Neonatal bone marrow transplantation prevents bone pathology in a mouse model of mucopolysaccharidosis type I

Neonatal bone marrow transplantation prevents bone pathology in a mouse model of mucopolysaccharidosis type I
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  Regular Article TRANSPLANTATION Q:A1  Neonatal bone marrow transplantation prevents bone pathology ina mouse model of mucopolysaccharidosis type I Alice Pievani, 1,2 Isabella Azario, 1,2 Laura Antolini, 3 Tsutomu Shimada, 4 Pravin Patel, 4 Cristina Remoli, 5 Benedetta Rambaldi, 2 Maria Grazia Valsecchi, 3 Mara Riminucci, 5 Andrea Biondi, 2,6 Shunji Tomatsu, 4 and Marta Serafini 1,2 Q:1Q:2 1 Dulbecco Telethon Institute,  2 Centro Ricerca M. Tettamanti, Department of Pediatrics,  3 Centro di Biostatistica per l’epidemiologia clinica, Department ofHealth Sciences, University of Milano-Bicocca, Monza, Italy;  4 Department of Biomedical Research, Alfred I. duPont Institute Hospital for Children,Wilmington, DE;  5 Department of Molecular Medicine, Sapienza University, Rome, Italy; and  6 Department of Pediatrics, University of Milano-Bicocca,San Gerardo Hospital/Fondazione MBBM, Monza, Italy Q:3 Key Points • Allogeneic BMT into newbornMPS I mice allows highdonor-derived hematopoieticengraftment and preventsbone deformities.• Bones of transplanted MPS Imice showed significantimprovements at radiographic,microcomputed tomography,and histological analyses. Neonatalbonemarrowtransplantation(BMT)couldofferanoveltherapeuticopportunityforgeneticdisordersby providingsustainablelevelsofthemissingproteinatbirth,thuspreventingtissuedamage.WetestedthisconceptinmucopolysaccharidosistypeI(MPSIH;Hurler syndrome), a lysosomal storage disorder caused by deficiency of  a - L -iduronidase.MPSIHischaracterizedbyabroadspectrumofclinicalmanifestations,includingseverepro-gressiveskeletalabnormalities.AlthoughBMTincreasesthelifespanofpatientswithMPSIH,musculoskeletal manifestations are only minimally responsive if the timing of BMT delays Q:4  ,suggestingalreadyirreversiblebonedamage.Inthisstudy,wetestedthehypothesisthattransplantingnormalbonemarrowintonewbornMPSImicesoonafterbirthcanpreventskeletaldysplasia.Weobservedthatneonatalbonemarrowtransplantationwaseffectiveat restoring a - L -iduronidase activity and clearing elevated glycosaminoglycans in bloodandmultipleorgans.At37weeksofage,weobservedanalmostcompletenormalizationofallbonetissueparameters,usingradiographic,microcomputedtomography,biochemical,and histological analyses.Overall, the magnitude of improvements correlated with the ex-tentofhematopoieticengraftment.WeconcludethatBMTataveryearlystageinlifemarkedlyreducessignsandsymptomsofMPSIbeforethey appear. ( Blood  . 2014;00(00):1-10) Introduction Bonemarrowtransplantation(BMT)isanef  fi cacioustherapeuticmo-dality for a number of nonmalignant conditions, both acquired andgeneticallydetermined.Inthetreatmentofgeneticdisorders,thehema-topoiesis transplanted from a healthy allogeneic donor can producethemissingproteinatlevelssuf  fi cienttocorrecttheunderlyingabnor-malities.However,thecurrentclinicalexperienceisonlypartiallysuccessful.Oneofthereasonsforthiscouldbethelimitedef  fi cacyof the protein provided through the hematopoietic cell secretome. Al-ternatively,thetimeatwhichBMTiscurrentlyperformedcouldbetoolate to prevent organ damage.This fundamental question is well exempli fi ed by the case of mucopolysaccharidosistypeI-Hurlersyndrome(MPSIH),alysosomalstorage disease in which allogeneic BMT, although considered fi rst-line treatment, has a limited effect on the skeletal abnormalities. InpatientswithMPSIH,theintracellularaccumulationofglycosamino-glycans(GAGs),dermatan sulfate (DS),and heparansulfate (HS)disruptsnormalcellfunctionsandgivesrisetoprogressivemultiorganmorbidity,whichistypicalforthedisease. 