Lentivirus Mediated Correction of Artemis-Deficient Severe Combined Immunodeficiency

14 Pages·2017·0.73 MB·English

UCSF
UC San Francisco Previously Published Works
Title
Lentivirus Mediated Correction of Artemis-Deficient Severe Combined Immunodeficiency
Permalink
https://escholarship.org/uc/item/8h27g83n
Journal
HUMAN GENE THERAPY, 28(1)
ISSN
1043-0342
Authors
Punwani, Divya
Kawahara, Misako
Yu, Jason
et al.
Publication Date
2017
DOI
10.1089/hum.2016.064

Peer reviewed
eScholarship.org Powered by the California Digital Library
University of California
Lentivirus Mediated Correction of Artemis-Deficient Severe
Combined Immunodeficiency
Divya Punwani,1,{ Misako Kawahara,1,{ Jason Yu,1,{ Ukina Sanford,1 Sushmita Roy,1
Kiran Patel,1 Denise A. Carbonaro,2 Andrea D. Karlen,3 Sara Khan,1 Kenneth Cornetta,4
Michael Rothe,5 Axel Schambach,5 Donald B. Kohn,2 Harry L. Malech,6 R. Scott McIvor,3
Jennifer M. Puck,1,*,{ and Morton J. Cowan1,*,{
1DepartmentofPediatrics,UniversityofCaliforniaSchoolofMedicineandUniversityofCaliforniaSanFranciscoBenioffChildren’sHospital,SanFrancisco,
SanFrancisco,California;2DepartmentsofMicrobiology,ImmunologyandMolecularGeneticsandPediatrics,UniversityofCaliforniaLosAngeles,LosAngeles,
California;3DepartmentofGenetics,CellBiologyandDevelopment,UniversityofMinnesota,Minneapolis,Minnesota;4DepartmentofMedicalandMolecular
Genetics,IndianaUniversity,andtheIndianaUniversityViralProductionFacility,Indianapolis,Indiana;5InstituteforExperimentalHematology,HannoverMedical
School,Hannover,Germany;6LaboratoryofHostDefenses,NationalInstituteofAllergyandInfectiousDiseases,DivisionofIntramuralResearch,
NationalInstitutesofHealth,Bethesda,Maryland.
{Theseauthorscontributedequallytothisstudy.
During B and T lymphocyte maturation, V(D)J recombination is initiated by creation of DNA double-
strand breaks. Artemis is an exonuclease essential for their subsequent repair by nonhomologous
end-joining. Mutations in DCLRE1C, the gene encoding Artemis, cause T-B-NK+ severe combined im-
munodeficiency (ART-SCID) and also confer heightened sensitivity to ionizing radiation and alkylating
chemotherapy. Although allogeneic hematopoietic cell transplantation can treat ART-SCID, conditioning
regimens are poorly tolerated, leading to early mortality and/or late complications, including short stat-
ure,endocrinopathies,anddentalaplasia.However,withoutalkylatingchemotherapyaspreconditioning,
patients usually have graft rejection or limited T cell and no B cell recovery. Thus, addition of normal
DCLRE1C cDNA to autologous hematopoietic stem cells is an attractive strategy to treat ART-SCID. We
designed a self-inactivating lentivirus vector containing human Artemis cDNA under transcriptional
regulation of the human endogenous Artemis promoter (AProArt). Fibroblasts from ART-SCID patients
transduced with AProArt lentivirus showed correction of radiosensitivity. Mobilized peripheral blood
CD34+ cells from an ART-SCID patient as well as hematopoietic stem cells from Artemis-deficient mice
demonstrated restored T and B cell development following AProArt transduction. Murine hematopoietic
cells transduced with AProArt exhibited no increase in replating potential in an in vitro immortalization
assay, and analysis of AProArt lentivirus insertions showed no predilection for sites that could activate
oncogenes.Theseefficacyandsafetyfindingssupportinstitutionofaclinicaltrialofgeneadditiontherapy
for ART-SCID.
Keywords: severe combined immunodeficiency, Artemis, gene therapy, lentivirus, radiation sensitivity
INTRODUCTION required for V(D)J rearrangement of the T and B
SEVERE COMBINED IMMUNODEFICIENCY (SCID) is a cellantigenreceptorscauseT-B-NK+SCID.These
group of genetic disorders characterized by mark- include lymphocyte specific recombinase activat-
edly decreased circulating T cell numbers with ing genes 1 and 2 (RAG1 and RAG2), as well as
impaired function plus absent or nonfunctional B ubiquitously expressed PRKDC, encoding a DNA
cells.1About20%ofSCIDpatientslackbothTand proteinkinasecsubunit,LIG4,encodingLigase-4,
Bcells,buthavenormalnumbersofnaturalkiller a nonhomologous end-joining DNA repair compo-
(NK) cells (T-B-NK+ SCID).2 Mutations in genes nent, and DCLRE1C, encoding Artemis. Because
*Correspondence:Dr.JenniferM.PuckorDr.MortonJ.Cowan,SmithCardiovascularResearchInstitute,UniversityofCaliforniaSanFrancisco,555MissionBayBoulevard
South,RmSC-252K,SanFrancisco,CA94158-9001.E-mail:[email protected]@ucsf.edu
112 j HUMANGENETHERAPY,VOLUME28NUMBER1 DOI:10.1089/hum.2016.064
ª2017byMaryAnnLiebert,Inc.
