US20130209426A1

US20130209426A1 - Method for genome modification

Info

Publication number: US20130209426A1
Application number: US13/594,053
Authority: US
Inventors: Allan Bradley; Kosuke Yusa; Sheikh Tamir Rashid
Original assignee: Cambridge Enterprise Ltd; Genome Research Ltd
Current assignee: Cambridge Enterprise Ltd; Genome Research Ltd
Priority date: 2010-08-25
Filing date: 2012-08-24
Publication date: 2013-08-15
Also published as: JP2013535980A; EP2609192A1; GB201014169D0; AU2011294869B2; CN103180436B; US9284576B2; CN103180436A; AU2011294869A1; CA2841641C; JP6063383B2; WO2012025725A1; CA2841641A1; DK2609192T3; US20130156743A1; EP2609192B1; SG188288A1; SG10201506704XA

Abstract

The present disclosure includes methods for manipulation of the genome, to products obtained or obtainable from such methods, and uses of these products.

Description

This application is a continuation in part of PCT/GB2011/001275, filed Aug. 25, 2011, which claims the benefit of GB Application No. 1014169.5, filed Aug. 25, 2010, both of which are herein incorporated by reference in its entirety.

BACKGROUND

A number of different techniques are available for genome modification. However, in many such methods the modification process leaves a residual sequence in the targeted genome. For example, Soldner et al (Cell, 146, 318-331, July 2011) disclose 4 targeting strategies for genome modification in an iPS cell. Two selection based strategies leave residual DNA (a IoxP site) in the genome. The non-selection based approaches have the obvious disadvantage that they are less efficient than selection based approaches.

SUMMARY OF DISCLOSURE

A method of modifying the genome of a cell, the method comprising recombining into the genome of the cell an exogenous nucleic acid, the nucleic acid comprising
(i) a modifying sequence and
(ii) a selection element flanked by transposon inverted repeats,
the method further comprising the steps of:
selection of insertion of the nucleic acid into the genome using the selection element, and
use of a transposase to excise the selection element, such that after transposase mediated excision the only exogenous nucleic acid remaining in the genome is the modifying sequence
A method of correcting or introducing a mutation in a genome of a cell, the method comprising
i recombining into the genome of the cell a nucleic acid comprising (i) a modifying sequence which corrects or introduces the mutation and (ii) a selection element flanked by transposon inverted repeats,
ii selection of insertion of the nucleic acid into the genome using the selection element, and
iii use of a transposase to excise the selection element.
A cell comprising an exogenous nucleic acid, the exogenous comprising (i) a modifying sequence and
(ii) a selection element flanked by transposon inverted repeats
A cell comprising a genome which has been modified by insertion of a modifying sequence using a method according to claim 1.
A modified cell comprising a modifying nucleic acid sequence, the cell having been selected for the insertion of the modifying nucleic acid sequence using a selectable element, and wherein the selectable element is then removed from the genome, such that genome of the modified cell is identical to the genome of the original starting cell except for the modifying nucleic acid.
A collection of cells, such as an organ or tissue, wherein the genomes of all, or a proportion, of the cells have been modified by insertion of a modifying sequence using a method as described above.
A collection of cells, such as an organ or tissue, wherein a mutation in the genomes of all, or a proportion, of the cells has been corrected by insertion of a modifying sequence using a method as described above.
A method for treating a disease in a patient, the method comprising modifying the genome of a cell using a method as described above to correct a mutation associated with the disease in the cell, and using the cell, or cells derived or differentiated therefrom, in disease treatment.
Use of a cell as described above in treatment of disease. A cell as described above for use in the treatment of disease.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Correction of the G290T mutation in the Tyr gene in mIPSCs.

a, The strategy for precise genome modification using the piggyBac transposon. Top line, structure of the Tyr gene; red line, 5′ external probe for Southern blot analysis; open arrow, piggyBac transposon carrying a PGK-puroΔtk cassette; P1, P2 and P3, PCR primers; B, BamHI; E, EcoNI. b, c, Southern blot (b) and PCR analyses (c) showing insertion (c/PB) and excision (c/Rev) of the piggyBac transposon. ES, mouse ESCs as a control. d, e, Sequence analyses revealed correction of the G290T mutation (d) and seamless excision of the piggyBac transposon (e). Note that two silent mutations (A and T indicated by arrowheads) introduced near the TTAA site were also detected. f, A chimeric mouse generated by injecting corrected Tyr c/Rev mIPSCs (left) displays black coat color. Right, a non-injected albino mouse.

FIG. 2. Correction of the Z mutation in A1ATD-hIPSCs.

a, The strategy for precise genome modification using ZFNs and the piggyBac transposon. Top line, structure of the A1AT gene; blue lines, Southern blot probes; thin and thick boxes, non-coding and coding exons, respectively; open arrow, piggyBac transposon; B, BamHI; A, AflIII. b, Sequences of wild-type (Reference), Z, PB, and Rev alleles. Amino acid position 342 (blue), recognition sites for ZFNs (green), piggyBac excision site (red) are shown. Sequence changes in Rev allele from Z allele were indicated by asterisks. c, Surveyor nuclease assay showing the cleavage of Z mutation in ZFNs-transfected K562 cells. Non-transfected cells were used as a control. d, Southern blot analysis showing bi-allelic piggyBac insertion (B-16) and bi-allelic excision (B-16-C2, -C3 and -C6) during correction of the A1ATD-hIPSCs line B. Genomic DNA was digested by BamHI (5′ and PB probes) or AlfIII (3′ probe). Genotype: ZZ, homozygous for Z allele; PP, homozygous for insertion of piggyBac; RR, homozygous for reverted allele. e, Sequence analysis showing correction of Z mutation in 3 corrected hIPSC lines. Wild-type sequence (top line) and A1ATD-hIPSC (second line).

FIG. 3. Functional analysis of restored A1AT in c-hIPSCs-derived hepatocyte-like cells.

a, Immunofluorescence showing the absence of polymeric A1AT protein in hepatocyte-like cells generated from c-hIPSCs. All forms of A1AT (left panels) and misfolded polymeric A1AT (middle panels). b, c, ELISA to assess the intracellular (b) and secreted (c) levels of polymeric A1AT protein in hepatocyte-like cells derived from A1ATD-hIPSCs (ZZ), c-hIPSCs (RR) and control hIPSCs (++). d, Endoglycosidase H (E) and peptide:N-glycosidase (P) digestion of A1AT immunoprecipitated from uncorrected (ZZ), corrected (RR) and control (++) hIPSC-derived hepatocyte-like cells (upper panels) and corresponding culture medium (lower panels). e, Chymotrypsin ELISA showing that corrected cells (RR) have A1AT enzymatic inhibitory activity that is superior to uncorrected cells (ZZ) and close to adult hepatocytes. f, g, Immunofluorescence of transplanted liver sections detecting human albumin (f) and A1AT (g). DNA was counterstained with DAPI. h, ELISA read-out of human albumin in the mouse serum longitudinally followed for each mouse. Asterisk, the mouse was subjected to histology analysis. Scale bars, 100 μm. Data in b, c and e are shown as mean±s.d. (n=3). Student's t-test was performed. NS, not significant.

FIG. 4. Southern blot analyses of bi-allelic targeting of the A1AT gene in 3 independent A1ATD-iPSC lines. a, The structures of the Z allele and the targeted PB allele. Thin and thick open boxes, non-coding and coding exon, respectively; B, BamHI. b, The result obtained with A1ATD-iPSCs from patient B. Both alleles in line B-16 were correctly targeted while line B-17 carried one correctly targeted allele and one abnormal allele. c-e, Similar results were obtained with A1ATD-iPSCs generated from patient A (c) and from patient C (d, e). Genomic DNA was digested by BamHI and hybridized with the 5′ external probe. The first lane on each panel is DNA from the parental A1ATD-iPSC line. Correct homozygous clones are highlighted in red.

FIG. 5. Characterization of genome modification in A1ATD-IPSC line C.

a, b, Southern blot (a) and sequence (b) analyses showing bi-allelic correction of Z mutation in A1ATD-iPSCs generated from patient C. See FIG. 2 for details.

FIG. 6. Pluripotency of corrected iPSCs.

a, Immunofluorescence showing the expression of pluripotency markers OCT4, SOX2, NANOG and TRA1-60 in corrected iPSCs grown for 20 passages after correction. DNAs were stained with DAPI (lower panels). b, Immunofluorescence of in vitro differentiated corrected iPSCs showing maintenance of differentiation capacity into three germ layers. Scale bars, 200 μm

FIG. 7. Detailed analysis of 20q11.21 amplification

a, aCGH result of the homozygous clone, C-B6. Top, an ideogram of chromosome 20; middle, genes in the region pointed by red in the chromosome ideogram; bottom, the log2 ratio plot of C-B6. The amplified region is highlighted in red. The log2 ratios in the adjacent region highlighted in gray are used as a normal copy number control for a comparison shown in c. b, SNP data showing B allele frequency (x axis on top) and smoothed log R ratio (x axis on bottom) of chromosome 20 of indicated cell lines. The 20q11.21 is highlighted in gray. c, Box plots showing log2 ratios of the affected (red) and the adjacent normal (gray) regions. Bars within the box plots represent median values. The ends of bars indicate the 25th and 75th percentiles, and the 10th and 90th percentiles are represented by error bars. Student' s t-test was performed. NS, not significant. d, Copy number and normalized copy number of the 20q11.21 region per diploid genome. Copy numbers were calculated from mean values of log2 ratio in the region. Copy numbers in the homozygous clones were further normalized with the calculated copy number of A1ATD-iPSC line C

FIG. 8. SNP analyses. a, b, SNP array data showing entire chromosome 14 (a) and 830-kb region around the A1AT (SERPINA1) gene (b). Data for fibroblasts and correctly targeted homozygous clones from each patient are shown. Blue dots represent B allele frequency (left y axis) of each SNP and red lines represent smoothed Log R ratio (right y axis). Note that heterozygosity is maintained in all 6 targeted clones.

