RESUMEN
Attempts to achieve a functional cure or amelioration of the severe X linked bleeding disorders haemophilia A (factor VIII deficiency) and haemophilia B (factor IX deficiency) using AAV-based vectors have been frustrated by immune responses that limit efficacy and durability. The immune responses include adaptive and innate pathways as well as cytokine mediated inflammation, especially of the target organ cells-hepatocytes. Immune suppression has only been partly effective in clinical trials at ameliorating the immune response and the lack of good animal models has delayed progress in identifying mechanisms and developing more effective approaches to controlling these effects of AAV gene transfer. Here we discuss the arguments for and against more potent immunosuppression to improve factor expression after AAV-mediated gene therapy.
Asunto(s)
Hemofilia A , Hemofilia B , Animales , Hemofilia A/genética , Hemofilia A/terapia , Hemofilia B/genética , Hemofilia B/terapia , Terapia Genética , Terapia de Inmunosupresión , InmunidadRESUMEN
BACKGROUND: Mathematical models of coagulation have been developed to mirror thrombin generation in plasma, with the aim of investigating how variation in coagulation factor levels regulates hemostasis. However, current models vary in the reactions they capture and the reaction rates used, and their validation is restricted by a lack of large coherent datasets, resulting in questioning of their utility. OBJECTIVES: To address this debate, we systematically assessed current models against a large dataset, using plasma coagulation factor levels from 348 individuals with normal hemostasis to identify the causes of these variations. METHODS: We compared model predictions with measured thrombin generation, quantifying and comparing the ability of each model to predict thrombin generation, the contributions of the individual reactions, and their dependence on reaction rates. RESULTS: We found that no current model predicted the hemostatic response across the whole cohort and all produced thrombin generation curves that did not resemble those obtained experimentally. Our analysis has identified the key reactions that lead to differential model predictions, where experimental uncertainty leads to variability in predictions, and we determined reactions that have a high influence on measured thrombin generation, such as the contribution of factor XI. CONCLUSION: This systematic assessment of models of coagulation, using large dataset inputs, points to ways in which these models can be improved. A model that accurately reflects the effects of the multiple subtle variations in an individual's hemostatic profile could be used for assessing antithrombotics or as a tool for precision medicine.
Asunto(s)
Coagulación Sanguínea , Trombina , Humanos , Trombina/metabolismo , Pruebas de Coagulación Sanguínea , Hemostasis , Reproducibilidad de los Resultados , Factores de Coagulación Sanguínea/metabolismo , Modelos Biológicos , Masculino , Femenino , Adulto , Persona de Mediana EdadRESUMEN
The single most important step on the path to our modern understanding of blood coagulation and haemophilia in the 20th century was taken by British pathologist Robert Gwyn Macfarlane with his 1964 publication 'An enzyme cascade in the blood clotting mechanism, and its function as a biochemical amplifier'. In the same year, Ratnoff and Davie in the USA reached the same conclusion. Macfarlane and Rosemary Biggs had previously, in 1952, discovered factor IX as the factor deficient in haemophilia B. In 1973, Arthur Bloom defined the distinct role of Factor VIII and von Willebrand factor in haemophilia A and von Willebrand's disease respectively. This inspired the efforts of Tuddenham and his group towards the purification of Factor VIII which reached homogeneity in 1982, leading to the cloning of the Factor VIII gene in 1984 in collaboration with US scientists at Genentech, which in turn enabled development of safe recombinant factor concentrates for patients with haemophilia. Brownlee cloned the factor IX gene in 1982 at the Sir William Dunn Institute of Pathology in Oxford. This led eventually to the first successful trial of gene therapy for haemophilia B in 2011 by the Nathwani group at UCL, which built on pioneering work of US groups and was partnered with St Jude in Memphis where Nathwani started the project. This trial has fuelled the current quest for a functional cure of haemophilia A and B. The UK has, therefore, made a rich contribution to advances in haemostasis over the last 60 years, often in partnership with other groups across the world.