GENE ADDITION THERAPY FOR ARTEMIS-DEFICIENT SCID 113
the latter three are required for repair of DNA vector with the human Artemis DCLRE1C cDNA
double-strand breaks, defects in these genes also under transcriptional regulation of its own human
causeincreasedsensitivitytoionizingradiationand Artemis promoter, designated AProArt. We used
alkylatingagentsusedaspreparativeregimensfor AProArttotransducehumanART-SCIDfibroblasts
hematopoieticcelltransplantation(HCT).1 and CD34+ HSC as well as HSC from Art-/- mice,
Early diagnosis of SCID by newborn screening achieving correction of radiation sensitivity in
using the T cell receptor excision circle assay,2–4 transduced human ART-SCID fibroblasts and suc-
followed by definitive treatment by allogeneic cessful in vivo and in vitro differentiation of trans-
HCT,leadstoexcellentratesofsurvival,exceeding ducedArtemis-deficientHSCintoTandBcells.
90%.5,6HCTfromhumanleukocyteantigen(HLA)-
identical siblings or unrelated donors results in
METHODS
the best outcomes, with 97% and 92% survival re-
spectively.7 However, T-depleted, haploidentical Animals
HCT may require preparative chemotherapy to Mice were housed in sterile isolator cages and
avoidrejectionandachieveBcellreconstitution.6,8 fed autoclaved chow, undergoing procedures ac-
Moreover,forrecipientsofHCTfromdonorsother cording to approved protocols at the University of
than HLA matched siblings, graft versus host dis- California, San Francisco. Art-/- mice have been
ease (GVHD) can be a major complication with described previously,20 and NSG mice were from
significantmorbidityandmortality.6 theJacksonLaboratory(BarHarbor,ME).
The DCLRE1C gene encoding Artemis was first
identified in patients with T-B-NK+ SCID (ART- Lentivirus assembly, production
SCID)whohadincreasedcellularradiosensitivity.9,10 and determination of titer
Artemis is an endonuclease essential for nonho- A 3750bp DNA fragment was synthesized to
mologousend-joiningofDNAdouble-strandbreaks includetheDCLRE1Cpromoter(APro)from-1000
thatarisefollowingexposuretoexternalagentsor to the translational start site (NG_007276.1, NM_
during V(D)J recombination of T and B cell recep- 1033855.2),23the1024bphumanDCLRE1Ccoding
tor genes.9 Navajo and Apache Native Americans sequence and 3¢ untranslated region, and a 590bp
from the southwestern United States have a fre- woodchuckpost-transcriptionalregulatoryelement
quent founder nonsense mutation in exon 8 of (WPRE). The putative protein-encoding sequence
DCLRE1C, Y192X.11,12 Homozygosity for this within the WPRE was modified to replace its
mutationcausesT-B-NK+SCIDin1/2000births, translational start site (GCTGAcgtcctttccAtg) with
or 25 times the incidence of SCID in the general a stop codon (ATCATcgtcctttccTtg) (modified bases
population.12–14 in uppercase letters). The synthetic MfeI–KpnI
WhileHCTcancureART-SCID,theheightened fragment was cloned into lentiviral vector pCSI-
sensitivities to irradiation and alkylator chemo- CDF-CG-PRE24betweenEcoRIandKpnItogener-
therapy conferred by DCLRE1C mutations are ate AProArt. A cytomegalovirus–green fluorescent
associated with increased early toxicity as well as protein (CMV-GFP) control vector with the same
late effects in survivors, including short stature, modified WPRE was generated by replacing the 5¢
absent or malformed permanent dentition, and end of pCS-AProArt through the DCLRE1C pro-
endocrinopathies.15,16Thus,usinggeneadditionto motor and coding sequences with a PvuI – XhoI
autologous hematopoietic stem cells (HSC) consti- fragment isolated from pCSI-CDF-CG-PRE, gener-
tutes an alternative strategy for the treatment of atingpCS-CMV-GFP.TheAProArtlentiviralvector
ART-SCID to avoid the observed risks of partially was produced under good manufacturing practice
matchedorunrelatedHSCallotransplant.17–19 conditions (Indiana University Vector Production
We previously described a mouse model of ART- Facility, Indianapolis, IN). Briefly, 293T cells were
SCID (Art-/-) that accurately represents both the transfected with plasmids encoding AProArt pro-
T and B cell lymphopenia and the resistance to virus and vesicular stomatitis virus envelope G
treatmentbyallogeneic mismatched HCT found in protein; lentiviral supernatant was harvested 48-
ART-SCIDpatients.20,21Usingthismodel,Multhap 72 hours after transfection and concentrated by
et al. demonstrated restoration of functional T and ultracentrifugation.ThecontrolGFPlentiviruswas
B lymphocytes by ex vivo complementation of the produced in-house using the same protocol. Lenti-
Artemis deficiency in murine stem cells using a viral titering in 293T cells was performed with se-
vectorcarryingmurineArtemiscDNAdrivenbythe rially diluted lentivirus; after 3 days the 293T cell
human Artemis promoter.22 Building upon these DNA was isolated for vector-specific quantitative
studies, we have now developed a novel lentiviral PCR(primersequencesavailableonrequest).
