Advances in AAV-CRISPR/Cas9-Mediated Hemophilia A Gene Therapy --Review / 中国实验血液学杂志

Hemophilia A(HA) is an X-linked recessive bleeding disorder caused by mutations in coagulation factor VIII. Nowadays, exogenous coagulation factor replacement therapy is the main treatment. With the continuous development of gene therapy, new research directions have been provided for the treatment of hemophilia A. CRISPR-Cas9 technology was applied to select suitable target sites, and mediate the targeted knock-in and efficient expression of exogenous B-domain-deleted FⅧ variant gene through corresponding vectors for the treatment of hemophilia A.CRISPR-Cas9 technology is an emerging gene editing tool with great efficiency, safety and effectiveness, and has been widely used in hemophilia gene therapy research. This paper reviews the vector selection, construction of therapeutic genes, gene editing technology and selection of expression target sites for hemophilia A gene therapy at this stage.

Hemophilia: a biography on therapeutical approaches

The history of hemophilia is ancient, with descriptions dated to the 2nd century AD. The first modern narratives appeared in 1800s, when total blood transfusion was the only available treatment and life expectancy was remarkably low. Advances occurred with the use of plasma and cryoprecipitate, but only the discovered of factor concentrates revolutionized the treatment. The implantation of prophylaxis allowed hemophilic patients to prevent bleeding and the development of chronic arthropathy, although with a significant burdensome with the regular infusions. In the past 20 years, this field has witnessed major improvements, including the development of gene therapy and other pharmacological approaches.

The existing scenario of haemophilia care in Canada and China - A review

ABSTRACT Hemophilia is an X-linked recessive genetic disorder which affects approximately 400,000 people globally. Differing healthcare reimbursement systems, budgetary constraints and geographical and cultural factors make it difficult for any country to fully deliver ideal care. Although developed countries have sufficient treatment products available, they are burdened by the higher expectation of outcomes, coupled with insufficient supportive care to monitor adherence and outcomes and to implement regular follow-up. In contrast, developing regions may not have ready access to factor replacement, but have developed excellent physiotherapy and rehabilitation programs. Although there are multiple studies that have attempted to assess country-specific variations in hemophilia care, very few compare hemophilia care between economically unequal countries and the challenges in achieving optimal hemophilia care. This literature review tries to bridge this gap and throws light on the country-specific differences in epidemiology, standard of hemophilia care and challenges faced in Canada and China. Data sources resulted in 20 studies (11 from Canada and 9 from China), which were reviewed. In a developed country, the main advantages are: the early treatment of bleeding episodes and the presence of a specialized interdisciplinary and comprehensive treatment concept. This is not the case in most developing countries, where the government does not have the resources to buy the necessary quantities of coagulation factors in the face of more urgent health priorities and hardly a few patients can afford to pay for their own treatment, even the on-demand home therapy.

A phase Ⅲ multi-center clinical trial on safety and efficacy of a domestic plasma derived factor Ⅸ for the treatment of patients with hemophilia B / 中华血液学杂志

Objective: To evaluate the efficacy and safety of a domestic human plasma derived coagulation Factor Ⅸ concentrate (pd-FⅨ) in patients with hemophilia B. Methods: The study was a multicenter, open-label and single-arm study. The efficacy of pd-F Ⅸ was evaluated by objective performance criteria. The doses of pd-FⅨ were calculated according to the bleeding symptom and disease severity. The infusion efficiency of pd-FⅨ and improvement of bleeding symptoms were measured at 30 minutes and (24±4) h after the first infusion, respectively. Adverse events were recorded. Viral infection and FⅨ inhibitor were detected 90 d after the first infusion. Results: All 36 subjects with hemophilia B were enrolled in the study. The median age of these patients was 31 years old and the median injection doses were 4 (1-17) times. The hemostatic effect of 27/36 (75.00%) and 9/36 (25.00%) acute bleeding events were rated as "excellent" and "better" , respectively. The recovery rate was 111.92% (65.55%-194.28%) at 30 minutes after infusion of FⅨ. There was no adverse event related to FⅨ. No reactivation of HBV, HCV or HIV and FⅨ inhibitor was detected at 90-104 d after the first FⅨ infusion. Conclusion: This domestically made human plasma derived FⅨ concentrate is safe and effective in the treatment of acute bleeding in patients with hemophilia B. Clinical trial registration: China food and Durg Administration, 2016L08027.

