Seroprevalence of Pre-Existing Nabs Against AAV1, 2, 5, 6 and 8 in South African Hemophilia B Patient Population

Introduction Several studies have shown that the induction of antibodies by natural exposure to various AAV serotypes can compromise the subsequent use of AAV as a gene therapy vector, limiting patient eligibility for AAV-delivered therapeutics. The implications of pre-existing antibodies to AAV serotypes are very different: Levels of anti-AAV2 or anti-AAV8 neutralizing antibodies (NABs) as low as 5 have been related to a decrease or even total impairment of AAV liver transduction after systemic delivery in humans. However, successful gene transfer has been reported in patients with anti-AAV5 NABs titers up to 340 and in non-human primates with titers up to 1030. Extensive surveys on the prevalence of anti-AAV antibodies in humans have been published. Results from these studies indicate that prevalence varies dependent on serotype, and that a significant proportion of individuals develop humoral immunity against various AAV serotypes early in life, starting around 2 years of age. Furthermore, the prevalence of antibodies to different AAV serotypes has been reported to vary according to geographical location. Study Objective We performed a NABs seroprevalence study in South African hemophilia B patient population (n=44) using a panel of AAV serotypes suitable for liver targeted therapy, to determine the AAV serotypes likely to be of greatest clinical applicability for the South African hemophilia B population. Methods Forty-four hemophilia B patient serum samples were obtained from Hemophilia Comprehensive Care Center in Johannesburg (South Africa). All the patient serum samples were analyzed for the presence of NABs against AAV serotypes 1, 2, 5, 6 and 8 with the use of highly sensitive luciferase-based bioassays. The assays entail incubation of the test serum samples dilution series with an AAV1, 2, 5, 6 or 8-based reporter vector that carries the luciferase gene. This incubation allows neutralizing antibodies in the test serum to bind to the reporter vector particles. These mixtures are subsequently transferred onto Hek293T cells, where reporter vector particles can transduce cells and mediate expression of luciferase. Anti-AAV1, 2, 5, 6 or 8 NABs titers were determined by calculation of the percentage of neutralization for each sample dilution and fitting the neutralization curve with a four-parameter method. Anti-AAV1, 2, 5, 6 or 8 NABs titer (IC50) is the dilution at which antibodies inhibit Hek293T cell transduction with AAV1, 2, 5, 6 or 8-LUC by 50%. The lowest patient serum dilution used in every assay was 8 and samples were considered positive when calculated anti-AAV NAB titer was ≥8. All analytical runs included proper negative and positive controls. Results and Discussion The presence of NABs against the AAV serotypes 1, 2, 5, 6 and 8 was determined in the serum of the hemophilia B patients (Fig. 1). The highest prevalence of NABs was found to be against the AAV2 serotype, 95% (n=42/44) followed by the AAV6 serotype, 82% (n=36/44), and the AAV1 serotype 77% (n=34/44). The prevalence of NABs against AAV5 and AAV8 was lower with 66% (n=29/44) for AAV5 and 64% (n=28/44) for AAV8. The serum samples positive for anti-AAV2 NABs had a high occurrence of titers above 1030 (39%) in comparison to anti-AAV1 NABs (20%), anti-AAV5 NABs (5%) or anti-AAV8 NABs (7%).The occurrence of samples with low titers (ranging from titer of 8 to titer of 50) was the highest for anti-AAV8 NABs (32%) and for anti-AAV5 NABs (27%), followed by anti-AAV2 (18%) and anti-AAV1 (5%) (Fig.1). Currently, an anti-AAV NABs titer of 5 is used as an exclusion criteria in most of the systemic AAV-based gene therapies. When applying a similar cut-off of 8, 23% of the analyzed patients could be treated with AAV1, 5% with AAV2, 34% with AAV5, 18% with AAV6 and 36% with AAV8-based therapeutics (Fig.2). However, we have previously reported that AAV5-neutralizing antibodies do not impair the efficacy of in vivo transduction of AAV5-based vector up to a measured titer of 340 in humans and 1030 in non-human primates. Therefore, applying the cut-off of 340 or 1030, either 84% or 95% of the South African Hemophilia B patients could benefit from treatment with AAV5-based gene therapy (Fig.2). Disclosures Majowicz: uniQure N.V.: Employment. van Waes:uniQure N.V.: Employment. Timmer:uniQure N.V.: Employment. van Deventer:uniQure Biopharma B.V.: Employment. Mahlangu:Takeda: Consultancy, Honoraria, Speakers Bureau; LFB: Consultancy; NovoNordisk: Consultancy, Research Funding, Speakers Bureau; Roche: Consultancy, Research Funding, Speakers Bureau; Baxalta: Consultancy, Research Funding, Speakers Bureau; Freeline Therapeutics: Research Funding; Pfizer: Consultancy, Research Funding, Speakers Bureau; Spark: Consultancy, Speakers Bureau; Chugai: Consultancy; Biomarin: Research Funding; CSL Behring: Consultancy, Research Funding, Speakers Bureau; Novartis: Research Funding; Sanofi Genzyme: Research Funding, Speakers Bureau; Shire: Consultancy, Research Funding, Speakers Bureau; Sobi: Research Funding, Speakers Bureau; uniQure: Research Funding; World Federation of Haemophilia: Speakers Bureau. Ferreira:uniQure N.V.: Employment.

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Employing Factor IX Variants to Avoid Limitations Imposed by Immune Recognition of AAV Vector in Hemophilia B Gene Therapy

Blood ◽

10.1182/blood.v118.21.3124.3124 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3124-3124 ◽

Cited By ~ 1

Author(s):

Paul E. Monahan ◽

Junjiang Sun ◽

Tong Gui ◽

David G Wichlan ◽

Scott W McPhee ◽

...

