Current Stem Cell Reports

, Volume 4, Issue 1, pp 52–60 | Cite as

In Utero Gene Therapy and Genome Editing

  • Heather A. Hartman
  • Avery C. Rossidis
  • William H. PeranteauEmail author
Prenatal Therapies (WH Peranteau, Section Editor)
Part of the following topical collections:
  1. Topical Collection on Prenatal Therapies


Purpose of Review

The purpose of this review is to summarize the current status of in utero gene therapy and postnatal in vivo genome editing with an eye toward prenatal genome editing.

Recent Findings

The rational for gene therapy and genome editing in utero is described, specifically the small size of the fetus, fetal immunologic immaturity, prenatal accessibility of stem and progenitor cells, and the ability to treat target diseases prior to birth. We review studies in normal and disease small and large animal models which demonstrate the feasibility and safety of in utero gene therapy using a variety of viral vectors. Postnatal in vivo genome editing with CRISPR-Cas9 in a number of murine disease models is discussed including the preference for nonhomologous end-joining compared to homology-directed repair in non-proliferating adult cells as a potential benefit to application of CRISPR-Cas9 genome editing to the fetus. Finally, the ethical challenges of human in utero gene therapy are discussed in the context of the EVERREST trial that is currently being designed.


In utero gene therapy and genome editing is a developing field with great potential to treat congenital monogenic diseases. More research in small and large animal models is required before clinical translation can occur.


Gene therapy Gene editing CRISPR-Cas9 Fetus In utero 


Compliance with Ethical Standards

Conflict of Interest

Heather A. Hartman, Avery C. Rossidis, and William H. Peranteau declare that they have no conflict of interest.

Human and Animal Rights and Informed Consent

This article does not contain any studies with human or animal subjects performed by any of the authors.


Papers of particular interest, published recently, have been highlighted as: • Of importance •• Of major importance

