Gene therapy technologies are inevitably required to boost the therapeutic performance of cell therapies; thus, validating the efficacy of gene carriers specifically used for preparing cellular therapeutics is a prerequisite for evaluating the therapeutic capabilities of gene and cell combinatorial therapies. Herein, the efficacy of a recombinant adeno-associated virus derivative (rAAVr3.45) was examined to evaluate its potential as a gene carrier for genetically manipulating interleukin-10 (IL10)-secreting human neural stem cells (hNSCs) that can potentially treat ischemic injuries or neurological disorders. Safety issues that could arise during the virus preparation or viral infection were investigated; no replication-competent AAVs were detected in the final cell suspensions, transgene expression was mostly transient, and no severe interference on endogenous gene expression by viral infection occurred. IL10 secretion from hNSCs infected by rAAVr3.45 encoding IL10 did not alter the transcriptional profile of any gene by more than threefold, but the exogenously boosted IL10 was sufficient to provoke immunomodulatory effects in an ischemic brain injury animal model, thereby accelerating the recovery of neurological deficits and the reduction of brain infarction volume. This study presents evidence that rAAVr3.45 can be potentially used as a gene carrier to prepare stem cell therapeutics.
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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.
Russell S, Bennett J, Wellman JA, Chung DC, Yu Z-F, Tillman A, et al. Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet. 2017;390:849–60.
Nathwani AC, Tuddenham EGD, Rangarajan S, Rosales C, McIntosh J, Linch DC, et al. Adenovirus-associated virus vector-mediated gene transfer in hemophilia B. N Engl J Med. 2011;365:2357–65.
Kaplitt MG, Feigin A, Tang C, Fitzsimons HL, Mattis P, Lawlor PA, et al. Safety and tolerability of gene therapy with an adeno-associated virus (AAV) borne GAD gene for Parkinson’s disease: an open label, phase I trial. Lancet. 2007;369:2097–105.
Maguire AM, High KA, Auricchio A, Wright JF, Pierce EA, Testa F, et al. Age-dependent effects of RPE65 gene therapy for Leber’s congenital amaurosis: a phase 1 dose-escalation trial. Lancet. 2009;374:1597–605.
Ginn SL, Amaya AK, Alexander IE, Edelstein M, Abedi MR. Gene therapy clinical trials worldwide to 2017: an update. J Gene Med. 2018;20:e3015.
Platt RJ, Chen S, Zhou Y, Yim MJ, Swiech L, Kempton HR, et al. CRISPR-Cas9 knockin mice for genome editing and cancer modeling. Cell. 2014;159:440–55.
Swiech L, Heidenreich M, Banerjee A, Habib N, Li Y, Trombetta J, et al. In vivo interrogation of gene function in the mammalian brain using CRISPR-Cas9. Nat Biotechnol. 2015;33:102–6.
Mingozzi F, High KA. Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nat Rev Genet. 2011;12:341–55.
Kotterman MA, Schaffer DV. Engineering adeno-associated viruses for clinical gene therapy. Nat Rev Genet. 2014;15:445–51.
Maheshri N, Koerber JT, Kaspar BK, Schaffer DV. Directed evolution of adeno-associated virus yields enhanced gene delivery vectors. Nat Biotechnol. 2006;24:198–204.
Jang JH, Koerber JT, Kim J-S, Asuri P, Vazin T, Bartel M, et al. An evolved adeno-associated viral variant enhances gene delivery and gene targeting in neural stem cells. Mol Ther. 2011;19:667–75.
Dalkara D, Byrne LC, Klimczak RR, Visel M, Yin L, Merigan WH, et al. In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous. Sci Transl Med. 2013;5:189ra76–189ra76.
Jang JH, Koerber JT, Gujraty K, Bethi SR, Kane RS, Schaffer DV. Surface immobilization of hexa-histidine-tagged adeno-associated viral vectors for localized gene delivery. Gene Ther. 2010;17:1384–9.
