Ciaran D. Scallan

Evidence for gene transfer and expression of factor IX in haemophilia B patients treated with an AAV vector.

Pre-clinical studies in mice and haemophilic dogs have shown that introduction of an adeno-associated viral (AAV) vector encoding blood coagulation factor IX (F.IX) into skeletal muscle results in sustained expression of F.IX at levels sufficient to correct the haemophilic phenotype. On the basis of these data and additional pre-clinical studies demonstrating an absence of vector-related toxicity, we initiated a clinical study of intramuscular injection of an AAV vector expressing human F.IX in adults with severe haemophilia B. The study has a dose-escalation design, and all patients have now been enrolled in the initial dose cohort (2×1011 vg/kg). Assessment in the first three patients of safety and gene transfer and expression show no evidence of germline transmission of vector sequences or formation of inhibitory antibodies against F.IX. We found that the vector sequences are present in muscle by PCR and Southern-blot analyses of muscle biopsies and we demonstrated expression of F.IX by immunohistochemistry. We observed modest changes in clinical endpoints including circulating levels of F.IX and frequency of F.IX protein infusion. The evidence of gene expression at low doses of vector suggests that dose calculations based on animal data may have overestimated the amount of vector required to achieve therapeutic levels in humans, and that the approach offers the possibility of converting severe haemophilia B to a milder form of the disease.

Improved coagulation in bleeding disorders by Non-Anticoagulant Sulfated Polysaccharides (NASP)

Additional therapeutic options are needed for patients with bleeding disorders such as hemophilia A, hemophilia B, severe von Willebrand disease, and other rare factor deficiencies. A novel approach to improve coagulation in such clotting disorders has been identified that, parodoxically, involves heparinlike sulfated polysaccharides. Select molecules of this broad class are largely devoid of anticoagulant activity and are here denoted Non-Anticoagulant Sulfated Polysaccharides (NASPs). A mechanism involving blockade of the extrinsic pathway downregulator, Tissue Factor Pathway Inhibitor (TFPI) by NASPs, was conceived as an approach for improving procoagulant behavior in hemophilic settings. A subset of NASPs, including pentosan polysulfate (PPS) and fucoidan inhibited both full-length and Kunitz 1 and 2 (K1K2) TFPI and, at concentrations from 4-500 nM, improved (i.e. accelerated) the clotting time of human hemophilia A and hemophilia B plasmas or plasma with reduced factor VII levels when tested in dilute prothrombin time (dPT) assays. Fucoidan did not reduce normal plasma APTT times implying specificity for extrinsic pathway control. Improved hemostasis in vivo was observed in mice with hemophilias A or B following low dose subcutaneous administration of PPS or fucoidan, or a combination of NASP plus factor supplement. Increased survival of factor deficient mice following a bleeding challenge was observed. Accordingly, administration of select NASP(s), via mechanism(s) not fully understood, represents a unique means of improving coagulation in bleeding disorders.

Novel Caprine Adeno-Associated Virus (AAV) Capsid (AAV-Go.1) Is Closely Related to the Primate AAV-5 and Has Unique Tropism and Neutralization Properties

ABSTRACT Preexisting humoral immunity to adeno-associated virus (AAV) vectors may limit their clinical utility in gene delivery. We describe a novel caprine AAV (AAV-Go.1) capsid with unique biological properties. AAV-Go.1 capsid was cloned from goat-derived adenovirus preparations. Surprisingly, AAV-Go.1 capsid was 94% identical to the human AAV-5, with differences predicted to be largely on the surface and on or under the spike-like protrusions. In an in vitro neutralization assay using human immunoglobulin G (IgG) (intravenous immune globulin [IVIG]), AAV-Go.1 had higher resistance than AAV-5 (100-fold) and resistance similar to that of AAV-4 or AAV-8. In an in vivo model, SCID mice were pretreated with IVIG to generate normal human IgG plasma levels prior to the administration of AAV human factor IX vectors. Protein expression after intramuscular administration of AAV-Go.1 was unaffected in IVIG-pretreated mice, while it was reduced 5- and 10-fold after administration of AAV-1 and AAV-8, respectively. In contrast, protein expression after intravenous administration of AAV-Go.1 was reduced 7.1-fold, similar to the 3.8-fold reduction observed after AAV-8administration in IVIG-pretreated mice, and protein expression was essentially extinguished after AAV-2 administration in mice pretreated with much less IVIG (15-fold). AAV-Go.1 vectors also demonstrated a marked tropism for lung when administered intravenously in SCID mice. The pulmonary tropism and high neutralization resistance to human preexisting antibodies suggest novel therapeutic uses for AAV-Go.1 vectors, including targeting diseases such as cystic fibrosis. Nonprimate sources of AAVs may be useful to identify additional capsids with distinct tropisms and high resistance to neutralization by human preexisting antibodies.

