Bradley L. Hodges

Development of Catheter-Based Procedures for Transducing the Isolated Rabbit Liver with Plasmid DNA

Rapid systemic injection of naked plasmid DNA (pDNA) in a large volume into a mouse tail vein has been shown to result in a high level of gene expression in the liver. However, the potential therapeutic benefit to humans embodied in hydrodynamic transfection of the liver cannot be realized until a clinically viable method for gene delivery is developed. In light of this fact, we have devised and evaluated several methods for delivering pDNA to the isolated rabbit liver using minimally invasive catheter-based techniques. Using a lobar technique, pDNA was delivered hydrodynamically to an isolated hepatic lobe using a balloon occlusion balloon catheter to occlude a selected hepatic vein. A whole organ technique was used wherein the entire hepatic venous system was isolated and the pDNA solution injected hydrodynamically into the vena cava between two balloons used to block hepatic venous outflow. Lobar delivery of a plasmid encoding a secreted alkaline phosphatase (SEAP) reporter gene resulted in significant levels of transgene product in the serum. A nonsecreted transgene product, chloramphenicol acetyltransferase (CAT), showed the highest levels of expression in the injected lobe distal to the injection site. Compared to lobar delivery, whole organ delivery yielded much higher serum levels of SEAP expression and a significantly broader hepatic parenchymal distribution of CAT expression. These preliminary studies suggest that catheter-mediated hydrodynamic delivery of pDNA to the isolated liver may provide a method for human gene therapy that is both therapeutically significant and clinically practicable.

Ability of Adeno-Associated Virus Serotype 8-Mediated Hepatic Expression of Acid α-Glucosidase to Correct the Biochemical and Motor Function Deficits of Presymptomatic and Symptomatic Pompe Mice

The availability of a murine model of Pompe disease has enabled an evaluation of the relative merits of various therapeutic paradigms, including gene therapy. We report here that administration of a recombinant adeno-associated virus serotype 8 (AAV8) vector (AAV8/DC190-GAA) encoding human acid alpha-glucosidase (GAA) into presymptomatic Pompe mice resulted in nearly complete correction of the lysosomal storage of glycogen in all the affected muscles. A relatively high dose of AAV8/DC190-GAA was necessary to attain a threshold level of GAA for inducing immunotolerance to the expressed enzyme and for correction of muscle function, coordination, and strength. Administration of AAV8/DC190-GAA into older Pompe mice with overt disease manifestations was also effective at correcting the lysosomal storage abnormality. However, these older mice exhibited only marginal improvements in motor function and no improvement in muscle strength. Examination of histologic sections showed evidence of skeletal muscle degeneration and fibrosis in aged Pompe mice whose symptoms were abated or rescued by early but not late treatment with AAV8/DC190-GAA. These results suggest that AAV8-mediated hepatic expression of GAA was effective at addressing the biochemical and functional deficits in Pompe mice. However, early therapeutic intervention is required to maintain significant muscle function and should be an important consideration in the management and treatment of Pompe disease.

Hydrodynamic delivery of DNA

Hydrodynamic delivery is an efficient and inexpensive procedure to deliver a wide range of nucleic acids to hepatic tissues and other organs in vivo. The successful application of hydrodynamic delivery is dependent on the rapid injection of a large aqueous volume containing DNA, RNA or other molecules into the vasculature of the liver. In this review, the development of the procedures for hydrodynamic delivery will be described and the parameters necessary for attaining maximal gene expression will be highlighted. A review of the mechanisms for transfecting hepatocytes, as well as potential uses of this approach in various research and clinical applications, will also be discussed.

Intracerebroventricular delivery of glucocerebrosidase reduces substrates and increases lifespan in a mouse model of neuronopathic Gaucher disease

Gaucher disease is caused by a deficit in the enzyme glucocerebrosidase. As a consequence, degradation of the glycolipids glucosylceramide (GluCer) and glucosylsphingosine (GluSph) is impaired, and their subsequent buildup can lead to significant pathology and early death. Type 1 Gaucher patients can be treated successfully with intravenous replacement enzyme, but this enzyme does not reach the CNS and thus does not ameliorate the neurological involvement in types 2 and 3 Gaucher disease. As one potential approach to treating these latter patients, we have evaluated intracerebroventricular (ICV) administration of recombinant human glucocerebrosidase (rhGC) in a mouse model of neuronopathic Gaucher disease. ICV administration resulted in enzyme distribution throughout the brain and alleviated neuropathology in multiple brain regions of this mouse model. Treatment also resulted in dose-dependent decreases in GluCer and GluSph and significantly extended survival. To evaluate the potential of continuous enzyme delivery, a group of animals was treated ICV with an adeno-associated viral vector encoding hGC and resulted in a further extension of survival. These data suggest that ICV administration of rhGC may represent a potential therapeutic approach for type 2/3 Gaucher patients. Preclinical evaluation in larger animals will be needed to ascertain the translatability of this approach to the clinic.

