Recombinant adeno-associated viruses (rAAVs) serve as vectors for in vivo gene delivery in both mice and humans, and have broad applicability for the treatment of genetic diseases. Clinical trials with AAV vectors have demonstrated promise and safety in several human diseases. However, the in vivo validation of novel AAV constructs expressing products that act specifically on human cells and tissues is limited by a paucity of effective translatable models. Humanized mice that are engrafted with human cells, tissues, and immune systems offer strong potential to test the biological effectiveness of AAV vectors on human cells and tissues. Using the BLT (bone marrow, liver, thymus) humanized NOD-scid Il2rgnull (NSG) mouse model, which enables efficient development of HLA-restricted effector and regulatory T cells (Tregs), we have evaluated the delivery and function of human interleukin (IL)-2 by an AAV vector. Humanized mice treated with an AAV vector expressing human IL-2 showed a significant and sustained increase in the number of functional human FOXP3+CD4+ Tregs. The expression of human IL-2 did not significantly change the levels or activation status of conventional T-cell subsets. Numbers of activated human natural killer cells were also increased significantly in humanized mice treated with the IL-2 vector. These data recapitulate observations in clinical trials of IL-2 therapy and collectively show that humanized mouse models offer a translational platform for testing the efficacy of AAV vectors targeting human immune cells.
KottermanMA, ChalbergTW, SchafferDV. Viral vectors for gene therapy: translational and clinical outlook. Annu Rev Biomed Eng, 2015; 17:63–89.
6.
KattenhornLM, TipperCH, StoicaL, et al.Adeno-associated virus gene therapy for liver disease. Hum Gene Ther, 2016; 27:947–961.
7.
MunchRC, MuthA, MuikA, et al.Off-target-free gene delivery by affinity-purified receptor-targeted viral vectors. Nat Commun, 2015; 6:6246.
8.
PepinD, SosulskiA, ZhangL, et al.AAV9 delivering a modified human Mullerian inhibiting substance as a gene therapy in patient-derived xenografts of ovarian cancer. Proc Natl Acad Sci U S A, 2015; 112:E4418–E4427.
9.
HashimotoH, MizushimaT, OguraT, et al.Study on AAV-mediated gene therapy for diabetes in humanized liver mouse to predict efficacy in humans. Biochem Biophys Res Commun, 2016; 478:1254–1260.
10.
SharmaA, WuW, SungB, et al.Respiratory syncytial virus (RSV) pulmonary infection in humanized mice induces human anti-RSV immune responses and pathology. J VIrol, 2016; 90:5068–5074.
11.
LiX, HuangJ, ZhangM, et al.Human CD8+ T cells mediate protective immunity induced by a human malaria vaccine in human immune system mice. Vaccine, 2016; 34:4501–4506.
12.
HuangJ, LiX, Coelho-dos-ReisJG, et al.Human immune system mice immunized with Plasmodium falciparum circumsporozoite protein induce protective human humoral immunity against malaria. J Immunol Methods, 2015; 427:42–50.
13.
HuangJ, LiX, Coelho-dos-ReisJG, et al.An AAV vector-mediated gene delivery approach facilitates reconstitution of functional human CD8+ T cells in mice. PLoS One, 2014; 9:e88205.
14.
BrunkowME, JefferyEW, HjerrildKA, et al.Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet, 2001; 27:68–73.
15.
CaudyAA, ReddyST, ChatilaT, et al.CD25 deficiency causes an immune dysregulation, polyendocrinopathy, enteropathy, X-linked-like syndrome, and defective IL-10 expression from CD4 lymphocytes. J Allergy Clin Immunol, 2007; 119:482–487.
16.
WildinRS, RamsdellF, PeakeJ, et al.X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat Genet, 2001; 27:18–20.
17.
HulmeMA, WasserfallCH, AtkinsonMA, et al.Central role for interleukin-2 in type 1 diabetes. Diabetes, 2012; 61:14–22.
JohnsonMC, GarlandAL, NicolsonSC, et al.Beta-cell-specific IL-2 therapy increases islet Foxp3+ Treg and suppresses type 1 diabetes in NOD mice. Diabetes, 2013; 62:3775–3784.
20.
ToddJA, EvangelouM, CutlerAJ, et al.Regulatory T cell responses in participants with type 1 diabetes after a single dose of interleukin-2: a non-randomised, open label, adaptive dose-finding trial. PLoS Med, 2016; 13:e1002139.
21.
PhamMN, von HerrathMG, VelaJL. Antigen-specific regulatory T cells and low dose of IL-2 in treatment of type 1 diabetes. Front Immunol, 2015; 6:651.
