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Background: Hemophilia A (HA) and hemophilia B (HB) are rare inherited bleeding disorders. Although causative genetic variants are clinically relevant, in 2012 only 20% of US patients had been genotyped., Objectives: My Life, Our Future (MLOF) was a multisector cross-sectional US initiative to improve our understanding of hemophilia through widespread genotyping., Methods: Subjects and potential genetic carriers were enrolled at US hemophilia treatment centers (HTCs). Bloodworks performed genotyping and returned results to providers. Clinical data were abstracted from the American Thrombosis and Hemostasis Network dataset. Community education was provided by the National Hemophilia Foundation., Results: From 2013 to 2017, 107 HTCs enrolled 11 341 subjects (68.8% male, 31.2% female) for testing for HA (n = 8976), HB (n = 2358), HA/HB (n = 3), and hemophilia not otherwise specified (n = 4). Variants were detected in most male patients (98.2%% HA, 98.1% HB). 1914 unique variants were found (1482 F8, 431 F9); 744 were novel (610 F8, 134 F9). Inhibitor data were available for 6986 subjects (5583 HA; 1403 HB). In severe HA, genotypes with the highest inhibitor rates were large deletions (77/80), complex intron 22 inversions (9/17), and no variant found (7/14). In severe HB, the highest rates were large deletions (24/42). Inhibitors were reported in 27.3% of Black versus 16.2% of White patients., Conclusions: The findings of MLOF are reported, the largest hemophilia genotyping project performed to date. The results support the need for comprehensive genetic approaches in hemophilia. This effort has contributed significantly towards better understanding variation in the F8 and F9 genes in hemophilia and risks of inhibitor formation., (© 2022 International Society on Thrombosis and Haemostasis.)

Gene therapy has the potential to maintain therapeutic blood clotting factor IX (FIX) levels in patients with hemophilia B by delivering a functional human F9 gene into liver cells. This phase 1/2, open-label dose-escalation study investigated BAX 335 (AskBio009, AAV8.sc-TTR-FIXR338Lopt), an adeno-associated virus serotype 8 (AAV8)-based FIX Padua gene therapy, in patients with hemophilia B. This report focuses on 12-month interim analyses of safety, pharmacokinetic variables, effects on FIX activity, and immune responses for dosed participants. Eight adult male participants (aged 20-69 years; range FIX activity, 0.5% to 2.0%) received 1 of 3 BAX 335 IV doses: 2.0 × 1011; 1.0 × 1012; or 3.0 × 1012 vector genomes/kg. Three (37.5%) participants had 4 serious adverse events, all considered unrelated to BAX 335. No serious adverse event led to death. No clinical thrombosis, inhibitors, or other FIX Padua-directed immunity was reported. FIX expression was measurable in 7 of 8 participants; peak FIX activity displayed dose dependence (32.0% to 58.5% in cohort 3). One participant achieved sustained therapeutic FIX activity of ∼20%, without bleeding or replacement therapy, for 4 years; in others, FIX activity was not sustained beyond 5 to 11 weeks. In contrast to some previous studies, corticosteroid treatment did not stabilize FIX activity loss. We hypothesize that the loss of transgene expression could have been caused by stimulation of innate immune responses, including CpG oligodeoxynucleotides introduced into the BAX 335 coding sequence by codon optimization. This trial was registered at www.clinicaltrials.gov as #NCT01687608., (© 2021 by The American Society of Hematology.)

