Numerous pediatric neurogenetic diseases may be optimally treated by in utero gene therapy (IUGT); but advancing such treatments requires animal models that recapitulate developmental physiology relevant to humans. One disease that could benefit from IUGT is the autosomal recessive motor neuron disease spinal muscular atrophy (SMA). Current SMA gene-targeting therapeutics are more efficacious when delivered shortly after birth, however postnatal treatment is rarely curative in severely affected patients. IUGT may provide benefit for SMA patients. In previous studies, we developed a large animal porcine model of SMA using AAV9 to deliver a short hairpin RNA (shRNA) directed at porcine survival motor neuron gene (Smn) mRNA on postnatal day 5. Here, we aimed to model developmental features of SMA in fetal piglets and to demonstrate the feasibility of prenatal gene therapy by delivering AAV9-shSmn in utero. Saline (sham), AAV9-GFP, or AAV9-shSmn was injected under direct ultrasound guidance between gestational ages 77–110 days. We developed an ultrasound-guided technique to deliver virus under direct visualization to mimic the clinic setting. Saline injection was tolerated and resulted in viable, healthy piglets. Litter rejection occurred within seven days of AAV9 injection for all other rounds. Our real-world experience of in utero viral delivery followed by AAV9-related fetal rejection suggests that the domestic sow may not be a viable model system for preclinical in utero AAV9 gene therapy studies.
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Feldkamp ML, Carey JC, Byrne JLB, Krikov S, Botto LD. Etiology and clinical presentation of birth defects: population based study. BMJ. 2017;8108:j2249.
Liu L, Oza S, Hogan D, Perin J, Rudan I, Lawn JE, et al. Global, regional, and national causes of child mortality in 2000–13, with projections to inform post-2015 priorities: an updated systematic analysis. Lancet. 2015;385:430–40.
Palanki R, Peranteau WH, Mitchell MJ. Delivery technologies for in utero gene therapy. Adv Drug Delivery Rev. 2021;169:51–62.
Peranteau WH, Flake AW. The future of in utero gene therapy. Mol Diagn Ther. 2020;24:135–42.
Hocquemiller M, Giersch L, Audrain M, Parker S, Cartier N. Adeno-associated virus-based gene therapy for CNS diseases. Human Gene Ther. 2016;27:478–96.
Martier R, Konstantinova P. Gene therapy for neurodegenerative diseases: slowing down the ticking clock. Front Neurosci. 2020;14:580179.
Foust KD, Nurre E, Montgomery CL, Hernandez A, Chan CM, Kaspar BK. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol. 2009;27:59–65.
Vandamme C, Adjali O, Mingozzi F. Unraveling the complex story of immune responses to AAV vectors trial after trial. Human Gene Ther. 2017;28:1061–74.
Verdera HC, Kuranda K, Mingozzi F. AAV vector immunogenicity in humans: a long journey to successful gene transfer. Mol Ther. 2020;28:723–46.
Prior TW, Snyder PJ, Rink BD, Pearl DK, Pyatt RE, Mihal DC, et al. Newborn and carrier screening for spinal muscular atrophy. Am J Med Genet A. 2010;152A:1608–16.
Kolb SJ, Kissel JT. Spinal muscular atrophy. Neurol Clin. 2015;33:831–46.
Lefebvre S, Bürglen L, Reboullet S, Clermont O, Burlet P, Viollet L, et al. Identification and characterization of a spinal muscular atrophy-determining gene. Cell. 1995;80:155–65.
Finkel RS, Mercuri E, Darras BT, Connolly AM, Kuntz NL, Kirschner J, et al. Nusinersen versus sham control in infantile-onset spinal muscular atrophy. N Engl J Med. 2017;377:1723–32.
De Vivo DC, Bertini E, Swoboda KJ, Hwu W-L, Crawford TO, Finkel RS, et al. Nusinersen initiated in infants during the presymptomatic stage of spinal muscular atrophy: Interim efficacy and safety results from the Phase 2 NURTURE study. Neuromuscul Disord. 2019;29:842–56.
Mendell JR, Al-Zaidy S, Shell R, Arnold WD, Rodino-Klapac LR, Prior TW, et al. Single-dose gene-replacement therapy for spinal muscular atrophy. N Engl J Med. 2017;377:1713–22.
Kariya S, Park G-H, Maeno-Hikichi Y, Leykekhman O, Lutz C, Arkovitz MS, et al. Reduced SMN protein impairs maturation of the neuromuscular junctions in mouse models of spinal muscular atrophy. Hum Mol Genet. 2008;17:2552–69.
Murray LM, Comley LH, Thomson D, Parkinson N, Talbot K, Gillingwater TH. Selective vulnerability of motor neurons and dissociation of pre- and post-synaptic pathology at the neuromuscular junction in mouse models of spinal muscular atrophy. Hum Mol Genet. 2008;17:949–62.