Keyword(s):

Clinical Trial ◽

Gene Therapy ◽

Board Of Directors ◽

Research Funding ◽

Specific Activity ◽

Factor Ix ◽

Hemophilia B ◽

Ongoing Clinical Trial ◽

Advisory Committees

Abstract Abstract 3124 Persistent factor IX expression and phenotypic improvement have been achieved in a human clinical trial for hemophilia B using liver-directed adeno-associated virus (AAV) gene therapy vectors. An ongoing clinical trial uses a vector incorporating self-complementing AAV (scAAV) genome form, factor IX codon optimization (FIXopt) and AAV serotype 8 capsid. As was seen in a previous single-strand AAV serotype 2 trial, dose escalation has been associated with apparent immune-mediated transient inflammation of vector-transduced liver, although in contrast to the previous trial persistent FIX expression has been maintained for the first time. Taken together, these important trials define a consistent threshold load of AAV capsid that has stimulated capsid-specific cytotoxic lymphocyte recognition and potential transaminitis. To advance the successes achieved in these trials while providing a clear margin of safety so that this immunogenic threshold need not be approached, we have pursued steps to limit further the AAV capsid load. Single amino acid substitutions at arginine 338 in the FIX catalytic domain generate FIX variants with increased specific activity. We separately substituted either R338A, R338Q, or R338L (FIX Padua) into a codon optimized human factor IX cDNA and evaluated F.IX expression in tissue culture following plasmid DNA transfection of HEK 293t cells. Each R338 substitution improved FIX specific activity, up to 10 times increased over wild type using the R338LFIXopt cDNA. We next generated scAAV8 vectors incorporating a liver-specific transthyretin (TTR) promoter to express optimized codon F.IX cDNA with or without the R338L substitution. FIX−/− mice receiving portal vein injection of 1 × 1010 vg/animal (4 ×1011 vg/kg) expressed 86.5% of normal FIX activity at 2 months post-transduction from the WTopt vector and 330% normal from the R338LFIXopt. Incorporation of R338Lopt variant resulted in at least 6 to 10 fold increase in FIX specific activity over a follow-up of > 40 weeks. At ten months following FIX gene delivery, mice underwent a tail transection bleeding challenge. FIX vector mice demonstrated therapeutic protection from this major bleeding challenge and furthermore all survived with no late rebleeding (a hallmark of hemophilic phenotype). Greater than 100% normal human FIX activity was maintained for >40 weeks following treatment with the R338LFIX vector (v. 26.3% at euthanasia in WTopt vector group). The prolonged follow-up permitted extended safety evaluation. Factor IX inhibitor antibodies were not detected in any mice throughout the follow-up; FIX-binding IgG1 and IgG2 were negative also. Thrombin/antithrombin III complexes (TAT) examined at 12 weeks and at >30 weeks of age in R338LFIXopt vector mice did not differ from levels in WTFIXopt vector-treated or age-matched C57Bl/6 hemostatically normal mice. Necropsy at 40–44 weeks after vector (1 year of age) showed only age-related changes with no microvascular or macrovascular thrombosis on H&E staining or specific immunostaining for fibrin/fibrinogen deposition; specific staining for fibrosis within myocardium or other sites was negative. We next synthesized a R338LFIXopt expression cassette containing the LP1 promoter/enhancer/intron sequence being used in the ongoing clinical trial and demonstrated equivalent FIX activity from either promoter construct. We then established that the R338LFIXopt vector gives a predictable dose-response across a range of doses as low as 1x 1010 vg/kg I.V. and as high as 4 × 1012 vg/kg I.V. Hemarthrosis is the most common bleeding complication in hemophilia and leads to chronic joint destruction. Bleeding was induced in the joint of FIX−/− mice that had been transduced 4 weeks earlier with the R338LFIX vector. Joints were collected at 2 weeks after induced bleed and the bleeding-induced joint damage was graded using an established histologic score. I.V. R338LFIXopt vector pretreatment resulted in protection against joint degeneration in a dose-dependent fashion in this most relevant clinical scenario. These preclinical studies demonstrate a safety :efficacy profile to advance hemophilia gene therapy using the scAAV8.R338LFIXopt vector. Disclosures: Monahan: Baxter: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Asklepios BioPharmaceutical: Patents & Royalties, Research Funding; CSL Behring: Honoraria; NovoNordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees; PharmaIN: Research Funding; Prolor-Biotech: Research Funding. McPhee:Asklepios Biopharmaceutical: Employment. Samulski:Asklepios Biopharmaceutical: Employment, Patents & Royalties.

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Prevalence of Neutralizing Antibodies to Adenoviral Serotypes 5 and 35 in the Adult Populations of The Gambia, South Africa, and the United States

Clinical and Diagnostic Laboratory Immunology ◽

10.1128/cdli.11.2.351-357.2004 ◽

2004 ◽

Vol 11 (2) ◽

pp. 351-357 ◽

Cited By ~ 155

Author(s):

Edward Nwanegbo ◽

Eftyhia Vardas ◽

Wentao Gao ◽

Hilton Whittle ◽

Huijie Sun ◽

...

Keyword(s):

United States ◽

South Africa ◽

Gene Therapy ◽

Neutralizing Antibodies ◽

Fluorescent Proteins ◽

The United States ◽

Neutralization Assay ◽

The Gambia ◽

Serum Samples ◽

Lung Carcinoma Cells

ABSTRACT One of the major limitations of the use of adenoviruses as gene therapy vectors is the existence of preformed immunity in various populations. Recent studies have linked failure of adenoviral gene therapy trials to the presence of antiadenoviral neutralizing antibodies (NAb). Understanding the distribution and specificity of such antibodies will assist in the design of successful recombinant adenoviral gene therapies and vaccines. To assess the prevalence of NAb to adenovirus serotypes 5 and 35 (Ad5 and Ad35), we analyzed serum samples from adult immunocompetent individuals living in The Gambia, South Africa, and the United States by using a neutralization assay. Serum samples were incubated with A549 lung carcinoma cells and adenoviruses encoding enhanced green or yellow fluorescent proteins; results were analyzed by fluorescence microscopy and flow cytometry. Using this technique, we found a high prevalence of NAb against Ad5 in Gambian, South African, and U.S. subjects at both low and high titers. Conversely, all subjects displayed a low prevalence of NAb to Ad35; when present, anti-Ad35 NAb were seen at low titers. Because of the ability of adenoviruses to elicit systemic and mucosal immune responses, Ad35 with its low NAb prevalence appears to be an attractive candidate vector for gene therapy applications.