Asunto(s)
Hemofilia A/epidemiología , Hemofilia A/terapia , Hemofilia B/epidemiología , Hemofilia B/terapia , Ensayos Clínicos como Asunto , Manejo de la Enfermedad , Susceptibilidad a Enfermedades , Hemofilia A/etiología , Hemofilia A/historia , Hemofilia B/etiología , Hemofilia B/historia , Historia del Siglo XX , Historia del Siglo XXI , Humanos , Resultado del TratamientoRESUMEN
The foundation of haemophilia A therapy in the last 35 years has been critically dependent on isolation of the Factor VIII (FVIII) protein and discovery of the cDNA sequence of the FVIII gene, published in 1984. Identification of the FVIII sequence resulted in a new era of recombinant concentrates and led to significant improvements in safety, set against the tragedy of widespread HIV and hepatitis infections in haemophilia patients from contaminated plasma-based products. We chronicle the scientific methods and race leading up to the publication of the FVIII DNA sequence and the legacy that follows through to revolutionary gene therapy treatment in clinical trials today.
Asunto(s)
Factor VIII/uso terapéutico , Terapia Genética/métodos , Hemofilia A/tratamiento farmacológico , Animales , Factor VIII/farmacología , Humanos , RatonesRESUMEN
The von Willebrand factor (VWF) and coagulation factor VIII (FVIII) are intricately involved in hemostasis. A tight, noncovalent complex between VWF and FVIII prolongs the half-life of FVIII in plasma, and failure to form this complex leads to rapid clearance of FVIII and bleeding diatheses such as hemophilia A and von Willebrand disease (VWD) type 2N. High-resolution insight into the complex between VWF and FVIII has so far been strikingly lacking. This is particularly the case for the flexible a3 region of FVIII, which is imperative for high-affinity binding. Here, a structural and biophysical characterization of the interaction between VWF and FVIII is presented with focus on two of the domains that have been proven pivotal for mediating the interaction, namely the a3 region of FVIII and the TIL'E' domains of VWF. Binding between the FVIII a3 region and VWF TIL'E' was here observed using NMR spectroscopy, where chemical shift changes were localized to two ß-sheet regions on the edge of TIL'E' upon FVIII a3 region binding. Isothermal titration calorimetry and NMR spectroscopy were used to characterize the interaction between FVIII and TIL'E' as well as mutants of TIL'E', which further highlights the importance of the ß-sheet region of TIL'E' for high-affinity binding. Overall, the results presented provide new insight into the role the FVIII a3 region plays for complex formation between VWF and FVIII and the ß-sheet region of TIL'E' is shown to be important for FVIII binding. Thus, the results pave the way for further high-resolution insights into this imperative complex.
Asunto(s)
Factor VIII/química , Factor VIII/metabolismo , Factor de von Willebrand/química , Factor de von Willebrand/metabolismo , Calorimetría , Espectroscopía de Resonancia Magnética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutación/genética , Unión Proteica , Dominios Proteicos , Factor de von Willebrand/genéticaRESUMEN
Haemophilia therapy has undergone very rapid evolution in the last 10 years. The major limitation of current replacement therapy is the short half-life of factors VIII and IX. These half-lives have been extended by the addition of various moieties, allowing less frequent infusion regimens. Entirely novel approaches have also entered the clinic, including a bispecific antibody that mimics factor VIII and strategies that rebalance the haemostatic mechanism by reducing antithrombin through inhibition of synthesis. These two treatments are available by subcutaneous injection at infrequent intervals and both can be used in patients with neutralising antibodies (inhibitors). Finally, a cure may be on the horizon with preliminary evidence of success for gene therapy in haemophilia B and A.
Asunto(s)
Anticuerpos Biespecíficos/uso terapéutico , Factor IX/uso terapéutico , Factor VIII/uso terapéutico , Terapia Genética , Hemofilia A/terapia , Hemofilia B/terapia , Anticuerpos Biespecíficos/farmacocinética , Antitrombinas/sangre , Factor IX/farmacocinética , Factor VIII/farmacocinética , Hemofilia A/sangre , Hemofilia A/genética , Hemofilia B/sangre , Hemofilia B/genética , HumanosRESUMEN
The best currently available treatments for hemophilia A and B (factor VIII or factor IX deficiency, respectively) require frequent intravenous infusion of highly expensive proteins that have short half-lives. Factor levels follow a saw-tooth pattern that is seldom in the normal range and falls so low that breakthrough bleeding occurs. Most hemophiliacs worldwide do not have access to even this level of care. In stark contrast, gene therapy holds out the hope of a cure by inducing continuous endogenous expression of factor VIII or factor IX following transfer of a functional gene to replace the hemophilic patient's own defective gene.
