The Light Chain of Rat Factor VIII Confers Its Superior Cofactor Activity

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2038-2038
Author(s):  
Qizhao Wang ◽  
Jenni Firrman ◽  
Katie A Pokiniewski ◽  
Wenjing Cao ◽  
Hongying Wei ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Light Chain ◽  
Hemophilia A ◽  
Specific Activity ◽  
Genetic Defect ◽  
High Dose ◽  
Coagulation Activity ◽  
Fviii Activity ◽  
Cofactor Activity

Abstract Hemophilia A is caused by genetic defect of human coagulation factor VIII (hFVIII) and patients have to take lifelong replacement therapy to prevent excessive bleedings upon hemostatic challenges. Due to the short half-life of hFVIII, replacement treatment has to be given frequently and inhibitors against infused hFVIII can be developed in about 20-30% of patients. These shortcomings have generated tremendous interest in developing HA gene therapies which is more efficient and long-lasting. However, early preclinical studies have shown FVIII activities were still limited after vector delivery. A Modified hFVIII with higher specific activity and pharmacodynamics properties is highly desirable to overcome the disadvantages of current protein replacement and gene therapy strategies. In the current study, we successfully constructed a B-domain deleted rat FVIII(rBDDF8) that contained a PACE/furin recognition site (RHQR) within a 14 amino acid linker between A2 and A3 domains. The rBDDF8 displayed significantly higher coagulation activity(~2.5-fold) than hBDDF8 after transfection into HEK 293 cells. In order to explore the mechanism for the observed superior cofactor activity, we constructed heavy chain(rHC) and light chain(rLC) of rFVIII. The rHC and rLC are able to reconstitute 5 times more FVIII activity than their human counter parts. However, when rHC is associated with human FVIII light chain (hLC), the reconstituted FVIII activity is lower that from hHC and hLC, suggesting that high coagulation activity of rFVIII is not mediated by its HC. On the contrary, when FVIII is constituted by hHC with rLC, we found that the activity is increased by 3~5-fold as against hHC and hLC. The hHC antigen level of FVIII reconstituted from hHC and rLC was 1.5-fold higher than that of hHC and hLC, suggesting that higher activity of FVIII with hHC and rLC is not through increased secretion. The specific activity deduced from activity/antigen ratio showed that FVIII with rLC is 3 times higher more than FVIII with hLC. To investigate the potential application of rFVIII in gene therapy, rBDDF8 was delivered in hemophilia A mouse model using AAV8 vectors. The high dose rBDDF8(4*1011 vg/mouse) resulted 2.5U FVIII activity at week 17, which is much higher(about 10-fold) than that of hBDDF8. When the rFVIII was delivered by dual chains strategy, i.e, administering vectors carrying only LC or HC simultaneously, it also showed 2-4 fold increased in FVIII activity. Interestingly, the combination of hHC and rLC also generated similar FVIII activity as rHC and rLC, further proving the rLC is the major contributor to the superior coagulation activity of rFVIII. Our results showed that the rFVIII has higher cofactor activity conferred by its LC. Our results suggest that rFVIII can be further exploited to make an ideal candidate for hemophilia gene therapy using AAV vectors. Disclosures No relevant conflicts of interest to declare.

A Novel Deletion in the Fviii B-Domain That Reduces Transgene Size While Preserving FVIII Activity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3182-3182
Author(s):  
Yi-Lin Liu ◽  
Hua Zhu ◽  
Alexander Schlachterman ◽  
Heesoon Chang ◽  
Rodney M. Camire ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Dose Group ◽  
Hemophilia A ◽  
Specific Activity ◽  
Post Injection ◽  
Vector Genome ◽  
Fviii Activity

