GTMB 7 - Gene Therapy & Molecular Biology

Gene Therapy and Molecular Biology Vol 7, page 185undergoes a marked drop in the postnatal period. Incontrast, SMN protein is greatly reduced in all tissuesfrom fetuses affected with SMA (Burlet et al, 1998). Theseobservations suggest that SMN protein may be requiredduring embryo-fetal development and as such, prenatalgene transfer may be more effective than adult treatment.Prenatal diagnosis is available using deletion analysis ofthe SMN 1 gene (Matthjis et al, 1998).F. HaemophiliasThe haemophilias A and B are also particularlysuitable for gene therapy in utero. Both are X-linkedhereditary haemorrhagic disorders which occur in 1 in10,000 and 1 in 25,000 males respectively and are causedby the absence or dysfunction of the respective humanfactor VIII (hFVIII) or IX (hFIX) clotting factors (Furie etal, 1994). Current treatment uses replacement therapy withhFVIII or hFIX. Unfortunately, a number of patientsdevelop antibodies to therapy leading to ineffectivetreatment and occasional anaphylaxis (Lusher, 2000).Indeed, the complications of haemophilia treatment havein some cases been far worse than the diseases themselves,increasing their morbidity and mortality (Soucie et al,2000).As the coagulation factors are required in the bloodand can be secreted functionally from a variety of tissues,the actual site of production is not so important as long astherapeutic plasma levels are realized. Adult gene therapystrategies have therefore concentrated on application to themuscle or the liver. Successful delivery and expression ofFIX has been achieved in adult animal models ofhaemophilia B following portal intravascularadministration of adenoviral (Kay et al, 1994) andretroviral vectors (Kay et al, 1993). Sustained FIXexpression was also observed after intramuscular injectionof adult haemophiliac dogs with adeno-associated viral(AAV) vectors expressing canine FIX (Chao et al, 1999;Herzog et al, 1999) and after intravascular injection ofadult haemophiliac mice with AAV vectors expressinghFIX (Snyder et al, 1999). These results have culminatedin the first clinical trial in humans that shows promisingresults although only low level hFIX expression has so farbeen observed (Kay et al, 2000). Successful delivery andexpression of therapeutic hFIX without formation ofantibodies has been achieved following administration ofretroviral vectors in neonatal animal models (Xu et al,2003). Prenatal gene therapy could be applied to the fetusvia a number of routes including muscle, peritoneal,hepatic, intravascular or skin application. More recentlyour group has demonstrated that in utero application canprovide long-term postnatal correction of the haemophiliacphenotype in FIX deficient mice (Waddington et al,submitted). Prenatal diagnosis is available early inpregnancy (Ljung, 1999).G. Haematopoietic diseases1. The thalassaemiasThe thalassaemias are inherited anaemias caused byover 200 mutations and globally are the commonestmonogenic disorders. They are most prevalent in theMediterranean region, the Middle East, the Indiansubcontinent and South-East Asia where gene frequenciesreach 3-10% of the population (Weatherall and Clegg,1996). β-thalassaemia is characterized by insufficientproduction of the β-globin peptide by erythroid cellswhich results in low levels of the major form of adulthaemoglobin, HbA, made up of two α- and two β-globinchains. The excess α-globin chains then precipitate in theerythroid cells, impair their maturation and this leads tohaemolysis and anaemia. Homozygotes or compoundheterozygotes suffer with the most severe form of thedisease, β-thalassaemia major. Similarly α-thalassaemiaresults in excess β-globin chains due to different degreesof α-globin chain deficiency. In the most severe form, α o -thalassaemia, all four α-globin chains are defective orabsent which leads to hydrops fetalis and intrauterinedeath. Patients with thalassaemia require regular lifelongblood transfusions to survive although this leads to ironoverload that affects the liver, heart and endocrine organs.Prevention of iron overload with iron-chelating therapysuch as parenteral deferoxamine is the mainstay of currentpatient management. Therapies aimed to increase theproduction of fetal haemoglobin have had disappointingresults (Olivieri and Weatherall, 1998). Allogeneichaematopoietic stem cell replacement offers the onlydefinitive cure and has been successful in over 1000patients worldwide (Olivieri, 1999). Outcomes depend onwhether the patient has hepatomegaly, portal fibrosis andhas effective chelating therapy before transplantation. The3 year disease-free survival falls from over 90% to 60% inchildren with the above risk factors.Gene therapy approaches have aimed to stablyintroduce a regulated human globin gene intohaemopoietic stem cells. Recently high expression oferythropoietin was found to improve the anaemia of β-thalassaemia in a mouse model by induction of high levelsof HbF synthesis (Johnston et al, 2003). Expression oftransgenic globin sequences would need to be sustained,finely regulated and at high levels since haemoglobinsynthesis represents 95% of all protein synthesis inreticulocytes. Initial attempts at gene therapy using the β-globin gene and a minimal locus control region (LCR)incorporated into a retroviral vector showed low levels andshort-term expression of β-globin after transplantation oftransduced haematopoietic stem cells into lethallyirradiated mice (Raftopoulos et al, 1997; Sadelain 2002).More recently lentiviral vectors containing the β-globingene and larger LCR elements have been used to transfectbone marrow from β-thalassaemic mice. This was thentransplanted into β o -thalassaemic heterozygote mice andresulted in therapeutically relevant levels of circulatinghaemoglobin (May et al, 2000). An advantage of prenatalgene therapy application in this context could be theaccess to rapidly dividing stem cell populations. Prenataldiagnosis for haemoglobinopathies can be done byassessment of globin-chain synthesis in fetal blood or bydirect analysis of fetal DNA obtained by chorionic-villussampling or amniocentesis.Sickle cell disease, another inherited disorder ofhaemoglobin may also be amenable to prenatal genetherapy. In this condition missense mutations in the β-185