gene editing in human stem cells using zinc finger …4 reproductive technologies laboratory, ciz,...

Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery

Angelo Lombardo1,2, Pietro Genovese1,2, Christian M. Beausejour3,6, Silvia Colleoni4, Ya-Li Lee3,

Kenneth A. Kim3, Dale Ando3, Fyodor D. Urnov3, Cesare Galli4,5, Philip D. Gregory3,7, Michael C. Holmes3,7, Luigi Naldini1,2,7

1 San Raffaele Telethon Institute for Gene Therapy and 2 “Vita Salute San Raffaele” University, via Olgettina, 58, 20132 Milan, Italy. 3 Sangamo BioSciences, Inc., Pt. Richmond Tech Center, 501 Canal Blvd., Suite A100 Richmond, California 94804, USA. 4 Reproductive Technologies Laboratory, CIZ, Istituto Sperimentale Italiano Lazzaro Spallanzani, via Porcellasco 7 /F, 26100 Cremona, Italy and 5 Dipartimento Clinico Veterinario, University of Bologna, Via Tolara di Sopra 50, 40064 Ozzano Emilia, Italy. 6 Present address: Département de Pharmacologie, Centre de Recherche, CHU Ste-Justine 3175, Côte Ste-Catherine, Montréal, Quebec H3T 1C5, Canada. 7These authors contributed equally to this work.

Supplementary Information Lombardo et al. 1

SUPPLEMENTARY FIGURES

GFP

82% 0.7%27%

7 Days 14 Days2 Days

80%

0.6 µg p24/ml IDLV 16 Days5 Days

GFP

0.04%

0.2 µg p24/ml IDLV

a

b

Supplementary Fig. 1. Transient transgene expression upon IDLV delivery.

Human Embryonic Kidney (HEK)293 cells (a) and cord blood CD34+ progenitor and stem cells (b)

were treated with the indicated dose in µg HIV-1 Gag p24/ml of IDLV containing a GFP expression

cassette, and analyzed by fluorescence-activated cell sorting (FACS) at the indicated times post-

infection. The percentage of GFP+ cells is indicated for each condition.


ZFNsSFFV

PGK

EF1α

∆U3-R-U5

RRE

∆GFP∆GFP ∆LNGFR PGK

Single (S)

∆GFP donor

Double (D)

U5-R ∆U3

∆LNGFR PGK ∆GFP

U5-R-∆U3

RRE

RRE

GF PGene Reporter Site

ZFNs site

GFP

ZFNs constructs (2x)

GF PGene reporter site

ZFNs sitea

0

0,2

0,4

0,6

0,8

1

1,2

0.2

0.4

0.6

0.8

1

0

%G

FP p

ositi

ve c

ells

ZFNs 0.1 ––––––µg/ml plasmid

–

Donor

1.2

PGK - ZFNs

SFFV - ZFNs(S) - Donor

(D) - Donor

– – –

– – –

– – ––––

– – – –––

– – ––––

0.1 0.2 0.3

0.1 0.2 0.3

– – ––––

– – – –––

– – –– ––

0.1 0.1 0.1 0.10.10.1– – –

–1 111111– – – –––

0.4 0.50.2

– – – 0.50.40.2

–

µg-p24/ml IDLV

1

b

Supplementary Fig. 2. IDLV delivery of ZFNs and donor DNA mediates gene correction in a

reporter cell line.

(a) Schematics of the vectors and the engineered reporter site used to measure gene correction. Top

drawing: the genomic reporter site in HEK293 cells, which contains a GFP gene disrupted by the

insertion of a portion of the IL2RG exon 51. The ZFNs pair targeting the exon 5 sequence can

deliver a DNA double-stand (ds) break in the defective gene. Middle drawings: the lentiviral

transfer construct used to express ZFNs, showing a choice of three internal promoters derived from

the Spleen Focus Forming Virus LTR (SFFV), the human phosphoglycerate kinase (PGK) and


Elongation Factor 1α (EF1α) genes. Because two different ZFNs are needed to induce a ds break at

the target site, two ZFN-expressing vectors must be used for each infection. Bottom drawings: the

lentiviral transfer vectors used to deliver a 5’ truncated GFP donor sequence (∆GFP, lacking the 5’

end of the GFP open reading frame and thus unable to express by itself) for homology-directed

repair of the defective GFP. The donor sequence was inserted either in internal position (Single, S)

or within the modified LTR; in the latter construct, duplication of the homology sequence occurs

upon reverse transcription (Double, D). For all transfer vectors the linear products of reverse

transcription are depicted and the constituent regions of the 5’ self-inactivating LTR are indicated

(∆U3-R-U5)2. The GFP donor constructs also contain a reporter expression cassette for the

truncated low-affinity Nerve Growth Factor Receptor (∆LNGFR) gene to monitor vector

integration. RRE: Rev-response element in the vector backbone. Representative schematic of

homology-driven correction of the genomic reporter site using the D donor vector leading to

restoration of GFP expression is also shown.