Abstract Hemophilia A is an inherited X-linked bleeding disorder caused by a deficiency in Factor VIII (FVIII). Clinically significant improvement of hemophilia phenotype can be achieved with low circulating factors, thus makes it a good target disease for gene therapy. Adeno-associated virus (AAV) vectors have proven successful for the delivery of the factor IX gene in humans with hemophilia B. For the treatment of hemophilia A, a problem in the packaging of the rFVIII cDNA or various B-domainless derivatives (i.e. rFVIII-SQ) in AAV vectors is the large size of the insert, which combined with required elements, can exceed the packaging capacity of AAV (~5 kb). This difficulty limits the choice of both promoter and regulatory elements when designing an expression cassette for AAV vectors. Here we developed strategies to overcome these limitations by (1) development of a novel FVIII B-domain deleted molecule (2) construction of a short liver-specific promoter. We further tested these vectors in a series of in vitro and in vivo experiments. Factor VIII-SQ is a well-characterized derivative of FVIII and has been used by several groups in a gene therapy setting; the recombinant protein is used clinically to treat hemophilia A. We have constructed a shorter version of FVIII-SQ, by deleting the entire B-domain. In addition, we have engineered this FVIII to be intracellularly processed using a PACE-furin recognition site such that the protein is secreted from cells as two chains (FVIII-RKR; fully processed heavy and light chains). This FVIII-RKR along with FVIII-SQ was transiently expressed in COS-1 cells and conditioned media was collected at 24, 48 and 72 hrs post transfection. Using a combination of ELISA and functional assays we were able to demonstrate that FVIII-RKR was efficiently secreted from these cells. The data also revealed that FVIII-RKR has a 4–8-fold increase in specific activity compared to FVIII-SQ. We further tested whether FVIII-RKR could function in an in vivo setting. Plasmid DNA (50μg) containing FVIII-RKR or FVIII-SQ with liver-specific mouse transthyretin (mTTR) promoter were introduced into hemophilia A (HA) mice hydrodynamically via tail vein. Two out of four mice in the SQ group and three out of four mice in the RKR group had significant shortening of the clotting time at days 1 and 3 post injection, indicating that this shortened version of FVIII is functional in vivo. To address FVIII long-term expression we synthesized AAV vectors and delivered to immuno-deficient HA mice through hepatic portal vein. AAV vectors containing an expression cassette of mTTR promoter and FVIII-SQ have been administered. Expression of physiological FVIII levels was observed in high dose group (4.0E+12 vector genome per animal, n=4). FVIII activity averages 1.88 U/ml by Coamatic assay or 0.81 U/ml by aPTT assay at 12 weeks post injection. In low dose group (1.0E+12 vector genome per animal, n=5) therapeutic level of FVIII is achieved, 0.59 U/ml by Coamatic assay or 0.23 U/ml by aPTT assay at 12 weeks post injection. Finally, AAV vectors with FVIII-RKR have been produced and shown to have similar packaging efficiency to AAV-FVIII-SQ. Studies are currently underway with AAV-FVIII-RKR to evaluate the ability of this vector to drive long-term expression of functional protein. In summary, we developed a novel FVIII molecule that has high specific activity and is suitable for efficiently packaging in the AAV vectors.

Identification of Activity Enhancement Residue of Human FVIII and Novel Factor VIII Variant for Human Gene Therapy

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4420-4420
Author(s):  
Jenni A. Firrman ◽  
Qizhao Wang ◽  
Weidong Xiao
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Light Chain ◽  
Transgene Expression ◽  
Human Factor ◽  
Specific Activity ◽  
Fold Increase ◽  
Coagulation Activity ◽  
Human Factor Viii

Abstract Gene therapy for Hemophilia A using the recombinant Adeno-associated virus (rAAV) offers an alternative to FVIII protein infusions; However, due to limitations associated with rAAV and the FVIII protein itself, the end result is transgene expression below therapeutic limits. One approach to improving the therapeutic value of rAAV gene therapy for HA is to engineer a more active FVIII protein through genetic modifications. Preliminary testing revealed that canine FVIII Light Chain (kLC) enhances human FVIII coagulation activity. Through the process of engineering, evaluation, positive and negative selection of kLC, key amino acids in canine factor VIII were identified. These amino acids were incorporated into human factor VIII and a new version of human factor VIII, hFVIII.JF12, was engineered. The hFVIII.JF12 is a human FVIII B-domain deleted construct containing 12 amino acid changes in the light chain that work together to enhance coagulation activity. In vitro, hFVIII.JF12 resulted in a 4.3 fold increase in clotting activity, but no increase in protein production. CD4KO/HA mice injected with rAAV vector carrying the hFVIII.JF12 gene produced an average 4.6 fold increase in clotting activity compared to those injected with hFVIIIBDD. An ELISA revealed no significant difference in protein production between these two groups of injected mice. In order to determine the mechanism of enhancement, the hFVIIIBDD and hFVIII.JF12 proteins were purified and functional properties analyzed. Results demonstrated that the hFVIII.JF12 protein produced a specific activity of 39,153.69 Units/m. This is a 6.28 fold increase over hFVIIIBDD specific activity, which was 6,237.92 Units/mg. Measurement of conversion from FX to FXa revealed that the hFVIII.JF12 protein generated a higher amount of FXa at a quicker rate. The hFVIII.JF12 construct is novel because it enhances FVIII activity both in vitro and in vivo through modifications to the light chain based on the kLC. This will be beneficial in the context of both gene and protein therapy because the protein is more specifically active. This research is also innovative because it demonstrates a novel method of enhancing transgene expression of FVIII delivered by an AAV vector through modifications to the gene itself. Disclosures No relevant conflicts of interest to declare.