Aims: Approximately 20,000 hematopoietic cell transplantation (HCT) procedures are performed in the US annually. This study aims to study the healthcare resource utilization and costs among commercially-insured patients with hematologic malignancies who received autologous HCT (auto-HCT) and allogeneic HCT (allo-HCT) in the US., Materials and Methods: Adult patients with hematologic malignancies undergoing auto- or allo-HCT between January 1, 2011 and June 30, 2014 were identified in the Truven Health MarketScan Research Databases. Patients with 12 months of continuous pharmacy and medical enrollment pre- and post-HCT were included. Patients with prior HCT were excluded. Controls were selected from patients without any claims for HCT and matched with HCT recipients in a 3:1 ratio based on age, gender, insurance type, and Deyo-Charlson Comorbidity Index categories. Total healthcare resource uses and costs were compared between auto- or allo-HCT recipients and controls., Results: In total, 10,527 patients (HCT, n = 2,672 vs control, n = 7,855) were included, with the majority of HCT recipients (63.6%) undergoing auto-HCT. During the 6-month pre-index and 12-month post-index period, auto-HCT recipients incurred $313,562 (p < .01) higher all-cause costs than controls, attributable to inpatient admission (54.1%), outpatient services (33.4%), and prescriptions (12.5%). The all-cause costs for allo-HCT recipients were $621,895 (p < .01) higher vs controls during the 18-month observation period, attributable to inpatient admissions (75.5%), outpatient services (22.1%), and prescriptions (2.4%)., Conclusions: The use of HCT among patients with hematologic malignancies is associated with considerable economic burden in direct healthcare costs in a commercially insured population. Incremental costs for HCT recipients were mainly driven by costs related to hospitalization and other medical services.

Approximately 20,000 hematopoietic cell transplantation (HCT) procedures are performed annually in the United States. Real-world data on the costs associated with post-transplantation complications are limited. Patients with hematologic malignancies aged ≥18 years undergoing autologous HCT (auto-HCT) or allogeneic HCT (allo-HCT) between January 1, 2011, and June 30, 2014, were identified in the Truven Health MarketScan Research Databases. Patients were required to have 12 months of continuous medical and pharmacy enrollment before and after HCT; patients who experience inpatient death within 12 months post-HCT were also included. Patients with previous HCT were excluded. Potential HCT-related complications were identified if they had a medical claim with a diagnosis code for relapse; infection; cardiovascular, renal, neurologic, pulmonary, hepatic, or gastrointestinal disease; secondary malignancy; thrombotic microangiopathy; or posterior reversible encephalopathy syndrome within 1 year post-HCT. Healthcare costs attributable to these complications were evaluated by comparing total costs in HCT recipients with complications and those without complications. The MarketScan Research Databases were further linked to the Social Security Administration's Master Death File to obtain patient death events in a subset of patients. A total of 2672 HCT recipients were included in the analysis. The mean ± SD age of recipients was 54.5 ± 11.6 years, and the majority of recipients (63.6%) underwent auto-HCT. Complications were identified in 81% of auto-HCT recipients and in 95.5% of allo-HCT recipients. Most complications occurred within 180 days post-HCT. Compared with Auto-HCT recipients without complications, those with complications incurred $51,475 higher adjusted total costs (P < .01). Compared with allo-HCT recipients without complications, those with complications incurred $181,473 higher adjusted total costs (P < .01). Among the patients with mortality data, auto-HCT recipients with complications had a higher mortality rate (13.4% vs 5.7%, P < .01) and a lower probability of survival (P < .01) compared with those without complications. In allo-HCT recipients, however, the mortality rate and probability of survival were not significantly different between those with complications and those without complications. HCT recipients with complications were associated with considerable economic burden in terms of direct healthcare costs in a commercially insured population, and in the case of auto-HCT, a higher mortality rate was observed in those with complications., (Copyright © 2017 The American Society for Blood and Marrow Transplantation. Published by Elsevier Inc. All rights reserved.)

Hemophilia A and B are rare, X-linked bleeding disorders. My Life, Our Future (MLOF) is a collaborative project established to genotype and study hemophilia. Patients were enrolled at US hemophilia treatment centers (HTCs). Genotyping was performed centrally using next-generation sequencing (NGS) with an approach that detected common F8 gene inversions simultaneously with F8 and F9 gene sequencing followed by confirmation using standard genotyping methods. Sixty-nine HTCs enrolled the first 3000 patients in under 3 years. Clinically reportable DNA variants were detected in 98.1% (2357/2401) of hemophilia A and 99.3% (595/599) of hemophilia B patients. Of the 924 unique variants found, 285 were novel. Predicted gene-disrupting variants were common in severe disease; missense variants predominated in mild-moderate disease. Novel DNA variants accounted for ∼30% of variants found and were detected continuously throughout the project, indicating that additional variation likely remains undiscovered. The NGS approach detected >1 reportable variants in 36 patients (10 females), a finding with potential clinical implications. NGS also detected incidental variants unlikely to cause disease, including 11 variants previously reported in hemophilia. Although these genes are thought to be conserved, our findings support caution in interpretation of new variants. In summary, MLOF has contributed significantly toward variant annotation in the F8 and F9 genes. In the near future, investigators will be able to access MLOF data and repository samples for research to advance our understanding of hemophilia., Competing Interests: Conflict-of-interest disclosure: J.M. is an employee of Bioverativ (which funded MLOF). The remaining authors declare no competing financial interests.