1,2 Thearrayofmusculoskel-etal abnormalities seen in Hurler syndrome is known as dysostosismultiplex, 3-6 consistingofabnormallyshapedvertebraeandribs,en-largedskull,spatulateribs,hypoplasticepiphyses,thickeneddiaphyses,bullet-shaped metacarpals, hip dysplasia, genu valgum, and spinalcordcompression. 7 Unfortunately,despitesuccessfulengraftmentof normal donor hematopoietic stem cells that ameliorate multiorganmorbidity,themusculoskeletalmanifestationsstilldeteriorateandaf-fect the quality of life in most transplanted patients with MPS IH. 8 Thisispresumablyaresultofthelimitedpenetrationoftheexpressed a - L -iduronidase (IDUA) enzyme into musculoskeletal tissues. 4 Al-though substantial clinical improvements of joint mobility, coarsefacial features, and claw hands were reported after transplantation, 9 with age, clinical and radiographic musculoskeletal abnormalitiesstill developed. The incomplete correction of the skeletal phenotypeproduced by BMT may also be because bone abnormalities are irre-versibleatthetimeofthetransplant(medianageatBMT,16months).However, during prenatal and perinatal life, infants with Hurler syn-drome have a nearly normal skeletal development, probably becauseof placental protection. 10,11 Therefore, we hypothesize that the  fi rst months of life represent the best window of opportunity for preventing bone deformities inHurlerchildren.Consistentwiththishypothesis,aretrospectiveanal-ysis demonstrated a superior long-term clinical outcome of Hurler patients when BMT was performed early in life. 12 In the same Submitted June 16, 2014; accepted September 25, 2014. Prepublished onlineas  Blood   First Edition paper, October 8, 2014; DOI 10.1182/blood-2014-06-581207.The online version of this article contains a data supplement.The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked “advertisement” in accordance with 18 USC section 1734.  ©   2014 by The American Society of HematologyBLOOD,  nnn nnn  2014  x  VOLUME 00, NUMBER 00 1  direction,recentimplementationofnewbornscreeningprogramsfor patients with MPS IH may offer an opportunity to consider BMT inthe  fi rst months of life. 13-15 Afewstudieshaveevaluatedwhetherperinatalinfusionofhema-topoietic stem cells could ameliorate the most critical features inMPSanimal models. 16-18 To date,neonatal BMT (nBMT)has never beentestedinMPSImice.ThemousemodelofMPSIwasproducedthrough the disruption of the IDUA gene 19 and presents skeletal ab-normalities similar to those seen in patients with MPS I, providing a valuable tool for the study of disease pathogenesis. MPS I mice de-velop extensive dysostosis detectable using histopathologic, radio-graphic, and microcomputed tomography (micro-CT) analyses at a laterstageoflife. 19-21 BMThaspreviouslybeentestedinadultMPSImicepreconditionedwithirradiation,providingonlyapartialbene fi t on skeletal abnormalities. 22 In the present study, we have evaluated whether neonataltransplantation of syngeneic BM from wild type (WT) donorscould rescue the severe skeletal phenotype in the mouse model of MPS I, using clinical, biochemical, radiographic, and patholog-ical analyses. Materials and methods Mouse model The MPS I mouse model (Idua  2  /  2 mice, C57BL/6 background) 19 was pur-chased from the Jackson Laboratory (Bar Harbor, ME). A breeding colonywas established from heterozygous mating pairs, and genotyping was per-formed on tail clip DNA, as described. 19 Pregnant dams obtained from thecolony (Charles River, Calco, Italy) were housed in the animal facility of theUniversity of Milano-Bicocca. Procedures involving animal handling and careconform to institutional guidelines, in compliance with national laws andpolicies. Bone marrow transplantation NeonatalBMTwasperformedas reported. 