Factor IX Knockout Mice Research Articles

Extravascular Factor IX Pool Fed By Prophylaxis Is a True Hemostatic Barrier Against Bleeding

Factor IX (FIX) can bind to type-IV collagen in the endothelial basement membrane and diffuse into extravascular spaces. Previous studies in rodents have reported a large biodistribution of FIX. However, the potential hemostatic activity of extravascular FIX and its role in protecting against joint bleeds have not been demonstrated. The capacity of 4 different FIX molecules (plasma derived [pd] and recombinants) to bind type I and type IV collagen was studied here. FIX molecules were also administered intravenously at doses of 50 to 3000 IU/kg in FIX knock-out (KO) mice. A specific FIX signal was detected in immunohistochemistry in the liver as well as in muscles and knee joints with recombinant FIX molecules injected at 1000 and 3000 IU/kg but not at the usual clinical doses of 50-100 IU/kg, while pdFIX generated a FIX signal at all doses including 50 IU/kg. After five 100 IU/kg daily infusions of rFIX, a clear accumulation of FIX was seen in the liver and joints of animals having received 5-day daily FIX infusions with all FIX molecules, with a higher FIX labelling intensity compared to that observed after a single infusion of FIX 100 IU/kg or a single infusion of FIX 500 IU/kg. The extravascular procoagulant activity of FIX was assessed in saphenous vein bleeding assays performed 7 days after a single infusion of FIX 100 IU/kg. An additional group of FIX KO mice received intravenously for 5 days a single daily dose of 100 IU/Kg of one the four types of FIX concentrate in order to mimic prophylaxis. The number of clots formed in 15 minutes was significantly greater in mice having received prophylaxis during 5 days with 100 IU/kg FIX compared to untreated FIX KO mice (p=0.009) and to mice having received a single infusion of FIX 100 IU/kg (p<0.0001) (Figure 1). Taken together, these results show that in individuals with severe hemophilia B receiving regular prophylaxis with FIX, extravascular accumulation of FIX over time may have a significant impact on the coagulation capacity and protection towards bleeding.

Development of an In Vitro Biopotency Assay for an AAV8 Hemophilia B Gene Therapy Vector Suitable for Clinical Product Release

Gene therapy product release requires reliable and consistent demonstration of biopotency. In hemophilia B vectors, this is usually determined in vivo by measuring the plasma levels of the expressed human factor IX (FIX) transgene product in FIX knockout mice. To circumvent this laborious assay, we developed an in vitro method in which the HepG2 human liver cell line was infected with the vector, and the resulting FIX activity was determined in the conditioned medium using a chromogenic assay. The initial low sensitivity of the assay, particularly toward adeno-associated viral serotype 8 (AAV8), increased approximately 100-fold and allowed linear measurement in a broad range of multiplicities of infection. Statistical parameters indicated high assay repeatability (relative standard deviation (RSD) < 5%) and intra-assay reproducibility (RSD < 20%). To compare the performance of the in vitro and in vivo biopotency assay, we applied statistical analyses including regression techniques and variation decomposition to the results obtained for 25 AAV8-FIX vector lots (BAX 335). These showed a highly significant correlation, with the cell culture-based assay demonstrating less variation than the in vivo test. The in vitro assay thus constitutes a viable alternative to using animals for lot release testing.

Evaluation of the Human Factor IX Gene Therapy Vector TAK-748 in Hemophilia: Results from Non-Clinical Studies in Factor IX Knockout Mice and Rhesus Monkeys

