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

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

10.1101/2021.03.18.435908 ◽

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.

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

10.21203/rs.3.rs-302699/v1 ◽

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.

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Effects of Heparin Oligosaccharides with High Affinity for Antithrombin III in Experimental Venous Thrombosis

Thrombosis and Haemostasis ◽

10.1055/s-0038-1657178 ◽

1982 ◽

Vol 47 (03) ◽

pp. 244-248 ◽

Cited By ~ 47

Author(s):

D P Thomas ◽

Rosemary E Merton ◽

T W Barrowcliffe ◽

L Thunberg ◽

U Lindahl

Keyword(s):

Antithrombin Iii ◽

Specific Activity ◽

High Specific Activity ◽

Factor Xa ◽

Blood Levels ◽

Thrombin Time ◽

High Affinity ◽

Very High

SummaryThe in vitro and in vivo characteristics of two oligosaccharide heparin fragments have been compared to those of unfractionated mucosal heparin. A decasaccharide fragment had essentially no activity by APTT or calcium thrombin time assays in vitro, but possessed very high specific activity by anti-Factor Xa assays. When injected into rabbits at doses of up to 80 ¼g/kg, this fragment was relatively ineffective in impairing stasis thrombosis despite producing high blood levels by anti-Xa assays. A 16-18 monosaccharide fragment had even higher specific activity (almost 2000 iu/mg) by chromogenic substrate anti-Xa assay, with minimal activity by APTT. When injected in vivo, this fragment gave low blood levels by APTT, very high anti-Xa levels, and was more effective in preventing thrombosis than the decasaccharide fragment. However, in comparison with unfractionated heparin, the 16-18 monosaccharide fragment was only partially effective in preventing thrombosis, despite producing much higher blood levels by anti-Xa assays.It is concluded that the high-affinity binding of a heparin fragment to antithrombin III does not by itself impair venous thrombogenesis, and that the anti-Factor Xa activity of heparin is only a partial expression of its therapeutic potential.

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

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.732234317.793540850 ◽

2017 ◽

Author(s):

Bruno Stieger

Keyword(s):

Gene Therapy ◽

Specific Activity ◽

High Specific Activity ◽

Factor Ix ◽

Hemophilia B ◽

Activity Factor

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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

Journal of Virology ◽

10.1128/jvi.79.24.15238-15245.2005 ◽

2005 ◽

Vol 79 (24) ◽

pp. 15238-15245 ◽

Cited By ~ 41

Author(s):

Alejandra E. Arbetman ◽

Michael Lochrie ◽

Shangzhen Zhou ◽

Jennifer Wellman ◽

Ciaran Scallan ◽

...

Keyword(s):

Protein Expression ◽

Neutralization Assay ◽

Biological Properties ◽

Scid Mice ◽

Factor Ix ◽

In Vivo Model ◽

Adeno Associated Virus ◽

Human Factor Ix

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.

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Factor IX variants improve gene therapy efficacy for hemophilia B

Blood ◽

10.1182/blood-2004-08-2990 ◽

2005 ◽

Vol 105 (6) ◽

pp. 2316-2323 ◽

Cited By ~ 45

Author(s):

Joerg Schuettrumpf ◽

Roland W. Herzog ◽

Alexander Schlachterman ◽

Antje Kaufhold ◽

Darrel W. Stafford ◽

...

Keyword(s):

Skeletal Muscle ◽

Intramuscular Injection ◽

Specific Activity ◽

Factor Ix ◽

Hemophilia B ◽

Promising Strategy ◽

B Mice ◽

Higher Doses ◽

Vector Delivery

Abstract Intramuscular injection of adeno-associated viral (AAV) vector to skeletal muscle of humans with hemophilia B is safe, but higher doses are required to achieve therapeutic factor IX (F.IX) levels. The efficacy of this approach is hampered by the retention of F.IX in muscle extracellular spaces and by the limiting capacity of muscle to synthesize fully active F.IX at high expression rates. To overcome these limitations, we constructed AAV vectors encoding F.IX variants for muscle- or liver-directed expression in hemophilia B mice. Circulating F.IX levels following intramuscular injection of AAV-F.IX-K5A/V10K, a variant with low-affinity to extracellular matrix, were 2-5 fold higher compared with wild-type (WT) F.IX, while the protein-specific activities remained similar. Expression of F.IX-R338A generated a protein with 2- or 6-fold higher specific activity than F.IX-WT following vector delivery to skeletal muscle or liver, respectively. F.IX-WT and variant forms provide effective hemostasis in vivo upon challenge by tail-clipping assay. Importantly, intramuscular injection of AAV-F.IX variants did not trigger antibody formation to F.IX in mice tolerant to F.IX-WT. These studies demonstrate that F.IX variants provide a promising strategy to improve the efficacy for a variety of gene-based therapies for hemophilia B.