In the phase 3 B-LONG (Recombinant Factor IX Fc Fusion Protein [rFIXFc] in Subjects With Haemophilia B) study, rFIXFc demonstrated a prolonged half-life compared with recombinant factor IX (rFIX), and safety and efficacy for prophylaxis and treatment of bleeding in subjects with moderately-severe to severe haemophilia B. In this B-LONG sub-analysis, rFIXFc was evaluated for efficacy in subjects requiring major surgery. Dosing was investigator-determined. Assessments included dosing, consumption, bleeding, transfusions and haemostatic response. A population pharmacokinetics model of rFIXFc was used to predict FIX activity. Twelve subjects underwent 14 major surgeries (including 11 orthopaedic surgeries); most subjects (11/12) received rFIXFc prophylaxis before surgery (range, ~2 weeks-12 months). Investigators/surgeons rated haemostatic responses as excellent (n = 13) or good (n = 1). In most surgeries (85·7%), haemostasis from the pre-surgical dose until the end of surgery was maintained with a single rFIXFc infusion. Blood loss was consistent with similar surgeries in subjects without haemophilia. The strong correlation (R(2) = 0·9586, P < 0·001) between observed and population pharmacokinetic model-predicted FIX activity suggests surgery did not impact rFIXFc pharmacokinetics. No unique safety concerns or inhibitors were observed. In conclusion, rFIXFc was safe and efficacious, with prolonged dosing intervals and low consumption, when used perioperatively in haemophilia B. Surgery did not appear to alter rFIXFc pharmacokinetics., (© 2014 Biogen Idec. British Journal of Haematology published by John Wiley & Sons Ltd.)

This phase 3 pivotal study evaluated the safety, efficacy, and pharmacokinetics of a recombinant FVIII Fc fusion protein (rFVIIIFc) for prophylaxis, treatment of acute bleeding, and perioperative hemostatic control in 165 previously treated males aged ≥12 years with severe hemophilia A. The study had 3 treatment arms: arm 1, individualized prophylaxis (25-65 IU/kg every 3-5 days, n = 118); arm 2, weekly prophylaxis (65 IU/kg, n = 24); and arm 3, episodic treatment (10-50 IU/kg, n = 23). A subgroup compared recombinant FVIII (rFVIII) and rFVIIIFc pharmacokinetics. End points included annualized bleeding rate (ABR), inhibitor development, and adverse events. The terminal half-life of rFVIIIFc (19.0 hours) was extended 1.5-fold vs rFVIII (12.4 hours; P < .001). Median ABRs observed in arms 1, 2, and 3 were 1.6, 3.6, and 33.6, respectively. In arm 1, the median weekly dose was 77.9 IU/kg; approximately 30% of subjects achieved a 5-day dosing interval (last 3 months on study). Across arms, 87.3% of bleeding episodes resolved with 1 injection. Adverse events were consistent with those expected in this population; no subjects developed inhibitors. rFVIIIFc was well-tolerated, had a prolonged half-life compared with rFVIII, and resulted in low ABRs when dosed prophylactically 1 to 2 times per week.