FIG. 9. Differentiation of corrected iPSCs into hepatocyte-like cells.

a, Quantitative RT-PCR analyses showing the expression of markers of liver development during corrected iPSC differentiation. Values are normalized to expression on day 0. b, Immunostaining showing the expression of specific proteins marking key stages of hepatocyte development during corrected iPSC differentiation (Day 3, endoderm; Day 10, hepatic endoderm;

Day

18 and 20, hepatic progenitor; Day 25, hepatocyte-like cells). c, Flow cytometric analyses showing that 80% of cells are positive for CXCR4 at 3 days of differentiation (endoderm stage) and 80% of cells are positive for Albumin at 25 days of differentiation. d-g, Corrected iPSC-derived hepatocyte-like cells display functional activity characteristic of primary human hepatocytes including presence of intracellular glycogen storage as shown by periodic acid Schiff staining (d), uptake of DiL-LDL (e), albumin secretion (f) and CytP450 metabolism (g). Scale bar, 100 μm

FIG. 10. Characterization of integration-free A1ATD-iPSC.

a, b, Pluripotency marker expression by RT-PCR (a) and immunostaining (b) analyses. Note that Sendai virus (SeV) genome were not detected in established iPSC lines. RV, retrovirus. c, Bisulfite sequencing of OCT4 and NANOG promoter regions. d, Southern blot analysis of the key pluripotency gene, OCT4, showing an evidence of integration-free iPSCs. Genomic DNA was digested with PstI. The OCT4 exon1 was used as a probe. Arrowhead, endogenous OCT4; asterisk, provirus-derived OCT4. e, CGH plot of primary A1ATD-iPSC line 3, showing no copy number change. f, Immunostaining of in vitro differentiated cells, showing maintenance of differentiation capacity. Scale bars, 500 μm (b), 200 μm(f)

FIG. 11. Correction of the Z mutation in the integration-free A1ATD-iPSC line B.

a, Validation of genetic modification by Southern blot analysis. See FIG. 2 for the detail. b, Sequencing analysis of gene correction. Note that the Z mutation (A) is corrected to wild-type sequence (G), while the piggyBac excisions produced TTAA as designed. c, CGH analysis showing that the 3-G5-A7 line kept the intact genome compared to the paretal fibroblasts. d, Immunostaining of 3-G5-A7-derived hepatocyte-like cells, showing that the corrected cells express normal A1AT porteins. Left, all forms of A1AT; middle, polymeric A1AT; right, DNA staining by DAPI. Scale bar, 50 μm.

