Role of L-Arginine on the Expression of Coagulation Factor VIII Gene.

Objective: To explore the key sites in which L-arginine affects the expression of human coagulation factor VIII gene, and to create new drug targets for the treatment of hemophilia. Methods: A total of 5 human FVIII genes (A1, A2, A3, C1 and C2) with B domain deletion were transfected into human umbilical vein endothelial cells (HUVECs) as promoters. Run-on assay and ELISA analysis were performed to observe the driving effect of each domain gene on chloramphenicol acetyl transferase (CAT) gene transcription and expression, and the effect of L-arginine on each promoter. Results: In co-culture with L-arginine, transcriptional expression of the CAT gene was not detected in the PCAT3-Basic group (negative control without promoters), PA3-CAT3-Enhancer group or PC1-CAT3-Enhancer group. The transcriptional expression of CAT gene in the PCAT3-Control group (positive control with promoters) and PA1-CAT3-Enhancer group was unchanged compared with the non-L-arginine intervention, while the transcriptional expression of CAT gene in the PA2-CAT3-Enhancer group was significantly enhanced. Conclusions: A1 and A2 domain genes had promoter function and could initiate the transcription and expression of CAT gene, but A3, C1 and C2 domain genes could not. Moreover, L-arginine can significantly enhance transcription and expression of human coagulation factor VIII via A2 domain.

The full-length Auxin Response Factor 8 isoform ARF8.1 controls pollen cell wall formation and directly regulates TDF1, AMS and MS188 expression.

Auxin Response Factor 8 plays a key role in late stamen development: its splice variants ARF8.4 and ARF8.2 control stamen elongation and anther dehiscence. Here, we characterized the role of ARF8 isoforms in pollen fertility. By phenotypic and ultrastructural analysis of arf8-7 mutant stamens, we found defects in pollen germination and viability caused by alterations in exine structure and pollen coat deposition. Furthermore, tapetum degeneration, a prerequisite for proper pollen wall formation, is delayed in arf8-7 anthers. In agreement, the genes encoding the transcription factors TDF1, AMS, MS188 and MS1, required for exine and pollen coat formation, and tapetum development, are downregulated in arf8-7 stamens. Consistently, the sporopollenin content is decreased, and the expression of sporopollenin synthesis/transport and pollen coat protein biosynthetic genes, regulated by AMS and MS188, is reduced. Inducible expression of the full-length isoform ARF8.1 in arf8-7 inflorescences complements the pollen (and tapetum) phenotype and restores the expression of the above transcription factors. Chromatin immunoprecipitation-quantitative polymerase chain reaction assay revealed that ARF8.1 directly targets the promoters of TDF1, AMS and MS188. In conclusion, the ARF8.1 isoform controls pollen and tapetum development acting directly on the expression of TDF1, AMS and MS188, which belong to the pollen/tapetum genetic pathway.

Generation of iPSC line from urine cells of hemophilia A with F8 (p. R814X) mutation.

The lack of coagulation factor VIII in patient with nonsense mutation hemophilia A leads to varying degrees of bleeding symptoms, and long-term use of alternative therapies can produce inhibitors that affect the efficacy. In this study, human induced pluripotent stem cells (iPSCs) of hemophilia A were generated by reprogramming of urine cells. Human urine cells (HUCs) were isolated by collecting patients' mid-stream urine, and cultured to good state in urine medium. Then, the HUCs were transfected with PEP4-EO2S-ET2K and pCEP4-M2L, and iPSCs were obtained in the medium without trophoblast cells and the composition was determined. Finally, alkaline phosphatase staining, karyotype analysis, immunofluorescence staining and teratoma were used to verify that we successfully reprogrammed hemophilia A-specific human induced pluripotent stem cells from patients' urine cells, providing a safe and effective cell model for the study of molecular mechanism and related treatment of hemophilia.

Early Phase Clinical Immunogenicity of Valoctocogene Roxaparvovec, an AAV5-Mediated Gene Therapy for Hemophilia A.

