV2/2-GDNF readministration results and extend this effect to a second transgene, green fluorescent protein (GFP). Unlike rAAV2/2-GDNF readministration, striatal rAAV2/2-GFP readministration leads to a loss of transgene in the second site in the absence of detectable circulating nAbs. In order to determine whether the transgene or the AAV2 capsid is the antigenic stimulus in brain for the immune response in the second site, we readministered rAAV2/2-GFP using two different rAAV serotypes (rAAV2/2 followed by rAAV2/5). In this case, there was no striatal inflammation or transgene loss detected in the second-injection site. In addition, striatal readministration of rAAV2/5-GFP also resulted in no detectable immune response. Furthermore, delaying rAAV2/2 striatal readministration to a 11-week interval abrogated the immune response in the second-injection site. Finally, while striatal readministration of rAAV2/2 leads to significant loss of transgene in the second-injection site, this effect is not due to loss of vector genomes as determined by quantitative real-time PCR. We conclude that intracellular processing of AAV capsids after transduction is the immunogenic antigen and capsid serotypes that are processed more quickly than rAAV2/2 are less immunogenic.

Neonatal intraperitoneal or intravenous injections of recombinant adeno-associated virus type 8 transduce dorsal root ganglia and lower motor neurons.

Targeting lower motor neurons (LMNs) for gene delivery could be useful for disorders such as spinal muscular atrophy and amyotrophic lateral sclerosis. LMNs reside in the ventral gray matter of the spinal cord and send axonal projections to innervate skeletal muscle. Studies have used intramuscular injections of adeno-associated virus type 2 (AAV2) to deliver viral vectors to LMNs via retrograde transport. However, treating large areas of the spinal cord in a human would require numerous intramuscular injections, thereby increasing viral titer and risk of immune response. New AAV serotypes, such as AAV8, have a dispersed transduction pattern after intravenous or intraperitoneal injection in neonatal mice, and may transduce LMNs by retrograde transport or through entry into the nervous system. To test LMN transduction after systemic injection, we administered recombinant AAV8 (rAAV8) carrying the green fluorescent protein (GFP) gene by intravenous or intraperitoneal injection to neonatal mice on postnatal day 1. Tissues were harvested 5 and 14 days postinjection and analyzed by real-time polymerase chain reaction and GFP immunohistochemistry to assess the presence of AAV genomes and GFP expression, respectively. Spinal cords were positive for AAV genomes at both time points. GFP immunohistochemistry revealed infrequent labeling of LMNs across all time points and injection routes. Somewhat surprisingly, there was extensive labeling of fibers in the dorsal horns and columns, indicating dorsal root ganglion transduction across all time points and injection routes. Our data suggest that systemic injection of rAAV8 is not an effective delivery route to target lower motor neurons, but could be useful for targeting sensory pathways in chronic pain.

Preclinical characterization of a recombinant adeno-associated virus type 1-pseudotyped vector demonstrates dose-dependent injection site inflammation and dissemination of vector genomes to distant sites.

To translate the potential advantages of recombinant adeno-associated virus type 1 (rAAV1) vectors into a clinical application for muscle-directed gene therapy for alpha1 -antitrypsin (AAT) deficiency, we performed safety studies in 170 C57BL/6 mice and 26 New Zealand White rabbits. A mouse toxicology study included 8 cohorts of 10 mice each (5 per sex). Mice were killed either 21 or 90 days after intramuscular injection of doses ranging up to 1x10(13)vector genomes (VG), equivalent to 4 x 10(14)VG/kg. A mouse biodistribution study was performed in 5 cohorts of 10 mice, receiving intramuscular injections at the same doses; as well as in a lower dose cohort (3 x 10(8) VG; equivalent to 1.2 x 10(10)VG/kg); and in 4 other cohorts (excluding the vehicle control) injected with identical doses intravenously. Finally, biodistribution was examined in rabbits, with serial collection of blood and semen, as well as terminal tissue collection. Two significant findings were present, both of which were dose dependent. First, inflammatory cell infiltrates were detected at the site of injection 21, 60, or 90 days after intramuscular injection of 1 x 10(13)VG. This was not associated with loss of transgene expression. Second, vector DNA sequences were detected in most animals, levels being highest with the highest doses and earliest time points. Vector DNA was also present in liver, spleen, kidneys, and a number of other organs, including the gonads of animals receiving the highest dose. Likewise, vector DNA was present in the semen of male rabbits at higher doses. The copy number of vector DNA in the blood and semen declined over time throughout the study. These two dose-dependent findings have served to guide to the design of a phase 1 human trial of rAAV1-AAT.