DETAILED DESCRIPTION

The present disclosure generally relates to method of modifying the genome of a cell, the method comprising recombining into the genome of the cell an exogenous nucleic acid, the nucleic acid comprising
(i) a modifying sequence and
(ii) a selection element flanked by transposon inverted repeats,
the method further comprising the steps of:
(a) selection of insertion of the nucleic acid into the genome using the selection element, and
(b) use of a transposase to excise the selection element, such that after transposase mediated excision the only exogenous nucleic acid remaining in the genome is the modifying sequence.
Recombination of the exogenous DNA sequence into the cell suitably occurs by homologous recombination. As such the exogenous nucleic acid is suitably in a form that can be incorporated into the genome of the cell by homologous recombination and the nucleic acid is provided to the cell under conditions that allow homologous recombination to occur. These conditions are well known in the art. In one aspect the exogenous nucleic acid is DNA, which may be provided as a circular plasmid or linear nucleic acid.
Homologous recombination is well known and understood. For example, the exogenous nucleic acid to be inserted suitably comprises two regions the same or a very similar sequence to the genome of the cell to be modified, such that homologous recombination between the homologous regions results in insertion of the exogenous nucleic acid into the chromosome, replacing the equivalent genomic sequence.
The exogenous nucleic acid to be inserted comprises a selectable marker and the insertion event can therefore be selected for by selection of the selection element under appropriate selection conditions.
In a further aspect of the invention the efficiency of targeting can be increased using Zinc Finger Nucleases (ZFNs) or Transcription activator like effector nucleases (TALENS) to affect double stranded breaks which can be repaired by, for example, homology directed DNA repair pathways using an exogenous donor plasmid or other exogenous nucleic acid sequence as a template. These approaches may be particularly useful in human cells where homologous recombination can be relatively inefficient. Thus the methods disclosed herein include those in which homologous recombination is used in combination with a technique for increasing the efficiency of the homologous recombination event. For example, the method of the invention also comprises providing to the cell a zinc finger nuclease or Transcription activator like effector nucleases (TALES), or nucleic acid encoding the same, to allow expression of the ZFN or TALES in the cell whose genome is to be modified.
By way of non-limiting example, we disclose herein that a combination of zinc finger nucleases (ZFNs)⁷and piggyBac^8,9technology in hIPSCs can achieve bi-allelic correction of a point mutation (Glu342Lys) in the α₁antitrypsin (A1AT, also called SERPINA1) gene that is responsible for α₁-antitrypsin deficiency (A1ATD). Genetic correction of hIPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene targeting technology that is currently available and prevents contamination of the host genome with residual non-human sequences. These results provide the first proof of principle for the potential of combining hIPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.
In one aspect the cell to be modified is a human cell or a non-human animal cell. In one aspect the cell is a pluripotent cell, such as a totipotent non-human cell, or a pluripotent human or non-human cell. The cell may also be an iPS cell, such as a human iPS cell or non-human iPS cell. An iPS cell may originate from a fibroblast cell, adipose tissue, bone marrow or other suitable somatic cell. iPS cells may be generated using methods well known in the art, such as expression of Yamanaka factors as disclosed in EP1970446 or as disclosed in Wang et at www.pnas.org/cgi/doi/10.1073/pnas.1100893108
iPS cells may be derived from individuals with diseases caused by genetic mutation, for example diseases affecting the epithelial, endothelial, or interstitial compartments of the lung, such as cystic fibrosis, a-1 antitrypsin deficiency-related emphysema, scleroderma, and sickle-cell disease.
In one aspect the cell to be modified is an iPS cell is derived from an individual in whom it is desired to re-transplant the iPS cell, or cells derived or differentiated therefrom.
In one aspect an iPS cell is reprogrammed using an integration-free method, for example as disclosed herein using Sendai virus vectors (ref 25). Suitably the reprogramming of an iPS cell does not leave any residual DNA in the genome of the iPS cell. Another example of suitable methodology includes that disclosed in Somers et al, Stem cells 2010;28:1728-1740. Another example of suitable integration free technology includes providing mRNA or DNA minicircle as disclosed in https://www.stemgent.com/applications/reprogramming.
In one aspect the iPS cell modified by the method of the invention maintains expression of pluripotency markers for at least 10, at least 15, or at least 20 passages and or the ability to differentiate into cells expressing markers of three germ layers.
The modifying sequence is the sequence of exogenous nucleic acid which is not the selection element flanked by the transposase inverted repeat sequences. This sequence of the exogenous nucleic acid generally remains in the genome after transposase excision.
The modifying sequence may comprise a nucleic acid sequence which is substantially identical to a part of the genome of the cell to be modified but which has an addition, substitution or deletion of nucleotides with respect to that genome. The modifying nucleic acid may be isogenic with the genome with the exception of the addition, substitution or deletion. The modifying nucleic acid may be isogenic with the genome with the exception of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides, for example, or even with the exception of up to 50 nucleotides, e.g. up to 100 nucleotides, up to 200 nucleotides, up to 500 nucleotides or up to 1 kb, or 2 kb.
Alternatively the modifying sequence may be a sequence which is only partly isogenic with the genomic DNA, for example at the ends of the nucleic acid sequence to allow for homologous recombination, but may comprise a region of non-isogenic DNA of some length, such as more than 20, 30 ,40 or 50 nucleotides, such as a gene from a species different to that of the cell to be modified.
In one aspect the modification causes a change in function of the cell, or the tissue or organism derivable from development of the cell. The modification need not be associated with a disease phenotype.
In one aspect the modification is correction of a mutation associated with disease, such as correction of a point mutation. A point mutation may be changed to a nucleotide that is not associated with a disease phenotype, for example, changing of the Lys 342 to Glu in the α1-antitrypsin (A1AT, also called SERPINA1) gene that is responsible for α 1-antitrypsin deficiency (A1ATD). There are many other diseases associated with mutations, such as point mutations, that can be addressed using the present methods, including Cystic fibrosis, Tay—Sachs disease, Sickle Cell Anemia and Neurofibromatosis.
In one aspect the modification restores an enzymatic function of the cell.
In one aspect the modification is a correction of one or both alleles of a gene. The modification may be a correction of one or both alleles of a point mutation associated with a disease phenotype.
As disclosed above, the method can be, for example:
A method of correcting a mutation in a genome of a cell, the method comprising
1) recombining into the genome of the cell an exogenous nucleic acid comprising (i) a modifying sequence which corrects the mutation and (ii) a selection element flanked by transposon inverted repeats,
2) selection of insertion of the nucleic acid into the genome using the selection element, and
3) use of a transposase to excise the selection element,
wherein after the excision process the genome of the corrected cell is identical to the genome of the original cell with the only exception being mono or biallelic correction of the mutation.
In such a method the modifying sequence comprises the wild type nucleic acid sequence that would be found in individuals without disease which is inserted into the genome to replace the genome region comprising the mutation.
It will be appreciated that the modifying sequence can also be used to introduce a mutation into a cell, and thus the invention also relates to a method of correcting or introducing a mutation into a genome of a cell, as disclosed herein.
It will also be appreciated that cells accumulate mutations every time they divide such that no two cells are alike. Accordingly in one aspect, after the excision process, the genome of the corrected cell is identical to the genome of the original cell with the only exception being mono or biallelic correction of the mutation or mono or biallelic introduction of a mutation—such as a point mutation—but wherein, in one aspect, there may be additional mutations in the genome that occur due to the natural mutation processes that occur in during cell division.
Thus the invention relates to a method of correcting or introducing a modification into the genome of a cell as disclosed herein, wherein after the transposon mediated excision process the genome of the corrected cell comprises the desired specific modification and may comprise a random mutation(s) but comprises no other nucleic acid sequence alteration, for example, no nucleic acid sequence alteration that might arise from insertion of, or recombination with, of a selectable element.
In one aspect, the exogenous nucleic acid may comprise a selection element located within transposon inverted repeats close to the site of the mutation to be corrected, for example the inverted repeat sequence may be between 3-100 nucleotides away from the site of the mutation to be corrected, such as 5-20 nucleotides, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides. The exogenous nucleic acid may be engineered to contain a transposon insertion site for insertion of a transposon carrying a selection element, to allow the selection element to be located close to the site of the mutation to be corrected. Alternatively the nucleic acid may be synthesized to contain a suitable site for insertion of a transposon carrying a selection element. Where the exogenous nucleic acid has been engineered to contain a site for transposon integration then in one aspect, which can be preferred, insertion of the transposon site does not change the protein encoded by the nucleic acid into which the site is engineered. In one aspect the site is the TTAA piggyBac insertion sequence.
Therefore, in another aspect, the method disclosed herein comprises use of an exogenous nucleic acid capable of recombination into the host genome, the nucleic acid comprising a transposon insertion site which is not found in the same relative location in the genome of the cell to be modified and which site does not affect any amino acid coding potential of the nucleic acid, such that—after transposase mediated excision—the genome of the cell comprises a mutation that has been corrected or introduced and additionally comprises a transposon insertion site.
As such, the insertion of the modifying sequence into the genome and subsequent excision of the selection element may result in a genome which has the native genomic sequence at the point of excision of the selection element, or may result in a genome which has a non-native genomic sequence at the point of excision of the selection element, suitably a nucleic acid sequence which does not change the sequence of any protein encoded by the nucleic acid.
In one aspect the inverted repeat sequences flanking the selectable marker are piggyBac inverted repeat sequences that can be excised by the action of the piggyBac transposase. The inverted repeat sequences are, in one aspect, about 200-350 base pairs, such as 250 or 300 bp, and may be unequal lengths, such as 300 bp for 5′ side and 250 bp for 3′ side, which are typical repeat length for piggyBac vector. In one aspect the minimla inverted repeat size is 311 and 236 base pairs for the 5′ and 3′ respectively. Other transposase sequences may be used. The action of a suitable transposase enzyme may provide for complete excision of the sequence comprised between those repeats to leave only a single transposon target site and leave no nucleotide footprint remaining in the genome, or may leave a single transposon target site which alters the nucleotide sequence of the genome of the cell but not the protein(s) encoded by that genome, for example.
In the present method, for example, an exogenous nucleic acid may be provided comprising a selection element located within piggyBac transposon inverted repeats, inserted into a piggyBac target site (TTAA). Insertion of the nucleic acid into the genome is followed by removal of the selectable element from the genome by action of a transposase such as the piggyBac transposase on the transposon inverted repeats, which will leave a TTAA remaining in the genome which TTAA could be either the wild type sequence or a variant of the genome if there is no naturally occurring TTAA sequence at that point in the genome. Transposase action may be provided by, for example, expression of the transposase gene from a plasmid within the cell.
Thus the method of the invention also comprises providing to the cell an expression vector comprising an expressable transposase. Expression vectors may be co-delivered with the exogenous DNA, for example by co-electroporation.
The invention is not limited to the inverted repeats of the piggyBac transposon and extends to any suitable transposon sequences that can be excised without leaving a footprint. In one aspect the transposase has a TTAA target site. In one aspect the transposon elements are from the superfamily of TTAA-specific mobile elements.
In one aspect the transposase for use in the method is the piggyBac transposase, which optionally may be engineered to have increased activity with respect to the wild type sequence, and may be a hyperactive piggyBac transposase as disclosed herein.
In one aspect the genome is modified on one allele of a gene after use of the method. In one aspect the genome is modified on both alleles of a gene after use of the method.
In one aspect the method can achieve at least, or greater than, 30%, 40%, 45% or 50% efficiency in targeting on one allele.
In one aspect the method is capable of simultaneous biallelic modification. In one aspect the method can achieve at least or greater than 1%, 2%, 3%, 4% biallelic targeting.
In the examples disclosed herein we demonstrate a very high efficiency of biallelic targeting which allows for the rapid isolation of cells with homozygous gene modification.
Any suitable selection element can be employed within the cell to select for insertion of the exogenous nucleic acid into the genome of the cell, and suitable selection systems are well known in the art. In one aspect the selectable element is a puro delta tk cassette, which can be selected by FIAU (1-(-2-deoxy-2-fluoro-1-b-D-arabinofuranosyl)-5-iodouracil (FIAU)).
Thus in one aspect the method is a method of modification of the genome of a cell comprising:
1 Delivering to a cell an exogenous nucleic acid comprising:
(i) a modifying sequence and
(ii) a selection element flanked by transposon inverted repeats
2 optionally delivering to the cell a zinc finger nuclease or TALES to enhance the efficiency of homologous recombination of the nucleic acid into the genome of the cell;
3 selection of the insertion of the nucleic acid into the genome using the selection marker
4 providing expression of a transposase within the cell to remove the selection element, such that after transposase mediated excision the only exogenous nucleic acid remaining in the genome is the modifying sequence.
In another aspect the method is a method of modification of the genome of an iPS cell, the genome of which has a mutation associated with a disease phenotype, the method comprising delivering to the cell:
1 an exogenous nucleic acid comprising:
(i) a modifying sequence and
(ii) a selection element flanked by piggyBac inverted repeats; and
2 a zinc finger nuclease or TALENS to enhance the efficiency of homologous recombination of the nucleic acid into the genome of the cell;
wherein delivery is followed by selection for the insertion of the nucleic acid into the genome using the selection marker; and then
providing expression of a piggyBac transposase within the cell to remove the selection element.
Suitably after transposase mediated excision the only exogenous nucleic acid remaining in the genome is the modifying sequence.
In a further step the genome of the ipS cell can then be analysed to confirm that no changes have been introduced into the genome of the cell other than those intended.
In all methods of the invention, where a pluripotent call line is used, the cell may be differentiated after removal of the selection element using methods known in the art.
In one aspect of the methods of the invention there is no transposase integrated into the genome of the cell. The transposase may be provided to the cell on an expression plasmid, for example. Suitable plasmid expression systems are well known in the art.
Also disclosed herein is a cell comprising an exogenous nucleic acid, the nucleic acid comprising (i) a modifying sequence and (ii) a selection element flanked by transposon inverted repeats. This cell represents the intermediate of the process described in more detail above.
Also disclosed is a cell comprising a genome which has been modified by insertion of a modifying sequence using a method according to claim 1. Such a cell represents the product of the method of the invention.
Also disclosed herein is a pair of cells or cell lines, one of which has been modified from the other as disclosed herein, such that the cells differ from one another only by one genetic modification, for example by a point mutation, insertion or deletion. An example includes for example a wild type cell and one with a disease causing mutation. In one aspect pairs of cells are isogenic.
Further disclosed is a modified cell comprising a modifying nucleic acid sequence, the cell having been selected for the insertion of the modifying nucleic acid sequence using a selectable element, and wherein the selectable element is then removed from the genome, such that the genome of the modified cell is identical to the genome of the original starting cell except for the modifying nucleic acid. Such a cell includes, for example, a cell selected for correction of a mutation and in which no other genetic changes have been made to the genome other than the correction event.
Also disclosed herein is a collection of cells, such as an organ or tissue, wherein the genomes of all, or a proportion, of the cells has been modified by insertion of a modifying sequence using a method as described above.
Also disclosed herein is a method for treating a disease in a patient, the method comprising modifying the genome of a cell using a method as described above to correct a mutation associated with the disease in the cell, and using the cell, or cells derived or differentiated therefrom, in disease treatment.
In particular the method for treatment of disease may include isolation of a cell from a individual, conversion of that cell to an iPS cell, modification of the genome of the cell as disclosed herein to effect a desired change to the genome of the cell, and then either delivery of that cell to the individual or differentiation of the cell to form a different cell type followed by delivery of the differentiated cell to the individual.
Also disclosed is a modified cell as described herein in treatment of disease, and use of a cell in the preparation of a medicament for the treatment of disease.
In one aspect the treatment is an autologous cell based therapy.
Cells of the invention may be used in combination with pharmaceutically acceptable excipients and/or carriers, such as buffers, examples of which are well known in the art.
By way of example, iPS and stem cell therapies have been suggested or tried in the following disease areas, all of which may be applicable for the present methods; brain degeneration, such as in Parkinson's and Alzheimer's disease, cancer, Cystic fibrosis, cystic fibrosis, a-1 antitrypsin deficiency-related emphysema, scleroderma, and sickle-cell disease.
The method of the invention can also be used ex-vivo. i.e. directly on cells isolated from a patient and then returned in their original differentiation state.
The method of the invention can also be used to directly modify the genome of cells in a tissue. The method of the invention therefore can comprise in vivo delivery of DNA and zinc-finger nucleases or TALENS to a tissue where the DNA is taken up, either as naked DNA or by an appropriate formulation, such as being packaged in a virus.
The present invention provides cells or tissues, such as iPS cell or tissues differentiated therefrom, into which specific modifications have been introduced, such as modifications that correct mutations.
Therefore in a further aspect the cells or tissues of the invention may be used as models for understanding disease, and the invention relates to uses of the modified cells of the invention or cells or tissues derived therefrom, optionally used in combination with the original unmodified cell, in the assessment of any of the following:
the effect of a therapeutic agent (e.g. drug screening);
the identification of targets for drug treatment;
the toxicological effect of a therapeutic agent;
the safety of a therapeutic agent;
the effect of an individual modification or modifications on drug activity;
the effect of an individual modification or modifications on likely patient responsiveness the effect of an individual modification or modifications on likely patient resistance.
The use of the modified cells of the invention, optionally used in combination with the original unmodified cell, may also be used to predict which patient sub-groups will respond to currently-available and future drug treatments.
The present invention provides pairs of cells or cell lines or tissues that differ only by a defined modification or modifications, and comparison of such cells or cell lines or tissues allows the precise effect of the modification(s) to be assessed.
It will be understood that particular embodiments described herein are shown by way of illustration and not as limitations of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine study, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims.
All publications and patent applications mentioned in the specification are indicative of the level of skill of those skilled in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” The use of the term “or” in the claims is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and “and/or.” Throughout this application, the term “about” is used to indicate that a value includes the inherent variation of error for the device, the method being employed to determine the value, or the variation that exists among the study subjects.
As used in this specification and claim(s), the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of including, such as “includes” and “include”) or “containing” (and any form of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. In one aspect such open ended terms also comprise within their scope a restricted or closed definition, for example such as “consisting essentially of”, or “consisting of”.
The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.
All of the compositions and/or methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
All documents referred to herein are incorporated by reference to the fullest extent permissible.
Any element of a disclosure is explicitly contemplated in combination with any other element of a disclosure, unless otherwise apparent from the context of the application.
The present invention is further described by reference to the following examples, not limiting upon the present invention.
The present invention is now illustrated in the following, non limiting example.

