Hemophilic Mice Research Articles

Clotting promotes glioma growth and infiltration through activation of focal adhesion kinase.

The tumor architecture of high-grade gliomas is shaped by tumor cell necrosis, invasive growth and the leakage of a fibrin-rich edema from poorly organized tumor blood vessels. Here, we demonstrate a marked upregulation of clot formation in the interstitial spaces of tumor tissues from patients with glioblastoma while tumor-free brain is essentially devoid of fibrin. The accumulation of fibrin in tumor interstitial spaces is functionally relevant as we demonstrate increased infiltration and growth of primary glioblastoma cells after embedding in a 3-dimensional matrix made of fibrin ex vivo. Additionally, we detected accelerated tumor growth after implanting glioblastoma cells together with clotted plasma in brains of immune deficient mice while glioblastoma development in clotting-deficient hemophilia mice was delayed. Glioblastoma growth correlated with the outgrowth of invadopodia and their adhesive interactions with the 3-dimensional clot matrix, which was mediated by integrins β1 and β3 and their common downstream target focal adhesion kinase (FAK). Knocking down FAK with CRISPR Cas9 caused an upregulation of p21/p27 cell cycle inhibitors, strong growth inhibition in cultured glioblastoma cells and sustained anti-tumorigenic effects in orthotopic glioblastoma xenografts in vivo. These results go hand in hand with genomic data from The Cancer Genome Atlas that indicate increased clotting activity and reduced patient survival in glioma subgroups with high integrin β1 and β3 expression. We therefore conclude that clotting in glioma interstitial spaces provides tumor cells with a potent proliferative stimulus that can be reversed by targeting the adhesive machinery of glioblastoma cells via inhibition of FAK.

Activated factor X delivered by adeno-associated virus significantly inhibited bleeding and alleviated hemophilic synovitis in hemophilic mice.

In hemophilia, deficiency of factor VIII or IX prevents the activation of the common coagulation pathway, and inhibits the conversion of FX to activated FXa, which is required for thrombin generation. We hypothesized that the direct expressed FXa has the potential to activate the common pathway and restore coagulation in hemophilia patients. In this study, the cassettes that expressed FXa, FXaop and FXa-FVII were packaged into an engineered AAV capsid, AAV843, and were delivered into hemophilia A and B mice by intravenous injection. AAV-FXaop could be stably expressed in vivo and showed the best immediate and prolonged hemostatic effects, similar to those of commercial drugs (Xyntha and Benefix). AAV-FXaop also significantly inhibited bleeding in hemophilia A mice with inhibitors. In addition, FXa expression in joints significantly alleviated the occurrence of hemophilic synovitis. AAV-delivered FXa may be a novel target for treating hemophilic and hemophilic synovitis.

Deletion of tissue factor pathway inhibitor isoform beta or gamma, but not alpha, improves clotting in hemophilic mice

BackgroundTissue factor pathway inhibitor (TFPI) regulates tissue factor–triggered coagulation. Humans and mice express transcripts encoding for multidistributed (endothelial, platelet, and plasma) 3-Kunitz domain TFPIα and endothelial membrane–anchored 2-Kunitz TFPIβ. Mice express a third transcript, γ, that encodes plasma lipoprotein–associated 2-Kunitz TFPI. In humans, proteolysis of α and/or β produces plasma lipoprotein–associated 2-Kunitz TFPI at lower levels. In clinical trials, monoclonal antibodies that target all TFPI isoforms extend coagulation and correct bleeding in hemophilic patients but with some thrombosis risks. ObjectivesTo determine the impact of TFPI isoform-specific deletions on promoting clotting in hemophilic mice. MethodsEngineered TFPI isoform–specific, hemophilic (factor VIII-null) mice were evaluated for clotting. ResultsMice expressing any single TFPI isoform were healthy. Thrombin generation assays identified TFPIγ as the dominant anticoagulation isoform in mouse plasma. Hemostasis was assessed by serial bleeding times from a tail vein laceration. Repeatedly, after a clot forms, it was manually disrupted; the number of clots/disruptions occurring over a 15-minute period were reported. C57BL/6 and hemophilic mice clot on average 25.6 vs 5.4 times, respectively. On a hemophilia background, TFPIβ or TFPIγ-specific deletion improved clotting to 14.6 and 15.2 times, respectively (P < .0001). TFPIα-specific deletion was without impact, clotting 5.1 times. Heterozygous deletion of TFPIβ was effective, clotting 11.8 times (P < .0001). Heterozygous deletion of TFPIα or TFPIγ alone was ineffective, clotting 3.0 and 6.1 times, respectively, but heterozygous TFPIαγ deletion improved clotting to 11.2 times (P < .001). ConclusionIn hemophilic mice, endothelial TFPIβ and plasma γ-derived 2-Kunitz TFPI individually contribute more to bleeding than total TFPIα.

Correlation of antigen expression with epigenetic modifications after rAAV delivery of a human factor IX variant in mice and rhesus macaques

We investigated long-term human coagulation factor IX (huFIX) expression of a novel variant when delivered into mice and rhesus macaques and compared transduction efficiencies using two different adeno-associated virus (AAV) capsids. In hemophilic mice injected with KP1-packaged recombinant AAV (rAAV) expressing the hyperactive FIX variant specific activity plasma levels were 10-fold or 2-fold enhanced when compared with wild-type or Padua huFIX injected mice, respectively. In rhesus macaques AAV-LK03 capsid outperformed AAV-KP1 in terms of antigen expression and liver transduction. Two animals from each group showed sustained low-level huFIX expression at 3months after administration, while one animal from each group lost huFIX mRNA and protein expression over time, despite comparable vector copies. We investigated whether epigenetic differences in the vector episomes could explain this loss of transcription. Cut&Tag analysis revealed lower levels of activating histone marks in the two animals that lost expression. When comparing rAAV genome associated histone modifications in rhesus macaques with those in mice injected with the same vector, the activating histone marks were starkly decreased in macaque-derived episomes. Differential epigenetic marking of AAV genomes may explain different expression profiles in mice and rhesus macaques, as well as the wide dose response variation observed in primates in both preclinical and human clinical trials.

