Posttranslational modifications of recombinant myotube-synthesized human factor IX

Blood ◽  
2001 ◽  
Vol 97 (1) ◽  
pp. 130-138 ◽  
Author(s):  
Valder R. Arruda ◽  
James N. Hagstrom ◽  
Jeffrey Deitch ◽  
Terry Heiman-Patterson ◽  
Rodney M. Camire ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Posttranslational Modifications ◽  
Half Life ◽  
Specific Activity ◽  
Factor Ix ◽  
Serine Phosphorylation ◽  
Hemophilia B ◽  
Tyrosine Sulfation ◽  
Aav Vector ◽  
Carbohydrate Analysis

Abstract Recent data demonstrate that the introduction into skeletal muscle of an adeno-associated viral (AAV) vector expressing blood coagulation factor IX (F.IX) can result in long-term expression of the transgene product and amelioration of the bleeding diathesis in animals with hemophilia B. These data suggest that biologically active F.IX can be synthesized in skeletal muscle. Factor IX undergoes extensive posttranslational modifications in the liver, the normal site of synthesis. In addition to affecting specific activity, these posttranslational modifications can also affect recovery, half-life in the circulation, and the immunogenicity of the protein. Before initiating a human trial of an AAV-mediated, muscle-directed approach for treating hemophilia B, a detailed biochemical analysis of F.IX synthesized in skeletal muscle was carried out. As a model system, human myotubes transduced with an AAV vector expressing F.IX was used. F.IX was purified from conditioned medium using a novel strategy designed to purify material representative of all species of rF.IX in the medium. Purified F.IX was analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), N-terminal sequence analysis, chemical γ-carboxyglutamyl analysis, carbohydrate analysis, assays for tyrosine sulfation, and serine phosphorylation, and for specific activity. Results show that myotube-synthesized F.IX has specific activity similar to that of liver-synthesized F.IX. Posttranslational modifications critical for specific activity, including removal of the signal sequence and propeptide, and γ-carboxylation of the N-terminal glutamic acid residues, are also similar, but carbohydrate analysis and assessment of tyrosine sulfation and serine phosphorylation disclose differences. In vivo experiments in mice showed that these differences affect recovery but not half-life of muscle-synthesized F.IX.

Factor IX variants improve gene therapy efficacy for hemophilia B

Blood ◽  
2005 ◽  
Vol 105 (6) ◽  
pp. 2316-2323 ◽  
Author(s):  
Joerg Schuettrumpf ◽  
Roland W. Herzog ◽  
Alexander Schlachterman ◽  
Antje Kaufhold ◽  
Darrel W. Stafford ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Intramuscular Injection ◽  
Specific Activity ◽  
Factor Ix ◽  
Hemophilia B ◽  
Promising Strategy ◽  
B Mice ◽  
Higher Doses ◽  
Vector Delivery

Abstract Intramuscular injection of adeno-associated viral (AAV) vector to skeletal muscle of humans with hemophilia B is safe, but higher doses are required to achieve therapeutic factor IX (F.IX) levels. The efficacy of this approach is hampered by the retention of F.IX in muscle extracellular spaces and by the limiting capacity of muscle to synthesize fully active F.IX at high expression rates. To overcome these limitations, we constructed AAV vectors encoding F.IX variants for muscle- or liver-directed expression in hemophilia B mice. Circulating F.IX levels following intramuscular injection of AAV-F.IX-K5A/V10K, a variant with low-affinity to extracellular matrix, were 2-5 fold higher compared with wild-type (WT) F.IX, while the protein-specific activities remained similar. Expression of F.IX-R338A generated a protein with 2- or 6-fold higher specific activity than F.IX-WT following vector delivery to skeletal muscle or liver, respectively. F.IX-WT and variant forms provide effective hemostasis in vivo upon challenge by tail-clipping assay. Importantly, intramuscular injection of AAV-F.IX variants did not trigger antibody formation to F.IX in mice tolerant to F.IX-WT. These studies demonstrate that F.IX variants provide a promising strategy to improve the efficacy for a variety of gene-based therapies for hemophilia B.