(b) Gene correction of the genomic reporter site. Three distinct IDLVs, two of them expressing the

cognate ZFNs targeting the disrupted GFP sequence and the third one carrying the ∆GFP donor and

the ∆LNGFR cassette, were packaged as IDLVs and added to the reporter cells at the indicated

doses (in µg HIV Gag p24/ml) and combinations. Transfection of standard donor and ZFN-

expressing plasmids by lipofectamine was used as positive control (at the indicated µg DNA/ml).

FACS analysis 14 days post-infection showed restoration of GFP expression in cells treated either

by standard co-transfection or IDLVs-mediated delivery of all the required components for gene

correction (n=4). The percentage of GFP+ cells for each condition is shown in the histogram. Note

that gene correction in this model requires resection of the intervening IL2RG sequence in the

defective GFP before its homology-directed repair, and thus occurs to lower level than that

observed for single base pair exchange. The correction efficiency increased with the ZFN-

expressing IDLVs dose and the double configuration of the donor sequence. Notably, treating

IDLV-infected cells with AZT (a reverse transcriptase inhibitor) abrogated gene correction,

indicating that the observed gene correction could not be due to plasmid or protein carryover from

the vector preparation (data not shown).

Overall, these data indicate that reverse transcribed IDLVs can drive sufficient gene

expression to induce ZFNs activity and act as proficient substrates for gene correction.


0 2 4 6 8 10 12

Days post trasduction

% M

ism

atch

es

0

5

10

15

20

25

30

35

0 2 4 6 8 10 12

1 µg-p24/ml ZFNs0.25 µg-p24/ml ZFNs 0.25 µg-p24/ml het.-ZFNs

1 µg-p24/ml het.-ZFNs

Supplementary Fig. 3. Transient ZFNs activity upon IDLV delivery.

IDLV-induced ZFNs activity in treated K-562 cells was measured by a mismatch sensitive

endonuclease assay that quantifies mutations (such as deletions consequent to NHEJ) occurring at

the ZFN genomic target site. Cells were treated either with 0.25 (circles) or 1 (triangles) µg HIV

Gag p24/ml of each of two IDLVs expressing a CCR5 ZFN set (these ZFNs will be introduced later

in the manuscript). Two different ZFNs pairs were used, either carrying a standard FokI domain

(filled symbols) or the obligate heterodimer FokI variants3 (open symbols). At the indicated days

post-infection a sample of the culture was collected, and genomic DNA was extracted for analysis

as described in the Supplementary Methods. The ratio of the uncleaved parental fragment to the

two lower migrating cleaved products was calculated using the formula (1-(parental

fraction)1/2)x1003.

In all experimental conditions we observed increasing accumulation of mutant alleles during

the first 3-4 days post-infection, followed by a steady plateau. These results are consistent with

transient ZFNs activity post-infection and stable maintenance of the ZFN-induced mutations in the

culture.


GFP-

GFP+

GFP

∆LN

GFR

GFP

15% ∆LNGFR+

0.6% ∆LNGFR+

∆LNGFR+

∆LNGFR-

1% GFP+

a

0.2 0.4 0.6 0.8 1 1.2 1.4 1.60

GFP-

GFP+

GFP+ ∆LNGFR-

GFP+ ∆LNGFR+

VCN per cell

RRE

∆LNGFR

Fok I

b

2 0.51

Standard curve

4 Kb

3 Kb

5 Kb

TI

GFP

-

GFP

+

GFP

+ ∆L

NG

FR-

GFP

+ ∆L

NG

FR+

Moc

k

Targeted integration ∆LNGFR GFPGFP

SpeI SpeI

PROBE

GF P

ZFNs site

∆GFP∆GFP ∆LNGFR PGK

Gene reporter site

Donor

PGK

SpeI

RRE

RRE

c

Supplementary Fig. 4. IDLV delivery of ZFNs and donor DNA mediates addition of a transgene

expression cassette into the ZFN target site.