Kong L, Wang X, Choe DW, Polley M, Burnett BG, Bosch-Marcé M, et al. Impaired synaptic vesicle release and immaturity of neuromuscular junctions in spinal muscular atrophy mice. J Neurosci. 2009;29:842–51.
Ling KKY, Lin M-Y, Zingg B, Feng Z, Ko C-P. Synaptic defects in the spinal and neuromuscular circuitry in a mouse model of spinal muscular atrophy. PLoS ONE. 2010;5:e15457.
Ruiz R, Casañas JJ, Torres-Benito L, Cano R, Tabares L. Altered intracellular Ca2+ homeostasis in nerve terminals of severe spinal muscular atrophy mice. J Neurosci. 2010;30:849–57.
Dachs E, Hereu M, Piedrafita L, Casanovas A, Calderó J, Esquerda JE. Defective neuromuscular junction organization and postnatal myogenesis in mice with severe spinal muscular atrophy. J Neuropathol Exp Neurol. 2011;70:444–61.
Kong L, Valdivia DO, Simon CM, Hassinan CW, Delestrée N, Ramos DM, et al. Impaired prenatal motor axon development necessitates early therapeutic intervention in severe SMA. Sci Transl Med. 2021;13:eabb6871.
Rashnonejad A, Amini Chermahini G, Gündüz C, Onay H, Aykut A, Durmaz B, et al. Fetal gene therapy using a single injection of recombinant AAV9 rescued SMA phenotype in mice. Mol Ther. 2019;27:2123–33.
Duque SI, Arnold WD, Odermatt P, Li X, Porensky PN, Schmelzer L, et al. A large animal model of spinal muscular atrophy and correction of phenotype. Ann Neurol. 2015;77:399–414.
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:e0171132.
David AL, McIntosh J, Peebles DM, Cook T, Waddington S, Weisz B, et al. Recombinant adeno-associated virus-mediated in utero gene transfer gives therapeutic transgene expression in the sheep. Human Gene Ther. 2011;22:419–26.
Conlon TJ, Mah CS, Pacak CA, Rucker Henninger MB, Erger KE, Jorgensen ML, et al. Transfer of therapeutic genes into fetal rhesus monkeys using recombinant adeno-associated type I viral vectors. Human Gene Ther Clin Dev 2016;27:152–9.
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;25:1843–53.
McCarty D, Monahan P, Samulski R. Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Ther. 2001;8:1248–54.
Foust KD, Wang X, McGovern VL, Braun L, Bevan AK, Haidet AM, et al. Rescue of the spinal muscular atrophy phenotype in a mouse model by early postnatal delivery of SMN. Nat Biotechnol. 2010;28:271–4.
Hadlock FP, Harrist RB, Martinez-Poyer J. In utero analysis of fetal growth: a sonographic weight standard. Radiology. 1991;181:129–33.
Hadlock FP, Harrist RB, Carpenter RJ, Deter RL, Park SK. Sonographic estimation of fetal weight. The value of femur length in addition to head and abdomen measurements. Radiology. 1984;150:535–40.
Gamble HJ. Further electron microscope studies of human foetal peripheral nerves. J Anat. 1966;100:487–502.
Langel SN, Paim FC, Alhamo MA, Buckley A, Van Geelen A, Lager KM, et al. Stage of gestation at porcine epidemic diarrhea virus infection of pregnant swine impacts maternal immunity and lactogenic immune protection of neonatal suckling piglets. Front Immunol. 2019;10:727.
Lozier JW, VanHoy GM, Jordan BA, Muir AJT, Lakritz J, Hinds CA, et al. Complications and outcomes of swine that underwent cesarean section for resolution of dystocia: 110 cases (2013‐2018). Vet Surg. 2021;50:38–43.
McClain LE, Davey MG, Zoltick PW, Limberis MP, Flake AW, Peranteau WH. Vector serotype screening for use in ovine perinatal lung gene therapy. J Pediatr Surg. 2016;51:879–84.
Chan YK, Wang SK, Chu CJ, Copland DA, Letizia AJ, Costa Verdera H, et al. Engineering adeno-associated viral vectors to evade innate immune and inflammatory responses. Sci Transl Med. 2021;13:eabd3438.
Mehta V, Peebles D, David AL. Animal models for prenatal gene therapy: choosing the right model. In: Coutelle C, Waddington SN, editors. Prenatal gene therapy. Totowa, NJ: Humana Press; 2012. p. 183–200. http://link.springer.com/10.1007/978-1-61779-873-3_9.
Engelhardt H, Croy BA, King GJ. Role of uterine immune cells in early pregnancy in pigs. J Reprod Fertil Suppl. 1997;52:115–31.
