Preclinical toxicology and biodistribution studies of recombinant adeno-associated virus 1 human acid α-glucosidase

Abstract

A biodistribution and toxicology study was performed to test the acute toxicities of intradiaphragmatic injection of a recombinant adeno-associated virus (rAAV) 2/1-human acid alpha-Glucosidase (hGAA) driven by a cytomegalovirus (CMV) promoter (rAAV1-CMV-hGAA) in New Zealand white rabbits and in the rodent Pompe disease model by injecting at the right quadriceps. Studies performed using fluoroscopy and AAV2-GFP demonstrated spread upon intradiaphragmatic injection, and the ability of AAV to infect and express acid α-glucosidase (GAA) throughout the diaphragm. For the preclinical study, 10 rabbits (5 male, 5 female) were divided into two groups, vehicle control (Lactated Ringer's) and test article (1.5×10(12) vector genomes [vg] rAAV1-CMV-hGAA), and euthanized on day 21. After direct visualization, the left hemidiaphragm was injected at three locations. There was up to a 2,500-fold increase in circulating anti-AAV1 antibodies directed to the vector capsids. In addition, up to an 18-fold increase in antibodies against the GAA protein was generated. Injection sites maintained up to 1.0×10(5) vg/μg genomic DNA (gDNA), while uninjected sites had up to 1.0×10(4) vg/μg gDNA. Vector DNA was present in blood at 24 hr postinjection at up to 1.0×10(6) vg/μg gDNA, followed by a decrease to 1.0×10(3) vg/μg gDNA at euthanization on day 21. Nominal amounts of vector DNA were present in peripheral organs, including the brain, spinal cord, gonads, and skeletal muscle. Upon histopathological examination, fibroplasias of the serosal surface were noted at diaphragm injections sites of both groups. In addition, an increase in mononuclear cell infiltration in the diaphragm and esophagus in vector-dosed animals was found. Elevated creatine phosphokinase levels, an indicator of muscle repair, was observed in all animals postprocedure but persisted in vector-injected rabbits until euthanization. A follow-up study suggested that this was directed against the human transgene expression in a foreign species. Overall, this study demonstrates diffusion of vector throughout the diaphragm after localized injections.

FIG. 1.

Ease of injection and vector spread throughout the rabbit diaphragm. A rabbit was injected at three sites (sections C, F, and I) by direct visualization with 3.3×10⁹ vg per site with AAV1-GFP. Three weeks after administration, samples were taken from 20 sites for GFP detection by immunohistochemistry and vector genomes by real-time qPCR. Substantial GFP was detected at the sites of injection and waned in distal sites. Vector genomes were detected in the highest copy number at the sites of injection and minimally at distal locations. Overall, the number of vector genomes decreased as the sampling moved further from the sites of injection (decrease in blush). AAV, adeno-associated virus; qPCR, quantitative polymerase chain reaction; vg, vector genomes.

FIG. 2.

Inflammatory cells in the diaphragm and distal esophagus. At euthanization on day 21, inflammatory cells were seen on the distal esophagus (A) and presented as mononuclear cell infiltrations in the outer longitudinal muscle layer of the tunica muscularis of the esophagus. In the diaphragm (C), at the sites of injection, an increase in severity of mononuclear cell infiltration, degeneration of skeletal muscle, and regeneration of skeletal muscle was observed in comparison to uninjected diaphragm muscle (B).

FIG. 3.

Real-time PCR for vector genomes in the diaphragm. The total copies of vector DNA/μg gDNA detected in the diaphragm are presented. The positive cutoff limit, ≥100 vg/μg gDNA, is presented by the horizon trend line (dashed). gDNA, genomic DNA.

FIG. 4.

Endoscopy-guided vector administration in the rabbit diaphragm. (A) Endoscopy was used from the thoracic side of the diaphragm to guide the injection needle through the chest wall. (B) Confirmation of vector administration by the formation of a bleb (circle) and potential leakage.

FIG. 5.

CPK levels in rAAV1-CMV-hGAA unilaterally diaphragm-injected rabbits. Serum CPK is plotted as groups mean±standard deviation for each group and upper limit of the normal reference range indicated (dashed trend line). Significance was determined on days 1 and 15 by unpaired t-test. CPK, creatine phosphokinase.