GTMB 7 - Gene Therapy & Molecular Biology

David et al: Current status and future direction of fetal gene therapyhepatocyte transduction however was observed afteradenoviral and retroviral mediated gene transfer into fetalsheep (David et al, 2003a) and primates (Tarantal et al,2001b).2. Intraperitoneal injectionIntraperitoneal injection has also been used forsuccessful gene transfer to multiple tissues including theliver in fetal mice (Lipshutz et al, 1999b, c) rats(Hatzoglou et al, 1990, 1995) and sheep (Tran et al, 2000).Persistent peritoneal expression was observed 18 monthsafter intraperitoneal injection of adeno-associated virusserotype 2 (AAV2) vectors containing the luciferase genein fetal mice (Lipshutz et al, 2001). Recent studies in thefetal mouse have shown that transgene expression couldbe increased by intraperitoneal injection of AAV5serotype vectors rather than AAV2 serotype vectors andby changing from the elongation factor 1α or CMVpromoter to the woodchuck hepatitis virusposttranscriptional regulatory element (Lipshutz et al,2003).In large animal models, retroviral vectors containingthe α-L-iduronidase gene were delivered by ultrasoundguided injection after exteriorisation of the uterus into theperitoneal cavity or yolk sac of mid-gestation fetal dogswith canine α-L-iduronidase deficiency (mucopolysaccharidosistype 1). Low level tissue transduction wasobserved but expression of the transgene did not persistbeyond the neonatal period (Meertens et al, 2002). In earlygestation fetal primates, ultrasound guided intraperitonealinjection of Moloney murine leukemia virus amphotrophicand vesicular stomatitis virus-G protein (VSV-G)pseudotyped retrovirus and VSV-G pseudotyped HIV-1lentiviral vectors resulted in only low level tissuetransduction (Tarantal et al, 2001b). In contrast long-termtransduction of hematopoietic stem cells in the bonemarrow and blood could be demonstrated 5 yearsfollowing delivery of retroviral vectors into the peritonealcavity of early gestation fetal sheep at laparotomy (Poradaet al, 1998). Delivery of adenoviral vectors containing thehFIX gene to early gestation fetal sheep by ultrasoundguided intraperitoneal injection had good fetal survival of77% and therapeutic hFIX production was achieved, albeittransiently (Figure 3) (David et al, 2003a).Immunohistochemical analysis after delivery of adenoviralvectors containing the lacZ gene showed positivetransgene expression on the surface of the umbilical cord,in the fetal small bowel serosa and in the hepatocytesbeneath the fetal liver capsule following intraperitonealinjection (Figure 4 A-C). The intraperitoneal route alsogave the most comprehensive spread of vector to fetaltissues as determined by PCR analysis but no vector wasdetectable by sensitive PCR analysis in the germline oflambs born after each route of administration (David et al,2003a).E. Intramuscular injectionThe main aim of intramuscular injection is to targetthe muscle for treatment of muscular dystrophies but thisroute may also be used for ectopic production of proteinssuch as hFIX in the treatment of haemophilias. In the fetalmouse, injection of adenoviral vectors containing the β-galactosidase gene into the shoulder or hindlimbmusculature resulted in persistent muscle and livertransgene expression for 16 and 8 weeks respectively afterinjection (Yang et al, 1999). Intramuscular injection oflentiviral vectors led to transduction of myocytes andcardiomyocytes indicating systemic spread of the virusfrom the site of injection (MacKenzie et al, 2002).Our group successfully achieved in vivo expressionof hFIX after injection of adenovirus and AAV hFIXvectors in adult and fetal mice (Schneider et al, 2002). Arecent study using EIAV lentivirus containing the lacZgene combined intrathoracic, supracostal, intraperitonealand intramuscular injection of three limbs and a singleflank in the fetal mouse. This resulted in widespread geneexpression in all injected muscles and also the diaphragmand heart which are the essential muscle groups to bereached for successful gene therapy of DMD (Gregory etal, 2003).Finally, delivery of adenoviral vectors into thehindlimb musculature by ultrasound guided injection hasbeen explored in one study in the early gestation fetalsheep. Fetal survival was 91% and therapeutic levels ofhFIX were also obtained after injection of adenovirushFIX vector (Figure 3).Figure 3. Time course of transgene expression after ultrasoundguided intraperitoneal, intramuscular, intrahepatic or intraamnioticdelivery of an adenoviral vector containing the humanfactor IX gene to early gestation sheep fetuses. Concentrations ofhuman factor IX in fetal or lamb plasma were determined byELISA analysis. Fetal samples were collected at post mortem(David et al 2003a). Republished with permission from MaryAnn Liebert Inc, Publishers.194