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Etranacogene Dezaparvovec (AAV5-Padua hFIX variant), an Enhanced Vector for Gene Transfer in Adults with Severe or Moderate-Severe Hemophilia B: Two Year Data from a Phase 2b Trial

Blood ◽

10.1182/blood-2020-139295 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 13-13

Author(s):

Annette von Drygalski ◽

Adam Giermasz ◽

Giancarlo Castaman ◽

Nigel S. Key ◽

Susan U. Lattimore ◽

...

Keyword(s):

Gene Therapy ◽

Board Of Directors ◽

Research Funding ◽

Factor Ix ◽

Hemophilia B ◽

Advisory Committees ◽

Adeno Associated Virus ◽

Human Factor Ix ◽

Virus Serotype ◽

Serotype 5

Background: Gene therapy for hemophilia offers the possibility to ameliorate disease severity to a mild or functionally curative state through a single administration. Etranacogene dezaparvovec (AMT-061) is an investigational gene therapy for hemophilia B comprising an adeno associated virus serotype 5 (AAV5) vector containing a codon-optimized Padua variant human factor IX (FIX) gene with liver specific promoter. Aims: We have previously shown a single dose of etranacogene dezaparvovec to provide sustained FIX activity into the mild-to normal range for up to 52 weeks post-dose in participants with severe or moderate-severe hemophilia B. This time, 2 years of follow-up data will be presented for the first time. Methods: A Phase 2b, open-label, single-dose, single-arm, multi-center trial (NCT03489291) in adult hemophilia B subjects. Interestingly, participants were not excluded based on neutralizing antibodies to AAV5. All subjects received a single intravenous dose of etranacogene dezaparvovec (2x1013 gc/kg) and will be followed for 5-years. The primary endpoint was FIX activity at Week 6. Secondary endpoints include e-diary recordings of bleeds and FIX concentrate use, laboratory parameters, joint health, patient reported outcomes, and adverse events (AEs). Results: All participants had FIX ≤1% (severe or moderately-severe FIX deficiency), required routine FIX prophylaxis, and had neutralizing activity to AAV5 at baseline. Following AMT-061 treatment, FIX activity increased rapidly to a mean of 31% at Week 6. At Week 52, mean FIX activity increased further to 41% with FIX activity levels of 50%, 31% and 41% in participants 1-3 respectively. There was no relationship between the presence of anti-AAV5 NAbs and response to etranacogene dezaparvovec. As of 52 weeks, there were no bleeds post-treatment and no requirement for FIX replacement aside from protocol-specified use for perioperative management in participant 3. There were no clinically significant elevations in liver enzymes and no participants required steroids related to the treatment. One participant experienced 2 mild AEs possibly related to treatment shortly after dosing (self-limiting headache and slightly elevated CRP). One patient had hip surgery due to worsening of pre-existing avascular necrosis deemed unrelated by investigator to etranacogene dezaparvovec and received FIX per protocol according to standard clinical practice. No participant developed inhibitors to FIX. Updated results to 2 years of follow-up will be presented with the main focus on FIX activity, FIX replacement therapy use and reported bleeds. Conclusions: Patients with AAV5 NAbs were included in the Phase 2b etranacogene dezaparvovec trial and have shown sustained FIX activity into the mild-to normal range. All participants were able to discontinue routine prophylaxis, and there have been no bleeds post-treatment with etranacogene dezaparvovec. Disclosures Giermasz: BioMarin: Consultancy, Research Funding, Speakers Bureau; Genentech/Roche: Consultancy, Research Funding, Speakers Bureau; uniQure: Consultancy, Research Funding; Sangamo Therapeutics: Research Funding; Bioverativ/Sanofi: Consultancy, Research Funding, Speakers Bureau. Castaman:Alexion: Honoraria; Roche: Consultancy, Honoraria, Speakers Bureau; CSL Behring: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Ablynx: Honoraria; Baxalta/Shire: Honoraria; Bayer: Honoraria; Uniqure: Honoraria, Membership on an entity's Board of Directors or advisory committees; Kedrion: Speakers Bureau; Werfen: Speakers Bureau; Sobi: Honoraria, Research Funding, Speakers Bureau; Novo Nordisk: Honoraria, Speakers Bureau. Key:Novo Nordisk: Other: Chair of Grants Committee; Takeda: Research Funding; Grifols: Research Funding; Uniqure: Consultancy. Miesbach:Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BioMarin Pharmaceutical Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; UniQure: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Recht:Spark: Research Funding; Novo Nordisk: Consultancy, Other: personal fees, Research Funding; uniQure: Consultancy, Other: personal fees, Research Funding; Takeda: Consultancy, Other: personal fees, Research Funding; BioMarin: Research Funding; Pfizer: Consultancy, Other: personal fees; Genentech: Consultancy, Other: personal fees, Research Funding; CSL Behring: Consultancy, Other: personal fees. Gomez:Global Blood Therapeutics: Speakers Bureau. Gut:uniQure: Current Employment. Pipe:Siemens: Research Funding; Medical and Scientific Advisory Council to the National Hemophilia Foundation; Medical Advisory Board to World Federation of Hemophilia: Membership on an entity's Board of Directors or advisory committees; Apcintex, Bayer, BioMarin, Catalyst Biosciences, CSL Behring, HEMA Biologics, Freeline, Novo Nordisk, Pfizer, F. Hoffmann-La Roche Ltd/Genentech, Inc., Sangamo Therapeutics, Sanofi, Takeda, Spark Therapeutics, uniQure: Consultancy. OffLabel Disclosure: Etranacogene dezaparvovec (AMT-061) is an investigational gene therapy for hemophilia B comprising an adeno associated virus serotype 5 (AAV5) vector containing a codon-optimized Padua variant human factor IX (FIX) gene with liver specific promoter.