scholarly journals A Spacer Sequence Inserted between the A1 and A2 Domains of Factor VIII Overcomes the Requirement for Proteolytic Cleavage at Arg 372 in the Generation of Cofactor Activity

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-21
Author(s):  
Manjunath Goolyam Basavaraj ◽  
Sriram Krishnaswamy
Keyword(s):  
Factor Viii ◽  
Western Blotting ◽  
Light Chain ◽  
Specific Activity ◽  
Factor X ◽  
Physiological Concentration ◽  
Proteolytic Activation ◽  
Linker Sequence ◽  
One Stage ◽  
Cofactor Activity

Factor VIII (FVIII) with a multi-domain structure (A1-a1-A2-a2-B-a3-A3-C1-C2) is a procofactor and precursor for the anti-hemophilic cofactor protein, FVIIIa. Following the intracellular processing within the B domain, secreted FVIII circulates as a heterodimer with variably sized (90K-200K) heavy chain (A1-a1-A2-a2-B) and an 80K light chain (a3-A3-C1-C2). Proteolytic activation of FVIII by thrombin that yields heterotrimeric FVIIIa (A1-a1/A2-a2/A3-C1-C2), the cofactor for intrinsic tenase, involves cleavage of three peptide bonds between Arg372-Ser373, Arg740-Ser741, and Arg1689-Ser1690. Cleavage at Arg740 removes the B-domain, and cleavage at Arg1689 removes the a3-acidic region and releases FVIII from vWF, its carrier protein, and exposes membrane binding sites within the FVIII light chain. Cleavage at Arg372 separates A1-a1 and A2-a2 domains and is implicated in the cofactor-dependent recognition and enhancement in the rate of factor X (FX) activation by intrinsic tenase. Subsequently, the separated A2-a2 domain dissociates spontaneously from the heterotrimeric FVIIIa resulting in the rapid loss of cofactor activity. We speculated that the requirement for cleavage at Arg372 might be obviated by the insertion of an optimized linker sequence between A1-a1 and A2-a2 domains on an uncleavable Gln372 backbone. To investigate this possibility, we prepared cDNA constructs of B-domain deleted FVIII variants; FVIII wild-type (FVIIIWT), FVIII372Q, and FVIII372Q followed by a rigid (Ala-Pro)5 linker sequence (FVIII372Q-AP5). All three FVIII constructs were stably transfected into BHK cells and high expressing clones were selected by one stage aPTT and western blotting of expression media. Selected stable clones were further expanded to collect 15L of expression media over 5-day period, and recombinant FVIII variants were purified using a three-step chromatographic approach. These FVIII variants were studied using SDS-PAGE, western blotting, aPTT assays, thrombin generation assay (TGA) and purified assays to assess kinetics of FX activation and spontaneous loss of cofactor activity. In contrast to FVIIIWT, FVIII372Q and FVIII372Q-AP5 were completely resistant to cleavage at Gln372 by thrombin, yielding bands corresponding to A1-a1-A2-a2 (90K) and A3-C1-C2 (73K). In one stage aPTT assays, FVIII372Q showed prolonged clotting times with specific activity in the range of 200-400 U/mg, while FVIIIWT and FVIII372Q-AP5 displayed comparable clotting times with specific activities ranging between 8000-10000 U/mg and 4500-5500 U/mg, respectively. In TGA initiated with either 0.1 pM tissue factor or 1 pM factor XIa, both FVIIIWT and FVIII372Q-AP5 displayed similar TGA profiles. In steady state kinetic studies of FX activation using limiting concentrations of factor IXa, saturating concentrations of FVIII variants pretreated with thrombin, membranes and increasing concentrations of FX, the cofactor function of thrombin-cleaved FVIII372Q was severely impaired. However, despite lack of cleavage at Gln372 in FVIII372Q-AP5, catalytic efficiency for FX activation by intrinsic tenase assembled by this variant was comparable to that seen with FVIIIaWT. At the physiological concentration of FX, the initial velocity for Xa formation (v/E) for intrinsic tenase assembled with FVIIIa372Q-AP5 was within a factor of 2 of that observed with FVIIIaWT while the rate observed with FVIIIa372Q was >10-fold lower. Following rapid activation with thrombin, loss of cofactor function was significantly slower for FVIIIa372Q-AP5(t1/2 ~ 10 min) compared to FVIIIaWT (t1/2 ~ 2 min). Our findings indicate that the requirement for cleavage at Arg372 for the development of full FVIIIa cofactor function can be overcome by modulating the A1-A2 connector with an optimized linker sequence. Failure to yield an infinitely stable cofactor in the case of FVIIIa372Q-AP5 suggests that cleavage at Arg372 does not solely explain the spontaneous loss of FVIIIa cofactor function. Disclosures Krishnaswamy: Bayer: Research Funding.