Petrs-Silva H, Dinculescu A, Li Q, Min S-H, Chiodo V, Pang J-J, et al. High-efficiency transduction of the mouse retina by tyrosine-mutant AAV serotype vectors. Mol Ther. 2009;17:463–71.
Horowitz ED, Weinberg MS, Asokan A. Glycated AAV vectors: chemical redirection of viral tissue tropism. Bioconjugate Chem. 2011;22:529–32.
Katrekar D, Moreno AM, Chen G, Worlikar A, Mali P. Oligonucleotide conjugated multi-functional adeno-associated viruses. Sci Rep. 2018;8:3589.
Koerber JT, Jang J-H, Yu JH, Kane RS, Schaffer DV. Engineering adeno-associated virus for one-step purification via immobilized metal affinity chromatography. Hum Gene Ther. 2007;18:367–78.
Pickar AK, Gersbach CA. Gene therapies for hemophilia hit the mark in clinical trials. Nat Med. 2018;24:121–2.
Kim Y, Kim E, Oh S, Yoon YE, Jang JH. Mutagenic analysis of an adeno-associated virus variant capable of simultaneously promoting immune resistance and robust gene delivery. Hum Gene Ther. 2018;29:25–41.
Jin G, Shin M, Kim S-H, Lee H, Jang J-H. SpONGE: spontaneous organization of numerous-layer generation by electrospray. Angew Chem Int Ed Engl. 2015;54:7587–91.
Kim E, Song IT, Lee S, Kim J-S, Lee H, Jang J-H. Drawing sticky adeno-associated viruses on surfaces for spatially patterned gene expression. Angew Chem Int Ed Engl. 2012;51:5598–601.
Kim S-H, Lee M, Cho M, Kim I-S, Park KI, Lee H, et al. Inverted quasi-spherical droplets on polydopamine–TiO2 substrates for enhancing gene delivery. Macromol Biosci. 2017;17:1700148.
Kim H-T, Kim I-S, Lee I-S, Lee J-P, Snyder EY, Park KI. Human neurospheres derived from the fetal central nervous system are regionally and temporally specified but are not committed. Exp Neurol. 2006;199:222–35.
Smith-Arica JR, Thomson AJ, Ansell R, Chiorini J, Davidson B, McWhir J. Infection efficiency of human and mouse embryonic stem cells using adenoviral and adeno-associated viral vectors. Cloning Stem Cells. 2003;5:51–62.
Dietrich WD, Busto R, Bethea JR. Postischemic hypothermia and IL-10 treatment provide long-lasting neuroprotection of CA1 hippocampus following transient global ischemia in rats. Exp Neurol. 1999;158:444–50.
Grilli M, Barbieri I, Basudev H, Brusa R, Casati C, Lozza G, et al. Interleukin-10 modulates neuronal threshold of vulnerability to ischaemic damage. Eur J Neurosci. 2000;12:2265–72.
Ouyang W, Rutz S, Crellin NK, Valdez PA, Hymowitz SG. Regulation and functions of the IL-10 family of cytokines in inflammation and disease. Annu Rev Immunol. 2011;29:71–109.
Saraiva M, O’Garra A. The regulation of IL-10 production by immune cells. Nat Rev Immunol. 2010;10:170–81.
Ooboshi H, Ibayashi S, Shichita T, Kumai Y, Takada J, Ago T, et al. Postischemic gene transfer of interleukin-10 protects against both focal and global brain ischemia. Circulation. 2005;111:913–9.
Knoblach SM, Faden AI. Interleukin-10 improves outcome and alters proinflammatory cytokine expression after experimental traumatic brain injury. Exp Neurol. 1998;153:143–51.
Huang BJ, Hu JC, Athanasiou KA. Cell-based tissue engineering strategies used in the clinical repair of articular cartilage. Biomaterials. 2016;98:1–22.
Cohen S, Samadikuchaksaraei A, Polak JM, Bishop AE. Antibiotics reduce the growth rate and differentiation of embryonic stem cell cultures. Tissue Eng. 2006;12:2025–30.
van Kuppeveld FJM, van der Logt JTM, Angulo AF, van Zoest MJ, Quint WGV, Niesters HGM, et al. Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification. Appl Environ Microbiol. 1992;58:2606–15.