Evaluation of immune responses to adeno-associated virus (AAV)-mediated gene therapies prior to and following dose administration plays a key role in determining therapeutic safety and efficacy. This report describes up to 3 years of immunogenicity data following administration of valoctocogene roxaparvovec (BMN 270), an AAV5-mediated gene therapy encoding human B domain-deleted FVIII (hFVIII-SQ) in a phase 1/2 clinical study of adult males with severe hemophilia A. Patients with pre-existing humoral immunity to AAV5 or with a history of FVIII inhibitors were excluded from the trial. Blood plasma and peripheral blood mononuclear cell (PBMC) samples were collected at regular intervals following dose administration for assessment of humoral and cellular immune responses to both the AAV5 vector and transgene-expressed hFVIII-SQ. The predominant immune response elicited by BMN 270 administration was largely limited to the development of antibodies against the AAV5 capsid that were cross-reactive with other common AAV serotypes. No FVIII inhibitor responses were observed within 3 years following dose administration. In a context of prophylactic or on-demand corticosteroid immunosuppression given after vector infusion, AAV5 and hFVIII-SQ peptide-specific cellular immune responses were intermittently detected by an interferon (IFN)-γ and tumor necrosis factor (TNF)-α FluoroSpot assay, but they were not clearly associated with detrimental safety events or changes in efficacy measures.

The Immune Response to the fVIII Gene Therapy in Preclinical Models.

Neutralizing antibodies to factor VIII (fVIII), referred to as "inhibitors," remain the most challenging complication post-fVIII replacement therapy. Preclinical development of novel fVIII products involves studies incorporating hemophilia A (HA) and wild-type animal models. Though immunogenicity is a critical aspect of preclinical pharmacology studies, gene therapy studies tend to focus on fVIII expression levels without major consideration for immunogenicity. Therefore, little clarity exists on whether preclinical testing can be predictive of clinical immunogenicity risk. Despite this, but perhaps due to the potential for transformative benefits, clinical gene therapy trials have progressed rapidly. In more than two decades, no inhibitors have been observed. However, all trials are conducted in previously treated patients without a history of inhibitors. The current review thus focuses on our understanding of preclinical immunogenicity for HA gene therapy candidates and the potential indication for inhibitor treatment, with a focus on product- and platform-specific determinants, including fVIII transgene sequence composition and tissue/vector biodistribution. Currently, the two leading clinical gene therapy vectors are adeno-associated viral (AAV) and lentiviral (LV) vectors. For HA applications, AAV vectors are liver-tropic and employ synthetic, high-expressing, liver-specific promoters. Factors including vector serotype and biodistribution, transcriptional regulatory elements, transgene sequence, dosing, liver immunoprivilege, and host immune status may contribute to tipping the scale between immunogenicity and tolerance. Many of these factors can also be important in delivery of LV-fVIII gene therapy, especially when delivered intravenously for liver-directed fVIII expression. However, ex vivo LV-fVIII targeting and transplantation of hematopoietic stem and progenitor cells (HSPC) has been demonstrated to achieve durable and curative fVIII production without inhibitor development in preclinical models. A critical variable appears to be pre-transplantation conditioning regimens that suppress and/or ablate T cells. Additionally, we and others have demonstrated the potential of LV-fVIII HSPC and liver-directed AAV-fVIII gene therapy to eradicate pre-existing inhibitors in murine and canine models of HA, respectively. Future preclinical studies will be essential to elucidate immune mechanism(s) at play in the context of gene therapy for HA, as well as strategies for preventing adverse immune responses and promoting immune tolerance even in the setting of pre-existing inhibitors.

Emicizumab Improves Ex Vivo Clotting Function in Patients with Mild/Moderate Hemophilia A.