Codon optimization of human factor VIII cDNAs leads to high-level expression

Blood ◽  
2011 ◽  
Vol 117 (3) ◽  
pp. 798-807 ◽  
Author(s):  
Natalie J. Ward ◽  
Suzanne M. K. Buckley ◽  
Simon N. Waddington ◽  
Thierry VandenDriessche ◽  
Marinee K. L. Chuah ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Viral Vectors ◽  
Hemophilia A ◽  
Lentiviral Vector ◽  
Wild Type ◽  
Fviii Activity ◽  
Human Factor Viii

Abstract Gene therapy for hemophilia A would be facilitated by development of smaller expression cassettes encoding factor VIII (FVIII), which demonstrate improved biosynthesis and/or enhanced biologic properties. B domain deleted (BDD) FVIII retains full procoagulant function and is expressed at higher levels than wild-type FVIII. However, a partial BDD FVIII, leaving an N-terminal 226 amino acid stretch (N6), increases in vitro secretion of FVIII tenfold compared with BDD-FVIII. In this study, we tested various BDD constructs in the context of either wild-type or codon-optimized cDNA sequences expressed under control of the strong, ubiquitous Spleen Focus Forming Virus promoter within a self-inactivating HIV-based lentiviral vector. Transduced 293T cells in vitro demonstrated detectable FVIII activity. Hemophilic mice treated with lentiviral vectors showed expression of FVIII activity and phenotypic correction sustained over 250 days. Importantly, codon-optimized constructs achieved an unprecedented 29- to 44-fold increase in expression, yielding more than 200% normal human FVIII levels. Addition of B domain sequences to BDD-FVIII did not significantly increase in vivo expression. These significant findings demonstrate that shorter FVIII constructs that can be more easily accommodated in viral vectors can result in increased therapeutic efficacy and may deliver effective gene therapy for hemophilia A.

Direct Comparison of Bioengineered Factor VIII Expression Constructs: Towards an Optimal Transgene for Gene Therapy of Hemophilia A.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 5477-5477
Author(s):  
Kerry L. Titus ◽  
Paul Lee ◽  
H. Trent Spencer ◽  
Christopher Doering
Keyword(s):  
Gene Therapy ◽  
Amino Acid ◽  
Factor Viii ◽  
Hemophilia A ◽  
Double Mutation ◽  
Specific Amino Acid ◽  
Stable Transfectants ◽  
Glycosylation Sites ◽  
Fviii Activity ◽  
A2 Domain

