Animal Models for Prenatal Gene Therapy: Choosing the Right Model

Part of the Methods in Molecular Biology book series (MIMB, volume 891)


Testing in animal models is an essential requirement during development of prenatal gene therapy for ­clinical application. Some information can be derived from cell lines or cultured fetal cells, such as the efficiency of gene transfer and the vector dose that might be required. Fetal tissues can also be maintained in culture for short periods of time and transduced ex vivo. Ultimately, however, the use of animals is unavoidable since in vivo experiments allow the length and level of transgene expression to be measured, and provide an assessment of the effect of the delivery procedure and the gene therapy on fetal and neonatal development. The choice of animal model is determined by the nature of the disease and characteristics of the animal, such as its size, lifespan, and immunology, the number of fetuses and their development, parturition, and the length of gestation and the placentation. The availability of a disease model is also critical. In this chapter, we discuss the various animal models that can be used and consider how their characteristics can affect the results obtained. The projection to human application and the regulatory hurdles are also presented.

Key words

Disease models Fetal size Fetal growth Fetal development Gestation length Lifespan Fetal immunity Immune responses Gene therapy Vectors Gene transfer Gene expression Fetal number Parturition Preterm birth Placenta Placentation Prenatal diagnosis 


  1. 1.
    Castillon N, Avril-Delplanque A, Coraux C et al (2004) Regeneration of a well-differentiated human airway surface epithelium by spheroid and lentivirus vector-transduced airway cells. J Gene Med 6:846–856PubMedCrossRefGoogle Scholar
  2. 2.
    Robinson V (2005) Finding alternatives: an overview of the 3Rs and the use of animals in research. Sch Sci Rev 87(319):1–4Google Scholar
  3. 3.
    David AL, Peebles DM, Gregory L et al (2006) Clinically applicable procedure for gene delivery to fetal gut by ultrasound-guided gastric injection: toward prenatal prevention of early-onset intestinal diseases. Hum Gene Ther 17:767–779PubMedCrossRefGoogle Scholar
  4. 4.
    Peebles D, Gregory LG, David A et al (2004) Widespread and efficient marker gene expression in the airway epithelia of fetal sheep after minimally invasive tracheal application of recombinant adenovirus in utero. Gene Ther 11:70–78PubMedCrossRefGoogle Scholar
  5. 5.
    Lim F-Y, Martin BG, Sena-Esteves M et al (2002) Adeno-associated virus (AAV)-mediated fetal gene transfer in respiratory epithelium and submucosal gland cells in human fetal tracheal organ culture. J Pediatr Surg 37:1051–1057PubMedCrossRefGoogle Scholar
  6. 6.
    Lim F-Y, Kobinger GP, Weiner DJ et al (2003) Human fetal trachea-SCID mouse xenografts: efficacy of vesicular stomatitis virus-G pseudotyped lentiviral-mediated gene transfer. J Pediatr Surg 38:834–839PubMedCrossRefGoogle Scholar
  7. 7.
    Dejneka NS, Surace EM, Aleman TS et al (2004) In utero gene therapy rescues vision in a murine model of congenital blindness. Mol Ther 9:182–188PubMedCrossRefGoogle Scholar
  8. 8.
    Williams ML, Coleman JE, Haire SE et al (2006) Lentiviral expression of retinal guanylate cyclase-1 (RetGC1) restores vision in an avian model of childhood blindness. PLoS Med 3:e201PubMedCrossRefGoogle Scholar
  9. 9.
    Seppen J, van der Rijt R, Looije N et al (2003) Long-term correction of bilirubin UDPglucuronyltransferase deficiency in rats by in utero lentiviral gene transfer. Mol Ther 8:593–599PubMedCrossRefGoogle Scholar
  10. 10.
    Waddington SN, Nivsarkar MS, Mistry AR et al (2004) Permanent phenotypic correction of hemophilia B in immunocompetent mice by prenatal gene therapy. Blood 104:2714–2721PubMedCrossRefGoogle Scholar
  11. 11.
    Karolewski BA, Wolfe JH (2006) Genetic correction of the fetal brain increases the lifespan of mice with the severe multisystemic disease mucopolysaccharidosis type VII. Mol Ther 14:14–24PubMedCrossRefGoogle Scholar
  12. 12.
    Wells DJ, Wells KE (2005) What do animal models have to tell us regarding Duchenne muscular dystrophy? Acta Myol 24:172–180PubMedGoogle Scholar
  13. 13.
