Browsing by Subject "Factor VIII"
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Item Curing Hemophilia: Repeated Treatments versus a One-Off Fix(Elsevier, 2020-05-06) Li, Ning; Kaczmarek, Radoslaw; Pediatrics, School of MedicineItem Factor VIII trafficking to CD4+ T cells shapes its immunogenicity and requires several types of antigen-presenting cells(American Society of Hematology, 2023) Kaczmarek, Radoslaw; Piñeros, Annie R.; Patterson, Paige E.; Bertolini, Thais B.; Perrin, George Q.; Sherman, Alexandra; Born, Jameson; Arisa, Sreevani; Arvin, Matthew C.; Kamocka, Malgorzata M.; Martinez, Michelle M.; Dunn, Kenneth W.; Quinn, Sean M.; Morris, Johnathan J.; Wilhelm, Amelia R.; Kaisho, Tsuneyasu; Munoz-Melero, Maite; Biswas, Moanaro; Kaplan, Mark H.; Linnemann, Amelia K.; George, Lindsey A.; Camire, Rodney M.; Herzog, Roland W.; Pediatrics, School of MedicineDespite >80 years of clinical experience with coagulation factor VIII (FVIII) inhibitors, surprisingly little is known about the in vivo mechanism of this most serious complication of replacement therapy for hemophilia A. These neutralizing antidrug alloantibodies arise in ∼30% of patients. Inhibitor formation is T-cell dependent, but events leading up to helper T-cell activation have been elusive because of, in part, the complex anatomy and cellular makeup of the spleen. Here, we show that FVIII antigen presentation to CD4+ T cells critically depends on a select set of several anatomically distinct antigen-presenting cells, whereby marginal zone B cells and marginal zone and marginal metallophilic macrophages but not red pulp macrophages (RPMFs) participate in shuttling FVIII to the white pulp in which conventional dendritic cells (DCs) prime helper T cells, which then differentiate into follicular helper T (Tfh) cells. Toll-like receptor 9 stimulation accelerated Tfh cell responses and germinal center and inhibitor formation, whereas systemic administration of FVIII alone in hemophilia A mice increased frequencies of monocyte-derived and plasmacytoid DCs. Moreover, FVIII enhanced T-cell proliferation to another protein antigen (ovalbumin), and inflammatory signaling-deficient mice were less likely to develop inhibitors, indicating that FVIII may have intrinsic immunostimulatory properties. Ovalbumin, which, unlike FVIII, is absorbed into the RPMF compartment, fails to elicit T-cell proliferative and antibody responses when administered at the same dose as FVIII. Altogether, we propose that an antigen trafficking pattern that results in efficient in vivo delivery to DCs and inflammatory signaling, shape the immunogenicity of FVIII.Item First hemophilia B gene therapy approved: More than two decades in the making(Elsevier, 2023) Herzog, Roland W.; VandenDriessche, Thierry; Ozelo, Margareth C.; Pediatrics, School of MedicineItem Influence of N-glycosylation in the A and C domains on the immunogenicity of factor VIII(American Society of Hematology, 2022) Vander Kooi, Amber; Wang, Shuaishuai; Fan, Meng-Ni; Chen, Alex; Zhang, Junping; Chen, Chun-Yu; Cai, Xiaohe; Konkle, Barbara A.; Xiao, Weidong; Li, Lei; Miao, Carol H.; Pediatrics, School of MedicineThe most significant complication in hemophilia A treatment is the formation of inhibitors against factor VIII (FVIII) protein. Glycans and glycan-binding proteins are central to a properly functioning immune system. This study focuses on whether glycosylation of FVIII plays an important role in induction and regulation of anti-FVIII immune responses. We investigated the potential roles of 4 N-glycosylation sites, including N41 and N239 in the A1 domain, N1810 in the A3 domain, and N2118 in the C1 domain of FVIII, in moderating its immunogenicity. Glycomics analysis of plasma-derived