Differences in venous clot structures between hemophilic mice treated with emicizumab versus factor VIII or factor VIIIFc.

Recombinant factor VIII (rFVIII), rFVIIIFc and emicizumab are established treatment options in the management of hemophilia A. Each has its unique mode of action, which can influence thrombin generation kinetics and therefore also the kinetics of thrombin substrates. Such differences may potentially result in clots with different structural and physical properties. A starting observation of incomplete wound closure in a patient on emicizumab prophylaxis led us to employ a relevant mouse model in which we noticed that emicizumab-induced clots appeared less stable compared to FVIII-induced clots. We therefore analyzed fibrin formation in vitro and in vivo. In vitro fibrin formation was faster and more abundant in the presence of emicizumab than in the presence of rFVIII/rFVIIIFc. Furthermore, the time-interval between the initiation of fibrin formation and factor XIII activation was twice as long for emicizumab than as for rFVIII/rFVIIIFc. Scanning electron microscopy and immunofluorescent spinning-disk confocal microscopy of in vivo-generated clots confirmed increased fibrin formation in the presence of emicizumab. Unexpectedly, we also detected a different morphology between rFVIII/rFVIIIFcand emicizumab-induced clots. Contrary to the regular fibrin mesh obtained with rFVIII/rFVIIIFc, fibrin fibers appeared to be fused into large patches upon emicizumab treatment. Moreover, fewer red blood cells were detected in regions in which these fibrin patches were present. The presence of highly dense fibrin structures associated with a diffuse fiber structure in emicizumab-induced clots was also observed when using super-resolution imaging. We hypothesize that the modified kinetics of thrombin, fibrin and factor XIIIa generation contribute to differences in structural and physical properties between clots formed in the presence of FVIII or emicizumab.

Tissue Factor Pathway Inhibitor (TFPI) Beta and Gamma Isoforms Contribute to Bleeding in Hemophilia Mice

Key Points: Expression of any single TFPI isoform (α, β, or γ) is sufficient for mouse survival.Two-Kunitz domain TFPI isoforms, anchored β and circulating γ, each contribute to hemophilia bleeding.In vivo, TFPIβ is a more potent anticoagulant than TFPIα. Upon injury, tissue factor (TF) triggers coagulation by complexing with factor (F)VIIa to activate FIX and FX, leading to thrombin generation and a fibrin clot. Tissue factor pathway inhibitor limits TF-triggered coagulation. Humans and mice express transcripts encoding for 3-Kunitz domain TFPIα and membrane-anchored 2-Kunitz TFPIβ. Mice also express transcripts encoding untethered, 2-Kunitz TFPIγ. In humans, proteolysis of TFPIα and/or β produces “γ-like” 2-Kunitz TFPI. Hemophilia A and B, caused by deficiency in factors VIII and IX, respectively, are congenital X-linked recessive bleeding disorders that affect an estimated 1.1 million males worldwide. Hemophilia can cause excessive bleeding spontaneously or in response to trauma that can be life-threatening or significantly decrease the quality of life. Approved treatments for hemophilia include factor replacement, bypassing agents, the bispecific antibody Emicizumab, and recently approved gene therapies. Also in development are agents that reduce endogenous anticoagulant activity to counter the procoagulant deficiency of hemophiliacs, including monoclonal antibodies (Mab) to TFPI. These Mab target all TFPI isoforms to improve coagulation and correct bleeding in hemophilia patients, but with some demonstrated thrombosis risk. We investigated if TFPI-isoform-specific inhibition could provide equivalent efficacy which might reduce thrombotic risk. We generated TFPI-isoform-specific exon deletions in mice bred into hemophilia (FVIII-null) background. We find mice expressing any single TFPI isoform (α, β, or γ) appear healthy and reproduce. Our in vivo tail-vein re-bleeding assay is based on serial clot disruptions over 15 minutes, which we have shown is sensitive to anticoagulant and antiplatelet treatment, as well as factor replacement therapy. In this assay, we find C57Bl/6 WT mice clot 25.6 + 0.8 times (mean + SEM), whereas FVIII-null hemophilia mice clot 5.3 +0.4 times. In hemophilia mice, TFPIα-specific deletion is without impact, forming clots 5.1 + 0.8 times, whereas TFPIβ-specific or TFPIγ-specific deletion improves clot formation to 16.8 + 2.0 and 15.2 + 1.5 times, respectively (p&amp;lt;0001). Even hemophilia mice with heterozygotic deletion of the β exon form clots 11.8 + 0.8 times (p&amp;lt;0001). Thus, in vivo 2-Kunitz TFPI isoforms, anchored β and untethered γ each provides greater anticoagulant activity and impact on bleeding than 3-Kunitz TFPIα. Ex vivo plasma-based thrombin generation assays show that TFPIγ provides more anticoagulant activity than TFPIα. As shown in the figure, the deletion of TFPIγ increases thrombin generation to a greater extent than the deletion of TFPIα. The addition of an anti-TFPI antibody that effectively blocks TFPI activity shows a significant increase in endogenous thrombin potential in the plasma of TFPIαβ-deleted (γ-only) mice (370 nM.min), versus TFPIβγ-deleted (α-only) plasma (70 nM.min) and TFPIαγ-deleted (β-only) mice plasma (22 nM.min), (p&amp;lt;0.001). As expected, membrane-anchored TFPIβ is not detected in plasma. It is well established that 3-Kunitz TFPIα is a potent anticoagulant in plasma-based clotting time assays, while truncated 2-Kunitz TFPI, like mouse TFPIγ, appears inactive. Based on such assays, it was unexpected that TFPIγ-only expressing mice thrive, and in vivo, TFPIα is not the dominant endogenous anticoagulant. If mouse models are to be useful in providing insights into human hemostasis and TF-associated diseases, then it is critical to understand the similarities and differences between mice and humans. As humans do not express TFPIγ, the observed impact of depleting circulating 2-Kunitz TFPI on bleeding and thrombin generation in hemophilic mice is unlikely to translate to man. By contrast, our results demonstrating that, in vivo, TFPIβ is a more potent anticoagulant than TFPIα in mice has potential therapeutic implications in humans.