Asunto(s)
Terapia Genética , Hemofilia A/genética , Hemofilia A/terapia , Hemofilia B/genética , Hemofilia B/terapia , Animales , Ensayos Clínicos como Asunto , Dependovirus/genética , Factor IX/genética , Factor VIII/genética , Terapia Genética/efectos adversos , Terapia Genética/economía , Terapia Genética/métodos , Vectores Genéticos/genética , Humanos , Resultado del TratamientoRESUMEN
Gene therapy provides hope for a cure for patients with hemophilia by establishing continuous endogenous expression of factor VIII or factor IX following transfer of a functional gene copy to replace the hemophilic patient's own defective gene. Hemophilia may be considered a "low-hanging fruit" for gene therapy because a small increment in blood factor levels (≥2% of normal) significantly improves the bleeding tendency from severe to moderate, eliminating most spontaneous bleeds. After decades of research, the first trial to provide clear evidence of efficiency after gene transfer in patients with hemophilia B using adeno-associated virus vectors was reported by the authors' group in 2011. This has been followed by unprecedented activity in this area, with the commencement of seven new early-phase trials involving >55 patients with hemophilia A or hemophilia B. These studies have, in large part, generated promising clinical data that lay a strong foundation for gene therapy to move forward rapidly to market authorization. This review discusses the data from the authors' studies and emerging results from other gene therapy trials in both hemophilia A and B.
Asunto(s)
Terapia Genética/tendencias , Vectores Genéticos/uso terapéutico , Hemofilia A/terapia , Hemofilia B/terapia , Dependovirus/genética , Factor IX/genética , Factor IX/uso terapéutico , Factor VIII/genética , Factor VIII/uso terapéutico , Vectores Genéticos/genética , Hemofilia A/genética , Hemofilia B/genética , HumanosRESUMEN
Low molecular weight heparin (LMWH) given to inhibit coagulation and reduce the risk of thrombosis, is typically monitored by anti-Xa assay. However, anti-Xa levels may not necessarily provide an accurate measure of coagulation inhibition. Moreover, pregnancy is associated with hypercoagulability, which may compromise the efficacy of LMWH. We looked at the association between anti-Xa levels and parameters of thrombin generation assay [TGA; area under the curve (AUC), peak height (PH) and time to peak (ttP)] using samples from 41 pregnant women receiving LMWH and 40 normal pregnant women controls. TGA results confirmed the physiological hypercoagulability of normal pregnancy (mean normalised values: AUC 119%; PH 157%; ttP 72%). Although anti-Xa measures correlated with all three TGA parameters, this group correlation masked significant inter-individual variability, demonstrated by the R(2) value or coefficient of determination. Anti-Xa levels contributed to 74% of variation in AUC values, 63% of variation in PH values and only 53% of variation in ttP values. The remainder reflects the contribution of patients' intrinsic coagulation status. Hence, some patients with 'safe' anti-Xa levels may potentially be under-anticoagulated, particularly in pregnancy. Measuring coagulability directly with TGA may lower the risk of adverse events due to under-anticoagulation in selected patients.