22.
FurtadoGC, Curotto de LafailleMA, KutchukhidzeN, et al.Interleukin 2 signaling is required for CD4+ regulatory T cell function. J Exp Med, 2002; 196:851–857.
23.
FloresRR, ZhouL, RobbinsPD. Expression of IL-2 in beta cells by AAV8 gene transfer in pre-diabetic NOD mice prevents diabetes through activation of FoxP3-positive regulatory T cells. Gene Ther, 2014; 21:715–722.
24.
ChurlaudG, JimenezV, RuberteJ, et al.Sustained stimulation and expansion of Tregs by IL2 control autoimmunity without impairing immune responses to infection, vaccination and cancer. Clin Immunol, 2014; 151:114–126.
25.
Grinberg-BleyerY, BaeyensA, YouS, et al.IL-2 reverses established type 1 diabetes in NOD mice by a local effect on pancreatic regulatory T cells. J Exp Med, 2010; 207:1871–1878.
26.
KorethJ, MatsuokaK, KimHT, et al.Interleukin-2 and regulatory T cells in graft-versus-host disease. N Engl J Med, 2011; 365:2055–2066.
27.
SaadounD, RosenzwajgM, JolyF, et al.Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis. N Engl J Med, 2011; 365:2067–2077.
28.
CastelaE, Le DuffF, ButoriC, et al.Effects of low-dose recombinant interleukin 2 to promote T-regulatory cells in alopecia areata. JAMA Dermatol, 2014; 150:748–751.
29.
HeJ, ZhangX, WeiY, et al.Low-dose interleukin-2 treatment selectively modulates CD4+ T cell subsets in patients with systemic lupus erythematosus. Nat Med, 2016; 22:991–993.
30.
HumrichJY, von Spee-MayerC, SiegertE, et al.Rapid induction of clinical remission by low-dose interleukin-2 in a patient with refractory SLE. Ann Rheum Dis, 2015; 74:791–792.
31.
LongSA, RieckM, SandaS, et al.Rapamycin/IL-2 combination therapy in patients with type 1 diabetes augments Tregs yet transiently impairs beta-cell function. Diabetes, 2012; 61:2340–2348.
32.
HartemannA, BensimonG, PayanCA, et al.Low-dose interleukin 2 in patients with type 1 diabetes: a phase 1/2 randomised, double-blind, placebo-controlled trial. Lancet Diabetes Endocrinol, 2013; 1:295–305.
33.
YuA, SnowhiteI, VendrameF, et al.Selective IL-2 responsiveness of regulatory T cells through multiple intrinsic mechanisms supports the use of low-dose IL-2 therapy in type 1 diabetes. Diabetes, 2015; 64:2172–2183.
34.
RosenzwajgM, ChurlaudG, MalloneR, et al.Low-dose interleukin-2 fosters a dose-dependent regulatory T cell tuned milieu in T1D patients. J Autoimmun, 2015; 58:48–58.
35.
WegeAK, MelkusMW, DentonPW, et al.Functional and phenotypic characterization of the humanized BLT mouse model. Curr Top Microbiol Immunol, 2008; 324:149–165.
36.
AryeeKE, ShultzLD, BrehmMA. Immunodeficient mouse model for human hematopoietic stem cell engraftment and immune system development. Methods Mol Biol, 2014; 1185:267–278.
37.
CovassinL, JangalweS, JouvetN, et al.Human immune system development and survival of non-obese diabetic (NOD)-scid IL2rγnull (NSG) mice engrafted with human thymus and autologous haematopoietic stem cells. Clin Exp Immunol, 2013; 174:372–388.
38.
HeY, WeinbergMS, HirschM, et al.Kinetics of adeno-associated virus serotype 2 (AAV2) and AAV8 capsid antigen presentation in vivo are identical. Hum Gene Ther, 2013; 24:545–553.
39.
McCartyDM, MonahanPE, SamulskiRJ. Self-complementary recombinant adeno-associated virus (scAAV) vectors promote efficient transduction independently of DNA synthesis. Gene Ther, 2001; 8:1248–1254.
40.
GriegerJC, ChoiVW, SamulskiRJ. Production and characterization of adeno-associated viral vectors. Nat Protoc, 2006; 1:1412–1428.
41.
SchneiderA, BucknerJH. Assessment of suppressive capacity by human regulatory T cells using a reproducible, bi-directional CFSE-based in vitro assay. Methods Mol Biol, 2011; 707:233–241.
42.
HoffmannP, EderR, BoeldTJ, et al.Only the CD45RA+ subpopulation of CD4+CD25high T cells gives rise to homogeneous regulatory T-cell lines upon in vitro expansion. Blood, 2006; 108:4260–4267.