    Arruda VR, Stedman HH, Nichols TC et al (2005) Regional intravascular delivery of AAV-2-FIX to skeletal muscle achieves long-term correction of hemophilia B in a large animal model. Blood 105:3458–3464PubMedCrossRefGoogle Scholar
  14. 14.
    Baldeschi C, Gache Y, Rattenholl A et al (2003) Genetic correction of canine dystrophic epidermolysis bullosa mediated by retroviral vectors. Hum Mol Genet 12:1897–1905PubMedCrossRefGoogle Scholar
  15. 15.
    Meertens L, Zhao Y, Rosic-Kablar S et al (2002) In utero injection of alpha-L-­iduronidase-carrying retrovirus in canine mucopolysaccharidosis type I: infection of ­multiple tissues and neonatal gene expression. Hum Gene Ther 13:1809–1820PubMedCrossRefGoogle Scholar
  16. 16.
    Leipprandt JR, Kraemer SA, Haithcock BE et al (1996) Caprine β-mannosidase: sequencing and characterization of the cDNA and identification of the molecular defect of caprine β-mannosidosis. Genomics 37:51–56PubMedCrossRefGoogle Scholar
  17. 17.
    Porada CD, Sanada C, Long CR et al (2010) Clinical and molecular characterization of a ­re-established line of sheep exhibiting hemophilia A. J Thromb Haemost 8:276–285PubMedCrossRefGoogle Scholar
  18. 18.
    Tessanne K, Long C, Spencer T et al (2011) 337 production of transgenic sheep using recombinant lentivirus microinjection of in vivo produced embryos. Reprod Fertil Dev 23:264CrossRefGoogle Scholar
  19. 19.
    Christensen G, Minamisawa S, Gruber PJ et al (2000) High-efficiency, long-term cardiac expression of foreign genes in living mouse embryos and neonates. Circulation 101:178–184PubMedCrossRefGoogle Scholar
  20. 20.
    Lipshutz GS, Gruber CA, Cao Y et al (2001) In utero delivery of adeno-associated viral vectors: intraperitoneal gene transfer produces long-term expression. Mol Ther 3:284–292PubMedCrossRefGoogle Scholar
  21. 21.
    Lipshutz GS, Flebbe-Rehwaldt L, Gaensler KML (1999) Adenovirus-mediated gene transfer in the midgestation fetal mouse. J Surg Res 84:150–156PubMedCrossRefGoogle Scholar
  22. 22.
    Gregory LG, Waddington SN, Holder MV et al (2004) Highly efficient EIAV-mediated in utero gene transfer and expression in the major muscle groups affected by Duchenne muscular dystrophy. Gene Ther 11:1117–1125PubMedCrossRefGoogle Scholar
  23. 23.
    Waddington SN, Buckley SMK, Berloehr C et al (2004) Reduced toxicity of F-deficient Sendai virus vector in the mouse fetus. Gene Ther 11:599–608PubMedCrossRefGoogle Scholar
  24. 24.
    Endo M, Henriques-Coelho T, Zoltick PW et al (2010) The developmental stage determines the distribution and duration of gene expression after early intra-amniotic gene transfer using lentiviral vectors. Gene Ther 17:61–71PubMedCrossRefGoogle Scholar
  25. 25.
    David AL, McIntosh J, Peebles DM et al (2011) Recombinant adeno-associated virus-mediated in utero gene transfer gives therapeutic transgene expression in the sheep. Hum Gene Ther 22:419–426PubMedCrossRefGoogle Scholar
  26. 26.
    Themis M, Waddington SN, Schmidt M et al (2005) Oncogenesis following delivery of a nonprimate lentiviral gene therapy vector to fetal and neonatal mice. Mol Ther 12:763–771PubMedCrossRefGoogle Scholar
  27. 27.
    Sabatino DE, MacKenzie TC, Peranteau WH et al (2007) Persistent expression of hFIX after tolerance induction by in utero or neonatal administration of AAV-1-F.IX in hemophilia B mice. Mol Ther 15:1677–1685PubMedCrossRefGoogle Scholar
  28. 28.
    Billingham RE, Brent L, Medawar PB (1953) Actively acquired tolerance of foreign cells. Nature 172:603–606PubMedCrossRefGoogle Scholar
  29. 29.
    Billingham RE, Brent L, Medawar PB (1956) Quantitative studies on tissue transplantation immunity III Actively acquired tolerance. Phil Trans R Soc Lond B B239:357–369CrossRefGoogle Scholar
  30. 30.