Liver Sinusoidal Endothelial Cells but Not Hepatocytes Express a Fluorescent Reporter Under the Endogenous F8 Promoter in a New Hemophilia a Mouse Model

Hemophilia A (HA) is an inherited bleeding disorder caused by mutations in the F8 gene leading to a deficiency in coagulation factor VIII (FVIII). Treatment of patients with HA has centered around replacement of FVIII protein. Gene therapy provides the possibility of permanently altering the HA phenotype. Gene therapy for HA induces FVIII expression in hepatocytes, despite the well supported belief that FVIII is not made within hepatocytes but rather in liver sinusoidal endothelial cells (LSECs). Ectopic expression of FVIII in hepatocytes has been implicated in mild toxicities and FVIII decline observed in adeno-associated viral (AAV) vector-based gene therapy, the causes of which remain unknown. Extrahepatic production of FVIII, e.g. in lymphoid tissues, has been controversial. A better understanding of where FVIII is produced is needed to improve gene therapy for HA and inform development of other therapeutic interventions. We developed in collaboration with Jackson Laboratory a new strain of hemophilic mice on the C57BL/6J background expressing the fluorescent protein mScarlet under the endogenous F8 promoter. Using the CRISPR/Cas9 gene editing system, we inserted a transgene encoding mScarlet into exon 1 of the mouse F8 gene through homology-directed repair. We selected mScarlet for its relative brightness and stability, to maximize the chances of detecting its expression despite weak F8 promoter activity. The donor DNA consisted of two homology arms flanking the mScarlet-encoding transgene followed by the bovine growth hormone polyadenylation [poly(A)] signal. Insertion of the transgene at the first F8 codon in exon 1 and inclusion of poly(A) ensured knock-out of F8. Male animals showed a bleeding phenotype consistent with other HA mouse strains, such as C57BL/6J HA and B6/129 HA. We performed retro-orbital bleeding and tested mouse plasma samples using the activated partial thromboplastin time (aPTT) assay. Animals expressing mScarlet (n=4) and C57BL/6J HA mice (n=3) had similarly prolonged aPTT (mean 57 seconds) compared to non-hemophilic C57BL/6J mice (n=5, 41 seconds). Production of mScarlet was first evaluated in the hepatocytes and LSECs of mScarlet mice (n=3) using flow cytometry. The livers were perfused in situ via the portal vein with a perfusion and collagenase medium. The livers were then processed into single cell suspensions, and hepatocytes and LSECs were separated by centrifugation. An anti-CD31 antibody was used to identify LSECs. Hepatocytes were identified by exclusion of CD45.2 + cells. CD31 +cells from mScarlet mice but not from control C57BL/6J mice expressed mScarlet. Neither mouse strain had detectable mScarlet in hepatocytes. Flow cytometry was then used to evaluate mScarlet in the splenic endothelial cells (ECs) and leukocytes. Using in situtranscardiac perfusion, mScarlet or control C57BL/6J mice received a perfusion and collagenase medium. Following processing into single cell suspensions, ECs were isolated from other splenic cells by magnetic separation using CD31 microbeads. Anti-CD31 and anti-CD45.2 antibodies were used to identify ECs and splenic leukocytes, respectively. Neither cell type from mScarlet or control mice had detectable mScarlet. We next evaluated mScarlet expression via fluorescent microscopy of liver and spleen sections. Before sectioning, tissues were fixed using in situ transcardiac perfusion fixation. Since mScarlet fluorescence was not detectable in tissue sections, we employed an anti-mScarlet antibody. Anti-mScarlet binding was detectable in liver sections from mScarlet but not control C57BL/6J mice. The staining pattern was consistent with the sinusoidal arrangement of hepatic capillaries running between rows of hepatocytes toward central venules. We found the same staining pattern using an anti-CD31 antibody, supporting that the mScarlet-expressing cells were LSECs but not hepatocytes. No anti-mScarlet binding was detected in the spleens from either mScarlet or C57BL/6J mice. We propose that FVIII is produced in LSECs but not in hepatocytes, splenic endothelial cells, splenic or hepatic leukocytes. Our novel mouse model expressing a well-defined and readily detectable reporter protein overcomes the limitations of immunohistochemistry using anti-FVIII antibodies, which complicated previous studies on the cellular origins of FVIII. Further investigation of extrahepatic FVIII production is ongoing.

In vivo genome editing using 244-cis LNPs and low-dose AAV achieves therapeutic threshold in hemophilia A mice

Gene therapy and rebalancing therapy have emerged as promising approaches for treating hemophilia A, but there are limitations, such as temporary efficacy due to individual differences. Genome editing for hemophilia has shown long-term therapeutic potential in preclinical trials. However, a cautious approach is necessary because genome editing is irreversible. Therefore, we attempted to induce low-level human factor 8 (hF8) gene knockin (KI) using 244-cis lipid nanoparticles and low-dose adeno-associated virus to minimize side effects and achieve a therapeutic threshold in hemophilia A mice. We selected the serpin family C member 1, SerpinC1, locus as a target to enable a combined rebalancing strategy with hF8 KI to augment efficacy. This strategy improved blood coagulation activity and reduced hemophilic complications without adverse effects. Furthermore, hemophilic mice with genome editing exhibit enhanced survival for 40weeks. Here, we demonstrate an effective, safe, and sustainable treatment for hemophilia A. This study provides valuable information to establish safe and long-term genome-editing-mediated treatment strategies for treating hemophilia and other protein-deficient genetic diseases.