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Adeno-Associated Virus 8 (AAV8) Vector Genome Biodistribution into Body Fluids Following Single Intravenous Administration of BAX 335 Gene Therapy for Hemophilia B

Blood ◽

10.1182/blood.v126.23.2040.2040 ◽

2015 ◽

Vol 126 (23) ◽

pp. 2040-2040 ◽

Cited By ~ 2

Author(s):

Boyan Litchev ◽

Paul E. Monahan ◽

Anne Prener

Keyword(s):

Gene Therapy ◽

Whole Blood ◽

Research Funding ◽

Body Fluids ◽

Hemophilia B ◽

Severe Hemophilia ◽

Adeno Associated Virus ◽

Vector Genome ◽

Post Infusion

Abstract Introduction: Gene therapy may provide long-term benefit in hemophilia B with a single administration. BAX 335 (AAV8.sc-TTR-FIXR338Lopt) delivers a self-complementary codon-optimized hyperactive F9 transgene (FIXR338Lopt), driven by the liver-specific transthyretin (TTR) promoter in an adeno-associated virus 8 (AAV8) capsid. Shedding is an area of concern with vectors of viral origin and must be evaluated as part of the regulatory application process. Objective: A secondary objective of this ongoing, phase 1/2, open-label study is to determine the duration of BAX 335 genome shedding into blood, saliva, semen, urine, and stool after single-dose administration in subjects with hemophilia B. Results of an unplanned preliminary analysis of data on shedding from this study are presented. Methods: Up to 16 adult men with severe hemophilia B are to be given a single intravenous dose of BAX 335 in up to 4 sequentially ascending dose cohorts: Cohort 1 (2.0x1011 vector genome [vg]/kg), Cohort 2 (1.0x1012 vg/kg), Cohort 3 (3.0x1012 vg/kg), and Cohort 4 (4.0x1012 vg/kg). A quantitative real time polymerase chain reaction (qPCR) is used to detect vector genomic DNA sequences (BAX 335 genomes) in body fluids collected from each subject. Whole blood, saliva, semen, urine, and stool samples are obtained before study drug administration; on day 1; at weeks 1, 2, and 3; and at weekly intervals thereafter until negative (2 consecutive samples below limit of detection). Results: The study is ongoing and preliminary results are available for 7 subjects (S1-S7; Table). All subjects were negative for BAX 335 genomes in all samples at screening. In blood, BAX 335 genomes were detected at the first weekly visit in all subjects, with peak levels achieved at that time. BAX 335 genomes were considered negative in blood between 5 and 9 months after treatment in the 4 subjects (S1-4) for whom follow-up is complete. The remaining 3 subjects (S5-S7) have not yet achieved a negative result in blood (S5) or have not yet reached the month 9 visit (S6 and S7) at the time of this abstract and are still being followed (Table). BAX 335 genomes were also identified at lower levels in saliva (n=7), semen (n=5), stool (n=7), and urine (n=4), with peak values achieved between 1 day and 2 weeks and persisting for 1-5 weeks. The safety profile for all subjects enrolled to date was acceptable, including no inhibitors to factor IX (FIX). Table 1. BAX 335 Genome in Whole Blood and Semen After Single Intravenous Dosing Subject No. First Time Detected* Peak Time Detected* Last Time Detected Time Negative Cohort 1 (2.0x1011 vg/kg) S1 Blood Semen D 1 (2908) ND D 1 (2908) ND M 4 (99) ND M 5 ND S2 Blood Semen D 1 (828513) D 1 (154) D 1 (828513) D 1 (154) M 4 (445) Wk 1 (67) M 5 Wk 2 Cohort 2 (1.0x1012 vg/kg) S3 Blood Semen D 1 (>1x106) D 1 (273) D 1 (>1x106) D 1 (273) M 5 (105) Wk 3 (137) M 6 Wk 4 S4 Blood Semen D 1 (>1x106) D 1 (4771) D 1 (>1x106) Wk 2 (6421) M 6 (93) Wk 3 (753) M 9 Wk 5 S5 Blood Semen D 1 (>1x106) ND D 1 (>1x106) ND M 12 (61)†ND NR†ND Cohort 3 (3.0x1012 vg/kg) S6 Blood Semen D 1 (>1x106) D 1 (2098) D 1 (>1x106) D 1 (2098) M 6 (185)†Wk 2 (751) NR†Wk 3 S7 Blood Semen D 1 (>1x106) D 1 (2236) D 1 (>1x106) Wk 1 (10680) Wk 12 (66815)†Wk 3 (196) NR†Wk 4 D=day; M=month; ND=not detected; NR=not reached; vg=vector genome; Wk=week. *Values in parentheses are the number of copies/10 mL blood or semen. †Subject still being followed. Conclusions: From these preliminary data for the first 7 subjects dosed in the study, a trend towards dose response in terms of both magnitude and duration of AAV shedding into whole blood, saliva, stools, urine, and semen was observed. Shedding has also been reported in other studies of AAV-mediated gene transfer in subjects with severe hemophilia B (Manno CS et al. Nat Med. 2006;12:342-7; Nathwani AC et al. N Engl J Med. 2014;371:1994-2004). The peak values for shedding in our study were observed between day 1 and week 2 in all subjects. Four subjects have completed the follow-up with 6 months post-infusion being the last time a positive sample was detected. Three subjects (S5-S7) are still being followed. S5 has for the past year had sustainable expression levels of FIX at 20% or above and 12 months post-infusion is persistently positive for vector genome in blood (all other fluids negative since week 3). Disclosures Litchev: Baxalta: Employment. Monahan:Pfizer: Honoraria; Baxter/Baxalta: Consultancy, Honoraria, Research Funding; Bayer: Consultancy; Asklepios BioPharmaceutical: Consultancy, Patents & Royalties: Author I.P. licensed by UNC to AskBio, Research Funding; Novo Nordisk: Consultancy, Honoraria, Research Funding; Prolor: Research Funding; Chatham LLC: Consultancy; CSL Behring: Consultancy, Honoraria. Prener:Novo Nordisk: Other: previous employee; Uniqure: Other: previous consultant; Baxalta: Employment.