Phase I trial of intramuscular injection of a recombinant adeno-associated virus serotype 2 alphal-antitrypsin (AAT) vector in AAT-deficient adults.

A phase I trial of intramuscular injection of a recombinant adeno-associated virus serotype 2 (rAAV2) alpha1-antitrypsin (AAT) vector was performed in 12 AAT-deficient adults, 10 of whom were male. All subjects were either homozygous for the most common AAT mutation (a missense mutation designated PI*Z) or compound heterozygous for PI*Z and another mutation known to cause disease. There were four dose cohorts, ranging from 2.1 x 10(12) vector genomes (VG) to 6.9 x 10(13) VG, with three subjects per cohort. Subjects were injected sequentially in a dose-escalating fashion with a minimum of 14 days between patients. Subjects who had been receiving AAT protein replacement discontinued that therapy 28 days before vector administration. There were no vector-related serious adverse events in any of the 12 participants. Vector DNA sequences were detected in the blood between 1 and 3 days after injection in nearly all patients receiving doses of 6.9 x 10(12) VG or higher. Anti-AAV2 capsid antibodies were present and rose after vector injection, but no other immune responses were detected. One subject who had not been receiving protein replacement exhibited low-level expression of wild-type M-AAT in the serum (82 nM), which was detectable 30 days after receiving an injection of 2.1 x 10(13) VG. Unfortunately, residual but declining M-AAT levels from the washout of the protein replacement elevated background levels sufficiently to obscure any possible vector expression in that range in most of the other individuals in the higher dose cohorts.

Gene delivery in renal tubular epithelial cells using recombinant adeno-associated viral vectors.

Gene therapy has the potential to provide a therapeutic strategy for numerous renal diseases such as diabetic nephropathy, chronic rejection, Alport syndrome, polycystic kidney disease, and inherited tubular disorders. In previous studies using cationic liposomes or adenoviral or retroviral vectors to deliver genes into the kidney, transgene expression has been transient and often associated with adverse host immune responses, particularly with the use of adenoviral vectors. The unique properties of recombinant adeno-associated viral (rAAV) vectors permit long-term stable transgene expression with a relatively low host immune response. The purpose of the present study was to evaluate gene expression in the rat kidney after intrarenal arterial infusion of a rAAV (serotype 2) vector encoding green fluorescence protein (GFP) induced by a cytomegalovirus-chicken beta-actin hybrid promoter. The left kidney of experimental animals was treated with either saline or transduced with rAAV2-GFP (0.125 ml/100 g body wt, 1 x 10(10)/ml infectious units) through the renal artery. A time-dependent expression of GFP was observed in all kidneys injected with rAAV2-GFP, with maximal expression observed at 6 wk posttransduction. The expression of GFP was restricted to cells in the S(3) segment of the proximal tubule and intercalated cells in the collecting duct, the latter identified by co-localization with H(+)-ATPase. No transduction was observed in the glomeruli or the intrarenal vasculature. These studies demonstrate successful transgene expression in tubular epithelial cells, specifically in the S(3) segment of the proximal tubule and intercalated cells, after intrarenal administration of a rAAV vector and provide the impetus for further studies to exploit its use as a tool for gene therapy in the kidney.

Intramuscular administration of recombinant adeno-associated virus 2 alpha-1 antitrypsin (rAAV-SERPINA1) vectors in a nonhuman primate model: safety and immunologic aspects.

We performed a series of studies in baboons to evaluate the safety of intramuscular administration of rAAV vector expressing the alpha-1 antitrypsin (AAT) gene (SERPINA1) in a nonhuman primate model. Initial experiments performed with an rAAV vector expressing the human SERPINA1 gene (at doses of up to 5 x 10(12) vector genomes/kg) resulted in the generation of anti-human AAT antibodies, which correlated with a loss of detectable transgene expression. Subsequent studies made use of the baboon SERPINA1 gene tagged with a short (10-amino-acid) c-myc tag. When animals were sacrificed, 4 months after vector injection, transduced myofibers showed efficient transgene expression without detectable humoral immune responses. Mild inflammation was observed in and near the sites of injection in some vector- and saline-injected animals, but serum creatine kinase (CK) values were normal in nearly every case. Real-time PCR was also performed 4 months after injection on gonadal tissue to evaluate the risk of germline transmission. No vector sequences were detected in the gonadal tissues from these animals. These studies indicate that the risks of immune reaction and germline transmission after intramuscular injection of rAAV-SERPINA1 in nonhuman primates are relatively low within the range of vector doses studied.