Safety and efficacy of an anti–human APC antibody for prophylaxis of congenital factor deficiencies in preclinical models

AbstractRebalance of coagulation and anticoagulation to achieve a hemostatic effect has recently gained attention as an alternative therapeutic strategy for hemophilia. We engineered a humanized chimeric antibody, SR604, based on a previously published murine antibody, HAPC1573, which selectively blocks the anticoagulant activity of human activated protein C (APC). SR604 effectively blocked the anticoagulation activities of APC in human plasma deficient in various coagulation factors in vitro with affinities ∼60 times greater than that of HAPC1573. SR604 exhibited prophylactic and therapeutic efficacy in the tail-bleeding and knee-injury models of hemophilia A and B mice expressing human APC (humanized hemophilic mice). SR604 did not interfere with the cytoprotection and endothelial barrier function of APC, nor were there obvious toxicity effects in humanized hemophilic mice. Pharmacokinetic study showed a high bioavailability (106%) of subcutaneously injected SR604 in cynomolgus monkeys. These results demonstrate that SR604 is expected to be a safe and effective therapeutic and/or prophylactic agent with a prolonged half-life for patients with congenital factor deficiencies including hemophilia A and B.

Activation of the Acute-Phase Response in Hemophilia.

To identify recurrent inflammation in hemophilia, we assessed the acute-phase response in the blood of patients with hemophilia A and B. Compared to age- and weight-matched controls, blood levels of interleukin-6 (IL-6), C-reactive protein (CRP), and LPS-binding protein (LBP) were significantly elevated in the entire cohort of hemophilia patients but exhibited a particularly pronounced increase in obese hemophilia patients with a body mass index (BMI) ≥30. Subgroup analysis of the remaining nonobese hemophilia patients (BMI: 18-29.9) revealed a significant spike of IL-6, CRP, and LBP in connection with a de-novo increase of soluble IL-6 receptor α (sIL-6Rα) in patients with bleeding events within the last month. Hemophilia patients who did not experience recent bleeding had IL-6, CRP, and sIL-6Rα blood levels similar to healthy controls. We did not find increased IL-6 or acute-phase reactants in hemophilia patients with arthropathy or infectious disease. The role of IL-6 as a marker of bleeding in hemophilia was confirmed in hemophilia patients with acute bleeding events as well as in transgenic hemophilia mice after needle puncture of the knee, which exhibited an extensive hematoma and a 150-fold increase of IL-6 blood levels within 7 days of the injury compared to needle-punctured control mice. Notably, IL-6 blood levels shrunk to a fourfold elevation in hemophilia mice over controls after 28 days, when the hematoma was replaced by arthrofibrosis. These findings indicate that acute-phase reactants in combination with sIL-6Rα could be sensitive biomarkers for the detection of acute and recent bleeding events in hemophilia.

Desferoxamine protects against hemophilic arthropathy through the upregulation of HIF-1α-BNIP3 mediated mitophagy

AimsHemophilic arthropathy (HA) is a typically iron overload induced joint disease secondary to continuous joint bleeding, however, the exact role of iron chelators in HA has not been fully elucidated. In the present study, we investigated whether desferoxamine (DFO), an iron chelator, could limit the development of HA and the underlying mechanisms. Materials and methodsA HA mice model was established by needle puncture in the left knees of FVIII-deficient hemophilic mice. HA progression was evaluated at 8 weeks after DFO administration. Moreover, chondrocytes were treated with ferric ammonium citrate (FAC) to mimic iron overload in vitro. Modulating effect of DFO on iron overload induced oxidative stress, chondrocytes apoptosis and extracellular matrix (ECM) degradation and the role of HIF-1α-BNIP3 mediated mitophagy were examined. Key findingsWe found that DFO limited the development of HA and protected iron overload induced ECM degradation, chondrocytes apoptosis and oxidative stress. Besides chelating Fe2+, we found that HIF-1α-BNIP3 mediated mitophagy played important roles in the protective effect of DFO. HIF-1α inhibition suppressed chondrocytes mitophagy process and partly diminished the protective effect of DFO on chondrocytes iron overload. SignificanceIn conclusion, DFO could protect against HA development via HIF-1α-BNIP3 mediated mitophagy, suggesting DFO might be a potential therapeutic supplement for HA treatment.

Blood-Induced Inflammation in Hemophilia

We previously showed that the acute-phase reactants IL-6, CRP and LBP are elevated in the blood of hemophilia patients in response to acute and recent bleeding. The systemic elevation of acute-phase markers in hemophilia patients was associated with increased activation of monocytes, which exhibit deficits of M2 markers such as CSF1-R, CD163 and Tie2. These deficits correlate with impaired differentiation of monocytes into macrophages thus resulting in the diminished ability of hemophilia macrophages to invade clot and phagocytose red blood cells. As macrophages are central to wound healing and tissue regeneration, we hypothesize that the diminished phagocytic activity of hemophilia macrophages contributes to blood-induced inflammation and joint degeneration in hemophiliacs with hemarthrosis. To assess the effect of joint bleeding on inflammation, we induced a needle-puncture injury of the knee in transgenic mice with hemophilia A. While this process produced only a minor injury in coagulation competent control mice, we observed an extensive hematoma on d1 and d7 in the knees of hemophilia mice as shown by gross and histological examination. By day 28, the hematoma was completely absorbed. The degenerative changes of the knee joints that followed the bleeds included synovia hyperplasia, fibrosis and decline in bone density. Joint bleeding reached a plateau on day 1-7 in hemophilia mice. Immunohistochemistry for myeloperoxidase revealed during this time a massive influx of neutrophil granulocytes into punctured joints that subsided at day 28 when the hematoma was replaced by arthrofibrosis. In addition, acute bleeding-induced inflammation was reflected by a steep increase of interleukin 6 in the blood circulation of knee-punctured hemophilia mice that reached a maximum at day 7 and then fell towards baseline levels at day 28. CD68-positive wound macrophages, on the other hand, were first observed in punctured knees on day 7 and persisted until the end of the observation period on day 28, when most of the macrophages were loaded up with hemosiderin. None of these complications occurred in punctured wildtype mice with normal clotting activity. The deposition of hemosiderin-loaded macrophages in the punctured joints of hemophilia mice suggests that failure to remove red blood cells from the wound area leads to inflammation and subsequent joint degeneration. This failure to clear red blood cells and their proinflammatory components appeared to be specific to hemophilia mice, as we observed rapid absorption of sizable hematomas after injection of anticoagulated blood into the knee joint of coagulation-competent wildtype mice, which in contrast to punctured hemophilia mice showed no signs of inflammation or joint destruction 7 days after blood injection. Together, these data show that hemarthrosis in hemophilia represents a relevant inflammatory stimulus that correlates strongly with joint degeneration. Moreover, the data indicate that inflammation in hemophilia is caused by impaired removal of red blood cells from injured joints, which may lead to the release of pro-inflammatory hemoglobin degradation products and subsequent arthrofibrosis.

