ZFN Mediated Targeting Of Albumin “Safe Harbor” Results In Therapeutic Levels Of Human Factor VIII In a Mouse Model Of Hemophilia A

Abstract Genome editing utilizing engineered zinc finger nucleases (ZFNs) is a promising approach to achieve long-term expression of therapeutic genes in vivo. We have previously demonstrated in vivotargeting of the endogenous murine albumin locus as a “safe harbor” for high levels of protein production, resulting in sufficient Factor IX to correct the disease phenotype in hemophilia B mice. Targeted insertion of the donor sequence into the genome offers multiple advantages. First, we are able to exploit the high transcriptional activity of the native albumin enhancer/promoter. Second, by obviating the need for these regulatory elements within the donor, we expand the effective carrying capacity of adeno-associated viral (AAV) vectors to enable delivery of larger transgenes that may not package efficiently, such as coagulation factor 8. B-Domain Deleted Factor VIII (BDD-F8) cDNA is approximately 4.4kb. Inclusion of required enhancer/promoter elements results in a construct that exceeds the ideal packaging limitations of rAAV vectors. However, since these regions are not required for our gene editing approach the promoterless hBDD-F8 donor remained below the AAV packaging capacity. Importantly, intravenous delivery of 5e11 vg of AAV8-mAlb-ZFN and 5e11 vg of AAV8-BDD-F8-Donor to hemophilia A mice resulted in 54.6% (±4.1%) FVIII activity in the blood 2 weeks following administration - confirming the potential of the albumin locus to express high levels of the targeted transgene. To further optimize in vivo ZFN-based genome editing with a view toward the ultimate clinical use of this technology, we sought methods to further increase ZFN potency (and thus limit the dose of AAV necessary for function). Of several strategies pursued (e.g. codon optimization and inclusion of a intron in the expression cassette) the most successful was to deploy separate vectors expressing each individual ZFN rather than a single vector encoding a dual expression cassette carrying both ZFNs separated by a 2A fusion peptide. Using next generation sequencing (Illumina’s MiSeq) technology to quantify insertions and deletions indicative of DNA cleavage and repair, we observed a >3-fold increase in ZFN potency in vivo by transitioning from the dual expression vector to two individual ZFN vectors at equivalent total vector doses. Given the encouraging results obtained in mice, we next sought to examine the effectiveness of targeting the albumin locus in non-human primates (NHPs). Importantly, a single intravenous co-injection of two individual AAV vectors encoding each of the NHP targeted albumin-specific ZFNs resulted in persistent levels of gene modification in liver biopsies from treated Rhesus macaques - demonstrating successful in vivocleavage in a large animal model. These data support the use of ZFN technology in the targeting of endogenous loci with large therapeutic transgenes that are not ideally suited for episomal AAV based expression (such as F.VIII). Together our results support the further investigation of genome editing at the albumin locus as a novel method for in vivo protein replacement. Disclosures: Doyon: Sangamo BioSciences, Inc.: Employment. Wechsler:Sangamo BioSciences, Inc.: Employment. Paschon:Sangamo BioSciences: Employment. Gregory:Sangamo BioSciences: Employment. Holmes:Sangamo BioSciences: Employment. Rebar:Sangamo BioSciences: Employment. High:Novo Nordisk: Consultancy, Member of a grant review committee, Member of a grant review committee Other; Intrexon: Consultancy; Genzyme, Inc.: Membership on an entity’s Board of Directors or advisory committees; Elsevier, Inc.: royalties from textbook, royalties from textbook Patents & Royalties; BristolMyersSquibb: Consultancy, membership on a Data Safety and Monitoring Board, membership on a Data Safety and Monitoring Board Other; bluebirdbio, Inc.: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; BioMarin: Consultancy; Alnylam Pharmaceuticals: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Shire : Consultancy; Benitec: Consultancy.

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In Vivo Genome Editing of Liver Albumin for Therapeutic Gene Expression: Rescue of Hemophilic Mice Via Integration of Factor 9

Blood ◽

10.1182/blood.v120.21.751.751 ◽

2012 ◽

Vol 120 (21) ◽

pp. 751-751 ◽

Cited By ~ 1

Author(s):

Xavier M Anguela ◽

Rajiv Sharma ◽

Yannick Doyon ◽

Sunnie Y Wong ◽

David E Paschon ◽

...

Keyword(s):