Glycopegylated Factor IX Enables Prolonged Efficacy After Subcutaneous Injection But Requires a Higher Than Expected Plasma Activity To Prevent Bleeding In Hemophilia B Mice

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1104-1104
Author(s):  
Derek S Sim ◽  
Alan Brooks ◽  
Cornell Mallari ◽  
Yifan Xu ◽  
Rick I Feldman ◽  
...  
Keyword(s):  
Half Life ◽  
Subcutaneous Injection ◽  
Specific Activity ◽  
Periodate Oxidation ◽  
Factor Ix ◽  
Hemophilia B ◽  
Current Therapy ◽  
B Mice ◽  
Trough Levels ◽  
Plasma Activity

Abstract Background Reduced frequency of administration as well as subcutaneous (s.c.) injection would improve the treatment of Hemophilia B. Conjugation to polyethylene glycol (PEG) has been shown to increase the half-life of i.v. dosed Factor IX (FIX), but s.c. dosing of PEGylated FIX was not previously evaluated. Because s.c. dosing is limited by volume and bioavailability, we evaluated the combination of PEGylation with the increased specific activity variant R338A to reduce the amount of protein needed to provide therapeutic levels of FIX. Methods FIX-R338A was PEGylated on N-linked glycans in the activation peptide by periodate oxidation of sialic acid residues followed by conjugation to amino-oxy functionalized PEG. Pharmacokinetic (PK) profiles were determined in hemophilia B mice and cynomologous monkeys. Allometric scaling was used to predict dose regimens in humans. Prophylactic efficacy was determined in a Hemophilia B mouse tail bleeding model. Results 60kDaPEG-R338A had prolonged terminal half-life in mice (3-fold) and monkeys (5-fold) and the s.c. bioavailability was 44% and 35%, respectively. The volume of distribution was reduced 5-fold. To achieve a trough level of 3% FIX activity, s.c. dosing at weekly, bi-monthly and monthly intervals was predicted to require doses of 7, 25 and 220 IU/kg in patients. However, in a tail vein transection injury model, approximately 10-fold higher plasma FIX activity levels of PEGylated proteins were found to be needed to protect hemophilia B mice against bleeding than was required for i.v. dosed un-PEGylated recombinant FIX. This difference was observed for PEGylated wild-type and R338A proteins, dosed i.v or s.c. We hypothesize that this is related to the reduced distribution of PEGylated FIX to the extravascular compartment. Trough levels of 30% FIX activity were predicted to be achievable in humans after weekly and bi-monthly s.c. dosing at 70 and 260 IU/kg. Conclusions The PEGylation of FIX led to a significant improvement in both i.v. and s.c. PK. Unexpectedly, a 10-fold higher plasma activity was needed for PEGylated FIX to provide protection against bleeding in Hemophilia B mice, suggesting that trough levels of 10 to 30% of PEGylated FIX activity may be needed in patients to provide efficacy equivalent to current therapy of recombinant or plasma derived FIX. Nevertheless, 60kDaPEG-R338A has the potential to treat hemophilia B patients with once weekly or twice monthly subcutaneous injection. Disclosures: Sim: Bayer HealthCare: Employment. Brooks:Bayer HealthCare: Employment. Mallari:Bayer HealthCare: Employment. Xu:Bayer HealthCare: Employment. Feldman:Bayer HealthCare: Employment. Schneider:Bayer HealthCare: Employment. Patel:Bayer HealthCare: Employment. Blasko:Bayer HealthCare: Employment. Ho:Bayer HealthCare: Employment. Su:Bayer HealthCare: Employment. Liu:Bayer HealthCare: Employment. Laux:Bayer HealthCare: Employment. Murphy:Bayer HealthCare: Employment.