An Antibody Targeting Human FV Promotes Thrombin Generation and Reduces Bleeding in a Hemophilic Mouse Model

Hemophilia is a bleeding disorder that results from a deficiency of factor VIII (Hemophilia A; HA) or factor IX (Hemophilia B; HB). Protein replacement therapy ameliorates bleeding but can result in the development of neutralizing antibodies. Innovative nonfactor therapies are in development for hemophilia which target anticoagulant proteins such as tissue factor pathway inhibitor (TFPI), antithrombin, and activated protein C (APC). While these approaches show promise, they influence multiple coagulation factors and pathways which could raise safety concerns. Considering its essential role in coagulation in enhancing thrombin generation (TG), we hypothesized that altering the functional properties of FV/FVa could be an attractive approach for HA and HB treatment. Activated FV (FVa) assembles with FXa on activated cellular surfaces and enhances the kinetics of TG by several orders of magnitude. Here we assess whether monoclonal antibodies targeting all forms of FV are effective in promoting TG in hemophilia using both in vitro and in vivo models in hemophilic mice. Using an initial TG assay screen, we identified a procoagulant antibody (GB5) that binds with high affinity (Kd <0.5 nM) to human (h)FV, FVa, and FV-short and investigated its mechanism of action. Using proteolytic fragments of FV and western blotting experiments, we found that GB5 likely binds in the middle of the light chain region. To evaluate the functional effects of GB5 in vitro, we first employed a plasma-based TG assay using HA or HB plasma triggered with low (0.2 pM) tissue factor. The presence of saturating GB5 increased TG about 3-fold in HA plasma. Similar results were observed with HB plasma. Interestingly, when TG was essentially eliminated in HA and HB plasma with addition of exogenous APC, GB5 restored TG. These results suggest that the procoagulant effect of GB5 neutralizes the anticoagulant effect of APC and enhanced TG in hemophilic plasma To begin to assess the mechanism, the effect of GB5 on FV activation in the TG assay was evaluated. TG assay samples were removed over time, quenched, and analyzed by immunoblotting using labeled FV specific antibodies. In the presence of GB5, ~95% of the starting full-length FV was converted to FVa within 3 min compared to 5 min in the absence of GB5. These results show that FV is activated faster in the presence of GB5 in the TG assay. To extend these data, we next assessed the influence of GB5 in the TG assays using FV-deficient plasma supplemented with either FV or FVa and TFPI⍺. The data show that GB5 had essentially no effect on the TG assay when FV deficient plasma was reconstituted with FVa, but enhanced TG at least 3-fold when supplemented with FV. Consistent with this, the kinetics of prothrombin activation with FXa-FVa-membranes in the presence or absence of GB5 were the same. These data suggest that GB5 impacts reactions that influence FV, with no direct effect on FVa procoagulant function. To evaluate whether GB5 has a procoagulant effect in vivo, a tail vein transection (TVT) model was employed in HA mice. In this model, one lateral tail vein is transected, and blood is collected over 40 min with clots at the vessel wall disrupted every 10 min as needed. Since GB5 interacts with human and not mouse FV, plasma derived human FV was infused into HA mice. We found that blood loss in the TVT model using HA mice infused with human FV alone (5 μg/mouse) or GB5 alone (20 μg/mouse) was no different than mice infused with PBS (n = 3-5 mice/group except GB5 alone where n = 1: ~600 μl blood loss). In contrast to these data, when human FV and GB5 were co-administered, blood loss was reduced ~3-fold (~200 μl) which was statistically significant relative to the controls but did not reach the level of wild-type mice (blood loss ~50 μl). Overall, the results show that GB5 is a procoagulant monoclonal antibody that binds with high affinity to human FV and influences reactions that lead to its activation and inactivation. We hypothesize that the net effect of GB5 is the enhanced production of FVa which has a major impact on TG. In plasma-based systems with and without the protein C system and an in vivo hemophilic model, GB5 is procoagulant and can reduce bleeding. Our work shows that targeting the common coagulation pathway at the level of FV has benefit in the context of hemophilia and may be a viable and safe therapeutic approach.

Tissue Factor Pathway Inhibitor Gamma Deletion Corrects the Bleeding Phenotype in Hemophilia Mice

