Faculty Opinions recommendation of Hemophilia B Gene Therapy with a High-Specific-Activity Factor IX Variant.

2017 ◽  
Author(s):  
Bruno Stieger
Keyword(s):  
Gene Therapy ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Ix ◽  
Hemophilia B ◽  
Activity Factor

scholarly journals Hemophilia B Gene Therapy with a High-Specific-Activity Factor IX Variant

2017 ◽  
Vol 377 (23) ◽  
pp. 2215-2227 ◽  
Author(s):  
Lindsey A. George ◽  
Spencer K. Sullivan ◽  
Adam Giermasz ◽  
John E.J. Rasko ◽  
Benjamin J. Samelson-Jones ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Ix ◽  
Hemophilia B ◽  
Activity Factor

scholarly journals Amelioration of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration

2021 ◽  
Author(s):  
Xi Chen ◽  
Xuran Niu ◽  
Yang Liu ◽  
Rui Zheng ◽  
Liren Wang ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Ix ◽  
Hemophilia B ◽  
Hereditary Diseases ◽  
Proliferating Cells ◽  
Promising Therapeutic Approach ◽  
Targeted Integration

Site-specific integration of exogenous gene through genome editing is a promising strategy for gene therapy. However, homology-directed repair (HDR) only occurring in proliferating cells is inefficient especially in vivo. To investigate the efficacy of Cas9-induced homology-independent targeted integration (HITI) strategy for gene therapy, a rat hemophilia B model was generated and employed. Through HITI, a DNA sequence encoding the last exon of rat Albumin (rAlb) gene fused with a high-specific-activity Factor IX variant (R338L) using T2A, was inserted into the last intron of rAlb via recombinant adeno-associated viral (rAAV). The knock-in efficiency reached up to 3.66% determined by ddPCR. The clotting time was reduced to normal level 4 weeks after treatment, and the circulating FIX level was gradually increased up to 52% of normal during 9 months even after partial hepatectomy, demonstrating the amelioration of hemophilia. Through PEM-seq, no significant off-targeting effect was detected. Moreover, this study provides a promising therapeutic approach for hereditary diseases.

scholarly journals Evolutionary insights into coagulation factor IX Padua and other high-specific-activity variants

2021 ◽  
Vol 5 (5) ◽  
pp. 1324-1332
Author(s):  
Benjamin J. Samelson-Jones ◽  
Jonathan D. Finn ◽  
Leslie J. Raffini ◽  
Elizabeth P. Merricks ◽  
Rodney M. Camire ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Amino Acid ◽  
Specific Activity ◽  
High Specific Activity ◽  
Coagulation Factor ◽  
Factor Ix ◽  
Activity Factor ◽  
New Variant ◽  
Active Variant

Abstract The high-specific-activity factor IX (FIX) variant Padua (R338L) is the most promising transgene for hemophilia B (HB) gene therapy. Although R338 is strongly conserved in mammalian evolution, amino acid substitutions at this position are underrepresented in HB databases. We therefore undertook a complete 20 amino acid scan and determined the specific activity of human (h) and canine (c) FIX variants with every amino acid substituted at position 338. Notably, we observe that hFIX-R338L is the most active variant and cFIX-R338L is sevenfold higher than wild-type (WT) cFIX. This is consistent with the previous identification of hFIX-R338L as a cause of a rare X-linked thrombophilia risk factor. Moreover, WT hFIX and cFIX are some of the least active variants. We confirmed the increased specific activity relative to FIX-WT in vivo of a new variant, cFIX-R338I, after gene therapy in an HB dog. Last, we screened 232 pediatric subjects with thromboembolic disease without identifying F9 R338 variants. Together these observations suggest a surprising evolutionary pressure to limit FIX activity with WT FIX rather than maximize FIX activity.

scholarly journals Amelioration of hemophilia B through CRISPR/Cas9 induced homology-independent targeted integration

2021 ◽  
Author(s):  
Dali Li ◽  
Xi Chen ◽  
Xuran Niu ◽  
Yang Liu ◽  
Rui Zheng ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Ix ◽  
Hemophilia B ◽  
Hereditary Diseases ◽  
Proliferating Cells ◽  
Promising Therapeutic Approach ◽  
Targeted Integration