Background: Prophylactic factor replacement in patients with hemophilia B improves outcomes but requires frequent injections. A recombinant factor IX Fc fusion protein (rFIXFc) with a prolonged half-life was developed to reduce the frequency of injections required., Methods: We conducted a phase 3, nonrandomized, open-label study of the safety, efficacy, and pharmacokinetics of rFIXFc for prophylaxis, treatment of bleeding, and perioperative hemostasis in 123 previously treated male patients. All participants were 12 years of age or older and had severe hemophilia B (endogenous factor IX level of ≤2 IU per deciliter, or ≤2% of normal levels). The study included four treatment groups: group 1 received weekly dose-adjusted prophylaxis (50 IU of rFIXFc per kilogram of body weight to start), group 2 received interval-adjusted prophylaxis (100 IU per kilogram every 10 days to start), group 3 received treatment as needed for bleeding episodes (20 to 100 IU per kilogram), and group 4 received treatment in the perioperative period. A subgroup of group 1 underwent comparative sequential pharmacokinetic assessments of recombinant factor IX and rFIXFc. The primary efficacy end point was the annualized bleeding rate, and safety end points included the development of inhibitors and adverse events., Results: As compared with recombinant factor IX, rFIXFc exhibited a prolonged terminal half-life (82.1 hours) (P<0.001). The median annualized bleeding rates in groups 1, 2, and 3 were 3.0, 1.4, and 17.7, respectively. In group 2, 53.8% of participants had dosing intervals of 14 days or more during the last 3 months of the study. In groups 1, 2 and 3, 90.4% of bleeding episodes resolved after one injection. Hemostasis was rated as excellent or good during all major surgeries. No inhibitors were detected in any participants receiving rFIXFc; in groups 1, 2, and 3, 73.9% of participants had at least one adverse event, and serious adverse events occurred in 10.9% of participants. These events were mostly consistent with those expected in the general population of patients with hemophilia., Conclusions: Prophylactic rFIXFc, administered every 1 to 2 weeks, resulted in low annualized bleeding rates in patients with hemophilia B. (Funded by Biogen Idec; ClinicalTrials.gov number, NCT01027364.).

Utilization of the synthetic vasopressin analogue (1-deamino-8-D-arginine-vasopressin, DDAVP) in treatment of mild haemophilia A (MHA, specific clotting factor VIII activity level 0.05-0.4 IU mL(-1) ) is convenient and effective for many but not all patients. Genetic testing for patients with MHA is increasingly recognized as providing valuable information for patient care beyond informing reproductive decisions, and as more patients are genotyped, mutation data can be utilized to individualize treatment decisions. To determine if genetic information informs response to DDAVP, a retrospective chart review was performed under Institutional Review Board approval to extract patient data with MHA, genetic mutation results, and response to DDAVP challenge. 62 patients met inclusion criteria. Complete responses (C) presented in mean value IU mL(-1) (range), were recorded for 32 of 62(52%) subjects: pre 0.19(0.04-0.45) and post 0.78(0.5-1.95); partial responses (P) were recorded for 15 of 62(24%) subjects: pre 0.1(0.06-0.15) and post 0.4(0.3-0.47); responses that were not clinically significant (N) were recorded for 15 of 62(24%) subjects: pre 0.17(0.02-0.34) and post 0.25(0.03-0.44). Subjects (related and unrelated) with the same mutation showed a trend towards a similar response to DDAVP. Eight genotypes were common to two or more subjects (n = 26). Two genotypes were concordant in all subjects [p.Ser2192Ile n = 3(C), p.Ala2220Pro n = 2(P)]. Of mutations in the C1 or C2 domains, 13 of 15(87%) subjects responded to DDAVP [C = 9(60%); P = 4(27%); n = 2(13%)]. Baseline FVIII:C did not predict magnitude of response to DDAVP. Genetic mutation results can assist with predicting DDAVP responsiveness, but baseline FVIII:C may not., (© 2013 John Wiley & Sons Ltd.)

Acquired von Willebrand syndrome (AVWS) is a rare bleeding disorder that typically presents as mucocutaneous bleeding in individuals with no personal or family history of bleeding disorder. Here we present a case in which a patient presented with profound epistaxis and was found to have AVWS in the setting of monoclonal gammopathy of undetermined significance (MGUS).

Evidence of antibody isotype/subtype switching may provide prognostic value regarding the state of immune responses to therapeutic proteins, e.g. anti-factor VIII (FVIII) antibodies that develop in many hemophilia A patients, clinically termed "inhibitors". A sensitive, high- information-content surface plasmon resonance (SPR) assay has been developed to quantify IgG subtype distributions and the domain specificity of anti-drug antibodies. Plasma samples from 22 subjects with an allo- or auto-immune reaction to FVIII were analyzed. Pre-analytical treatment protocols were developed to minimize non-specific binding and specific matrix interference due to von Willebrand factor-FVIII interactions. The dynamic range for IgG quantification was 0.2-5 µg/ml (∼1-33 nM), allowing characterization of inhibitor-positive samples. Subtype-specific monoclonal antibodies were used to quantify the IgG subtype distribution of FVIII-specific antibodies. Most samples obtained from multiply-infused inhibitor subjects contained IgG₄ antibodies. Several distinct phenotypes were assigned based on the IgG subtype distribution: IgG₁, IgG₄, IgG₁ & IgG₄, and IgG₁, IgG₂ & IgG₄. An IgG₁-only response was found in mild/moderate HA subjects during early FVIII infusions, and analysis of serial samples followed antibody class switching as several subjects' immune responses developed. Competition studies utilizing a recombinant FVIII-C2 domain indicated 40-80% of FVIII-specific antibodies in most samples were directed against this domain.

