Supplementary Materials for

Preclinical modeling highlights the therapeutic potential of

hematopoietic stem cell gene editing for correction of SCID-X1

Giulia Schiroli, Samuele Ferrari, Anthony Conway, Aurelien Jacob, Valentina Capo,

Luisa Albano, Tiziana Plati, Maria C. Castiello, Francesca Sanvito, Andrew R. Gennery,

Chiara Bovolenta, Rahul Palchaudhuri, David T. Scadden, Michael C. Holmes,

Anna Villa, Giovanni Sitia, Angelo Lombardo, Pietro Genovese,* Luigi Naldini*

*Corresponding author. Email: genovese.pietro@hsr.it (P.G.); naldini.luigi@hsr.it (L.N.)

Published 11 October 2017, Sci. Transl. Med. 9, eaan0820 (2017)

DOI: 10.1126/scitranslmed.aan0820

The PDF file includes:

Materials and Methods

Fig. S1. Humanized SCID-X1 mice.

Fig. S2. Phenotypical and functional characterization of humanized SCID-X1

mice.

Fig. S3. Hematopoietic reconstitution and functional studies of WT/SCID-X1

competitive transplants.

Fig. S4. Phenotypical characterization of lymphoblastic T lymphomas developing

in mice transplanted without irradiation.

Fig. S5. Molecular and functional characterization of T lymphoma.

Fig. S6. Depletion of hematopoietic compartments with CD45-SAP in SCID-X1

mice.

Fig. S7. Development of a gene editing strategy for mouse HSPCs.

Fig. S8. Functionality of gene-edited lymphoid cells from transplanted mice.

Fig. S9. Functional validation of IL2RG-edited primary human T cells.

Fig. S10. Tailoring of gene editing protocol for human HSPCs.

Table S1. Phenotypical characterization of humanized SCID-X1 mice.

Table S2. Intron 1 IL2RG ZFNs off-target list.

Table S3. List of genomic gRNA target sequences.

Table S4. List of primers and probes.

Table S5. List of antibodies for flow cytometry.

References (37–43)

www.sciencetranslationalmedicine.org/cgi/content/full/9/411/eaan0820/DC1

Other Supplementary Material for this manuscript includes the following:

(available at

www.sciencetranslationalmedicine.org/cgi/content/full/9/411/eaan0820/DC1)

Table S6. Raw data for Table 2 (provided as an Excel file).

Supplementary Materials

Materials and Methods

Mice

Humanized SCID-X1 embryonic stem cells (ESCs) were generated by performing homologous

recombination of the human IL2RG into the mouse Il2rg locus, followed by neomycin selection of

targeted cells and subsequent deletion of the neomycin expression cassette via Cre-lox recombination

by InGenious Targeting Laboratories. Successfully targeted ESC clones were sequenced to confirm the

presence of the introduced human sequence and the deletion of the neomycin resistance cassette.

Selected ESC clones were then microinjected into Balb/c blastocysts. Resulting chimeras with a high

percentage of black coat color were mated to wild-type C57BL/6N mice to generate heterozygous

offspring.

C57BL/6N, C57BL/6-Ly5.1, Gt(ROSA)26Sortm1.1(CAG-cas9*,-EGFP)Fezh/J, and NOD-SCID-

IL2Rg-/- (NSG) mice were purchased from The Jackson Laboratory. Gt(ROSA)26Sortm1.1(CAG-

cas9*,-EGFP)Fezh/J mice were then crossed to humanized SCID-X1 C57BL/6N mice. All the mice

were maintained in specific-pathogen-free (SPF) conditions, and the procedures involving animals

were designed and performed with the approval of the Animal Care and Use Committee of the San

Raffaele Hospital (IACUC #528, 817) and communicated to the Ministry of Health and local

authorities according to Italian law.

Vectors and nucleases

LV donor templates for HDR were generated using HIV-derived, third-generation self-inactivating

transfer constructs. IDLV stocks were prepared and titered as previously described (3). Where

indicated, IDLVs were purified through endonuclease treatment and anion exchange chromatography

with gradient elution and gel filtration.

AAV6 donor templates for HDR were generated from a construct containing AAV2 inverted terminal

repeats, produced by triple-transfection method and purified by ultracentrifugation on a cesium

chloride gradient as previously described (8).

ZFNs that target the AAVS1 locus or exon 5 of IL2RG were previously described (7, 30). Lead ZFNs

targeting intron 1 of IL2RG were identified after several rounds of optimization, including changes to

DNA binding modules within the ZFP and inter-module linkers. A final pair of lead ZFNs was then

determined after a final round of optimizations for enhanced specificity at the 7 off-target sites as well

as enhanced on-target activity in human mobilized PB CD34+ cells.

ZFNs were transiently expressed from in vitro transcribed mRNAs as previously described (7). For

gene editing experiments in mouse cells and in human cells, when indicated, modified nucleotides (5-

mC and U, TriLink Biotechnologies) were incorporated during the in vitro transcription procedure.

ZFN mRNAs were purified by reverse phase dHPLC (Transgenomic) when indicated with a protocol

adapted from (37).

Sequences of the gRNAs were designed using an online CRISPR design tool (38) and selected for

optimal predicted specificity score. Genomic sequences recognized by the gRNAs are indicated in table

S3. gRNA3 (IL2RG exon 5) and gRNA8 (IL2RG intron 1) were used for further mouse and human

experiments based on highest activity.

Molecular analyses

For molecular analyses, genomic DNA was isolated with DNeasy Blood & Tissue Kit or QIAamp

DNA Micro Kit (QIAGEN). Nuclease activity (IL2RG exon 5, IL2RG intron 1, GhR, AAVS1) was

measured by mismatch-sensitive endonuclease assay by PCR-based amplification of the targeted locus

followed by digestion with T7 Endonuclease I (NEB) according to the manufacturer’s instructions.

Digested DNA fragments were resolved and quantified by capillary electrophoresis on LabChip GX

Touch HT (Perkin Elmer) according to the manufacturer’s instructions. For mouse genotyping,

genomic DNA was extracted from the tail using the REDExtract-N-Amp Tissue PCR Kit (Sigma

Aldrich). PCR amplicons were resolved on agarose gel and visualized by ethidium bromide staining.

TCR rearrangement analysis on genomic DNA was performed as previously described (22).