Moffett-King A. Natural killer cells and pregnancy. Nat Rev Immunol. 2002;2:656–63.
Tayade C, Black GP, Fang Y, Croy BA. Differential gene expression in endometrium, endometrial lymphocytes, and trophoblasts during successful and abortive embryo implantation. J Immunol. 2006;176:148–56.
Tayade C, Fang Y, Croy BA. A review of gene expression in porcine endometrial lymphocytes, endothelium and trophoblast during pregnancy success and failure. J Reprod Dev. 2007;53:455–63.
Rogers GL, Martino AT, Aslanidi GV, Jayandharan GR, Srivastava A, Herzog RW. Innate immune responses to AAV vectors. Front Microbiol. 2011;2:194.
McDonald EA, Pond-Tor S, Jarilla B, Sagliba MJ, Gonzal A, Amoylen AJ, et al. Schistosomiasis Japonica during pregnancy is associated with elevated endotoxin levels in maternal and placental compartments. J Infect Dis. 2014;209:468–72.
Kondratova L, Kondratov O, Ragheb R, Zolotukhin S. Removal of endotoxin from rAAV samples using a simple detergent-based protocol. Mol Ther Methods Clin Dev. 2019;15:112–9.
Oliviero C, Junnikkala S, Peltoniemi O. The challenge of large litters on the immune system of the sow and the piglets. Reprod Domest Anim. 2019;54:12–21.
Mattison JA, Vaughan KL. An overview of nonhuman primates in aging research. Exp Gerontol. 2017;94:41–5.
Reynolds LP, Caton JS, Redmer DA, Grazul-Bilska AT, Vonnahme KA, Borowicz PP, et al. Evidence for altered placental blood flow and vascularity in compromised pregnancies. J Physiol. 2006;572:51–8.
Hoy S, Lutter C, Wähner M, Puppe B. The effect of birth weight on the early postnatal vitality of piglets. Dtsch Tierarztl Wochenschr. 1994;101:393–6.
Islas-Fabila P, Mota-Rojas D, Martínez-Burnes J, Mora-Medina P, González-Lozano M, Roldán-Santiago P, et al. Physiological and metabolic responses in newborn piglets associated with the birth order. Anim Reprod Sci. 2018;197:247–56.
Langendijk P, Fleuren M, van Hees H, van Kempen T. The course of parturition affects piglet condition at birth and survival and growth through the nursery phase. Animals. 2018;8:60.
Hinderer C, Katz N, Buza EL, Dyer C, Goode T, Bell P, et al. Severe toxicity in nonhuman primates and piglets following high-dose intravenous administration of an adeno-associated virus vector expressing human SMN. Human Gene Ther. 2018;29:285–98.
Hordeaux J, Wang Q, Katz N, Buza EL, Bell P, Wilson JM. The neurotropic properties of AAV-PHP.B are limited to C57BL/6J mice. Mol Ther. 2018;26:664–8.
Ramsingh AI, Gray SJ, Reilly A, Koday M, Bratt D, Koday MT, et al. Sustained AAV9-mediated expression of a non-self protein in the CNS of non-human primates after immunomodulation. PLoS ONE. 2018;13:e0198154.
Van Alstyne M, Tattoli I, Delestrée N, Recinos Y, Workman E, Shihabuddin LS, et al. Gain of toxic function by long-term AAV9-mediated SMN overexpression in the sensorimotor circuit. Nat Neurosci. 2021. http://www.nature.com/articles/s41593-021-00827-3.
Prabhu N, Saylam E, Louis C, Moss M, Millner R, Douglass D, et al. Thrombotic microangiopathy (TMA): a potential adverse reaction post Zolgensma (onasemnogene abeparvovec-xioi) therapy for Spinal Muscular Atrophy (SMA) (5483). Neurology. 2020;94 15 Supplement:5483.
We would like to express our profound gratitude for the generosity and altruism of donors and their families, whose gifts of tissues serve an integral role in advancing medical research and education. We are grateful to Arthur H.M. Burghes, Ph.D. and his laboratory for guidance and advice. We would like to thank Jose Otero and Denise Gamble for their pathology input.
This project was funded by CureSMA, the SMA Foundation and the NIH (R01 NS062269) to CJS.
SJK has received compensation for consultation from AveXis, Biogen, and Genentech. CJS receives grant support from Roche Ltd and has served as a paid advisor, consultant, and/or speaker to the SMA Foundation, Biogen, Ionis Pharmaceuticals, PTC Therapeutics, Roche/Genentech, Sarepta, NuraBio, AveXis, and Novartis. All other authors report no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Rich, K.A., Wier, C.G., Russo, J. et al. Premature delivery in the domestic sow in response to in utero delivery of AAV9 to fetal piglets. Gene Ther (2021). https://doi.org/10.1038/s41434-021-00305-2