Asunto(s)
Anticoagulantes , Inhibidores del Factor Xa/sangre , Heparina de Bajo-Peso-Molecular , Monitoreo Fisiológico , Complicaciones Hematológicas del Embarazo , Trombofilia , Adulto , Anticoagulantes/administración & dosificación , Anticoagulantes/farmacocinética , Femenino , Heparina de Bajo-Peso-Molecular/administración & dosificación , Heparina de Bajo-Peso-Molecular/farmacocinética , Humanos , Embarazo , Complicaciones Hematológicas del Embarazo/sangre , Complicaciones Hematológicas del Embarazo/tratamiento farmacológico , Estudios Retrospectivos , Tiempo de Trombina/métodos , Trombofilia/sangre , Trombofilia/tratamiento farmacológicoRESUMEN
BACKGROUND: In patients with severe hemophilia B, gene therapy that is mediated by a novel self-complementary adeno-associated virus serotype 8 (AAV8) vector has been shown to raise factor IX levels for periods of up to 16 months. We wanted to determine the durability of transgene expression, the vector dose-response relationship, and the level of persistent or late toxicity. METHODS: We evaluated the stability of transgene expression and long-term safety in 10 patients with severe hemophilia B: 6 patients who had been enrolled in an initial phase 1 dose-escalation trial, with 2 patients each receiving a low, intermediate, or high dose, and 4 additional patients who received the high dose (2×10(12) vector genomes per kilogram of body weight). The patients subsequently underwent extensive clinical and laboratory monitoring. RESULTS: A single intravenous infusion of vector in all 10 patients with severe hemophilia B resulted in a dose-dependent increase in circulating factor IX to a level that was 1 to 6% of the normal value over a median period of 3.2 years, with observation ongoing. In the high-dose group, a consistent increase in the factor IX level to a mean (±SD) of 5.1±1.7% was observed in all 6 patients, which resulted in a reduction of more than 90% in both bleeding episodes and the use of prophylactic factor IX concentrate. A transient increase in the mean alanine aminotransferase level to 86 IU per liter (range, 36 to 202) occurred between week 7 and week 10 in 4 of the 6 patients in the high-dose group but resolved over a median of 5 days (range, 2 to 35) after prednisolone treatment. CONCLUSIONS: In 10 patients with severe hemophilia B, the infusion of a single dose of AAV8 vector resulted in long-term therapeutic factor IX expression associated with clinical improvement. With a follow-up period of up to 3 years, no late toxic effects from the therapy were reported. (Funded by the National Heart, Lung, and Blood Institute and others; ClinicalTrials.gov number, NCT00979238.).
Asunto(s)
Factor IX/genética , Terapia Genética , Vectores Genéticos/administración & dosificación , Hemofilia B/terapia , Adulto , Alanina Transaminasa/sangre , Dependovirus/genética , Factor IX/metabolismo , Estudios de Seguimiento , Expresión Génica , Terapia Genética/efectos adversos , Hemofilia B/sangre , Hemofilia B/genética , Humanos , Infusiones Intravenosas , Masculino , Persona de Mediana Edad , Transgenes , Adulto JovenRESUMEN
Although much of the function of von Willebrand factor (VWF) has been revealed, detailed insight into the molecular structure that enables VWF to orchestrate hemostatic processes, in particular factor VIII (FVIII) binding and stabilization in plasma, is lacking. Here, we present the high-resolution solution structure and structural dynamics of the D' region of VWF, which constitutes the major FVIII binding site. D' consists of 2 domains, trypsin-inhibitor-like (TIL') and E', of which the TIL' domain lacks extensive secondary structure, is strikingly dynamic and harbors a cluster of pathological mutations leading to decreased FVIII binding affinity (type 2N von Willebrand disease [VWD]). This indicates that the backbone malleability of TIL' is important for its biological activity. The principal FVIII binding site is localized to a flexible, positively charged region on TIL', which is supported by the rigid scaffold of the TIL' and E' domain ß sheets. Furthermore, surface-charge mapping of the TIL'E' structure reveals a potential mechanism for the electrostatically guided, high-affinity VWFâ FVIII interaction. Our findings provide novel insights into VWFâ FVIII complex formation, leading to a greater understanding of the molecular basis of the bleeding diathesis type 2N VWD.