114 PUNWANI ET AL.
In vitro assays with human fibroblasts and murine cytokines: interleukin-3 (20ng/mL,
R&D Systems) and Flt3L, SCF, and TPO (100ng/
Immunoprecipitation and Western blotting. Skin
mL, R&D Systems) for 24h. AProArt or GFP len-
fibroblasts from Artemis-deficient (ART-SCID) and
Ligase-4 deficient patients and healthy controls tiviruswasaddedatafinalconcentrationof5·107
wereculturedinDulbecco’smodifiedEaglemedium IU/mL for 24h at 37(cid:2)C. One hundred thousand
transducedArt-/-HSCor104or105WTHSCwere
with 10% fetal calf serum and antibiotics. Fibro-
injected into Art-/- CD45.2 recipient mice pre-
blasts were incubated with GFP or AProArt lenti-
treated with 500lg of ultra-purified anti-mouse
virusatmultiplicityofinfection100for24h.Protein
CD117(anti-c-kit,ACK2,Biolegend)9dayspriorto
wasisolated3or8dayspost-transductionandpre-
transplant.
cipitated using a rabbit anti-Artemis antibody
(kindly provided by Steve Yannone, Laurence Ber-
Functional assays with murine lymphocytes
keleyLaboratory,Berkeley,CA).ForWesternblot,
achickenpolyclonalanti-Artemisantibody(Abcam) Tcellproliferation. Splenocytesfromtransplant
wasusedfollowedbyaperoxidase-conjugateddonkey recipientswereisolatedandresuspendedinRPMI
anti-chickensecondantibody(GallusImmunotech) 1640 with 10% fetal bovine serum and antibiotics.
todetectprecipitatedprotein. One hundred thousand splenocytes per well in a
96-well plate were stimulated with 4 or 10lg/mL
Proliferation. Fibroblasts were exposed to 1, 3, ofConcanavalin-A(ConA,Sigma-Aldrich),5lg/mL
or 5 Gy c-radiation 3 days after lentivirus trans- plate-bound anti-mouse CD3e (Tonbo), or medium
duction. After 24h, the cells were labeled with onlyat37(cid:2)Cfor4days.Proliferationwasmeasured
BrdU for 24h, harvested, fixed, and stained with by EdU incorporation (Click-iT EdU imaging kit,
anti-BrdUantibody(BDBiosciences)followingthe Life Technologies), following the manufacturer’s
manufacturer’s instructions. Cell cycle distribu- instructions using a FACSVerse flow cytometer
tion and BrdU incorporation were determined by (BD Biosciences). The assays were carried out in
flowcytometryusingBDFACSVerseandanalyzed triplicate.
usingFlowJosoftware.
Enzyme-linked immunosorbent assay for serum
cH2AX foci. Fibroblastswereseededoncham- immunoglobulin M and immunoglobulin G anti-
ber slides (Nunc Lab-Tek) and exposed to 4 Gy body. ELISA using serum from transplanted
c-radiation 3 days after transduction. After 6, 24, mice injected intraperitoneally with 100lg of ei-
48, and 168h (7 days) the cells were fixed, per- ther NP -Ficoll or NP -keyhole limpet hemocya-
41 24
meabilized, blocked with 5% goat serum, and in- nin (KLH) (Biosearch Technologies, Novato, CA)
cubatedwithmouseanti-cH2AX(Ser139)(Upstate wascarriedoutasdescribed.20
Antibodies) and rabbitanti-53BP1 (Bethyl Labora-
tories)overnightat4(cid:2)C.Thecellswerewashed,in- Transduction and differentiation
cubated with fluorochrome-conjugated secondary of human CD34+ HSC
antibodies (Jackson ImmunoResearch) for 45min With informed consent cytokine-mobilized pe-
at room temperature, washed thrice, stained with ripheral blood stem cells (PBSC) were collected
DAPI, mounted in Vectashield (Vector Labora- from an ART-SCID patient, under an internal re-
tories) and analyzed using a fluorescence micro- view board–approvedprotocol(Laboratoryof Host
scope(Keyence). Defenses, National Institute of Allergy and In-
fectious Diseases, National Institutes of Health),
Isolation, transduction, and transplantation and CD34+HSCwereisolated(CliniMACS, Milte-
of murine bone marrow hematopoietic nyiBiotechnology).De-identifiedCD34+cellsfrom
stem cells PBSCofhealthydonorswereusedascontrolHSC.