For digital droplet PCR analysis, 5-50 ng of genomic DNA were analyzed in duplicate using the

QX200 Droplet Digital PCR System (Biorad) according to the manufacturer’s instructions. Primers and

probes were designed on the junction between the vector sequence and the targeted locus and on

control sequences used for normalization (mouse Sema3a or human TTC5 genes). Thermal conditions

for annealing and extension were adjusted for each specific application as follows: Exon 5 IL2RG HDR

5’ integration junction ddPCR: 60°C for 30 sec, 72°C for 4 min; Intron 1 IL2RG HDR 3’ integration

junction ddPCR: 55°C for 30 sec, 72°C for 2 min. Primers and probes for PCR and ddPCR

amplifications are shown in table S4.

For gene expression analyses, total RNA was extracted using the RNeasy Plus Micro Kit (QIAGEN).

cDNA was synthetized with SuperScript VILO cDNA Synthesis Kit (Invitrogen) and used for Q-PCR

in a Viia7 Real-time PCR thermal cycler using TaqMan Gene Expression Assays (Applied Biosystems)

mapping to IRF7, OAS1, RIG-I, and HPRT as normalizer. The relative expression of each gene was

first normalized to HPRT expression and then represented as fold change relative to the mock-treated

sample.

Flow cytometry

For immunophenotypic analyses (performed on FACSCanto II; BD Pharmingen), we used the

antibodies listed in table S5. Single stained and Fluorescence Minus One stained cells were used as

controls. LIVE/DEAD Fixable Dead Cell Stain Kit (Thermo Fisher), 7-aminoactinomycin (Sigma

Aldrich), or propidium iodide (Thermo Fisher) were included in the sample preparation for flow

cytometry according to the manufacturer’s instructions to exclude dead cells from the analysis.

Apoptosis analysis was performed as previously described (7). Cell sorting was performed using

MoFlo XDP Cell Sorter (Beckman Coulter).

Gene editing of mouse Lin- cells and transplantation

Donor mice between 6 and 10 weeks of age were euthanized by CO2, and BM cells were retrieved from

femurs, tibias, and humeri. HSPCs were purified by Lin- selection using the mouse Lineage Cell

Depletion Kit (Miltenyi Biotec) according to the manufacturer’s instructions. Cells were then cultured

in serum-free StemSpan medium (StemCell Technologies) containing penicillin, streptomycin,

glutamine, 200 ng/ml B18R recombinant protein (eBiovision), and a combination of mouse cytokines

(20 ng/ml IL-3, 100 ng/ml SCF, 100 ng/ml Flt-3L, 50 ng/ml TPO all from Peprotech), at a

concentration of 106 cells/ml. For the “Electro ZFN” gene editing protocol, SCID-X1 Lin- cells were

pre-stimulated for 2-3 hours and then infected with the indicated IDLVs at MOI 100. 16 hours later,

cells were washed and electroporated with 175 g/ml ZFN encoding modified mRNAs (P3 Primary

Cell 4D-Nucleofector X Kit, program DK-100; Lonza). 16 hours after electroporation, cells were

washed and injected into recipient mice. For the “LV gRNA” gene editing protocol, Cas9+/- / SCID-X1

Lin- cells were pre-stimulated for 2 hours and then infected with the indicated IDLVs at MOI 100. 12

hours later, cells were washed for 2 hours and exposed to a second hit of vector at MOI 100 for

additional 2 hours. Cells were then washed and injected into recipient mice. Gene-edited cells were

transplanted at a dose of 106 via tail injection into 8-week-old lethally irradiated SCID-X1 mice (8 Gy

total, fractionated into two doses). A fraction of edited cells was cultured in vitro for a minimum of 3

more days to quantify the fraction of edited cells by flow cytometry and perform ddPCR analysis and

NHEJ assay. For CFC assays, 1,000 cells/plate were seeded one day after electroporation in

methylcellulose-based medium (MethoCult GFM3434; StemCell Technologies). For GhR targeting

experiments, edited Cas9+/+ cells were injected (106 cells/mouse) into congenic lethally irradiated

CD45 mismatched recipients.

For competitive transplants, C57BL/6-Ly5.1 and SCID-X1 (CD45.2) Lin- cells were cultured for 16

hours in the medium described above, mixed at the indicated ratios, and transplanted at a total dose of

106 cells/mouse into 8-week-old lethally irradiated or non-irradiated SCID-X1 mice.

For chemotherapy conditioning, treosulfan (Medac), was administered to the mice i.p. at a total dose of

7000 mg/kg (fractionated in 6 doses from day -4 to day -1) as previously described (39).

For biological conditioning, the CD45.2-SAP immunotoxin was prepared as described (23) by

combining a biotinylated CD45.2 antibody (clone 104, Biolegend) with streptavidin–SAP conjugate

(2.7 saporin molecules per streptavidin, Advanced Targeting Systems) in a 1:1 molar ratio and diluted

in PBS immediately before use. In vivo administration was performed by intravenous injections of a

dose of 3 mg/kg. Transplantation of Lin- cells was performed 3 days after CD45-SAP administration.

Mice were monitored weekly for body weight and signs of suffering, and euthanized when showing

≥15% weight loss and/or labored breathing, followed by necropsy and pathology analysis of selected

organs. Serial collections of blood from the mouse tail were performed to monitor the hematological

parameters, donor cell engraftment and the presence of edited cells. At the end of the experiment, BM

thymus and spleen were harvested and analyzed.

In vivo immune studies

For Pneumovax challenge, mice were injected intraperitoneally with 115 μg/mouse of Pneumovax

vaccine (Sanofi Pasteur), containing highly purified capsular polysaccharides from 23 serotypes of

Streptococcus pneumoniae, and then blood was collected weekly for 3 weeks. Specific anti-Pneumovax

IgM antibodies were evaluated in serum by ELISA assay as previously described (40).

For LCMV infection experiments, mice were intravenously injected with 106 pfu of LCMV Armstrong

virus. Viral stocks were produced and titrated as previously described (41). Viral titers were measured

by extracting viral RNA from the serum of the infected mice using QIAamp Viral RNA Mini Kit

(QIAGEN). cDNA was synthetized using SuperScript VILO cDNA Synthesis Kit (Invitrogen) and

used for Q-PCR in a Viia7 real-time PCR thermal cycler. Specific primers are listed in table S4.