43.
BruskoTM, WasserfallCH, HulmeMA, et al.Influence of membrane CD25 stability on T lymphocyte activity: implications for immunoregulation. PLoS One, 2009; 4:e7980.
44.
WeberT. Do we need marker gene studies in humans to improve clinical AAV gene therapy?. Gene Ther, 2017; 24:72–73.
45.
TheocharidesAP, RongvauxA, FritschK, et al.Humanized hemato-lymphoid system mice. Haematologica, 2016; 101:5–19.
46.
WalshNC, KenneyLL, JangalweS, et al.Humanized mouse models of clinical disease. Annu Rev Pathol, 2017; 12:187–215.
47.
ShultzLD, BrehmMA, Garcia-MartinezJV, et al.Humanized mice for immune system investigation: progress, promise and challenges. Nat Rev Immunol, 2012; 12:786–798.
48.
LongSA, BucknerJH, GreenbaumCJ. IL-2 therapy in type 1 diabetes: “Trials” and tribulations. Clin Immunol, 2013; 149:324–331.
49.
CovassinL, JangalweS, JouvetN, et al.Human immune system development and survival of non-obese diabetic (NOD)-scid IL2rγnull (NSG) mice engrafted with human thymus and autologous haematopoietic stem cells. Clin Exp Immunol, 2013; 174:372–388.
50.
LanP, WangL, DioufB, et al.Induction of human T-cell tolerance to porcine xenoantigens through mixed hematopoietic chimerism. Blood, 2004; 103:3964–3969.
51.
MelkusMW, EstesJD, Padgett-ThomasA, et al.Humanized mice mount specific adaptive and innate immune responses to EBV and TSST-1. Nat Med, 2006; 12:1316–1322.
52.
ItoM, HiramatsuH, KobayashiK, et al.NOD/SCID/\documentclass{aastex}\usepackage{amsbsy}\usepackage{amsfonts}\usepackage{amssymb}\usepackage{bm}\usepackage{mathrsfs}\usepackage{pifont}\usepackage{stmaryrd}\usepackage{textcomp}\usepackage{portland, xspace}\usepackage{amsmath, amsxtra}\usepackage{upgreek}\pagestyle{empty}\DeclareMathSizes{10}{9}{7}{6}\begin{document}$$\gamma_{\rm c}^{\rm null}$$ \end{document} mouse: an excellent recipient mouse model for engraftment of human cells. Blood, 2002; 100:3175–3182.
53.
ShultzLD, LyonsBL, BurzenskiLM, et al.Human lymphoid and myeloid cell development in NOD/LtSz-scid IL2Rγ null mice engrafted with mobilized human hemopoietic stem cells. J Immunol, 2005; 174:6477–6489.
54.
RochmanY, SpolskiR, LeonardWJ. New insights into the regulation of T cells by γc family cytokines. Nat Rev Immunol, 2009; 9:480–490.
55.
TraggiaiE, ChichaL, MazzucchelliL, et al.Development of a human adaptive immune system in cord blood cell-transplanted mice. Science, 2004; 304:104–107.
56.
GoyamaS, WunderlichM, MulloyJC. Xenograft models for normal and malignant stem cells. Blood, 2015; 125:2630–2640.
57.
BrehmMA, CuthbertA, YangC, et al.Parameters for establishing humanized mouse models to study human immunity: analysis of human hematopoietic stem cell engraftment in three immunodeficient strains of mice bearing the IL2rγnull mutation. Clin Immunol, 2010; 135:84–98.
58.
LepusCM, GibsonTF, GerberSA, et al.Comparison of human fetal liver, umbilical cord blood, and adult blood hematopoietic stem cell engraftment in NOD-scid/γc–/–, Balb/c-Rag2–/–γc–/–, and C.B-17-scid/bg immunodeficient mice. Hum Immunol, 2009; 70:790–802.
59.
McDermottSP, EppertK, LechmanER, et al.Comparison of human cord blood engraftment between immunocompromised mouse strains. Blood, 2010; 116:193–200.
60.
TakenakaK, PrasolavaTK, WangJCY, et al.Polymorphism in Sirpa modulates engraftment of human hematopoietic stem cells. Nat Immunol, 2007; 8:1313–1323.
61.
StoddartCA, MaidjiE, GalkinaSA, et al.Superior human leukocyte reconstitution and susceptibility to vaginal HIV transmission in humanized NOD-scid IL-2Rγ–/– (NSG) BLT mice. Virology, 2011; 417:154–160.
62.
