GTMB 7 - Gene Therapy & Molecular Biology

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David et al: Current status and future direction of fetal gene therapyglobin gene lead to haemoglobin polymerization causingthe red blood cells to become deformed or ‘sickled’. Theability of gene therapy to correct the pathophysiology hasbeen demonstrated in a study in transgenic sickle Hbmouse models. Bone marrow transduced with lentiviralvectors containing a β A globin gene variant that preventshaemoglobin polymerization was transplanted into twomouse sickle cell disease models resulting in therapeuticcorrection of the disease (Pawliuk et al, 2001).2. Immunodeficiency disordersThe greatest success of gene therapy so far has beenin the treatment of congenital severe combinedimmunodeficiency disorders (SCID). These represent themost severe form of primary immunodeficiencies and theyoccur in approximately 1 in 75,000 births. The mostcommon types of SCID are X-linked (Xl-SCID) and theautosomal recessive adenosine deaminase deficiency(ADA) found in 50% and 15% of sufferers respectively. Inboth conditions the genetic defect causes a profound blockin T cell differentiation which leads to absent T cell andhumoral responses. Xl-SCID is due to a deficiency of theγc chain, an essential component of cytokine receptorswhich is necessary for T cell and natural killer celldevelopment. In ADA deficiency there is selectiveaccumulation of the toxic metabolite deoxyATP in T cells.Clinically the patients present with chronic diarrhoea andfailure to thrive with recurrent respiratory andopportunitstic infections leading to death within the firstyear of life (Cavazzana-Calvo et al, 2001).Histocompatible bone marrow transplantation(BMT) has been used to treat both conditions with somesuccess. Survival after transplantation with HLA-identicalbone marrow is over 90% but matched sibling donors areusually not available. Haploidentical BMT with T-celldepletion is commonly performed instead, with survivalrates of up to 78% although many patients require lifelongimmunoglobulin replacement therapy because ofinadequate humoral activity (Buckley RH et al, 1999). Inutero haematopoietic stem cell transplantation has beenachieved in fetuses with Xl-SCID by ultrasound guidedintraperitoneal or intravenous injection (Flake et al, 1996;Touraine 1992; Wengler et al, 1996; Westgren et al,2002). A selective T-cell and natural killer cellreconstitution can be achieved but B cell engraftment hasnot been detected. In ADA deficiency, a long-circulatingform of bovine ADA conjugated with polyethylene glycol(PEG-ADA) has been used to correct the metabolicabnormalities and prevent life-threatening opportunisticinfections.The strategy for gene therapy of SCID is based onthe concept that genetically corrected autologous T-cellprecursors should have a selective survival advantage overnon-corrected cells. In addition, patients are unable tomount an effective immune response to the transgenewhich has proved to be a major problem in gene therapytreatment of other genetic diseases. In ADA-SCID, clinicaltrials have used infusion of autologous peripheral T-cells,CD34 + bone marrow or umbilical cord blood cellstransduced with a retroviral vector containing ADAcDNA. The earlier trials did not use conditioning of thebone marrow and PEG-ADA treatment was continued inall patients during and after treatment which made itdifficult to evaluate immune function (Blaese et al, 1995;Bordignon et al, 1995; Kohn et al, 1995). Some patientsshowed long term persistence of the transduced cellsalthough at low level. A more recent trial was performedin two infants with nonmyeloablative conditioning usingbusulfan and without concurrent PEG-ADA treatment.Both patients showed sustained engraftment of geneticallycorrected haematopoietic stem cells with differentiationinto multiple lineages and improvement in their clinicalcondition (Aiuti et al, 2002).In a similar way Xl-SCID has been treated usingautologous transplantation of CD34 + bone marrowtransduced ex vivo with retroviral vectors containing the γcgene. Fifteen patients have now been treated and effectiveimmune reconstitution has been achieved in thirteenpatients (Friedmann, 2003). Unfortunately because of aserious