Tratamiento profiláctico en la hemofilia en países de la región Latinoamericana. Un reporte del Grupo Latinoamericano para el Impulso del Tratamiento de la Hemofilia (GLAITH) / Prophylaxis in hemophilia: situation analysis and call-to-action in Latin America. A report from the GLAITH group

La profilaxis en el tratamiento de la hemofilia ha sido crucial en la mejoría del pronóstico y calidad de vida en las personas con hemofilia (PCH). A pesar de ello, no está globalmente implementado y no ha sido ejecutado satisfactoriamente en Latinoamérica, donde es difícil evaluar la situación, y el manejo de las PCH no se ajusta a los estándares ideales. El grupo GLAITH (Grupo Latino Americano para el Impulso del Tratamiento de la Hemofilia) discutió el problema a través de una encuesta entre sus integrantes. Los hallazgos fueron discutidos en Bogotá en mayo del 2013 en donde los participantes definieron los puntos esenciales a comunicar en un llamado a la acción. Las proporciones de casos de hemofilia A reportados fueron entre 75 y 90% y entre 10 y 25%, los de hemofilia B. La hemofilia grave representó entre el 26 y el 55% de los casos. Un alto porcentaje de PCH tiene artropatía hemofílica. La atención de PCH varía en cada país, sólo se cubre entre el 50 y 60% del tratamiento, que es a demanda en el 85 a 95% de los casos. Sólo 5 a 15% reciben profilaxis, la mayoría secundaria. Pocos países tienen programa nacional o registros homogéneos. En llamado a la acción y conclusión para la región se recomienda: establecimiento de un registro latinoamericano unificado; estudios prospectivos de costo efectividad y evaluación de criterios en profilaxis secundaria; estudios comparativos de calidad de vida, individualización del tratamiento e implementación de la profilaxis en forma global en Latinoamérica.

Prophylactic treatment in the management of hemophilia has been a crucial factor in improving the prognosis and quality of life for people with hemophilia (PCH). However, it is not globally implemented. In Latin America it is difficult to assess the status of PCH and the its management does not conform to ideal standards. The GLAITH group discussed the problem in Latin America. A survey of its members and its findings were discussed at a meeting in Bogota in May 2013. Proportions of hemophilia A and B were 75-90% and 10-25% respectively. Severe hemophilia represents 26-55% of cases. A high percentage of PCH have hemophilic arthropathy. The general care and specific treatments of PCH vary by country, only 50-60% of the treatment is covered and in 85-95% of the cases are performed on an ondemand basis. Just 5-15% receives prophylaxis, most of them secondary. Few countries have a national program or homogeneous records. Finally the GLAITH group proceeded to develop a conclusion and call to action for the region where the following points are recommended: the establishment of a unified Latin American registry; prospective cost-effectiveness studies and evaluation criteria related to secondary prophylaxis; comparative studies of quality of life with and without prophylaxis in the region; promotion of individualization of treatment and, the increase of primary and secondary prophylaxis globally in Latin America.

Resultados de operación acceso y del Programa Nacional de Hemostasia y Trombosis / The results of operation access and the National Hemostasis and Thrombosis Program

En el decenio que va desde 1996 hasta 2006, se establecieron en Chile las políticas sanitarias programáticas que se hicieron cargo de la dramática situación que vivían hasta ese momento las personas afectadas de hemofilia. En dicho período fue fundamental la implementación de la Operación Acceso y del Programa Nacional de Hemostasia y Trombosis, ambos a cargo del Ministerio de Salud con el apoyo de la Sociedad Chilena de la Hemofilia y de la Facultad de Medicina de la Universidad de Chile. Este proceso trajo aparejado además la sistematización en cuanto a registro de casos, un avance cualitativo y cuantitativo en cuanto al acceso a tratamientos, así como a medicamentos seguros, efectivos y oportunos para la comunidad hemofílica del país con independencia de su sistema previsional. El artículo anterior de esta serie describe los programas, y este los resultados en el mencionado período.

During the time span going from 1996 to 2006 the healthcare policies that address hemophilic patient’s dramatic needs were set up in Chile. Operation Access and the National Hemostasis and Thrombosis Program were implemented, both in charge of the Ministry of Health, with the support of the Chilean Society of Hemophilia and the School of Medicine of the University of Chile. The process was coupled with the systematization of case registries, a qualitative and quantitative advance in access to treatment options, including timely access to safe and effective medications, regardless of the health insurance system to which patients belonged. The previous article of this series describes both programs, while the present article describes the results in that the ten-year period.

Cloning of coagulant VII factor from hepatoma cell line

Factor VII, is a coagulant protease; it begins the proteolytic cascade reactions and produces thrombin. The use of recombinant human factor VII, [rhFVII] is effective for the treatment of patients with hemophilia A or B. It is a target for gene therapy. This study was done to clone factor VII from HepG2 cell line. RNA was extracted from the hepatoma, [HepG2], cell line. On reverse transcription FVII cDNA was amplified by RT-PCR. PCR product was cloned into the pTZ57R/T vector and transported into the E-coli cells. By amplification of the FVII gene, the PCR band was observed and cloning into the vector was confirmed by restriction analysis. In this paper we report the cloning of factor VII from HepG2 cell line

Improving purification of coagulation F IX using heparin affinity chromatography and its comparison with ion exchange chromatography