  1. 1.
    Naldini L. Gene therapy returns to centre stage. Nature. 2015;526(7573):351–60. Scholar
  2. 2.
    Maeder ML, Gersbach CA. Genome-editing technologies for gene and cell therapy. Mol Ther. 2016;24(3):430–46. Scholar
  3. 3.
    Dilworth MR, Kusinski LC, Baker BC, Renshall LJ, Greenwood SL, Sibley CP, et al. Defining fetal growth restriction in mice: a standardized and clinically relevant approach. Placenta. 2011;32(11):914–6. Scholar
  4. 4.
    Owen RD. Immunogentic consequences of vascular anastomoses between bovine twins. Science. 1945;102(2651):400–1. Scholar
  5. 5.
    Witt R, MacKenzie TC, Peranteau WH. Fetal stem cell and gene therapy. Semin Fetal Neonatal Med. 2017;22(6):410–4. Scholar
  6. 6.
    Billingham RE, Brent L, Medawar PB. Actively acquired tolerance of foreign cells. Nature. 1953;172(4379):603–6.CrossRefPubMedGoogle Scholar
  7. 7.
    Simonsen M. The acquired immunity concept in kidney homotransplantation. Ann N Y Acad Sci. 1955;59(3):448–52. Scholar
  8. 8.
    Calcedo R, Griesenbach U, Dorgan DJ, Soussi S, Boyd AC, Davies JC, et al. Self-reactive CFTR T cells in humans: implications for gene therapy. Hum Gene Ther Clin Dev. 2013;24(3):108–15. Scholar
  9. 9.
    Mingozzi F, Maus MV, Hui DJ, Sabatino DE, Murphy SL, Rasko JE, et al. CD8(+) T-cell responses to adeno-associated virus capsid in humans. Nat Med. 2007;13(4):419–22. Scholar
  10. 10.
    Calcedo R, Morizono H, Wang L, McCarter R, He J, Jones D, et al. Adeno-associated virus antibody profiles in newborns, children, and adolescents. Clin Vaccine Immunol. 2011;18(9):1586–8. Scholar
  11. 11.
    Wang D, Mou H, Li S, Li Y, Hough S, Tran K, et al. Adenovirus-mediated somatic genome editing of Pten by CRISPR/Cas9 in mouse liver in spite of Cas9-specific immune responses. Hum Gene Ther. 2015;26(7):432–42. Scholar
  12. 12.
    Sabatino DE, Mackenzie TC, Peranteau W, Edmonson S, Campagnoli C, Liu YL, et al. Persistent expression of hF.IX after tolerance induction by in utero or neonatal administration of AAV-1-F.IX in hemophilia B mice. Mol Ther. 2007;15(9):1677–85. Scholar
  13. 13.
    Davey MG, Riley JS, Andrews A, Tyminski A, Limberis M, Pogoriler JE, et al. Induction of immune tolerance to foreign protein via adeno-associated viral vector gene transfer in mid-gestation fetal sheep. PLoS One. 2017;12(1):e0171132. Scholar
  14. 14.
    Colletti E, Lindstedt S, Park PJ, Almeida-Porada G, Porada CD. Early fetal gene delivery utilizes both central and peripheral mechanisms of tolerance induction. Exp Hematol. 2008;36(7):816–22. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Tran ND, Porada CD, Almeida-Porada G, Glimp HA, Anderson WF, Zanjani ED. Induction of stable prenatal tolerance to beta-galactosidase by in utero gene transfer into preimmune sheep fetuses. Blood. 2001;97(11):3417–23. Scholar
  16. 16.
    Meertens L, Zhao Y, Rosic-Kablar S, Li L, Chan K, Dobson H, et al. 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. 2002;13(15):1809–20. Scholar
  17. 17.
    Garrett DJ, Larson JE, Dunn D, Marrero L, Cohen JC. In utero recombinant adeno-associated virus gene transfer in mice, rats, and primates. BMC Biotechnol. 2003;3(1):16. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Yin H, Kauffman KJ, Anderson DG. Delivery technologies for genome editing. Nat Rev Drug Discov. 2017;16(6):387–99. Scholar
  19. 19.
    Potter H, Heller R. Transfection by electroporation. Curr Protoc Immunol. 2017;117:10.5.1–9. Scholar
  20. 20.
    Han X, Liu Z, Jo MC, Zhang K, Li Y, Zeng Z, et al. CRISPR-Cas9 delivery to hard-to-transfect cells via membrane deformation. Sci Adv. 2015;1(7):e1500454. Scholar
  21. 21.
    Wu Y, Liang D, Wang Y, Bai M, Tang W, Bao S, et al. Correction of a genetic disease in mouse via use of CRISPR-Cas9. Cell Stem Cell. 2013;13(6):659–62. Scholar
  22. 22.