23 Brie fl y,1-to2-day-old pups(Ly5.2 [CD45.2] MPS I or Ly5.2 WT mice) were treated with a singleintraperitoneal injection of busulfan (20 mg/kg; Busilvex, Pierre Fabre,Boulogne, France). Eight- to 12-week-old C57BL/6-Ly5.1 (CD45.1) WTdonor mice (Charles River) were killed with CO 2 , and the BM washarvestedby fl ushing femurs and tibiae.Newbornmice were transplanted24 hours after busulfan administration by intravenous (via temporal vein)injectionof2 3 10 6 donorBMcells.At37weeksofage,tissuesfromrecipient killed mice were harvested and preserved for analyses, as speci fi ed here and insupplemental  Methods . IDUA and  b -hexosaminidase activity assay IDUA and  b -hexosaminidase ( b -Hex) activities were measured  fl uoro-metrically, as previously described. 24,25 See supplemental  Methods  for details. Analysis of glycosaminoglycans in tissues and plasma GAGs were quanti fi ed as described. 26,27 See supplemental  Methods  for details. Histopathology For the evaluation of long bone morphology, the hind limbs were decalci fi edinEDTA(Sigma-Aldrich)for3weeksandlongitudinallyembeddedinparaf  fi n.Sections4 m mthickweredeparaf  fi nized,rehydrated,andstainedwithhematoxylin/ eosin by standard procedures.For evaluation of growth plate by light microscopy, bone was decalci fi edand toluidine blue-stained sections 0.5 m m thick were examined. Statistical analysis Thedistributionofcontinuousvariableswascontrastedingroupsbythenon-parametric test on equality of the medians (Wilcoxon).  P  values were con-sidered signi fi cant when in 2-sided alternative or 1-sided alternative for explorative purpose (micro-CT) were below 5%  Q:5 . The relation between en-graftment and treatment in the MPS I BMT group was assessed by Pearson ’ scorrelation coef  fi cient and relative  P -value for absence of correlation. Seesupplemental  Methods  for details. Results Donor hematopoietic engraftment in MPS I mice after nBMT Afterconditioningwithbusulfan,2-to3-day-oldWTCD45.2 1 andMPSICD45.2 1 miceweretransplantedwithBMcellsderivedfrom adultCD45.1 1 WTmice (Figure1A).Wecomparedtheengraftment levelsofthetransplantedgroups,consistingofWTrecipients(hereafter named WT nBMT; n 5 8 mice [3 males, 5 females]) and MPS Irecipients (hereafter named MPS I nBMT; n 5 14 mice [7 males, 7females]).Quanti fi cationofCD45.1 1 donor-derivedcellsinperiph-eralblood(PB)revealednosigni fi cantdifferencesbetweenthe2groupsofmiceat4weeksposttransplant(medianWTnBMT,54.72%[range,22.18%to87.23%]vsmedianMPSInBMT,59.18%[range,0.93%to86.34%];  P 5 .609). Both transplanted groups maintained stable en-graftment levels until euthanasia at 37 weeks of age (PB, median WTnBMT 67.45% [range, 16.00% to 95.86%] vs median MPS I nBMT,81.30% [range, 0.80% to 95.80%];  P  5  .714; spleen, median WTnBMT, 83.95% [range, 53.30% to 97.50%] vs median MPS I nBMT,64.10% [range, 1.37% to 98.30%];  P  5  .304) (Figure 1B). Amongtransplanted mice, 13 (8 MPS I nBMT and 5 WT nBMT) of 22 micepresented a high hematopoietic chimerism, de fi ned as more than50%donorCD45.1 1 cellsinPB(median,92.60% ’ range,54.70%to95.86% CD45.1 1 ). Hence, we included this subgroup of highlyengrafted mice (hereafter named MPS I nBMT-hi) in all studiesreported. The remaining transplanted mice showed an engraftment leveloflessthan50%ofCD45.1 1 withinPBcells(median,16.00%;range, 0.80% to 46.40% CD45.1 1 ). In both WT nBMT and MPS InBMT mice, similar engraftment levels were also observed inhematopoietic organs, such as  Q:6 spleen and thymus (Figure 1C), inwhich we could also appreciate a comparable presence of myeloid(CD11b,Gr-1),B(CD45R),andT(CD3)lymphoidcells,indicatingmultilineage donor-derived engraftment (Figure 1C).At 37 weeks, the median body weight of nontransplanted MPS Imice was similar to that of age-matched WTmice (median MPS I,33.32 g [range, 18.70 to 37.90 g] vs median WT, 34.68 g [range,21.84 to 36.46 g];  P 5 .534). Because of busulfan toxicity, thebody weight of transplanted MPS I nBMTmice (median, 21.30 g;range, 16.40 to 40.30 g) and WT nBMT mice (median, 23.55 g;range, 16.77 to 34.35 g) was lower compared with nontransplantedWT mice. IDUA activity markedly