Tissue factor (TF) is a transmembrane receptor that initiates coagulation and, via protease-activated receptors, enhances inflammation. Tissue factor pathway inhibitor (TFPI) is the endogenous regulator of TF activities. Anti-TFPI antibodies show promise as a hemophilia treatment but must be tittered to avoid potential thrombogenic side effects. From a single gene, through alternative splicing, humans express TFPI transcripts that encode for α and β isoforms; while mice express transcripts that encode for α and β, and also a third isoform γ, which is the most abundant. It is hypothesized that TFPIα is the dominant isoform anti-coagulant in man, whereas cell-anchored TFPIβ is the dominant anti-inflammatory. The main goal of our study is to further understand the impact of the different TFPI isoforms in hemophilic mice. To assess the functions of the α, β, and γ mouse-specific TFPI isoforms, using CRISPR/Cas9 technology, we generated TFPI α-, β-, γ-, α/β-, α/γ-, and β/γ- deleted mouse strains. Each knock-out strain is viable, and the offspring were born with expected Mendelian ratios. To evaluate the hemostatic function of the individual isoforms, all TFPI mice strains were bred on a hemophilia (FVIII-null) background and evaluated with a tail-vein rebleeding assay (TVRB). In this TVRB, a lateral tail vein is transected to cause bleeding; time to clot formation is recorded, then the clot is immediately dislodged, and the process is repeated over 15 minutes. In this assay, C57/Bl6 mice clot on average 26 +1.3 (mean+SD) times, whereas F8 null mice clot 5 + 2.6 times on average. Surprisingly, in the F8 null background, deletion of TFPIγ (14 + 6.5), alone or in combination as α/γ-deleted or β/γ-deleted, significantly reduced bleeding (p < 0.001), whereas TFPIα deletion had no impact on hemophilia bleeding (5 + 3). We next performed ex-vivo thrombin generation assays (TGA). The blood was collected via the inferior vena cava into corn trypsin inhibitor plus citrate syringes. Platelet-poor plasma was extracted and added into 96-well plates in addition to TF, phospholipids, calcium, and substrate. Endogenous thrombin potential was calculated from the area under the curve for generated thrombin for each TFPI mice strain on a hemophilia background. In agreement with the in vivo model, TFPIγ-deletion on the F8 null background significantly improved endogenous thrombin potential (P=0.037), while additional deletion of TFPIα (α/γ-deletion) did not provide any additional procoagulant effect. In conclusion, both in vivo and ex vivo hemostasis mouse models showed that, rather than three Kunitz TFPIα or cell-anchored TFPIβ, soluble two-Kunitz domain TFPIγ is the dominant mouse plasma isoform with respect to anti-coagulant function. Studies using mouse models that investigate the role of TFPI in hemostasis, thrombosis, and inflammation must carefully consider and address the impact of mouse TFPIγ on outcomes before extrapolating findings to human TFPI in health and disease.

Platelet αIIbβ3 Receptor Antagonist Impedes the Hemostatic Efficacy of Platelet-Specific FVIII Gene Therapy in Hemophilia a Mice

Hemophilia A is a prime model for gene therapy. Data from the current human clinical trial using liver-specific AAV-mediated FVIII gene therapy are encouraging, but FVIII levels steadily decline, raising serious concerns about durability and safety. Furthermore, pediatric and adult patients who have severe liver disease, neutralizing antibodies to AAV, which are present in 30-50% of the population, are excluded from AAV liver gene therapy. Our previous studies have demonstrated that platelet-targeted FVIII expression under the control of the platelet-specific αIIb promoter (2bF8) is effective in rescuing the bleeding phenotype of hemophilia (FVIII-/-) mice even in the presence of anti-FVIII inhibitory antibodies (inhibitors). Currently, our gene therapy approach is in Phase I clinical trial. Since antiplatelet drugs are commonly used in the clinic, it is important to investigate how platelet antagonists affect the clinical efficacy of platelet-derived FVIII. The αIIbβ3 complex is a receptor for fibrinogen and VWF, with a crucial role in platelet activation and platelet plug formation at the site of injury. The αIIbβ3 complex is the target of several drugs used in cardiovascular medicine. Thus, we propose to use an αIIbβ3 antibody to block the integrin αIIbβ3 receptor to investigate how platelet aggregation impacts the clinical efficacy of platelet-derived FVIII in FVIII-/- mice. We used anti-mouse integrin αIIbβ3 antibody (Leo.H4) to block the αIIbβ3 complex, inhibiting its binding to fibrinogen and therefore inhibiting platelet aggregation. Hemostatic properties of platelet-FVIII were assessed by ex vivo ROTEM and native whole blood TGA (nWB-TGA) analysis of whole blood and in vivo tail FeCl3-induced carotid artery injury model and lateral tail vein transection (TVT) injury model.In 2bF8/FVIII-/- mice, platelet-FVIII levels were 6.89±0.65 mU/108 platelets, with all parameters from ROTEM analysis comparable to those in WT control mice. However, all parameters, including clotting time, clot formation time, maximal clot firmness, α-angle, and A10 were significantly impaired in the presence of Leo.H4 compared to native samples from 2bF8 mice (8.5±1.96 min, 3.1±0.41min, 67.25±2.63mm, 55.75±3.59°, 44.25±4.03mm in native samples versus 10.68±1.77min, 13.5±3.11min, 28.5±0.58mm, 33±2.83°, 18.25±1.26mm in those with Leo.H4, respectively). In contrast, in WT mice, only α-angle and A10 were significantly tempered in the presence of Leo.H4. Interestingly, in 2bF8/FVIII-/- mice, peak thrombin and endogenous thrombin potential determined by TGA were significantly enhanced in the presence of Leo.H4 compared to native samples (58.4±23.72nM and 600.8±185.83nM vs. 97.93±13.89nM and 921.03±72.95nM, respectively), but there were no significant differences in other parameters. Leo.H4 did not impact the hemostatic parameters determined by nWB-TGA in WT mice. Complete occlusion occurred in the FeCl3-induced carotid injury in all 2bF8 and WT mice, but none occluded when Leo.H4 was infused. In the TVT injury model, when Leo.H4 was infused, none of 2bF8/FVIII-/- mice stopped bleeding in the 10-minute bleeding test, and animals lost a significant amount of blood (525.98 ± 92.06 μL). In contrast, when animals were infused with IgG isotype control, the bleeding time was 0.99 ± 0.05 min, and blood loss in the IgG control group was 17.05 ± 9.37 μL, which was 30-fold lower than in the Leo.H4 group. There were no significant differences in the bleeding time and blood loss between the 2bF8 infused with IgG and WT groups in the TVT test. In conclusion, our results demonstrated that αIIbβ3-mediated platelet aggregation is critical in maintaining the hemostatic functions and clinical efficacy of platelet-derived FVIII in hemophilia A. Anti-αIIbβ3 antibody, Leo.H4, impedes the hemostatic efficacy of platelet-FVIII in FVIII-/- mice.