Femurs,tibias,andfibulasfromdonorwild-type HumanHSCwereprestimulatedinrectronectin-
(WT) or Art-/- CD45.1 mice were flushed and cru- coatedsix-wellplates(1–2·106cells/mL),inX-Vivo-
shed to isolate bone marrow cells. After depleting 15(Lonza)withhumanSCF,Flt3L,thrombopoietin
lineage committed cells (mouse depletion kit, Mil- (Peprotech, all 100ng/mL), IL-3 (Peprotech, 20ng/
tenyi), cells were stained with c-kit and Sca-1 mL), 1% human albumin, and gentamicin (50lg/
(clones2B8andD7,respectively,eBioscience)and mL)for24h.AProArtorGFPlentiviruswasadded
lineagenegative,Sca1positive,c-kitpositive(LSK) at a final concentration of 1·108 IU/mL for 8h at
HSC were sorted (FACSJazz, BD Biosciences). 37(cid:2)C. The cells were then washed and cultured
LSK cells were plated in Rectronectin-coated 96- overnight in fresh medium, followed by a second
well plates in X-Vivo-15 (Lonza) with L-glutamine transduction with fresh lentivirus for 8h. Aliquots
GENE ADDITION THERAPY FOR ARTEMIS-DEFICIENT SCID 115
wereusedforprogenitorcolonyformationassaysin ence, human Artemis cDNA driven by its own en-
semisolidmediumwithcytokines(Methocult,Stem dogenous promoter was cloned into self-
CellTechnologies),25and106transducedcellswere inactivating lentiviral vector CSII with promoter
transplantedbyintrahepaticinjectioninto150cGy and enhancer sequences deleted from the U3 re-
irradiatednewbornto3dayoldNSGmice.Themice gionofthe3¢LTR(Fig.1).24Thecentralpolypurine
werebledevery4weeksandsacrificedat3months tract was included to increase the virus titer and
of age for analysis of peripheral blood, bone mar- the frequency of integration into the host cell ge-
row, and spleen for human cell chimerism by flow nome. This AProArt vector included a modified
cytometry. WPREelementtoincreasetransgeneexpressionin
which the putative X protein start codon was mu-
tated to a stop to reduce the risk of insertional
Determination of vector copy number
mutagenesis. A control CMV-GFP vector was con-
and lentivirus integration site analysis
Lentiviral vector copy number for transduced structedwiththesamebackbone(Fig.1).Thetiter
murine LSK cells and human CD34+ cells was de- ofeachlentiviruswasabout2·109IU/mL.
termined as described.25,26 The virus integration
site assay was carried out as described.27 Briefly, Human Artemis expression and rescue from
genomicDNAfromlentivirus-transducedcellswas radiation toxicity in ART-deficient fibroblasts
TodemonstrateArtemisproteinexpressionand
digestedandligatedwithadaptors,andtworounds
function following transduction with AProArt len-
ofnestedPCRwereperformed.PCRproductswere
tivirus, we transduced control (WT), Artemis-
cloned, analyzed by Sanger or next generation se-
deficient(ART-SCID)andligase-4deficient(LIG4)
quencing (HiSeq2500, Illumina), mapped to the
humanfibroblastcelllineswithAProArtorcontrol
human genome, and examined for oncogenic po-
GFPlentiviruses.LikeArtemisdeficiency,ligase-4
tentialbyGeneOntologysoftware.
deficiency results in radiation-sensitive SCID;31,32
therefore, LIG4 fibroblasts provided a control for
In vitro immortalization assay
The in vitro immortalization (IVIM) assay was specificityoftheeffectsofAproArttransduction.At
carried out as described.25,28 Briefly, prestimu- 3 days post transduction, Artemis protein was
lated, lineage negative murine bone marrow cells identified by Western blot in WT and LIG4 fibro-
were transduced with AProArt lentivirus; a long blasts and in AProArt-transduced ART-SCID fi-
terminal repeats (LTR)-regulated c-retroviral pos- broblasts,butnotinuntreatedorGFP-transduced
itive control (RSF91); or an intermediate positive ART-SCIDfibroblasts(Fig.2a).Similarexpression
control, a self-inactivating-lentivirus with an in- wasseenat8days(datanotshown).
ternal strong spleen focus-forming virus element. Wealsoevaluatedproliferationofthefibroblasts
Thesegene-modifiedcellswereexpandedalongside following exposure to 0–5 Gy of c-irradiation (IR)
anontransducednegativecontrol.After14daysof (Fig. 2b). Without transduction, both the LIG4
expansion, the cultures were harvested and re- deficient (middle panel) and untransduced ART-
platedin96-wellplateswith100cells/well.After2 SCID (Fig. 2b, right panel) fibroblasts were radia-
weeks, the wells were scored for immortalized
clones.Differencesintheincidence(Fisher’sexact)
and replating frequency (Mann-Whitney) were
consideredsignificantatp<0.05.