Introduction: Adeno-associated virus (AAV)-based factor IX (FIX) gene therapy has the potential to provide clinical benefit in patients with hemophilia B. TAK-748 is a novel next-generation AAV vector for FIX gene therapy. The vector design includes the insertion of 3 hepatocyte-specific cis-regulatory elements to increase the strength of the liver-specific transthyretin promoter driving expression of a human FIX transgene. Aims: The objectives of this study were to investigate the TAK-748 dose-response relationship for FIX activity, and evaluate its efficacy, in FIX knockout (KO) mice and rhesus monkeys. Methods: Male FIX KO mice (N=12/group) received single intravenous doses of TAK-748 (7.4×1010, 1.5×1011, 7.4×1011, or 1.5×1012 vector genomes [vg]/kg) or buffer. Blood samples were taken on days 7, 14, 28, 42, and 56 for the analysis of plasma FIX activity. At the end of the observation period, the bleeding phenotype was assessed by a tail-tip bleeding assay. The viral transduction efficiency of TAK-748 in liver tissue was analyzed by quantitative real-time polymerase chain reaction. Safety assessments included monitoring for clinical signs, and histopathological analysis of selected organs (liver, spleen, kidney, and heart). Male rhesus monkeys (N=3/group) were administered single TAK-748 intravenous bolus injections (3.8×1011, 9.5×1011, or 1.9×1012 vg/kg). Blood samples were collected before dosing and weekly after dosing up to week 18. Plasma FIX activity, human (hu)FIX antigen, and anti-hu-Padua FIX neutralizing antibodies were analyzed. Results: No clinical signs or deaths were recorded in animals treated with TAK-748, and there were no TAK-748-related histopathological findings in the tissues collected from the mice. FIX activity levels in plasma from FIX KO mice treated with buffer were below the lower limit of quantification. Administration of TAK-748 resulted in a dose-dependent increase in mean plasma FIX activity, and supraphysiologic mean FIX levels up to 41.0 IU/mL (1.5×1012 vg/kg). In the tail-tip bleeding assay, blood loss was significantly reduced in the TAK-748 groups at dose levels above 7.4×1010 vg/kg (P&lt;0.05 vs. buffer control). Administration of TAK-748 to rhesus monkeys resulted in a dose-dependent increase in mean plasma FIX activity and antigen, and peak levels of huFIX expression were detected 2-4 weeks after treatment. Mean huFIX activity was 0.3, 0.6, and 1.9 IU/mL after treatment with 3.8×1011 vg/kg, 9.5×1011 vg/kg, and 1.9×1012 vg/kg TAK-748, respectively. A significant reduction in FIX activity and huFIX protein was observed in most of the animals starting about 4 weeks after dosing with TAK-748. In most animals, anti-huFIX Padua neutralizing antibody titers were detected at about week 6 of the study and correlated with the preceding reductions in huFIX expression. Conclusions: Treatment with TAK-748 resulted in dose-dependent increases in plasma FIX activity and was efficacious in FIX KO mice and rhesus monkeys. There were no treatment-related safety findings. Disclosures Weiller: Baxalta Innovations GmbH, a Takeda company: Employment. Wang:Shire US Inc., a Takeda company: Employment, Equity Ownership. Coulibaly:Baxalta Innovations GmbH, a Takeda company: Employment. Schuster:Baxalta Innovations GmbH, a Takeda company: Employment. Rottensteiner:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Sun:Shire US Inc., a Takeda company: Employment. Chuah:Shire, a Takeda company: Consultancy, Research Funding; Catalyst Bio: Consultancy, Research Funding; Pfizer: Research Funding. Vandendriessche:Shire, a Takeda company: Consultancy, Honoraria, Research Funding; Catalyst Bio: Consultancy, Research Funding; Pfizer: Research Funding; Biotest: Honoraria. Scheiflinger:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Höllriegl:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership.

227. A Dose-Escalating Preclinical Study to Determine the Efficacy, MED, and Safety of a Clinical Candidate Vector in a Mouse Model of Hemophilia B

Despite the availability of safe and effective recombinant FIX therapeutics, patients with Hemophilia B could obtain additional clinical benefit by having longer-acting therapeutic options. Although longer-acting recombinant protein FIX therapies are becoming available, the ultimate long-acting FIX therapy could be provided by a gene therapy approach, for example with rAAV vectors encoding FIX. Promising clinical data has been generated over the last few years in an academic setting, but these results must be replicated and extended in a commercial setting for these new therapies to become broadly available to patients.Dose-dependent inflammatory responses that impact the transgene expression have been reported in clinical trials with rAAV FIX vectors, highlighting the need for clinical rAAV vectors to have the highest possible potency such that the overall vector dose can be reduced. We have developed a potent AAV serotype rh10 vector containing a wild-type FIX gene under the control of a highly active liver specific promoter with the goal of achieving approximately 10% of normal FIX levels in patients.In preparation for a clinical trial, we tested this rAAVrh10FIX vector for aspects of efficacy and safety in the factor IX knockout (FIX-KO) mouse model of Hemophilia B. A dose-dependent increase in FIX protein and activity was observed following intravenous administration at vector doses between 1.6×1010 and 5.0×1013 GC/kg. FIX-KO mice that received AAVrh10-hFIXco at 1.6 × 1010 GC/kg achieved between 5% and 8% of normal hFIX levels at 2 weeks post-dose and maintained this level for the 90-day study period. FIX-KO mice that received rAAVrh10FIX vector at 5.0 × 1010 GC/kg achieved between 30% and 42% of normal hFIX levels at 14 days post-dose and maintained this level for the 90-day study period. From these results we can estimate that the vector dose required to achieve the stated goal of 10% of normal FIX levels of 10% would be between 1.6 × 1010 GC/kg and 5.0 × 1010 GC/kg. Expression of hFIX antigen correlated well with measured activity of hFIX antigen, with doses between 1.6 × 1010 GC/kg and 5.0 × 1010 GC/kg providing between 8% and 35% activity of normal hFIX levels.FIX-KO mice provide an excellent model to examine the effects of AAVrh10-hFIXco on hemophilia B-related complications. FIX KO mice are prone to spontaneous bleeds and often suffer from fatal hemorrhages after handling or trauma. In this study, 4 out of 7 animals in the Day 90 control group succumbed to hemophilia-related complications. Single dose intravenous administration of AAVrh10-hFIXco at doses of 1.6 × 1010, 5.0 × 1010, 1.6 × 1011, 5.0 × 1011, 5.0 × 1012, and 5.0 × 1013 GC/kg to FIX-KO mice did not result in test article-related mortality, clinical pathology, gross pathology, or histopathologic findings.