Abstract Site-specific integration of exogenous gene through genome editing is a promising strategy for gene therapy. However, homology-directed repair (HDR) only occurring in proliferating cells is inefficient especially in vivo. To investigate the efficacy of Cas9-induced homology-independent targeted integration (HITI) strategy for gene therapy, a rat hemophilia B model was generated and employed. Through HITI, a DNA sequence encoding the last exon of rat Albumin (rAlb) gene fused with a high-specific-activity Factor IX variant (R338L) using T2A, was inserted into the last intron of rAlb via recombinant adeno-associated viral (rAAV). The knock-in efficiency reached up to 3.66% determined by ddPCR. The clotting time was reduced to normal level 4 weeks after treatment, and the circulating FIX level was gradually increased up to 52% of normal during 9 months even after partial hepatectomy, demonstrating the amelioration of hemophilia. Through PEM-seq, no significant off-targeting effect was detected. Moreover, this study provides a promising therapeutic approach for hereditary diseases.

scholarly journals Factor IX Padua: From Biochemistry to Gene Therapy

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. SCI-9-SCI-9
Author(s):  
Valder Arruda ◽  
Ben J. Samelson-Jones
Keyword(s):  
Gene Therapy ◽  
Clinical Trials ◽  
Amino Acid ◽  
Early Phase ◽  
Specific Activity ◽  
Factor Ix ◽  
Hemophilia B ◽  
Amino Acid Substitutions ◽  
Wild Type ◽  
Active Variant

Abstract Novel approaches to enhance the biologic activity of therapeutic proteins have the potential to improve protein- and gene-based therapy for hemophilia. We have identified the variant Factor IX Padua (FIX) (R338L) with 8-fold increase in specific activity compared to wild-type FIX as well as additional strategies to identify other modifications with a positive effect on the biological activity of FIX. FIX-Padua is already in early phase gene therapy clinical trials for hemophilia B. However, it is notable that the field is moving forward even though the molecular basis for its enhanced function has remained elusive. The increased specific activity of FIX Padua compared to FIX wild-type resides in the activated protease as purified recombinant FIX Padua displays enhanced clotting activity as both a zymogen and activated protease. This augmentation of FIX Padua zymogen and protease is observed in both clotting and thrombin generation assays. However, preliminary biochemical characterization suggests that that the increased activity is most pronounced in plasma-based assays, while differences in enzyme kinetic parameters measured in reconstituted systems are more modest. Intriguingly, we have found that most amino acid substitutions at position 338, result in a FIX variant with comparable or enhanced clotting activity with the Padua substitution resulting in the most active variant, suggesting that R338 in FIX wild-type forms an unfavorable interaction that can be relieved by most amino acid substitutions. The wild-type variant is actually the least active variant at this position that is not known to cause hemophilia B. Since, R338 is strictly conserved among mammalian FIX orthologues, there may be an evolutionary pressure to maintain the unfavorable interactions of R338 and limit FIX activity. The corollary to this speculation is that other FIX mutations that relieve deleterious interactions will also increase clotting activity. The characterization of FIX Padua suggests small biochemical improvements may result in substantial increases in plasma based clotting activity. Promising preclinical studies on efficacy and safety, including thrombogenicity and immunogenicity, in small and large animal models provide the basis for translational studies using these proteins. These studies support the concept that the thrombotic risk of FIX Padua activity is similar to FIX wild-type activity. The immunogenicity of FIX Padua is comparable to FIX wild-type in either an adeno-associated virus-based muscle- or liver-directed gene therapy in canine models of hemophilia B. In the last 18 months, results from first 10 men with severe hemophilia B enrolled in two ongoing AAV liver-directed gene therapy clinical trials using a FIX Padua as a transgene were reported. No subject in either study developed inhibitors to FIX Padua or thrombotic complications. In subjects with sustained FIX Padua expression, FIX activity was greater than 10%. These promising early phase results demonstrate the potential of utilizing variants with increased specific activity in gene therapy allowing for lower therapeutic vector doses. It remains to be seen if curative factor levels can be safely achieved with further vector refinements including improved FIX variants. Disclosures Arruda: Pfizer: Research Funding.