RESULTS
Lentiviral vector design and production
Previous studies demonstrated that high-level Figure 1. Schematic representations of AProArt and control cytomega-
lovirus–greenfluorescentprotein(CMV-GFP)lentivirusvectors.Thehuman
Artemis protein expression was associated with
Artemis (DLCRE1C) cDNA (Hu Artemis), with its endogenous promoter
DNA damage, increased apoptosis, and cell cycle (APro) was introduced into self-inactivating lentiviral vector pCS with
arrestatG1.29TheendogenoushumanDCLRE1C promoterandenhancersequencesdeleted(X)fromtheU3regionofthe3¢
minimal 5¢ promoter (APro) was hypothesized to LTR to generate pCS-AProArt. A central polypurine tract (CPPT) was in-
cluded in the vector backbone, preceded by the packaging site (w). The
producemoremoderateandphysiologicexpression
vectorincludesamodifiedwoodchuckpost-transcriptionalregulatoryele-
of Artemis; reduced toxicity had been demon- ment(WPRE)anda5¢CMVpromotertogeneratefull-lengthvectormes-
strated in murine Artemis expression comparing sageforpackaging.Thecontrollentiviralvectorwiththesamebackbone,
APro versus the strong human elongationfactor 1 pCS-CMV-GFP, was constructed to express GFP under transcriptional
regulationoftheCMVpromoter.LTR,longterminalrepeats.
alpha (EF1a) promoter.30 Building on this experi-
116 PUNWANI ET AL.
Figure2. Invitroassayswithhumanskinfibroblasts.(a)ImmunoprecipitationWesternblottodetectArtemisprotein(upperarrow)infibroblastsfromcontrol
(wildtype,WT),Artemisdeficient(ART-SCID)orLigase-4deficient(LIG4)skinfibroblasts,untransduced(left)ortransducedwithGFP(middle)orAProArtlentivirus
(right).(b)BrdUfibroblastproliferationwithWT(left),LIG4deficient(middle),orART-SCID(right)cells,followingexposuretoionizingradiation(IR).Thefibroblasts
wereuntransduced(brokenline)ortransducedwithcontrol(grayline)orAProArtlentivirus(blackline).Mean–standarddeviation(SD),n=3replicates;*p<0.05.
(c)EnumerationofcH2AXfociindicatingDNAdouble-strandbreaks,inWT(left),LIG4deficient(middle),orART-SCID(right)fibroblasts,followingexposureto4Gy
IR.Thefibroblastswereeitheruntransduced(brokenline)ortransducedwithAProArt(solidline).Mean–SD,n=3replicates;*p<0.05;**p<0.001.
tion sensitive, exhibiting up to 10-fold reduced leftpanel),atalltimepointspostIR.ThusAProArt
proliferation compared to WT (Fig. 2b, left panel) lentivirus specifically reversed the DNA repair
followingexposuretogradedIRdoses.TheWTand defectofArtemis-deficienthumanfibroblasts.
LIG4cellsshowednochangeinproliferationwhen
transduced with either control vector or AProArt; Determination of optimal AProArt lentivirus
however, ART-SCID cells following AProArt concentration for transduction of human
transduction proliferated significantly more than CD34+ HSC
untransduced or GFP-transduced ART-SCID cells Previous studies suggested that overexpression
(p<0.05), achieving a level comparable to that of of DCLRE1C could cause cellular cytotoxicity.23
untransducedWTfibroblasts. Moreover, Art-/- murine HSC transduced with
Finally,weevaluatedcH2AXfociasanindicator murine Artemis cDNA under transcriptional reg-
of ongoing repair of DNA damage; elevated num- ulationofastrongEF1apromoterfailedtoengraft
bersoffocipersistingovertimeindicateincomplete in Art-/- recipients.22 To determine the optimal
DNArepair(Fig.2c).AProArttransductiondidnot concentration of AProArt lentivirus for trans-
change the number of cH2AX foci either at peak duction of human CD34+ HSC, we examined the
production (6 hours) or after 1 to 7 days in either relationshipbetweenlentivirusconcentration,trans-
theWTor theradiationsensitiveLIG4fibroblasts duction rates and resulting vector copy number
(Fig. 2c, left and middle panels); in contrast, DNA (VCN) per cell. Human mobilized PBSC from nor-
repair was normalized in AProArt-transduced mal,Artemis-repletedonorsweretransducedwith
ART-SCID fibroblasts (Fig. 2c, right panel). The increasing concentrations (1.25·107 to 2·108 IU/
numbers of cH2AX foci in transduced ART-SCID mL)ofAProArtlentivirusandculturedfor14days
cellswerecomparabletothoseofWTcells(Fig.2c, in either semi-solid medium for colony forming
GENE ADDITION THERAPY FOR ARTEMIS-DEFICIENT SCID 117
assays or in liquid bulk cultures. With increasing evidencedbythepresenceofdonor-derivedCD45.1
AProArt lentivirus concentration, theproportion of lymphocytes in their peripheral blood, five of six
transduced colonies increased, reaching approxi- transplanted Art-/- mice engrafted with 105
mately50%atboth1·108IU/mLand4·108IU/mL AProArt-transduced Art-/- LSK HSC and all
(Fig.3a).With1·108IU/mLofAProArtlentivirus, transplantedArt-/-miceengraftedwithWTdonor
an average VCN of 3 was achieved (Fig. 3b). cells (Fig. 4a, black squares). Chimerism for T, B,
Therefore, this concentration of lentivirus was se- and myeloid lineages in Table 1 indicated multi-
lectedforsubsequentexperiments. lineageengraftment.