    Howard JG, Michie D (1962) Induction of transplantation immunity in the newborn mouse. Transplant Bull 29:1–6PubMedCrossRefGoogle Scholar
  31. 31.
    Holladay SD, Smialowicz RJ (2000) Development of the murine and human immune system: differential effects of immunotoxicants depend on time of exposure. Environ Health Perspect 108:463–473PubMedGoogle Scholar
  32. 32.
    Morris B (1986) The ontogeny and comportment of lymphoid cells in fetal and neonatal sheep. Immunol Rev 91:219–233PubMedCrossRefGoogle Scholar
  33. 33.
    Schinkel PG, Ferguson KA (1953) Skin transplantation in the foetal lamb. Aust J Biol Sci 6:533Google Scholar
  34. 34.
    Silverstein AM, Prendergast RA, Kraner KL (1964) Fetal response to antigenic stimulus IV. Rejection of skin homografts by the fetal lamb. J Exp Med 119:955–964PubMedCrossRefGoogle Scholar
  35. 35.
    Neiderhuber JE, Shermeta D, Turcotte JG, Pikas PW (1971) Kidney transplantation in the foetal lamb. Transplantation 12:161–166CrossRefGoogle Scholar
  36. 36.
    McCullagh P (1988) Immunological tolerance of sheep to skin allografts. Transplantation 46:280–285PubMedCrossRefGoogle Scholar
  37. 37.
    McCullagh P (1989) Inability of fetal skin to induce allograft tolerance in fetal lambs. Immunology 67:489–495PubMedGoogle Scholar
  38. 38.
    Moore NW, Rowson LEA (1961) Attempts to induce tissue tolerance in sheep. Res Vet Sci 2:1Google Scholar
  39. 39.
    Mitchell RM (1959) Attempts to induce tolerance of renal homografts in sheep by intra-embryonic injection of spleen cells. Transplant Bulletin 6:424–426CrossRefGoogle Scholar
  40. 40.
    Nettleton PF (2000) Border disease. In: Martin WB, Aitken ID (eds) Diseases of sheep. Blackwell Science, Oxford, pp 95–102Google Scholar
  41. 41.
    McClure S, McCullagh P, Parsonson IM et al (1988) Maturation of immunological reactivity in the fetal lamb infected with Adabane virus. J Comp Pathol 99:133–143PubMedCrossRefGoogle Scholar
  42. 42.
    Silverstein AM, Uhr JW, Lukes RJ (1963) Fetal response to antigenic stimulus II. Antibody production by the fetal lamb. J Exp Med 117:799–812PubMedCrossRefGoogle Scholar
  43. 43.
    Silverstein AM, Thorbecke GJ, Kraner KL, Lukes RJ (1963) Fetal response to antigenic stimulus. III. Gamma-globulin production in normal and stimulated fetal lambs. J Immunol 91:384–395PubMedGoogle Scholar
  44. 44.
    Fahey KJ, Morris B (1978) Humoral immune responses in foetal sheep. Immunology 35:651–661PubMedGoogle Scholar
  45. 45.
    Fahey KJ, Morris B (1974) Lymphopoiesis and immune reactivity in the fetal lamb. Ser Haematol 7:548–567PubMedGoogle Scholar
  46. 46.
    Stites DP, Carr MC, Fudenberg HH (1974) Ontogeny of cellular immunity in the human fetus: development of responses to phytohemagglutinin and to allogeneic cells. Cell Immunol 11:257–271PubMedCrossRefGoogle Scholar
  47. 47.
    Toivanen P, Uksila J, Leino A et al (1981) Development of mitogen responding T cells and natural killer cells in the human fetus. Immunol Rev 57:89–105PubMedCrossRefGoogle Scholar
  48. 48.
    Velardi A, Cooper MD (1984) An immunofluorescence analysis of the ontogeny of myeloid, T, and B lineage cells in mouse hemopoietic tissues. J Immunol 133:672–677PubMedGoogle Scholar
  49. 49.
    Tyan ML, Herzenberg LA (1968) Studies on the ontogeny of the mouse immune system. II. Immunoglobulin-producing cells. J Immunol 101:446–450PubMedGoogle Scholar
  50. 50.
    Grossi CE, Velardi A, Cooper MD (1985) Postnatal liver hemopoiesis in mice: generation of pre-B cells, granulocytes, and erythrocytes in discrete colonies. J Immunol 135:2303–2311PubMedGoogle Scholar
  51. 51.