Board Of Directors ◽

Transgene Expression ◽

Factor Ix ◽

Safe Harbor ◽

Wild Type ◽

Advisory Committees ◽

Aav Vector ◽

Children's Hospital ◽

Children’S Hospital

Abstract Abstract 751 Gene correction using zinc finger nuclease (ZFN) technology can be applied to target virtually any locus in the human genome. Beyond correcting mutated genes causative of disease, ZFNs can also be utilized to target transgene insertion into genomic “safe harbors.” Ideally, specific gene targeting to such “safe harbor” sites would (i) ensure therapeutically relevant levels of transgene expression and (ii) tolerate transgene addition without deleterious effect on the host organism. For liver-derived protein replacement, albumin represents an attractive target locus. Firstly, albumin is very highly expressed exclusively in the liver, thus targeting of a relatively small percentage of alleles should yield therapeutically relevant levels of liver-specific transgene expression. Second, the reduction or complete absence of albumin in animals and even humans (analbuminemia) produces surprisingly few symptoms. Here, we sought to investigate whether ZFN-mediated targeted insertion of a promoter-less copy of the human F9 cDNA at the mouse albumin locus could result in human Factor IX production and successfully correct the hemophilic phenotype in mice. To address this question, we constructed an AAV vector encoding a pair of ZFNs targeting intron 1 of the mouse albumin locus (AAV8-mAlb-ZFN) and a donor AAV vector (AAV8-Donor) harboring a partial cDNA cassette containing exons 2–8 of the wild-type human F9 gene flanked by sequences lacking significant homology to the mouse genome. Co-delivery of 1e11 vg of AAV8-mAlb-ZFN along with 5e11vg of AAV8-Donor resulted in stable (>12wk) circulating F.IX levels of 1600–3200 ng/mL (32–64% of normal). As a control, mice injected with the AAV8-Donor along with an AAV vector encoding a ZFN pair targeting an unrelated locus exhibited background F.IX levels (∼50 ng/mL). A dose-response study was performed by administering a fixed dose of donor (5e11 vg/mouse) with decreasing doses of AAV8-mAlb-ZFN (1e11, 1e10 and 1e9 vg/mouse). Human F.IX levels increased as a function of ZFN dose in the range tested (3260±480, 225±43 and 31±4 ng/mL at the high, medium and low dose, respectively). Importantly, these results showed that donor homology to the target site is not required to achieve robust levels of gene addition to the albumin locus in adult mice, thus permitting the design of donor vectors harboring corrective copies of transgenes up to the maximum AAV packaging capacity of ∼4.7 Kb. Albumin and factor IX are both synthesized as pre-propeptides and turned into propeptides after the signal peptide is removed. Expression of human F9 exons 2–8 spliced with mouse albumin exon 1 is expected to yield a chimeric propeptide. The first 2 N-terminal amino acids would originate from proalbumin, followed by a Val to Leu mutation at position −17 of the hF.IX propeptide and 16 aa encoded by human F9. To evaluate whether this chimeric human F.IX derived from gene addition to the albumin locus would be processed correctly and normalize the prolonged clotting times in hemophilia B (HB) mice, we injected 1e11 vg of AAV8-mAlb-ZFN and 5e11vg of AAV8-Donor into HB animals. Two weeks post-treatment, hF.IX antigen levels were in the range of 20% of normal and activated partial thromboplastin time, a measurement of clot formation, was corrected to wild-type levels (42 seconds), from an average of 70 seconds pre-treatment. Thus expression of a therapeutic protein (F.IX) from the albumin locus is shown to correct the HB disease phenotype in vivo. In summary, these data provide the first demonstration of ZFN-mediated in vivo genome editing of a safe harbor locus for therapeutic protein production. While we provide here a proof of principle establishing phenotypic correction of hemophilia B, appropriately designed donors could expand this strategy. Most importantly the magnitude of albumin expression (>15 g / day) should enable production of a diverse range of transgenes at therapeutically consequential levels. Disclosures: Anguela: The Children's Hospital of Philadelphia: Patents & Royalties. Sharma:The Children's Hospital of Philadelphia: Patents & Royalties. Doyon:Sangamo BioSciences, Inc.: Employment. Wong:Sangamo BioSciences, Inc.: Employment. Paschon:Sangamo BioSciences, Inc.: Employment. Gregory:Sangamo BioSciences, Inc.: Employment. Holmes:Sangamo BioSciences, Inc.: Employment. Rebar:Sangamo BioSciences, Inc.: Employment. High:Shire Pharmaceuticals: Consultancy; Sangamo Biosciences, Inc: Collaborator, Collaborator Other; Novo Nordisk: Visiting Professor, Visiting Professor Other; Genzyme, Inc: Membership on an entity's Board of Directors or advisory committees; The Children's Hospital of Philadelphia: Patents & Royalties; Bluebird Bio, Inc: Membership on an entity's Board of Directors or advisory committees.

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Treatment Of Serious Bleeds With a B-Domain Deleted Recombinant Porcine Sequence Factor VIII (OBI-1) In Patients With Acquired Hemophilia A: A Prospective Clinical Trial

Blood ◽

10.1182/blood.v122.21.206.206 ◽

2013 ◽

Vol 122 (21) ◽

pp. 206-206 ◽

Cited By ~ 1

Author(s):

Rebecca Kruse-Jarres ◽

Jean St. Louis ◽

Anne Greist ◽

Amy D. Shapiro ◽

Hedy Smith ◽

...

Keyword(s):

Factor Viii ◽

Board Of Directors ◽

Research Funding ◽

Hemophilia A ◽

Positive Response ◽

Acquired Hemophilia A ◽

Advisory Committees ◽

Baxter Healthcare ◽

Bayer Healthcare ◽

Travel Grant

Abstract Introduction Acquired hemophilia A (AHA) is a rare bleeding disorder, resulting from auto-antibodies to human factor VIII (hFVIII). The challenges created by the management of AHA and the co-morbidities present in this typically elderly population, can be managed by a recombinant, highly pure, B-domain deleted, porcine sequence FVIII (OBI-1) that is not generally susceptible to the inhibitory activity of anti-human FVIII antibodies. Treatment with OBI-1 allows for monitoring of FVIII levels which provides a reproducible and objective surrogate predictor of hemostasis. Eradication of hFVIII inhibitors with immunosuppressive therapy is critical for disease management. During immunosuppression, the patient transitions from a bleeding state at initial presentation to a relative hypercoagulable state which can be an issue in patients who are susceptible to thromboembolic events due to their comorbidities. This transition period is of most concern especially when using traditionally utilized bypassing agents that cannot be monitored. OBI-1 enables measurement of FVIII levels, guiding dosing and enhancing treatment safety during this critical period. Methods This global, prospective, multi-center phase 2/3 open label clinical trial investigates the efficacy and safety of OBI-1 in the treatment of serious bleeds in adults with AHA conducted under ICH guidelines and local IRB/Ethics Committee oversight. Primary efficacy endpoint was assessed at 24 hours (eg. effective, partially effective). All subjects (N= 18) presented with a serious bleed and were treated with an initial dose of OBI-1 (200 U/kg), followed by additional doses based on the subject's target factor VIII levels, anti-OBI-1 titer, and clinical factors. Results In all 18 subjects, a positive response (14 effective/4 partially effective) to treatment was observed at 24 hours. This positive response to OBI-1 treatment was seen by 8 hours in 14/18 of the subjects and at 16 hours in 16/18 of the subjects. Median total exposure to OBI-1 per subject was 1782.5 U/kg. The median total first dose was 14,000 U. For subjects who received additional doses of OBI-1, the median dose was reduced from the initial dose, but did not differ considerably over subsequent doses (9180 to 13561 U; median 11000 U). The majority of subjects (17/18) received concomitant immunosuppressive therapies. No related serious adverse reactions occurred. Non-serious adverse events related to treatment were noted in 5/18 (27.8%) subjects. One subject had mild tachycardia, hypotension and constipation. One subject had 2 instances of mild PICC line occlusion. One subject had a mild hypofibrogenemia. All of these adverse effects completely resolved. Three subjects developed anti-porcine inhibitors after infusion of study drug (range 8-108 BU) and two were discontinued from treatment. Anti-porcine inhibitors were detected prior to infusion in 6/18 patients (range 0.8-29 BU). All of these subjects had a favorable clinical response at 24 hours post-OB-1 infusions. Conclusions Data from this prospective study demonstrate OBI-1 as a safe and effective treatment of bleeding episodes in patients with AHA, with the added advantage over other bypass therapies of allowing FVIII monitoring throughout treatment and healing phase. Disclosures: Kruse-Jarres: Baxter Healthcare: Consultancy; Bayer HealthCare: Consultancy; Biogen IDEC: Consultancy; Grifols: Consultancy; Kedrion: Consultancy; Novo Nordisk: Consultancy. St. Louis:CSL Behring: Research Funding; Octapharma: Consultancy, Research Funding; Baxter: Consultancy; Novo Nordisk: Honoraria. Shapiro:Kedrion Biopharma: Consultancy; Chugai Pharma USA: Consultancy; Biogen IDEC: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Bayer HealthCare: Membership on an entity’s Board of Directors or advisory committees; Novo Nordisk: Consultancy, Membership on an entity’s Board of Directors or advisory committees; Baxter Healthcare: Consultancy, Membership on an entity’s Board of Directors or advisory committees. Chowdary:Baxter Healthcare: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Travel grant Other; Novo Nordisk: Honoraria, Research Funding, Travel grant, Travel grant Other; Bayer HealthCare: Honoraria, Travel grant, Travel grant Other; Pfizer: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Travel grant, Travel grant Other; CSL Behring: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding, Travel grant Other; Biogen IDEC: Honoraria, Travel, Travel Other. Drebes:Octapharma: Travel grant Other; CSL Behring: Travel grant, Travel grant Other; Leo-pharma: Travel grant, Travel grant Other; Bayer Healthcare: Consultancy, Honoraria. Gomperts:Baxter Healthcare: Consultancy; Asklepios Biopharmaceutoicals Inc: Consultancy; Cangene Inc: Consultancy. Chapman:Baxter Healthcare: Employment. Mo:Baxter Healthcare: Employment. Novack:Baxter Healthcare: Employment. Farin:Baxter Healthcare: Employment.