Asunto(s)
Factor VIII/química , Factor de von Willebrand/química , Sitios de Unión , Cristalografía por Rayos X , Factor VIII/genética , Factor VIII/metabolismo , Humanos , Estabilidad Proteica , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Electricidad Estática , Factor de von Willebrand/genética , Factor de von Willebrand/metabolismoRESUMEN
Recombinant adeno-associated virus (rAAV) vectors encoding human factor VIII (hFVIII) were systematically evaluated for hemophilia A (HA) gene therapy. A 5.7-kb rAAV-expression cassette (rAAV-HLP-codop-hFVIII-N6) containing a codon-optimized hFVIII cDNA in which a 226 amino acid (aa) B-domain spacer replaced the entire B domain and a hybrid liver-specific promoter (HLP) mediated 10-fold higher hFVIII levels in mice compared with non-codon-optimized variants. A further twofold improvement in potency was achieved by replacing the 226-aa N6 spacer with a novel 17-aa peptide (V3) in which 6 glycosylation triplets from the B domain were juxtaposed. The resulting 5.2-kb rAAV-HLP-codop-hFVIII-V3 cassette was more efficiently packaged within AAV virions and mediated supraphysiologic hFVIII expression (732 ± 162% of normal) in HA knock-out mice following administration of 2 × 10(12) vector genomes/kg, a vector dose shown to be safe in subjects with hemophilia B. Stable hFVIII expression at 15 ± 4% of normal was observed at this dose in a nonhuman primate. hFVIII expression above 100% was observed in 3 macaques that received a higher dose of either this vector or the N6 variant. These animals developed neutralizing anti-FVIII antibodies that were abrogated with transient immunosuppression. Therefore, rAAV-HLP-codop-hFVIII-V3 substantially improves the prospects of effective HA gene therapy.
Asunto(s)
Dependovirus/genética , Factor VIII/farmacología , Terapia Genética , Variación Genética/genética , Vectores Genéticos/administración & dosificación , Hemofilia A/terapia , Animales , Western Blotting , Factor VIII/genética , Factor VIII/inmunología , Glicosilación , Hemofilia A/genética , Humanos , Tolerancia Inmunológica , Hígado/metabolismo , Macaca mulatta , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Regiones Promotoras Genéticas/genéticaRESUMEN
We explored adeno-associated viral vector (AAV)-mediated gene transfer in the perinatal period in animal models of severe congenital factor VII (FVII) deficiency, a disease associated with early postnatal life-threatening hemorrhage. In young adult mice with plasma FVII < 1% of normal, a single tail vein administration of AAV (1 × 10(13) vector genomes [vg]/kg) resulted in expression of murine FVII at 266% ± 34% of normal for ≥ 67 days, which mediated protection against fatal hemorrhage and significantly improved survival. Codon optimization of human FVII (hFVIIcoop) improved AAV transgene expression by 37-fold compared with the wild-type hFVII cDNA. In adult macaques, a single peripheral vein injection of 2 × 10(11) vg/kg of the hFVIIcoop AAV vector resulted in therapeutic levels of hFVII expression that were equivalent in males (10.7% ± 3.1%) and females (12.3% ± 0.8%). In utero delivery of this vector in the third trimester to fetal monkeys conferred expression of hFVII at birth of 20.4% ± 3.7%, with a gradual decline to > 1% by 7 weeks. Re-administration of an alternative serotype at 12 months postnatal age increased hFVII levels to 165% ± 6.2% of normal, which remained at therapeutic levels for a further 28 weeks without toxicity. Thus, perinatal AAV-mediated gene transfer shows promise for disorders with onset of pathology early after birth.