Chemical Chaperones Improve Protein Secretion and Rescue Mutant Factor VIII in Mice with Hemophilia A

Inefficient intracellular protein trafficking is a critical issue in the pathogenesis of a variety of diseases and in recombinant protein production. Here we investigated the trafficking of factor VIII (FVIII), which is affected in the coagulation disorder hemophilia A. We hypothesized that chemical chaperones may be useful to enhance folding and processing of FVIII in recombinant protein production, and as a therapeutic approach in patients with impaired FVIII secretion. A tagged B-domain-deleted version of human FVIII was expressed in cultured Chinese Hamster Ovary cells to mimic the industrial production of this important protein. Of several chemical chaperones tested, the addition of betaine resulted in increased secretion of FVIII, by increasing solubility of intracellular FVIII aggregates and improving transport from endoplasmic reticulum to Golgi. Similar results were obtained in experiments monitoring recombinant full-length FVIII. Oral betaine administration also increased FVIII and factor IX (FIX) plasma levels in FVIII or FIX knockout mice following gene transfer. Moreover, in vitro and in vivo applications of betaine were also able to rescue a trafficking-defective FVIII mutant (FVIIIQ305P). We conclude that chemical chaperones such as betaine might represent a useful treatment concept for hemophilia and other diseases caused by deficient intracellular protein trafficking.

Long-term phenotypic correction in factor IX knockout mice by using phiC31 integrase-mediated gene therapy

Hemophilia B, a hereditary bleeding disorder caused by a deficiency of coagulation factor IX (FIX), is an excellent candidate for gene therapy. However, to date, success in hemophilia gene therapy clinical trials has been limited due to failure to achieve or sustain therapeutic levels of factor expression. The ΦC31 integrase system efficiently integrates plasmid DNA carrying a transgene and an attB site into a limited number of endogenous pseudo attP sites in mammalian genomes, leading to robust, sustained transgene expression. A strategy utilizing plasmid DNA integrated with ΦC31 integrase may offer a facile and safe alternative for sustained human FIX (hFIX) expression. Hydrodynamic tail vein injection was used for delivery of plasmids encoding ΦC31 integrase and hFIX to the liver of FIX knockout mice. We demonstrated prolonged therapeutic levels of hFIX in this knockout mouse model of hemophilia B over a 6-month time course when ΦC31 integrase was used. Additionally, we observed sustained FIX activity in plasma and phenotypic correction of bleeding after tail clip in ΦC31-treated mice. In the livers that received integrase, we also demonstrated prolonged hFIX expression in hepatocytes by immunohistochemistry and documented sequence-specific genomic integration of the hFIX plasmid. These studies suggest the possibility that a similar approach in large animals and humans could lead to a simple and successful gene therapy for hemophilia.

Genetic Modification of Donor Hepatocytes Improves Therapeutic Efficacy for Hemophilia B in Mice

Hepatocyte transplantation (Tx) holds promise for curing genetic liver diseases. However, a limited number of donor hepatocytes can be transplanted into the host liver. Recipient preconditioning and donor cell engineering are under investigation to improve cell engraftment. In theory, genetically engineered cells secreting therapeutic proteins with superior function could compensate for poor engraftment efficiency. We have generated a bioengineered human coagulation factor IX (FIX) with augmented specific activity (named FIX-Triple). The aim of this study was to evaluate therapeutic efficacy of cell therapy using hemophilia B (HB) as a disease model by transplanting FIX-Triple-secreting hepatocytes. The donor hepatocytes were isolated from FIX-Triple knock-in (KI) or FIX-WT (wild-type) KI mice and transplanted intrasplenically into FIX knock-out (KO) mice. FIX-Triple KI recipients exhibited fourfold higher plasma FIX clotting activity than FIX-WT KI recipients. By repeated Txs, the clotting activity of FIX-Triple KI recipients even increased to more than 10% of normal mouse plasma. The engraftment and FIX production efficiencies of transplanted cells were equivalent between the FIX-WT KI and FIX-Triple KI donors. A hemostatic function assay showed that FIX-Triple KI recipients with repeated Txs had more enhanced clot kinetics and a greater maximum rate of thrombus generation than those with a single Tx. Moreover, FIX inhibitors in these recipients rarely developed. In conclusion, hepatocyte Tx with genetically engineered donor cells is an effective therapeutic strategy for HB.