Many risk factors for falls identified in the general population are found in patients with haemophilia. Furthermore, fall risk increases with age and patients with haemophilia are increasingly entering the over 65 age group. After a fall occurs, there are often behavioural changes that have significant health consequences and further increase fall risk. Fall risk can be quickly assessed in the clinical setting with specific questions in the medical history and by a variety of performance-based screening tools. Identification of fall risk enables early intervention, thereby preventing injury and fear of physical activity, both of which have been associated with falling and may carry an increased risk in patients with haemophilia. Review of the existing literature on assessment of fall risk reveals the importance of screening in the clinical setting, which is commonly done via a fall history and performance-based assessment tools. Selecting appropriate fall risk screening tools is an important step in identifying and providing optimal interventions for those at risk. Assessments of fall history, fear of falling, gait velocity, gait variability and vestibular dysfunction are suggested as screening tools for patients with haemophilia. Additional research is needed to determine the optimal screening, evaluation and treatment techniques for these patients. The longitudinal physical therapy care provided by Haemophilia Treatment Centres presents a unique opportunity for instituting measures that will reduce the incidence of falling in patients with haemophilia., (© 2012 Blackwell Publishing Ltd.)

Women with factor X deficiency (FXD) who want to become pregnant face uncertain risks to themselves and to an unborn infant from haemorrhagic complications during pregnancy and at parturition. Women with FXD may also experience difficulty achieving pregnancy secondary to haemorrhagic symptoms of the reproductive organs. Case reports describe differences in bleeding phenotypes and pregnancy outcomes that are not easily correlated with prepregnancy bleeding symptoms or factor X levels. The aim of this article is to identify factors for consideration and information to assist the physician in counselling women with FXD who want to become pregnant, and to offer guidelines for management where appropriate. We identified cases of pregnancy among women with FXD and their outcomes from the literature; 15 women with 24 pregnancies were identified and 18 were successful. The women in this small cohort did not have an increased rate of spontaneous abortion, (8.3% vs. 13.5% in the general US population) but did have a 2.5-fold increased risk of preterm labour (37.5% vs. 12.2% in the general US population). The role of prophylaxis to control reproductive haemorrhagic symptoms, including haemorrhagic complications of pregnancy has not yet been defined, but use of prophylaxis may allow more women to be able to attempt pregnancy. Women who had access to a tertiary care centre with a multidisciplinary team including an obstetrician with high-risk obstetric training, a haematologist, a perinatologist, and access to a reference laboratory and blood bank were able in most cases to successfully deliver healthy, term infants., (© 2011 Blackwell Publishing Ltd.)

Current factor VIII (FVIII) products display a half-life (t(1/2)) of ∼ 8-12 hours, requiring frequent intravenous injections for prophylaxis and treatment of patients with hemophilia A. rFVIIIFc is a recombinant fusion protein composed of a single molecule of FVIII covalently linked to the Fc domain of human IgG(1) to extend circulating rFVIII t(1/2). This first-in-human study in previously treated subjects with severe hemophilia A investigated safety and pharmacokinetics of rFVIIIFc. Sixteen subjects received a single dose of rFVIII at 25 or 65 IU/kg followed by an equal dose of rFVIIIFc. Most adverse events were unrelated to study drug. None of the study subjects developed anti-rFVIIIFc antibodies or inhibitors. Across dose levels, compared with rFVIII, rFVIIIFc showed 1.54- to 1.70-fold longer elimination t(1/2), 1.49- to 1.56-fold lower clearance, and 1.48- to 1.56-fold higher total systemic exposure. rFVIII and rFVIIIFc had comparable dose-dependent peak plasma concentrations and recoveries. Time to 1% FVIII activity above baseline was ∼ 1.53- to 1.68-fold longer than rFVIII across dose levels. Each subject showed prolonged exposure to rFVIIIFc relative to rFVIII. Thus, rFVIIIFc may offer a viable therapeutic approach to achieve prolonged hemostatic protection and less frequent dosing in patients with hemophilia A. This trial was registered at www.clinicaltrials.gov as NCT01027377.