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Target Joint Bleeding in Pediatric Patients with Hemophilia Α Receiving Twice Weekly Prophylaxis with a Pegylated Full-Length Recombinant Factor VIII with Extended Half-Life

Blood ◽

10.1182/blood.v128.22.3778.3778 ◽

2016 ◽

Vol 128 (22) ◽

pp. 3778-3778

Author(s):

Amy L. Dunn ◽

Alexis A. Thompson ◽

Werner Engl ◽

Marlies Sharkhawy ◽

Brigitt E. Abbuehl

Keyword(s):

Factor Viii ◽

Board Of Directors ◽

Half Life ◽

Research Funding ◽

Pediatric Patients ◽

Hemophilia A ◽

Point Estimate ◽

Severe Hemophilia ◽

Advisory Committees ◽

The Mean

Abstract Introduction: Patients with hemophilia A are at risk for acute bleeding which may affect muscles and other soft tissues but characteristically involves joints. Prophylaxis with factor VIII (FVIII) is the optimal treatment to prevent bleeding into joints and, when begun at a young age, may prevent arthropathy. BAX 8551, a polyethylene glycol (peg)ylated, full-length, recombinant FVIII built on ADVATE2, demonstrated extended half-life, efficacy, and safety for prophylaxis and treatment of bleeding in patients with severe hemophilia A. Methods: A phase 3, prospective, uncontrolled, multicenter study was performed in pediatric patients with severe hemophilia A without history of inhibitors. To be eligible, patients aged <6 years had to have ≥50, those aged 6 to <12 years ≥150 previous exposure days (EDs) to FVIII. Patients received twice weekly infusions of 50 ±10 IU/kg of BAX 855 over a period of 6 months or ≥50 EDs. The prevalence of target joints, defined as a single joint with ≥3 spontaneous bleeding episodes in any consecutive 6-month period, was assessed at baseline. Annualized rates of target joint bleeds and the course of target joints were evaluated by age (<6 and 6 to <12 years). The study was performed in accordance with the principles of the Declaration of Helsinki of the World Medical Association. Results:Sixty-six patients were treated with a mean (SD) BAX 855 dose of 51.1 (5.5) IU/kg at a mean (SD) frequency of 1.8 (0.2) infusions/week. Fourteen of 66 patients (21.2%), 3/32 (9.4%) in the younger and 11/34 (32.4%) in the older cohort, had a total of 23 target joints at screening. The number of target joint bleeds decreased during a mean (SD) of 48.5 (7.7; median: 49.0) prophylactic EDs/patient. Five of 66 (7.6%) patients had at least 1 target joint bleed, 1/32 (3.1%) in the younger and 4/34 (11.8%) in the older cohort. The point estimate for the mean (95% CI) annualized rate of target joint bleeds was 0 (0 - infinity; median: 0) compared to an annualized rate of all joint bleeds of 1.1 (0.6 - 1.9; median: 0) and an annualized rate of all bleeds of 3.0 (2.2 - 4.2; median: 2.0) (Table 1). The point estimate for the mean (95% CI) annualized bleeding rate (ABR) in 52 patients without target joints was 2.9 (2.0 - 4.2; median: 2.0) and was similar in 14 patients with target joints at screening at 3.5 (1.9 - 6.6; median: 2.1). In the younger cohort, the ABR was lower in patients with than those without target joints. However, the number of patients <6 years with target joints (N = 3) was too small to draw any conclusions (Table 1). During BAX 855 prophylaxis, no new target joints developed in any patient. Ten of 14 patients had at least 1 target joint revert to a non-target joint. In 8 of these 10 patients, 4 with 1 and 4 with 2 target joints, all target joints resolved. Conclusions:These results suggest that twice weekly infusion of BAX 855 is effective in the prevention of bleeding into target joints and may revert target to non-target joints in pediatric patients with severe hemophilia A. 1BAX 855 (Baxalta US Inc., now part of Shire) is licensed in the US and Japan under the trade name ADYNOVATE. 2ADVATE is a trade mark of Baxalta US Inc., now part of Shire. Disclosures Dunn: NovoNordisk: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Kedrion: Research Funding; Pfizer: Research Funding; CSL Behring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Biogen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxalta (now part of Shire): Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Octapharma: Research Funding. Thompson:Eli Lily: Research Funding; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Research Funding; bluebird bio: Consultancy, Research Funding; ApoPharma: Consultancy, Membership on an entity's Board of Directors or advisory committees; Mast: Research Funding; Amgen: Research Funding; Baxalta (now part of Shire): Research Funding. Engl:Shire: Employment, Equity Ownership. Sharkhawy:Baxalta (now part of Shire): Employment. Abbuehl:Baxalta (now part of Shire): Employment.

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Early Prophylaxis Provides Continued Joint Protection in Severe Hemophilia A: Results of the Joint Outcome Continuation Study

Blood ◽

10.1182/blood-2018-99-117413 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 382-382 ◽

Cited By ~ 3

Author(s):

Beth Boulden Warren ◽

Dianne Thornhill ◽

Jill Stein ◽

Michael Fadell ◽

Sharon Funk ◽

...