Abstract A major obstacle for gene therapy of hemophilia A is the achievement of adequate factor VIII (fVIII) expression. Bioengineering strategies have targeted specific sequences within human fVIII that are thought to be responsible for its generally poor expression. Specific amino acid substitutions, L303E/F309S herein referred to as double mutation (DM), function to decrease fVIII binding to BiP, a resident ER chaperone, which results in increased fVIII secretion (Swaroop, Moussalli et al. 1997). Furthermore, addition of 6 N-linked glycosylation sites, designated 226/N6, located within the human B domain also increases human fVIII expression (Miao, Sirachainan et al. 2004). We previously demonstrated that porcine and certain hybrid human/porcine fVIII constructs are expressed at 10 – 14-fold greater levels than human fVIII (Doering, Healey et al. 2002; Doering, Healey et al. 2004). The aim of the current study was to directly compare various fVIII expression constructs in order to determine an optimal transgene for gene therapy applications. The following fVIII constructs were generated: human B-domain-deleted fVIII (HBDD-fVIII), HBDD-fVIII with a 14 amino acid linker between the A2 domain and the activation peptide (HSQ-fVIII), porcine fVIII containing a 24 amino acid linker (HEP-fVIII), hybrid human/porcine-fVIII which has porcine A1 and A3 domains (HP47), and modified HBDD, HSQ and HEP-fVIII constructs containing DM and/or 226/N6. Each construct was transiently transfected into BHK-M cells, and fVIII production between 48 – 72 hrs post-transfection was measured using a one-stage clotting assay. Under these conditions, the addition of the DM and 226/N6 significantly increased fVIII expression for HBDD (P = 0.003), though not for HSQ. Addition of DM or 226/N6 alone did not significantly increase the expression of either human fVIII construct, and furthermore, the addition of DM to HEP-fVIII decreased its expression 98%. HEP-fVIII was expressed at 8-fold or greater levels than any of the other human constructs. Next, ~25 stably transfected BHK-M clones were isolated following transfection with each of the fVIII expression constructs and the rate of fVIII production for each clone was determined. Several clones did not express detectable fVIII activity (<0.01 units/mL) and were excluded from the analysis. Approximately 14% of the total number of clones were excluded, ranging from 0 – 42% for the different constructs. HEP-DM-fVIII was the exception, where 82% of the clones had activity <0.01 units/mL. Mean HEP-fVIII expression was 3.93 ± 3.22 units/mL/24 hr (n = 19) (Figure 1), and HP47 was similarly expressed at 3.21 ± 2.31 units/mL/24 hr (n = 19). All of the HSQ-based constructs and HBDD-DM/226/N6 showed similar mean expression levels (0.28 ± 0.03 units/mL/24 hr) and were significantly higher than HBDD and HBDD-DM, which had a mean of 0.12 ± 0.01 units/mL. In the current study, we provide experimental evidence that the expression of HEP-fVIII and HP47 is superior to other bioengineered fVIII expression constructs, which should eliminate the expression barrier that has hampered the clinical translation of gene therapy for hemophilia A. Figure 1: Stable Transfectants Figure 1:. Stable Transfectants

Circulating factor VIII immune complexes in patients with type 2 acquired hemophilia A and protection from activated protein C–mediated proteolysis

Blood ◽  
2001 ◽  
Vol 97 (3) ◽  
pp. 669-677 ◽  
Author(s):  
Keiji Nogami ◽  
Midori Shima ◽  
John C. Giddings ◽  
Kazuya Hosokawa ◽  
Masanori Nagata ◽  
...  
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Immune Complexes ◽  
Protein C ◽  
Hemophilia A ◽  
Activated Protein C ◽  
Acquired Hemophilia A ◽  
Acquired Hemophilia ◽  
Fviii Activity

Abstract Factor VIII (FVIII) inhibitor antibodies are classified into 2 groups according to the kinetic pattern of FVIII inactivation. Type 2 antibodies are more commonly observed in patients with acquired hemophilia A and do not completely inhibit FVIII activity; in most cases, substantial levels of circulating FVIII are detected. Three type 2 autoantibodies from patients who had normal levels of FVIII antigen despite having low levels of FVIII activity were studied. The antibodies reacted exclusively with the light chain of FVIII but not with the C2 domain, and their epitopes were therefore ascribed to the regions in the A3-C1 domains. Heavy and light chains of FVIII were detected in plasma-derived immune complexes extracted by using protein G Sepharose. Direct binding assays using anhydro-activated protein C (anhydro-APC), a catalytically inactive derivative of activated protein C (APC) in which the active-site serine is converted to dehydroalanine, were used to examine the relation between immune complexes and APC. The intact FVIII, 80-kd light chain, and 72-kd light chain bound in a dose-dependent manner to anhydro-APC, with Kdvalues of 580, 540, and 310 nM, respectively, whereas no appreciable binding was detected for the heavy chain. The 3 autoantibodies blocked FVIII binding to anhydro-APC by approximately 80% and consequently inhibited APC-induced FVIII proteolytic inactivation. These antibodies also bound to a synthetic peptide, His2009-Val2018, which contains the APC binding site. The findings suggest that binding of type 2 autoantibodies, recognizing residues His2009 to Val2018, protects FVIII from APC-mediated proteolysis and might contribute to the presence of FVIII immune complexes in the circulation.