EXAMPLE 1

Human induced pluripotent stem cells (hIPSCs) represent a unique opportunity for regenerative medicine since they offer the prospect of generating unlimited quantities of cells for autologous transplantation as a novel treatment for a broad range of disorders^1,23,4. However, the use of hIPSCs in the context of genetically inherited human disease will require correction of disease-causing mutations in a manner that is fully compatible with clinical applications^3,5. The methods currently available, such as homologous recombination, lack the necessary efficiency and also leave residual sequences in the targeted genome⁶. Therefore, the development of new approaches to edit the mammalian genome is a prerequisite to delivering the clinical promise of hIPSCs. Here, we show that a combination of zinc finger nucleases (ZFNs)⁷and piggyBac^8,9technology in hIPSCs can achieve bi-allelic correction of a point mutation (Glu342Lys) in the α₁antitrypsin (A1AT, also called SERPINA1) gene that is responsible for α₁-antitrypsin deficiency (A1ATD). Genetic correction of hIPSCs restored the structure and function of A1AT in subsequently derived liver cells in vitro and in vivo. This approach is significantly more efficient than any other gene targeting technology that is currently available and crucially prevents contamination of the host genome with residual non-human sequences. Our results provide the first proof of principle for the potential of combining hIPSCs with genetic correction to generate clinically relevant cells for autologous cell-based therapies.
Currently available methods for gene targeting rely on positive selection to isolate rare clones that have undergone homologous recombination. To remove the unwanted selection cassettes, Cre/IoxP or Flp/FRT recombination systems are used, which leave behind single IoxP or FRT sites^10,11. These small ectopic sequences have the potential to interfere with transcriptional regulatory elements of surrounding genes¹², most of which are not fully characterized in the human genome. An alternative method to remove selection cassettes is to convert them into transposons. The most suitable transposon for this purpose is piggyBac, a moth-derived DNA transposon, which can transpose efficiently in mammalian cells including human embryonic stem cells (hESCs)^9,13. A remarkable feature of this mobile element is seamless excision, which enables removal of transgenes flanked by piggyBac inverted repeats without leaving any residual sequences^9,14.
To explore the use of piggyBac for the correction of point mutations, we designed a vector to correct an albino mutation (G290T substitution in the Tyr gene) in mouse induced pluripotent stem cells (mIPSCs) isolated from fibroblasts of the C57B16-Tyr^c-Brdstrain¹⁵. The targeting vector was constructed, carrying a wild-type 290G sequence and a PGK-puroΔtk cassette flanked by piggyBac repeats into the TTAA site (FIG. 1 a). Following isolation of targeted clones, the selection cassette was excised from the mIPSCs genome by transient expression of the piggyBac transposase and subsequent FIAU selection. Genomic modification was verified by Southern blot and PCR analyses (FIGS. 1 b, c). The correction of the G290T mutation and seamless piggyBac excision were confirmed by sequence analyses (FIGS. 1 d, e). Two introduced silent mutations were observed, confirming that the T290G substitution was mediated by gene correction, not by spontaneous reversion (FIG. 1 e). The function of the reverted allele was tested by injecting the corrected mIPSCs into albino mouse blastocysts. The resulting chimeric mice displayed a black coat color, indicating phenotypic correction of the albino mutation (FIG. 1 f). These results collectively demonstrate that the piggyBac transposon can be used as a versatile tool for highly precise modification (e.g. correction or mutation) of the mammalian genome at a single base-pair level.
We next explored whether this approach could be used to correct a mutation in hIPSCs derived from individuals with α₁-antitrypsin deficiency (A1ATD)¹⁶. A1ATD is an autosomal recessive disorder found in 1 out of 2000 individuals of North European descent and represents the most common inherited metabolic disease of the liver^17,18. It results from a single point mutation in the MAT gene (the Z allele; Glu342Lys) that causes the protein to form ordered polymers within the endoplasmic reticulum of hepatocytes^17,18. The resulting inclusions cause cirrhosis for which the only current therapy is liver transplantation. The increasing shortage of donors and harmful effects of immunosuppressive treatments impose major limitations on organ transplantation, making the potential of hIPSC-based therapy highly attractive. Since homologous recombination is relatively inefficient in hESCs⁶, we employed ZFN technology, which stimulates gene targeting in hESCs as well as hIPSCs^7,10,19. ZFN pairs were designed to specifically cleave the site of the Z mutation (FIGS. 2 a-c, Table 1). Amino acid sequences of left (L) and right (R) ZFNs are shown. Zinc finger protein regions are underlined.

ZFN-L
(SEQ ID NO: 1)
MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAMAERPFQCRICMRNFSQSGSLTRHIRTHTG
EKPFACDICGRKFAQSADRTKHTKIHTGSQKPFQCRICMRNFSRSDHLSTHIRTHTGEKPFACDICGRKF
AQSAHRITHTKIHLRGSQLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVY
GYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWK
VYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGE
INFRS

ZFN-R
(SEQ ID NO: 2)
MDYKDHDGDYKDHDIDYKDDDDKMAPKKKRKVGIHGVPAAMAERPFQCRICMRKFAQSSDLRRHTKIHTG
EKPFQCRICMRNFSQSSDLSRHIRTHTGEKPFACDICGRKFAQSGNLARHTKIHTPNPHRRTDPSHKPFQ
CRICMRNFSQSGHLARHIRTHTGEKPFACDICGRKFARLDNRTAHTKIHLRGSQLVKSELEEKKSELRHK
LKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKA
YSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITN
CNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS

A targeting vector was constructed from isogenic DNA with piggyBac repeats flanking the PGK-puroΔtk cassette (FIG. 2 a). To minimize the distance between the mutation and the piggyBac transposon, a CTG leucine codon, 10 bp upstream of the mutation, was altered to a TTA leucine codon, generating the TTAA sequence, which would be left in the genome following piggyBac excision (FIG. 2 b).