(a) The cells treated with 0.2 µg D-donor IDLV from Supplementary Figure 2 were sorted

according to GFP expression (i.e. correction, 1% GFP+ in pre-sorted cells) and analyzed by FACS

for ∆LNGFR expression (i.e. vector integration, 0.6% ∆LNGFR+ in pre-sorted cells). Whereas the

percentage of ∆LNGFR-expressing cells was very low in the GFP-negative sorted population, it

was highly enriched in the GFP-positive sorted cells (up to 15%, n=2). To determine whether this

finding indicated higher permissiveness of gene-corrected cells to random vector integration or

targeted integration of the ∆LNGFR expression cassette into to the ZFNs site, which is flanked on

both sides by GFP homology sequences in the D-donor vector, we sorted from the GFP-positive

population the ∆LNGFR-positive and -negative cells, and analyzed their genomic DNA for the

content (b) and location (c) of vector DNA.

(b) Quantitative PCR (Q-PCR) using primers and probe specific for the donor (∆LNGFR), ZFN-

expressing (FokI), or either IDLVs (RRE). The vector copy number (VCN) was below 0.03 per cell

in the GFP+∆LNGFR− cells, indicating that gene correction in these cells occurred without

incorporation of vector sequences outside of the homology region, as expected for gene correction.

On the other hand, VCN was approximately 1 per cell in the GFP+∆LNGFR+ cells, with matching

estimates using primers detecting the vector backbone (RRE) or the ∆LNGFR transgene. Because

we measured a very low content of ZFN-expressing vector in all cell populations (using primers

specific for the FokI nuclease), enhanced integration was specifically observed for the vector

bearing homology sequences to the ZFNs target site.

(c) Genomic DNA extracted from the sorted cells was analyzed by Southern blot using the

restriction enzyme SpeI. This analysis showed the expected band for integration of the ∆LNGFR

expression cassette at the ZFNs target site in the GFP+∆LNGFR+ cells (see schematic below) to an

amount corresponding to 1 copy per genome, as measured by the copy number standards on the left

(n=2). Because the total vector content of these cells was 1 VCN (b) and all the cells were GFP+,

vector integration must have occurred by homology-driven insertion of the ∆LNGFR cassette into

the target site, concomitantly leading to reconstitution of the GFP reporter.

Overall, these results provided proof-of-principle that IDLV-mediated delivery of ZFNs and

donor DNA enable both gene correction and specific integration at the ZFNs target site of a

transgene expression cassette co-delivered by the vector and embedded in the donor homology

sequence. Both of these events appear to be the outcome of homology-directed repair of a ZFN-

delivered ds break, and their relative occurrence may be influenced by experimental design.


0

1,000

2,000

4,000

3,000

5,000

6,000

7,000

23 3 4 14 20 12 9 1 2 5 18 17 21 7 13 25 8 24 10 22 15 6 11 19

M.F

.I. (a

rbitr

ary

unit)

Clone number

Integration site analysis

a

23 3 14 20 12 1 2 5 17 21 7 25 8 24 22 15 6 19

Clone number

K-5

62

TI

HT(n)-TI

IL2RG

GFP

P

GFP

P

n = 1,2,3..PROBE

RRE

GFP

PP

PROBE

P P

PROBE

6 Kb

10 Kb

2.5 Kb

2 Kb

3.5 Kb

8 Kb

b

23 3 14 20 12 1 2 5 17 21 7 25 8 24 22 15 6 19

Clone number

NTC

3’ TI

5’ TI1 Kb

1 Kb

4 Kb HT GFP GFPRRE

GFP

c


0

1

2

4

3

5

6

23 3 14 20 12 1 2 5 17 21 7 25 8 24 22 15 6 19

Clone number

Cop

ies

perc

ell

GFPRRE Fok I

d

Head-to-tail concatemertargeted integration

6GFPpolyA

PGK

IL2RG locus

ZFNs site

Head-to-Tail donorIDLVs concatemer

3 4 5 876

RRE

HDR-mediated

6GFPpolyA

PGKRRE

6GFPpolyA

PGKRRE

GFPpolyA

PGK4 76

e

Supplementary Fig. 5. Integration analysis of the GFP expression cassette in randomly isolated

clones isolated from K-562 cells treated with IL2RG-targeting donor and ZFN-expressing IDLVs.