FVIII revealed that sites N41, N239, and N1810 contain mostly sialylated complex glycoforms, while high mannose glycans dominate at site N2118. A missense variant that substitutes asparagine (N) to glutamine (Q) was introduced to eliminate glycosylation on each of these sites. Following gene transfer of plasmids encoding B domain deleted FVIII (BDD-FVIII) and each of these 4 FVIII variants, it was found that specific activity of FVIII in plasma remained similar among all treatment groups. Slightly increased or comparable immune responses in N41Q, N239Q, and N1810Q FVIII variant plasmid-treated mice and significantly decreased immune responses in N2118Q FVIII plasmid-treated mice were observed when compared with BDD-FVIII plasmid-treated mice. The reduction of inhibitor response by N2118Q FVIII variant was also demonstrated in AAV-mediated gene transfer experiments. Furthermore, a specific glycopeptide epitope surrounding the N2118 glycosylation site was identified and characterized to activate T cells in an FVIII-specific proliferation assay. These results indicate that N-glycosylation of FVIII can have significant impact on its immunogenicity.Item Inhibitor development according to concentrate after 50 exposure days in severe hemophilia: data from the European HAemophilia Safety Surveillance (EUHASS)(Elsevier, 2024-05-27) Fischer, Kathelijn; Lassila, Riitta; Peyvandi, Flora; Gatt, Alexander; Gouw, Samantha C.; Hollingsworth, Rob; Lambert, Thierry; Kaczmarek, Radek; Carbonero, Diana; Makris, Mike; European HAemophilia Safety Surveillance (EUHASS) participants; Pediatrics, School of MedicineBackground: Patients with hemophilia have a life-long risk of developing neutralizing antibodies (inhibitors) against clotting factor concentrates. After the first 50 exposure days (EDs), ie, in previously treated patients (PTPs), data on inhibitor development are limited. Objectives: To report inhibitor development according to factor (F)VIII or FIX concentrate use in PTPs with severe hemophilia A and B. Methods: Inhibitor development in PTPs was collected since 2008 from 97 centers participating in European HAemophilia Safety Surveillance. Per concentrate, inhibitors were reported quarterly and the number of PTPs treated annually. Incidence rates (IRs)/1000 treatment years with 95% CIs were compared between concentrate types (plasma derived FVIII/FIX, standard half-life recombinant FVIII/FIX, and extended half-life recombinant (EHL-rFVIII/IX) concentrates using IR ratios with CI. Medians and IQRs were calculated for inhibitor characteristics. Results: For severe haemophilia A, inhibitor rate was 66/65,200 treatment years, IR 1.00/1000 years (CI 0.80-1.30), occurring at median 13.5 years (2.7-31.5) and 150 EDs (80-773). IR on plasma-derived pdFVIII (IR, 1.13) and standard half-life recombinant FVIII (IR, 1.12) were similar, whereas IR on EHL-rFVIII was lower at 0.13 (incidence rate ratio, 0.12; 95% CI, <0.01-0.70; P < .01).For severe hemophilia B, inhibitor rate was 5/11,160 treatment years and IR was 0.45/1000 years (95% CI, 0.15-1.04), at median 3.7 years (95% CI, 2.1-42.4) and 260 EDs (95% CI, 130 to >1000). Data were insufficient to compare by type of FIX concentrates. Conclusion: Low inhibitor rates were observed for PTPs with severe hemophilia A and B. Data suggested reduced inhibitor development on EHL-rFVIII, but no significant difference between plasma-derived FVIII and standard half-life recombinant FVIII. FIX inhibitor rates were too low for robust statistical analysis.Item Inhibitor development according to concentrate in severe hemophilia: reporting on 1392 Previously Untreated Patients from Europe and Canada(Elsevier, 