Drexler HG, Uphoff CC. Mycoplasma contamination of cell cultures: incidence, sources, effects, detection, elimination, prevention. Cytotechnology. 2002;39:75–90.
Hay RJ, Macy ML, Chen TR. Mycoplasma infection of cultured cells. Nature. 1989;339:487–8.
Thorne BA, Takeya RK, Peluso RW. Manufacturing recombinant adeno-associated viral vectors from producer cell clones. Hum Gene Ther. 2009;20:707–14.
Allay JA, Sleep S, Long S, Tillman DM, Clark R, Carney G, et al. Good manufacturing practice production of self-complementary serotype 8 adeno-associated viral vector for a hemophilia B clinical trial. Hum Gene Ther. 2011;22:595–604.
Alkan C, Coe BP, Eichler EE. Genome structural variation discovery and genotyping. Nat Rev Genet. 2011;12:363–76.
Metzker ML. Sequencing technologies - the next generation. Nat Rev Genet. 2010;11:31–46.
Karlsson S, Nienhuis AW. Developmental regulation of human globin genes. Annu Rev Biochem. 1985;54:1071–108.
DeKelver RC, Choi VM, Moehle EA, Paschon DE, Hockemeyer D, Meijsing SH, et al. Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome. Genome Res. 2010;20:1133–42.
Tiyaboonchai A, Mac H, Shamsedeen R, Mills JA, Kishore S, French DL, et al. Utilization of the AAVS1 safe harbor locus for hematopoietic specific transgene expression and gene knockdown in human ES cells. Stem Cell Res. 2014;12:630–7.
Calvert JW, Yin W, Patel M, Badr A, Mychaskiw G, Parent AD, et al. Hyperbaric oxygenation prevented brain injury induced by hypoxia–ischemia in a neonatal rat model. Brain Res. 2002;951:1–8.
Kaeppel C, Beattie SG, Fronza R, van Logtenstein R, Salmon F, Schmidt S, et al. A largely random AAV integration profile after LPLD gene therapy. Nat Med. 2013;19:889–91.
McCarty DM, Young SM, Samulski RJ. Integration of adeno-associated virus (AAV) and recombinant AAV vectors. Annu Rev Genet. 2004;38:819–45.
Logan GJ, Dane AP, Hallwirth CV, Smyth CM, Wilkie EE, Amaya AK, et al. Identification of liver-specific enhancer–promoter activity in the 3′ untranslated region of the wild-type AAV2 genome. Nat Genet. 2017;49:1267.
Weitzman MD, Kyöstiö SR, Kotin RM, Owens RA. Adeno-associated virus (AAV) Rep proteins mediate complex formation between AAV DNA and its integration site in human DNA. Proc Natl Acad Sci USA. 1994;91:5808.
Nakai H, Montini E, Fuess S, Storm TA, Grompe M, Kay MA. AAV serotype 2 vectors preferentially integrate into active genes in mice. Nat Genet. 2003;34:297–302.
Cummings BJ, Uchida N, Tamaki SJ, Salazar DL, Hooshmand M, Summers R, et al. Human neural stem cells differentiate and promote locomotor recovery in spinal cord-injured mice. Proc Natl Acad Sci USA. 2005;102:14069–74.
Bernstock JD, Peruzzotti-Jametti L, Ye D, Gessler FA, Maric D, Vicario N, et al. Neural stem cell transplantation in ischemic stroke: a role for preconditioning and cellular engineering. J Cereb Blood Flow Metab. 2017;37:2314–9.
Drago D, Cossetti C, Iraci N, Gaude E, Musco G, Bachi A, et al. The stem cell secretome and its role in brain repair. Biochimie. 2013;95:2271–85.
Martino G, Pluchino S. The therapeutic potential of neural stem cells. Nat Rev Neurosci. 2006;7:395–406.
Amor S, Puentes F, Baker D, can der Valk P. Inflammation in neurodegenerative diseases. Immunology. 2010;129:154–69.
