scholarly journals A Novel Lysine to Arginine Substitution at Position 301 Enhances Activity of Factor IX

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3772-3772
Author(s):  
Daniel Verhoef ◽  
Jonathan H. Foley ◽  
Andrew Goodale ◽  
Emma Macrae ◽  
Jenny McIntosh ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Specific Activity ◽  
Factor Ix ◽  
Wild Type ◽  
Gain Of Function ◽  
Employment Equity ◽  
Knock Out ◽  
Genome Integration ◽  
Equity Ownership ◽  
Knock Out Mice

Abstract Introduction: AAV-mediated gene transfer of blood coagulation Factor IX (FIX) has been established as a safe and long-term treatment for patients suffering from severe hereditary Haemophilia B. A gain-of-function F9 transgene (F9-R338L; Padua) has recently been used to achieve higher functional levels of FIX, effectively eliminating the need for regular prophylaxis. The naturally-occurring R338L Padua mutation is situated in the catalytic domain of FIX on a helical side loop (region 332-339) that is involved in FVIIIa-mediated stimulation of substrate turnover. Here, we examined if a single amino acid substitution of a lysine at position 301 leads to gain of function. This basic residue sits adjacent to the 332-339 loop on an exposed helical segment (292-303) that has been implicated to interact with the FVIIIa A2 domain in the FIXa-FVIIIa tenase complex. Methods: We examined the lysine at position 301 (numbering based on mature polypeptide chain) in more detail by conservative mutation to arginine (K301R) and non-conservative mutation to leucine (K301L). To assess specific FIX activity, F9-K301 variants were transiently expressed in HEK293T cells and tested for antigenic FIX levels and chromogenic activity 48 hours post transfection. To assess specific activity in plasma, AAV-mediated gene transfer (1x1010vg/mouse) of F9-K301 variants in hemophilia B knock-out mice (CL57B6) was carried out. In addition, we investigated whether the F9-K301R mutation enhances specific activity in combination with the F9-R338L Padua mutation via site-specific genome integration. Results: Transient transfection of F9-K301 variants in HEK293T cells showed a 25% increase in specific activity with F9-K301R but a 50% reduction in activity with F9-K301L as compared to wild type F9 (WT-F9). Validation of gain-of-function was done by AAV-mediated gene transfer in hemophilia B knock-out mice. Four weeks post injection, plasma FIX antigen levels were similar in mice transduced with either F9-K301R (0.91±0.3 U/ml; N=3), F9-K301L (0.93±0.0 U/ml; N=2) or WT-F9 (0.94±0.19 U/ml; N=4) constructs. Interestingly, specific chromogenic activity in plasma from F9-K301R mice (2.71±0.66 U/ml) was more than 2-fold higher compared to plasma from mice in the WT-F9 cohort (1.25±0.2 U/ml). On the other hand, specific activity in the F9-K301L cohort (0.37±0.07 U/ml) was reduced compared to wild type F9, consistent with a haemophilic phenotype. Next, we investigated whether the F9-K301R mutation enhances activity in combination with the F9-R338L Padua mutation. To do so, we stably expressed wild type FIX (WT-FIX) and three FIX gain-of-function variants (FIX-K301R, FIX-R338L and FIX-K301R/R338L) in HEK293 cells via site-specific genome integration. Interestingly, higher FIX antigen levels were observed in conditioned media from cells (1.5x106) stably expressing FIX-K301R (0.14±0.01 U/ml) FIX-R338L (0.11±0.01 U/ml) and FIX-K301R/R338L (0.10±0.01 U/ml) relative to cells expressing WT-FIX (0.08±0.01 U/ml). Similar to previous results, specific chromogenic activity was more than 2-fold higher in FIX-K301R (1.25±0.08 U/ml) compared to WT-FIX (0.54±0.06 U/ml). In addition, specific activity was higher in FIX-K301R/R338L (7.71±0.35 U/ml) compared to FIX-R338L (6.69±0.32 U/ml), suggesting molecular synergism between both gain-of-function mutations. Ongoing studies are focused on characterizing these recombinant FIX variants in purified and plasma-based activity assays and unraveling the mechanism(s) leading to increased expression/secretion of these gain-of-function variants. Conclusion: In summary, these results show that the K301R mutation enhances catalytic activity of FIX in vitro and in vivo and synergistically enhances activity in combination with the R338L Padua mutation. As such, this gain-of-function mutation could potentially serve to facilitate higher levels of FIX activity in the plasma of Haemophilia B patients following AAV-mediated gene transfer. Disclosures Verhoef: Freeline: Employment, Equity Ownership. Foley:Freeline: Employment, Equity Ownership. Goodale:Freeline: Employment, Equity Ownership. Macrae:Freeline: Employment, Equity Ownership. McIntosh:BioMarin: Patents & Royalties; Freeline: Consultancy, Equity Ownership. Corbau:Freeline: Employment, Equity Ownership. Nathwani:Freeline: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