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A New Technic for the Production of an in Vivo Labeled Fibrinogen

Blood ◽

10.1182/blood.v29.4.517.517 ◽

1967 ◽

Vol 29 (4) ◽

pp. 517-525 ◽

Cited By ~ 3

Author(s):

HENRY GANS ◽

JAMES MC LEOD ◽

JAMES T. LOWMAN

Keyword(s):

Amino Acid ◽

Specific Activity ◽

High Specific Activity ◽

Entire Period ◽

Turnover Rates ◽

Prolonged Infusion ◽

Slow Infusion

Abstract The fact that in vitro labeled proteins, as a rule, exhibit faster turnover rates than in vivo labeled materials led us to explore means of obtaining in vivo labeled fibrinogen of high specific activity. It was found that defibrination of the rat provides a stimulus for the liver to regenerate fibrinogen at an accelerated rate. Administration of seleno75 methionine shortly after thrombin-induced defibrination of the animal resulted in the incorporation of large quantities of the label. The rate of incorporation was further increased if the amino acid was administered as a slow infusion during the entire period of fibrinogen regeneration. In addition, prior nephrectomy of the animal would appear to result in a slight increase in specific activity of the fibrinogen preparation obtained. The results of these studies indicate that defibrination, nephrectomy, and the prolonged infusion of the labeled amino acid selenomethionine provided us with a technic for obtaining a biosynthetically labeled, γ-emitting, fibrinogen preparation of high specific activity.

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Delivery of human factor IX in mice by encapsulated recombinant myoblasts: a novel approach towards allogeneic gene therapy of hemophilia B

Blood ◽

10.1182/blood.v87.12.5095.bloodjournal87125095 ◽

1996 ◽

Vol 87 (12) ◽

pp. 5095-5103 ◽

Cited By ~ 114

Author(s):

G Hortelano ◽

A Al-Hendy ◽

FA Ofosu ◽

PL Chang

Keyword(s):

Gene Therapy ◽

Human Factor ◽

Ex Vivo ◽

Cost Effective ◽

Factor Ix ◽

Hemophilia B ◽

Therapy Strategy ◽

Human Factor Ix

A potentially cost-effective strategy for gene therapy of hemophilia B is to create universal factor IX-secreting cell lines suitable for implantation into different patients. To avoid graft rejection, the implanted cells are enclosed in alginate-polylysine-alginate microcapsules that are permeable to factor IX diffusion, but impermeable to the hosts' immune mediators. This nonautologous approach was assessed by implanting encapsulated mouse myoblasts secreting human factor IX into allogeneic mice. Human factor IX was detected in the mouse plasma for up to 14 days maximally at approximately 4 ng/mL. Antibodies to human factor IX were detected after 3 weeks at escalating levels, which were sustained throughout the entire experiment (213 days). The antibodies accelerated the clearance of human factor IX from the circulation of the implanted mice and inhibited the detection of human factor IX in the mice plasma in vitro. The encapsulated myoblasts retrieved periodically from the implanted mice up to 213 days postimplantation were viable and continued to secrete human factor IX ex vivo at undiminished rates, hence suggesting continued factor IX gene expression in vivo. Thus, this allogeneic gene therapy strategy represents a potentially feasible alternative to autologous approaches for the treatment of hemophilia B.

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Copper transport in rats involving a new plasma protein

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.1985.249.1.e77 ◽

1985 ◽

Vol 249 (1) ◽

pp. E77-E88 ◽

Cited By ~ 7

Author(s):

K. C. Weiss ◽

M. C. Linder

Keyword(s):

Time Course ◽

Specific Activity ◽

High Specific Activity ◽

Apparent Molecular Weight ◽

Female Rats ◽

Whole Body ◽

Peripheral Tissues ◽

Chelex 100

The time course of distribution of high-specific activity 67CuCl2 to tissues and plasma components was followed in adult, female rats. Immediately after intubation or injection, tracer 67Cu associated with two components of the blood plasma separable on columns of Sephadex G-150: albumin and another (larger) component, which was not ceruloplasmin. The latter, tentatively named transcuprein, had an apparent molecular weight of 270,000 and a high affinity for Cu2+, as judged by processing through Chelex-100, dilution, and exchange with albumin copper, in vitro and in vivo. It was capable of donating copper to tumor cells in serum-free medium. Analysis of "cold" plasma by furnace atomic absorption confirmed the presence of 10-15% of plasma copper in this peak. Plots of percent dose and 67Cu specific activity against time showed that copper followed a very specific pathway after binding to albumin and transcuprein, entering mainly the liver, then reappearing in the plasma on ceruloplasmin, and then achieving peak distribution in peripheral tissues (muscles, brain, etc.). 67Cu disappeared from liver and kidney with an apparent half-life of 4.5 days, the same exponential rate found for whole body turnover. Apparent turnover of ceruloplasmin copper was more rapid. Even after 7-12 days, tracer copper in plasma was still found exclusively with ceruloplasmin. The results indicate that copper follows a carefully prescribed path, on entering the blood and binding to a new transport protein.

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