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Structure-guided evolution of antigenically distinct adeno-associated virus variants for immune evasion

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1704766114 ◽

2017 ◽

Vol 114 (24) ◽

pp. E4812-E4821 ◽

Cited By ~ 62

Author(s):

Longping Victor Tse ◽

Kelli A. Klinc ◽

Victoria J. Madigan ◽

Ruth M. Castellanos Rivera ◽

Lindsey F. Wells ◽

...

Keyword(s):

Gene Therapy ◽

Clinical Trials ◽

Immune Evasion ◽

Neutralizing Antibodies ◽

Rhesus Macaques ◽

Transduction Efficiency ◽

Serum Samples ◽

Antibody Recognition ◽

Serotype 1 ◽

High Degree

Preexisting neutralizing antibodies (NAbs) against adeno-associated viruses (AAVs) pose a major, unresolved challenge that restricts patient enrollment in gene therapy clinical trials using recombinant AAV vectors. Structural studies suggest that despite a high degree of sequence variability, antibody recognition sites or antigenic hotspots on AAVs and other related parvoviruses might be evolutionarily conserved. To test this hypothesis, we developed a structure-guided evolution approach that does not require selective pressure exerted by NAbs. This strategy yielded highly divergent antigenic footprints that do not exist in natural AAV isolates. Specifically, synthetic variants obtained by evolving murine antigenic epitopes on an AAV serotype 1 capsid template can evade NAbs without compromising titer, transduction efficiency, or tissue tropism. One lead AAV variant generated by combining multiple evolved antigenic sites effectively evades polyclonal anti-AAV1 neutralizing sera from immunized mice and rhesus macaques. Furthermore, this variant displays robust immune evasion in nonhuman primate and human serum samples at dilution factors as high as 1:5, currently mandated by several clinical trials. Our results provide evidence that antibody recognition of AAV capsids is conserved across species. This approach can be applied to any AAV strain to evade NAbs in prospective patients for human gene therapy.

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AMT-060 Gene Therapy in Adults with Severe or Moderate-Severe Hemophilia B Confirm Stable FIX Expression and Durable Reductions in Bleeding and Factor IX Consumption for up to 5 Years

Blood ◽

10.1182/blood-2020-139225 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 26-26

Author(s):

Frank W.G. Leebeek ◽

Karina Meijer ◽

Michiel Coppens ◽

Peter Kampmann ◽

Robert Klamroth ◽

...

Keyword(s):

Gene Therapy ◽

Board Of Directors ◽

Research Funding ◽

Factor Ix ◽

Hemophilia B ◽

First Year ◽

Severe Hemophilia ◽

Advisory Committees ◽

The Mean

Background: Gene therapy aims to provide long-term therapeutic benefit from a single administration. AMT-060 is an adeno-associated virus serotype 5 (AAV5) vector with a codon-optimized wildtype human factor IX (FIX) gene and liver-specific promoter. AMT-060 is being evaluated in an ongoing study of 10 participants with severe/moderate-severe hemophilia B (Phase 1/2 study, NCT02396342) over 5 years. Aim: To describe efficacy and safety outcomes from an analysis at up to 5-years post-AMT-060. Methods: Adult males with FIX activity ≤2% and a severe bleeding phenotype received a single intravenous infusion of AMT-060 (5x1012 gc/kg, Cohort 1, n=5) or (2×1013 gc/kg, Cohort 2, n=5). Assessments included FIX activity, FIX replacement use, annualized bleeding rate (ABR), treatment-related adverse events (TRAE), immunological and inflammatory biomarkers up to 5 years (Cohort 1) and 4.5 years (Cohort 2). Results: As of November 2019, for Cohort 1 the mean FIX activity (at 4.0 years) was 5.1% as compared to 4.4% in the first year, 6.8% in the second year, 7.3% in the third year and 7.0% in the fourth year. Mean FIX activity for Cohort 2 was 7.5% as compared to 7.1% in the first year, 8.4% in the second year 7.9% in the third year, and 7.4% in the fourth year. Eight of 9 participants using prophylaxis at baseline were able to discontinue use. During the last 12, and 6 months of observation respectively, the mean annualized bleed rate (ABR) was 3.3. for Cohort 1 and 0.0 for Cohort 2. These represent, respectively, a reduction in mean ABR to the year prior to treatment of 77% and 100% for Cohort 1 and Cohort 2. During this same period the consumption of FIX replacement therapy declined 90% and 100% relative to pre-treatment, respectively for Cohort 1 and Cohort 2. No participants developed FIX inhibitors or signs of sustained AAV5 capsid-specific T-cell activation. As previously reported, TRAE were mainly reported in the first 3.5 months after treatment, including three participants who experienced transient mild elevations in alanine aminotransferase. One additional TRAE (joint swelling post-exercise) was observed during the last 12 months of observation post-treatment. Updated data, up to 5-years of observation, will be presented for the first time. Conclusions: Long-term stable endogenous FIX activity and reductions in ABR and FIX replacement use were sustained over multiple years following a single treatment with AMT-060. There were no additional safety concerns with longer term follow-up. This data supports the ongoing Phase 3 study of the enhanced construct etranacogene dezaparvovec (AMT-061), which encodes the highly active Padua FIX variant. Disclosures Meijer: Bayer: Research Funding; Sanquin: Research Funding; Pfizer: Research Funding; Bayer: Speakers Bureau; Sanquin: Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; BMS: Speakers Bureau; Aspen: Speakers Bureau; Uniqure: Consultancy. Kampmann:Uniqure: Speakers Bureau; Shire Pharmaceuticals: Speakers Bureau. Klamroth:CSL Behring: Research Funding, Speakers Bureau; Novo Nordisk: Consultancy, Research Funding, Speakers Bureau; Octapharma: Consultancy, Research Funding, Speakers Bureau; Pfizer: Consultancy, Research Funding, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Takeda/Shire: Consultancy, Research Funding, Speakers Bureau; Sobi: Consultancy, Speakers Bureau; Biotest: Speakers Bureau; Grifols: Speakers Bureau; Biomarin: Consultancy, Research Funding, Speakers Bureau; Bayer: Consultancy, Research Funding, Speakers Bureau. Castaman:Novo Nordisk: Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Speakers Bureau; Pfizer: Honoraria, Research Funding; Ablynx: Honoraria; Alexion: Honoraria; Bayer: Honoraria; CSL Behring: Honoraria, Research Funding; Kedrion: Speakers Bureau; Sobi: Honoraria, Research Funding, Speakers Bureau; Uniqure: Honoraria, Membership on an entity's Board of Directors or advisory committees; Werfen: Speakers Bureau; Baxalta/Shire: Honoraria. Bönig:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees; Genzyme: Consultancy, Membership on an entity's Board of Directors or advisory committees; Healthineers: Current equity holder in publicly-traded company; Sandor-Hexal: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Polyphor: Research Funding; Miltenyi: Honoraria, Research Funding; Erydel: Research Funding; Chugai: Honoraria, Research Funding; Bayer: Research Funding; Terumo BCT: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kiadis: Honoraria; Uniqure: Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Stage: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Fresenius: Honoraria; medac: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Sawyer:uniQure: Current Employment. Miesbach:UniQure: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BioMarin Pharmaceutical Inc: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. OffLabel Disclosure: AMT-060 = AAV5 vector gene therapy in subjects with moderate to severe hemophilia B