Asunto(s)
Dependovirus , Deficiencia del Factor VII/terapia , Factor VII/uso terapéutico , Terapia Genética/métodos , Vectores Genéticos , Hemorragia/prevención & control , Atención Perinatal , Animales , Animales Recién Nacidos , Codón , Dependovirus/genética , Factor VII/análisis , Factor VII/biosíntesis , Factor VII/genética , Deficiencia del Factor VII/sangre , Deficiencia del Factor VII/genética , Deficiencia del Factor VII/fisiopatología , Femenino , Terapias Fetales/efectos adversos , Expresión Génica , Terapia Genética/efectos adversos , Vectores Genéticos/administración & dosificación , Vectores Genéticos/efectos adversos , Hemorragia/etiología , Células Hep G2 , Humanos , Inyecciones Intravenosas , Macaca mulatta , Masculino , Ratones , Embarazo , Caracteres Sexuales , Análisis de SupervivenciaRESUMEN
BACKGROUND: Hemophilia B, an X-linked disorder, is ideally suited for gene therapy. We investigated the use of a new gene therapy in patients with the disorder. METHODS: We infused a single dose of a serotype-8-pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein in six patients with severe hemophilia B (FIX activity, <1% of normal values). Study participants were enrolled sequentially in one of three cohorts (given a high, intermediate, or low dose of vector), with two participants in each group. Vector was administered without immunosuppressive therapy, and participants were followed for 6 to 16 months. RESULTS: AAV-mediated expression of FIX at 2 to 11% of normal levels was observed in all participants. Four of the six discontinued FIX prophylaxis and remained free of spontaneous hemorrhage; in the other two, the interval between prophylactic injections was increased. Of the two participants who received the high dose of vector, one had a transient, asymptomatic elevation of serum aminotransferase levels, which was associated with the detection of AAV8-capsid-specific T cells in the peripheral blood; the other had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of these two participants received a short course of glucocorticoid therapy, which rapidly normalized aminotransferase levels and maintained FIX levels in the range of 3 to 11% of normal values. CONCLUSIONS: Peripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype, with few side effects. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression. (Funded by the Medical Research Council and others; ClinicalTrials.gov number, NCT00979238.).
Asunto(s)
Dependovirus , Factor IX/genética , Terapia Genética , Vectores Genéticos , Hemofilia B/terapia , Adulto , Dependovirus/genética , Factor IX/uso terapéutico , Terapia Genética/efectos adversos , Vectores Genéticos/inmunología , Humanos , Infusiones Intravenosas , Persona de Mediana Edad , Transgenes/inmunologíaRESUMEN
An archive of congenital human diseases is presented, aiming to contain all those where recessive (biallelic) can be compared with X-linked and/or dominant (monoallelic) inheritance. A significant deficit of recessive inheritance is evident, both in disease inheritance and in contribution to inheritance per known disease gene. The deficit contrasts with expectation derived from the cell biology of mutation, and from the importance of recessive mutation in evolution and its preponderance in N-ethyl-N-nitrosourea (ENU) mutagenesis. The deficit fits well with the standard model of demographic change since the neolithic era, and may also reflect natural selection acting on heterozygotes.
Asunto(s)
Genes Recesivos , Enfermedades Genéticas Congénitas/genética , Demografía , Genes Dominantes , Humanos , Patrón de Herencia , Mutación , Selección GenéticaRESUMEN
Hemophilia is a bleeding disorder with X-linked inheritance. Current prenatal diagnostic methods for hemophilia are invasive and pose a risk to the fetus. Cell-free fetal DNA analysis in maternal plasma provides a noninvasive mean of assessing fetal sex in such pregnancies. However, the disease status of male fetuses remains unknown if mutation-specific confirmatory analysis is not performed. Here we have developed a noninvasive test to diagnose whether the fetus has inherited a causative mutation for hemophilia from its mother. The strategy is based on a relative mutation dosage approach, which we have previously established for determining the mutational status of fetuses for autosomal disease mutations. In this study, the relative mutation dosage method is used to deduce whether a fetus has inherited a hemophilia mutation on chromosome X by detecting whether the concentration of the mutant or wild-type allele is overrepresented in the plasma of heterozygous women carrying male fetuses. We correctly detected fetal genotypes for hemophilia mutations in all of the 12 studied maternal plasma samples obtained from at-risk pregnancies from as early as the 11th week of gestation. This development would make the decision to undertake prenatal testing less traumatic and safer for at-risk families.
Asunto(s)
ADN/sangre , Hemofilia A/diagnóstico , Técnicas Analíticas Microfluídicas/métodos , Madres , Reacción en Cadena de la Polimerasa/métodos , Diagnóstico Prenatal/métodos , Algoritmos , Cromosomas Humanos X , ADN/análisis , Femenino , Genotipo , Edad Gestacional , Hemofilia A/sangre , Hemofilia A/genética , Humanos , Masculino , Reacción en Cadena de la Polimerasa/instrumentación , Polimorfismo de Nucleótido Simple , Embarazo , Diagnóstico Prenatal/instrumentación , Sensibilidad y Especificidad , Procesamiento de Señales Asistido por ComputadorRESUMEN
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.