Ex vivo functional studies of lymphoid cells

Intracellular IFN staining was performed on PB-derived mononuclear cells of mice infected with

LCMV in the presence or absence of GP33 peptide stimulation as previously described (41).

For T and B cell proliferation assays, 5x105 splenocytes were labeled with CellTrace Violet or FarRed

cell proliferation kits (Thermo Fisher) according to the manufacturer’s instructions. Labeled cells were

stimulated with Concanavalin A (2.5 μg/ml, Sigma), anti-mouse CD3e antibody (1 μg/ml, BD

Pharmingen) and human IL-2 (100 U, Thermo Fisher), or PMA (50 ng/ml, Calbiochem) and ionomycin

(1 μg/ml, Calbiochem), to measure T cell proliferation, or CpG (1μg/ml, InvivoGen) to measure B cell

proliferation, and cultured in RPMI medium supplemented with 10% FBS and 50 μM β-

mercaptoethanol (Gibco). Cells were analyzed after 3 days by flow cytometry. Proliferation index was

calculated according to FlowJo software rules. Analysis of the phosphorylation state of downstream

effectors of IL2RG pathway on human primary T cells was performed as previously described (7).

Gene editing of human cells

Human B lymphoblastoid cells were cultured in IMDM medium (GIBCO-BRL) supplemented with

penicillin, streptomycin, glutamine, and 10% FBS. 3x105 cells were electroporated (SF Cell Line 4D-

Nucleofector X Kit, program EW 113; Lonza) with 50 µg/ml of plasmids encoding for donor DNA

template, Cas9, and cognate gRNA, or ZFN. Cells were then expanded to perform flow cytometry and

molecular analyses.

Primary T lymphocytes from healthy donors’ PB mononuclear cells were isolated and activated using

magnetic beads conjugated to anti-human CD3 and CD28 antibodies (Dynabeads human T-activator

CD3/CD28; Invitrogen) in IMDM medium (GIBCO-BRL) supplemented with penicillin, streptomycin,

glutamine, 10% FBS, and 5 ng/ml of IL-7 and IL-15 (PeproTech) as described (42). After 2 days of

stimulation, T cells were infected with the indicated IDLVs at MOI of 100. The following day,

transduced T cells were electroporated with mRNAs encoding for ZFNs (106 cells/condition, 100 µg/ml

of ZFNs; P3 Primary Cell 4D-Nucleofector X Kit, program EO 115; Lonza) and then expanded to

perform flow cytometry and molecular analyses.

CD34+ cells were either freshly purified from human CB after obtaining informed consent and upon

approval by the Ospedale San Raffaele Bioethical Committee, or purchased frozen from Lonza. 106

CD34+ cells/ml were stimulated in serum-free StemSpan medium (StemCell Technologies)

supplemented with penicillin, streptomycin, glutamine, 1 µM SR-1 (Biovision), 50 nM UM171

(STEMCell Technologies), 10 µM dmPGE2 added only at the beginning of the culture (Cayman), and

human early-acting cytokines (for CB-derived cells: SCF 100 ng/ml, Flt3-L 100 ng/ml, TPO 20 ng/ml,

and IL-6 20 ng/ml; for BM-derived cells: SCF 300 ng/ml, Flt3-L 300 ng/ml, TPO 100 ng/ml, and IL-3

60 ng/ml; all purchased from Peprotech) for the indicated times. Transduction with IDLV was

performed at MOI 100-200. Transduction with AAV6 was performed at 104 vg/cell. Cells were

electroporated with 175 µg/ml ZFN encoding mRNAs (P3 Primary Cell 4D-Nucleofector X Kit,

program EO-100; Lonza). For CRISPR/Cas9 editing in human CD34+ cells, 2.5 µM of

ribonucleoproteins (RNP) were electroporated. RNPs were made by incubating Cas9 protein

(Integrated DNA Technologies) with synthetic 2’O-methyl-phosphorotyoate modified (10) sgRNA

(Metabion, HPLC purified, intron 1 IL2RG guide 8) at 1:1.5 molar ratio for 10 min at 25°C. For large

scale experiments, electroporation was performed using Lonza 4D-Nucleofector LV Unit (P3 primary

cell nucleofector kit, program EO 100, Lonza). For IFN gene expression studies, 10 µg/ml polyI:C

(Tocris) was electroporated or 200 ng/ml B18R Recombinant Protein (eBiovision) was added to the

culture medium 15 min after electroporation, as indicated.

CD34+ HSPC xenotransplantation studies

For transplantation, 3x105 CD34+ cells treated for editing at day 5 of culture were injected

intravenously into NSG mice after sub-lethal irradiation (180-200 cGy). Sample size was determined

by the total number of available treated cells. Mice were attributed to each experimental group

randomly. Human CD45+ cell engraftment and the presence of gene-edited cells were monitored by

serial collection of blood from the mouse tail and, at the end of the experiment (>20 weeks after

transplantation), BM and spleen were harvested and analyzed. Secondary transplantation was

performed upon injection of 106 purified human CD34+ harvested from the BM of primary engrafted

NSG mice.

Intron 1 IL2RG ZFN off-target analysis

Several lead ZFN pairs were then subjected to unbiased identification of candidate off-target sites using

methods similar to those previously described (29) in K562 cells. Briefly, K562 cells were

electroporated with mRNA encoding the ZFNs as well as barcoded ssDNA oligos using the BTX ECM

830 electroporator device (Harvard Apparatus, #45-0052) to allow for unbiased identification of sites

which have undergone double-stranded DNA cleavage and NHEJ-mediated integration of the ssDNA

oligos. The top 65 sites identified by Miseq next-generation sequencing (NGS) to contain integrated

oligos were then confirmed in human mobilized PB CD34+ cells, which have undergone a similar

electroporation of the same ZFN mRNAs. Genomic DNA from ZFN-treated CD34+ cells was

amplified by PCR, generating amplicons surrounding the potential ZFN binding site. PCR products

were purified using Agencourt AMPure XP beads (Beckman Coulter), and adaptors were added by

TruSeq DNA LT Sample Prep Kit (Illumina). To build an equimolar library, PCR products were

quantified with KAPA Library Quantification Kit for Illumina sequencing platforms

(KAPABIOSYSTEMS) on C1000 Thermal Cycler (BIO-RAD) and sequenced on MiSeq Illumina

Platform using MiSeq Reagent v.3 (Illumina). Quantification of insertions and deletions (indels) was

performed using methods similar to those previously described (43). Briefly, raw paired-end reads were

joined and aligned to the specific genomic target sequences. Sequences with indels of ≥1 bp located

within a 40 bp region encompassing the ZFN target site were considered nuclease-induced genome

modifications (table S6). All sites that produced a Bonferroni p value ≤0.01 in comparison to a GFP-

encoding mRNA electroporated control, were deemed off-target sites (table S2).