BACKGROUND: Emicizumab prophylaxis is a promising treatment that reduces bleeding events in severely affected patients with hemophilia A (PwHA). It is anticipated that emicizumab could be similarly effective in mild/moderate PwHA (PwMHA) although this effect has not been investigated. AIM: We evaluated ex vivo coagulant effects of emicizumabin PwMHA. METHODS: Clot waveform analysis (CWA) triggered by prothrombin time/activated partial prothrombin time-mixed reagents was utilized to examine coagulant effects of emicizumabin factor (F)VIII-deficient plasma mixed with recombinant (r)FVIIIand in native plasmas from 16 PwMHA. The CWA parameter, adjusted-|min1| (Ad|min1|), was used. Increases in Ad|min1| (ΔAd|min1|) mediated by emicizumab were calculated from the slopes of regression lines in the presence of rFVIII. RESULTS: Ad|min1| in FVIII-deficient plasma with various concentrations of rFVIII negatively correlated with ΔAd|min1|by adding emicizumab, and these data were defined as standard reference values. Ad|min1| (4.57 ± 0.50) in 16 PwMHA increased to 5.05 ± 0.54 and 5.37 ± 0.60 by adding emicizumab at 50 and 100 µg/mL, respectively, but remained lower than the normal range (7.22 ± 0.21). ΔAd|min1| levels were 1.5 to 2-fold higher in five cases and 0.4 to 0.6-fold lower in four cases, compared with reference values determined by rFVIII. In some cases, genetic analyses suggested that specific point mutations could have contributed to these findings. Further studies using rFVIII mutants indicated, however, that the differences in ΔAd|min1| were not related to individual FVIII gene defects. CONCLUSION: Emicizumab enhances coagulation potential in PwMHA. Assessment of ex vivo coagulant activity of emicizumab could be helpful for predicting coagulant potentials prior to treatment in these patients.

Preclinical Development of a Hematopoietic Stem and Progenitor Cell Bioengineered Factor VIII Lentiviral Vector Gene Therapy for Hemophilia A.

Genetically modified, autologous hematopoietic stem and progenitor cells (HSPCs) represent a new class of genetic medicine. Following this therapeutic paradigm, we are developing a product candidate, designated CD68-ET3-LV CD34+, for the treatment of the severe bleeding disorder, hemophilia A. The product consists of autologous CD34+ cells transduced with a human immunodeficiency virus 1-based, monocyte lineage-restricted, self-inactivating lentiviral vector (LV), termed CD68-ET3-LV, encoding a bioengineered coagulation factor VIII (fVIII) transgene, termed ET3, designed for enhanced expression. This vector was shown capable of high-titer manufacture under clinical scale and Good Manufacturing Practice. Biochemical and immunogenicity testing of recombinant ET3, as well as safety and efficacy testing of CD68-ET3-LV HSPCs, were utilized to demonstrate overall safety and efficacy in murine models. In the first model, administration of CD68-ET3-LV-transduced stem-cell antigen-1+ cells to hemophilia A mice resulted in sustained plasma fVIII production and hemostatic correction without signs of toxicity. Patient-derived, autologous mobilized peripheral blood (mPB) CD34+ cells are the clinical target cells for ex vivo transduction using CD68-ET3-LV, and the resulting genetically modified cells represent the investigational drug candidate. In the second model, CD68-ET3-LV gene transfer into mPB CD34+ cells isolated from normal human donors was utilized to obtain in vitro and in vivo pharmacology, pharmacokinetic, and toxicology assessment. CD68-ET3-LV demonstrated reproducible and efficient gene transfer into mPB CD34+ cells, with vector copy numbers in the range of 1 copy per diploid genome equivalent without affecting clonogenic potential. Differentiation of human CD34+ cells into monocytes was associated with increased fVIII production, supporting the designed function of the CD68 promoter. To assess in vivo pharmacodynamics, CD68-ET3-LV CD34+ cell product was administered to immunodeficient mice. Treated mice displayed sustained plasma fVIII levels and no signs of product related toxicity. Collectively, the findings of the current study support the preclinical safety and efficacy of CD68-ET3-LV CD34+.

Expression and characterization of a codon-optimized blood coagulation factor VIII.