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One Year Data from a Phase 2b Trial of AMT-061 (AAV5-Padua hFIX variant), an Enhanced Vector for Gene Transfer in Adults with Severe or Moderate-Severe Hemophilia B

Blood ◽

10.1182/blood-2019-128765 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3348-3348

Author(s):

Steven Pipe ◽

Adam Giermasz ◽

Giancarlo Castaman ◽

Nigel S. Key ◽

Susan U Lattimore ◽

...

Keyword(s):

Gene Therapy ◽

Board Of Directors ◽

Research Funding ◽

Hemophilia B ◽

Severe Hemophilia ◽

Advisory Committees ◽

Human Factor Ix ◽

Virus Serotype ◽

Patient Reported

Background: Gene therapy for hemophilia offers the possibility of ameliorating the disease severity to a milder or functionally curative state through a single treatment. AMT-061 is an investigational gene therapy for hemophilia B comprising an adeno-associated virus serotype 5 (AAV5) vector containing a codon-optimized Padua variant human factor IX (FIX) gene with liver-specific promoter. Aims: Confirm that a single dose of AMT-061 will provide a minimum-therapeutic response of FIX activity 6-weeks post-dose in participants with severe or moderate-severe hemophilia B. Here, 1 year of follow-up will be presented for the first time. Methods: Phase 2b, open-label, multi-center trial (NCT03489291) in adult males with FIX ≤2% and without active hepatitis or uncontrolled HIV. Participants were not excluded based on neutralizing antibodies to AAV5. Participants received a single intravenous dose of AMT-061 (2x1013 gc/kg) and will be followed for 5-years. The primary endpoint was FIX activity at Week 6. Secondary endpoints include e-diary recordings of bleeds and FIX concentrate use, laboratory parameters, joint health, patient-reported outcomes, and adverse events (AEs). Results: All participants had FIX ≤1% (severe or moderately-severe FIX deficiency), required routine FIX prophylaxis, and had neutralizing activity to AAV5 at baseline. Following AMT-061 treatment, FIX activity increased rapidly (Figure) to a mean of 31% at Week 6. At Week 36, mean FIX activity increased further to 45% with FIX activity levels of 54%, 30% and 51% in participants 1-3 respectively. As of 36 weeks, there were no bleeds post-treatment and no requirement for FIX replacement aside from protocol-specified use for perioperative management in participant 3. There were no clinically significant elevations in liver enzymes and no participants required steroids related to the treatment. One participant experienced 2 mild AEs possibly related to treatment shortly after dosing (self-limiting headache and slightly elevated CRP). One patient had hip surgery due to worsening of pre-existing avascular necrosis deemed unrelated by investigator to AMT-061 and received FIX per protocol according to standard clinical practice. No participant developed inhibitors to FIX. Updated results to 52 weeks of follow-up will be presented. Conclusions: Sustained elevation of FIX activity into the mild-to-normative range were observed in all participants 36 weeks after treatment with AMT-061. AMT-061 was safe and well-tolerated with no requirement for immunosuppression. These data support the ongoing Phase 3 study. Figure Disclosures Pipe: Novo Nordisk: Consultancy; Apcintex: Consultancy; Roche/Genentech: Consultancy; BioMarin: Consultancy; Shire: Consultancy; Sanofi: Consultancy; uniQure: Consultancy; Pfizer: Consultancy; HEMA Biologics: Consultancy; Catalyst Bioscience: Consultancy; Freeline: Consultancy; CSL Behring: Consultancy; Bayer: Consultancy; Spark Therapeutics: Consultancy. Giermasz:Genentech/Roche: Consultancy, Other: Research, Speakers Bureau; Sangamo: Other: Research; BioMarin: Consultancy, Other: Research; uniQure: Consultancy, Other: Research; Bioverativ/Sanofi: Consultancy, Speakers Bureau. Castaman:Roche: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Kedrion: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Sanofi: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Takeda (SHIRE): Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Uniqure: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Werfen: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bayer: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Research Funding; Sobi: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Key:Uniqure BV: Research Funding. Leebeek:CSL Behring: Research Funding; Baxalta/Shire: Research Funding; uniQure BV: Consultancy, Research Funding. Miesbach:Bayer, Chugai, Novo Nordisk, Octapharma, Pfizer, Takeda/Shire, UniQure: Speakers Bureau; Bayer, BioMarin, CSL Behring, Chugai, Freeline, Novo Nordisk, Octapharma, Pfizer, Roche, Takeda/Shire, UniQure: Consultancy; Bayer, Novo Nordisk, Octapharma, Pfizer, Takeda/Shire: Research Funding. Recht:Bioverativ, CSL Behring, Genentech, Kedrion, NovoNordisk, Pfizer, Shire, Uniqure: Consultancy; American Thorombosis & Hemostasis Network: Other: Immediate Past Chair; Bioverativ, Genentech, NovoNordisk Shire: Research Funding. Gomez:Alnylam: Consultancy; Novo Nordisk, Novartis, Pfizer, Sanofi, Takeda, UniQure: Research Funding. Long:Uniqure BV: Employment. Gut:Uniqure BV: Employment. von Drygalski:University of California San Diego: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties; UniQure, Bayer, Bioverativ/Sanofi, Pfizer, Novo Nordisk, Biomarin, Shire, CSL Behring: Consultancy; Hematherix Inc.: Membership on an entity's Board of Directors or advisory committees, Other: Founder.

