GTMB 7 - Gene Therapy & Molecular Biology

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David et al: Current status and future direction of fetal gene therapymodel (Ponder et al, 2002). Some aspects of bone diseasewere not prevented however, which may be due toabnormal bone formation in utero. There was also concernthat systemic gene therapy administration may not reachthe brain even in neonatal dogs when the blood-brainbarrier is still forming. The immature blood-brain andblood –cerebrospinal fluid (CSF) barrier is morepermeable to small proteins than in mature brains andthere is a developmentally regulated mechanism thatselectively transfers some larger proteins from the blood tothe CSF (Dziegielewska et al, 2001). Thus a prenatal genetransfer approach may be more effective and alsoapplicable to other disorders that affect the brain, such asthe glycosphingolipid lysosomal storage diseases (Gaucherand Tay-Sachs disease) (Jeyakumar et al, 2002).D. Muscular dystrophiesDuchenne muscular dystrophy (DMD) is thecommonest form of muscular dystrophy, a group ofcongenital disorders characterised by muscle wasting andweakness. This X-linked recessive disease has anincidence of 1 in 3500 live male births. Affected boys areusually diagnosed aged 3-4 years and characteristically,skeletal muscle degeneration after repeated rounds ofnecrosis is followed by the onset of fibrosis that eventuallyleads to muscle weakness and death (Emery, 1993).Patients are usually confined to a wheelchair by age 11years, and although improved nursing care and positivepressure ventilation to aid breathing allows some patientsto reach the 3rd decade, respiratory or cardiac failure is thecommon cause of death (Simonds et al, 2000). Prenataldiagnosis is available for almost all muscular dystrophiesincluding Duchenne (Emery, 2002). Current treatmentincludes supportive measures such as surgery forcorrection of contractures and prevention of respiratoryinfections. The disease is caused by mutations in the DMDgene that encodes the 427kDA protein dystrophin,associated with the sarcolemma in muscle. Skeletal andcardiac muscle biopsies from DMD patients arecharacterized by absent or abnormal dystrophin. Genetransfer into muscle cells has been explored usingnaturally occurring animal models of muscular dystrophythat involve mutations in the DMD gene (Wells and Wells,2000). The large size of dystrophin cDNA (14kb)precludes insertion into conventional vectors with theexception of gutless adenovirus. Consequently themajority of viral constructs incorporate mini ormicrodystrophin cassettes based on a 6.3kb truncateddystrophin gene resulting from a large inframe deletion inthe rod domain which was isolated from a Beckermuscular dystrophy patient with very mild symptoms.Adenoviral transfer of minidystrophin results in goodtransduction of neonatal mdx mouse muscle with reduceddegeneration and improved muscle mechanics (Deconincket al, 1996; Vincent et al, 1993). In the neonatal and adultmdx mouse, injection of an adeno-associated viruscontaining a minidystrophin into the leg muscle led tonormal myofiber histology and protected membraneintegrity (Wang B et al, 2000). The early onset of thisdisease, which begins to be visible histologically by the18th-20th week of gestation (Vassilopoulos and Emery,1977; Turkel et al, 1981) and presents clinically between2-4 years of age, complicates postnatal gene therapy. Thusa prenatal approach to treatment might prevent the diseaseprocess.Prenatal gene transfer may offer advantages overneonatal or adult treatment. Efficient gene delivery toseveral affected muscles groups is technically difficult andthe alternative may be efficient gene transfer to a largepercentage of existing and rapidly expanding muscle cellsin utero. Postnatal gene delivery is also complicated by therisk of cellular immune responses against the transgenicproteins as demonstrated in the dystrophin-deficient mdxmouse model by loss of transgenic dystrophin-expressingfibres following dystrophin gene transfer (Wells andWells, 2000; Chamberlain 2002). In contrast in utero genetransfer may avoid the development of immune reactionsto the vector or transgene product and enable repeatinjection postnatally. Furthermore immune responses havebeen reported in several adenovirus-mediated genetransfer studies although it was not possible to determinethe relative contribution of the immune response to thevector or transgene. In most DMD patients, there is a lackof dystrophin expression which could lead to a functionalcopy of the dystrophin protein being recognised as aforeign antigen. Gene transfer during fetal life could leadto immunological tolerance to the dystrophin or allowrepeated injection post-natally. Similar conditions such asthe congenital Emery-Dreifuss and Fukuyama musculardystrophies (Emery, 2002) could also potentially betreated using a prenatal gene transfer approach.E. Neurological disordersSpinal muscular atrophy (SMA) is one of the mostcommon inherited causes of childhood mortality, with anincidence of 1 in 10,000 live births. It is characterized byprogressive degeneration of alpha motor neurons withinthe spinal cord and results in proximal, symmetrical limband trunk muscle paralysis that leads to death (Crawfordand Pardo, 1996). SMA is caused by homozygous loss ormutation in the survival motor neuron gene 1 (SMN 1)which is telomeric. Humans and primates also have acentromeric copy called the SMN 2 gene but this fails toprovide sufficient full-length SMN protein to maintainmotor neurons. Evidence from family studies and animalmodels of SMA suggest that the number of copies of theSMN 2 gene may modify the severity of the disease. Genetherapy strategy would have to provide and express afunctional copy of the SMN gene in the relevant neuronalcells. Efficient expression of the SMN gene wasdemonstrated recently after adenovirus-mediated deliveryof the SMN gene to human primary fibroblasts from SMApatients in vitro (DiDonato et al, 2003). Intraspinal orintramuscular application of a vector targeting neuronalcells will be required for in vivo therapy and other diseasesrequiring this targeting include amyotrophic lateralsclerosis.Immunohistochemical analysis of normal fetal tissuehas demonstrated that the expression of SMN protein isrelatively high in skeletal muscle, heart and brain and184
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
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