2023-11-20) Fischer, Kathelijn; Lassila, Riitta; Peyvandi, Flora; Gatt, Alexander; Hollingsworth, Rob; Lambert, Thierry; Kaczmarek, Radek; Bettle, Amanda; Samji, Nasrin; Rivard, Georges-Étienne; Carcao, Manuel; Iorio, Alfonso; Makris, Mike; Pediatrics, School of MedicineBackground: Clotting factor concentrates have been the mainstay of severe hemophilia treatment over the last 50 years. Differences in risk of neutralizing antibody (inhibitor) formation according to concentrate used remain clinically relevant. Objectives: To assess inhibitor development according to type of clotting factor concentrate in previously untreated patients (PUPs) with severe hemophilia A and B. Methods: The European Haemophilia Safety Surveillance (EUHASS) and Canadian Bleeding Disorders Registry (CBDR) have been monitoring adverse events overall and according to concentrate for 11 and 8 years, respectively. Inhibitors were reported quarterly, and PUPs completed 50 exposure days without inhibitor development annually. Cumulative inhibitor incidences and 95% confidence intervals (CIs) were compared without adjustment for other risk factors. Results: Fifty-six European and 23 Canadian centers reported inhibitor development in 312 of 1219 (26%; CI, 23%-28%) PUPs with severe hemophilia A and 14 of 173 (8%; CI, 5%-13%) PUPs with severe hemophilia B. Inhibitor development was lower on plasma-derived factor (F)VIII (pdFVIII, 20%; CI, 14%-26%) than on standard half-life recombinant FVIII (SHL-rFVIII, 27%; CI, 24%-30% and odds ratio, 0.67; CI, 0.45%-0.98%; P = .04). Extended half-life recombinant FVIII (EHL-rFVIII, 22%; CI, 12%-36%) showed an intermediate inhibitor rate, while inhibitor rates for Advate (26%; CI, 22%-31%) and Kogenate/Helixate (30%; CI, 24%-36%) overlapped. For other SHL-rFVIII concentrates, inhibitor rates varied from 3% to 43%. Inhibitor development was similar for pdFIX (11%; CI, 3%-25%), SHL-rFIX (8%; CI, 3%-15%), and EHL-rFIX (7%; CI, 1%-22%). Conclusion: While confirming expected rates of inhibitors in PUPs, inhibitor development was lower in pdFVIII than in SHL-rFVIII. Preliminary data suggest variation in inhibitor development among different SHL-rFVIII and EHL-rFVIII concentrates.Item Minimal Essential Human Factor VIII Alterations Enhance Secretion and Gene Therapy Efficiency(Elsevier, 2020-10-22) Cao, Wenjing; Dong, Biao; Horling, Franziska; Firrman, Jenni A.; Lengler, Johannes; Klugmann, Matthias; de la Rosa, Maurus; Wu, Wenman; Wang, Qizhao; Wei, Hongying; Moore, Andrea R.; Roberts, Sean A.; Booth, Carmen J.; Hoellriegl, Werner; Li, Dong; Konkle, Barbara; Miao, Carol; Reipert, Birgit M.; Scheiflinger, Friedrich; Rottensteiner, Hanspeter; Xiao, Weidong; Pediatrics, School of MedicineOne important limitation for achieving therapeutic expression of human factor VIII (FVIII) in hemophilia A gene therapy is inefficient secretion of the FVIII protein. Substitution of five amino acids in the A1 domain of human FVIII with the corresponding porcine FVIII residues generated a secretion-enhanced human FVIII variant termed B-domain-deleted (BDD)-FVIII-X5 that resulted in 8-fold higher FVIII activity levels in the supernatant of an in vitro cell-based assay system than seen with unmodified human BDD-FVIII. Analysis of purified recombinant BDD-FVIII-X5 and BDD-FVIII revealed similar specific activities for both proteins, indicating that the effect of the X5 alteration is confined to increased FVIII secretion. Intravenous delivery in FVIII-deficient mice of liver-targeted adeno-associated virus (AAV) vectors designed to express BDD-FVIII-X5 or BDD-FVIII achieved substantially higher plasma FVIII activity levels for BDD-FVIII-X5, even when highly efficient codon-optimized F8 nucleotide sequences were employed. A comprehensive immunogenicity assessment using in vitro