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Bakgrund - Hemofili A och B är X-kromosombundna blödarsjukdomar, som beror på genetiska avvikelser i de gener som kodar för koagulationsfaktor VIII respektive IX. I dagsläget förlitar sig hemofilipatienter på kontinuerliga intravenösa injektioner med faktorkoncentrat, för att förhindra att potentiellt livshotande blödningar uppstår. Genterapi med rekombinanta adeno-associerade virus (AAV) skulle kunna erbjuda ett kurativt behandlingsalternativ, genom införandet av friska arvsanlag i hepatocyter. Syfte - Syftet med den här litteraturstudien var att undersöka huruvida genterapi medierad av AAV-vektorer är en effektiv och säker behandling mot hemofili A och B ur ett långsiktigt perspektiv. Metod - Studien är genomförd som en systematisk litteraturstudie och är baserad på sex originalartiklar framsökta via databasen PubMed, med sökorden "hemophilia AND gene therapy". Specificerade sökkriterier användes för att underlätta relevansbedömning och valet av artiklar. Resultat - En ökad endogen koagulationsfaktorproduktion kunde påvisas hos majoriteten av studiedeltagarna efter genterapibehandlingarna. Sammantaget observerades också en väsentlig blödningsreducering och en minskad faktorkoncentratanvändning. Störst förbättring noterades i de kohorter som erhållit högre genterapidoser eller den muterade faktor IX Padua-genen. Ingen immunrespons mot transgenprodukten detekterades i någon studie. Däremot sågs ett humoralt immunsvar mot AAV-kapsiden hos samtliga studiedeltagare. En mycket stor variation i T-cellssvar mot AAV-kapsiden kunde noteras. Förhöjda nivåer av alaninaminotransferas (ALAT) var den vanligast förekommande incidenten, men samtliga fall kunde framgångsrikt behandlas med glukokortikoidpreparat. Slutsats - Genterapibehandling med rekombinanta AAV-vektorer mot hemofili A och B förefaller effektiv och säker. Förhöjda ALAT-nivåer återstår dock som en behandlingsproblematik. Längre uppföljningar av fler genterapibehandlade hemofilipatienter krävs, för att kunna dra några definitiva slutsatser, väga risker mot nytta, samt optimera och individanpassa doser. Background - Hemophilia A and B are X-linked bleeding disorders, resulting from defects in the genes encoding coagulation factors VIII and IX respectively. The current treatment for hemophiliacs entails frequent intravenous injections of coagulation factor concentrates, to prevent potentially life-threatening hemorrhages. Gene therapy utilizing recombinant adeno-associated viruses (AAV) could offer a potentially curative treatment option through the introduction of healthy genes into hepatocytes. Aim - The aim of this literature study was to investigate the long-term efficacy and safety of AAV vector-mediated gene therapy for the treatment of hemophilia A and B. Methods - The study is conducted as a systematic literature study and is based on six original articles retrieved from the search engine PubMed, using the key words "hemophilia AND gene therapy". Specific search criteria were used to facilitate the relevance assessment and selection of articles. Results - An increased endogenous coagulation factor synthesis was noted in the majority of the study participants after the gene therapy. Overall, a significant reduction in bleeding episodes and the use of factor concentrates were observed. The greatest improvements were noted in the cohorts that received the higher gene therapy doses or the mutated factor IX Padua gene. None of the study participants had an immunologic response to the transgene product. A humoral immune response against the AAV capsid was seen in all participants though. Large differences in AAV capsid-specific T-cell activation were observed. The most common adverse event was an elevation in the alanine aminotransferase (ALT) level. However, these events could be controlled with glucocorticoids. Conclusions - AAV vector-mediated gene therapy for the treatment of hemophilia A and B had a positive efficacy and safety profile. Although increased ALT levels remain a concern. Monitoring of larger numbers of study participants for longer follow-up periods is necessary for any definite conclusions to be drawn, to weigh risks against benefits and to optimize individual dosing.

Hemostasis is essential to life; it is the ability of organism to stop bleeding and to maintain the fluidity of the blood in an intact vascular bed at one time. Factor VIII is called antihemophilic globulin or antihemophilic factor. It is a plasma glycoprotein composed of two noncovalently associated chains. One chain is a heavy one, comprising domains A1- A2- B and the light chain composed of the domains A3- C1-C2. FVIII activity was measured in two ways from historical reasons a) a one-phase method, where a mixture of FVIII deficient plasma and patient plasma is analyzed using APTT assay; the absence of FVIII in the patient plasma leads to lengthening of the time, b) two-phase method, where the first step leads to formation of FVIIIa and FXa and in the second phase there are thrombin and fibrin created. Two-phase method is difficult to implement in routine laboratory, and therefore it was stopped using during time. This method was substituted by the chromogenic method after development of the chromogenic substrates, where there is in the first step created FVIIIa and FXa in the presence of FIXa, phospholipids and Ca2+, and in the second step there is formed a yellow coloration by the addition of the chromogenic substrate. FVIII deficiency causes a severe bleeding disorder, hemophilia A. I performed measurements from May to September 2013 at Coagulation laboratory at the Institute of Hematology and Blood Transfusion in Prague, where I was employed. I examined in total 76 patients, hemophiliacs A, who were at least 8 days without any treatment or substitution. I assigned numbers to patient's samples to ensure anonymity of patients. I worked with the automatic coagulation analyzer STA- R Evolution? from Diagnostica Stago, which works on the principle of photometry and chronometry. I determined the factor VIII by one-phase method and two-phase method and I compared the results. The genetic part of the work was analyzed in the genetic laboratory, which is part of our department. I worked up the results obtained from both methods in 76 patients to the table and the graph. The group included 14 (18 %) moderate and 56 (74 %) of mild hemophiliacs, then 6 (8%) hemophiliacs who did not meet the criterion of a mild hemophilia A, but clinically they belonged into mild hemophiliacs. Based on the stated criteria, we found out that 15 (20 %) patients had a ratio of FVIII: C1st/FVIII: Chr or FVIII: Chr / FVIII: C1st 0.6, they differed significantly in their values set by one-stage clotting FVIII and FVIII set by the chromogenic method. A total of 11 patients with FVIII activity were higher in the single-phase method. At three patients FVIII: C1st was even on the upper limit of the normal value, while FVIII chromogenic method gave on average 16%. We managed to find a causal mutation in the FVIII in 14 patients with "the different results", we could not investigate 1 patient genetically because of the missing genetic material. Mutations in patients with lower activity of FVIII set up by the chromogenic two-phase method were concentrated predominantly into the A3 domain; mutations in patients with FVIII a lower activity set up by one-phase clotting assay were concentrated in the A2 domain. The results presented show, that diagnostic of any patient with mild or moderate hemophilia A should include determination of FVIII by both methods; FVIII: C1st and FVIII: Chr.