(a) Mean Fluorescence Intensity (M.F.I. in arbitrary units) of GFP in 24 clones isolated from the

sorted GFP-positive K-562 cells shown in Figure 3c of the main text (sample treated with 1 µg

p24/ml ZFN IDLVs). Clone ID number is indicated on the bottom. Black bars indicate the clones

selected for the integration site analysis reported below.

(b) Genomic DNA extracted from the indicated clones was analyzed by Southern blot using the

restriction enzyme PstI (P) and the IL2RG probe, which are both located outside of the homology

region included in the vector. Schematics (not on scale) on the right of the blot depict targeted

integration into the IL2RG locus of the sole GFP cassette (TI) or of head-to-tail vector concatemers

[HT(n)-TI, where n represents an integer ≥1 of the indicated subunit]. Solid gray line represents the

IL2RG gene, while dotted gray line represents vector sequences. sinLTRs are shown as gray boxes.

The wild-type IL2RG band is also indicated. K-562: un-treated cells.


(c) PCR analyses on genomic DNA from the indicated clones using primers specific for the 3’ and

5’ GFP cassette-IL2RG integration junctions, and for the presence of head-to-tail vector

concatemer. Black arrows in the schematics on the right indicate the location of the primers. NTC:

no template control.

(d) Q-PCR on genomic DNA from the indicated clones to determine the copy number of integrated

vector backbone (RRE), GFP (GFP) and ZFN cDNA (FokI).

(e) Representation of the proposed mechanism for targeted integration of vector concatemers into

the ZFN-target site.

Overall, Southern blot analysis showed a single common band with the expected size for

targeted GFP integration into the Il2RG locus (TI band) in 15 out of the 18 clones analyzed. In

these clones, PCR analysis confirmed the presence of both 5’ and 3’ GFP-IL2RG integration

junctions. Moreover, Q-PCR showed that 13 clones contained 1 copy of the sole GFP cassette and 2

clones (clone 15 and 20) contained 2 copies. In the latter two clones Southern analysis confirmed

biallelic targeting. These data indicate homology-driven integration of the sole GFP expression

cassette into one or both IL2RG loci. In clones 23, 3 and 14 Southern blot showed a single high

molecular weight band; PCR detected both the GFP-IL2RG junctions and the presence of head-to-

tail vector repeats (HT) and Q-PCR detected 2 or more copies of GFP and 1 or more copies of

vector sequences. These data are consistent with homology-driven addition of a concatemer of 2 or

5 GFP cassettes with intervening vector sequence into the IL2RG locus [HT(n)-TI bands, where

n=2 or 5, respectively]. The only exception to this analysis was clone 20, which is one of the two

clones showing biallelic targeting. In this clone the upper band is consistent with homology-directed

insertion of the GFP cassette at the IL2RG locus at the 5’ integration junction and a concomitant

NHEJ of vector terminal sequences at the 3’ junction, as confirmed by Q-PCR. The presence of

ZFN DNA was detected in one of the clone (clone 22).

In conclusion, in all clones analyzed, the integrated GFP cassette was found at the IL2RG

locus, either as a single copy or in the form of head-to-tail concatemer with intervening vector

sequences of increasing size, suggesting that the vast majority of the transgene in the GFP+ ZFN-

treated cells is specifically found at the ZFNs target site. The mechanism of integration was

consistent with homology-directed repair at both junctions in all cases except for one. Head-to-tail

donor concatemers may form by intermolecular recombination or ligation at the vector LTR

sequences, and may become integrated through strand annealing at the two outer homology regions

of the concatemer, as shown by the schematic in (e). Alternatively, intermolecular recombination or

ligation of the vector LTRs might have occurred upon targeted integration of two separate vectors

into the ZFNs site.


14% GFP+MFI=580

17% GFP+MFI= 1100

0.6% GFP+MFI=470

FSC

0.7% GFP+MFI=450

14% GFP+MFI= 1300

21% GFP+MFI=562

GFP

K-562 cells

U-937 cells

Donor - IDLV +

ZFN - IDLVsDonor - IDLV Donor - LV

Supplementary Fig. 6. GFP expression pattern in the indicated cell lines treated with donor-IDLV

containing a PGK-GFP expression cassette flanked by CCR5 homology sequences, and with or

without CCR5-targeting ZFN-IDLVs.