adverse event in two of the patients, all genetherapy trials involving retroviral vectors inhaematopoietic stem cells were initially halted in the US(Gansbacher and European Society of Gene Therapy2003) (see VI Ethical and safety issues) and have nowbeen restricted to case by case reviewed permission(Friedmann, 2003). Nevertheless this study has shown theability of gene therapy to cure such conditions. Because ofthe survival advantage of genetically corrected cells andthe ineffective immune response in SCID patients, it isunlikely that prenatal gene transfer would provide aparticular benefit over postnatal treatment of thiscondition.H. Skin disordersFetal gene delivery into the amniotic cavity mayhave unique benefits for treatment of inherited skindisorders. Epidermolysis bullosa is a group of inheritedblistering diseases characterized by epidermal-dermalseparation resulting from mutations that affect the functionof critical components of the basement membrane zone.The dystrophic form of epidermolysis bullosa (DEB) isdue to mutations in COL7A1, the gene encoding type VIIcollagen and has a prevalence of up to 2.4 per 100,000population (Horn and Tidman, 2002). The clinicalpresentation varies from a mild dominantly inheriteddisease characterized by skin and oral blisters and naildystrophy to a severe recessive subtype in which patientssuffer from contractures, severe dental caries, dysphagia,anal fissures and squamous cell carcinoma. Currenttherapy involves management of the diseasemanifestations with proper wound care, surgical release ofskin contractures, balloon dilatation of oesophagealstrictures and graft skin therapy (Pai and Marinkovich,2002).Easy accessibility and visualization of skin make itan attractive target for gene therapy. Gene delivery can bein vivo by direct introduction to the skin by injection,electroporation or a ‘gene gun’. Alternatively a skinsample could be removed from the patient, and epidermalkeratinocytes cultured and transduced ex vivo to insertgenetic material and the genetically engineered cells186
Gene Therapy and Molecular Biology Vol 7, page 187returned in the form of a skin graft (Uitto and Pulkkinen,2000). Preliminary studies show keratinocytes andfibroblasts from patients with DEB can be successfullytransduced using lentiviral vectors containing theCOL7A1 transgene in vitro resulting in long-termexpression and synthesis of type VII collagen (Chen et al,2002). In a canine animal model of DEB, transduction ofkeratinocytes with a retrovirus containing the collagentype VII cDNA corrected the observable defects in in vitroreconstructed skin (Baldeschi et al, 2003). A non-viralgene transfer approach has been used for junctionalepidermolysis bullosa (JEB) in which there is severelaminin-5 deficiency. Integration of an attB-containinglaminin 5 β3 expression plasmid using φC31 integrase intohuman keratinocytes from JEB patients produced skintissue with no histological evidence of subepidermalblistering when regenerated on SCID mice (Ortiz-Urda etal, 2003).Epidermolysis bullosa however, is a generalizeddisorder affecting the entire skin and the extracutaneoustissues. Prenatal therapy delivered into the amniotic fluidwould bathe the entire skin surface and reach thegastrointestinal system by fetal swallowing. Injection intothe amniotic cavity can be performed safely at relativelyearly gestation, but the timing of intra-amniotic deliverywill be important from developmental considerations.Even at 20 weeks gestation, the fetal epidermis isincompletely keratinized and this would aid gene tranfer.However there is a high rate of apotosis in fetalkeratinocytes and therefore the ideal strategy would be totarget stem cells (Haake and Cooklis, 1997). Prenataldiagnosis for epidermolysis bullosa can now be performedwith a 98% success rate in at risk families, paving the wayfor preliminary studies into prenatal treatment (Pfendner etal, 2003). Disorders of defective keratinisation such asharlequin ichthyosis, an autosomal recessive severe andusually fatal congenital ichthyosis (Akiyama, 1998), mayalso be amenable to prenatal gene transfer.I. Perinatal diseasePulmonary hypoplasia is another important cause ofneonatal morbidity and mortality. In this condition, thefetal