Asunto(s)
Codón/genética , Factor VIII/genética , Terapia Genética/métodos , Hemofilia A/terapia , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Ensayo de Inmunoadsorción Enzimática , Factor VIII/metabolismo , Femenino , Expresión Génica , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Células HEK293 , Hemofilia A/sangre , Hemofilia A/genética , Humanos , Inyecciones Intravenosas , Lentivirus/genética , Masculino , Ratones , Ratones de la Cepa 129 , Ratones Noqueados , Datos de Secuencia Molecular , Mutación , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Virus Formadores de Foco en el Bazo/genéticaRESUMEN
STUDY OBJECTIVES: We reviewed the management and treatment outcomes of menorrhagia in adolescents with inherited bleeding disorders and assessed the impact of menorrhagia on their quality of life. DESIGN: Retrospective review of case notes and a questionnaire study. SETTING: Comprehensive-care hemophilia treatment center. PARTICIPANTS: Adolescents with inherited bleeding disorders who had registered at the center and were attending the multidisciplinary hemophilia and gynecology clinic for management of menorrhagia. INTERVENTIONS: Review of medical records and assessment of menstrual blood loss using the pictorial blood assessment chart and quality of life measurements during menstruation using a questionnaire. MAIN OUTCOME MEASURES: Scores on pictorial blood assessment charts and quality of life measurements before and after treatment. RESULTS: Of 153 girls aged 12 to 19 years who had registered at the center and had an inherited bleeding disorder, 42 (27%) attended the multidisciplinary clinic for management of menorrhagia. The majority (38/42; 90%) had experienced menorrhagia since menarche. Of the group, 5 (12%) required hospital admission for acute menorrhagia and severe anemia. Treatment options for menorrhagia included tranexamic acid, desmopressin, combined oral contraceptive pills, clotting factor concentrate, and the levonorgestrel intrauterine system. These treatment modalities, alone or in combination, were associated with a reduction in menstrual blood loss (median pre- and posttreatment pictorial blood assessment chart scores were 215 and 88, respectively) and improvement in quality of life scores (median pre- and posttreatment were 26 and 44, respectively). CONCLUSIONS: Menorrhagia is a common symptom in adolescents with inherited bleeding disorders. It can present acutely, and it adversely affects quality of life. Treatment options include hemostatic and/or hormonal therapies and can improve the quality of life of affected girls.
Asunto(s)
Trastornos de la Coagulación Sanguínea Heredados/complicaciones , Trastornos de las Plaquetas Sanguíneas/complicaciones , Menorragia/etiología , Calidad de Vida , Adolescente , Niño , Anticonceptivos Hormonales Orales/uso terapéutico , Femenino , Hemostáticos/uso terapéutico , Humanos , Menorragia/tratamiento farmacológico , Estudios Retrospectivos , Adulto JovenRESUMEN
Factor XI (FXI) functions in blood coagulation. FXI is composed of four apple (Ap) domains and a serine protease (SP) domain. Deficiency of FXI leads to an injury-related bleeding disorder, which is remarkable for the lack of correlation between bleeding symptoms and FXI coagulant activity (FXI:C). The number of mutations previously reported in our interactive web database (http://www.FactorXI.org) is now significantly increased to 183 through our new patient studies and from literature surveys. Eight novel missense mutations give a total of 120 throughout the FXI gene (F11). The most abundant defects in FXI are revealed to be those from low-protein plasma levels (Type I: CRM-) that originate from protein misfolding, rather than from functional defects (Type II: CRM+). A total of 70 Ap missense mutations were analysed using a consensus Ap domain structure generated from the FXI dimer crystal structure. This showed that all parts of the Ap domain were affected. The 47 SP missense mutations were also distributed throughout the SP domain structure. The periphery of the Ap beta-sheet structure is sensitive to structural perturbation caused by residue changes throughout the Ap domain, yet this beta-sheet is crucial for FXI dimer formation. Residues located at the Ap4:Ap4 interface in the dimer are much less directly involved. We conclude that the abundance of Type I defects in FXI results from the sensitivity of the Ap domain folding to residue changes within this, and discuss how structural knowledge of the mutations improves our understanding of FXI deficiencies.