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Factor IX Expression within the Normal Range Prevents Spontaneous Bleeds Requiring Treatment Following FLT180a Gene Therapy in Patients with Severe Hemophilia B: Long-Term Follow-up Study of the B-Amaze Program

Blood ◽

10.1182/blood-2021-146546 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 3967-3967

Author(s):

Pratima Chowdary ◽

Susan Shapiro ◽

Mike Makris ◽

Gillian Evans ◽

Sara Boyce ◽

...

Keyword(s):

Gene Therapy ◽

Research Funding ◽

Transgene Expression ◽

Factor Ix ◽

Hemophilia B ◽

Normal Range ◽

Follow Up Study ◽

Long Term Follow Up

Abstract Introduction: FLT180a (verbrinacogene setparvovec) is an investigational, liver-directed AAV gene therapy for the treatment of patients with hemophilia B (HB). FLT180a consists of a novel, potent, engineered capsid (AAVS3) containing an expression cassette encoding a Factor IX (FIX) gain-of-function protein variant ('Padua'; FIX-R338L). The B-AMAZE study was designed to identify a dose of FLT180a that maintains FIX activity within the normal range (50-150%) and thereby protect patients with severe HB from spontaneous and traumatic bleeds. Methods: B-AMAZE was a multicentre, open-label Phase 1/2 clinical trial (NCT03369444; sponsored by UCL) that evaluated FLT180a dose levels using an escalating/descending adaptive design in patients with severe (FIX activity <1%) or moderately severe (FIX activity 1-2%) HB who were negative for AAVS3 neutralizing antibodies. A novel regimen of prophylactic corticosteroids with/without tacrolimus was implemented to mitigate the impact of vector-related transaminitis on FIX expression. Patients who completed the 26-week B-AMAZE study were eligible for the ongoing long-term follow-up study (NCT03641703; sponsored by Freeline). Results: Ten HB patients received a single dose of FLT180a. Four FLT180a doses ranging from 3.84e11 vg/kg to 1.28e12 vg/kg were assessed. As of the data cut-off date, all patients have been followed for ≥16 months. FLT180a demonstrated a favorable safety profile, without evidence of inhibitors against FIX, infusion-related or allergic reactions. The most common treatment-related adverse event was transient elevation in alanine aminotransferase. An event of AV fistula thrombosis occurred in a 67-year-old patient who received the highest dose of 1.28e12 vg/kg (total dose of 1.15e14 vg) and had supranormal FIX levels; this patient was treated with anticoagulants. While these FIX levels demonstrate the potency of our proprietary AAVS3 capsid, this dose will not be used in future hemophilia studies. At Week 26 after FLT180a administration, a dose-response relationship was observed with mean FIX activity of 45.0%, 35.5%, 141.5%, and 175.5% for 3.84e11, 6.4e11, 8.32e11, and 1.28e12 vg/kg doses, respectively (Table); FIX activity levels ≥50% were achieved in 7 of 8 patients treated with the three highest doses. One patient (Patient 4) who received 6.4e11 vg/kg lost transgene expression early due to transaminitis and resumed routine factor prophylaxis. The 8.32e11 vg/kg cohort received an extended immune management regimen (9-18 weeks) with prophylactic tacrolimus in addition to prednisolone to prevent breakthrough vector-related transaminitis. However, after cessation of the immune management regimen, transaminitis with concomitant reductions in FIX activity was observed in all patients in the 8.32e11 vg/kg cohort. The combination of prophylactic tacrolimus and prednisolone appeared to have suppressed immune-mediated transaminitis while administered, but recurrence of transaminitis developed soon after cessation. This unique and previously unreported observation suggests that the longer-duration prophylactic immune management regimen may have prevented tolerization to the vector because this was not observed in earlier cohorts where a brief course of tacrolimus was given reactively for breakthrough transaminitis. All patients (including the 8.32e11 vg/kg cohort) have achieved steady state. Patients in the earliest cohort who received the lowest dose (3.84e11 vg/kg) have shown stable FIX activity for >3 years. There were no spontaneous bleeds that required FIX supplementation in patients who maintained FIX activity above 50%; Patient 4 in the 6.4e11 vg/kg cohort experienced two bleeds (cause unknown) after he lost transgene expression, which were treated with exogenous FIX. One patient received exogenous FIX for treatment of a traumatic bleed, but his FIX activity level was 57% at the time of the event. Additional efficacy and safety results with >3.5 years of follow-up will be presented. Conclusions: B-AMAZE is the first HB gene therapy study to achieve normal levels of FIX activity using relatively low vector doses. Results suggest that a dose of 7.7e11 vg/kg, coupled with a short course of prophylactic immune management, has the potential to achieve durable FIX activity in the normal range (50-150%) and thereby prevent spontaneous bleeds and normalize hemostasis in the event of traumatic bleeds. Figure 1 Figure 1. Disclosures Chowdary: Sanofi: Honoraria; Roche: Honoraria; CSL Behring: Honoraria, Research Funding; Freeline: Honoraria, Research Funding; Novo Nordisk: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; SOBI: Honoraria, Research Funding; Takeda: Honoraria, Research Funding; Boehringer Ingelheim: Honoraria; Chugai: Honoraria; Spark: Honoraria; Bayer: Honoraria, Research Funding. Shapiro: Roche: Honoraria; CSL Bering: Honoraria; Takeda: Honoraria, Speakers Bureau; Pfizer: Consultancy, Speakers Bureau. Makris: Freeline: Consultancy. Dolan: Takeda: Speakers Bureau; Roche-Chugai: Speakers Bureau; Spark Therapeutics: Speakers Bureau; Octapharma: Speakers Bureau; CSL: Speakers Bureau; Biomarin: Speakers Bureau; Bayer: Research Funding, Speakers Bureau; Novo Nordisk: Research Funding, Speakers Bureau; Pfizer: Research Funding. Tuddenham: Freeline: Consultancy, Current holder of individual stocks in a privately-held company. Long: Freeline: Current Employment. Krop: Freeline: Current Employment. Nathwani: Freeline: Current holder of individual stocks in a privately-held company, Other: Board of directors.