Systemic administration of AAV8-α-galactosidase A induces humoral tolerance in nonhuman primates despite low hepatic expression.

In mice, liver-restricted expression of lysosomal enzymes from adeno-associated viral serotype 8 (AAV8) vectors results in reduced antibodies to the expressed proteins. To ask whether this result might translate to patients, nonhuman primates (NHPs) were injected systemically with AAV8 encoding α-galactosidase A (α-gal). As in mice, sustained expression in monkeys attenuated antibody responses to α-gal. However, this effect was not robust, and sustained α-gal levels were 1-2 logs lower than those achieved in male mice at the same vector dose. Because our mouse studies had shown that antibody levels were directly related to expression levels, several strategies were evaluated to increase expression in monkeys. Unlike mice, expression in monkeys did not respond to androgens. Local delivery to the liver, immune suppression, a self-complementary vector and pharmacologic approaches similarly failed to increase expression. While equivalent vector copies reached mouse and primate liver and there were no apparent differences in vector form, methylation or deamination, transgene expression was limited at the mRNA level in monkeys. These results suggest that compared to mice, transcription from an AAV8 vector in monkeys can be significantly reduced. They also suggest some current limits on achieving clinically useful antibody reduction and therapeutic benefit for lysosomal storage diseases using a systemic AAV8-based approach.

531. Balloon Catheter-Mediated Hepatic Vein Delivery of a Viral Vector Mitigates Neutralization by Anti-Viral Antibodies and Results in Efficient Transduction of Rabbit Liver

The liver, and in particular, hepatocytes, represent an attractive target for both viral and non-viral gene transfer vectors. However, there are certain delivery issues associated with both of these vector systems that make their use in humans problematic. In particular, for viral vectors, the existence of anti-viral antibodies in the general population represents a potential barrier to their effective use in the clinic. Building upon our earlier success using balloon catheters to deliver plasmid DNA by way of the hepatic venous circulation to rabbit liver, we have used this same model to ask if we could use a similar approach to deliver a model viral vector. Specifically, we have compared a local, balloon catheter-mediated hepatic vein delivery protocol to systemic delivery of a CMV-driven adenoviral vector expressing b-galactosidase in terms of liver transduction efficiency, toxicity, and cell types transduced. We have also made this comparison in the presence of defined anti-AdV antibody titers in the recipient rabbits. In the naive animal, local balloon catheter-based delivery conferred an advantage in overall liver transduction when compared to systemic delivery of an identical dose of virus. In rabbits bearing anti-AdV antibody titers equivalent to those found in pooled human serum, this difference in expression between local and systemic delivery was even more striking. Importantly, in the presence of passively-administered anti-AdV antibodies, balloon-catheter mediated delivery resulted in expression levels that were comparable to those obtained by systemic delivery of an equivalent dose in a naive animal. Since in general, systemic delivery of AdV in naive animal models results in expression levels of secreted proteins regarded as therapeutic, the present results predict that retrograde delivery by a hepatic vein route using a balloon catheter should result in therapeutic levels of expression, even in the presence of average human levels of anti-AdV antibodies. Further support for this hypothesis is the finding that this local hepatic vein delivery approach resulted in the majority of expression originating from hepatocytes even in the passively immunized animals. In contrast, systemic delivery of AdV in passively immunized animals resulted in the majority of expression originating from non-hepatocytes. This latter finding implies that this local approach should help minimize the transduction of antigen-presenting cells, thereby reducing any immune consequences of viral transduction. Taken together, these data suggest that coupled with additional features such as a hepatocyte-specific expression cassette, balloon catheter viral-mediated transduction of the liver may represent a practical clinical approach for treating indications for which hepatocytes can be used as a depot for therapeutic protein production.