increases in peripheral organs of nBMTMPS I mice, resulting in reduction of GAG levels The tissues (spleen, liver, heart, lung, and kidney) harvested from MPSInBMTmicewereevaluatedforIDUAactivitycomparedwiththose from 37-week-old WT and untreated MPS I mice. All homog-enatesfromMPSImicehadnoIDUAactivity,asshowninFigure2A. 2 PIEVANI et al BLOOD,  nnn nnn  2014  x  VOLUME 00, NUMBER 00  MPS I nBMT-hi mice displayed partial restoration of IDUA activityin all evaluated tissues, particularly high in spleen (70% of WT mice)(Figure 2A). IDUA activity in liver and kidney in MPS I nBMT-himice increased to up to 40% of values found in WT mice. IDUAactivityinthe heartand the lung of MPS I nBMT-himice was20%of the enzyme activity in WT mice. Furthermore, we evaluated IDUAactivity levels in each organ compared with the percentage of donor engraftmentinPBforallMPSInBMTmice.Enzymeactivityinallthetissues,excepttheheart,signi fi cantlycorrelatedwiththelevelsofdonor cells in PB (supplemental Figure 1). In addition, the levels of the lyso-somalenzyme b -hexosaminidase,whichresultselevatedinMPSI,weresigni fi cantly reduced in the same tissues after nBMT (Figure 2B).To con fi rm these data, we also quanti fi ed GAG levels in spleen,liver, heart, lung, and kidney from WT, MPS I, and MPS I nBMT-himice (Figure 2C). MPS I mice exhibited higher levels of GAGsstorageinallorgans,incomparisonwithWTmice( P , .0001forallorgans).Inaddition,MPSInBMT-hianimalsexhibitedastatisticallysigni fi cant reduction in GAGs storage material in the spleen, liver,heart, lung, and kidney (MPS I nBMT-hi vs MPS I;  P 5  .0003)(Figure2C).GAGlevelsinspleen,liver,andlungofMPSInBMT-himicewerenormalized(MPSInBMT-hivsWT,respectively, P 5 .30, P 5 .68,and P 5 .30),suggestingthatnBMT-hicompletelycorrectedan error of metabolism at these tissues.Levels of plasma GAGs [ D DiHS-0S,  D DiHS-NS,  D Di-6S, andmono-sulfated-Gal b 1-4GlcNAc  Q:7 (6S) (mono-sulfated KS)] in un-treatedMPSImiceweresigni fi cantlyhighercomparedwiththoseinWTmice( P , .0006forallGAGs)(Figure 2D).ThelevelsoftheseGAGs in MPS I nBMT-hi mice followed-up for 37 weeks weresigni fi cantly reduced compared with untreated MPS I mice (MPS InBMT-hi vs MPS I;  P  ,  .002) and were normalized in mono-sulfatedKS(MPSInBMT-hivsWT; P 5 .09).Elevationof  D DiHS-0S, D DiHS-NS,and D Di-6SderivedfromHSwasaresultofprimarystorage substrate, whereas KS was elevated secondarily because of the magnitude of skeletal dysplasia.AnimportantfactorcontributingtothereductionofGAGlevelsinthe examined organs and plasma was the magnitude of engraftment. Figure 1. Donor hematopoietic engraftment intonewborn transplanted MPS I mice.  (A) Experimentalscheme. Newborn WT and MPS I (CD45.2) mice wereconditioned with busulfan (20 mg/kg) on day 1 to 2 afterbirth and transplanted with 2 3 10 6 adult BM nucleatedcells (CD45.1) on the next day, as indicated. Groupswere WT untreated (WT), MPS I untreated (MPS I),MPS I mice transplanted with CD45.1 donor BM cells(MPS I nBMT), and WT mice transplanted with CD45.1donor BM cells (WT nBMT). Chimerism was determinedat 4 weeks of age, and at 37 weeks of age, mice werekilled for detection of long-term engraftment, biochemical,radiographic, and histological analyses. (B) Long-termdonor chimerism (engraftment of CD45.1 1  cells) wasdetermined using flow cytometry in PB and spleen at37 weeks of age. Upper horizontal line of box representsthe 75th percentile, the lower horizontal line of boxrepresents the 25th percentile, the horizontal bar withinthe box represents the median, the square within boxrepresents the mean, and vertical lines outside the boxrepresent the minimum and maximum.  