Essentials Recombinant factor VIII (FVIII) is known to be expressed at a low level in cell culture. To increase expression, we used codon-optimization of a B-domain deleted FVIII (BDD-FVIII). This resulted in 7-fold increase of the expression level in cell culture. The biochemical properties of codon-optimized BDD-FVIII were similar to the wild-type protein. SUMMARY: Background Production of recombinant factor VIII (FVIII) is challenging because of its low expression. It was previously shown that codon-optimization of a B-domain-deleted FVIII (BDD-FVIII) cDNA resulted in increased protein expression. However, it is well recognized that synonymous mutations may affect the protein structure and function. Objectives To compare biochemical properties of a BDD-FVIII variants expressed from codon-optimized and wild-type cDNAs (CO and WT, respectively). Methods Each variant of the BDD-FVIII was expressed in several independent Chinese hamster ovary (CHO) cell lines, generated using a lentiviral platform. The proteins were purified by two-step affinity chromatography and analyzed in parallel by PAGE-western blot, mass spectrometry, circular dichroism, surface plasmon resonance, and chromogenic, clotting and thrombin generation assays. Results and conclusion The average yield of the CO was 7-fold higher than WT, whereas both proteins were identical in the amino acid sequences (99% coverage) and very similar in patterns of the molecular fragments (before and after thrombin cleavage), glycosylation and tyrosine sulfation, secondary structures and binding to von Willebrand factor and to a fragment of the low-density lipoprotein receptor-related protein 1. The CO preparations had on average 1.5-fold higher FVIII specific activity (activity normalized to protein mass) than WT preparations, which was attributed to better preservation of the CO structure as a result of considerably higher protein concentrations during the production. We concluded that the codon-optimization of the BDD-FVIII resulted in significant increase of its expression and did not affect the structure-function properties.

Current and future prospects for hemophilia gene therapy.

Here we review the recent literature on Hemophilia gene transfer/therapy. Gene therapy is one of several new technologies being developed as a treatment for bleeding disorders. We will discuss current and pending clinical efforts and attempt to relate how the field is trending. In doing so, we will focus on the use of recombinant Adeno-associated viral (rAAV) vector-mediated gene transfer since all currently active trials are using this vector. Recent exciting results embody nearly 20 years of preclinical and translational research. After several early clinical attempts, therapeutic factor levels that can now be achieved reflect several modifications of the original vectors. Patterns of results are slowly starting to emerge as different AAV vectors are being tested. As with any new technology, there are drawbacks, and the potential for immune/inflammatory and oncogenic risks have emerged and will be discussed.

Spinal Epidural Hematoma Following Cupping Glass Treatment in an Infant With Hemophilia A.

A 6 months old infant, diagnosed with a rare mutation causing severe hemophilia A, presented with spinal epidural hematoma. Parents later admitted the infant had glass cupping therapy performed within 2 weeks of the onset of symptoms. The rare mutation, rare bleeding complication, and the eventual course of therapy applied in this case will be discussed in our case report.

Current management of the hemophilic child: a demanding interlocutor. Quality of life and adequate cost-efficacy analysis.

Hemophilias are the most known inherited bleeding disorders. The challenges in the management of hemophilic children are different from those in adults: prophylaxis regimen removed the hallmark of crippling disease with lifelong disabilities; individualized regimens are being implemented in order to overcome venous access problems. Presently, at least in high-income countries, advances in treatment of hemophilia resulted in continuous improvement of the patients' quality of life and life expectancy. Inhibitors remain the most severe complication of hemophilia therapy. The treatment' compliance is the key to achieve a successful management. The patient, his family, the medical and psychological team are the players of a comprehensive care system. The current management of hemophilic children is the example of huge resource investments enabling long-term benefits in particular quality of life as a primary objective of the healthcare process.

Turoctocog alfa (NovoEight®)--from design to clinical proof of concept.