Modulating the microenvironment during FVIII uptake influences the nature of FVIII-peptides presented by antigen-presenting cells.

Hemophilia A is a severe bleeding disorder caused by the deficiency of functionally active coagulation factor VIII (FVIII). The induction of neutralizing anti-drug antibodies is a major complication in the treatment of hemophilia A patients with FVIII replacement therapies. Why some patients develop neutralizing antibodies (FVIII inhibitors) while others do not is not well understood. Previous studies indicated that the induction of FVIII inhibitors requires cognate interactions between FVIII-specific B cells and FVIII-specific CD4+ T cells in germinal center reactions. In this study, we investigated the FVIII peptide repertoire presented by antigen-presenting cells (APCs) under different microenvironment conditions that are expected to alter the uptake of FVIII by APCs. The aim of this study was to better understand the association between different microenvironment conditions during FVIII uptake and the FVIII peptide patterns presented by APCs. We used a FVIII-specific CD4+ T cell hybridoma library derived from humanized HLA-DRB1*1501 (human MHC class II) hemophilic mice that were treated with human FVIII. APCs obtained from the same mouse strain were preincubated with FVIII under different conditions which are expected to alter the uptake of FVIII by APCs. Subsequently, these preincubated APCs were used to stimulate the FVIII-specific CD4+ T cell hybridoma library. Stimulation of peptide-specific CD4+ T-cell hybridoma clones was assessed by analyzing the IL-2 release into cell culture supernatants. The results of this study indicate that the specific microenvironment conditions during FVIII uptake by APCs determine the peptide specificities of subsequently activated FVIII-specific CD4+ T cell hybridoma clones. Incubation of APCs with FVIII complexed with von Willebrand Factor, FVIII activated by thrombin or FVIII combined with a blockade of receptors on APCs previously associated with FVIII uptake and clearance, resulted in distinct peptide repertoires of subsequently activated hybridoma clones. Based on our data we conclude that the specific microenvironment during FVIII uptake by APCs determines the FVIII peptide repertoire presented on MHC class II expressed by APCs and the peptide specificity of subsequently activated FVIII-specific CD4+ T cell hybridoma clones.

Blocking human protein C anticoagulant activity improves clotting defects of hemophilia mice expressing human protein C

AbstractHemophilia A and B are hereditary coagulation defects resulting in unstable blood clotting and recurrent bleeding. Current factor replacement therapies have major limitations such as the short half-life of the factors and development of inhibitors. Alternative approaches to rebalance the hemostasis by inhibiting the anticoagulant pathways have recently gained considerable interest. In this study, we tested the therapeutic potential of a monoclonal antibody, HAPC1573, that selectively blocks the anticoagulant activity of human activated protein C (APC). We generated F8−/− or F9−/− hemophilia mice expressing human protein C by genetically replacing the murine Proc gene with the human PROC. The resulting PROC+/+;F8−/− or PROC+/+;F9−/− mice had bleeding characteristics similar to their corresponding F8−/− or F9−/− mice. Pretreating the PROC+/+;F8−/− mice with HAPC1573 shortened the tail bleeding time. HAPC1573 pretreatment significantly reduced mortality and alleviated joint swelling, similar to those treated with either FVIII or FIX, of either PROC+/+;F8−/− or PROC+/+;F9−/− mice in a needle puncture–induced knee-joint bleeding model. Additionally, we found that HAPC1573 significantly improved the thrombin generation of PROC+/+;F8−/− mice but not F8−/− mice, indicating that HAPC1573 enhanced the coagulant activity of hemophilia mice by modulating human APC in vivo. We further documented that HAPC1573 inhibited the APC anticoagulant activity to improve the clotting time of human plasma deficient of FVIII, FIX, FXI, FVII, VWF, FV, or FX. These results demonstrate that selectively blocking the anticoagulant activity of human APC may be an effective therapeutic and/or prophylactic approach for bleeding disorders lacking FVIII, FIX, or other clotting factors.

Hemophilia: The Past, the Present, and the Future.

Hemophilia: The Past, the Present, and the Future.

Lipid Nanoparticle-mRNA Formulations for Therapeutic Applications.

After decades of extensive fundamental studies and clinical trials, lipid nanoparticles (LNPs) have demonstrated effective mRNA delivery such as the Moderna and Pfizer-BioNTech vaccines fighting against COVID-19. Moreover, researchers and clinicians have been investigating mRNA therapeutics for a variety of therapeutic indications including protein replacement therapy, genome editing, and cancer immunotherapy. To realize these therapeutics in the clinic, there are many formidable challenges. First, novel delivery systems such as LNPs with high delivery efficiency and low toxicity need to be developed for different cell types. Second, mRNA molecules need to be engineered for improved pharmaceutical properties. Lastly, the LNP-mRNA nanoparticle formulations need to match their therapeutic applications.In this Account, we summarize our recent advances in the design and development of various classes of lipids and lipid derivatives, which can be formulated with multiple types of mRNA molecules to treat diverse diseases. For example, we conceived a series of ionizable lipid-like molecules based on the structures of a benzene core, an amide linker, and hydrophobic tails. We identified N1,N3,N5-tris(3-(didodecylamino)propyl)benzene-1,3,5-tricarboxamide (TT3) as a lead compound for mRNA delivery both in vitro and in vivo. Moreover, we tuned the biodegradability of these lipid-like molecules by introducing branched ester or linear ester chains. Meanwhile, inspired by biomimetic compounds, we synthesized vitamin-derived lipids, chemotherapeutic conjugated lipids, phospholipids, and glycolipids. These scaffolds greatly broaden the chemical space of ionizable lipids for mRNA delivery. In another section, we highlight our efforts on the research direction of mRNA engineering. We previously optimized mRNA chemistry using chemically-modified nucleotides to increase the protein expression, such as pseudouridine (ψ), 5-methoxyuridine (5moU), and N1-methylpseudouridine (me1ψ). Also, we engineered the sequences of mRNA 5' untranslated regions (5'-UTRs) and 3' untranslated regions (3'-UTRs), which dramatically enhanced protein expression. With the progress of LNP development and mRNA engineering, we consolidate these technologies and apply them to treat diseases such as genetic disorders, infectious diseases, and cancers. For instance, TT3 and its analog-derived lipid-like nanoparticles can effectively deliver factor IX or VIII mRNA and recover the clotting activity in hemophilia mouse models. Engineered mRNAs encoding SARS-CoV-2 antigens serve well as vaccine candidates against COVID-19. Vitamin-derived lipid nanoparticles loaded with antimicrobial peptide-cathepsin B mRNA enable adoptive macrophage transfer to treat multidrug resistant bacterial sepsis. Biomimetic lipids such as phospholipids formulated with mRNAs encoding costimulatory receptors lead to enhanced cancer immunotherapy.Overall, lipid-mRNA nanoparticle formulations have considerably benefited public health in the COVID-19 pandemic. To expand their applications in clinical use, research work from many disciplines such as chemistry, engineering, materials, pharmaceutical sciences, and medicine need to be integrated. With these collaborative efforts, we believe that more and more lipid-mRNA nanoparticle formulations will enter the clinic in the near future and benefit human health.