scholarly journals Adeno-Associated Viral Vector Delivery of Optimized Human Factor VIII Achieves Therapeutic Factor VIII Levels in Non-Human Primates

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 199-199 ◽  
Author(s):  
Xavier M. Anguela ◽  
Liron Elkouby ◽  
Raffaella Toso ◽  
Marti DiPietro ◽  
Robert J. Davidson ◽  
...  
Keyword(s):  
Gene Transfer ◽  
Factor Viii ◽  
Human Factor ◽  
Hemophilia A ◽  
Factor Ix ◽  
Large Animal ◽  
Wild Type ◽  
Employment Equity ◽  
Human Factor Viii ◽  
Equity Ownership

Abstract Clinical studies of adeno-associated viral (AAV)-mediated gene transfer of factor IX for hemophilia B have demonstrated long term expression of therapeutic levels of factor IX but revealed that the AAV vector dose may be limiting due to anti-AAV immune responses (Nathwani, 2011). While there is significant interest in moving this approach forward for hemophilia A, it is challenging to express high levels of human factor VIII (hFVIII) due to its intrinsic properties that result in lower expression levels compared to similarly sized proteins (Lynch, 1993). Approaches using codon optimization and variants of hFVIII with enhanced function (increased activity, stability and/or secretion) may provide strategies to increase hFVIII expression to support AAV clinical studies for hemophilia A. For example, we previously developed a codon-optimized hFVIII (CO3) that expressed 5-8-fold higher protein levels than wild type hFVIII after AAV delivery in the context of an optimized expression cassette utilizing a modified transthyretin (TTRm) promoter. Introduction of a PACE-furin (P/F) variant (Siner, 2013) that deletes residues 1645-47 (Δ3) or 1645-48 (Δ4) of the PACE-furin recognition site in CO3 resulted in hFVIII expression after AAV delivery that was 18 (Δ3) or 12-fold (Δ4) better than wild type hFVIII. To date, only one published study has reported clinically relevant levels of human FVIII following AAV treatment in a large animal model. This study used a hFVIII variant that contained a 17 amino acid synthetic sequence flanked by 14-amino acid SQ residues from the N- and C-terminal ends of the B domain (McIntosh, 2013). While the presence of the synthetic spacer allowed for an increase in circulating hFVIII levels, the use of a non-wild-type FVIII sequence in hemophilia A patients may increase the risk of development of neutralizing antibodies to FVIII due to its potential neo-antigenicity. Our goal in this study was to generate an AAV-hFVIII vector capable of expressing therapeutic doses of FVIII at a clinically relevant vector dose without adding any neoantigens to the protein. To this end, we generated 26 codon-optimized hFVIII-SQ constructs under the control of the TTRm promoter. Hydrodynamic delivery of the pAAV-TTRm-hFVIII plasmids identified 11 candidates that expressed FVIII 2-7 fold higher than CO3. Nine of these FVIII expression constructs were made into AAV vectors and delivered to hemophilia A/CD4 KO mice (1x1011 vg/mouse) using a novel capsid, AAV-Spark100. At 4 weeks post vector administration, 2/9 constructs were similar to CO3, 5/9 were 3-4 fold higher than CO3 and 2/9 (SPK-8003 and SPK-8005) were 4-6 fold higher than CO3. To determine if the deletion of the PACE-furin site would result in higher FVIII expression, the Δ4 P/F deletion was introduced into SPK-8003. The levels of FVIII expression after AAV-TTRm-SPK-8003-Δ4 P/F delivery were 2-fold higher than AAV-TTRm-SPK-8003. In order to evaluate the potency of these novel cassettes in a large animal model, SPK-8005 was administered as a single dose via intravenous infusion to male cynomolgus macaques and followed for 8 weeks of observation. At two weeks after gene transfer, NHPs transduced with 2x1012 vg/kg of SPK-8005 expressed hFVIII antigen levels of 12.7 ± 2.1% (average ± standard error of the mean, n=3). Average FVIII expression after treatment with 5x1012 vg/kg was 22.6 ± 0.8% (n=2). Finally, at the highest tested dose of 1x1013 vg/kg, hFVIII antigen levels of 54.1 ± 15.6% were observed two weeks after AAV infusion (n=3). As anticipated, hFVIII expression declined in approximately one third of the animals around week 4, concomitant with the appearance of inhibitory antibodies to human factor VIII in these macaques. In summary, these data using highly active, novel codon-optimized FVIII constructs devoid of potential neoantigens demonstrate the feasibility of lowering the AAV capsid load for a gene-based therapeutic approach for hemophilia A to a dosage level that appears to be efficacious and safe in the treatment of hemophilia B. Disclosures Anguela: Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties. Elkouby:Spark Therapeutics, Inc.: Employment, Equity Ownership. Toso:Spark Therapeutics, Inc.: Employment, Equity Ownership. DiPietro:Spark Therapeutics, Inc.: Employment, Equity Ownership. Davidson:Spark Therapeutics: Consultancy. High:Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties: AAV gene transfer technology. Sabatino:Spark Therapeutics, Inc.: Research Funding.