Purification and characterization of factor VIII 1,689-Cys: a nonfunctional cofactor occurring in a patient with severe hemophilia A

Blood ◽  
1989 ◽  
Vol 73 (8) ◽  
pp. 2117-2122
Author(s):  
DP O'Brien ◽  
EG Tuddenham
Keyword(s):  
Factor Viii ◽  
Light Chain ◽  
Hemophilia A ◽  
Specific Activity ◽  
Exchange Chromatography ◽  
Heavy Chains ◽  
Thrombin Cleavage ◽  
Before And After ◽  
Functional Defect ◽  
Thrombin Activation

We have purified the factor VIII from a CRM+ Hemophilia A plasma (90 U/dL VIII:Ag but 0 U/dL VIII:C) and analyzed the protein before and after thrombin activation by Western blotting with monoclonal antibodies (MoAbs). Normal or patient citrated plasma was ultracentrifuged, cryo-ethanol-precipitated and chromatographed on Sepharose 6B. The void volume fractions were reduced and subjected to ion exchange chromatography yielding material of specific activity approximately 1,000 U/mg protein (VIII:C or VIII:Ag). Factor VIII purified in this way from normal plasma is fully activatable by thrombin with proteolytic fragmentation as previously described by F. Rotblat et al (Biochemistry 24: 4294, 1985). Factor VIII 1,689-Cys has the normal distribution of factor VIII light and heavy chains prior to thrombin activation. After exposure to thrombin the heavy chain polypeptides were fully proteolysed but the light chain was totally resistant to cleavage. This is consistent with the demonstration in the patient's leucocyte DNA of a C to T transition in codon 1,689 converting Arg to Cys at the light chain thrombin cleavage site as previously described by J. Gitschier et al (Blood 72:1022, 1988). Uncleaved light chain of Factor VIII 1,689-Cys is not released from von Willebrand factor (vWF) by thrombin, but this is not the sole cause of the functional defect since the protein purified free of vWF has no coagulant activity. We conclude that the functional defect in factor VIII 1,689-Cys is a consequence of failure to release the acidic peptide from the light chain upon thrombin activation.

scholarly journals Ultrasound Combined with Microbubbles Mediated Factor VIII Plasmid Gene Therapy in Hemophilia a Mice

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2039-2039
Author(s):  
Shuxian Song ◽  
James Harrang ◽  
Bryn Smith ◽  
Carol H. Miao
Keyword(s):  
Gene Expression ◽  
Gene Therapy ◽  
Gene Transfer ◽  
Factor Viii ◽  
Hemophilia A ◽  
Liver Perfusion ◽  
Transfer Efficiency ◽  
Clotting Factor ◽  
Gene Transfer Efficiency ◽  
Fviii Activity

Abstract Hemophilia A is a genetic bleeding disorder resulted from a deficiency of blood clotting factor VIII. In order to develop the efficient approach to gene therapy for hemophilia A, we previously explored reporter gene transfer mediated by ultrasound (US) combined with microbubbles (MBs). It was demonstrated that US/MB can significantly enhance gene transfer efficiency and serve as an efficient non-viral physical delivery strategy. In this study, we further delivered a therapeutic FVIII plasmid into the livers of hemophilia A (HA) mice. In consideration of FVIII synthesis from multiple tissues/cell lines, we first explored the distribution of gene expression using a pGL4.13 [luc2/SV40] luciferase plasmid driven by a ubiquitous promoter. One day following gene transfer, hepatocytes and endothelia cells were isolated from treated lobes by liver perfusion and centrifuge method. Evaluation of luciferase levels in two cell populations indicated that luciferase predominantly expressed in hepatocytes (5.35´104 RLU/107 cells vs. 1.46´103 RLU/107 cells in endothelia cells). Furthermore, gene transfer of pGFP (driven by a ubiquitous CMV promoter) mediated by US/MB also showed fluorescence distribution mostly in hepatocytes. These results indicate that hepatocyte is the predominant site of gene expression following US/MB mediated gene transfer into the liver. Based on these results, a hepatocyte-specific human FVIII plasmid (pBS-HCRHP-hFVIII/N6A) was used for US/MB mediated gene transfer in HA mice. In the short-term experiment, FVIII activity levels of treated HA mice ranged from 4-40% of normal FVIII activity. To follow FVIII expression for longer term, HA mice were pretreated with IL-2/IL-2 mAb (JES6-1) complexes on day −5, −4, and −3 to prevent immune response. In addition, the mice were infused with normal mouse plasma and human FVIII protein prior to gene transfer to maintain hemostasis. Subsequently, FVIII plasmids and 5 Vol% NUVOX MBs were injected into the mouse liver under simultaneous US exposure (1.1MHz transducer H158A driven by a pulse generator and high-power radio frequency amplifier capable of generating up to 1000W). Blood and liver samples were collected at serial time points after treatment to determine FVIII activity in plasma and liver damage. Following gene transfer, 10-30% of FVIII activity was achieved on day 4 and persisted in the average level of 20% by day 28. In a separate long-term follow-up experiment (n=3), 2 of 3 mice still maintained 10-30% activity after 120 days. Both transaminase levels (alanine aminotransferase and aspartate aminotransferase) and histological examination showed that the procedure of plasmid/MBs portal-vein injection and pulse-train acoustic exposure produced transiently localized liver damages however the damages were repaired and the liver recovered rapidly. Phenotypic correction of HA mice was further examined by tail clip assay. Blood loss of US/MB treated mice was significantly reduced compared with naive HA mice. Furthermore, a novel plasmid encoding a B domain-deleted FVIII variant containing mutations of 10 amino acids in the A1 domain (BDDFVIII-X10, a kind gift from Weidong Xiao) was constructed. Preliminary results from ongoing study showed that the gene transfer efficiency could be further improved with better plasmid and more efficient immune modulation. Together all the results indicate that US/MB mediated gene transfer is highly promising for efficient and safe gene therapy of hemophilia A. Disclosures No relevant conflicts of interest to declare.