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

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3124-3124 ◽  
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.

scholarly journals The efficacy and the risk of immunogenicity of FIX Padua (R338L) in hemophilia B dogs treated by AAV muscle gene therapy

Blood ◽  
2012 ◽  
Vol 120 (23) ◽  
pp. 4521-4523 ◽  
Author(s):  
Jonathan D. Finn ◽  
Timothy C. Nichols ◽  
Nikolaos Svoronos ◽  
Elizabeth P. Merricks ◽  
Dwight A. Bellenger ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Antibody Formation ◽  
Specific Activity ◽  
Factor Ix ◽  
Hemophilia B ◽  
Wild Type ◽  
Cell Responses ◽  
Thrombotic Complications ◽  
Bleeding Episodes ◽  
Muscle Gene

Abstract Studies on gene therapy for hemophilia B (HB) using adeno-associated viral (AAV) vectors showed that the safety of a given strategy is directly related to the vector dose. To overcome this limitation, we sought to test the efficacy and the risk of immunogenicity of a novel factor IX (FIX) R338L associated with ∼ 8-fold increased specific activity. Muscle-directed expression of canine FIX-R338L by AAV vectors was carried out in HB dogs. Therapeutic levels of circulating canine FIX activity (3.5%-8%) showed 8- to 9-fold increased specific activity, similar to humans with FIX-R338L. Phenotypic improvement was documented by the lack of bleeding episodes for a cumulative 5-year observation. No antibody formation and T-cell responses to FIX-R338L were observed, even on challenges with FIX wild-type protein. Moreover, no adverse vascular thrombotic complications were noted. Thus, FIX-R338L provides an attractive strategy to safely enhance the efficacy of gene therapy for HB.

scholarly journals Activity of a FIX-Padua Transgene Product in Commonly Used FIX:C One-Stage and Chromogenic Assay Systems Following PF-06838435 (SPK-9001) Gene Delivery

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2198-2198 ◽  
Author(s):  
Mary Robinson ◽  
Lindsey A. George ◽  
Benjamin J. Samelson-Jones ◽  
Valder R Arruda ◽  
Katherine A. High ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Specific Activity ◽  
Factor Ix ◽  
Hemophilia B ◽  
Employment Equity ◽  
Siemens Healthcare ◽  
Buffer Solutions ◽  
One Stage ◽  
Chromogenic Assay ◽  
Equity Ownership