SCID-X1 cells

A sample of cord blood CD34+ cells from a subject with SCID-X1 was collected after obtaining

informed consent and upon approval by the Medical Ethics Committee of the Great North Children’s

Hospital, Newcastle Upon Tyne, UK.

Supplementary Figures

Fig. S1. Humanized SCID-X1 mice.

(A) Schematics of the knock-in strategy used to generate humanized SCID-X1 mice. Briefly, a cassette

containing a R226H mutant IL2RG replaced the murine Il2rg. (B) Representative hematoxylin and

eosin stained sections of 2-month-old humanized SCID-X1 (top) and wild-type C57BL/6 (bottom)

mouse tissues. In the humanized SCID-X1 bone marrow, no obvious abnormalities were observed. The

humanized SCID-X1 spleen showed a decreased lymphoid cellularity in the white pulp, and moderate

extramedullary hematopoiesis in the red pulp, possibly accounting for the increased size of the organ

observed macroscopically (see table S1). In the humanized SCID-X1 thymus, a marked decrease of

cellularity was observed in the cortical and medullary regions. Scale bar, 100 m for bone marrow; 200

m for spleen and thymus.

Fig. S2. Phenotypical and functional characterization of humanized SCID-X1 mice.

Hematopoietic populations were analyzed for 2-month-old humanized SCID-X1 mice and WT

littermates as control. (A) Total counts of white blood cells (WBCs), CD4+ and CD8+ T cells, CD19+

B220+ B cells, NK cells, and CD11b+ myeloid cells. WT, n=11; SCID-X1, n=10; *p<0.05, ** p<0.01,

*** p<0.001 (Mann-Whitney test). (B) Percent composition of myeloid and lymphoid lineages in PB

(WT, n=11; SCID-X1, n=10). (C) Total counts of BM hematopoietic progenitor cells (HPC), gated

within Lin- cells. Populations were defined according to signaling lymphocytic activation molecule

(SLAM) markers. HPC-2: Kit+ Sca1+ CD48+ CD150+; HPC-1: Kit+ Sca1+ CD48+ CD150-; MPP

(multipotent progenitors): Kit+ Sca1+ CD48- CD150-; HSC: Kit+ Sca1+ CD48- CD150+. WT, n=10;

SCID-X1, n=10; *p<0.05 (Mann-Whitney test). (D) (Left) Total counts of BM nucleated cells (WT,

n=6; SCID-X1, n=5); p>0.05 (Mann-Whitney test). (Right) Percentage of B cell progenitors within

CD45+ BM cells (WT, n=6; SCID-X1, n=5). Populations were defined within CD45+ cells as

following: pro-B cells: B220+ CD43+ CD24+; pre-pro-B cells: B220+ CD43+ CD24-; pre-B cells:

B220low CD43- IgM-; immature B cells: B220low CD43- IgM+; mature B cells: B220high CD43- IgM+.

The lack of mature B cells reflects the strict requirement of IL7R-α signaling in early pro-B cell

development stage. (E) (Left) Total counts of nucleated cells harvested from the thymus (WT, n=6;

SCID-X1, n=5); **p<0.01 (Mann-Whitney test). (Right) Percent composition of T cell progenitors in

thymus (WT, n=5; SCID-X1, n=5). Populations were defined within CD45+ cells as following: DN

cells: CD3- CD4- CD8-; DP cells: CD3- CD4+ CD8+; SP CD4+: CD3+ CD4+ CD8-; SP CD8+: CD3+

CD4- CD8+. (F) (Left) Total counts of nucleated cells within the spleen (WT, n=6; SCID-X1, n=5),

p>0.05 (Mann-Whitney test). (Right) Percent composition of myeloid and lymphoid lineages in spleen

(WT, n=6; SCID-X1, n=5). (G) Ratio of CD4 to CD8 T cells in spleen (WT, n=6; SCID-X1, n=5);

**p<0.01 (Mann-Whitney test). (H) Proportion of phenotypically defined T cell subsets in the spleen,

according to CD62L and CD44 markers: CD62L+ CD44- naïve, CD62L+ CD44+ memory, and

CD62L- CD44+ effectors (WT, n=6; SCID-X1, n=5) (I) Concentration of IgM measured by ELISA in

the serum of WT (n=5) and humanized SCID-X1(n=5) mice at indicated times after Pneumovax (Pnx)

injection (B cell-dependent vaccine) or PBS as control. (J) Percentage (normalized to unstimulated

cells) of proliferating CD4+ T cells (left) and CD8+ T cells (right), harvested from 2-month-old

humanized SCID-X1 and WT mice and stimulated for 3 days as indicated with anti-CD3 antibodies

and/or IL-2 (WT, n=5; SCID-X1, n=5), (K), Percentage of CD8+ T cells producing IFNγ from the PB

of SCID-X1 or WT mice, 8 days after infection with LCMV Armstrong (WT, n=6; SCID-X1, n=7),

**p<0.01 (Mann-Whitney test).

Fig. S3. Hematopoietic reconstitution and functional studies of WT/SCID-X1 competitive

transplants.

(A) Chimerism of WT and SCID-X1 cells observed within BM Lin- HSPCs 25 weeks after transplant

(100% WT and 1% WT, n=3; 10% WT, n=4). (B) Percentage of B cell progenitors within CD45+ BM

cells (100% WT, 1% WT, and SCID, n=6; 10% WT, n=7, pooled from 2 independent experiments).

(C) Percent composition of myeloid and lymphoid lineages in the spleen of mice from B. (D) T cell

phenotype within CD4+ (left) and CD8+ (right) T cells in the spleen of the transplanted mice.

Populations were classified as in fig. S2H (100% WT, 1% WT, and SCID, n=3; 10% WT, n=4). (E)

Phenotype of B cells in the spleen of the transplanted mice (100% WT and 1% WT, n=3; 10% WT,

n=4). Populations were defined as in fig. S2. Of note, the fractions of transitional B cells and follicular

B cells decreased with the dose of WT HSPCs infused, suggesting reduced output from the BM.