The post-transplant splenocytes were cultured
Correction of immunodeficiency in medium alone or with ConA (4 or 10lg/mL) or
in Artemis-deficient mice anti-mouse CD3e (5lg/mL), with proliferation of
To determine whether human AProArt trans- gated populations measured by flow cytometry
ductioncouldrescuetheabilityofArt-/-LSKHSC 4dayslater(Fig.4b).Donor-derivedCD45.1Tcells
to differentiate into T and B lymphocytes, CD45.2 in Art-/- recipients of either WT (hatched bars) or
Art-/- mice pretreated with 500lg of anti-ckit an- AProArt-transduced Art-/- HSC (Fig. 4a, black
tibody9dayspreviously,toopenmarrowniches,33 bars) proliferated in response to stimuli. Analysis
were injected with either 105 CD45.1 Art-/- LSK of absolute numbers of CD4+ and CD8+ T cells in
cells transduced with AProArt, or 104 or 105 un- the spleens showed that Art-/- mice transplanted
transduced CD45.1 WT LSK cells (Fig. 4a). As with WT or AProArt-transduced Art-/- HSC had
comparable numbers of CD4 helper and CD8 cy-
totoxicTcells,significantlymorethanthoseseenin
Art-/-controls(Fig.4c).
Art-/-micetransplantedwithAProArt-transduced
Art-/- HSC showed B cell development in the bone
marrow, as did those receiving WT HSC, while
untransplanted Art-/- mice had undetectable im-
matureBcells(Fig.4d).Toassessspecificantibody
production in vivo, mice were immunized 16–24
weeks following transplant with 100lg of either
NP -Ficoll or NP -KLH. Serum ELISA assays
41 24
42 days after immunization revealed that Art-/-
micetransplantedwithAProArt-transducedArt-/-
HSC produced antigen-specific IgM and IgG in
amounts comparable to those produced by Art-/-
mice transplanted with WT HSC (Fig. 4e). Un-
transplanted Art-/- mice produced no immuno-
globulin M or immunoglobulin G.
Colony forming assays with the AProArt-
transduced Art-/- LSK HSC used for the above
experiments showed a transduction efficiency of
87.5% and a mean vector copy number of 3–2 per
cell in positive colonies. The average VCN in the
different lineages of the Art-/- mice transplanted
withgenecorrectedcellswas3–2inTcells,3–1in
Bcells,and2–2ingranulocytes.
Transduction and in vivo differentiation
of human HSC
Figure 3. Relationship between vector copy number or transduction ef-
ficiency, and lentivirus concentration. Healthy control human peripheral Cytokine-mobilized PBSC were obtained from
bloodmobilizedCD34+cellstransducedwithincreasingconcentrationsof an ART-SCID patient, homozygousfor the Navajo
AProArtlentivirus(blackbars)wereculturedfor14days.(a)Percentageof
foundermutation,whowas20yearspostHCTand
lentivirus transduced colonies per total hematopoietic colonies isolated
had partial T cell and no B cell reconstitution.34
from semisolidmedium, determined by quantitativePCR of individual col-
onies.Mean–SD,n=3replicates.(b)Averagevectorcopynumberpertwo Isolated CD34+ HSC had no detectable cells of do-
copiesofahumangenomiccontrolincellsgrowninliquidbulkcultures, nororigin(datanotshown).TheseART-SCIDHSC
determinedbyquantitativePCR.Mean–SD,n=3replicates. were transduced with 1·108 IU/mL AProArt or
Figure4. AnalysisofArt-/-mice,transplantedwithCD45.1donorcells.(a)Percentageofdonor-derivedCD45.1lymphocytesinperipheralbloodat4,8,12,16,
and20weeksposttransplantationintoArt-/-CD45.2recipientspretreatedwith500lgofanti-c-kitantibody.Micereceived105CD45.1AProArt-transduced
Art-/-(blacksquares,n=5),or104(X’s,n=5)or105untransducedwildtype(WT,graycircles,n=3)hematopoieticstemcells(HSC).Horizontallinesindicate
meanchimerism.(b)ProliferationofCD3+splenocytesfromuntransplantedcontrolArt-/-(clearbars,n=5);WT(graybars,n=7);orArt-/-micetransplanted