SUPPLEMENTARY TABLE 1

ZFN design

	Target sequence ^a	Triplet subsites	Finger designs ^b
ZFN name	(5′-3′)	(5′-3′)	−1123456

ZFN-L	gtCGATGGTCAGCAca	CGA	QSAHRIT	F4
		TGG	RSDHLST	F3
		TCA	QSADRTK	F2
		GCA	QSGSLTR	F1

ZFN-R	gaAAGGGActGAAGCTGCTgg	AAG	RLDNRTA	F5
		GGA	QSGHLAR	F4
		GAA	QSGNLAR	F3
		GCT	QSSDLSR	F2
		GCT	QSSDLRR	F1

^aThe core DNA target sequences (capital letters) and 2 flanking bases on each side (small letters) are shown.
^bThe amino acid residues at positions ‘−1’ to ‘+6’ of the recognition alpha helix^31,32of each of the zinc finger DNA-binding domain for each DNA triplet target are shown.

Puromycin-resistant hIPSC colonies obtained after co-electroporation of ZFN expression vectors and the targeting vector were screened for targeted clones by PCR. A1ATD-hIPSC lines derived from 3 different patients yielded targeted clones (Table 2). Remarkably, 54% of the puromycin-resistant colonies were targeted on one allele, while 4% were the result of simultaneous targeting of both alleles (FIG. 4).

TABLE 2

Summary of PCR genotyping of ZFN-stimulated gene targeting

				Het. +	Homo./Hemi. +
A1ATD-	Clones		Homo./	additonal	additonal
iPSC line	analyzed	Het.^a	Hemi.^b	integrations^c	integrations^c	Non-targeted^d

A	84	45	3	23	8	5
B	18	10	2	3	3	0
C^e	216	112	9	52	21	22

Mean frequency [%]	54	6	23	12	5

To remove the piggyBac-flanked selection cassette from these modified clones, we transiently transfected two homozygously targeted clones (B-16 and C-G4) with a hyperactive form of the piggyBac transposase⁸and subjected them to FIAU selection. The genotype of the resulting FIAU-resistant colonies was analyzed by PCR and confirmed by Southern blot (FIG. 2 d and FIG. 5 a). Bi-allelic excision was observed in 11% of FIAU-resistant colonies (Table 3). Sequence analyses demonstrated that the Z mutation was corrected on both alleles and that transposon excision yielded a TTAA sequence as initially planned (FIG. 2 b, e and FIG. 5 b). The resulting corrected A1ATD-hIPSC (c-hIPSC) lines maintained the expression of pluripotency markers for more than 20 passages and their abilities to differentiate into cells expressing markers of the three germ layers (FIG. 6), indicating that genome modification did not alter the pluripotency of c-hIPSCs.

TABLE 3

Frequencies of bi-allelic piggyBac excision

	Bi-allelic excision	Bi-allelic excision
	w/o re-integration	w/re-integration

Cell		No. of	Frequency	No. of	Frequency
line	analyzed	clones	[%]	clones	[%]

B-16	88	15	17	33	38
C-G4	94	5	5	19	20
		Mean frequency [%]	11		29

Genomic instability is known to be associated with prolonged culture of hESCs^20,21and those arising during genome modification would be another concern for clinical application of hIPSCs. Therefore, we analyzed the genomic integrity of the hIPSC lines using comparative genomic hybridization (CGH) (Table 4a-c). Two out of three A1ATD-hIPSC primary lines differed from their parental fibroblasts, showing amplifications or deletions ranging from 20 kb to 1.3 Mb, including a gain of 20q11.21, a frequently amplified region in hESCs^22,23(see FIG. 7). Line A retained a normal genome content compared to its parental fibroblast. Reassuringly, we found that after ZFN-stimulated targeting, four out of six homozygous clones had unaltered genomes compared to their parental hIPSC lines. Sixteen cell lines with bi-allelic piggyBac excision were compared with their corresponding primary hIPSCs and 12 had unaltered genomes. We also analyzed the hIPSC lines by SNP arrays to check for loss of heterozygosity and found that all lines analyzed retained heterozygosity throughout their genome (FIG. 8). This observation demonstrates that bi-allelic gene correction was the result of simultaneous homologous recombination followed by simultaneous excision at both alleles and that mitotic recombination was not involved in this process.

SUPPLEMENTARY TABLE 2

aCGH analyses of primary A1ATD-hIPSCs and their derivatives

	A1AT	Reference						No. of affected
Cell line	genotype	Genome	Abnormality	Chr.	Band	Size	Mean log2 ratio	genes

a. Primary A1ATD-hIPSCs

A	Z/Z	A-Fibroblast	—
B	Z/Z	B-Fibroblast	Gain		1	p36.11	300 kb	0.60	8
			Loss	8	q22.2	20 kb	−0.93	1
C	Z/Z	C-Fibroblast	Loss		2	p22.3	207 kb	−0.96	1
			Loss	17	q23.1	142 kb	−0.76	3
			Gain*	20	q11.21	1.34 Mb	0.21	30

b. Homozygously targeted clones

A-A9	PB/PB	A	—
A-G6	PB/PB	A	Loss		14	q23.13	23 kb	−0.87	0
B-16	PB/PB	B	—
C-B6	PB/PB	C	Gain*	20	q11.21	1.34 Mb	0.71	30
C-C11	PB/PB	C	—
C-G4	PB/PB	C	—

c. Clones with bi-allelic piggyBac excision

B-16-C2	Rev/Rev	B	—
B-16-C3	Rev/Rev	B	—
B-16-C6	Rev/Rev	B	—
B-16-D2	Rev/Rev	B	—
B-16-E4	Rev/Rev	B	Gain	22	q11.23	47 kb	0.89	1
B-16-E5	Rev/Rev	B	Gain		15	q26.2	4.7 kb	1.15	1
B-16-F4	Rev/Rev	B	—
B-16-F10	Rev/Rev	B	Gain		17	q23.3-q24.2	4.7 Mb	0.62	56
B-16-G8	Rev/Rev	B	—
B-16-G10	Rev/Rev	B	Gain		4	q31.3	262 kb	0.57	1
B-16-H4	Rev/Rev	B	—
C-G4-B3	Rev/Rev	C	—
C-G4-C2	Rev/Rev	C	—
C-G4-C10	Rev/Rev	C	—
C-G4-D7	Rev/Rev	C	—
C-G4-E9	Rev/Rev	C	—

d. in vitro differentiated hepatocytes

B-16-C2-hep.	Rev/Rev	B-16-C2	—

*See Supplementary FIG. 5 and Supplementary Analysis for more detail.

ZFN off-target cleavage and imprecise excision after multiple piggyBac transposition might introduce mutations into the genome. In order to investigate these possibilities at a single basepair resolution, we sequenced exomes of the corrected B-16-C2 line and its parental fibroblast. Comparison of these exomes identified 29 mutations (Table 5). The genesis of these mutations was determined by analysis of the primary hIPSC line and the homozygously targeted intermediate. Twenty-four point mutations and one 1-bp deletion were detected in the primary hIPSC line and four mutations arose during genetic correction: one during targeting and three during piggyBac excision. These mutations appeared to arise during culture since their genomic signatures were inconsistent with ZFN off-target sites or piggyBac integration sites. Taken together, we conclude that the combination of ZFNs with piggyBac provides a new method for rapid and clean correction of a point mutation in hIPSCs without affecting their basic characteristics.