753. Evaluation of AAV Neutralizing Antibodies Using a Passive Immunity Model in SCID Mice

The existence of pre-existing antibodies in the human population represents one of the greatest challenges to gene delivery using viral vectors. To evaluate the effect of neutralizing antibodies on the intra-hepatic delivery of AAV vectors in vivo, a passive immunity (PI) model was developed by intravenous (IV) injection of purified human immunoglobulin (IVIg) into SCID mice. IVIg was chosen as a source of anti-AAV antibodies as it is purified from thousands of individuals with presumably different AAV antibody characteristics. AAV human FIX (AAV2-hFIX) vectors were then delivered via the portal vein, tail vein or by direct liver injection. FIX expression was used as a measure of vector escape from neutralization. Even in the presence of low neutralizing titers (<1:10), viral transduction was either completely eliminated in the case of IV delivered vector (5 × 1011 vg/mouse) or nearly so for vector delivered via the portal vein or direct liver injection (1 × 1011 vg/mouse). This neutralization capacity was not predicted by an in vitro neutralizing assay. Changing the rate of vector delivery and the injection volume did not reduce the level of neutralization. However, AAV pseudotypes 8 and 6 were more resistant to neutralization than AAV2 (96% neutralization for AAV2 compared to 59% for AAV8 and 76% for AAV6, at titers of 1:4, 1:7 and 1:6 respectively, and at a dose of 1 × 1011 vg/mouse). Furthermore, the neutralization effect was partially overcome if the injected vector also contained empty particles suggesting that immuno-absorption of antibodies might be a useful strategy to circumvent viral neutralization. In summary, we have established an animal model to evaluate the neutralization effect on AAV liver transduction and are applying it to the evaluation of AAV serotypes and delivery approaches.

102. Catheter-Based Intrahepatic rAAV Gene Delivery: Influence of Infusion Flow Rate and Volume on Uptake

Catheter-based, fluoroscopy-aided interventional radiology approaches have been utilized in a variety of clinical applications. Safe, consistent and efficient methods for liver directed gene delivery need to be developed. In this study, we examined the effects of infusion flow rate and infusion volume on intrahepatic catheter-based delivery of recombinant adeno-assocated virus 2 vectors (rAAV2) in dogs. To study the efficiency of intrahepatic gene transfer, vector (rAAV-lac Z (nonfunctional promoter)) was delivered through the hepatic artery with an occlusion balloon catheter with a protocol mimicking the clinical angiography procedure of an ongoing clinical gene transfer study in hemophilia B subjects. Three groups of beagle dogs (n=5/group) utilizing three different intrahepatic injection parameters were utilized: A) 8 ml injected at 0.1 ml/min/kg (most similar to ongoing clinical trial), B) 8 ml injected at 8 ml/min/kg and C) 40 ml injected at 8 ml/min/kg. Reproducibility and extent of gene transfer was studied by determining vector distribution and vector copy number in the liver, including individual lobes, 4 weeks post injection. Quantitative PCR using linearized standards was employed to examine vector levels in the liver. Liver histology, serum chemistries and hematology parameters were also examined in all animals over the course of 4 weeks to determine the safety of the various delivery parameters. The results indicate that intrahepatic transgene delivery was influenced by the infusion rate. On average, the rAAV2 copy number per hepatocyte with fast infusion was twice that of slow infusion (e.g. 2.8 copy/cell group B vs 1.4 copy/cell group A). Likewise, vector copy numbers per liver were approximately two times higher in the fast infusion groups (p=0.04, one-way ANOVA). Groups B (higher rate) and C (higher rate and higher volume) did not significantly differ. AAV2 vector tended to be more evenly distributed in individual livers in the fast infusion groups. No changes in chemistries nor hematology values were detected in any dogs over the course of study. Histological analysis of the liver for capsid immunoflourescence is ongoing. This study demonstrates that the infusion flow rate, in particular, can be an important variable in efficient and safe hepatic delivery of AAV.