scholarly journals Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer

Blood ◽  
2012 ◽  
Vol 119 (13) ◽  
pp. 3038-3041 ◽  
Author(s):  
George Buchlis ◽  
Gregory M. Podsakoff ◽  
Antonetta Radu ◽  
Sarah M. Hawk ◽  
Alan W. Flake ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Gene Transfer ◽  
Muscle Tissue ◽  
Viral Vector ◽  
Factor Ix ◽  
Hemophilia B ◽  
Immunofluorescent Staining ◽  
Severe Hemophilia ◽  
Aav Vector ◽  
Human Factor Ix

AbstractIn previous work we transferred a human factor IX–encoding adeno-associated viral vector (AAV) into skeletal muscle of men with severe hemophilia B. Biopsy of injected muscle up to 1 year after vector injection showed evidence of gene transfer by Southern blot and of protein expression by IHC and immunofluorescent staining. Although the procedure appeared safe, circulating F.IX levels remained subtherapeutic (< 1%). Recently, we obtained muscle tissue from a subject injected 10 years earlier who died of causes unrelated to gene transfer. Using Western blot, IHC, and immunofluorescent staining, we show persistent factor IX expression in injected muscle tissue. F.IX transcripts were detected in injected skeletal muscle using RT-PCR, and isolated whole genomic DNA tested positive for the presence of the transferred AAV vector sequence. This is the longest reported transgene expression to date from a parenterally administered AAV vector, with broad implications for the future of muscle-directed gene transfer.

scholarly journals Prolonged half-life and preserved enzymatic properties of factor IX selectively PEGylated on native N-glycans in the activation peptide

Blood ◽  
2011 ◽  
Vol 118 (8) ◽  
pp. 2333-2341 ◽  
Author(s):  
Henrik Østergaard ◽  
Jais R. Bjelke ◽  
Lene Hansen ◽  
Lars Christian Petersen ◽  
Anette A. Pedersen ◽  
...  
Keyword(s):  
Half Life ◽  
Specific Activity ◽  
Factor Ix ◽  
Hemophilia B ◽  
Duration Of Effect ◽  
B Mice ◽  
Bleeding Episodes ◽  
Activation Peptide ◽  
Extended Circulation

Abstract Current management of hemophilia B entails multiple weekly infusions of factor IX (FIX) to prevent bleeding episodes. In an attempt to make a longer acting recombinant FIX (rFIX), we have explored a new releasable protraction concept using the native N-glycans in the activation peptide as sites for attachment of polyethylene glycol (PEG). Release of the activation peptide by physiologic activators converted glycoPEGylated rFIX (N9-GP) to native rFIXa and proceeded with normal kinetics for FXIa, while the Km for activation by FVIIa–tissue factor (TF) was increased by 2-fold. Consistent with minimal perturbation of rFIX by the attached PEG, N9-GP retained 73%-100% specific activity in plasma and whole-blood–based assays and showed efficacy comparable with rFIX in stopping acute bleeds in hemophilia B mice. In animal models N9-GP exhibited up to 2-fold increased in vivo recovery and a markedly prolonged half-life in mini-pig (76 hours) and hemophilia B dog (113 hours) compared with rFIX (16 hours). The extended circulation time of N9-GP was reflected in prolonged correction of coagulation parameters in hemophilia B dog and duration of effect in hemophilia B mice. Collectively, these results suggest that N9-GP has the potential to offer efficacious prophylactic and acute treatment of hemophilia B patients at a reduced dosing frequency.

scholarly journals Peripheral transvenular delivery of adeno-associated viral vectors to skeletal muscle as a novel therapy for hemophilia B

Blood ◽  
2010 ◽  
Vol 115 (23) ◽  
pp. 4678-4688 ◽  
Author(s):  
Valder R. Arruda ◽  
Hansell H. Stedman ◽  
Virginia Haurigot ◽  
George Buchlis ◽  
Stefano Baila ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Viral Vectors ◽  
High Titer ◽  
Factor Ix ◽  
Hemophilia B ◽  
Novel Therapy ◽  
Spontaneous Bleeding ◽  
Aav Vector ◽  
Efficient Vector ◽  
Bleeding Episodes