    Mestas J, Hughes CC (2004) Of mice and not men: differences between mouse and human immunology. J Immunol 172:2731–2738PubMedGoogle Scholar
  52. 52.
    Jerebtsova M, Batshaw ML, Ye X (2002) Humoral immune response to recombinant adenovirus and adeno-associated virus after in utero administration of viral vectors in mice. Pediatr Res 52:95–104PubMedCrossRefGoogle Scholar
  53. 53.
    Seppen J, van Til NP, van der Rijt R et al (2006) Immune response to lentiviral bilirubin UDP-glucuronosyltransferase gene transfer in fetal and neonatal rats. Gene Ther 13:672–677PubMedCrossRefGoogle Scholar
  54. 54.
    Merianos DJ, Tiblad E, Santore MT et al (2009) Maternal alloantibodies induce a postnatal immune response that limits engraftment following in utero hematopoietic cell transplantation in mice. J Clin Invest 119:2590–2600PubMedGoogle Scholar
  55. 55.
    Nijagal A, Wegorzewska M, Jarvis E et al (2011) Maternal T cells limit engraftment after in utero hematopoietic cell transplantation in mice. J Clin Invest. doi: 10.1172/JCI44907
  56. 56.
    Manno CS, Pierce GF, Arruda VR et al (2006) Successful transduction of liver in hemophilia by AAV-factor IX and limitations imposed by the host immune response. Nat Med 12:342–347PubMedCrossRefGoogle Scholar
  57. 57.
    Goldenberg RL, Culhane JF, Iams JD, Romero R (2008) Epidemiology and causes of preterm birth. Lancet 371:75–84PubMedCrossRefGoogle Scholar
  58. 58.
    Harrison MR, Keller RL, Hawgood SB et al (2003) A randomised trial of fetal endoscopic tracheal occlusion for severe fetal congenital diaphragmatic hernia. N Eng J Med 349:1916–1924CrossRefGoogle Scholar
  59. 59.
    Mitchell BF, Taggart MJ (2009) Are animal models relevant to key aspects of human parturition? Am J Physiol Regul Integr Comp Physiol 297:R525–R545PubMedCrossRefGoogle Scholar
  60. 60.
    Merlino AA, Welsh TN, Tan H et al (2007) Nuclear progesterone receptors in the human pregnancy myometrium: evidence that parturition involves functional progesterone withdrawal mediated by increased expression of progesterone receptor-A. J Clin Endocrinol Metab 92:1927–1933PubMedCrossRefGoogle Scholar
  61. 61.
    Kalkhoven E, Wissink S, van der Saag PT, van der Burg B (1996) Negative interaction between the RelA(p65) subunit of NF-kappaB and the progesterone receptor. J Biol Chem 271:6217–6224PubMedCrossRefGoogle Scholar
  62. 62.
    Goldenberg RL, Hauth JC, Andrews WW (2000) Intrauterine infection and preterm delivery. N Engl J Med 342:1500–1507PubMedCrossRefGoogle Scholar
  63. 63.
    Romero R (2006) The preterm parturition syndrome. BJOG 113:17–42PubMedGoogle Scholar
  64. 64.
    Fidel PI Jr, Romero R, Maymon E, Hertelendy F (1998) Bacteria-induced or bacterial product-induced preterm parturition in mice and rabbits is preceded by a significant fall in serum progesterone concentrations. J Matern Fetal Med 7:222–226PubMedCrossRefGoogle Scholar
  65. 65.
    Mitchell BF, Zielnik B, Wong S, Roberts CD, Mitchell JM (2005) Intraperitoneal infusion of proinflammatory cytokines does not cause activation of the rat uterus during late gestation. Am J Physiol Endocrinol Metab 289: E658–E664PubMedCrossRefGoogle Scholar
  66. 66.
    Baggia S, Gravett MG, Witkin SS, Haluska GJ, Novy MJ (1996) Interleukin-1 beta intra-amniotic infusion induces tumor necrosis ­factor-alpha, prostaglandin production, and preterm contractions in pregnant rhesus monkeys. J Soc Gynecol Investig 3:121–126PubMedCrossRefGoogle Scholar
  67. 67.
    Chwalisz K, Fahrenholz F, Hackenberg M, Garfield R, Elger W (1991) The progesterone antagonist onapristone increases the effectiveness of oxytocin to produce delivery without changing the myometrial oxytocin receptor concentrations. Am J Obstet Gynecol 165:1760–1770PubMedGoogle Scholar
  68. 68.
    Benirschke K, Kaufmann P (1990) Placental types in pathology of the human placenta. Springer, New YorkGoogle Scholar
  69. 69.