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Strategies for Selection of AAV Vectors for Administration to Liver: Studies in Nonhuman Primates

Blood ◽

10.1182/blood.v128.22.2316.2316 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2316-2316 ◽

Cited By ~ 1

Author(s):

Lili Wang ◽

James M Wilson ◽

Roberto Calcedo ◽

Peter Bell ◽

Zhenning He ◽

...

Keyword(s):

Gene Therapy ◽

Clinical Trials ◽

Gene Transfer ◽

Board Of Directors ◽

Research Funding ◽

Nonhuman Primates ◽

Hemophilia B ◽

Advisory Committees ◽

Equity Ownership ◽

Liver Gene

Abstract Vectors based on adeno-associated virus (AAV) have demonstrated early promise in clinical trials, including published reports in patients with hemophilia B where therapeutic levels of factor IX have been achieved using AAV serotype 8, a member of the clade E family. With the success of these hemophilia B clinical trials utilizing AAV8, numerous alternative AAV capsid types that target the liver have begun to enter clinical testing across multiple disease indications. Specific properties of the various AAV capsids should be taken into account such as overall efficiency of hepatocyte gene transfer, differential gene transfer across the porto-central axis within the liver, durability of gene expression and the potential for vector re-administration. In this study, we evaluated these aspects of AAV-directed liver gene transfer in rhesus macaques across various capsid serotypes. Animals were injected with vectors expressing the secreted reporter gene rhesus bhCG and produced using different capsids [AAV5, AAV3b and two clade E vectors (AAVrh10 and AAV8)]. Nonhuman primates (NHPs) injected with clade E vectors expressing bhCG were administered 3 months later with AAV5 or AAV3b vectors expressing rhesus derived AFP. The key findings were: 1) in naïve animals, clade E vectors demonstrated the highest levels of periportal gene transfer with AAV5 vectors having the lowest levels of periportal gene transfer; 2) AAVrh10 and AAV5 elicited higher levels of neutralizing antibodies (NAb) than AAV8 and AAV3b; 3) significant animal-to-animal variation in transgene expression was noted with AAV3b in seronegative animals; and 4) within the short time frame tested NAb elicited from AAVrh10 appears to have inhibited subsequent in vivo transduction with the serologically distinct AAV3b serotype; prior exposure to AAV8 did not interfere with AAV3b transduction. These studies highlight the influence that capsids can play in efficiency and immunogenicity of AAV vectors for liver gene therapy. Disclosures Wilson: Dimension Therapeutics: Consultancy, Equity Ownership, Patents & Royalties, Research Funding; Solid Gene Therapy: Consultancy, Membership on an entity's Board of Directors or advisory committees; REGENXBIO: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding. Kattenhorn:Dimension Therapeutics: Employment. Wadsworth:Dimension Therapeutics: Employment, Equity Ownership.

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Extravascular Administration of Factor IX: Potential for Replacement Therapy of Canine and Human Hemophilia B

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656082 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0944-0948 ◽

Cited By ~ 20

Author(s):

Darla Liles ◽

Charles N Landen ◽

Dougald M Monroe ◽

Celeste M Lindley ◽

Marjorie s Read ◽

...

Keyword(s):

Gene Therapy ◽

Vitamin K ◽

Absorption Rate ◽

Venous Access ◽

Factor Ix ◽

Hemophilia B ◽

Current Therapy ◽

Home Therapy ◽

Routes Of Administration ◽

Plasma Compartment

SummaryCurrent therapy for hemophilia B requires large intravenous doses of factor IX (F.IX) given in the clinic or at home. Although home therapy is possible for many patients, it is often complicated by factors such as the lack of good venous access. Very little is known about extravascular routes for administering proteins like F.IX (57 kD) or other vitamin K-dependent procoagulant factors into the circulation. Questions about the absorption rate from extravascular administration as well as plasma recovery and bioavailability have arisen recently with the growing availibility of highly purified procoagulant proteins and increased interest in gene therapy of hemophilia B. Therefore, a group of studies were undertaken to determine the absorption rate, plasma recovery, and bioavailability of high purity, human plasma-derived F.IX concentrates administered via extravascular routes in hemophilia B dogs and in one human hemophilia B subject. Five hemophilia B dogs were given human F.IX via either a subcutaneous (SC), intramuscular (IM), intra- peritoneal (IP) or intravenous (IV) route. In a subsequent study, a single SC administration of human F.IX was compared to an identical IV dose of F.IX in the human hemophilia B subject. All extravascular routes of F.IX administration in both the canine and human gave lower levels of circulating plasma F.IX than the IV route, however all routes resulted in measurable F.IX activity. Of the extravascular routes, the IM injection in the canine resulted in a bioavailibility of 82.8%, while the SC injection resulted in a bioavailability of 63.5%. F.IX reached the plasma compartment by all extravascular routes used, confirming that F.IX can be absorbed extravascularly. The duration of measurable F.IX activity following extravascular administration is prolonged beyond that typically seen with IV administration. These data show that significant levels of F.IX may be obtained via SC injection in canine and ‘ human hemophilia B subjects and further highlight the potential of extravascular routes of administration for future experimental and clinical uses of F.IX and other procoagulant proteins.

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