Abstract Muscle represents an important tissue target for adeno-associated viral (AAV) vector-mediated gene transfer of the factor IX (FIX) gene in hemophilia B (HB) subjects with advanced liver disease. Previous studies of direct intramuscular administration of an AAV-FIX vector in humans showed limited efficacy. Here we adapted an intravascular delivery system of AAV vectors encoding the FIX transgene to skeletal muscle of HB dogs. The procedure, performed under transient immunosuppression (IS), resulted in widespread transduction of muscle and sustained, dose-dependent therapeutic levels of canine FIX transgene up to 10-fold higher than those obtained by intramuscular delivery. Correction of bleeding time correlated clinically with a dramatic reduction of spontaneous bleeding episodes. None of the dogs (n = 14) receiving the AAV vector under transient IS developed inhibitory antibodies to canine FIX; transient inhibitor was detected after vector delivery without IS. The use of AAV serotypes with high tropism for muscle and low susceptibility to anti-AAV2 antibodies allowed for efficient vector administration in naive dogs and in the presence of low- but not high-titer anti-AAV2 antibodies. Collectively, these results demonstrate the feasibility of this approach for treatment of HB and highlight the importance of IS to prevent immune responses to the FIX transgene product.

scholarly journals Real World Factor Dispensation and Expenditures in us Patients with Hemophilia B: Standard Half-Life vs. Extended Half-Life Factor IX Replacement Products

2018 ◽  
Vol 21 ◽  
pp. S111
Author(s):  
A Chhabra ◽  
D Spurden ◽  
BJ Tortella ◽  
PF Fogarty ◽  
A Pleil ◽  
...  
Keyword(s):  
Real World ◽  
Half Life ◽  
Factor Ix ◽  
Hemophilia B ◽  
Life Factor

scholarly journals In silico evaluation of limited sampling strategies for individualized dosing of extended half-life factor IX concentrates in hemophilia B patients

2021 ◽  
Author(s):  
T. Preijers ◽  
M. W. F. van Spengler ◽  
K. Meijer ◽  
K. Fijnvandraat ◽  
K. Fischer ◽  
...  
Keyword(s):  
In Silico ◽  
Half Life ◽  
Factor Ix ◽  
Hemophilia B ◽  
Weekly Dose ◽  
Sampling Strategies ◽  
Time Profiles ◽  
Limited Sampling ◽  
Individualized Dosing ◽  
Life Factor

Abstract Purpose Hemophilia B is a bleeding disorder, caused by a factor IX (FIX) deficiency. Recently, FIX concentrates with extended half-life (EHL) have become available. Prophylactic dosing of EHL-FIX concentrates can be optimized by assessment of individual pharmacokinetic (PK) parameters. To determine these parameters, limited sampling strategies (LSSs) may be applied. The study aims to establish adequate LSSs for estimating individual PK parameters of EHL-FIX concentrates using in silico evaluation. Methods Monte Carlo simulations were performed to obtain FIX activity versus time profiles using published population PK models for N9-GP (Refixia), rFIXFc (Alprolix), and rIX-FP (Idelvion). Fourteen LSSs, containing three or four samples taken within 8 days after administration, were formulated. Bayesian analysis was applied to obtain estimates for clearance (CL), half-life (t1/2), time to 1% (Time1%), and calculated weekly dose (Dose1%). Bias and precision of these estimates were assessed to determine which LSS was adequate. Results For all PK parameters of N9-GP, rFIXFc and rIX-FP bias was generally acceptable (range: −5% to 5%). For N9-GP, precision of all parameters for all LSSs was acceptable (< 25%). For rFIXFc, precision was acceptable for CL and Time1%, except for t1/2 (range: 27.1% to 44.7%) and Dose1% (range: 12% to 29.4%). For rIX-FP, all LSSs showed acceptable bias and precision, except for Dose1% using LSS with the last sample taken on day 3 (LSS 6 and 10). Conclusion Best performing LSSs were LSS with samples taken at days 1, 5, 7, and 8 (N9-GP and rFIXFc) and at days 1, 4, 6, and 8 (rIX-FP), respectively.

scholarly journals Hemophilia B Gene Therapy with a High-Specific-Activity Factor IX Variant