    •• Ma H, Marti-Gutierrez N, Park SW, Wu J, Lee Y, Suzuki K, et al. Correction of a pathogenic gene mutation in human embryos. Nature. 2017;548(7668):413–9. This is the first described in vitro CRISPR-Cas9 homology-directed repair of human embryonic cells with correction in all cells.CrossRefPubMedGoogle Scholar
  23. 23.
    Zuris JA, Thompson DB, Shu Y, Guilinger JP, Bessen JL, Hu JH, et al. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo. Nat Biotechnol. 2015;33(1):73–80. Scholar
  24. 24.
    Ahi YS, Bangari DS, Mittal SK. Adenoviral vector immunity: its implications and circumvention strategies. Curr Gene Ther. 2011;11(4):307–20. Scholar
  25. 25.
    Zincarelli C, Soltys S, Rengo G, Rabinowitz JE. Analysis of AAV serotypes 1-9 mediated gene expression and tropism in mice after systemic injection. Mol Ther. 2008;16(6):1073–80. Scholar
  26. 26.
    Lisowski L, Tay SS, Alexander IE. Adeno-associated virus serotypes for gene therapeutics. Curr Opin Pharmacol. 2015;24:59–67. Scholar
  27. 27.
    Majowicz A, Salas D, Zabaleta N, Rodriguez-Garcia E, Gonzalez-Aseguinolaza G, Petry H, et al. Successful repeated hepatic gene delivery in mice and non-human primates achieved by sequential administration of AAV5ch and AAV1. Mol Ther. 2017;25(8):1831–42. Scholar
  28. 28.
    Mattar CN, Nathwani AC, Waddington SN, Dighe N, Kaeppel C, Nowrouzi A, et al. Stable human FIX expression after 0.9G intrauterine gene transfer of self-complementary adeno-associated viral vector 5 and 8 in macaques. Mol Ther. 2011;19(11):1950–60. Scholar
  29. 29.
    Sugano H, Matsumoto T, Miyake K, Watanabe A, Iijima O, Migita M, et al. Successful gene therapy in utero for lethal murine hypophosphatasia. Hum Gene Ther. 2012;23(4):399–406. Scholar
  30. 30.
    Dejneka NS, Surace EM, Aleman TS, Cideciyan AV, Lyubarsky A, Savchenko A, et al. In utero gene therapy rescues vision in a murine model of congenital blindness. Mol Ther. 2004;9(2):182–8. Scholar
  31. 31.
    Endo M, Henriques-Coelho T, Zoltick PW, Stitelman DH, Peranteau WH, Radu A, et al. The developmental stage determines the distribution and duration of gene expression after early intra-amniotic gene transfer using lentiviral vectors. Gene Ther. 2010;17(1):61–71. Scholar
  32. 32.
    Joyeux L, Danzer E, Limberis MP, Zoltick PW, Radu A, Flake AW, et al. In utero lung gene transfer using adeno-associated viral and lentiviral vectors in mice. Hum Gene Ther Methods. 2014;25(3):197–205. Scholar
  33. 33.
    Stitelman DH, Brazelton T, Bora A, Traas J, Merianos D, Limberis M, et al. Developmental stage determines efficiency of gene transfer to muscle satellite cells by in utero delivery of adeno-associated virus vector serotype 2/9. Mol Ther Methods Clin Dev. 2014;1:14040. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    • Mattar CNZ, Gil-Farina I, Rosales C, Johana N, Tan YYW, McIntosh J, et al. In utero transfer of adeno-associated viral vectors produces long-term factor IX levels in a cynomolgus macaque model. Mol Ther. 2017;25(8):1843–53. In utero primate gene therapy for hemophilia results in long term therapeutic human factor IX gene expression. CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Shen JS, Meng XL, Yokoo T, Sakurai K, Watabe K, Ohashi T, et al. Widespread and highly persistent gene transfer to the CNS by retrovirus vector in utero: implication for gene therapy to Krabbe disease. J Gene Med. 2005;7(5):540–51. Scholar
  36. 36.
    Reay DP, Bilbao R, Koppanati BM, Cai L, O'Day TL, Jiang Z, et al. Full-length dystrophin gene transfer to the mdx mouse in utero. Gene Ther. 2008;15(7):531–6. Scholar
  37. 37.
    Abi-Nader KN, David AL. Fetal muscle gene therapy/gene delivery in large animals. Methods Mol Biol. 2011;709:239–56. Scholar
  38. 38.