Abstract Background PF-06838435 (SPK-9001), a gene therapy candidate containing a high specific-activity factor IX variant (R338L, FIX-Padua), is currently in phase 3 of clinical development for the treatment of Hemophilia B. Initial data with this vector is promising with significant reductions in bleeding episodes and FIX consumption (George LA et al, NEJM 2017; 377:2215-2217). To date, little is known about the activity of the expressed transgene product as measured in FIX:C one-stage and chromogenic assay systems commonly used to monitor FIX replacement therapy in patients with Hemophilia B. Aim The goal of the study was to assess the activity of the PF-06838435 expressed transgene product in plasma samples collected from participants in the Phase 1/2 trial using four commonly used FIX:C aPTT reagents. For comparison, the activity of the expressed FIX-Padua gene product was also assessed in the ROX FACTOR IX chromogenic assay. In addition, the activity of the Padua variant in congenital FIX deficient plasma spiked with increasing concentrations of purified recombinant human FIX Padua protein (rHFIXp - Samelson-Jones Lab, CHOP/UPenn), as well as recombinant human FIX (rHFIX, BeneFIX®, Pfizer Inc.), was assessed in the same FIX:C assay procedures. Methods FIX:C, in four samples collected from two different patients who received FIX gene therapy, was tested in four in vitro diagnostic (IVD) approved FIX:C one-stage assay systems, STA®-PTT Automate and STA®-C.K. Prest® on the STA-R Evolution® (Diagnostica Stago Inc.), Dade Actin® FSL on the BCS®XP (Siemens Healthcare), and HemosIL® SynthASil® on the ACL TOP® (Instrumentation Laboratory). In addition, samples were also tested in the ROX FACTOR IX (Rossix AB) chromogenic assay on the BCS®XP (Siemens Healthcare). The aPTT reagents selected for this study correspond to 90% of the FIX:C assay reagents currently used in CAP accredited laboratories in the US(2014 CAP Proficiency Survey) and represent the three main types of activator commonly used in the FIX one-stage clot assay: silica (STA®-PTT Automate, HemosIL® SynthASil®), ellagic acid (Dade Actin® FSL) and kaolin (STA®-C.K. Prest®). For comparison, FIX:C in each of the five FIX:C assay systems was also determined in samples spiked with purified rHFIXp (provided by Dr. Samelson-Jones) or rHFIX (provided by Spark Therapeutics Inc.) in 20X buffer solutions. On the day of testing rHFIXp, and rHFIX 20X buffer solutions were diluted 1:20 in congenital FIX deficient plasma to achieve approximate final FIX:C concentrations of 40, 30 and 20%, extrapolated from an estimated 8-fold specific activity to antigen ratio. Results A consistent pattern in the measured FIX:C for the PF-06838435 transgene product was observed in the five FIX:C assay systems (Fig. 1). For the aPTT based FIX:C assays, Actin FSL, gave the lowest FIX:C values, whereas PTT Automate measured the highest FIX:C levels. In all cases, the chromogenic FIX:C assay gave the lowest activity values for the transgene product. A similar FIX:C assay dependent pattern was observed for the purified rHFIXp spiked at 40, 30 and 20% FIX:C (Fig. 2). In all FIX:C assays tested, rHFIXp was under-recovered to a varying degree from target, with recoveries for the 20% FIX:C samples ranging between -26.5% (STA®-PTT Automate) and -73.5% (Dade Actin® FSL) in the aPTT based FIX:C assays and -85% in the ROX FIX chromogenic assay. In contrast, rHFIX (BeneFIX®) was recovered within ±25% of expected values in all aPTT based FIX:C assays and, consistent with previously reported data, modestly under-recovered in the ROX FIX chromogenic assay (Fig. 3). Conclusion This study found differences in the FIX:C results obtained for a Padua FIX variant transgene product and recombinant human FIX-Padua when tested in commonly used IVD approved FIX:C assays in North America. These results suggest that FIX:C assay selection is important for measuring FIX-Padua activity, which will be particularly relevant in hemophilia B gene therapy following FIX-Padua gene transfer. Disclosures George: University of Pennsylvania: Equity Ownership; Pfizer: Consultancy. High:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. Carr:Sparks Therapeutics Inc.: Consultancy. Tiefenbacher:Laboratory Corporation of America: Employment, Equity Ownership; Siemens Healthcare: Consultancy.

Recombinant Adeno-Associated Viral Vectors Expressing Human Coagulation FIX-E456H Variant in Hemophilia B Mice

2019 ◽  
Vol 119 (12) ◽  
pp. 1956-1967
Author(s):  
Sandra Le Quellec ◽  
Allison P. Dane ◽  
Elena Barbon ◽  
Jean-Claude Bordet ◽  
Federico Mingozzi ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Liver Toxicity ◽  
Specific Activity ◽  
Factor Ix ◽  
Activity Level ◽  
Hemophilia B ◽  
Suitable Alternative ◽  
Chromogenic Assay ◽  
Human Factor Ix

