Jennifer B. Nietupski

Comprehensive analysis of the acute toxicities induced by systemic administration of cationic lipid:plasmid DNA complexes in mice.

A major limitation associated with systemic administration of cationic lipid:plasmid DNA (pDNA) complexes is the vector toxicity at the doses necessary to produce therapeutically relevant levels of transgene expression. Systematic evaluation of these toxicities has revealed that mice injected intravenously with cationic lipid:pDNA complexes develop significant, dose-dependent hematologic and serologic changes typified by profound leukopenia, thrombocytopenia, and elevated levels of serum transaminases indicative of hepatocellular necrosis. Vector administration also induced a potent inflammatory response characterized by complement activation and the induction of the cytokines IFN-gamma, TNF-alpha, IL-6, and IL-12. These toxicities were found to be transient, resolving with different kinetics to pretreatment levels by 14 days posttreatment. The toxic syndrome observed was independent of the cationic lipid:pDNA ratio, the cationic lipid species, and the level of transgene expression attained. Mechanistic studies determined that neither the complement cascade nor TNF-alpha were key mediators in the development of these characteristic toxicities. Administration of equivalent doses of the individual vector components revealed that cationic liposomes or pDNA alone did not generate the toxic responses observed with cationic lipid:pDNA complexes. Only moderate leukopenia was associated with administration of cationic liposomes or pDNA alone, while only mild thrombocytopenia was noted in pDNA-treated animals. These results establish a panel of objective parameters that can be used to quantify the acute toxicities resulting from systemic administration of cationic lipid:pDNA complexes, which in turn provides a means to compare the therapeutic indices of these vectors.

Preexisting Immunity and Low Expression in Primates Highlight Translational Challenges for Liver-directed AAV8-mediated Gene Therapy

Liver-directed gene therapy with adeno-associated virus (AAV) vectors effectively treats mouse models of lysosomal storage diseases (LSDs). We asked whether these results were likely to translate to patients. To understand to what extent preexisting anti-AAV8 antibodies could impede AAV8-mediated liver transduction in primates, commonly preexposed to AAV, we quantified the effects of preexisting antibodies on liver transduction and subsequent transgene expression in mouse and nonhuman primate (NHP) models. Using the highest viral dose previously reported in a clinical trial, passive transfer of NHP sera containing relatively low anti-AAV8 titers into mice blocked liver transduction, which could be partially overcome by increasing vector dose tenfold. Based on this and a survey of anti-AAV8 titers in 112 humans, we predict that high-dose systemic gene therapy would successfully transduce liver in >50% of human patients. However, although high-dose AAV8 administration to mice and monkeys with equivalent anti-AAV8 titers led to comparable liver vector copy numbers, the resulting transgene expression in primates was ~1.5-logs lower than mice. This suggests vector fate differs in these species and that strategies focused solely on overcoming preexisting vector-specific antibodies may be insufficient to achieve clinically meaningful expression levels of LSD genes using a liver-directed gene therapy approach in patients.

AAV8-mediated expression of glucocerebrosidase ameliorates the storage pathology in the visceral organs of a mouse model of Gaucher disease

Gaucher disease is the most common of the lysosomal storage disorders. The primary manifestation is the accumulation of glucosylceramide (GL‐1) in the macrophages of liver and spleen (Gaucher cells), due to a deficiency in the lysosomal hydrolase glucocerebrosidase (GC). A Gaucher mouse model (D409V/null) exhibiting reduced GC activity and accumulation of GL‐1 was used to evaluate adeno‐associated viral (AAV)‐mediated gene therapy.

Iminosugar-based inhibitors of glucosylceramide synthase increase brain glycosphingolipids and survival in a mouse model of Sandhoff disease

The neuropathic glycosphingolipidoses are a subgroup of lysosomal storage disorders for which there are no effective therapies. A potential approach is substrate reduction therapy using inhibitors of glucosylceramide synthase (GCS) to decrease the synthesis of glucosylceramide and related glycosphingolipids that accumulate in the lysosomes. Genz-529468, a blood-brain barrier-permeant iminosugar-based GCS inhibitor, was used to evaluate this concept in a mouse model of Sandhoff disease, which accumulates the glycosphingolipid GM2 in the visceral organs and CNS. As expected, oral administration of the drug inhibited hepatic GM2 accumulation. Paradoxically, in the brain, treatment resulted in a slight increase in GM2 levels and a 20-fold increase in glucosylceramide levels. The increase in brain glucosylceramide levels might be due to concurrent inhibition of the non-lysosomal glucosylceramidase, Gba2. Similar results were observed with NB-DNJ, another iminosugar-based GCS inhibitor. Despite these unanticipated increases in glycosphingolipids in the CNS, treatment nevertheless delayed the loss of motor function and coordination and extended the lifespan of the Sandhoff mice. These results suggest that the CNS benefits observed in the Sandhoff mice might not necessarily be due to substrate reduction therapy but rather to off-target effects.