Current factor IX (FIX) products display a half-life (t(1/2)) of ∼ 18 hours, requiring frequent intravenous infusions for prophylaxis and treatment in patients with hemophilia B. This open-label, dose-escalation trial in previously treated adult subjects with hemophilia B examined the safety and pharmacokinetics of rFIXFc. rFIXFc is a recombinant fusion protein composed of FIX and the Fc domain of human IgG(1), to extend circulating time. Fourteen subjects received a single dose of rFIXFc; 1 subject each received 1, 5, 12.5, or 25 IU/kg, and 5 subjects each received 50 or 100 IU/kg. rFIXFc was well tolerated, and most adverse events were mild or moderate in intensity. No inhibitors were detected in any subject. Dose-proportional increases in rFIXFc activity and Ag exposure were observed. With baseline subtraction, mean activity terminal t(1/2) and mean residence time for rFIXFc were 56.7 and 71.8 hours, respectively. This is ∼ 3-fold longer than that reported for current rFIX products. The incremental recovery of rFIXFc was 0.93 IU/dL per IU/kg, similar to plasma-derived FIX. These results show that rFIXFc may offer a viable therapeutic approach to achieve prolonged hemostatic protection and less frequent dosing in patients with hemophilia B. The trial was registered at www.clinicaltrials.gov as NCT00716716.

Every other day (qod) factor VIII prophylaxis prevents joint bleeds in children with severe haemophilia A. Although three times weekly or qod prophylaxis is recommended by the National Hemophilia Foundation (NHF), how widely these practices have been adopted is not known. We sought to define current prophylaxis practices at US haemophilia treatment centres (HTCs). An email survey was distributed to US HTCs, utilizing web-based membership rosters of the Centers for Disease Control (CDC) and the Hemostasis Thrombosis Research Society (HTRS). Of 62 HTCs responding, prophylaxis is initiated on a three times weekly schedule in 29 (46.8%), twice weekly in 13 HTCs (21.0%) and once weekly in 20 HTCs (32.2%). Central venous catheters are used to infuse factor prophylactically at 55 HTCs (88.7%), including in 100% of children initiating prophylaxis at 19 HTCs (30.6%) and in 50% of those at 41 HTCs (66.1%), but avoided altogether at seven HTCs (11.3%). Prophylaxis is initiated after one or more bleeds in 56 HTCs (90.3%), but after the first bleed in only 28 HTCs (25.2%). Among 226 newborns with severe haemophilia A in 62 HTCs, 1.82 births/HTC/year, the median age at first bleed, excluding circumcision, is 7 months. Of the 113 (53.5%) newborns who underwent circumcision, 62 (54.9%) bled. Despite a recommended standard of three times weekly prophylaxis, over half of surveyed HTCs do not follow these guidelines, and nearly one-third begin prophylaxis on a once weekly schedule to delay or avoid the need for central venous access., (© 2011 Blackwell Publishing Ltd.)