scholarly journals The Factor VIII Variant X5 Enhances Hemophilia a Gene Therapy Efficiency By Its Improved Secretion

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3356-3356
Author(s):  
Franziska Horling ◽  
Johannes Lengler ◽  
Wenjing Cao ◽  
Biao Dong ◽  
Bagirath Gangadharan ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Liver Cell ◽  
Human Liver ◽  
Hemophilia A ◽  
Employment Equity ◽  
Fviii Activity ◽  
Human Liver Cell ◽  
Equity Ownership

Introduction. Adeno-associated virus (AAV)-based factor VIII (FVIII) gene therapy holds great promise to provide clinical benefit in patients with hemophilia A. However, very high doses are currently needed to achieve therapeutic factor levels and the durability appears to be limited to a couple of years. Vector efficiency could be improved by employing more potent liver-specific promoters, but this might come at the price of overstraining the cellular protein folding capacity, causing FVIII to misfold in the lumen of the Endoplasmic Reticulum (ER). This event would in turn activate the unfolded protein response, cause oxidative stress, and if not resolved may even induce cell death. Aims. The objective of the presented study was to test whether the B-domain deleted (BDD)-FVIII-X5 variant can overcome the secretion challenge of high level FVIII expression in the context of hepatic gene therapy. Methods. The human FVIII variant BDD-FVIII-X5 harboring 5 amino acid exchanges in the A1 domain was previously isolated in a screen aimed at identifying those residues in porcine FVIII that are critical for efficient secretion. BDD-FVIII and BDD-FVIII-X5 were produced in Chinese Hamster Ovary (CHO) cells and purified to apparent homogeneity using standard procedures. The preparations were assayed for total protein by UV absorbance at 280 nm and FVIII activity by a chromogenic assay. Both FVIII variants were vectorized using AAV8 and tested in the human liver cell line HepG2 and FVIII knockout mice (E17) at various doses. Resulting samples were assayed for FVIII chromogenic activity. The potential immunogenic risk was evaluated in three hemophilic mouse strains (E17, human FVIII transgenic, humanized HLA-DRB1*1501). Results. A characterization of purified recombinant Refacto-like BDD-FVIII and the corresponding X5 variant revealed similarity of the two proteins and their specific activities in particular, indicating that introduction of the 5 amino acids from porcine FVIII did not alter functionality of human BDD-FVIII. In vitro expression of BDD-FVIII-X5 in a human liver cell line resulted in substantially increased FVIII activity levels in the supernatant compared with the non-modified BDD-FVIII, commensurate with enhanced secretion of the X5 variant. Intravenous delivery of liver-targeted AAV8 vectors carrying the BDD-FVIII-X5 transgene achieved substantial increases in plasma coagulation activity over BDD-FVIII in FVIII-deficient mice, even when highly efficient codon-optimized F8 nucleotide sequences were employed. Evaluation of the immunogenicity of the BDD-FVIII-X5 variant by an immunological risk assessment did not reveal any increased immunogenic risk compared to BDD-FVIII. Conclusions: The fully active BDD-FVIII-X5 variant demonstrated improved secretion in vitro and in vivo, resulting in substantially higher FVIII levels in a hemophilia A mouse model. No signs of enhanced immunogenicity were noted in a comparative immunogenicity study. The results obtained warrant further exploration of the BDD-FVIII-X5 variant for a next generation hemophilia A gene therapy. Disclosures Horling: Baxalta Innovations GmbH, a Takeda company: Employment. Lengler:Baxalta Innovations GmbH, a Takeda company: Employment. Gangadharan:Baxalta Innovations GmbH, a Takeda company: Employment. De La Rosa:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Hoellriegl:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Reipert:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Scheiflinger:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership. Xiao:Ivygen: Other: Patent application on FVIII-X5 has been submitted. Rottensteiner:Baxalta Innovations GmbH, a Takeda company: Employment, Equity Ownership.