Iminosugar-based inhibitors of glucosylceramide synthase prolong survival but paradoxically increase brain glucosylceramide levels in Niemann–Pick C mice

Niemann Pick type C (NPC) disease is a progressive neurodegenerative disease caused by mutations in NPC1 or NPC2, the gene products of which are involved in cholesterol transport in late endosomes. NPC is characterized by an accumulation of cholesterol, sphingomyelin and glycosphingolipids in the visceral organs, primarily the liver and spleen. In the brain, there is a redistribution of unesterified cholesterol and a concomitant accumulation of glycosphingolipids. It has been suggested that reducing the aberrant lysosomal storage of glycosphingolipids in the brain by a substrate reduction therapy (SRT) approach may prove beneficial. Inhibiting glucosylceramide synthase (GCS) using the iminosugar-based inhibitor miglustat (NB-DNJ) has been reported to increase the survival of NPC mice. Here, we tested the effects of Genz-529468, a more potent iminosugar-based inhibitor of GCS, in the NPC mouse. Oral administration of Genz-529468 or NB-DNJ to NPC mice improved their motor function, reduced CNS inflammation, and increased their longevity. However, Genz-529468 offered a wider therapeutic window and better therapeutic index than NB-DNJ. Analysis of the glycolipids in the CNS of the iminosugar-treated NPC mouse revealed that the glucosylceramide (GL1) but not the ganglioside levels were highly elevated. This increase in GL1 was likely caused by the off-target inhibition of the murine non-lysosomal glucosylceramidase, Gba2. Hence, the basis for the observed effects of these inhibitors in NPC mice might be related to their inhibition of Gba2 or another unintended target rather than a result of substrate reduction.

Cationic Lipid Structure and Formulation Considerations for Optimal Gene Transfection of the Lung

Abstract Enhanced gene transduction to the lung using cationic lipids could be attained through optimization of the structure of the lipids and the formulation of the cationic lipid : plasmid DN A (pDNA) complexes. We have expanded on our earlier observation of the importance of the structural orientation of the cationic lipid headgroup. Through the synthesis of a number of matched pairs of cationic lipids differing only in the configuration of their headgroup, we confirmed that those harboring a T-shape headgroup are more active than their linear counterparts, at least when tested in the lungs of BALB/c mice. Additionally, we demonstrated that not only are the structural considerations of these cationic lipids important, but also their protonation state, the free base being invariably more active than its salt counterpart. The salt forms of cationic lipids bound pDNA with greater avidity, which may have affected their subsequent intracellular dissolution and transit of the pDNA to the nucleus. Inclusion of a number of frequently used solutes in the vehicle severely inhibited the gene transfection activity of the cationic lipids. The selection of neutral co-lipids was also an important factor for overall transfection activity of the formulation, with significant gains in transfection activity realized when diphytanoylphosphatidylethanol-amine or dilinoleoylphosphatidylethanolamine were used in lieu of dioleoylphosphati-dylethanolamine. Finally, we showed that a transacylation reaction could occur between the cationic lipid and neutral co-lipid which reduced the transfection activity of the complexes. It is the hope that as our understanding of the many factors that influence the activity of these cationic lipid: pDNA complexes improves, formulations with much greater potency can be realized for use in the treatment of pulmonary diseases.

Efficacy of Enzyme and Substrate Reduction Therapy with a Novel Antagonist of Glucosylceramide Synthase for Fabry Disease.