    Wooding FB (1992) Current topic: the synepitheliochorial placenta of ruminants: binucleate cell fusions and hormone production. Placenta 13:101–113PubMedCrossRefGoogle Scholar
  70. 70.
    Enders AC (1965) A comparative study of the fine structure of the trophoblast in several hemochorial placentas. Am J Anat 116:29–67PubMedCrossRefGoogle Scholar
  71. 71.
    Hamilton WJ, Boyd JD (1970) The human placenta. Heffer and Sons, CambridgeGoogle Scholar
  72. 72.
    Kaufmann P, Davidoff M (1977) The guinea pig placenta. Adv Anat Embryol Cell Biol 53:1–90Google Scholar
  73. 73.
    Scott VL, Burgess SC, Shack LA, Lockett NN, Coats KS (2008) Expression of CD134 and CXCR4 mRNA in term placentas from FIV-infected and control cats. Vet Immunol Immunopathol 123:90–96PubMedCrossRefGoogle Scholar
  74. 74.
    Bergelson JM (1997) Isolation of a common receptor for Coxsackie B viruses and adenoviruses 2 and 5. Science 275:1320–1323PubMedCrossRefGoogle Scholar
  75. 75.
    Koi H, Zhang J, Makrigiannakis A et al (2001) Differential expression of the coxsackievirus and adenovirus receptor regulates adenovirus infection of the placenta. Biol Reprod 64:1001–1009PubMedCrossRefGoogle Scholar
  76. 76.
    MacCalman CD, Furth EE, Omigbodun A et al (1996) Transduction of human trophoblast cells by recombinant adenoviruses is differentiation dependent. Biol Reprod 54:682–691PubMedCrossRefGoogle Scholar
  77. 77.
    Parry S, Holder J, Strauss JR (1997) Mechanisms of trophoblast-virus interaction. J Reprod Immunol 37:25–34PubMedCrossRefGoogle Scholar
  78. 78.
    Committee for Medicinal Products for Human Use (2006) Guideline on non-clinical testing for inadvertent germline transmission of gene transfer vectors (273974). European Medicines Agency, LondonGoogle Scholar
  79. 79.
    Committee for Medicinal Products for Human Use (2008) Guideline on the non-clinical studies required before first clinical use of gene therapy medicinal products (125459). European Medicines Agency, LondonGoogle Scholar
  80. 80.
    Siman CM, Sibley CP, Jones CJ et al (2001) The functional regeneration of syncytiotrophoblast in cultured explants of term placenta. Am J Physiol Regul Integr Comp Physiol 280:R1116–R1122PubMedGoogle Scholar
  81. 81.
    Crocker IP, Tansinda DM, Baker PN (2004) Altered cell kinetics in cultured placental villous explants in pregnancies complicated by pre-eclampsia and intrauterine growth restriction. J Pathol 204:11–18PubMedCrossRefGoogle Scholar
  82. 82.
    Brownbill P, Edwards D, Jones C et al (1995) Mechanisms of alphafetoprotein transfer in the perfused human placental cotyledon from uncomplicated pregnancy. J Clin Invest 96:2220–2226PubMedCrossRefGoogle Scholar
  83. 83.
    Brownbill P, Mills TA, Soydemir DF, Sibley CP (2008) Vasoactivity to and endogenous release of vascular endothelial growth factor in the in vitro perfused human placental lobule from pregnancies complicated by preeclampsia. Placenta 29:950–955PubMedCrossRefGoogle Scholar
  84. 84.
    Sibley CP, Birdsey TJ, Brownbill P et al (1998) Mechanisms of maternofetal exchange across the human placenta. Biochem Soc Trans 26:86–91PubMedGoogle Scholar
  85. 85.
    Wells D, Delhanty JD (2001) Preimplantation genetic diagnosis: applications for molecular medicine. Trends Mol Med 7:23–30PubMedCrossRefGoogle Scholar
  86. 86.
    Snowdon C, Green JM (1997) Preimplantation diagnosis and other reproductive options: attitudes of male and female carriers of recessive disorders. Hum Reprod 12:341–350PubMedCrossRefGoogle Scholar
  87. 87.
    Benirschke K, Kaufmann P, Baergen RN (2006) Pathology of the human placenta. Springer, New YorkGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC. 2012

Authors and Affiliations

  1. 1.Prenatal Cell and Gene Therapy Group, EGA Institute for Women’s HealthUniversity College LondonLondonUK

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