with104untransducedWTHSC(hatchedbars,n=4)orAProArtlentivirustransducedArt-/-HSC(blackbars,n=6),measuredasapercentageofEdUpositive
cellsafter4daysinmedium,4lg/mLor10lg/mLConcavalinA(ConA),or5lg/mLanti-mouseCD3e.Mean–standarderrorofthemean(SEM),n=3replicates;
*p<0.05,**p<0.001.(c)CD8+(blackbars)andCD4+(graybars)TcellnumbersincontrolArt-/-(n=5)andWT(n=5)mice,andArt-/-micetransplantedwith
104WTHSC(n=7)orAProArtlentivirustransducedArt-/-HSC(n=6).Mean–SEM;*p<0.05,**p<0.001,comparedtoArt-/-control.(d)Bcelldevelopmentin
bonemarrowofuntransducedcontrolArt-/-(clearbars,n=3);WT(graybars,n=3);andArt-/-mice3monthsaftertransplantwith104WTHSC(hatchedbars,
n=5)orAProArt-transducedArt-/-HSC(blackbars,n=5).Bcellprogenitorsubsetswere:CD45+B220+CD43+proBcells;CD45+B220+CD43-IgM-preB
cells; and CD45+ B220+ CD43- IgM+ immature B cells,17 as a percentage of CD45+ B220+ B cells. Mean–SEM; *p<0.05. (e) Antigen-specific serum
immunoglobulin(Ig)MandIgGantibodyresponsestoimmunizationwithNP-KLH(minuspre-immuneresponse)inWT(blackdiamonds,n=3)andArt-/-(black
squares,n=3)controlmice,andArt-/-recipientsofArt-/-HSCtransducedwithAProArtlentivirus(blacktriangles,n=3)or104WTHSC(grayrectangles,
n=3).Mean–SD,n=3replicates;*p<0.05comparedtoArt-/-controls.NP-KLH, NP24-keyholelimpethemocyanin.
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GENE ADDITION THERAPY FOR ARTEMIS-DEFICIENT SCID 119
Table1. T,B,andmyeloidchimerism(mean–SD)inArt-/-CD45.2recipientsofCD45.1,AProArttransducedArt-/-,
orcontroluntransducedlineagenegative,Sca1positive,c-kitpositivehematopoieticstemcells
%Donor %Donor %Donor %Donor %Donor
DonorCells WeeksPostHCT (CD45.1)a Lymphocytes CD3+TCells B220+Bcells Granulocytes
105Art-/-transducedwithAProArt 12 19.47–10.48 51.58–8.32 36.57–2.01 0.35–0.53 6.37–4.39
16 26.56–18.98 52.72–21.31 37.85–20.70 0.73–0.46 8.77–7.10
20 36.08–14.29 60.32–17.34 45.28–17.34 0.97–0.70 11.83–3.03
104WTuntransduced 12 23.38–7.83 56.94–14.98 44.76–12.51 3.02–3.29 8.58–4.54
16 34.92–11.03 70.72–9.01 43.63–8.67 3.11–2.35 13.71–7.67
20 42.96–13.11 71.82–12.14 44.45–10.84 3.49–2.19 11.51–9.45
105WTuntransduced 12 34.43–18.57 63.97–14.9 58.35–16.93 1.15–0.79 16.35–11.76
16 42–19.41 66.37–17.35 61.66–19.85 2.5–2.05 12.91–4.35
20 50.43–9.29 87.07–4.60 81.58–5.62 2.50–1.74 17.87–1.99
aMean–SDofatleastfivemicepergroup.
HCT,hematopoieticstemcelltransplantation;WT,wildtype(control).
GFP lentivirus and injected into irradiated, new- contrast, mice receiving GFP-transduced ART-
born to 3 day old NSG mice to assess whether SCID CD34+ cells had no evidence of Ig j re-
transduction with AProArt would correct the arrangement.
DCLRE1Cdefect(Fig.5).CD34+HSCfromhealthy RNA from chimeric NSG mouse spleens was
donors weresimilarly transduced with the control subjectedtoTcellreceptorspectratypingtomeasure
GFP virus and injected into irradiated recipients. the diversity of human T cell receptor rearrange-
At 4, 6, 8, and 10 weeks, peripheral blood was an- ment(representativeexamplesinFig.5d).36Again,
alyzed by flow cytometry for the appearance of mice with GFP-transduced normal or AProArt-
human lymphocytes bearing the human pan- transducedART-SCIDHSCshowedseveralhuman
leukocyte marker CD45, and at 12 weeks bone recombinants yielding oligoclonal and polyclonal
marrow and spleens were harvested. NSG recipi- tracingswithinthe24Tcellreceptor,Vbeta(TCR
ents of GFP-transduced control HSC, as well as Vb) families tested, while mice transplanted with
those transplanted with AProArt-transduced ART- GFP-transduced ART-SCID CD34+ cells had only
SCIDHSC,exhibitedsustainedhumanlymphocyte minimalTCRVbrearrangement.