Keyword(s):

Soft Tissue ◽

Factor Viii ◽

Board Of Directors ◽

Research Funding ◽

Hemophilia A ◽

Joint Damage ◽

Severe Hemophilia ◽

Advisory Committees ◽

Episodic Treatment ◽

Bayer Healthcare

Abstract Background: The Joint Outcome Study (JOS) was a randomized controlled trial showing that, in severe hemophilia A, prophylactic factor VIII every other day starting prior to age 30 months leads to better joint outcomes at age 6 years than enhanced episodic treatment with factor VIII for bleeding1. After conclusion of the JOS, all participants were encouraged to continue on, or to transition to, prophylaxis. Here we describe the results of the Joint Outcome Continuation Study (JOS-C), which followed the participants of the JOS to age 18 years. Methods: All participants of the JOS were eligible for the JOS-C. MRIs of 6 index joints (right and left ankles, knees, and elbows), index joint physical exam scores using the Colorado Haemophilia Paediatric Joint Physical Examination Scale2 , estimates of joint bleeding episodes, and surgery information were collected. The primary endpoint, as in the initial JOS analysis, was evidence of hemophilia-related osteochondral joint damage on MRI, scored using the extended MRI scale3. Results: Of the 65 previous participants of the JOS, 37 gave informed consent for the JOS-C study, including 18 initially randomized to prophylaxis prior to age 30 months ("early prophylaxis"), and 19 initially randomized to enhanced episodic treatment who started prophylaxis at a mean age of 7.5 years (median 6.1, range 2.7-17.1, "delayed prophylaxis"). All initially on prophylaxis in the JOS continued on prophylaxis through the JOS-C. One participant (early prophylaxis arm) failed to complete an MRI, and four others (2 early and 2 delayed prophylaxis) had their MRIs excluded for technical reasons. Four participants (3 early prophylaxis and 1 delayed prophylaxis) developed high titer inhibitors during or shortly after the JOS and were analyzed separately. Osteochondral joint damage was defined as evidence of osteochondral damage on MRI or a need for joint surgery. The relative risk of osteochondral damage in those on delayed prophylaxis as compared to those on early prophylaxis was 6.5 (95% CI 1.3, 33.6; p=0.029). At age 18, 67% of those on early prophylaxis, and only 24% of those on delayed prophylaxis had zero index joints with osteochondral damage (Figure 1). Twenty-five percent of early prophylaxis and 47% of delayed prophylaxis participants had osteochondral damage to more than one joint. Most participants had some soft tissue changes on MRI, defined as effusion, synovial hypertrophy, or hemosiderin deposition. There was no difference in risk of soft tissue damage between initial treatment groups (p=0.48). Osteochondral damage scores were available for 3 patients with inhibitors: two with refractory inhibitors had osteochondral changes on at least one joint, and one with an inhibitor that tolerized within 3 months had no osteochondral damage. Total physical exam scores were also higher in the delayed prophylaxis arm (mean 22.6, standard deviation (SD) 15.5) than in the early prophylaxis arm (mean 16.2, SD 10.5), but this difference was not statistically significant (p=0.19). Conclusion: The JOS-C demonstrates that, in severe hemophilia A, initiation of prophylaxis prior to age 30 months provides continued protection against joint damage throughout childhood. Those who started on prophylaxis later in childhood had higher risk of joint damage at age 18. Initiation of factor VIII prophylaxis in the toddler years is critical to preventing osteochondral joint damage and should not be delayed. ReferencesManco-Johnson MJ, Abshire TC, Shapiro AD, et al. Prophylaxis versus episodic treatment to prevent joint disease in boys with severe hemophilia. N Engl J Med. 2007;357(6):535-544.Hacker MR, Funk SM, Manco-Johnson MJ. The Colorado Haemophilia Paediatric Joint Physical Examination Scale: normal values and interrater reliability. Haemophilia. 2007;13(1):71-78.Hong W, Raunig D, Lundin B. SPINART study: validation of the extended magnetic resonance imaging scale for evaluation of joint status in adult patients with severe haemophilia A using baseline data. Haemophilia. 2016;22(6):e519-e526. Figure 1: Percentage of participants with zero joints with osteochondral damage at JOS exit (age 6 years) and JOS-C exit (age 18 years), excluding participants with inhibitors. Disclosures Warren: Bayer Healthcare: Research Funding; HTRS/Novo Nordisk: Research Funding; Bayer Hemophilia Awards Program Fellowship Project Award: Research Funding; CSL Behring Heimburger Award: Research Funding. Shapiro:Genetech: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Prometic Life Sciences: Consultancy, Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding; Daiichi Sankyo: Research Funding; Kedrion Biopharma: Consultancy, Research Funding; Bio Products Laboratory: Consultancy; Bioverativ, a Sanofi Company: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer Healthcare: Other: International Network of Pediatric Hemophilia; Sangamo Biosciences: Consultancy; Octapharma: Research Funding; Shire: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; OPKO: Research Funding; BioMarin: Research Funding. Recht:Shire: Research Funding; Biogen: Research Funding; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding; Kedrion: Membership on an entity's Board of Directors or advisory committees. Manco-Johnson:Bayer AG: Honoraria, Research Funding; Novo Nordisk: Honoraria; Biogentek: Honoraria; CSL Behring: Honoraria; Baxalta, now part of Shire: Honoraria.

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Tolerance Induction To FIX Padua With AAV Liver Gene Transfer In Inhibitor-Prone Hemophilia B Dogs

Blood ◽

10.1182/blood.v122.21.4203.4203 ◽

2013 ◽

Vol 122 (21) ◽

pp. 4203-4203

Author(s):

Julie M Crudele ◽

Jonathan D Finn ◽

Nicholas B Martin ◽

Joshua I Siner ◽

Yifeng Chen ◽

...

Keyword(s):