stimulation assays and various in vivo preclinical models of hemophilia A demonstrated that the BDD-FVIII-X5 variant does not exhibit an increased immunogenicity risk compared to BDD-FVIII. In conclusion, BDD-FVIII-X5 is an effective FVIII variant molecule that can be further developed for use in gene- and protein-based therapeutics for patients with hemophilia A.Item Optimizing liver health before and after gene therapy for hemophilia A(American Society of Hematology, 2024) Ragni, Margaret V.; Mead, Henry; de Jong, Ype P.; Kaczmarek, Radoslaw; Leavitt, Andrew D.; Long, Brian; Nugent, Diane J.; Sabatino, Denise E.; Fong, Sylvia; von Drygalski, Annette; Walsh, Christopher E.; Luxon, Bruce A.; Pediatrics, School of MedicineGene therapy for severe hemophilia A uses an adeno-associated virus (AAV) vector and liver-specific promoters that depend on healthy hepatocyte function to achieve safe and long-lasting increases in factor VIII (FVIII) activity. Thus, hepatocyte health is an essential aspect of safe and successful gene therapy. Many people living with hemophilia A have current or past chronic hepatitis C virus infection, metabolic dysfunction-associated steatosis or steatohepatitis, or other conditions that may compromise the efficacy and safety of AAV-mediated gene therapy. In addition, gene therapy may induce an immune response to transduced hepatocytes, leading to liver inflammation and reduced FVIII activity. The immune response can be treated with immunosuppression, but close monitoring of liver function tests and factor levels is necessary. The long-term risk of hepatocellular carcinoma associated with gene therapy is unknown. Routine screening by imaging for hepatocellular carcinoma, preferable every 6 months, is essential in patients at high risk and recommended in all recipients of hemophilia A gene therapy. This paper describes our current understanding of the biologic underpinnings of how liver health affects hemophilia A gene therapy, and provides practical clinical guidance for assessing, monitoring, and managing liver health both before and after gene therapy.Item Ultrasound-mediated gene delivery of factor VIII plasmids for hemophilia A gene therapy in mice(Elsevier, 2022-01-10) Song, Shuxian; Lyle, Meghan J.; Noble-Vranish, Misty L.; Min-Tran, Dominic M.; Harrang, James; Xiao, Weidong; Unger, Evan C.; Miao, Carol H.; Pediatrics, School of MedicineGene therapy offers great promises for a cure of hemophilia A resulting from factor VIII (FVIII) gene deficiency. We have developed and optimized a non-viral ultrasound-mediated gene delivery (UMGD) strategy. UMGD of reporter plasmids targeting mice livers achieved high levels of transgene expression predominantly in hepatocytes. Following UMGD of a plasmid encoding human FVIII driven by a hepatocyte-specific promoter/enhancer (pHP-hF8/N6) into the livers of hemophilia A mice, a partial phenotypic correction was achieved in treated mice. In order to achieve persistent and therapeutic FVIII gene expression, we adopted a plasmid (pHP-hF8-X10) encoding an FVIII variant with significantly increased FVIII secretion. By employing an optimized pulse-train ultrasound condition and immunomodulation, the treated hemophilia A mice achieved 25%–150% of FVIII gene expression on days 1–7 with very mild transient liver damage, as indicated by a small increase of transaminase levels that returned to normal within 3 days. Therapeutic levels of FVIII can be maintained persistently without the generation of inhibitors in mice. These results indicate that UMGD can significantly enhance the efficiency of plasmid DNA transfer into the liver. They also demonstrate the potential of this novel technology to safely and effectively treat hemophilia A.