Turoctocog alfa (NovoEight®) is a recombinant factor VIII (rFVIII) with a truncated B-domain made from the sequence coding for 10 amino acids from the N-terminus and 11 amino acids from the C-terminus of the naturally occurring B-domain. Turoctocog alfa is produced in Chinese hamster ovary (CHO) cells without addition of any human- or animal-derived materials. During secretion, some rFVIII molecules are cleaved at the C-terminal of the heavy chain (HC) at amino acid 720, and a monoclonal antibody binding C-terminal to this position is used in the purification process allowing isolation of the intact rFVIII. Viral inactivation is ensured by a detergent inactivation step as well as a 20-nm nano-filtration step. Characterisation of the purified protein demonstrated that turoctocog alfa was fully sulphated at Tyr346 and Tyr1664, which is required for optimal proteolytic activation by thrombin. Kinetic assessments confirmed that turoctocog alfa was activated by thrombin at a similar rate as seen for other rFVIII products fully sulphated at these positions. Tyr1680 was also fully sulphated in turoctocog alfa resulting in strong affinity (low nm Kd ) for binding to von Willebrand factor (VWF). Half-lives of 7.2 ± 0.9 h in F8-KO mice and 8.9 ± 1.8 h haemophilia A dogs supported that turoctocog alfa bound to VWF after infusion. Functional studies including thromboelastography analysis of human haemophilia A whole blood with added turoctocog alfa and effect studies in mice bleeding models demonstrated a dose-dependent effect of turoctocog alfa. The non-clinical data thus confirm the haemostatic effect of turoctocog alfa and, together with the comprehensive clinical evaluation, support the use as FVIII replacement therapy in patients with haemophilia A.

Structure-activity relationship of the pro- and anticoagulant effects of Fucus vesiculosus fucoidan.

Fucoidan is a highly complex sulfated polysaccharide commonly extracted from brown seaweed. In addition to their many biological activities, fucoidans have recently been demonstrated to inhibit or increase coagulation at different concentration ranges. Their structural features, i.e. molecular weight (Mw), Mw distribution, degree of sulfation, monosaccharide composition, and different linkages, are known to affect these activities. Therefore, structure-activity relationship (SAR) analysis of fucoidan is crucial for its potential use as a procoagulant. In this study, Fucus vesiculosus (F.v.) fucoidan was fractionated by charge and size as well as over- and desulfated to different degrees to yield preparations with various structural properties. The fractions' pro- and anticoagulant activities were assessed by calibrated automated thrombography (CAT) and activated partial thromboplastin time(aPTT) assays. Binding to and inhibition of the anticoagulant protein tissue factor pathway inhibitor (TFPI) and the ability to activate coagulation via the contact pathway were also investigated. This paper discusses the impact of charge density, size, and sugar composition on fucoidan's pro- and anticoagulant activities. Fucoidan requires a minimal charge density of 0.5 sulfates per sugar unit and a size of 70 sugar units to demonstrate desired procoagulant activities for improvement of haemostasis in factor VIII/factor IX-deficient plasma.

Advances in hemophilia: experimental aspects and therapy.

This article describes recent clinical and research advances in hemophilia therapy. Different prophylactic regimens for the management of severe hemophilia are described along with the use of adjuvant treatment options to achieve hemostasis. The safety and efficacy of radionuclide synovectomy with phosphorus 32-sulfur colloid to treat existing joint arthropathy also are described. The development of inhibitors to factor VIII or IX remains a challenge for hemophilia care and recent approaches to achieve immune tolerance induction are discussed. Finally, recent advances in hemophilia are mentioned, including the role of iron, inflammation, and angiogenesis in the pathogenesis of hemophilic arthropathy.

Acquired hemophilia first manifesting as life-threatening intracranial hemorrhage: case report.

A 74-year-old man presented with life-threatening intracranial hemorrhage and prolonged activated partial thromboplastin time (APTT). The massive subdural hematoma was removed, but multiple intracranial hemorrhages occurred despite the administration of factor VIII and factor IX concentrates. Subdural hematoma, intracerebral hemorrhage in the left temporal lobe, and thalamic hemorrhage subsequently occurred with further prolongation of APTT. He died of enlargement of the thalamic hemorrhage. Acquired hemophilia was diagnosed caused by factor VIII inhibitor. Acquired hemophilia may cause life-threatening hemorrhage, and should be considered in patients with intracranial hemorrhages associated with unexplained prolongation of APTT.