Same samples as those shown in Figure 5b of the main text. Representative density dot

plots of FACS analysis performed at least 1 month post-infection. For comparison, cells transduced

with the same donor construct but packaged with integrase-competent LV are shown. MFI: mean

fluorescent intensity (in arbitrary units) of the GFP+ cells.

The mean fluorescence intensity of GFP differs between cells treated with IDLV/ZFN and

LV, consistent with ZFN-mediated targeted addition of the PGK-GFP cassette into the CCR5 locus.


0

1,000

2,000

3,000

4,000

6,000

5,000

7,000

8,000

9,000

10,000

35 50 43 54 8 36 2 47 19 34 17 31 67 51 52 30 64 37 1 59 57 53 48 4 44 6 26 62 66 21

M.F

.I. (a

rbitr

ary

unit)

Clone number

Integration site analysis

a

50 54 47 17 31 67 64 5957 26 62 21

Clone number

10 Kb

4 Kb5 Kb

8 KbPROBE PROBE

PROBE

TI

HT(n)-TI GFP

S

GFP

N

n = 1,2,3..PROBE

RRE

GFP

NS

PROBE

b

50 54 47 17 31 67 64 5957 26 62 21

Clone number

GFPRRE

GFP GFPRRE

0.7 Kb

1.5 Kb 3’ TI

5’ TI

1.5 Kb NHEJ

5 Kb HT

GFP

c

50 54 47 17 31 67 64 5957 26 62 21Clone number

0

1

2

3

4

6

5

7

8

Cop

ies

perc

ell

GFPRRE Fok I

d


Supplementary Fig. 7. Integration analysis of the GFP expression cassette in cell clones isolated

from K-562 cells treated with CCR5-targeting donor and ZFN-expressing IDLVs.

We randomly isolated 67 cell clones from the treated K-562 cells in Figure 5b of the main text

(sample treated with 0.25 µg p24/ml ZFN IDLVs) and found that, in accordance with the

percentage of the GFP-positive cells in the parental population, 45% of them expressed GFP

although to different intensities.

(a) Mean Fluorescence Intensity (M.F.I. in arbitrary units) of the 30 GFP+ clones. Clone ID number

is indicated on the bottom. Black bars indicate the clones selected for the integration site analysis

reported below. We selected 12 GFP-positive clones representative of the whole GFP expression

range for further molecular analysis.

(b) Genomic DNA extracted from the indicated clones was analyzed by Southern blot using the

restriction enzymes StuI (S), NdeI (N) (both cutting outside of the homology regions present in the

donor vector) and the GFP probe. Schematics (not on scale) on the right of the blot depict targeted

integration into the CCR5 locus of the sole GFP cassette (TI) or of head-to-tail vector concatemers

[HT(n)-TI, where n represents an integer ≥1 of the indicated subunit]. Solid gray line represents the

CCR5 gene, while dotted gray line represents vector sequences. sinLTRs are shown as gray boxes.

(c) PCR analyses on genomic DNA from the indicated clones using primers specific for the 3’ and

5’ GFP cassette-CCR5 integration junctions, and for the presence of head-to-tail vector concatemer.

Black arrows in the schematics on the right indicate the location of the primers.

(d) Q-PCR on genomic DNA from the indicated clones to determine the copy number of integrated

vector backbone (RRE), GFP (GFP) and ZFN cDNA (FokI).

Overall, in 5 out of 12 clones (Clone 64, 57 59, 62 and 21) we detected only the GFP

sequence, with a range of copies per cell that varied from 1 to 3 (note that K-562 cells are tetraploid

for the CCR5 gene4). In these clones, Southern blot analysis showed a single common band with the

expected size for targeted GFP integration into the CCR5 locus (TI band), and PCR analysis

detected the presence of both 5’ and 3’ integration junctions. These data indicated homology-driven

integration of the sole GFP expression cassette into one or more CCR5 loci. In clones 54, 17 and 31,

Q-PCR analysis detected multiple copies of GFP and vector sequences, while Southern blot

analysis showed a single high molecular weight band that was of different size in each clone. In

these clones, PCR analysis detected both the GFP-CCR5 junctions and the presence of head-to-tail

vector repeats (HT). These data indicated homology-driven addition of a vector concatemer of

different size into the CCR5 locus [HT(n)-TI bands]. In the remaining clones, where Q-PCR

analysis detected multiple copies of GFP and vector sequences, Southern blot analysis showed 2 to

3 bands per clone, corresponding to the TI (Clones 50, 47 and 26) and the HT(n)-TI (Clones 50, 47,