scholarly journals Spk-9001: Adeno-Associated Virus Mediated Gene Transfer for Hemophilia B Achieves Sustained Mean Factor IX Activity Levels of >30% without Immunosuppression

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3-3 ◽  
Author(s):  
Lindsey A. George ◽  
Spencer K. Sullivan ◽  
Adam Giermasz ◽  
Jonathan M. Ducore ◽  
Jerome M. Teitel ◽  
...  
Keyword(s):  
Immune Response ◽  
Gene Transfer ◽  
Board Of Directors ◽  
Research Funding ◽  
Factor Ix ◽  
Hemophilia B ◽  
Employment Equity ◽  
Advisory Committees ◽  
Equity Ownership

Abstract Background: Earlier data demonstrated long-term expression of factor IX (mean FIX:C ~5.1%) following AAV8-mediated gene transfer at 2 x1012 vg/kg in hemophilia B (Nathwani et al., 2014). While the clinical improvement imparted by stable FIX levels is clear, these levels of expression fall short of trough values obtained by long-acting FIX prophylaxis (Santagostino et al. 2016), and of natural history data suggesting that levels of ~12% are required to eliminate spontaneous hemarthroses (den Uijl et al. 2011). Achieving higher levels of FIX:C with dose escalation has not been possible without eliciting a dose-dependent, capsid-specific immune response that may prevent sustained expression and efficacy (Mingozzi et al. 2007, Monahan et al. 2015). We sought to develop a highly efficient vector capsid and expression cassette that could be administered at low doses to achieve hemostatic FIX expression without need for immunosuppression. Methods: The investigational product, SPK-9001, utilizes a bioengineered AAV capsid (Spark100) with liver specific tropism. The prevalence of neutralizing antibodies (NAb) to Spark100 among sampled hemophilia B sera was 40% (Anguela et al. 2015). The expression cassette is a codon-optimized, single-stranded transgene encoding FIX Padua, a naturally occurring variant with a single amino acid substitution (R338L) that confers ~8-fold greater specific activity compared to wild-type FIX (Simioni et al. 2009). Data on bleeding and factor infusions in the year prior to enrollment were retrospectively compiled. Laboratory values, bleeding frequency, FIX consumption, changes in activity and quality-of-life via Haem-A-QoL were prospectively evaluated after vector infusion. Results: We enrolled 9 subjects, of whom 2 failed screening for liver fibrosis and 7 were infused with SPK-9001 at a dose of 5 x1011 vg/kg. Infused subjects were adult males ages 18-52 years with baseline FIX:C </=2% and Spark100 NAb titer of <1:1 or 1:1. Table 1 outlines infused subject data with a follow up interval of >2-34 weeks after vector infusion. Figure 1 outlines subject vector-derived FIX:C for the first 12 weeks. There have been no vector or procedure related adverse events. Steady-state FIX expression is reached by 12 weeks after vector infusion, resulting in a mean FIX:C of 32.3% ±6.5%. To date, no subjects required immunosuppression or demonstrated evidence of a cytotoxic immune response (characterized by loss of FIX activity, elevation of transaminase values >/=1.5-times the upper limit of normal, and positive IFN-gammaELISPOT response to capsid peptides). No subjects developed a FIX inhibitor or demonstrated ELISPOT reactivity to the FIX (R338L) gene product. Subject 3 infused with FIX concentrate for a suspected ankle bleed 2 days after vector infusion. Beyond this, no subjects required factor or experienced any bleeding events. The 4 subjects previously maintained on prophylaxis safely stopped without break-through bleeding. As of today (cumulative 724 days post vector infusion), total factor consumption was reduced by 543,589 IU, tantamount to a cumulative savings of $1,182,298 USD.Six of 7 subjects report increased physical activity and improved quality of life. Conclusion: As of 8/4/2016, we report the highest and most consistent levels of sustained vector-derived FIX:C following FIX gene transfer. Levels of FIX:C achieved by SPK-9001 permitted termination of prophylaxis, prevention of bleeding, and nearly complete cessation of factor use. Despite the heterogeneity in subjects with respect to presence and extent of hemophilic arthropathy, age, and co-morbidities, consistency of transgene expression and clinical outcomes have been observed in all participants studied to date. A vector dose of 5x1011 vg/kg is the lowest dose currently reported in hemophilia gene transfer trials; the absence of any observed CD8+ T cell immune response supports the hypothesis that lowering the dose can reduce or eliminate the risk of a capsid-specific immune response and maximize efficacy. In summary, preliminary data suggest SPK-9001 safely and consistently produces sustained elevation in FIX:C levels sufficient to prevent spontaneous hemarthroses without the need for factor consumption or immunosuppression. Disclosures Ducore: Octapharama: Membership on an entity's Board of Directors or advisory committees; LFB: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees; 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 (Shire): Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees. Cuker:Biogen-Idec: Consultancy, Research Funding; T2 Biosystems: Research Funding; Genzyme: Consultancy; Stago: Consultancy; Amgen: Consultancy. McGuinn:Spark: Research Funding; Biogen: Research Funding; Novo Nordisk: Research Funding; Baxalta: Research Funding. Luk:Spark Therapeutics, Inc.: Employment. Wright:Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties: SPK-9001. Chen:Spark Therapeutics, Inc.: Employment. Hui:Spark Therapeutics, Inc.: Employment. Wachtel:Spark Therapeutics, Inc.: Employment. Urich:Spark Therapeutics, Inc.: Employment. Takefman:Spark Therapeutics, Inc.: Employment. Couto:Spark Therapeutics, Inc.: Employment. Carr:Pfizer, Inc.: Research Funding. Anguela:Spark Therapeutics, Inc.: Employment, Patents & Royalties: SPK-9001. High:Spark Therapeutics, Inc.: Employment, Equity Ownership, Patents & Royalties: SPK-9001.

scholarly journals Activity of a FIX-Padua Transgene Product in Commonly Used FIX:C One-Stage and Chromogenic Assay Systems Following PF-06838435 (SPK-9001) Gene Delivery