Efficacy and safety of a new-class hemostatic drug candidate, AV513, in dogs with hemophilia A.

AV513 is a select fucoidan, a sulfated polysaccharide of botanical origin. It inhibits tissue factor pathway inhibitor (TFPI) activity and accelerates clotting of human hemophilia A and B plasma. In prior work, subcutaneous administration of AV513 to mice with hemophilia A improved hemostasis. The current studies were designed to evaluate potential efficacy and safety in dogs with hemophilia A (hemophilia A dogs) with minimally increased hemostasis after adenoassociated viral-FVIII gene transfer and in treatment-naive severe hemophilia A dogs. AV513 administered subcutaneously to low-FVIII dogs for multiple weeks improved hemostasis as exhibited in thromboelastography (TEG) and cuticle bleeding time (CBT) tests. Moreover, AV513 administered orally to AAV-FVIII dogs and treatment-naive severe hemophilia A dogs for a multiweek dose-escalating period yielded correction to normal ranges in both TEG and CBT end points at 5 to 15 mg/kg and 15 to 20 mg/kg dose levels, respectively. In all 3 separate studies, throughout their duration, AV513 was well tolerated by the dogs without any adverse events. Additional pharmacologic characterization of AV513 included intravenous pharmacokinetic analysis in rats. In summary, the combination of safety and efficacy in 2 global tests of hemostasis in the hemophilia A dog model indicate that further evaluation of AV513 as a hemostatic agent in hemophilia A patients is warranted.

[Effect of L-arginine on human coagulant factor VIII gene expression].

OBJECTIVE: To investigate the effect of L-arginine on expression of human FVIII gene. METHODS: Plasmid pcDNA6/V5-HisA-BDDhF VIII containing B domain deleted human coagulant factor VIII cDNA (BDDhF VIII cDNA) was constructed and transfected into human umbilical vein endothelial cells (HUVEC). After 72 h incubation with L-arginine (final concentration was 10 mmol/L) , the supernatant was collected for determining the antigen and clotting activity of human FVIII (FVIII: Ag and FVIII: C ) with ELISA and one stage clotting assay respectively. HUVECs were harvested for detecting human FVIII mRNA by Northern blot analysis. The five functional domains of BDDhFVIII cDNA including A1, A2, A3, C1 and C2 were amplified with PCR and inserted into pcDNA6/V5-HisA to construct the expression plasmids pcDNA6/V5-Hi-sA-BDDhFVIII-A1, pcDNA6/V5-HisA-BDDhFVIII-A2, pcDNA6/V5-HisA-BDDhFVIII-A3, pcDNA6/V5-HisA-BDDhFVIII-C1 and pcDNA6/V5-HisA-BDDhFVIII-C2, respectively. HUVEC were transfected with the five plasmids respectively and incubated with L-arginine (at the final concentration of 10 mmol/L) for 72 h. Nucleoli were then isolated and underwent run-on assay. RESULTS: After 24 h incubation with L-arginine, FVIII: Ag and FVIII: C were increased markedly in the supernatant of HUVEC [FVIII: Ag was (146.08 +/- 4.78) ng/ ml, and FVIII: C (0.752 +/- 0.009) U/ml/10(6) cells x 24 h, while in control supernatant without L-arginine, FVIII: Ag was (34.66 +/- 3.98) ng/ml, and FVIII: C (0.171 +/- 0.006) U/ml/10(6) cell x 24 h, P < 0.01]. Northern blot analysis indicated that, after adding L-arginine, the transcription of human FVIII mRNA was intensified remarkably in HUVEC transfected with pcDNA6/V5-HisA-BDDhFVIII, but no any transcription in those transfected with pcDNA6/V5-HisA. Run-on assay demonstrated that with L-arginine induction, A1 and A2 domains transcription was increased obviously, while no change in A3, C1 and C2 domains transcription. CONCLUSION: L-arginine increases expression of human FVIII gene in HUVEC through enhancing its transcription, particularly, domain A1 and A2 within FVIII gene.