Gene Therapy ◽

Board Of Directors ◽

Neutralizing Antibodies ◽

Tolerance Induction ◽

Igg Antibodies ◽

Review Committee ◽

Advisory Committees ◽

Expression Levels ◽

Liver Gene ◽

Grant Review

Abstract Emerging data from early phase clinical studies of AAV gene therapy for hemophilia B (HB) (factor IX [FIX] deficiency) show sustained expression of therapeutic levels of FIX and phenotypic improvement. However, the safety and efficacy of in vivo gene therapy is limited by the vector dose. Recently, we reported a naturally occurring, hyperfunctional FIX (FIX Padua) caused by a single amino acid change of arginine 338 to leucine that exhibits an 8-fold increase in specific activity in humans (N Engl J Med 2009), making it a potential candidate for HB gene therapy with reduced vector doses. However, to take advantage of FIX Padua for HB gene therapy, it is critical to first define the risk of immunogenicity of this variant in preclinical models of severe HB. We have previously shown that delivery of AAV-cFIX-Padua to skeletal muscle in HB dogs with a missense mutation in the canine (c) F9 gene resulted in no anti-FIX neutralizing antibodies (inhibitors), non-neutralizing antibodies (IgG) or FIX-specific T-cell response (Blood 2012). While promising, these dogs express FIX RNA and have a pre-existing tolerance to cFIX due to the nature of their mutation, and so do not represent the most rigorous model for immunogenicity studies. Here, we tested the efficacy and immunogenicity of cFIX Padua in a severe HB dog colony with an early stop codon mutation. This mutation results in no FIX RNA transcript, and the dogs are prone to develop cFIX inhibitors upon exposure to protein concentrates. Three dogs were infused peripherally with a liver-specific AAV8-cFIX-Padua at two different doses, and monitored for cFIX antigen and activity levels and inhibitors. The first dog, which received 3 x 1012 vg/kg, showed average plateaued expression levels of 3.98 ± 1.44% antigen and 24.5 ± 4.1% activity, with no development of anti-cFIX inhibitors or IgG antibodies. Whole blood clotting time (WBCT) and aPTTs returned to normal by day 3 post-vector administration and have remained stable for >20 months (ongoing observations). A second dog was treated with a lower dose of 1 x 1012 vg/kg and showed average plateaued expression levels of 2.41 ± 0.05% antigen and 22.0 ± 0.4% activity, with no development of anti-cFIX inhibitors or IgG antibodies. WBCT and aPTTs returned to normal by day 3 post-vector administration and have remained stable for >3 months. An additional dog, upon previous exposure to recombinant human (h) FIX protein, had developed inhibitors that cross-reacted with cFIX. This immune response was ongoing at the time of vector administration (3 x 1012 vg/kg). Anti-cFIX antibodies peaked at day 14 post-AAV, with 4.7 BUs and 3643 ng/mL IgG2, but dropped to undetectable levels by day 70. There was a concurrent rise in cFIX Padua expression levels, suggesting successful tolerization to the cFIX Padua. Antigen levels plateaued at 14.6 ± 4.3% and activity at 51.7 ± 23.5%, with ongoing normalization of WBCT and aPTTs for >18 months. In all three dogs, cholesterol, albumin and total protein were within normal limits with no clinical or laboratory evidence of nephrotic syndrome (a potential complication in FIX inhibitor patients that have undergone immune tolerance induction with frequent FIX protein injections). The safety of FIX Padua was further confirmed using a mouse model of HB. Mice (n=8-12/group) were treated with 5 x 1010 vg/kg liver-directed AAV8-hFIX-WT or AAV8-hFIX-Padua, resulting in expression levels of 1076 ± 343 ng/mL (21.5 ± 6.9% antigen, 67.5 ± 10.1% activity) and 797 ± 255 ng/mL (15.9 ± 5.1% antigen, 274.8 ± 73.8% activity), respectively. In cross-over experiments, the mice were then were immunologically challenged 10-15 weeks after gene delivery with 100 ug/kg of the reciprocal recombinant protein (ie mice expressing hFIX Padua were challenged with hFIX WT, and visa versa). Challenges were administered subcutaneously alone or with adjuvant (CFA) weekly for 4 weeks. In no instance did mice develop antibodies to either FIX, suggesting that tolerance was successfully induced in all cases. Together, these date show that FIX Padua shows no increase in immunogenicity compared to FIX WT and is capable not only of preventing inhibitor formation, but also of eradicating pre-existing inhibitory antibodies to FIX in an inhibitor-prone HB dog model. Thus, FIX Padua is an attractive transgene that will allow for decreased vector doses in human HB gene therapy, improving the safety profile of AAV liver gene therapy without increased immunogenicity. Disclosures: High: Alnylam Pharmaceuticals: Consultancy; BioMarin: Consultancy; bluebirdbio, Inc.: Consultancy, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees; BristolMyersSquibb: Consultancy, membership on a Data Safety and Monitoring Board, membership on a Data Safety and Monitoring Board Other; Elsevier, Inc.: royalties from textbook, royalties from textbook Patents & Royalties; Genzyme, Inc.: Membership on an entity’s Board of Directors or advisory committees; Intrexon: Consultancy; Novo Nordisk: Consultancy, Member of a grant review committee, Member of a grant review committee Other; Shire : Consultancy; Benitec: Consultancy.

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Large Cohort of Symptomatic Female Carriers of Hemophilia in an Extended Native American Family

Blood ◽

10.1182/blood.v126.23.4700.4700 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4700-4700

Author(s):

Becki Berkowitz ◽

Amber Federizo ◽

Garrett E. Bergman ◽

Paula J. Ulsh

Keyword(s):

Soft Tissue ◽

Factor Viii ◽

Board Of Directors ◽

Gene Mutation ◽

Hemophilia A ◽

Clotting Factor ◽

Factor Level ◽

Advisory Committees ◽

Low Levels ◽

Female Carriers

Abstract Hemophilia A is an X-linked recessive genetic bleeding disorder resulting in a lack of clotting factor VIII. Although this disorder primarily affects males, a female who inherits one affected X chromosome from a parent becomes a carrier of hemophilia. While it is widely believed that carriers are asymptomatic, some of these women have mild hemophilia, defined by ISTH as a circulating factor VIII level > 0.5 to 0.40 IU/ml or 5 - 40 % of normal. (White et al Thromb Haemost 2001) Data demonstrates hemophilia A carriers have the same risk for bleeding as a male with mild hemophilia A at the corresponding factor level. Carriers report significantly more bleeding events than non-carriers from small wounds and after invasive procedures, and their bleeding tendency is inversely correlated to their factor level. (Plug et al Blood 2006) Carriers have been shown to demonstrate decreased joint range of motion, soft tissue and osteochondral changes on MRI, consistent with subclinical joint bleeds leading to structural abnormalities in their joints. (Gilbert et al Haemophilia 2014). Additionally, carriers have been shown to report higher scores on pictorial blood assessment charts, a semi-quantitative measure of menstrual blood loss. (Kadir et al Haemophilia 1999) We report here a unique patient population from our Owyhee Indian Health Hemophilia Treatment Center Outreach Clinic on the Duck Valley Indian Reservation in Owyhee, NV. On this reservation, a German Immigrant with hemophilia A married 2 women of the Shoshone Indian Tribe, and they had 14 children (8 females and 6 males). The family tree reveals after four generations there are currently 162 descendants with the same hemophilia A gene mutation, which has been identified. Factor VIII levels in the female family members range from 7% to 50%. The male hemophilia A patients are treated on demand with plasma-derived factor VIII products, currently Koate-DVI, for traumatic events, and prophylactically for medical or dental procedures, or surgery. Approximately 20-25% of the female carriers in this population have been treated with plasma-derived FVIII concentrates, currently Koate-DVI, for childbirth and surgeries. Additionally, all female carriers from teenage years to age 30 are treated with desmopressin acetate nasal spray (Stimate) for menorrhagia and are treated with oral aminocaproic acid (Amicar) for nose bleeds and soft tissue bleeds. Carriers of hemophilia A with factor VIII levels in the range observed in this family, particularly when symptomatic, should receive the diagnosis of "mild hemophilia". Their propensity for developing subclinical as well as clinical bleeding needs to be recognized to assure the receive treatment appropriate to their symptomatology. The low levels of FVIII observed in this family are likely due to extreme lyonization associated with their particular gene mutation. Familial low levels of FVIII can also be seen in some forms of type 2 von Willebrand Disease secondary to poor FVIII binding and a shortened half-life. However, since VWD is inherited in an autosomal recessive pattern, males would not selectively have the severity observed here. Optimal diagnostic and therapeutic strategies as well as many other aspects concerning mild hemophilia remain to be clarified. Additional studies to define these findings are underway. Disclosures Berkowitz: Pfizer: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Kedrion: Membership on an entity's Board of Directors or advisory committees; NovoNordisk: Speakers Bureau; Baxter: Speakers Bureau. Federizo:Emergent: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Pfizer: Membership on an entity's Board of Directors or advisory committees; Baxalta: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Biogen Idec: Membership on an entity's Board of Directors or advisory committees; Octapharma: Membership on an entity's Board of Directors or advisory committees; Kedrion: Membership on an entity's Board of Directors or advisory committees. Bergman:Kedrion Biopharma: Employment. Ulsh:Kedrion Biopharma: Employment.