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2198-2198 ◽  
Author(s):  
Mary Robinson ◽  
Lindsey A. George ◽  
Benjamin J. Samelson-Jones ◽  
Valder R Arruda ◽  
Katherine A. High ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Specific Activity ◽  
Factor Ix ◽  
Hemophilia B ◽  
Employment Equity ◽  
Siemens Healthcare ◽  
Buffer Solutions ◽  
One Stage ◽  
Chromogenic Assay ◽  
Equity Ownership

Abstract Background PF-06838435 (SPK-9001), a gene therapy candidate containing a high specific-activity factor IX variant (R338L, FIX-Padua), is currently in phase 3 of clinical development for the treatment of Hemophilia B. Initial data with this vector is promising with significant reductions in bleeding episodes and FIX consumption (George LA et al, NEJM 2017; 377:2215-2217). To date, little is known about the activity of the expressed transgene product as measured in FIX:C one-stage and chromogenic assay systems commonly used to monitor FIX replacement therapy in patients with Hemophilia B. Aim The goal of the study was to assess the activity of the PF-06838435 expressed transgene product in plasma samples collected from participants in the Phase 1/2 trial using four commonly used FIX:C aPTT reagents. For comparison, the activity of the expressed FIX-Padua gene product was also assessed in the ROX FACTOR IX chromogenic assay. In addition, the activity of the Padua variant in congenital FIX deficient plasma spiked with increasing concentrations of purified recombinant human FIX Padua protein (rHFIXp - Samelson-Jones Lab, CHOP/UPenn), as well as recombinant human FIX (rHFIX, BeneFIX®, Pfizer Inc.), was assessed in the same FIX:C assay procedures. Methods FIX:C, in four samples collected from two different patients who received FIX gene therapy, was tested in four in vitro diagnostic (IVD) approved FIX:C one-stage assay systems, STA®-PTT Automate and STA®-C.K. Prest® on the STA-R Evolution® (Diagnostica Stago Inc.), Dade Actin® FSL on the BCS®XP (Siemens Healthcare), and HemosIL® SynthASil® on the ACL TOP® (Instrumentation Laboratory). In addition, samples were also tested in the ROX FACTOR IX (Rossix AB) chromogenic assay on the BCS®XP (Siemens Healthcare). The aPTT reagents selected for this study correspond to 90% of the FIX:C assay reagents currently used in CAP accredited laboratories in the US(2014 CAP Proficiency Survey) and represent the three main types of activator commonly used in the FIX one-stage clot assay: silica (STA®-PTT Automate, HemosIL® SynthASil®), ellagic acid (Dade Actin® FSL) and kaolin (STA®-C.K. Prest®). For comparison, FIX:C in each of the five FIX:C assay systems was also determined in samples spiked with purified rHFIXp (provided by Dr. Samelson-Jones) or rHFIX (provided by Spark Therapeutics Inc.) in 20X buffer solutions. On the day of testing rHFIXp, and rHFIX 20X buffer solutions were diluted 1:20 in congenital FIX deficient plasma to achieve approximate final FIX:C concentrations of 40, 30 and 20%, extrapolated from an estimated 8-fold specific activity to antigen ratio. Results A consistent pattern in the measured FIX:C for the PF-06838435 transgene product was observed in the five FIX:C assay systems (Fig. 1). For the aPTT based FIX:C assays, Actin FSL, gave the lowest FIX:C values, whereas PTT Automate measured the highest FIX:C levels. In all cases, the chromogenic FIX:C assay gave the lowest activity values for the transgene product. A similar FIX:C assay dependent pattern was observed for the purified rHFIXp spiked at 40, 30 and 20% FIX:C (Fig. 2). In all FIX:C assays tested, rHFIXp was under-recovered to a varying degree from target, with recoveries for the 20% FIX:C samples ranging between -26.5% (STA®-PTT Automate) and -73.5% (Dade Actin® FSL) in the aPTT based FIX:C assays and -85% in the ROX FIX chromogenic assay. In contrast, rHFIX (BeneFIX®) was recovered within ±25% of expected values in all aPTT based FIX:C assays and, consistent with previously reported data, modestly under-recovered in the ROX FIX chromogenic assay (Fig. 3). Conclusion This study found differences in the FIX:C results obtained for a Padua FIX variant transgene product and recombinant human FIX-Padua when tested in commonly used IVD approved FIX:C assays in North America. These results suggest that FIX:C assay selection is important for measuring FIX-Padua activity, which will be particularly relevant in hemophilia B gene therapy following FIX-Padua gene transfer. Disclosures George: University of Pennsylvania: Equity Ownership; Pfizer: Consultancy. High:Spark Therapeutics: Employment, Equity Ownership, Patents & Royalties. Carr:Sparks Therapeutics Inc.: Consultancy. Tiefenbacher:Laboratory Corporation of America: Employment, Equity Ownership; Siemens Healthcare: Consultancy.