AbstractGene therapy using recombinant adeno-associated virus (AAV) has induced sustained long-term coagulation human factor IX (hFIX) levels in hemophilia B (HB) patients. However, asymptomatic transient liver toxicity was observed at high vector doses, highlighting the need to improve the potency of these vectors. We report the generation of an AAV transgene cassette containing the hyperfunctional hFIX-E456H variant showing improved binding to platelets, with a comparison to wild-type hFIX (hFIX-WT) and hFIX-R384L variant (Padua) transgenes, containing F9 truncated-intron 1 (I1). In vitro specific activity was increased by 3.2- and 4.2-fold with hFIX-E456H and hFIX-R384L variants compared with hFIX-WT, using chromogenic assay, and by 7-and 8.6-fold with hFIX-E456H and hFIX-R384L variants compared with hFIX-WT, using one-stage assay. The transgenes were packaged into single-stranded AAV2/8 vectors that were tail vein injected at 5 × 109, 2 × 1010, and 5 × 1010 vg per mouse in HB mice. Plasma FIX activity level, assessed by chromogenic assay, was up to fourfold higher for hFIX-E456H compared with hFIX-WT and was not different compared with hFIX-R384L, among the three dose cohorts. Overall, the in vivo specific activity was increased by threefold for hFIX-E456H and 4.9-fold for hFIX-R384L compared with hFIX-WT. At the lower dose of 5 × 109 vg, the blood loss was significantly lower for hFIX-E456H compared with hFIX-WT, but did not differ compared with hFIX-R384L. The results found for the hFIX-E456H variant indicate that it might be a suitable alternative for gene therapy of HB.

scholarly journals Pharmacokinetics of Subcutaneously Administered CB2679d/ISU304 in Wild-Type and Hemophilia B Mice

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1389-1389 ◽  
Author(s):  
Seung-Beom Hong ◽  
Howard Levy ◽  
Jae Yong Jung ◽  
Minkyung Park ◽  
A Rim Seo ◽  
...  
Keyword(s):  
Specific Activity ◽  
Phase 1 ◽  
Sandwich Elisa ◽  
High Specific Activity ◽  
Biological Properties ◽  
Factor Ix ◽  
Hemophilia B ◽  
Wild Type

Abstract The rapid clearance of factor IX (FIX) necessitates frequent intravenous IV administrations to achieve effective prophylaxis for patients with hemophilia B (HB). Subcutaneous (SC) administration would be a preferred route of administration but has been limited by low bioavailability and potency of the marketed FIX products. CB2679d/ISU304 with enhanced biological properties was developed using a rational protein design approach and has resistance to inhibition by ATIII, increased affinity for FVIIIa, increased catalytic activity and resultant 20-fold enhanced potency in vitro (clotting activity) and in vivo (the tail clip model) and 8-fold increased duration of aPTT activity in vivo compared to recombinant wild-type FIX dosed at the same mass. ISU304 (4622 IU/mg) was injected into HB mice SC with a single dose ISU304 at 0.02, 0.05 or 0.15 mg/kg and sampled at 4, 6, 8, 24 hours. Groups of wild-type mice received ISU304 0.02, 0.05 or 0.15 mg/kg SC and BeneFIX (273 IU/mg) 0.15 mg/kg SC, and sampled at 0.25, 1, 4, 8, 24, 48, 72, and 96 hours. Daily SC injection in HB mice of ISU304 at 0.05 mg/kg was sampled at 24, 48, 72 and 96 hours. FIX antigen was measured using a sandwich ELISA and FIX activity was measured using a one-stage clotting assay on Stago Compact. Pharmacokinetics of FIX was performed using PKSolver. There was a dose-dependent increase of plasma FIX antigen with SC ISU304. Mass-based pharmacokinetic profiles of ISU304 (t1/2, 18 hours; Tmax,8 hours, Bioavailability, 19-22%) were similar to those of BeneFIX (t1/2, 20 hours; Tmax, 8 hours, Bioavailability, 16%). Due to ISU304 high specific activity, SC dose of ISU304 yields much higher FIX activities in mouse plasma compared with the same mass dose of BeneFIX. Daily SC dosing of ISU304 230 IU/kg reached steady-state plateau FIX 8% activity after three injections. The bioavailability and increased potency of CB2679d/ISU304 facilitates the initiation of the Phase 1 subcutaneous dosing study in individuals with hemophilia B. Figure 1 Figure 1. Table 1 Table 1. Figure 2 Figure 2. Disclosures Hong: ISU Abxis: Employment. Levy:Catalyst Biosciences: Employment. Jung:ISU Abxis: Employment. Park:ISU Abxis: Employment. Seo:ISU Abxis: Employment. Seo:ISU Abxis: Employment. Madison:Catalyst Biosciences: Employment, Equity Ownership, Patents & Royalties.

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