Hemophilia B is a genetic disorder due to deficiency or complete absence of factor IX coagulation factor. Treatment of choice for these patients is use of factor IX concentrates. Therefore, purification of plasma proteins and separation of factor IX have been major objectives for scientists involved in this field. In this respect, purification procedure using ion exchange chromatography is widely used, but in the past decade affinity chromatography was also introduced. The objective of the present study has been to apply both techniques for the purification of factor IX and compare the quality and yield of the product. For the purification procedure, chromatography columns [XK-16], containing DEAE sepharose and Heparin sepharose were used. Factor IX coagulation activity was measured using a one-stage coagulation assay and factor IX antigen was quantified using ELISA technique. The specific activity and relative increase in purity of factor IX was calculated and it was demonstrated that specific activity improved from 3.1 IU/mg using DEAE ion exchange to 29 IU/mg when affinity chromatography was added and purity was increased from 155 to 1450 respectively. The present study demonstrates that addition of an affinity chromatography step using heparin sepharose is a major improvement in the purification of factor IX, where both specific activity and purity are increased considerably

Royal Hospital management of haemophilia patients

The following is the Royal Hospital protocol for managing haemophilia. It was adopted in july 2000. After one year we have encountered minor problems. It was well received by administration and all concerned departments. Colleagues around the Sultanate can use part or whole of this protocol and adapt it to their local needs

Hepatite pelo vírus C (HCV) em crianças e adolescentes hemofílicos / Hepatitis C virus (HCV) in children and adolescent hemophiliacs

Objetivo: Avaliar a soroprevalência de infecçäo pelo HCV entre hemofílicos no Estado do Pará e a possível relaçäo com nível sérico de enzimas hepáticas, tipo de hemofilia, idade, gravidade, combinaçäo e tipo de tratamento e data de início de tratamento como hemoderivados. Métodos: Estudo epidemiológico com corte transversal, analisando 62 pacientes hemofílicos do Centro de Hemoterapia do Pará (HEMOPA), nascidos a partir de 01/01/80, através de revisäo de prontuário, exame físico e exames laboratoriais: Anti-HCV (ELISA 3ª geraçäo). Reaçäo de cadeia de polimerase - PCR - (HCV-RNA) e dosagem de nível séricos de trasaminases (ALT e AST). A análise estatística dos dados foi feita aplicando os testes do qui-quadrado e o teste exato de Fisher, considerando-se significantes os resultados de p<_ 0,05. Resultados: Dos 62 pacientes analisados, 48,4 por cento (n=30) eram soropositivos para o HCV. Dentre estes pacientes, 43,3 por cento (n=13) apresentavam viremia com detecçäo de RNA viral pela técnica de PCR. A infecçäo pelo HCV se relacionou com data de início de tratamento anterior a 1993 (p= 0,0005); com o tipo hemofilia, sendo o tipo A mais freqüente (p= 0,028); com gravidade, sendo mais freqüente na forma moderada (p= 0,026); e com faixa etária, sendo mais freqüente acima dos 5 anos de idade (p= 0,025). Conclusöes: A infecçäo pelo HCV entre hemofílico no Estado do Pará é elevada (48,4 por cento) e se relacionou com início do tratamento anterior a 1993...

HIV seroconversion in Thai hemophiliacs up to 1991.

In Thailand, the anti-HIV screening in the donor blood was started in 1987 and was compulsory nationwide in February 1989. Sixty-six hemophilia A and 10 hemophilia B patients who received approximately six million units of factor VIII and IX in the form of fresh frozen plasma, frozen cryoprecipitate, cryoprecipitate removed plasma, fresh dry plasma and factor concentrate during 1976 to 1991 were tested for anti-HIV since 1987. The age ranged from 1-39 year (mean +/- SD = 15 +/- 7.3). The anti-HIV test was performed by ELISA and/or gel agglutination and confirmed by Western blot analysis. The patients would be checked 1-2 times per year and as necessary. A total of 174 tests for the first, second, third, fourth, fifth and sixth tests were studied in 76, 49, 27, 14, 5 and 3 patients respectively during 1987 to 1991. The prevalence of HIV seroconversion in the year 1987, 1988, 1989, 1990 and 1991 was 2.2% (1/45), 1.9% (1/53), 1.6% (1/63), 1.5% (1/67) and 3.9% (3/76) respectively. Three HIV seroconversion were found in the first, fourth and fifth anti-HIV test in 3 hemophilia A patients who received massive infusion of blood components during orthopedic corrective surgery. One case of HIV seroconversion found in 1987 was transmitted by HIV unscreened blood while 2 cases in 1991 by anti-HIV seronegative blood whose donors were in the window period of HIV infection. The prevalence of HIV seroconversion in Thai hemophiliacs is much lower than those in western countries.(ABSTRACT TRUNCATED AT 250 WORDS)