Fabry disease, an X-linked glycosphingolipid storage disorder, is caused by the deficient activity of α-galactosidase A (α-Gal A). This results in the lysosomal accumulation in various cell types of its glycolipid substrates, including globotriaosylceramide (GL-3) and lysoglobotriaosylceramide (globotriaosyl lysosphingolipid, lyso-GL-3), leading to kidney, heart, and cerebrovascular disease. To complement and potentially augment the current standard of care, biweekly infusions of recombinant α-Gal A, the merits of substrate reduction therapy (SRT) by selectively inhibiting glucosylceramide synthase (GCS) were examined. Here, we report the development of a novel, orally available GCS inhibitor (Genz-682452) with pharmacological and safety profiles that have potential for treating Fabry disease. Treating Fabry mice with Genz-682452 resulted in reduced tissue levels of GL-3 and lyso-GL-3 and a delayed loss of the thermal nociceptive response. Greatest improvements were realized when the therapeutic intervention was administered to younger mice before they developed overt pathology. Importantly, as the pharmacologic profiles of α-Gal A and Genz-682452 are different, treating animals with both drugs conferred the greatest efficacy. For example, because Genz-682452, but not α-Gal A, can traverse the blood-brain barrier, levels of accumulated glycosphingolipids were reduced in the brain of Genz-682452-treated but not α-Gal A-treated mice. These results suggest that combining substrate reduction and enzyme replacement may confer both complementary and additive therapeutic benefits in Fabry disease.

CNS-accessible Inhibitor of Glucosylceramide Synthase for Substrate Reduction Therapy of Neuronopathic Gaucher Disease.

Gaucher disease (GD) is caused by a deficiency of glucocerebrosidase and the consequent lysosomal accumulation of unmetabolized glycolipid substrates. Enzyme-replacement therapy adequately manages the visceral manifestations of nonneuronopathic type-1 Gaucher patients, but not the brain disease in neuronopathic types 2 and 3 GD. Substrate reduction therapy through inhibition of glucosylceramide synthase (GCS) has also been shown to effectively treat the visceral disease. Here, we evaluated the efficacy of a novel small molecule inhibitor of GCS with central nervous system (CNS) access (Genz-682452) to treat the brain disease. Treatment of the conduritol β epoxide-induced mouse model of neuronopathic GD with Genz-682452 reduced the accumulation of liver and brain glycolipids (>70% and >20% respectively), extent of gliosis, and severity of ataxia. In the genetic 4L;C* mouse model, Genz-682452 reduced the levels of substrate in the brain by >40%, the extent of gliosis, and paresis. Importantly, Genz-682452-treated 4L;C* mice also exhibited an ~30% increase in lifespan. Together, these data indicate that an orally available antagonist of GCS that has CNS access is effective at attenuating several of the neuropathologic and behavioral manifestations associated with mouse models of neuronopathic GD. Therefore, Genz-682452 holds promise as a potential therapeutic approach for patients with type-3 GD.

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.

Liposome-mediated gene transfer to lung isografts

OBJECTIVES Our objective were to determine the feasibility, efficacy, and safety of in vivo and ex vivo liposome-mediated gene transfer to lung isografts. METHODS Fischer rats were divided into three main groups: (1) Nontransplant setting: Liposome-chloramphenicol acetyl transferase cDNA was intravenously injected, and lungs were harvested at different time points: 2, 6, 12, and 24 hours; 2, 5, 8, and 21 days (n = 3). Chloramphenicol acetyl transferase activity was determined in lungs, hearts, livers, and kidneys. The distribution and type of transfected cells were evaluated by in situ hybridization. Lung toxicity was assessed by arterial oxygen tension, histology, and tumor necrosis factor-alpha levels. (2) In vivo graft transfection: Left lungs were transplanted 6 hours, 4 hours, and 15 minutes after intravenous injection and were assessed for chloramphenicol acetyl transferase activity and arterial oxygen tension on postoperative day 2. (3) Ex vivo graft transfection: Grafts were infused ex vivo with either 660 micrograms (n = 3) or 330 micrograms (n = 3) of DNA complexed to liposomes and stored at 10 degrees C for 4 hours. Chloramphenicol acetyl transferase activity was assessed 44 hours after transplantation. RESULTS Transgene expression was detected in endothelial cells, macrophages, and interstitial cells. Chloramphenicol acetyl transferase activity was present as early as 2 hours, increased significantly between 6 hours and 8 days, and then decreased to minimal levels by 21 days. Chloramphenicol acetyl transferase activity was greatest in donor lungs and hearts and minimal in livers and kidneys. Arterial oxygen tension was normal in treated animals. Inflammation was minimal, and tumor necrosis factor-alpha levels increased only sevenfold in treated animals. CONCLUSION In vivo and ex vivo liposome-mediated gene transfer to lung isografts allows significant transgene expression with minimal effects on graft function.