67 and 26) bands. These data indicated targeted addition of the sole GFP cassette and of vector

concatemers into multiple CCR5 alleles in these clones. The only exception to this analysis was

clone 67. In this clone, the lower band was consistent with homology-directed insertion of the

cassette at the CCR5 locus at the 5’ integration junction and a concomitant NHEJ of vector terminal

sequences with the CCR5 locus at the 3’ junction, as indicated by PCR analysis [(see PCR lane in

(c)]. Re-probing of the Southern blot with a CCR5 probe confirmed that all bands labelled with the

GFP probe were derived from targeted integration into the CCR5 locus (data not shown). Q-PCR

analysis of 12 GFP-negative clones did not show any vector, GFP sequences and FokI integrated

(data not shown).

In conclusion, in all clones analyzed, the integrated GFP cassettes were found at the CCR5

loci, either as a single copy or in the form of head-to-tail concatemer with intervening vector

sequences of increasing size. The mechanism of integration was consistent with homology-driven

integration at both junctions in all cases except for one.


Donor IDLV Delivery of Donor and ZFNs

GFP

SSC

3% GFP+ 0.3% GFP+ 5.1% GFP+

FSC

SSC

7-A

AD

FSC

SS

EA-4

FSC

R1

R2

R3

Gated on R1 Gated on R1+ R2

Gated on R1+ R2 + R3

HUES-3

Supplementary Fig. 8. Representative FACS analyses to indicate the gates and markers used to

identify viable human ES cells in the culture. SSC: side scatter. FSC: forward scatter. 7-AAD: 7-

aminoactinomicin D. SSEA-4: stage-specific embryonic antigen 4.


>CCR5; ZFN-L

MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAMAERPFQCRICMRNFSDRSNLSRH

IRTHTGEKPFACDICGRKFAISSNLNSHTKIHTGSQKPFQCRICMRNFSRSDNLARHIRTHTGEKPFAC

DICGRKFATSGNLTRHTKIHLRGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMK

VMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEEN

QTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMI

KAGTLTLEEVRRKFNNGEINF

>CCR5; ZFN-R

MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAMAERPFQCRICMRNFSRSDNLSVH

IRTHTGEKPFACDICGRKFAQKINLQVHTKIHTGEKPFQCRICMRNFSRSDVLSEHIRTHTGEKPFAC

DICGRKFAQRNHRTTHTKIHLRGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMK

VMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEEN

QTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMI

KAGTLTLEEVRRKFNNGEINF

Supplementary Fig. 9. The complete amino acid sequences of the CCR5-targeting ZFNs.

Underlined are the recognition α-helices (from position –1 to position +6). The design and

characterization of these CCR5-targeting ZFNs will be described in detail elsewhere (E. Perez, C.

June, PDG, MCH, FDU, KAK, DA, YL submitted).


SUPPLEMENTARY METHODS

Vectors. ZFN-expressing and donor transfer constructs were generated from the HIV-derived self-

inactivating transfer construct pCCLsin.cPPT.hPGK.eGFP.Wpre or from related constructs

containing the U3 region of the SFFV LTR5 or the human Elongation Factor 1α (EF1α) promoter-

intron in place of the hPGK promoter; detailed cloning information is available upon request.

Briefly, for the donor vectors, the following sequences from the ZFN-targeted genes were cloned

either into the MfeI site (combined vector shown in Fig. 1a of the main text and S donor in

Supplementary Fig. 2a), in place of the hPGK.eGFP cassette (Fig. 3a, 4d and 5a of the main

text), or within the BbsI site of the sinLTR (D donor in Supplementary Fig. 2a) in antisense

orientation relative to vector transcription: the IL2RG donor contains a PCR-generated 1,543 bp

fragment of the IL2RG locus (pos. 70245100-70246642 on the "-" strand of the X chromosome;

March 2006 UCSC human genome assembly) with or without a hPGK-GFP-polyA cassette inserted

into the DraIII site; the CCR5 donor contains a PCR-generated 1881 bp fragment of the CCR5

locus (pos. 46389088-46390968 on the "-" strand of chromosome 3; March 2006 UCSC human

genome assembly) with a hPGK-GFP/PuroR-polyA cassette cloned into a BglI site that was inserted

between the CCR5-ZFN binding sites.