Engineering Liver Tissues under the Kidney Capsule Site Provides Therapeutic Effects to Hemophilia B Mice

Recent advances in liver tissue engineering have encouraged further investigation into the evaluation of therapeutic benefits based on animal disease models. In the present study, liver tissues were engineered in coagulation factor IX knockout (FIX-KO) mice, a mouse model of hemophilia B, to determine if the tissue engineering approach would provide therapeutic benefits. Primary hepatocytes were isolated from the liver of wild-type mice and suspended in a mixture of culture medium and extracellular matrix components. The hepatocyte suspension was injected into the space under the bilateral kidney capsules of the FIX-KO mice to engineer liver tissues. The plasma FIX activities (FIX:C) of the untreated FIX-KO mice were undetectable at any time point. In contrast, the liver tissue engineered FIX-KO mice achieved 1.5-2.5% of plasma FIX activities (FIX:C) and this elevated FIX:C level persisted throughout the 90 day experimental period. Significant FIX mRNA expression levels were found in the engineered liver tissues at levels similar to the wild-type livers. The present study demonstrates that liver tissue engineering could provide therapeutic benefits in the treatment of hemophilia B.

Therapeutic Effects of Hepatocyte Transplantation on Hemophilia B

Hepatocyte transplantation offers an alternative therapeutic approach in the treatment of liver-related diseases. Hemophilia B is a bleeding disorder lacking factor IX (FIX) production in the liver, and achieving more than 1% coagulation activity results in significant improvement in the quality of life of the patients. The aim of this study was to investigate the efficacy of hepatocyte transplantation in the mouse model of hemophilia B. We transplanted isolated normal mouse hepatocytes into the liver of FIX knock-out mice. In some recipient mice, additional hepatocyte transplantations were performed 15 days after the first transplant. The recipient plasma FIX activities increased at 1% to 2% and persisted throughout the experimental period. An additional increase was achieved by the repeated transplantation. Close correlation between FIX messenger RNA levels of the liver and plasma FIX activity levels was observed. These results demonstrate that hepatocyte transplantation can provide therapeutic benefits in the treatment of hemophilia B.

Factor IX: Insights from knock-out and genetically engineered mice

The study of coagulation factors has been rapidly advanced by studies performed in genetically engineered mouse strains. Investigation of factor IX (FIX) has benefited from excellent gene-deleted mouse models that recapitulate many of the features of human haemophilia B. Moreover, advanced positional cloning techniques and availability of technology to allow not only knock-out mice, but also knock-in and knock-down mice, provide new opportunities to observe genotype-phenotype and structure-function correlations regarding FIX, as well as the interaction of FIX with inflammatory, immune, and tissue repair systems. In this paper, available FIX knock-out mice and additional haemophilia B mouse models are reviewed specifically in regards to observations these models have facilitated concerning:factor IX gene expression and factor IX protein pharmacokinetics; the role of FIX in haemostasis, thrombosis and wound healing; insights into coagulation FIX arising out of gene therapy applications in haemophilia mouse models; immunology of tolerance or loss of tolerance of FIX and inhibitor antibody formation.

Nicht-viraler Gentransfer führt zu therapeutischen Faktor-IX-Spiegeln im Hämophilie-B-Mausmodell

Safety issues concerning the risk of malignancy formation and immune response to viral vectors were raised in initial gene therapy trials. In contrast, non-viral gene delivery methods have long been offside. We therefore explore a non-viral gene transfer approach for the treatment of hemophilia B. First, we constructed a strong liver-specific expression plasmid for human factor IX (FIX). Next, we tested the vector by injecting two doses under hydrodynamic conditions into the tail veins of FIX knockout mice. A single injection resulted in an increase in FIX expression over 100% of normal plasma levels. The FIX resulted fully functional. Further, no anti-FIX antibodies were observed and expression levels were vector dose dependent. The high expression obtained in small animals give hope for further development of non-viral gene transfer for the treatment of hemophilia B in humans.