chimerism(Fig.5a).Theaveragepercentofhuman
CD45 cells detected in the periphery of mice that AProArt lentiviral vector copy number
receivedgenecorrectedcellsfrom8weeksonwards, and integration site analysis
represented 27% of the cells detected in NSG mice Colony forming assays with the AProArt-
transplanted with WT HSC, corresponding to the transduced ART-SCID HSC used for the above
25% transduction efficiency of the ART-SCID cells. experiments showed a transduction efficiency of
GFP lentivirus-transduced ART-SCID HSC did not 25.4%andameanvectorcopynumberof2–1per
differentiate into mature lymphocytes, however, re- cell in positive colonies, confirming that human
flectingtheiruncorrectedDCLRE1Cdefect.AProArt transduced HSC were within an acceptable in-
transduced ART-SCID CD34+ cells gave rise to dif- tegrant range for clinical application.
ferentiatedhumanCD3+TandCD19+Bcells,while Lentivirus integration site analysis in bulk cul-
GFP-transducedART-SCIDCD34+cellsfailed todo turedAProArtorGFPtransducedCD34+cellsfrom
so(Fig.5b).ControlCD34+cells,whethertransduced healthydonorsshowedavarietyofinsertionsites.27
withAProArtorwithGFP,differentiatedintoTand A total of 1,468 unique integration sites were re-
Bcellsinperipheralblood,bonemarrow,andspleen covered in AproArt-transduced samples, of which
ofNSGmice. 924 (63%) were located within the promoter or ge-
DNA from the bone marrow of NSG recipient nomic span of human reference gene sequences.37
micewasusedtoconfirmdenovopolyclonalBcell InGFP-transducedsamples,atotalof1,529unique
developmentbyassayingforIgj-receptorexcision integration sites were found, with 976 (64%) map-
circles and rearranged linear mature gene coding pingtoreferencegenes.Todeterminewhetherthere
joints (Kde CJ), by quantitative PCR (Fig. 5c). 35 wereanypreferredvirusintegrationsites,thegene
Mice receiving GFP-transduced normal HSC or lists were examined using the DAVID Bioinfor-
AProArt-transduced ART-SCID HSC had equiva- maticsResources6.8,38,39forgeneontologyanalysis.
lent j-receptor excision circles and coding joints, Following functional annotation (Biological Process
indicating that the human CD34+ cells had differ- Database,‡3-foldenrichment,Benjamini-Hochberg
entiated into mature Ig j expressing B cells. In corrected p value <0.01), the gene list from the
Figure5. AnalysisofNSGmicetransplantedwithhumanCD34+HSC.(a)Percentofhumancells,identifiedbyexpressionofhuman-specificpan-leukocyte
markerCD45,detectedinperipheralblood(PB),bonemarrow(BM)orspleen,analyzedbyflowcytometryatweeks(w)indicatedposttransplantationwith
GFP-transducedcontrol(WT)HSC(clearbars,n=6),AProArt-transducedART-SCIDHSC(blackbars,n=7)orGFP-transducedART-SCIDHSC(graybars,
n=5).Mean–SD;*p<0.05.(b)FractionofhumanCD19+Bcells(leftpanel)orCD3+Tcells(rightpanel)analyzedbyflowcytometry,intotalhumanCD45+cells
detectedintheperipheralblood(PB),bonemarrow(BM)andspleenofNSGmicetransplantedwithGFP-transducedcontrolHSC(clearbars,n=6),AProArt-
transducedART-SCIDHSC(blackbars,n=7)orGFP-transducedART-SCIDHSC(graybars,n=5).Mean–SD;*p<0.05.(c)QuantificationofhumanBcellIgj-
chain rearrangement by quantitative PCR, identified as j-chain restriction circles (KRECs) and Kde coding joints (Kde CJ), using DNA from NSG mice
transplantedwithGFP-transducedWTHSC(clearbars,n=6),AProArt-transducedART-SCIDHSC(blackbars,n=7)orGFP-transducedART-SCIDHSC(gray
bars,n=5).Mean–SD;*p<0.05;**p<0.001.(d)Tcellreceptordiversityin24humanVbTcellreceptor(TCR)familiesbyspectratyping,usingRNAfrom
spleensofNSGmicetransplantedwithGFP-transducedcontrolHSC(left),AProArtlentivirustransducedART-SCIDHSC(middle)orGFP-transducedART-SCID
HSC (right). Monoclonal pattern, single peak per Vb TCR family; oligoclonal, 1–4 peaks; polyclonal, ‡5 peaks. Color images available online at www
.liebertpub.com/hum
120 j