    Lin S, Staahl BT, Alla RK, Doudna JA. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. elife. 2014;3:e04766. PubMedPubMedCentralGoogle Scholar
  39. 39.
    Roybal JL, Endo M, Radu A, Gray L, Todorow CA, Zoltick PW, et al. Early gestational gene transfer with targeted ATP7B expression in the liver improves phenotype in a murine model of Wilson’s disease. Gene Ther. 2012;19(11):1085–94. Scholar
  40. 40.
    Han XD, Lin C, Chang J, Sadelain M, Kan YW. Fetal gene therapy of alpha-thalassemia in a mouse model. Proc Natl Acad Sci U S A. 2007;104(21):9007–11. Scholar
  41. 41.
    Endo M, Zoltick PW, Radu A, Jiang Q, Qiujie J, Matsui C, et al. Early intra-amniotic gene transfer using lentiviral vector improves skin blistering phenotype in a murine model of Herlitz junctional epidermolysis bullosa. Gene Ther. 2012;19(5):561–9. Scholar
  42. 42.
    Waddington SN, Buckley SM, Nivsarkar M, Jezzard S, Schneider H, Dahse T, et al. In utero gene transfer of human factor IX to fetal mice can induce postnatal tolerance of the exogenous clotting factor. Blood. 2003;101(4):1359–66. Scholar
  43. 43.
    David A, Cook T, Waddington S, Peebles D, Nivsarkar M, Knapton H, et al. Ultrasound-guided percutaneous delivery of adenoviral vectors encoding the beta-galactosidase and human factor IX genes to early gestation fetal sheep in utero. Hum Gene Ther. 2003;14(4):353–64. Scholar
  44. 44.
    Keswani SG, Balaji S, Katz AB, King A, Omar K, Habli M, et al. Intraplacental gene therapy with Ad-IGF-1 corrects naturally occurring rabbit model of intrauterine growth restriction. Hum Gene Ther. 2015;26(3):172–82. Scholar
  45. 45.
    Carr DJ, Wallace JM, Aitken RP, Milne JS, Martin JF, Zachary IC, et al. Peri- and postnatal effects of prenatal adenoviral VEGF gene therapy in growth-restricted sheep. Biol Reprod. 2016;94(6):142. CrossRefPubMedGoogle Scholar
  46. 46.
    Carr DJ, Wallace JM, Aitken RP, Milne JS, Mehta V, Martin JF, et al. Uteroplacental adenovirus vascular endothelial growth factor gene therapy increases fetal growth velocity in growth-restricted sheep pregnancies. Hum Gene Ther. 2014;25(4):375–84. Scholar
  47. 47.
    • Sheppard M, Spencer RN, Ashcroft R, David AL, Consortium E. Ethics and social acceptability of a proposed clinical trial using maternal gene therapy to treat severe early-onset fetal growth restriction. Ultrasound Obstet Gynecol. 2016;47(4):484–91. This paper explores ethical considerations for the implementation of the EVERREST trial.CrossRefPubMedGoogle Scholar
  48. 48.
    Krishnan T, David AL. Placenta-directed gene therapy for fetal growth restriction. Semin Fetal Neonatal Med. 2017;22(6):415–22. Scholar
  49. 49.
    Lieber MR, Gu J, Lu H, Shimazaki N, Tsai AG. Nonhomologous DNA end joining (NHEJ) and chromosomal translocations in humans. Subcell Biochem. 2010;50:279–96. Scholar
  50. 50.
    Heyer WD, Ehmsen KT, Liu J. Regulation of homologous recombination in eukaryotes. Annu Rev Genet. 2010;44(1):113–39. Scholar
  51. 51.
    Rosen LE, Morrison HA, Masri S, Brown MJ, Springstubb B, Sussman D, et al. Homing endonuclease I-CreI derivatives with novel DNA target specificities. Nucleic Acids Res. 2006;34(17):4791–800. Scholar
  52. 52.
    Grizot S, Smith J, Daboussi F, Prieto J, Redondo P, Merino N, et al. Efficient targeting of a SCID gene by an engineered single-chain homing endonuclease. Nucleic Acids Res. 2009;37(16):5405–19. Scholar
  53. 53.
    Arnould S, Perez C, Cabaniols JP, Smith J, Gouble A, Grizot S, et al. Engineered I-CreI derivatives cleaving sequences from the human XPC gene can induce highly efficient gene correction in mammalian cells. J Mol Biol. 2007;371(1):49–65. Scholar
  54. 54.
    Wang J, Exline CM, DeClercq JJ, Llewellyn GN, Hayward SB, Li PW, et al. Homology-driven genome editing in hematopoietic stem and progenitor cells using ZFN mRNA and AAV6 donors. Nat Biotechnol. 2015;33(12):1256–63. Scholar