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Epitope Dependent Augmentation of the Immune Response in Hemophilia Α Mice Immunized with Factor VIII/Antibody Immune Complexes

Blood ◽

10.1182/blood-2019-127436 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2387-2387

Author(s):

Glaivy Batsuli ◽

Seema R Patel ◽

Courtney Cox ◽

Wallace H. Baldwin ◽

John S. Lollar ◽

...

Keyword(s):

Immune Response ◽

Dendritic Cells ◽

Board Of Directors ◽

Hemophilia A ◽

Advisory Board ◽

Inhibitory Antibody ◽

Advisory Committees ◽

Antibody Titers

Introduction: The immune response to factor VIII (fVIII) is a CD4+ T cell dependent process initiated by fVIII recognition and processing by antigen presenting cells. The C1 and C2 domains have been proposed as the primary domains that mediate fVIII internalization by dendritic cells. Our prior studies demonstrate that fVIII pre-bound to anti-C1 and C2 domain monoclonal antibodies (MAbs) reduces fVIII internalization by murine derived dendritic cells. However, anti-A1 and A3 domain antibodies increase fVIII endocytosis by dendritic cells. In this study, we analyzed the antibody titers of hemophilia A mice immunized with binary fVIII and MAb complexes to determine whether changes in fVIII internalization observed in vitro correspond to changes in the immune response to fVIII in vivo. Methods: Exon16 (E16) knockout mice deficient in fVIII were immunized with B-domain deleted fVIII in the presence of either anti-fVIII MAb 2-116 (anti-A1, IgG2a), 4A4 (anti-A2, IgG2a), 2-113 (anti-A3, IgG1), B136 (anti-C1, IgG2a), or 3D12 (anti-C2, IgG2b) versus fVIII alone. Mice were immunized by weekly retro-orbital injections of 0.1 µg fVIII incubated with 1 µg anti-fVIII MAb for 4 weeks followed by a boost dose of 0.2 µg fVIII and 2 µg anti-fVIII MAb one week later at week 5. Anti-fVIII ELISA titers (in arbitrary units, AU) were analyzed from plasma samples collected at week 7 to account for IgG half-life. A separate cohort of mice immunized with anti-fVIII MAbs alone served as controls to normalize ELISA titers in each fVIII/MAb group to account for residual injected MAbs in the plasma samples. Results: Hemophilia A mice immunized with fVIII/2-116 and fVIII/4A4 complexes significantly increased anti-fVIII ELISA titers compared to mice immunized with fVIII alone (figure 1). Mice immunized with fVIII/2-116 and fVIII/4A4 had median ELISA titers of 7,401 AU (interquartile range, IQR, 2,654 - 11,910 AU) and 3,620 AU (IQR 1,062 - 7,969 AU), respectively, compared to mice immunized with fVIII (median titer 1,063 AU, IQR 402 - 2,476 AU). MAb 2-116 is a non-inhibitory antibody with a titer of <1 Bethesda Unit (BU)/mg IgG, while 4A4 is a highly inhibitory antibody with a titer of 40,000 BU/mg IgG. Neither MAb interferes with fVIII binding to von Willebrand factor (VWF) or phospholipid vesicles. Mice immunized with fVIII/2-113 (median titer 2,210 AU, IQR 402 - 8,318 AU), fVIII/B136 (median titer 123 AU, IQR 0 - 9,709 AU), and fVIII/3D12 (median titer 3,244 AU, IQR 0 - 5,180 AU) did not have significantly different anti-fVIII ELISA titers compared to mice immunized with fVIII. However there was a trend towards reduced anti-fVIII titers with fVIII/B136 and fVIII/3D12 injections. MAbs B136 and 3D12 inhibit fVIII binding to VWF and have been shown to significantly increased fVIII clearance in hemophilia A mice compared to fVIII in a VWF-dependent manner. Median titers for mice immunized with MAbs alone to account for residual injected MAbs were 13 AU (2-116), 12 AU (4A4), 18 AU (2-113), 16 AU (B136), and 4 AU (3D12). Conclusions: Immunization of hemophilia A mice with fVIII/MAb complexes, specifically anti-A1 MAb 2-116 and anti-A2 MAb 4A4, enhance the immune response to fVIII. MAb 2-116 significantly increased anti-fVIII antibody titers in vivo, which correlates with increased fVIII internalization by immature dendritic cells observed in vitro. A better understanding of the effect of anti-fVIII antibodies on fVIII conformational changes could provide insight into whether these changes alter fVIII recognition by immune cells and subsequently propagate the immune response to fVIII at the onset of inhibitor formation or during immune tolerance induction. Disclosures Batsuli: Genentech: Other: Advisory board participant; Bayer: Other: Advisory board participant; Octapharma: Other: Advisory board participant. Meeks:HEMA Biologics: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Genentech: Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Takeda-Shire: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees.

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Breakthrough Bleeding in Hemophilia a Patients on Prophylaxis

Blood ◽

10.1182/blood.v128.22.2581.2581 ◽

2016 ◽

Vol 128 (22) ◽

pp. 2581-2581 ◽

Cited By ~ 2

Author(s):

Beth Boulden Warren ◽

Taylor Blades ◽

Natalie L Smith ◽

Michael Wang ◽

Marilyn J. Manco-Johnson

Keyword(s):