HUES differentiation. Embryoid Bodies (EBs) were derived from HUES colonies cultured in

suspension medium without growth factors. After four days of culture, EBs were plated on matrigel

(BD Bioscience)-coated dishes and grown for 8-10 days in DMEM-F12 medium supplemented with

transferrin, insulin, sodium selenite, heparin, putrescine, progesterone and 20 ng/ml bFGF . The

plated EBs originated columnar cells organized in neural rosettes by day 5. The rosettes were

isolated from the surrounding cells by collagenase, cultured in the same medium supplemented with

20 ng/ml EGF and induced to differentiate into neural derivatives by growth factors withdrawal for

4-6 weeks, then fixed and analysed by confocal fluorescence microscopy.

6

7

Confocal immunofluorescence analysis. Cells were fixed in 4% paraformaldehyde and

permeabilized/blocked for 1 hour in 10% FBS, 1% bovine serum albumin, 0.1% Triton in PBS. The

cells were then incubated for 2 hours with TO-PRO®-3 iodide (InvitrogenTM) and the following

primary antibodies: anti-GFP (InvitrogenTM) or anti-human Oct-3/4 (Santa Cruz Biotechnology,

Inc.) or TUJ-1 (COVANCE). Fluorescent signals from single optical sections were acquired by 3-

laser confocal microscope (Radiance 2100; Bio-Rad, Hercules, CA).


Mismatch selective endonuclease assay. This assay was used to measure the extent of mutations

consequent to NHEJ at the ZFN target site3. Briefly, PCR was performed using primers flanking the

ZFN-recognition site in the CCR5 gene (primers and conditions are indicated below in the PCR

analyses section). The PCR product was denaturated, allowed to re-anneal and digested with

Surveyor nuclease assay (Transgenomic). Because this enzyme cuts DNA at sites of duplex

distortions, the products of re-annealing between wild type and mutant alleles - carrying mutations

or deletions consequent to ZFN activity - are specifically digested. The reaction products were

separated on a Spreadex EL1200 Wide Mini gel (Elchrom Scientific), stained by ethydium bromide

and the bands quantified by ImageQuant TL software. The ratio of the uncleaved parental fragment

to the two lower migrating cleaved products was calculated using the formula (1-(parental

fraction)1/2)x100.

PCR analyses.

Editing of the IL2RG gene.

Forward IL2RG primer: 5’-GCTAAGGCCAAGAAAGTAGGGCTAAAG-3’

Reverse IL2RG primer: 5’-TTCCTTCCATCACCAAACCCTCTTG-3’

Amplification conditions: 94 °C for 10 min, then 20 cycles of 94 °C for 1 min, 60 °C for 30 sec and

72 °C for 1 min and 40 sec, followed by extension at 72 °C for 7 min. The expected amplicon

length is 1.7 Kb.

Targeted gene addition into the IL2RG gene (5’ junction).


Reverse hPGK primer: 5’-ACGTGAAGAATGTGCGAGACCCAG-3’.



length is 980 bp.

Targeted gene addition into the IL2RG gene (3’ junction).

Forward BGHpA primer: 5’-ATGCGGTGGGCTCTATGG-3’

Reverse IL2RG primer: 5’-TTCCTTCCATCACCAAACCCTCTTG-3’.



length is 860 bp.

Amplification of the DCL1 gene.

Forward ΨA: 5’-GGGTGGATCAGGTATTGTCTGG-3’

Reverse ΨA: 5’-GTTCTGTACTGTAGTTGAGAGAGC-3’



72 °C for 50 sec, followed by extension at 72 °C for 10 min.

The expected amplicon length is 600 bp.

Targeted gene addition into the IL2RG gene (from outside the homology region spanning the

insertion).


Reverse IL2RG primer: 5’-TTCCTTCCATCACCAAACCCTCTTG-3’

Amplification conditions: 94 °C for 10 min, then 20-30 cycles of 94 °C for 1 min, 60 °C for 30 sec

and 72 °C for 4 min, followed by extension at 72 °C for 10 min. The expected amplicon length for

targeted gene addition of the GFP cassette is 3.2 Kb, while the amplicon length for the IL2RG half-

gene cDNA is 2.5 Kb.

Detection of the head-to-tail concatemers into the IL2RG gene.

Forward GFP primer: 5’-ACAACCACTACCTGAGCACC-3’

Reverse hPGK primer I: 5’-ACGTGAAGAATGTGCGAGACCCAG-3’


72 °C for 5 min, followed by extension at 72 °C for 10 min. The expected amplicon length is 4 Kb

for the 1 LTR repeat or 4.25 Kb for the 2 LTR repeat.