  55. 55.
    Modares M, Shariati L, Hejazi Z, Shahbazi M, Tabatabaiefar MA, Khanahmad H. Inducing indel mutation in the SOX6 gene by zinc finger nuclease for gamma reactivation: an approach towards gene therapy of Beta thalassemia. J Cell Biochem. 2017;
  56. 56.
    Sorek R, Kunin V, Hugenholtz P. CRISPR--a widespread system that provides acquired resistance against phages in bacteria and archaea. Nat Rev Microbiol. 2008;6(3):181–6. Scholar
  57. 57.
    Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816–21. Scholar
  58. 58.
    Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J. GRNA-programmed genome editing in human cells. elife. 2013;2:e00471. CrossRefPubMedPubMedCentralGoogle Scholar
  59. 59.
    Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science. 2013;339(6121):823–6. Scholar
  60. 60.
    Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339(6121):819–23. Scholar
  61. 61.
    Kemaladewi DU, Maino E, Hyatt E, et al. Correction of a splicing defect in a mouse model of congenital muscular dystrophy type 1A using a homology-directed-repair-independent mechanism. Nat Med. 2017;
  62. 62.
    Tabebordbar M, Zhu K, Cheng JKW, Chew WL, Widrick JJ, Yan WX, et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science. 2016;351(6271):407–11. Scholar
  63. 63.
    Nelson CE, Hakim CH, Ousterout DG, Thakore PI, Moreb EA, Castellanos Rivera RM, et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science. 2016;351(6271):403–7. Scholar
  64. 64.
    Ouellet DL, Cherif K, Rousseau J, Tremblay JP. Deletion of the GAA repeats from the human frataxin gene using the CRISPR-Cas9 system in YG8R-derived cells and mouse models of Friedreich ataxia. Gene Ther. 2017;24(5):265–74. Scholar
  65. 65.
    • Yin H, Xue W, Chen S, Bogorad RL, Benedetti E, Grompe M, et al. Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat Biotechnol. 2014;32(6):551–3. This is one of the first in vivo studies using CRISPR-Cas9 to demonstrate disease model correction postnatally in a murine model of hereditary tyrosinemia, an otherwise fatal disease.CrossRefPubMedPubMedCentralGoogle Scholar
  66. 66.
    Ah Mew N, Krivitzky L, McCarter R, Batshaw M, Tuchman M. Network UCDCotRDCR. Clinical outcomes of neonatal onset proximal versus distal urea cycle disorders do not differ. J Pediatr. 2013;162(2):324–9.e1. Scholar
  67. 67.
    Hodges PE, Rosenberg LE. The spfash mouse: a missense mutation in the ornithine transcarbamylase gene also causes aberrant mRNA splicing. Proc Natl Acad Sci U S A. 1989;86(11):4142–6. Scholar
  68. 68.
    Yang Y, Wang L, Bell P, McMenamin D, He Z, White J, et al. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat Biotechnol. 2016;34(3):334–8. Scholar
  69. 69.
    Bennett J. Taking stock of retinal gene therapy: looking back and moving forward. Mol Ther. 2017;25(5):1076–94. Scholar
  70. 70.
    George LA, Fogarty PF. Gene therapy for hemophilia: past, present and future. Semin Hematol. 2016;53(1):46–54. Scholar
  71. 71.
    Porada CD, Park PJ, Tellez J, Ozturk F, Glimp HA, Almeida-Porada G, et al. Male germ-line cells are at risk following direct-injection retroviral-mediated gene transfer in utero. Mol Ther. 2005;12(4):754–62. Scholar
  72. 72.
    Park PJ, Colletti E, Ozturk F, Wood JA, Tellez J, Almeida-Porada G, et al. Factors determining the risk of inadvertent retroviral transduction of male germ cells after in utero gene transfer in sheep. Hum Gene Ther. 2009;20(3):201–15. Scholar

Copyright information

© Springer International Publishing AG, part of Springer Nature 2018

Authors and Affiliations

  • Heather A. Hartman
    • 1
  • Avery C. Rossidis
    • 1
  • William H. Peranteau
    • 1
    Email author
  1. 1.The Center for Fetal ResearchThe Children’s Hospital of PhiladelphiaPhiladelphiaUSA

Personalised recommendations