Restoration of Porcine Factor VIII Procoagulant Activity to Hemophilia A Mice with Pre-Existing Immunity to Human Factor VIII.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 3284-3284
Author(s):  
Christopher B. Doering ◽  
Bagirath Gangadharan ◽  
Hillary Z. Dukart ◽  
H. Trent Spencer
Keyword(s):  
Gene Therapy ◽  
Factor Viii ◽  
Hemophilia A ◽  
Donor Cell ◽  
Activity Levels ◽  
Cell Engraftment ◽  
Fviii Activity ◽  
Human Factor Viii ◽  
Total Body ◽  
High Level

Abstract The development of inhibitory antibodies directed against human factor VIII (hfVIII) remains the most significant clinical complication associated with the treatment of hemophilia A. Recently, we demonstrated that transplantation of genetically-modified hematopoietic stem cells (HSCs) containing a high-expression porcine (HEP) fVIII transgene promoted sustained, high-level fVIII expression in naïve, genetically-immunocompetent hemophilia A mice (Gangadharan et al. 2006. Blood 107, 3859–64). In the current study, HEP-fVIII HSC transplantation (HSCT)-based gene therapy was tested in the setting of hemophilia A complicated by the presence of circulating anti-hfVIII inhibitory antibodies. Following a series of intravenous injections with recombinant hfVIII, hemophilia A mice developed anti-hfVIII antibodies with clinically-significant inhibitory titers. The median ELISA and Bethesda (inhibitory) titers were 2938 and 55 (n = 18), respectively, and a significant correlation between the anti-hfVIII ELISA and Bethesda (inhibitory) titers was observed (P < 0.001). With the exception of two mice, all plasma samples analyzed contained anti-HEP-fVIII cross-reactive antibodies by ELISA, and 5 of 16 displayed detectable anti-HEP-fVIII inhibitory activity. HSCs were transduced ex vivo with recombinant retrovirus carrying a HEP-fVIII transgene and transplanted into lethally-irradiated (11 Gy total body irradiation) hemophilia A mice (n = 11). Twelve wks post-HSCT, all mice displayed high-level donor cell engraftment in peripheral blood mononuclear cells (PBMCs) at 81 ± 22% (mean ± SD) and expressed therapeutic levels of fVIII with a mean activity of 3 ± 1.3 units/ml (Figure 1). A second cohort of mice (n = 7) underwent a similar HSCT gene therapy procedure with the exception that they received a sub-lethal dose of total body irradiation (5.5 Gy). These mice exhibited lower-level donor cell engraftment at 6.5 ± 6% (range 0.7 – 13%) in PBMCs at 12 weeks post-HSCT. At day 9 post-HSCT, the mice in this cohort contained circulating fVIII activity levels at 1 ± 0.6 units/ml (Figure 1). However in 6 of 7 mice, fVIII activity levels returned to baseline (≤0.01 units/ml) by day 28 post-HSCT, and in 4 of 7 mice, fVIII activity remained below the level of detection. The remaining 3 mice displayed 0.22 ± 0.3 (range 0.04 – 0.57) units/ml fVIII activity at 12 weeks post-HSCT and showed a trend towards greater donor PBMC engraftment, 12.8 ± 0.2% versus 1.8 ± 1.3% (P = 0.057). Analysis of the anti-hfVIII ELISA titers post-HSCT in both cohorts of mice revealed that while the titers steadily decreased in the lethally-irradiated mice (initial t1/2 ~ 12 days), the titers remained unchanged in the sub-lethally-irradiated mice possibly explaining the differences observed for donor cell engraftment and fVIII expression between the 2 groups. These data provide proof-of-concept that HSCT-based gene therapy incorporating a HEP-fVIII transgene could be utilized for the treatment of high-risk patients with refractory anti-hfVIII inhibitors. Figure Figure

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