SUPPLEMENTARY TABLE 3

Validated mutations in protein coding regions indentified by exome sequencing

Wt

Mut

cDNA

Protein

Detected in

SIFT functional

Expression in

Chr

Position

Allele

Gene_ID

Transcript_ID

Mutation type

annotation

Fibro-B

iPSC-B

B-16

B-16-C2

prediction

hepatocytes***

Chr1	35223302	G	A	GJB5	ENST00000338513_r62	MISSENSE	c.371G > A	p.R124Q	TOLERATED	−

Chr1	173499158	C	A	SLC9A11	ENST00000367714_r62	SILENT	c.2199G > T	p.V733V	N/A	−

Chr1	233314872	C	A	PCNXL2	ENST00000258229_r62	MISSENSE	c.3116G > T	p.G1039V	DAMAGING	−

Chr3	42705381	C	T	ZBTB47	ENST00000457842_r62	SILENT	c.702C > T	p.G234G	N/A	+

Chr3	100962755	C	T	IMPG2	ENST00000193391_r62	MISSENSE	c.2420G > A	p.R807K	TOLERATED	−

Chr3	189586471	G	A	TP63	ENST00000264731_r62	SILENT	c.1095G > A	p.S365S	N/A	−

Chr3	189586472	G	A	TP63	ENST00000264731_r62	MISSENSE	c.1096G > A	p.D366N	TOLERATED

Chr4	2831347	G	A	SH3BP2	ENST00000452765_r62	SILENT	c.714G > A	p.P238P	N/A	+

Chr4	57182264	C	A	KIAAI211	ENST00000264229_r62	MISSENSE	c.2596C > A	p.Q866K	TOLERATED	−

Chr4	122254096	G	T	QRFPR	ENST00000394427_r62	MISSENSE	c.677C > A	p.P226H	DAMAGING	−

Chr5	14692993	C	T	FAM105B	ENST00000284274_r62	MISSENSE	c.895C > T	p.R299C	TOLERATED	+

Chr5	33936907	T	C	RXFP3	ENST00000330120_r62	MISSENSE	c.62T > C	p.L21P	DAMAGING*	−

Chr6	166883328	C	A	RPS6KA2	ENST00000265678_r62	SPLICE	Exon11 + 1G > T	N/A	N/A	+

Chr7	5567522	T	C	ACTB	ENST00000331789_r62	MISSENSE	c.985A > G	p.I329V	DAMAGING*	+

Chr9	124083640	A	C	GSN	ENST00000373823_r62	MISSENSE	c.1286A > C	p.Y429S	DAMAGING	+

Chr9	125424828	C	T	OR1L1	ENST00000373686_r62	SILENT	c.984C > T	p.T328T	N/A	−

Chr11	4143385	G	A	RRM1	ENST00000300738_r62	MISSENSE	c.1053G > A	p.M351I	DAMAGING	+

Chr11	28119199	C	T	KIF18A	ENST00000263181_r62	MISSENSE	c.296G > A	p.R99H	DAMAGING	−

Chr11	76909568	C	A	MYO7A	ENST00000409709_r62	SILENT	c.4470C > A	p.I1490I	N/A	+

Chr12	129160382	C	T	TMEM132C	ENST00000315208_r62	MISSENSE	c.511C > T	p.R171C	DAMAGING	−

Chr13	99361826	G	A	SLC15A1	ENST00000376503_r62	MISSENSE	c.1067C > T	p.T356I	DAMAGING	+

Chr14	69521906	A	G	DCAF5	ENST00000341516_r62	MISSENSE	c.1597T > C	p.S533P	DAMAGING	+

Chr14	94844957	C	T	SERPINA1**	ENST00000440909_r62	SILENT	c.1086G > A	p.L362L	N/A	+

Chr14	94844959	G	A	SERPINA1**	ENST00000440909_r62	SILENT	c.1084C > T	p.L362L	N/A

Chr14	106378125	G	T	IGHD5-5	ENST00000390588_r62	MISSENSE	c.11C > A	p.A4D	Not scored	−

Chr15	68497606	C	T	CALML4	ENST00000448060_r62	MISSENSE	c.109G > A	p.G37S	TOLERATED	+

Chr15	86225404	A	C	AKAP13	ENST00000361243_r62	MISSENSE	c.5129A > C	p.H1710P	DAMAGING	+

Chr17	79428053	C	G	BAHCC1	ENST00000307745_r62	MISSENSE	c.6364C > G	p.L2122V	TOLERATED	−

Chr18	52899761	G	—	TCF4	ENST00000356073_r62	FRAMESHIFT	c.1628 C > −	p.S543fs*1	N/A	+

ChrX	12938569	C	A	TLR8	ENST00000218032_r62	MISSENSE	c.1410C > A	p.F470L	TOLERATED	−

ChrX	107823778	C	A	COL4A5	ENST00000328300_r62	SILENT	c.796C > A	p.R266R	N/A	−

N/A, not appricable
*Low confidence
**These two silent mutations were intensionally introduced though genetic correction.
***Expression in hepatocyte was analyzed using control samples in GSE 11942. Genes with ‘present’ call in more than 50% of samples are considered as expressed.

To confirm that the genetic correction of hIPSCs resulted in the expected phenotypic correction, hIPSCs were differentiated in vitro into hepatocyte-like cells, the main cell type affected by the disease A1ATD. Differentiation of the corrected lines occurred as expected, resulting in a near homogenous population of hepatocyte-like cells (FIGS. 9 a-c). Remarkably, CGH analysis of differentiated cells showed that hepatic differentiation neither increases the number of genetic abnormalities nor selects for cells with abnormal karyotype (Table 4d). The resulting cells shared key functional attributes of their in vivo counterparts including glycogen storage, LDL-cholesterol uptake, albumin secretion and Cytochrome P450 activity (FIGS. 9 d-g). Importantly, immunofluorescence and ELISA both confirmed the absence of mutant polymeric A1AT in c-hIPSCs-derived hepatocyte-like cells that instead efficiently secreted normal endoglycosidase-H-insensitive monomeric A1AT (FIGS. 3 a-d). In addition, secreted A1AT displayed an enzymatic inhibitory activity that was comparable to that obtained from normal adult hepatocytes (FIG. 3 e), thereby suggesting that physiological restoration of enzyme inhibitory activity could be achieved.
Finally, the in vivo function of c-hIPSCs-derived hepatocyte-like cells (B-C16-2 line) was assessed following transplantation into the liver of Alb-uPA^+/+;Rag2^−/−;II2rg^−/−mice via intra-splenic injection. Livers harvested 14 days after injection were colonized by human cells identified using antibodies specific to human albumin and A1AT (FIGS. 3 f, g). These human hepatocyte-like cells were distributed throughout the liver lobes and were seen to be integrated into the existing mouse parenchyma (FIGS. 3 f, g). In addition, human albumin was detected in the serum of transplanted animals for at least 5 weeks (FIG. 3 h), while no tumor formation was detected in any mice. Therefore, c-hIPSCs-derived hepatocyte-like cells were able to colonize the liver in vivo and display functional activities characteristic of their human ESC-derived counterparts²⁴. Collectively these analyses demonstrate that genetic correction of the Z mutation resulted in functional restoration of A1AT in patient-derived cells.
All experimental evidence above strongly support the applicability of genetic correction in patient-specific iPSCs for cell-based therapy of A1ATD. We therefore repeated the genetic correction in more clinically relevant cells using patient-specific iPSCs reprogrammed from fibroblasts with Sendaiviral vectors, an integration-free method²⁵(FIGS. 10 a-f). One primary hIPSC line with an intact genome by CGH analysis (FIG. 10 e and Table 6) was corrected by the method described above. The final product, iPSC-3-G5-A7, had the corrected A1AT, had an intact genome compared to the parental fibroblast, and expressed normal A1AT protein when differentiated to hepatocyte-like cells (FIG. 11 and Table 6). This is the first demonstration of the generation of mutation-corrected patient-specific iPSCs, which could realize the therapeutic promise of hIPSCs.

SUPPLEMENTARY TABLE 4

aCGH analyses of integration-free iPSCs and their derivatives.

a. Primary factor-free A1ATD-hIPSCs

SeV-iPSC1	Z/Z	Fibroblast-B	Gain		6	q22.1	271 kb	0.52	2
SeV-iPSC2	Z/Z	Fibroblast-B	Loss		2	q23.3	18 kb	−0.93	1
SeV-iPSC3	Z/Z	Fibroblast-B	—
SeV-iPSC4	Z/Z	Fibroblast-B	Gain		10	q21.1	1.1 Mb	0.52	2
			Gain	16	p13.12	76 kb	0.61	0
			Gain	22	q13.33	86 kb	0.63	3
SeV-iPSC5	Z/Z	Fibroblast-B	Gain		18	p11.31-p11.23	951 kb	0.6	5
SeV-iPSC6	Z/Z	Fibroblast-B	Loss	22	q13.1	10 kb	−0.81	1

b. Homozygously targeted clones

iPSC1-C3*	PB/PB	Fibroblast-B	—
iPSC3-G5	PB/PB	Fibroblast-B	—

c. Clones with bi-allelic piggyBac excision

iPSC1-C3-A8*	Rev/Rev	Fibroblast-B	—
iPSC1-C3-A9*	Rev/Rev	Fibroblast-B	—
iPSC3-G5-A7	Rev/Rev	Fibroblast-B	—
iPSC3-G5-C8	Rev/Rev	Fibroblast-B	Loss	20	p12.1	421 kb	−1.00	1

*A deletion on Chr.6 found in the parental line is not shown.