The strength of the streptavidin/biotin interaction poses challenges for the recovery of biotinylated molecules from streptavidin resins. As an alternative to high-temperature elution in urea-containing buffers, we show that mono-biotinylated proteins can be released with relatively gentle heating in the presence of biotin and 2% SDS/Rapigest, avoiding protein carbamylation and minimizing streptavidin dissociation. We demonstrate the utility of this mild elution strategy in two studies of the human androgen receptor (AR). In the first, in which formaldehyde cross-linked complexes are analyzed in yeast, a mass spectrometry-based comparison of the AR complex using SILAC reveals an association between the androgen-activated AR and the Hsp90 chaperonin, while Hsp70 chaperonins associate specifically with the unliganded complex. In the second study, the endogenous AR is quantified in the LNCaP cell line by absolute SILAC and MRM-MS showing approximately 127,000 AR copies per cell, substantially more than previously measured using radioligand binding., (Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Current methods for eradicating clinically significant inhibitory antibodies to human factor VIII (hFVIII) in patients with hemophilia A rely on repeated delivery of high doses of factor concentrates for a minimum of many months. We hypothesize that tolerance can be induced more efficiently and reliably through hFVIII antigen presentation by tolerogenic dendritic cells (tDCs). In this study, we generated tDCs from hemophilia A mice and modified them with a foamy virus vector expressing a bioengineered hFVIII transgene. Naive and preimmunized mice infused with hFVIII expressing tDCs showed suppression of the T cell and inhibitor responses to recombinant hFVIII (rhFVIII). Treatment with hFVIII expressing tDCs was also associated with a higher percentage of splenocytes demonstrating a regulatory T cell phenotype in immunized mice. Furthermore, CD4(+) T cells harvested from recipients of hFVIII expression vector-modified tDCs were able to mediate antigen-specific immune suppression in naive secondary recipients. We also demonstrated a trend for improved suppression of inhibitor formation by coexpressing interleukin-10 (IL-10) and hFVIII from a bicistronic vector. These preclinical results demonstrate the potential for employing vector modified ex vivo generated tDCs to treat high titer inhibitors in patients with hemophilia A.

Foamy viruses (FVs), or spumaviruses, are nonpathogenic retroviruses that have been developed as integrating viral vectors. This protocol presents methods for producing high-titer FV vector stocks, free of contaminating replication-competent retrovirus, to be used for transducing hematopoietic stem cells. FV vector stocks are produced by transfecting 293 cells, harvesting and filtering the culture medium, and concentrating vector virions by ultracentrifugation. The resulting stocks are free of replication-competent helper virus, as indicated by a sensitive marker rescue assay. A typical stock made from 23 10-cm dishes has a final volume of 2 mL with a titer of 10(7) to 10(8) transducing units/mL. Potential advantages of FV vectors include a lack of pathogenicity of the wild-type virus, a wide host range, stable virions that can be concentrated by centrifugation, a double-stranded DNA genome that is reverse-transcribed in the vector-producing cells, and the largest packaging capacity of any retrovirus. FV vectors are especially useful for transducing hematopoietic cells. Because hematopoietic stem cells have the ability to self-renew, proliferate, and repopulate the bone marrow after transplantation, efficient transduction of these cells offers the promise to cure many inherited and acquired diseases.

The development of inhibitory antibodies to factor VIII (FVIII) is currently the most significant complication of FVIII replacement therapy in the management of patients with severe hemophilia A. Immune tolerance protocols for the eradication of inhibitors require daily delivery of intravenous FVIII for at least 6 months and are unsuccessful in 20-40% of treated patients. We hypothesize that tolerance can be induced more efficiently and reliably by delivery of FVIII antigen within autologous apoptotic cells (ACs). In this study, we demonstrated suppression of the T cell and inhibitor responses to FVIII by infusion of FVIII expression vector modified apoptotic syngeneic fibroblasts in both naive and preimmunized hemophilia A mice. ACs without FVIII antigen exerted modest generalized immune suppression mediated by anti-inflammatory signals. However, FVIII expressing apoptotic syngeneic fibroblasts produced much stronger antigen-specific immune suppression. Mice treated with these fibroblasts generated CD4+ T cells that suppressed the immune response to FVIII after adoptive transfer into naive recipients and antigen-specific CD4+CD25+ regulatory T cells (Tregs) that inhibited the proliferation of FVIII responsive effector T cells in vitro. These preclinical results demonstrate the potential for using FVIII vector modified autologous ACs to treat high-titer inhibitors in patients with hemophilia A.