Factor Viii ◽

Board Of Directors ◽

Research Funding ◽

Hemophilia A ◽

Tolerance Induction ◽

Severe Hemophilia ◽

Advisory Committees ◽

Breakthrough Bleeding ◽

University Of Colorado ◽

The University

Abstract Background: Patients with severe hemophilia A (factor VIII (FVIII)<1%), as well as many patients with moderate hemophilia A (FVIII 1-5%), are treated intravenously with FVIII prophylaxis to prevent bleeding. However, breakthrough bleeding occurs in many patients. Methods: To better understand breakthrough bleeding, we evaluated the effect of hemophilia severity, prophylaxis adherence, and replacement FVIII utilization on bleeding rates in patients with hemophilia A on prophylaxis. Data on hemophilia severity and bleeding rates were collected from subjects with hemophilia A, ages 2-30 years, treated at the University of Colorado for hemophilia A, using the University of Colorado Clinical Research Bleeding Disorders Database (UCBDD). Adherence and FVIII utilization data were collected from a subset of subjects with hemophilia A on prophylaxis without active inhibitors who participated in the Centers for Disease Control/American Thrombosis and Hemostasis Network Community Counts (CDCCC) registry between December 2013 and June 2016, which surveyed participants about their estimated percentage of missed prophylaxis doses. The CDCCC registry was also used to corroborate bleeding rates. The effect of hemophilia severity and missed prophylaxis doses (percentage of prescribed doses) on bleeding rates were analyzed using logistic regression, with bleeding rates dichotomized as high or low relative to study population median bleeding rates. The relationship between weekly factor utilization and annualized bleeding rates was evaluated using Pearson's correlation. Results: Of 89 patients with severe hemophilia A in the UCBDD, 86.5% of patients were on continuous prophylaxis, with an additional 7.9% on immune tolerance induction. The 5.6% of patients with severe hemophilia on episodic treatment had been encouraged to use prophylaxis but had declined. Of 37 patients with moderate hemophilia A, 48.7% were on continuous prophylaxis. Sixty-nine subjects on prophylaxis had data in the UCBDD and the CDCCC registry collected during the defined time period. Bleeding rates are shown in Table 1. Prophylaxis doses in this population had an interquartile range of 74.3 to 120 units/kg/week (mean 96.9 units/kg/week), dosed 2-7 times per week depending on activities and historic bleeding patterns. Eighty-two percent of patients rated their percentage of missed prophylaxis doses at <10%, 11.6% rated their missed doses at 10-20%, 2.9% rated their missed doses at 21-50%, and 2.9% rated missed doses at >50%. There was not a statistically significant relationship between any bleeding rate and percentage of missed doses, hemophilia severity, or factor utilization, as shown graphically in figures 1 and 2. Conclusion: Although prophylaxis usage and adherence were excellent, breakthrough bleeding was common, with breakthrough joint bleeding occurring in 36% of subjects, and was not related to FVIII dose per week. Prospective studies are needed to better determine individually tailored prophylaxis regimen using dose, product class, and timing with activities, in order to achieve more effective prophylaxis. Table 1 Bleeding Rates on Continuous Prophylaxis. No intracranial or gastrointestinal hemorrhages were recorded in this population. Table 1. Bleeding Rates on Continuous Prophylaxis. No intracranial or gastrointestinal hemorrhages were recorded in this population. Figure 1 Relationship between Bleeding Rates, Hemophilia Severity and Adherence Figure 1. Relationship between Bleeding Rates, Hemophilia Severity and Adherence Figure 2 No correlation was found between bleeding rates and factor VIII utilization as measured in units per kg per week. Figure 2. No correlation was found between bleeding rates and factor VIII utilization as measured in units per kg per week. Disclosures Warren: HTRS/Novo Nordisk: Research Funding; Bayer Hemophilia Awards Program: Research Funding. Wang:Biogen: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees; Baxalta: Membership on an entity's Board of Directors or advisory committees; HEMA Biologics: Membership on an entity's Board of Directors or advisory committees; LFB: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees. Manco-Johnson:Baxalta: Honoraria; NovoNordisk: Honoraria; BiogenIdec: Honoraria; Bayer: Honoraria, Research Funding; CSL Behring: Honoraria.

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Blocking Protein S Improves Hemostasis in Hemophilia a and B

Blood ◽

10.1182/blood.v128.22.79.79 ◽

2016 ◽

Vol 128 (22) ◽

pp. 79-79

Author(s):

Luca Bologna ◽

Raja Prince ◽

Mirko Manetti ◽

Daniela Melchiorre ◽

Irene Rosa ◽

...

Keyword(s):

Blood Loss ◽

Board Of Directors ◽

Tissue Factor ◽

Research Funding ◽

Hemophilia A ◽

Protein S ◽

Advisory Committees ◽

Intense Staining ◽

Preliminary Results

Abstract Introduction&Aim Hemophilia A and B are X-linked disorders caused by an absence or a reduction in coagulation FVIII and respectively, FIX. Patients with hemophilia often suffer from spontaneous bleeding within the musculoskeletal system, such as hemarthrosis. Here, we investigated whether targeting protein S (PS) could promote hemostasis in hemophilia by re-balancing coagulation. PS is a natural anticoagulant, acting as non-enzymatic cofactor for activated protein C (APC) in the inactivation of FVa and FVIIIa, and for tissue factor pathway inhibitor (TFPI) in the inhibition of FXa. This dual role makes PS a key regulator of the inhibition of thrombin generation (TG). Methods & Results Hemophilic (F8-/- or F9-/-) Pros1+/- mice were intercrossed. F8-/-Pros1-/- and F9-/- Pros1-/- mice were viable and found at the expected Mendelian frequency with no increased mortality compared to hemophilic mice. F8-/-Pros1-/- mice did not show DIC onset neither an increased mortality once challenge with tissue factor (TF). Ex vivo evaluation of TG potential showed that F8-/-Pros1-/- mice were APC-resistant, they had an improved low TF induced-TG and a 20% more dense fibrin network with larger fibrin fibers diameter as compared to F8-/-mice. Comparable results were found in human HA plasma where blocking PS raised TG even in the presence of high inhibitor titer. To assess the in vivo effect of PS inhibition on HA mice hemostasis, two tail- clipping (TC) assays were used. In both, mild TC model (2 mm cut) and severe TC (4 mm cut), blood loss significantly decreased in F8-/-Pros1-/- compared to F8- /- mice (mild TC: 407±21 vs 661±29ul, p<0.0001; severe TC:173±14 vs 249±24ul, p<0.05). In addition, the infusion of anti-hPS antibody on F8-/-Pros1+/- mice, reduced the blood loss compared to F8-/-Pros1+/- mice infused with an isotype IgG (196±10 vs 302±25ul, p=0.005). As recurrent joint bleeding is the most common manifestation of HA, we challenged F8-/-Pros1-/- mice in an acute hemarthrosis (AH) model. Joint swelling 72 hours after injury was reduced in F8-/-Pros1-/- compared to F8-/- (0.11±0.03 vs 1.02±0.07mm, p<0.0001, n=10). These results were also confirmed by s.c. injection of anti-hPS antibody (0.46±0.0 vs 0.78±0.09mm isotype IgG, p=0.02, n=9) and by i.v. injection of PS-siRNA prior to AH challenge in F8-/-Pros1+/- (0.29±0.09 vs 0.92±0.12mm siRNA control, p=0.03, n=5) and F8-/- mice(0.35±0.08 vs 0.78±0.09mm siRNA control, p=0.05, n=5). Similar results were obtained in F9-/-Pros1-/- mice. Histological analysis of joint showed joint bleeding reduction in F8-/-Pros1-/- compared to F8-/- and an increased fibrin staining comparable to F8+/+ mice.To understand the intra-articular hemostatic effect of blocking PS, joint sections were stained for TFPI. Preliminary results indicate a massive staining in the synovia of F8-/- mice, while F8-/-Pros1-/- and F8+/+ mice present a less intense signal. These data suggest that the intra-articular space is a modulable anticoagulant environment. Human HA joint tissues were then analyzed for both PS and TFPI. A strong signal was found for TFPI and PS in the synovial lining and sublining layers of HA patients on demand (n=7). Interestingly, PS and TFPI stainings were remarkably decreased in HA patients under prophylaxis (n=5). Joint section from osteoarthritis patients (n=7) did not show an intense staining for TFPI and PS similarly to hemophilic patients under prophylaxis. To understand the improved phenotype of F8-/-Pros1-/- after AH, the function of macrophages were investigated. At the steady state, F8-/-Pros1-/- presented significantly 2-fold increased levels of inflammatory macrophages (M1) than in F8-/- mice. In addition bone marrow derived macrophages from F8-/-Pros1-/- exhibit 10-fold higher RBC phagocytic activity than F8-/- . Preliminary results indicate an increase of a monocyte attractant MCP-1 level, in knee lavage after AH in F8-/-Pros1-/- than F8-/- mice. Conclusion These data provide the first evidence that blocking PS has the ability to ameliorate hemophilia as judged by in vivo improvement of bleeding phenotype in the TC assay as well as in the AH model, suggesting a new valuable tool for hemophilia therapy. In addition, the modulable presence of PS and TFPI in human and mice joints is a novel pathophysiological aspect of hemarthrosis and constitutes a potential therapeutical target. Disclosures Kremer Hovinga: NovoNordisk: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL-Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bayer: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Real-World Experience Using Emicizumab Prophylaxis for Hemophilia a: Single-Center Experience