P   value wascalculated using Wilcoxon nonparametric unpaired test,2 sides. (C) Representative multilineage donor-derived re-constitution was analyzed, using flow cytometry in spleen(myeloid, B and T lymphoid cells) and thymus (T lymphoidcells) of recipient WT nBMT and MPS I nBMT mice. Q:14 BLOOD,  nnn nnn  2014  x  VOLUME 00, NUMBER 00 SKELETAL OUTCOME OF MPS I MICE AFTER NEONATAL BMT 3  We observed an inverse correlation between the extent of hematopoi-etic chimerism and GAG levels (data not shown). Correction of skeleton deformities in MPS I mice after nBMT MarkedskeletaldeformitiesintheMPSImousemodelbeganat16to24 weeks of age and included craniofacial abnormalities and thick-eningofthezygomaticarchesandthelongbones. 19 At37weeks,theMPS I mice manifested an evident skeletal dysplasia. At this age,untreatedMPSImicehadashort,broadface(Figure3A).Incontrast,the facial morphology of the MPS I nBMT-hi mice was overall nor-mal and was similar to that of WT siblings (Figure 3A). Mice withlower engraftment presented an intermediate phenotype (data not shown). In agreement with the macroscopic features, X-ray analysisdemonstrated that the width of the skull and the zygomatic archeswassigni fi cantlyhigherinMPSImiceincomparisonwithWTmice,whereasasigni fi cantreductioninbothparameterswasobservedinMPSI Figure 2. Lysosomal IDUA enzyme activity,  b -hexosaminidase enzyme activity, and GAG levels.  (A) IDUA activity was measured in spleen, liver, heart, kidney, andlung of WT (n 5 9), MPS I (n 5 8), and MPS I nBMT-hi mice (n 5 7) at 37 weeks of life. (B) The  b -hexosaminidase activity was measured in the same organs of WT (n 5 9),MPS I (n 5  8), and MPS I nBMT-hi mice (n  5 7) at 37 weeks of life. (C) At 37 weeks of age, the level of sulfated GAGs was determined, using the Blyscan assay in theindicated organs of WT (n 5 9), MPS I (n 5 8), and MPS I nBMT-hi (n 5 7). (D) At 37 weeks of age, the levels of  D DiHS-0S,  D DiHS-NS,  D Di-6S, and mono-sulfated Gal b 1-4GlcNAc(6S)(mono-sulfatedKS)weredeterminedusingLC-MS/MS Q:15  intheplasmaofWT(n 5 9),MPSI(n 5 8),andMPSInBMT-hi(n 5 7).Median,mean,andminimum/maximumvaluesareshown. Statistical comparison between groups was performed using Wilcoxon nonparametric unpaired test, 2 sides. 4 PIEVANI et al BLOOD,  nnn nnn  2014  x  VOLUME 00, NUMBER 00  nBMT-himice(skullwidth, P 5 .0014;zygomaticwidth Q:8  , P 5 .0003;vsuntreated MPS I mice) (Figure 3A-B). In addition, radiographs of MPSImiceshowedthatlongbonesofthefore-andhind-limbswerethickened and sclerotic, whereas these features were consistentlyreduced in MPS I nBMT-hi mice, as shown in Figure 3C. As ex-pected,thewidthofthehumerus,radius/ulna,femur,andtibiaofMPSImicewasalsosigni fi cantlylargerthaninWTlittermates(Figure3D).For MPS I nBMT-hi mice, the width of long bones, including the humerus Figure 3. Neonatal BMT prevents bone defects in MPS I mice.  (A) Skull morphology (on the left) and radiographs (on the right) of 37-week-old WT, MPS I, WT nBMT, andMPS I nBMT-hi mice. Signs of skull abnormalities include blunted snouts with a loss of the fine taper to the face. (B) Measurements of maximum skull width and zygomaticwidth. All measures were performed on radiographic images of WT (n 5 8; 4 males and 4 females), MPS I (n 5 8; 4 males and 4 females), WT nBMT (n 5 8; 5 males and 3females), and MPS I nBMT-hi mice (n 5 7; 4 males and 3 females), as shown on the right side of each chart. The median values relative to WT mice and minimum/maximumvalues are shown. * P  , .05; ** P  , .01; *** P  , .001 with Wilcoxon nonparametric unpaired test, 2 sides. (C) Representative examples of femur radiographs of WT, MPS I,WT nBMT, and MPS I nBMT-hi mice. (D) Measurements of femur, humerus, tibia, and radius-ulna thicknesses. The pictures on the right side of each graph indicate howmeasures were calculated. The median values relative to WT mice and minimum/maximum values are shown. * P  , .05; ** P  , .01; *** P  , .001 with Wilcoxon nonparametricunpaired test, 2 sides. BLOOD,  nnn nnn  2014  x  VOLUME 00, NUMBER 00 SKELETAL OUTCOME OF MPS I MICE AFTER NEONATAL BMT 5
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