In the present study, we demonstrate that ZFNs and piggyBac transposon enable simultaneous bi-allelic correction of diseased hIPSCs. No residual ectopic sequences remain at the site of correction and the genome appears to be undisturbed elsewhere. Although we could readily obtain cell lines without large genomic alterations during genetic modification, the resulting corrected hIPSCs carry 29 mutations in protein coding exons, of which 22 were non-synonymous or splice site mutations. The likely impact of this mutation load needs to be considered in the context of their likely functional impact, taking into account the normal germ-line load, accumulated somatic variation, the presence of compensating normal gene copies and the requirement for the gene product in the derived differentiated cells. From this point of view, only eight mutations might affect gene functions in hepatocyte-like cells (Table 5). Nevertheless, the corrected iPSCs could efficiently differentiate to hepatocyte-like cells and engraft into the animal model for liver injury without tumor formation. Therefore, limited genomic abnormalities might have restricted biological consequences. Careful screening of primary and corrected hiPSCs using deep sequencing analyses would contribute to the safe use of hIPSCs in clinical applications.
hIPSCs derived from different patients were effectively corrected, demonstrating that this method could be applied to a large number of A1ATD-hIPSC lines. Since the bi-allelic correction could be carried out in less than 4 months, our approach may be compatible with large-scale production of corrected patient-specific hIPSCs not only for A1ATD but also for other monogenic disorders.
Method Summary
A1ATD-hIPSCs were described previously¹⁶. 2×10⁶hIPSCs were co-transfected with ZFN expression vectors and the donor template, and subjected to puromycin selection (1 μg ml⁻¹) initiated 4 days after transfection. For transposon excision, targeted cells were transfected with pCMV-hyPBase⁸, cultured for 4 days, replated and selected in 250 nM 1-(2-Deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-indouracil (FIAU). To increase clonogenicity, cells were treated with ROCK inhibitor²⁶, Y-27632 (10 μM) 4 hours prior to dissociation and 24 hours post plating. Resulting colonies were picked 2 weeks later, analyzed by PCR and further verified by Southern blot analysis. Primer sequences are listed in Supplementary Table 7.

SUPPLEMENTARY TABLE 5

Primer sequences

Construction of pPB-R1R2-NP	attR1-F	CTAGCTAGCACAAGTTTGTACAAAAAAGCTGAAC
	attR1-R	CCCAAGCTTGAATTCGGATCCCATAGTGACTGGATATGTTGTGTT
	attR2-F	CCGCTCGAGTCTAGACATAGTGACTGGATATGTTGTGTT
	attR2-R	GACTAGTACCACTTTGTACAAGAAAGCTGAA
	EM7neo-F	CCCAAGCTTGTTGACAATTAATCATCGGCATAG
	EM7neo-R	CCGCTCGAGTCAGAAGAACTCGTCAAGAAGGCG

Construction of the Tyr-mini	Left arm-F	GGCGCGCCATGGAACAGTGAAGTTCTCATCCCCAG
targeting vector	Left arm-R	TTTAACGTACGTCACAATATGATTATCTTTCTAGGGTTAATGATATCAACATCTACGAC
		CTCTTTGTATG
	Right arm-F	AAGAATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAAACATAGGTGTTGA
		TCCATTGTTCATTTGGCCATA
	Right arm-R	CCTTAATTAAGCGGAAACTGTAAGTTTGGATTTG

Construction of the Tyr-	Left arm-F	CCGCTCGAGTGCCCATGCCTAAACACAGATGTA
retrieving vector	Left arm-R	CCCAAGCTTTTTTGGATTCGGATTCTGAGGCT
	Right arm-F	CCCAAGCTTGTACAAGATTTTCATATGTAAGC
	Right arm-R	AGGCGCGCCAGGAATTCATGCCCCAGTTGACA

Homologous recombination	F	GTTGGCGCCTACCGGTGGATGTGGAATGTG
detection at Tyr	R	GAAATCTCTTCAAGCTGGGAAAACCTAAAG

Tyr genotyping	P1	TCCTCGAGCCTGTGCCTCCTCTAAG
	P2	CCCAAGTACTCATCTGTGCAAATGTC
	P3	GCGACGGATTCGCGCTATTTAGAAAG

Tyr
5′ probe	F	ACTCATTATTCCAGGATACTTGAGTG
	R	ATTTACCACATGCCCAAACTAATAAC

A1AT 2-KB fragment	F	GTGGTGGGTCCCAGAAGAACAAGAGGATGC
	R	CATAGCTGAGGAGTCCTTGCAATGGCCTTCC

Sequencing primers of the	Seq1	AGGGGCCGAGGGAAACAAATGAAGA
A1AT 2-kb region	Seq2	GCTAAAGATGACACTTATTTTGGAA
	Seq3	ATGTGACAGGGAGGGAGAGGATGTG
	Seq4	GAAAAGTGGTGAATCCCACCCAAA
	Seq5	GCCCATCTGTTTCTGGAGGGCTCCA
	Seq6	TCACCTAACCAGACTCGGGCCCTGC
	Seq7	TACCAGGGTGCAACAAGGTCGTCAG

Construction of the A1AT-donor	Left arm-F	GGCGCGCCTCTGCACGACAGGTCTGCCAGCTTAC
template vector	Left arm-R	AAGAATGCATGCGTCAATTTTACGCAGACTATCTTTCTAGGGTTAACACAGCCTTATGC
		ACGGCCTGGAGGGGAG
	Right arm-F	TTAACGTACGTCACAATATGATTATCTTTCTAGGGTTAACCATCGACGAGAAAGGGACT
		GAAGCTGCTGG
	Right arm-R	CCTTAATTAATGGTCTTCTGGGGCCTGCTGGGGC

Genotyping after ZFN-	V4	TGGAGTGACGATGCTCTTCCCTGTTC
mediated targeting	L1	GGTCAATGGGTGATGTGCTTCCTCTC
	PB5P2	CGTCAATTTTACGCATGATTATCTTTAAC

Detection of backbone	F	ACACAGGAAACAGCTATGACCATGATTACG
integration	R	CGTCAATTTTACGCATGATTATCTTTAAC

Genotyping after transposon	U4	TGGAGTGACGATGCTCTTCCCTGTTC
excision	L2	GCAGTTATTTTTGGGTGGGATTCAC
	PB-P2	GCGACGGATTCGCGCTATTTAGAAAG

Detection of transposon re-	F	CTGCTGCAACTTACCTCCGGGATG
integration	R	CCAATCCTCCCCCTTGCTGTCCTG