Marginal zone B cells, which have the highest homeostatic proliferation potential in lymphopenic

conditions, were instead increased.

Fig. S4. Phenotypical characterization of lymphoblastic T lymphomas developing in mice

transplanted without irradiation.

(A) Percent chimerism with WT and SCID-X1 cells measured within BM Lin- cells in mice

transplanted with the indicated % of WT cells without irradiation and surviving until the end of the

experiment (100% WT n=4, 10% WT n=3). (B) Percentage of naïve CD3+ T cells measured in the

spleen of mice from A (No irradiation, n=7), or transplanted after TBI (n=8); ** p<0.01 (Mann-

Whitney test). (C) Total counts of nucleated cells from the thymus of mice which developed or did not

develop lymphoblastic T lymphoma (tumor n=12, no tumor n=7), *** p<0.001 (Mann-Whitney test).

(D) Hematoxylin and eosin stained sections of the indicated organs showing diffuse infiltration of

lymphoma. Transformed lymphoblasts appear as a homogeneous sheet of medium-sized cells, with

scant cytoplasm and round nuclei, with numerous mitotic figures and a starry-sky appearance with

tingible body macrophages and apoptotic cells. Scale bars, 50 m for bone marrow, kidney, and liver;

100 m for lung and spleen. (E) (Left) Percentage of mice presenting with the indicated macroscopic

abnormalities of thymus and spleen among the mice that developed lymphoblastic T lymphoma (n=17).

(Right) Proportion of mice showing tumor spreading outside of the thymus (100% WT, n=6; 10% WT,

n=11). (F) (Left) Proportion of tumors showing the indicated immunophenotype according to the

surface expression of CD4 and CD8 (100% WT, n=6; 10% WT, n=11). (Right) Representative plots

showing expression of CD4 and CD8 markers in tumor samples or control thymocytes. (G) (Left)

Proportion of tumors showing the indicated immunophenotype according to the surface expression of

CD3 and TCRβ markers (100% WT, n=6; 10% WT, n=11). (Right) Representative plots showing

expression of CD3 and TCRβ markers from tumor samples.

Fig. S5. Molecular and functional characterization of T lymphoma.

(A) Multiplex PCR analyses for TCR rearrangements on genomic DNA extracted from thymi of mice

that developed lymphoma. The primers are located on different variable regions of the β locus(Vβ) and

on J region 1.7 (Jβ1.7, top) or 2.7 (Jβ2.7, bottom). (B) Survival curve of C57BL/6 (n=5) or SCID-X1

(n=5) mice challenged intravenously with 106 primary lymphoma cells. * p<0.05 (log-rank test). (C)

(Top) Image of the lead screen designed to protect the thymus from irradiation. (Bottom)

Representative plots showing apoptosis measurement (Annexin V and 7-aad staining) in cells harvested

from the thymus of irradiated mice, mice irradiated and shielded during the procedure, or untreated

mice as control. (D) Graph showing the chimerism of WT and SCID-X1 cells measured at 15 weeks

within PB myeloid cells of SCID-X1 mice transplanted with the same dose of WT Lin- cells (10%)

without irradiation (No irradiation, n=8), after TBI (n=13) or after TBI with the thymus screen (n=13).

Fig. S6. Depletion of hematopoietic compartments with CD45-SAP in SCID-X1 mice.

(A) Number of the indicated primitive populations measured in the BM of SCID-X1 mice conditioned

with 3 mg/kg of CD45-SAP 3 days after treatment, TBI 1 day after treatment, or untreated (UT) (pool

from n=4 mice per group), normalized to untreated. (B) Weight (g) of SCID-X1mice conditioned as

indicated, normalized to untreated controls (n=4 mice per group; * p<0.05, Kruskal-Wallis test). (C)

Absolute number of myeloid cells (CD11b), B cells (CD19), CD4+, and CD8+ T cells in BM (left) and

spleen (right) of mice treated as indicated (n=3 to 4 per group). (D) Percent chimerism of WT cells

within the indicated populations in the PB of SCID-X1 mice from Fig. 3C (n=5 per group) at different

times after transplant.

Fig. S7. Development of a gene editing strategy for mouse HSPCs.

(A) Schematics of the donor DNA templates for exon 5 or intron 1 IL2RG editing delivered by IDLV.

(B) Flow chart of the gene editing protocols developed for mouse HSPCs and cell analyses. (C-H)

Procedure based on the delivery of donor DNA template by IDLV and the transfection of mRNAs

encoding ZFNs (“Electro ZFN protocol”). (C) Percentage of HDR (measured by stable GFP expression

for exon 5 or digital droplet PCR for intron 1) and of NHEJ 3 days after editing exon 5 (n=35) or intron

1 IL2RG (n=3). (D) Fold change in the number of live cells measured 1 day after treatment of Lin- cells

with mock electroporation (Electro, n=1), electroporation with GFP-encoding mRNA (GFP mRNA,

n=1), electroporation with ZFN encoding mRNAs (n=2) or Cas9 encoding mRNA (n=1), or

electroporation with ZFN mRNAs after IDLV transduction (IDLV+ZFN, n=12). (E) Percentages of

HDR and NHEJ as in C within sorted Sca1+ or Sca1- cells (Electro ZFN, n=7). (F) Percentage of

GFP+ cells in PB after transplant (exon 5 IL2RG edited cells treated with Electro ZFN protocol, n=36).

Each line represents a single mouse. (G) Percentage of GFP+ cells within the PB populations (B, T, or

myeloid, ≥15 weeks after transplant) or BM (KLS, measured at the end-point (>21 weeks) of mice

transplanted with exon 5 IL2RG edited HSPC (Electro ZFN, n=12); median is plotted. (H) Percentage

of NHEJ measured in vitro 3 days after editing, and in the PB of mice ≥12 weeks after transplant