2017 ◽  
Vol 377 (23) ◽  
pp. 2215-2227 ◽  
Author(s):  
Lindsey A. George ◽  
Spencer K. Sullivan ◽  
Adam Giermasz ◽  
John E.J. Rasko ◽  
Benjamin J. Samelson-Jones ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Specific Activity ◽  
High Specific Activity ◽  
Factor Ix ◽  
Hemophilia B ◽  
Activity Factor

Characterization of the Immune Response to Canine Factor IX Following AAV-Mediated Intravascular Gene Delivery to Skeletal Muscle in Hemophilia B Dogs.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1297-1297
Author(s):  
Daniel J. Hui ◽  
Federico Mingozzi ◽  
Denise E. Sabatino ◽  
Stephanie McCorquodale ◽  
Aaron Dillow ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Immune Response ◽  
T Cell ◽  
T Cell Responses ◽  
Route Of Administration ◽  
Factor Ix ◽  
Hemophilia B ◽  
Cell Responses ◽  
Chapel Hill ◽  
Ifn Γ

Abstract Progress towards an effective gene therapy for hemophilia B (HB) has been facilitated by large animal studies. Previous work has shown intravascular delivery of an adeno-associated viral (AAV) serotype 2 vector expressing canine factor IX (cF.IX) to skeletal muscle in HB dogs resulted in long-term expression of cF.IX at levels of 4–20% of normal, which nearly corrected the disease phenotype. However, occurrence of inhibitors to F.IX in some animals raises concerns of a potential immune response to the transgene product, prompting a more thorough examination of T cell responses in this setting. Early work revealed that transient immunosuppression (IS) with cyclophosphamide was required to avoid inadvertent antibody formation to F.IX. Here we report in detail the nature and the duration of T cell responses against the transgene product. Six HB dogs from the Chapel Hill colony received AAV2 vector at three different doses (1x1012 vg/kg, n=3; 3 x 1012 vg/kg, n=2; 8 x 1012 vg/kg, n=1) in addition to weekly infusion with cyclophosphamide (6 doses total). PBMCs were isolated from whole blood prior to vector infusion, during IS and after removal from IS, and used to measure the T cell response to F.IX by ELISpot assay for IL-10 and IFN-γ secretion using a cF.IX peptide library composed of 15-mers overlapping by 10 amino acids, spanning the entire protein sequence. Peptides were arranged into a matrix of pools, such that each peptide was contained in two orthogonal pools. Interestingly, in the IL-10 assay, one common T cell epitope corresponding to peptide 68 in the cF.IX library was found in all intravascularly-administered dogs from each of the three dose groups. The same epitope was also detectable in naïve HB dogs. Another epitope, corresponding to peptide 84, was found in a dog injected with the highest dose of vector after it developed a non-neutralizing antibody response against the cF.IX transgene product. Peptide 84 spans the region of the protein that contains the missense mutation responsible for HB (Chapel Hill mutation, Glu379 → Gly), which is a key difference in the newly introduced transgene product. Furthermore, the lack of any IFN-γ secretion coupled with the marked IL-10 response gives a cytokine profile that is characteristic of a Th2 response. This is in contrast with the Th1 response seen in previous studies with direct intramuscular injection of an AAV serotype 1 expressing cF.IX in dogs from the same colony. IS successfully reduced T cell responses to undetectable levels, while IL-10 secretion was detectable in PBMCs before vector delivery and one month after IS was discontinued. Overall circulating cF.IX levels did not seem to be affected by late restoration of T cells responses. No T cell responses against AAV capsid were detectable by ELISpot on PBMCs in any of the dogs studied. Interestingly, the relatively mild IS regimen also appeared to reduce the formation of neutralizing antibodies against AAV capsid, regardless of the route of administration. In summary, the nature of T cell responses (Th2 vs. Th1) suggests that route of administration, and/or AAV serotype, may play a role in the determination of the immune response elicited. We conclude that IS may provide a means to decrease T cell responses to the transgene following intravascular delivery of AAV-F.IX to skeletal muscle.

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