Krishnamurthy P, Thal M, Verma S, Hoxha E, Lambers E, Ramirez V, et al. Interleukin-10 deficiency impairs bone marrow-derived endothelial progenitor cell survival and function in ischemic myocardium. Circ Res. 2011;109:1280–9.
Hagberg H, Mallard C, Ferriero DM, Vannucci SJ, Levison SW, Vexler ZS, et al. The role of inflammation in perinatal brain injury. Nat Rev Neurol. 2015;11:192–208.
Lorek A, Takei Y, Cady EB, Wyatt JS, Penrice J, Edwards AD, et al. Delayed (“secondary”) cerebral energy failure after acute hypoxia-ischemia in the newborn piglet: continuous 48-hour studies by phosphorus magnetic resonance spectroscopy. Pediatr Res. 1994;36:699–706.
Sato Y, Nakanishi K, Hayakawa M, Kakizawa H, Saito A, Kuroda Y, et al. Reduction of brain injury in neonatal hypoxic-ischemic rats by intracerebroventricular injection of neural stem/progenitor cells together with chondroitinase ABC. Reprod Sci. 2008;15:613–20.
Wang L, Jiang F, Li Q, He X, Ma J. Mild hypothermia combined with neural stem cell transplantation for hypoxic-ischemic encephalopathy: neuroprotective effects of combined therapy. Neural Regen Res. 2014;9:1745–52.
Lin M-T, Storer B, Martin PJ, Tseng L-H, Grogan B, Chen P-J, et al. Genetic variation in the IL-10 pathway modulates severity of acute graft-versus-host disease following hematopoietic cell transplantation: synergism between IL-10 genotype of patient and IL-10 receptor β genotype of donor. Blood. 2005;106:3995–4001.
Kanehisa M, Goto S. KEGG: Kyoto Encyclopedia of Genes and Genomes. Nucleic Acids Res. 2000;28:27–30.
Habedanck R, Stierhof Y-D, Wilkinson CJ, Nigg EA. The Polo kinase Plk4 functions in centriole duplication. Nat Cell Biol. 2005;7:1140–6.
Iorio MV, Croce CM. MicroRNA dysregulation in cancer: diagnostics, monitoring and therapeutics. A comprehensive review. EMBO Mol Med. 2012;4:143.
Grochowski C, Radzikowska E, Maciejewski R. Neural stem cell therapy—brief review. Clin Neurol Neurosurg. 2018;173:8–14.
Park KI, Teng YD, Snyder EY. The injured brain interacts reciprocally with neural stem cells supported by scaffolds to reconstitute lost tissue. Nat Biotechnol. 2002;20:1111–7.
Lee IS, Koo KY, Jung K, Kim M, Kim IS, Hwang K, et al. Neurogenin-2-transduced human neural progenitor cells attenuate neonatal hypoxic-ischemic brain injury. Transl Res. 2017;183:121–36 e9.
Cheng H, Huang SS, Lin SM, Lin MJ, Chu YC, Chih CL, et al. The neuroprotective effect of glial cell line-derived neurotrophic factor in fibrin glue against chronic focal cerebral ischemia in conscious rats. Brain Res. 2005;1033:28–33.
Kelly S, Bliss TM, Shah AK, Sun GH, Ma M, Foo WC, et al. Transplanted human fetal neural stem cells survive, migrate, and differentiate in ischemic rat cerebral cortex. Proc Natl Acad Sci USA. 2004;101:11839–44.
This research was supported by Basic Science Research Program through the National Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future Planning (MSIP) (2018R1A2A2A05020786), the Bio&Medical Technology Development Program of NRF funded by the Korean government, MSIP (2017M3A9B4061968), and the grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (HI14C1564 & HI16C1089).
The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.
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Cho, M., Jung, K., Kim, SH. et al. Safety and efficacy evaluations of an adeno-associated virus variant for preparing IL10-secreting human neural stem cell-based therapeutics. Gene Ther 26, 135–150 (2019). https://doi.org/10.1038/s41434-019-0057-8