(Electro ZFN, n=29, 23, 4, and 3 for in vitro exon 5, PB exon 5, in vitro intron 1, and PB intron 1,

respectively); median is plotted. (I-M) Targeted gene editing within the non-coding region of the Ghr

gene in mouse HSPCs constitutively expressing S.p.Cas9 (28). (I) Percentage of GFP-expressing cells

in the indicated hematopoietic populations of Cas9 homozygous (Cas9+/+) and heterozygous (Cas9+/-)

mice. These mice constitutively express a Cas9-GFP transgene with intervening 2A self-cleaving

peptide, therefore GFP expression corresponds to Cas9 expression. (J) Schematic of the IDLV or LV

vectors used to express gRNAs in Cas9+/+ transgenic Lin- cells. BFP: blue fluorescent protein. (K) Fold

change in the number of Lin- Cas9+/+ cells recovered 1 day after editing the Ghr gene by IDLV or LV

delivery of the cognate gRNA, compared to untreated samples (n=12). (L) Percentage of NHEJ

measured in the PB after transplantation of Cas9+/+ HSPCs treated for Ghr disruption with LV (n=3) or

IDLV (n=5) expressing the cognate gRNA. (M) Percentage of NHEJ at the targeted Ghr gene

measured in the transplanted mice from L upon sorting CD11b+, CD3+, and CD19+ populations from

the spleen, and Kit+, Sca1+, and KLS+ populations from BM Lin- cells (IDLV, n=5; LV n=3). (N-U)

Optimized gene correction protocol based on a single IDLV comprising either of the two IL2RG donor

templates described above and expressing the cognate gRNA targeting a region within either ZFN

cleavage site (“LV gRNA protocol”). (N) Exon 5 and intron 1 IL2RG sequences targeted by the gRNAs

tested. Bases in red correspond to the sequences recognized by either ZFN heterodimer. Sequences

within the left and right homology arms of the donor vectors are indicated on the top. (O) Male B

lymphoblastoid cells were electroporated with plasmids encoding exon 5 or intron 1 IL2RG gRNAs,

the donor template, and Cas9. The histogram shows the percentage of HDR-mediated integration

measured by flow cytometry (GFP) and the percentage of NHEJ measured 2 weeks after editing. Cells

electroporated without the gRNA plasmid are shown as negative controls. For further experiments,

gRNA 3 and 8 were selected for exon 5 and intron 1 IL2RG, respectively. (P) Schematics of the IDLV

vectors used to deliver the donor DNA template and express the cognate gRNA. (Q) Analysis as in C,

performed using LV gRNA protocol (n=20 and 16 for exon 5 and intron 1, respectively). (R) Analysis

as in E, performed using LV gRNA protocol (n=4). (S) Analysis as in F, performed using LV gRNA

protocol (n=21). (T) Analysis as in G, performed using LV gRNA protocol (n=21). (U) Analysis as in

H, performed using LV gRNA protocol (n=17, 12, 15, and 10 for in vitro exon 5, PB exon 5, in vitro

intron 1, and PB intron 1, respectively; Mann-Whitney test). * p<0.05, ** p<0.01, *** p<0.001, ****

p<0.0001.

Fig. S8. Functionality of gene-edited lymphoid cells from transplanted mice.

(A) Percent composition of myeloid and lymphoid lineages measured in the spleen of mice from Fig.

4A (1% WT, n=6; SCID, n=8; Electro ZFN, n= 11; LV gRNA, n=21) Significance shown in

comparison to SCID-X1 group, * p<0.05, *** p<0.001 (Kruskal-Wallis test). (B) Proliferation index of

CD8+ (left) and CD4+ (right) T cells harvested from the spleens of transplanted mice at the end of the

experiment and treated for 3 days with the indicated stimuli. Exon 5 IL2RG edited cells (n=12) and

intron 1 IL2RG edited cells (n=4) are shown pooled; cells from WT (n=7) and SCID-X1 mice (n=2) are

used as control. (C) Long-term follow-up of mice from Fig. 4A transplanted with HSPCs treated as

indicated (Electro ZFN protocol, n=12; LV gRNA protocol, n=21). No development of thymic

lymphomas was found during the observation time and at necropsy at the end of the experiment. Some

mice were sacrificed earlier (≥126 days) to perform other analyses.

Fig. S9. Functional validation of IL2RG-edited primary human T cells.

(A) Validation of the ddPCR used to quantify HDR at the 3’ junction between the donor sequence and

the targeted intron 1 IL2RG in human cells. (Left) DNA of a male B-lymphoblastoid clone harboring

the intended targeted integration by HDR in intron 1 IL2RG was mixed with DNA from untreated cells

(WT) at the indicated ratios. Quantification of HDR at the locus in the mix by ddPCR is plotted on the

y axis (linear regression, R=0.9984). (Right) Correlation between the percentage of GFP+ cells

measured by flow cytometry (the donor construct also contained a GFP reporter cassette for this

experiment) and the percentage of HDR measured by ddPCR (n=45; linear regression, R=0.9744). (B)

Representative plots showing the expression of IL2RG on the surface of untreated male B-

lymphoblastoid cells (left) and upon editing using a donor template (depicted on top) containing an

exogenous splicing acceptor followed by a 2A.GFP cassette within the intron 1 IL2RG homology

region. All GFP+ cells lost expression of IL2RG, demonstrating exhaustive splicing of the integrated

construct. (C) Percentage of human male primary T cells edited by HDR (n=10) or NHEJ (n=1) at exon

5 IL2RG. (D) Growth curve in culture of primary T cells from Fig. 5A. (E) Flow cytometry plots

showing the expression of IL2RG in male primary T cells edited with wild-type (left) or codon usage-

optimized (right) IL2RG intron 1 corrective cDNA donor coupled to a PGK.GFP reporter cassette.

IL2RG mean fluorescence intensity (MFI) is indicated above each quadrant of GFP+ and GFP- cells.

(F) Heat map representing fold changes in pAKT (S473), pSTAT5 (Y694), and pSTAT1 (Y701) after

different times of exposure (min) to the indicated amounts of IL-2 and IL-15 in male primary T cells

edited with IL2RG intron 1 corrective cDNA donor coupled to a PGK.GFP reporter cassette.

Comparison between GFP+ edited cells and GFP- non-edited cells is shown.

Fig. S10. Tailoring of gene editing protocol for human HSPCs.