Tolerance to factor VIII in a transgenic mouse expressing human factor VIII cDNA carrying an Arg(593) to Cys substitution.

Inhibitory antibodies develop in approximately 25% of patients with severe hemophilia. A following treatment with factorVIII. In E-16KO or E-17KO mice, in which the factor VIII gene has been inactivated by insertion of a neo cassette, inhibitors develop following administration of factor VIII. Here, we describe the generation of transgenic mice expressing human factor VIII-R593C (huFVIII-R593C). Human factor VIII-R593C cDNA under control of a mouse albumin enhancer/promoter was injected into fertilized oocytes. Analysis of transgenic mice revealed that human factor VIII-R593C was expressed in the liver. Transgenic mice were crossed with factor VIII-deficient mice (E-16KO mice). In plasma of E-16KO mice antibodies were detected after five serial intravenous injections of factor VIII, while plasma of huFVIII-R593C/E-16KO mice did not contain detectable levels of antibodies. No antibody secreting cells were observed in either spleen or bone marrow of huFVIII-R593C/E-16KO mice. Also, factor VIII-specific memory B cells were not observed in the spleen of huFVIII-R593C/E-16KO mice. Analysis of T cell responses revealed that splenocytes derived of E-16KO mice secreted IL-10 and IFN-gamma following restimulation with factor VIII in vitro. In contrast, no factor VIII-specific T cell responses were observed in huFVIII-R593C/E-16KO mice. These results indicate that huFVIII-R593C/E-16KO mice are tolerant to intravenously administered factor VIII. It is anticipated that this model may prove useful for studying immune responses in the context of factor VIII gene therapy.

Haemophilic factors produced by transgenic livestock: abundance that can enable alternative therapies worldwide.

Haemophilia replacement factors, both plasma-derived and recombinant, are in relatively short supply and are high-cost products. This has stymied the study and development of alternative methods of administration of haemophilia therapy even in the most economically advanced countries, owing to the large amounts of material needed because bioabsorption and bioavailability of haemophilic factors can be less than 10% when using non-intravenous routes of delivery. There is therefore a need to increase access to therapy worldwide by decreasing the cost and increasing the abundance so that therapy can be achieved through simplified, alternative delivery methods. Transgenic livestock have been used to produce haemophilic factors in milk. Only the pig mammary gland has been shown to carry out the post-translational processing necessary to enable both the biological activity and long circulation half-life needed for therapeutic glycoproteins. Furthermore, the large amounts of recombinant protein that can be produced from pig milk make feasible the use of alternative delivery methods such as oral, intratracheal, subcutaneous, and intramuscular administration.

The preparation and phospholipid binding property of the C2 domain of human factor VIII.

The C2 domain of human factor VIII was expressed in a yeast secretion system and its binding properties were studied. A cDNA coding the C2 domain sequence of human factor VIII with a N-terminal six amino acids extension (C-C2) was constructed, transformed into Pichia pastoris cells and expressed. The product was purified by ammonium sulfate fractionation and anion exchange chromatography. It emerged as a single peak from both ion exchange and gel filtration columns, indicating C-C2 is a homogenous monomer. The binding activity of C-C2 to phosphatidylserine-containing phospholipid vesicles was measured by competitive binding with annexin V. The values of IC50 were approximately 70nM for both factor VIII and its light chain, but were about 7000nM for C-C2. These results indicated C-C2 has 100-fold less binding affinity than factor VIII or the light chain. Direct binding to solidified phosphatidyl-serine-containing phospholipids also showed that C-C2 has approximately 50-fold less binding affinity than does the light chain. C-C2 poorly inhibited Xase activity. These results together clearly show that the C2 domain alone does not have full membrane binding activity, and suggest that the other light chain domains, A3 and/or C1, are also involved in the phospholipid binding activity of factor VIII.