lungs fail to develop resulting in respiratoryinsufficiency at birth. Current neonatal management issupportive and involves surfactant replacement, carefulmechanical ventilation avoiding barotrauma and treatmentof pulmonary hypertension. Pulmonary hypoplasia canoccur when there is reduced or no liquor surrounding thefetus (oligo or anhydramnios) prior to 22 weeks gestation,most commonly because of preterm premature rupture ofthe membranes (PPROM). Serial amnioinfusion has beenused for the prevention of pulmonary hypoplasia withsome success but has a high complication rate (Tan et al,2003). Space occupying lesions that compress the lungswithin the chest cavity also result in pulmonaryhypoplasia. Examples of such conditions include pleuraleffusion associated with congenital cardiac defects andcongenital diaphragmatic hernia (CDH) in which thebowel herniates through the diaphragmatic defect. Fetalinterventions such as drainage of pleural effusions can beused to treat the underlying cause of the pulmonaryhypoplasia. Temporary occlusion of the trachea with anexpandable balloon for treatment of CDH results inimpressive expansion of the hypoplastic lung with trachealfluid. However ‘plugging’ has yet to be shown to improveoutcome in the long term (Harrison et al, 1998). Studiessuggest that pulmonary hypoplasia in CDH begins duringembryogenesis as an abnormality in growth factorsignalling and actually precedes the development of theanatomical defect (Jesudason 2002). Prenatal gene therapycould be envisaged in the future to enhance antenatal lunggrowth and maturation by the targeted delivery of growthfactors at specific times during lung development.J. Infectious diseaseInfectious diseases with pathogens such as Group Bstreptococcus, human immunodeficiency virus, hepatitis Bvirus and herpes simplex virus are a major cause ofneonatal morbidity and mortality. Transmission of thesediseases from mother to infant often occurs shortly before,during, or after birth by early rupture of the amnioticmembranes or direct contact with infectious secretionsduring labor and delivery. Delivery by caesarean section toprevent such contact, and antibiotic and maternal antiviraltreatments have been used with some success, particularlyin the prevention of vertical HIV transmission.Immunisation of the fetus with DNA vaccines in latepregnancy has been proposed as an alternative approach toprevent neonatal infection (Gerdts et al, 2000; Sarzotti etal, 1996; Watts et al, 1999). The mucosal surfaces of theeyes, respiratory and gastrointestinal tract are the primarysite of entry for infectious agents during birth and theneonatal period. Thus intra-amniotic or intra-oral deliveryof antigen would probably provide the best diseaseprotection. Studies in the fetal mouse (Sarzotti et al, 1996),sheep (Gerdts, et al, 2000) and baboon (Watts et al, 1999)have shown that fetal immunisation can induce activeimmunity in the newborn. In particular, in the fetal sheep,intra-oral administration of hepatitis B surface antigenDNA resulted in a higher protective antibody titre than anintramuscular injection of the recombinant protein vaccine(Gerdts, et al, 2003). The timing of such an intervention iscrucial since exposure of the fetus to the antigen beforeimmune competence is reached may result in tolerance. Inaddition a single in utero injection may not be sufficient tomaintain immunity. At present there is no clinicalindication for such a prenatal immunization strategy.K. Placental disordersPre-eclampsia/eclampsia is one of the leading causesof maternal and fetal morbidity and mortality. Theunderlying defect is believed to be inadequate deepplacentation that fails to transform the spiral arteries intouteroplacental vessels and thus limits placental blood flow(Brosens et al, 2002). Secondary damage such as fibrindeposition and thrombosis then limit placental perfusionfurther and there is also widespread activation of thematernal vascular endothelium leading to decreasedformation of vasodilators such as nitric oxide (Walker,2000). Gene therapy could be used to improve uteroplacentalperfusion by for example, temporary expressionof nitric oxide synthase or placental growth factor. This187
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