Expression of Mpl is restricted to hematopoietic cells in the megakaryocyte lineage and to undifferentiated progenitors, where it initiates critical cell survival and proliferation signals after stimulation by its ligand, thrombopoietin (TPO). As a result, a deficiency in Mpl function in patients with congenital amegakaryocytic thrombocytopenia (CAMT) and in mpl(-/-) mice produces profound thrombocytopenia and a severe stem cell-repopulating defect. Gene therapy has the potential to correct the hematopoietic defects of CAMT by ectopic gene expression that restores normal Mpl receptor activity. We rescued the mpl(-/-) mouse with a transgenic vector expressing mpl from the promoter elements of the 2-kb region of DNA just proximal to the natural gene start site. Transgene rescued mice exhibit thrombocytosis but only partial correction of the stem cell defect. Furthermore, they show very low-level expression of Mpl on platelets and megakaryocytes, and the transgene-rescued megakaryocytes exhibit diminished TPO-dependent kinase phosphorylation and reduced platelet production in bone marrow chimeras. Thrombocytosis is an unexpected consequence of reduced Mpl expression and activity. However, impaired TPO homeostasis in the transgene-rescued mice produces elevated plasma TPO levels, which serves as an unchecked stimulus to drive the observed excessive megakaryocytopoiesis.

Foamy virus (FV) vectors are a promising gene delivery system for use in hematopoietic stem cell gene therapy. Previous FV vector marking studies in the NOD/SCID xenotransplantation model used umbilical cord blood (UCB)-derived SCID repopulating cells (SRCs) that were assayed 5-10 weeks posttransplantation. We now report efficient FV vector transduction (>65%) of UCB-derived primitive, long-term SRCs engrafted for 18 weeks. In addition, we evaluated gene transfer into mobilized peripheral blood (MPB)-derived SRCs by improved, deleted FV vectors containing minimal cis-acting sequences and packaged by split helper constructs that would be appropriate for use in clinical trials. When used at a multiplicity of infection of 1 in a 10-hr transduction protocol, these improved vectors transduced 34% of engrafted MPB-derived SRCs.

The efficiency of gene transfer into human hematopoietic stem cells by oncoretroviral vectors is too low for effective gene therapy of most hematologic diseases. Retroviral vectors based on the nonpathogenic foamy viruses (FV) are an alternative gene-transfer system. In this study, human umbilical cord blood CD34(+) cells were transduced with FV vectors by a single 10-h exposure to vector stocks and then injected into sublethally irradiated nonobese diabetic-severe combined immunodeficiency (NOD/SCID) mice. At 5-7 weeks after transplantation, high transgene expression rates were observed in engrafted human hematopoietic cells, including over 60% of clonogenic progenitors. Significant transgene silencing did not occur. We developed an approach for expanding human cell populations derived from transplanted mice to show that multiple SCID repopulating cells (SRCs) had been transduced, including some that were capable of both lymphoid and myeloid differentiation. These findings demonstrate for the first time that human pluripotent (lympho-myeloid) hematopoietic stem cells repopulate NOD/SCID mice and can be efficiently transduced by FV vectors.

Gene transfer into hematopoietic stem cells (HSCs) is an ideal treatment strategy for many genetic and hematologic diseases. However, progress has been limited by the low HSC transduction rates obtained with retroviral vectors based on murine leukemia viruses. This study examined the potential of vectors derived from the nonpathogenic human foamy virus (HFV) to transduce human CD34(+) cells and murine HSCs. More than 80% of human hematopoietic progenitors present in CD34(+) cell preparations derived from cord blood were transduced by a single overnight exposure to HFV vector stocks. Mice that received transduced bone marrow cells expressed the vector-encoded transgene long term in all major hematopoietic cell lineages and in over 50% of cells in some animals. Secondary bone marrow transplants and integration site analysis confirmed that gene transfer occurred at the stem cell level. Transgene silencing was not observed. Thus vectors based on foamy viruses represent a promising approach for HSC gene therapy. (Blood. 2001;98:604-609)

The domestic cat is an outbred species with many identified analogues of human genetic diseases. Therefore, it has the potential to serve as a large animal model for evaluating the feasibility of hematopoietic stem cell gene therapy. This study compared gene transfer rates into feline hematopoietic progenitors by oncoretroviral vectors pseudotyped with the subgroup A feline leukemia virus (FeLV-A), the gibbon ape leukemia virus (GALV), and the murine amphotropic virus. Gene transfer rates were superior with the FeLV-A pseudotypes, which were then tested for their ability to transduce a cat hematopoietic repopulating cell. At more than 1 year posttransplantation, persistent marking was seen in both lymphoid and myeloid lineages of a myeloablated domestic cat that had received autologous marrow cells transduced with an FeLV-A pseudotyped vector.