Discrimination between the wild type and the mutated IL2RG cDNA.

Forward IL2RG Exon 5 Wt primer: 5’-TTTAACCCACTCTGTGGAAGT-3’

Forward IL2RG Exon 5 mutated primer: 5’-TTTAACCCACTCTGTGGCTCC-3’

Reverse IL2RG 3’UTR primer: 5’-TGGGGTGAGGTGAGTATGAGACG-3’

Amplification conditions: 95 °C for 10 min, then 35 cycles of 95 °C for 45 sec, 60 °C for 30 sec and

72 °C for 1 min, followed by extension at 72 °C for 10 min. The expected amplicon length is 607

bp.

Targeted gene addition into the CCR5 gene (3’ junction).


Reverse CCR5 primer: 5’-ATCTAGCCTTGTCCTTCCTCC-3’

Amplification conditions: 94 °C for 10 min, then 30-40 cycles of 94 °C for 1 min, 60 °C for 30 sec

and 72 °C for 1 min and 30 sec, followed by extension at 72 °C for 10 min. The expected amplicon

length is 1.5 Kb.

Targeted gene addition into the CCR5 gene (5’ junction).

Forward CCR5 primer: 5’-TTGGAGGGGTGAGGTGAGAGG-3’

Reverse hPGK primer: 5’-TGAAGAATGTGCGAGACCCAGG-3’




bp.

Detection of the head-to-tail concatemers into the CCR5 gene.


Reverse hPGK primer: 5’-TGAAGAATGTGCGAGACCCAGG-3’


72 °C for 5 min, followed by extension at 72 °C for 10 min. The expected amplicon length is 4.9 Kb

for the 1 LTR repeat or 5.1 Kb for the 2 LTR repeat.

NHEJ of the vector into the CCR5 gene (3’ junction).

Reverse U5 PBS primer: 5’-GGCGCCACTGCTAGAGATTTT-3’

Reverse CCR5 primer I: 5’-ACAAGTCTCTCGCCTGGTTCTAAG-3’



length is 1.7 Kb.

Detection of FokI cDNA by Q-PCR.

Forward primer: 5’-CCTGACGGCGCCATCTAT-3’ 600 nM

Reverse primer: 5’-CGATCACGCCGTAATCGAT-3’ 600 nM

Probe: 5’-6-FAM-CAGTGGGCAGCCC-MGB-3’ 200 nM

Standard TaqMan amplification conditions were used.

Mismatch selective endonuclease assay.

CCR5_Cel1_160_F1 primer: 5’-AAGATGGATTATCAAGTGTCAAGTCC-3’

CCR5_Cel1_160_R1 primer: 5’-CAAAGTCCCACTGGGCG-3’



bp.


SUPPLEMENTARY REFERENCES

1. Urnov, F.D. et al. Highly efficient endogenous human gene correction using designed zinc-

finger nucleases. Nature 435, 646-651 (2005).

2. Follenzi, A., Ailles, L.E., Bakovic, S., Geuna, M. & Naldini, L. Gene transfer by lentiviral

vectors is limited by nuclear translocation and rescued by HIV-1 pol sequences. Nat Genet

25, 217-222 (2000).

3. Miller, J.C. et al. An improved zinc-finger nuclease architecture for highly specific genome

editing. Nat Biotechnol 25, 778-785 (2007).

4. Naumann, S., Reutzel, D., Speicher, M. & Decker, H.J. Complete karyotype characterization

of the K562 cell line by combined application of G-banding, multiplex-fluorescence in situ

hybridization, fluorescence in situ hybridization, and comparative genomic hybridization.

Leuk Res 25, 313-322 (2001).

5. Demaison, C. et al. High-level transduction and gene expression in hematopoietic

repopulating cells using a human immunodeficiency [correction of imunodeficiency] virus

type 1-based lentiviral vector containing an internal spleen focus forming virus promoter.

Hum Gene Ther 13, 803-813 (2002).

6. Zhang, S.C., Wernig, M., Duncan, I.D., Brustle, O. & Thomson, J.A. In vitro differentiation

of transplantable neural precursors from human embryonic stem cells. Nat Biotechnol 19,

1129-1133 (2001).

7. Lazzari, G. et al. Direct derivation of neural rosettes from cloned bovine blastocysts: a

model of early neurulation events and neural crest specification in vitro. Stem Cells 24,

2514-2521 (2006).


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