Protection of Human Factor V from Activated Protein C Using a Monoclonal Antibody-Biochemical Characterization and Evaluation Using a Mouse Injury Model

Hemophilia is an X-linked bleeding disorder resulting in deficiency of FVIII (hemophilia A; HA) or FIX (HB). Despite treatment with infused FVIII or FIX, people with hemophilia still experience joint bleeding and impaired quality of life. Furthermore, the development of neutralizing antibodies to FVIII or FIX remains a major complication of therapy. To this end, several innovative therapeutic advances which rebalance the coagulation system by targeting anticoagulant pathways (antithrombin, tissue factor pathway inhibitor, and activated protein C (APC)) are particularly interesting as they should be effective for both HA and HB, with and without inhibitors, and may also be useful to treat certain rare bleeding disorders. Many of these approaches are in clinical trials and show promise. However, each of these anticoagulant pathways target multiple coagulation factors, and APC plays an important cellular signaling role. In the current study, we developed a monoclonal antibody (GB5) that targets human FV/FVa which could serve as an alternative therapeutic approach to enhance thrombin generation and prevent bleeding in different clinical situations.Monoclonal antibody GB5 was identified following screening of hybridoma clones derived from mice immunized with human FV. The initial screen identified GB5 as an antibody that could promote thrombin generation (TG) in the presence of APC. To characterize the antibody, purified GB5 was attached to a biosensor tip and evaluated using biolayer interferometry. Analysis of biosensor tracings revealed GB5 bound specifically and with high affinity to both FV (K d = 0.6 nM; 2 experiments) and FVa (K d = 0.7 nM; 2 experiments). A TG assay with pooled normal human plasma (NHP) or HA plasma in the absence or presence of APC was used to assess the functional effects of GB5. In the absence of APC, GB5 had a minimal effect on the TG profile initiated with low tissue factor (0.2 pM). As expected, the addition of APC (2 nM, final) to NHP or HA plasma substantially reduced peak thrombin. However, GB5 dose-dependently increased peak thrombin in the presence of APC in NHP or HA plasma and restored TG to normal levels at ~20 nM. GB5 also protected FV from APC when assessed using a purified prothrombin activation assay. Western blotting experiments using FV proteolyzed with thrombin, APC, or plasmin revealed that GB5 binds the light chain A3-C1-C2.Preliminary experiments revealed that GB5 does not cross react with mouse FV. To evaluate the effect of GB5 in vivo, wild-type (WT) mice were treated with a liver targeted antisense oligonucleotide (ASO) to knockdown mouse plasma FV (ASO-FV; 40 mg/kg; FV-ASO mice). Mouse platelet FV was not impacted. To supplement the plasma compartment with FV, the FV-ASO mice were administered human FV (hFV-mice). This experimental set-up allowed for clot formation to largely be dependent on hFV using the mouse laser injury model. In these studies, we compared the kinetics of thrombus formation in WT mice, FV-ASO mice, or hFV-mice (n=2-3/group; 7-10 thrombi per group) for 10-15 minutes following laser injury. We found that platelet and fibrin accumulation was robust in WT mice and almost undetectable in FV-ASO mice (consistent with the lack of plasma FV), while clot formation was low but detectable in hFV-mice. There was no obvious difference in thrombus size when GB5 was given to WT or FV-ASO mice. However, in hFV-mice (200 μg/kg hFV), GB5 increased platelet and fibrin accumulation to levels seen with WT mice. Quantitative analysis revealed that compared to hFV mice alone, GB5 increased platelet accumulation 3-fold and fibrin (3-6-fold accumulation. Significantly greater accumulation of platelets and fibrin (~30-fold) was observed when higher amounts of hFV (400 μg/kg) were co-infused with GB5 compared to hFV-mice without antibody.Together, these results demonstrate that monoclonal antibody GB5 binds to hFV/FVa with high affinity and confers APC resistance. Using a unique mouse model to assess human FV, we found that GB5 enhanced clot formation in vivo using the laser injury model. From a mechanistic standpoint, these data show that protecting FV from APC translates to greater thrombin generation in vivo and suggest this approach may be useful to treat bleeding disorders such as HA and HB. Additional injury models using the hFV-mice and hemophilic mice to assess the effectiveness of GB5 are ongoing. DisclosuresCrosby: Ionis Pharmaceuticals: Other: Jeffrey is a current employee of Ionis Pharmaceuticals. Revenko: Ionis Pharmaceuticals: Other: Alexey is a current employee of the company.. MacLeod: Ionis Pharmaceuticals: Other: Robert is a current employee of the company. Monia: Ionis Pharmaceuticals: Other: Brett is a current employee of the company. .