(A) Time course analysis of the expression of the indicated IRG (IRF7, OAS1, RIG-I) performed in

CB-derived CD34+ cells treated for gene editing with IDLV as donor (IDLV+ ZFN, n=3), or

electroporated with polyinosinic:polycytidylic acid (pI:C) as positive control (n=3). The 12-hour time

point after electroporation was used for subsequent analyses. (B) Fold expression of the indicated IRG

compared to untreated control in CB-derived CD34+ cells untreated (UT) or treated with pI:C, mock

electroporated (E), IDLV transduced, IDLV transduced +E, electroporated with ZFN mRNA, or gene

edited with or without B18R (n= 9, 3, 2, 2, 3, 2, 9, 4, respectively). (C) Fold change in expression of

the indicated IRG compared to untreated control in CB-derived CD34+ cells treated for intron 1 IL2RG

editing with ZFN mRNAs containing or not containing modified bases (mod) and/or HPLC-purified, as

indicated (n=2). (D) Percentage of GFP+ cells within the bulk treated cells (left) or the indicated

subpopulations (right) measured three days after intron 1 IL2RG editing using ZFN mRNAs with or

without HPLC purification (n=4); *p<0.05 (Mann-Whitney test). (E) Percentage of GFP+ cells within

the bulk treated cells or the indicated subpopulations measured 3 days after intron 1 IL2RG editing

performed with AAV6 donor template, added at the indicated times with respect to electroporation. (F)

(Left) Percentage of GFP+ cells measured within the indicated subpopulations three days after intron 1

IL2RG gene editing of BM-derived HSPCs performed with IDLV or AAV6 donor template vector

added 24 hours before or 15 min after electroporation, respectively (IDLV -24 hours, n=4; AAV6 +15

min, n=4), *p<0.05 (Mann-Whitney test). (Right) Composition of BM HSPCs at the experimental time

points shown, three days after gene targeting. (G) Percentage of human CD45+ B (CD19+), T (CD3+),

and myeloid (CD13+) cell populations measured at 15 weeks in PB of mice transplanted with medium

scale and large scale IL2RG edited cells and untreated cells (UT) from Fig. 5I.

Table S1. Phenotypical characterization of humanized SCID-X1 mice.

↓ low; ↓↓ very low; ↑ high; Nd: not detectable; np: analysis not performed; LN: lymph nodes.

Mice Mutation

WBC Organs WBC

proliferat

ion

Ref CD4

T

CD8

T B NK

Granu

locyte

s

Thymus Spleen LN

Il2rg-/-

ex 2-6 del

ex 3-8 del

ex 7-8 del

↓

↓

↓

↓

↓↓

↓↓

↓↓

↓↓

↓

nd

nd

nd

np

↑

↑

small

small

small

small

enlarged

enlarged

small

nd

nd

no

no

no

14

15

16

Humanized

SCID-X1

226R>H

IL2RG

knock-in

↓ ↓↓ nd nd ↑ small enlarged nd no current

study

Table S2. Intron 1 IL2RG ZFNs off-target list.

65 candidate off-target (OT) sites were identified for the 57629/57718 ZFN lead pair by unbiased

genome-wide screening in K562 cell line, and then 7 were confirmed (Bonferroni p-value ≤ 0.01

compared to a control sample) in CD34+ cells. Genome note indicates whether indels are located

within RefSeq genes or are intergenic. DHS: DNase I hypersensitive site.

Designation Locus (hg38 coordinates) Genome note

On chrX:71111318-71111358 IL2RG, intron 1

OT1 chr1:171443656-171443696 Intergenic

OT2 chr16:70011144-70011184 PDXDC2P, intron 15

OT3 chr5:151135158-151135198 ANXA6, intron 6

OT4 chr12:109775484-109775524 Intergenic (on CD34+ DHS)

OT5 chr3:37865600-37865640 CTDSPL, intron 1

OT6 chr6:170554282-170554322 TBP, promoter

OT7 chr6:150741780-150741820 PLEKHG1, intron 3

Table S3. List of genomic gRNA target sequences.

Protospacer adjacent motif (PAM) sequence is indicated in red.

Description DNA Sequence (5’3’)

Exon5 IL2RG g1 AACGCTACACGTTTCGTGTTCGG

Exon5 IL2RG g2 AGCCGCTTTAACCCACTCTGTGG

Exon5 IL2RG g3 TTCCACAGAGTGGGTTAAAGCGG

Exon5 IL2RG g4 GCTGAGCACTTCCACAGAGTGGG

Exon5 IL2RG g5 TGCTGAGCACTTCCACAGAGTGG

Intron 1 IL2RG g6 GAAGGGGCCGTACAGAGATCTGG

Intron 1 IL2RG g7 CACTGGCCATTACAATCATGTGG

Intron 1 IL2RG g8 ACTGGCCATTACAATCATGTGGG

Intron 1 IL2RG g9 CACATGATTGTAATGGCCAGTGG

Intron 1 IL2RG g10 TGATTGTAATGGCCAGTGGCAGG

Intron 1 IL2RG g11 TTCTGCCCACATGATTGTAATGG

Intron 1 IL2RG g12 GCAGGCACCAGATCTCTGTACGG

Intron 1 IL2RG g13 AGAGATCTGGTGCCTGCCACTGG

Ghr g1 TAGCTCCGGGGCCGCTGCGGGG

Table S4. List of primers and probes.

Description Orientation DNA Sequence (5’3’)

IL2RG R226H

genotyping FW ACTTTCCCTCATCCTCTTTCTCC

RV GAGGGAGCAGGAGCACATAGG

Il2rg genotyping FW TGGATGAGCTGAAACGGTACACATTTCGGG

RV GCATGGAACCTCTAGCTTCCTCTCAACACT

Rosa26 CAS9

genotyping FW AAGGGAGCTGCAGTGGAGTA

RV CGGGCCATTTACCGTAAGTTAT

Rosa26 WT

genotyping FW AAGGGAGCTGCAGTGGAGTA

RV GGAGCGGGAGAAATGGATATGAAG

5’ HDR integration

junction (exon 5

IL2RG)

FW GCTAAGGCCAAGAAAGTAGGGCTAAAG

RV AGCCAGAAGTACACGCACAGC

3’ HDR integration

junction (exon 5

IL2RG)

FW ACCTCTACAAATGTGGTATGGCTG

RV TTCCTTCCATCACCAAACCCTCTTG

NHEJ AAVS1 FW CTTCAGGACAGCATGTTTGC

RV ACAGGAGGTGGGGGTTAGAC

NHEJ Intron 1 IL2RG FW CACCCTCTGTAAAGCCCTGG

RV AAGAAATCTAGATTGGGGAG

NHEJ Ghr FW TTCTTTGGGGGAACCCGATG

RV CAGCTCGTGGGTTGTCAGG

NHEJ Exon 5 IL2RG FW TTCTCCCTTCTCTCATAGACACCC

RV CTCATGGATTGGGTCATGTGG

LCMV Q-PCR FW CTCCTTTCCCAAGAGAAGACTAAG

RV TCCATTTGGTCAGGCAATAAC

Exon 5 IL2RG 5’

integration junction

ddPCR

FW GCTAAGGCCAAGAAAGTAGGGCTAAAG

RV AGCCAGAAGTACACGCACAGC

Probe 1

(FAM)