BergesBK, RowanMR. The utility of the new generation of humanized mice to study HIV-1 infection: transmission, prevention, pathogenesis, and treatment. Retrovirology, 2011; 8:65.
63.
BiswasS, ChangH, SarkisPT, et al.Humoral immune responses in humanized BLT mice immunized with West Nile virus and HIV-1 envelope proteins are largely mediated via human CD5+ B cells. Immunology, 2011; 134:419–433.
64.
AkkinaR. Human immune responses and potential for vaccine assessment in humanized mice. Curr Opin Immunol, 2013; 25:403–409.
65.
BrehmMA, WilesMV, GreinerDL, et al.Generation of improved humanized mouse models for human infectious diseases. J Immunol Methods, 2014; 410:3–17.
ItoR, TakahashiT, KatanoI, et al.Current advances in humanized mouse models. Cell Mol Immunol, 2012; 9:208–214.
68.
KalscheuerH, DanzlN, OnoeT, et al.A model for personalized in vivo analysis of human immune responsiveness. Sci Transl Med, 2012; 4:125ra130.
69.
BirdBH, SpenglerJR, ChakrabartiAK, et al.Humanized mouse model of Ebola virus disease mimics the immune responses in human disease. J Infect Dis, 2016; 213:703–711.
70.
OnoeT, KalscheuerH, DanzlN, et al.Human natural regulatory T cell development, suppressive function, and postthymic maturation in a humanized mouse model. J Immunol, 2011; 187:3895–3903.
71.
HahnSA, BellinghausenI, TrinschekB, et al.Translating Treg therapy in humanized mice. Front Immunol, 2015; 6:623.
72.
LongSA, RieckM, SandaS, et al.Rapamycin/IL-2 combination therapy in patients with type 1 diabetes augments Tregs yet transiently impairs beta-cell function. Diabetes, 2012; 61:2340–2348.
73.
BrehmMA, ShultzLD, LubanJ, et al.Overcoming current limitations in humanized mouse research. J Infect Dis, 2013; 208(Suppl 2):S125–S130.
74.
KatanoI, TakahashiT, ItoR, et al.Predominant development of mature and functional human NK cells in a novel human IL-2-producing transgenic NOG mouse. J Immunol, 2015; 194:3513–3525.
75.
ItoR, KatanoI, KawaiK, et al.A novel xenogeneic graft-versus-host disease model for investigating the pathological role of human CD4+ or CD8+ T cells using immunodeficient NOG mice. Am J Transplant, 2017; 17:1216–1228.
76.
AbrahamS, PahwaR, YeC, et al.Long-term engraftment of human natural T regulatory cells in NOD/SCID IL2rγcnull mice by expression of human IL-2. PLoS One, 2012; 7:e51832.
77.
KlevornLE, Berrien-ElliottMM, YuanJ, et al.Rescue of tolerant CD8+ T cells during cancer immunotherapy with IL2:antibody complexes. Cancer Immunol Res, 2016; 4:1016–1026.
78.
MinamiY, KonoT, MiyazakiT, et al.The IL-2 receptor complex: its structure, function, and target genes. Annu Rev Immunol, 1993; 11:245–268.
79.
BoymanO, SprentJ. The role of interleukin-2 during homeostasis and activation of the immune system. Nat Rev Immunol, 2012; 12:180–190.
80.
LiaoW, LinJX, LeonardWJ. Interleukin-2 at the crossroads of effector responses, tolerance, and immunotherapy. Immunity, 2013; 38:13–25.
81.
HirakawaM, MatosT, LiuH, et al.Low-dose IL-2 selectively activates subsets of CD4+ Tregs and NK cells. JCI Insight, 2016; 1:e89278.
82.
KimM, KimTJ, KimHM, et al.Multi-cellular natural killer (NK) cell clusters enhance NK cell activation through localizing IL-2 within the cluster. Sci Rep, 2017; 7:40623.
83.
BoymanO, KovarM, RubinsteinMP, et al.Selective stimulation of T cell subsets with antibody–cytokine immune complexes. Science, 2006; 311:1924–1927.
84.
LevinAM, BatesDL, RingAM, et al.Exploiting a natural conformational switch to engineer an interleukin-2 “superkine.”. Nature, 2012; 484:529–533.
85.
SeokJ, WarrenHS, CuencaAG, et al; Inflammation and Host Response to Injury, Large Scale Collaborative Research Program. Genomic responses in mouse models poorly mimic human inflammatory diseases. Proc Natl Acad Sci U S A, 2013; 110:3507–3512.
86.
MestasJ, HughesCC. Of mice and not men: differences between mouse and human immunology. J Immunol, 2004; 172:2731–2738.
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