Functional characterization of the H+/K+ ATPase in wild type and gastrin knock-out mice

2007 ◽  
Vol 45 (05) ◽  
Author(s):  
A Schnur ◽  
P Hegyi ◽  
V Venglovecz ◽  
Z Rakonczay ◽  
I Ignáth ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Wild Type ◽  
Knock Out ◽  
Knock Out Mice

scholarly journals Postnatal changes of interleukin-18 receptor immunoreactivity in neurons of the retrosplenial cortex in wild-type and interleukin-18 knock out mice

2017 ◽  
Vol 94 (3) ◽  
pp. 93-99
Author(s):  
Tetsu HAYAKAWA ◽  
Masaki HATA ◽  
Sachi KUWAHARA-OTANI ◽  
Hideshi YAGI ◽  
Haruki OKAMURA
Keyword(s):  
Retrosplenial Cortex ◽  
Wild Type ◽  
Interleukin 18 ◽  
Knock Out ◽  
Knock Out Mice

scholarly journals Retinol Saturase Knock-Out Mice are Characterized by Impaired Clearance of Apoptotic Cells and Develop Mild Autoimmunity

Biomolecules ◽  
2019 ◽  
Vol 9 (11) ◽  
pp. 737 ◽  
Author(s):  
Zsolt Sarang ◽  
Tibor Sághy ◽  
Zsófia Budai ◽  
László Ujlaky-Nagy ◽  
Judit Bedekovics ◽  
...  
Keyword(s):  
Transglutaminase 2 ◽  
Tissue Homeostasis ◽  
Apoptotic Cells ◽  
Wild Type ◽  
Integrin Receptors ◽  
Macrophage Differentiation ◽  
Phagocytic Capacity ◽  
Knock Out ◽  
Knock Out Mice ◽  
Number Of Cells

Apoptosis and the proper clearance of apoptotic cells play a central role in maintaining tissue homeostasis. Previous work in our laboratory has shown that when a high number of cells enters apoptosis in a tissue, the macrophages that engulf them produce retinoids to enhance their own phagocytic capacity by upregulating several phagocytic genes. Our data indicated that these retinoids might be dihydroretinoids, which are products of the retinol saturase (RetSat) pathway. In the present study, the efferocytosis of RetSat-null mice was investigated. We show that among the retinoid-sensitive phagocytic genes, only transglutaminase 2 responded in macrophages and in differentiating monocytes to dihydroretinol. Administration of dihydroretinol did not affect the expression of the tested genes differently between differentiating wild type and RetSat-null monocytes, despite the fact that the expression of RetSat was induced. However, in the absence of RetSat, the expression of numerous differentiation-related genes was altered. Among these, impaired production of MFG-E8, a protein that bridges apoptotic cells to the αvβ3/β5 integrin receptors of macrophages, resulted in impaired efferocytosis, very likely causing the development of mild autoimmunity in aged female mice. Our data indicate that RetSat affects monocyte/macrophage differentiation independently of its capability to produce dihydroretinol at this stage.

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