AGCAACACCAGCAAAGAGAA

Probe 2

(FAM)

AGCACTGGTCCGAGTGGA

Intron 1 IL2RG 3’

integration junction

ddPCR

FW CTAGATTGGGGAGAAAATGA

RV GTGGGAAGGGGCCGTACAG

Probe (FAM) GTAGCTCCTATGCTAGGCGTAGCC

Mouse Sema3a

ddPCR FW ACCGATTCCAGATGATTGGC

RV TCCATATTAATGCAGTGCTTGC

Probe (HEX) AGAGGCCTGTCCTGCAGCTCATGG

Human TTC5 ddPCR (HEX)

PrimePCR ddPCR Copy Number Assay: TTC5, Human

(Biorad)

mVβ1 FW CAGACAGCTCCAAGCTACTTTTAC

mVβ2 FW ATGAGCCAGGGCAGAACCTTGTAC

mVβ3 FW GAAATTCAGTCCTCTGAGGCAGGA

mVβ4 FW CTAAAGCCTGATCACTCGGCCACA

mVβ5.1 FW CCTTGGAGCTAGAGGACTCTGCCG

mVβ5.2 FW CCTTGGAACTGGAGGACTCTGCTA

mVβ5.3 FW CCTTGGACCTAGAGGACTTTACTG

mVβ6 FW GCCCAGAAGAACGAGATGGCCGTT

mVβ7 FW GGATTCTGCTAAAACAAACCAGACATC

mVβ8.1 FW GCTTCCCTTTCTCAGACAGCTGTA

mVβ8.2 FW GCTACCCCCTCTCAGACATCAGTG

mVβ8.3 FW GGCTTCTCCCTCTCAGACATCTT

mVβ9 FW CTCTCTCTACATTGGCTCTGCAGG

mVβ10 FW CTTCGAATCAAGTCTGTAGAGCCGG

mVβ11 FW TGAAGATCCAGAGCAGCGGGCCCC

mVβ12 FW CCACTCTGAAGATTCAACCTACAGAACCC

mVβ13 FW CAAGATCCAGTCTGCAAAGCAGGG

mVβ14 FW GCACGGAGAAGCTGCTTCTCAGCC

mVβ15 FW GCATATCTTGAAGACAGAGGC

mVβ16 FW CTCTGAAAATCCAACCCACAGCACTGG

mVβ17 FW TCTGAAGAAGACGACTCAGCACTG

mVβ18 FW GCAAGGCCTGGAGACAGCAGTATC

mJb1.7 RV CCAAGACCATGGTCATCCAAC

mJb2.7 RV TGAGAGCTGTCTCCTACTATCGATT

Table S5. List of antibodies for flow cytometry.

Antibody Fluorochrome Clone Company

Anti-mouse antibodies

Annexin V PB

Biolegend

CD117 APC 2B8 BD

CD117 APC780 2B8 eBioscience

CD11b FITC, APC, PE M1/70 BD

CD11b APC-Cy7, PB M1/70 Biolegend

CD16/32 none 2.4G2 BD

CD150 APC TC15-12F12.2 Biolegend

CD19 PE, APC 1D3 BD

CD19 PB, PeCy7 6D5 Biolegend

CD24 Percp5.5 BD

M1/69 BD

CD25 APC, Percp5.5 PC61 BD

CD3 PB 17A2 Biolegend

CD3e FITC, PE, APCH7 145-2C11 BD

CD4 PeCy7 GK1.5 eBioscience

CD4 PB, PE, APC RM4-5 BD

CD43 APC-Cy7 1B11 Biolegend

CD44 APC, Pecy7 IM7 BD

CD45 PE, PerCP 30-F11 BD

CD45.1 APC780 A20 eBioscience

CD45.1 FITC A20 BD

CD45.1 PB, PeCy7 A20 Biolegend

CD45.2 FITC, PerCP-Cy5.5 104 BD

CD45.2 PE, PB 104 Biolegend

CD45R/B220 PE, PB RA3-6B2 BD

CD45R/B220 APC RA3-6B2 Biolegend

CD48 PB, PE HM48-1 Biolegend

CD62L PE, APC MEL-14 BD

CD69 FITC H12F3 BD

CD8a FITC, PB, PE, PeCy7 53-6.7 BD

CD8a APC780 53-6.7 eBioscience

Gr1 FITC, PB RB6-8C5 Biolegend

IgM Biotin, APC II/41 BD

IFN- APC XMG1.2 Biolegend

Lineage Cocktail PE

Biolegend

Lineage Cocktail Biotin Miltenyi Biotec

NK.1.1 FITC PK136 BD

NK-1.1 PB PK136 Biolegend

TCR APC H57-597 Biolegend

Sca1 PB D7 Biolegend

Sca1 PeCy7 D7 BD

Streptavidin PE, PerCP, PE

BD

Streptavidin Pacific Orange

Invitrogen

Streptavidin APC780

eBioscience

Ter119 PE TER-119 BD

Anti-human antibodies

Annexin V PB Biolegend

CD16/32 none Miltenyi Biotec

CD133/2 PE 293C3 Miltenyi Biotec

CD34 PB AC136 Miltenyi Biotec

CD34 PECy7 8G12 BD

CD90 APC 5E10 BD

CD132 APC TUGm2 Biolegend

CD45 PB HI30 Biolegend

CD45 APCH7 HI30 eBioscience

CD19 PE HIB19 BD

CD19 Pecy7 HIB19 Biolegend

CD3 Pecy7 HIT3a Biolegend

CD3 PE SK7 BD

CD13 APC WM15 BD

CD33 PeCy7 P67.6 BD

CD38 APC HB7 BD

CD38 Percp5.5 HB7 Biolegend

CD4 PB RPA-T4 BD

CD8 APCH7 SK1 BD

pAKT (S473) Alexa647 D9E Cell Signaling

pSTAT1 (Y701) Alexa647 58D6 Cell Signaling

pSTAT5 (Y694) Pecy7 47 BD