Blood ◽

10.1182/blood-2020-139568 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 36-36

Author(s):

Anshul Vagrecha ◽

Joseph Stanco ◽

Daphenee Ulus ◽

Suchitra Acharya

Keyword(s):

Factor Viii ◽

Board Of Directors ◽

Knee Arthroplasty ◽

Real World ◽

Hemophilia A ◽

Recombinant Factor Viii ◽

Phase Iii ◽

Advisory Committees ◽

Fviii Inhibitor ◽

Bypassing Agents

INTRODUCTION: Emicizumab is a recombinant humanized bispecific antibody that mimics Factor VIII by bridging Factor IXa and X. It is an important breakthrough in the therapeutic armamentarium for Hemophilia A. Recently concluded Phase III HAVEN trials demonstrated that prophylaxis with emicizumab is superior to recombinant Factor VIII (rFVIII). Patients with Hemophilia A with inhibitors had a 79% reduction in the Annualized Bleeding Rate (ABR) while on emicizumab prophylaxis vs other bypassing agents. Patients without inhibitors had a 68% reduction in ABR on emicizumab vs rFVIII prophylaxis. However, there is limited data on its use in the post-marketing era. METHODS: We report our experience with emicizumab in children and adults with Hemophilia A with and without inhibitors at a single Hemophilia Treatment Center. This Northwell IRB approved retrospective analysis (Jan 2017 - Jun 2020) included data from 12 months prior to starting emicizumab prophylaxis RESULTS: A total of 38 patients on emicizumab prophylaxis were included. 35 patients (92.1%) had severe and 3 (7.9%) had moderate Hemophilia A. 11 patients (28.9%) had a FVIII inhibitor. The mean age was 16.4 yrs with 18 patients (47%) below the age of 12 (age limit used in HAVEN trials). 54.1% of patients were Caucasian, 18.9% were Asian and 16.2% were African American. Thirty patients (78.9%) were on prophylaxis with either rFVIII (71.5%) or FEIBA (21.4%) before starting emicizumab. All 38 patients were started on emicizumab with a loading dose of 3 mg/kg once weekly for 4 weeks followed by a maintenance dose of 1.5 mg/kg weekly. The ABR decreased by 52% for inhibitor patients (3.6 events before and 1.7 events after starting emicizumab). Similarly, there was a dramatic 70.6% reduction for non-inhibitor patients (1.5 events before and 0.44 events after starting emicizumab). Patients on emicizumab prophylaxis experienced 35 bleeding events over 24 months. Nine out of these 35 events occurred in one patient alone with a high titer FVIII inhibitor. 56.5% of these events were joint bleeds, 26.1% (muscle bleeds), 13% (soft tissue bleeds) and 4.4% were mucocutaneous bleeds. Barring 2 events, all episodes (93.9%) were managed outpatient. A majority of these bleeds (72.1%) in inhibitor patients were treated with recombinant Factor VIIa (rVIIa, 16-24 doses for documented hemarthroses) and 3.4% were treated with FEIBA after nonresponse to rVIIa (9-15 doses) due to delayed treatment. Remaining non -inhibitor patients were treated with rFVIII (2-5 doses) with a good response. 52 % of these bleeds were trauma-related. Five surgeries were conducted in the inhibitor patients while on emicizumab prophylaxis and none experienced perioperative bleeding. RVIIa was used as a bypassing agent for 2 of these surgeries - a hemispherectomy and a port removal. No bypassing agent was used for the remaining 3 port removals. Five surgeries (1 total knee arthroplasty and 4 port removals) were performed in patients without a FVIII inhibitor. Recombinant Factor VIII was used as the replacement agent and the patient with knee arthroplasty experienced post-operative bleeding. There were no thrombotic episodes or deaths and all but one continued on emicizumab. The patient post knee arthroplasty discontinued emicizumab after recurrent bleeds into the replaced joint despite aggressive replacement with rFVIII and absence of an inhibitor. DISCUSSION: We report a real-world experience on the use of emicizumab prophylaxis for Hemophilia A with and without inhibitors in children and adults with 78.9% of our cohort below the age of 21 years. Our study demonstrated a similar decrease in ABR for non-inhibitor patients comparable to the HAVEN clinical trials. However, our inhibitor patients experienced a lower bleed reduction rate on emicizumab and all patients still experienced a considerable number of trauma-related bleeds (likely due to increased participation of inhibitor patients in sports). Two patients with traumatic muscle/joint bleeds were treated with aPCC due to non-response to rVIIa suggesting the need for early treatment of suspected bleeds on emicizumab. No thrombotic events or thrombotic microangiopathy was observed in our patient cohort. Our data suggest the need for ongoing patient education for early bleed recognition on emicizumab prophylaxis, prompt treatment of breakthrough bleeds and pre-sports prophylaxis with FVIII or bypassing agents. Disclosures Acharya: BPL: Membership on an entity's Board of Directors or advisory committees; Novonordisk: Membership on an entity's Board of Directors or advisory committees; Bayer Pharma Inc.: Research Funding.

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