A1AT
5′ probe	F	GATTCCCCAACCTGAGGGTGACCAAG
	R	AAGTGACAGAGAAACGCAAGCCTTC

A1AT
3′ probe	F	CGAGGGTCACGCTAAACTTCTGCAG
	R	CTCTGGGAGGTTATGCTAGTTTGAA

Surveyor Nuclease assay	F	AGGAGCAAGGCCTATGTGA
	R	GAGGAGCGAGAGGCAGTTAT

a Het., clones heterozygous for PB allele. b Homo./Hemi., clones homozygous or hemizygous for PB allele. Cells with one targeted allele and deletion of the other allele are undistinguishable from correctly targeted homozygous clones by PCR. Such cells are designated as hemizygotes. c Vector backbone integration was analyzed by PCR. d Clones showing incorrect PCR bands are included. e A sum of 2 independent experiments.
Methods
Plasmid Construction
Gateway-adapted piggyBac transposon vectors: A destination vector pPB-R1R2-NP was constructed as follows. The attR1 and attR2 sites were PCR-generated and digested by NheI/HindIII and XhoI/SpeI, respectively. EM7-neo was PCR-generated and digested by HindIII/XhoI. These 3 fragments were then cloned into the NheI-SpeI site of pPB-LR527, resulting in pPB-R1R2-Neo. An EcoRI-XbaI fragment containing PheS was excised from pR6K-R1R2-ZP28, blunt-ended and cloned into the blunted XhoI site of pPB-R1R2-Neo, resulting in pPB-R1R2-NP. An entry vector pENTR-PGKpuroΔtk was constructed by cloning a KpnI-NotI PGK-puroitk fragment into the KpnI-NotI site of pENTR-2B.
A targeting vector for Tyr: The targeting vector was constructed using BAC recombineering. A BAC clone RP24-221M7 was introduced into Escherichia coli
strain EL350²⁹. A mini targeting vector was first constructed to modify the Tyr gene on the BAC. Left and right homology arms were PCR-generated and digested by AscI/BsiWI and NsiI/PacI, respectively. The transposon fragment was excised from pPB-R1R2-NP by NsiI/BsiWI digestion. These 3 fragments were then cloned into AscI/Pact site of pMCS, resulting in pMCS-Tyr-NP. An AscI-PacI fragment was excised from pMSC-Tyr-NP and used for BAC targeting. A retrieving vector was constructed by cloning PCR-generated left and right homology arm into the XhoI/AscI site of pMSC-DTA, following AscI/HindIII and XhoI/HindIII digestion of the left and right arm, respectively. The retrieving vector was linearized by HindIII digestion and used to retrieve 3.0-kb 5′ arm, the transposon and 6.5-kb 3′ arm. Finally, the Neo-PheS cassette was replaced with the PGK-puroΔtk cassette by Gateway cloning, resulting in pDTA-TyrPB. The targeting vector was linearized by AscI prior to electroporation into the albino mIPSCs. A donor template vector for A1AT: A 2-kb fragment, which contained 1 kb at both side of Z mutation, was first PCR-amplified using genomic DNA from A1ATD-hIPSC line B as a template and cloned into pCR4-blunt-TOPO (Invitrogen), resulting in pCR4-AAT_Z. To construct a donor template with corrected sequence and a piggyBac transposon, the 5′ arm and 3′ arm were PCR-amplified and digested with AscI/NsiI and BsiWI/PacI, respectively. The NsiI-BsiWI fragment containing a piggyBac transposon with the Neo-PheS cassette was excised from pPB-R1R2-NP. The digested fragments were cloned into the AscI-Pact site of pMCS, resulting in pMCS-AAT-PB:NP. The Neo-PheS cassette was subsequently replaced with a PGK-puroΔtk cassette by Gateway cloning, resulting in the final donor vector, pMCS-AAT-PB:PGKpuroΔtk.
The plasmids (pPB-R1R2-NP, pENTR-PGKpuroΔtk, pMCS-AAT-PB:PGKpuroΔtk) have been deposited in the Wellcome Trust Sanger Institute Archives and available upon request (http://www.sanger.ac.uk/technology/clonerequests/).
Cell Culture
Appropriate ethical approval and patient consent have been obtained (Ethics reference no. 08/H0311/201; R&D no. A091485). A1ATD-hIPSCs (ref. 16; A, patient 2 line 1; B, patient 1 line1; C, patient 3 line1) were cultured on MEF-feeder layers in hESC medium: DMEM/F12 supplemented with 20% knockout serum replacement, 1 mM GlutaMax, 0.1 mM 2-mercaptoethanol, 1× non-essential amino acid and 4 ng ml-1 FGF2 (Invitrogen). Subculture was performed every 5-7 days by detaching hIPSCs by incubation in 0.5 mg ml-1 dispase and 0.5 mg ml-1 collagenase type IV for 1 hr at 37 ° C., collecting detached hIPSC colonies, breaking down into small clamps and plating them onto new feeder plates. Mouse embryonic fibroblasts (CF1 or B6129F1) were cultured in DMEM containing 10% FCS, 2 mM Glutamine, 0.1 mM 2-melcaptoethanol and 1× non-essential amino acid. mIPSCs (iPS25A1; ref. 15) were cultured on MEF-feeder layers in mESC medium: KO-DMEM supplemented with 15% FBS, 1 mM GlutaMax, 0.1 mM 2-mercaptoethanol, 1× non-essential amino acid and 1000 unit ml-1 LIF (Millipore).
Gene Targeting and Transposon Excision in Mouse IPSCs
1×107 cells were electroporated with 25 μg of a linearized targeting vector in 800 μl of HEPES-buffered saline using a Gene Pulser II electroporator (230 V, 500 μF) and plated onto three 10-cm dishes. The next day, puromycin selection (1 μg ml-1) was initiated. Resulting colonies were picked and screened by PCR. Targeted clones were expanded and further verified by Southern blot analysis. Correctly targeted clones were then subjected to transposon excision. 2×106 cells were electroporated with 40 μg of pCMV-hyPBase in 800 μl of HEPES-buffered saline using a Gene Pulser II electroporator (230 V, 500 μF) and plated onto one well of a 6-well plate. After passage once, cells were replated on day 4 at 5×105 cells per 10-cm dish. On the following day, FIAU (0.2 μM) selection was initiated. On day 5 of selection, FIAU was withdrawn. Resulting colonies were picked at day 7 and screened by PCR. Primer sequences to detect homologous recombination are shown in Table 7.
ZFNs-Mediated Gene Targeting in A1ATD-hIPSCs.
On the day of electropolation (day 0), near-confluent cells were pre-treated with a ROCK inhibitor26 (Y-27632, Sigma) at 10 μM for 3-4 hrs prior to electroporation. Cells were then washed with PBS once, detached by Accutase (Millipore; 10 min at 37° C.) and mixed with DMEM/F12 containing 10% FCS. Cells were dissociated into single-cell suspension by vigorous pipetting and counted. 2×106 cells were pelleted and mixed with 5 μg of a 5′-ZFN expression vector, 5 μg of a 3′-ZFN expression vector and 2 μg of the donor template in 100 μl of hESC solution 1 (Lonza). The cell suspension was transferred to a cuvette and electroporated using the Amaxa Nucleofector device (Lonza) with program A23. The electroporated cells were plated onto one or two 10-cm feeder dishes in MEF-conditioned hESC medium containing 10 μM Y-27632. hESC medium without any drug was used for daily medium change between day 1-3. On day 4, puromycin selection (1 μg ml-1) was started. On day 6, medium was changed to MEF-conditioned hESC medium containing 0.5 μg ml-1 puromycin, which was used for medium change at every other day until picking colonies. Resulting colonies were picked on day 13-17. Colonies was cut into 2 pieces. One half was transferred onto a well of 24-well feeder plate and the other half was lysed and used for PCR-genotyping. PCR-positive clones were further expanded and homologous recombination was verified by Southern blot analysis.
Transposon Excision in Homozygously Targeted hIPSCs.
Homozygously targeted clones (B-16, C-G4, SeV-1-C3 and SeV-3-G5) were used for transposon removal. Line A-derived clones were omitted because this line displayed a lower capability of differentiating into endodermal lineages. Cells were prepared as described above. 2×106 cells were mixed with 10 μg of the hyperactive piggyBac transposase expression vector (pCMV-hyPBase8) in 100 μl of hESC solution 1 and electroporated using the Nucleofector device with the program A23. Electroporated cells were plated onto 6-well plate in 1:2, 1:3 and 1:6 dilutions in MEF-conditioned hESC medium containing 10 μM Y-27632. Note that ROCK inhibitor was added to the culture medium until day 6 in this experiment. On day 2, cells with ˜80% confluency were passaged using Accutase at a split ratio 1:2, 1:3 and 1:6 into 6-well plates. On day 4, cells with ˜80% confluency were washed with PBS, detached with Accutase, suspended in hESC medium and pelleted. Cells were resuspended in hESC medium into single-cell level and counted. 1×104 cells were then plated onto one 10-cm dish in hESC medium containing 10 μM Y-27632. 16-18 hrs after plating (day 5), medium was changed to hESC medium containing 0.25 μM FIAU and 10 μM Y-27632. On day 6, medium was changed to hESC medium containing 0.25 μM FIAU and then medium was changed every other day. Genotype and deletion of the piggyBac transposon were analyzed by PCR and further verified by Southern blot analysis.
CGH Analysis
Genomic DNA was extracted using a DNeasy kit (Qiagen). Agilent 244K human genome arrays were used following the manufacturer's protocol. The arrays were scanned with an Agilent microarray scanner and data were generated by Agilent Feature Extraction software. CGH calls were made with Agilent's DNA analytics software using the ADM2 algorithm (6.0 threshold) with a minimum of 5 probes in the region as a filter.
SNP Analysis
An Illumina HumanCytoSNP-12 SNP array was used following the manufacturer's protocol. Genotype calls were performed by Illumina's GenomeStudio. B allele frequency and log R ratio were analyzed by KaryoStudio. CNVpatition v2.4.4 bundled in KaryoStudio was used for copy number analysis.
ZFN Design
ZFNs were designed against a region containing Z mutation in the A1AT gene (see FIGS. 2 a, b) and assembled as previously described³⁰. The amino acid residues at positions ‘−1’ to ‘+6’ of the recognition alpha helix^31,32of each of the zinc finger DNA-binding domain for each DNA triplet target are shown in Table 4. The ZFNs were linked to wild type FokI catalytic domain. The activity of the ZFN at the endogenous target site was determined using the Surveyor Nuclease assay as previously described³³.
hIPSCs-derived hepatocyte-like cell transplantation in immunodeficient uPA transgenic mice
All mice were housed in pathogen-free conditions and animal studies were approved by the committee on animal experimentation of the Institut Pasteur and by the French Ministry of Agriculture. Differentiated cells (5×105 cells per animal in 50 μl DMEM) were injected into the spleens of 3- to 4-week-old Alb-uPA+/+;Rag2−/−;II2rg−/− mice (n=7). The recipient mouse was sacrificed 2 weeks after transplantation for histological analysis. Blood samples were collected and human albumin in plasma was quantified by ELISA (Bethy Laboratories). Frozen liver sections were analyzed by immunofluorecence with human albumin (Dako) or human A1AT (Dako) specific antibodies. Non-transplanted mice were used as controls.
Exome Sequencing
The corrected iPSC line, B-16-C2, and its parental fibroblasts were analyzed. Exome sequencing and analysis were performed as described previously34 with minor modification. Exome pulldown was performed using an Agilent SureSelect Human All Exon 50 Mb Kit according to the manufacturer's instructions. Enriched DNA was sequenced on an Illumina HiSeq2000 (75-bp paired-end sequencing). 90.32% (Fibroblast-B) and 90.72% (B-16-C2) of total targeted regions were covered with more than 10× sequencing depth, covering 93.01% and 93.35% of CCDS exons, respectively. Substitutions in the coding sequence were called as positions with at least 20% of reads reporting a different base with respect the reference human sequence (GRCh37). Additionally, somatic mutations were identified by comparing the sequence with the control fibroblasts, and removing the common polymorphisms described in dbSNP and in the 1000 Genomes Project35. Small insertions and deletions were identified using samtools, as the ones not present in the control cell line and that had at least 20× of coverage and 20% of the reads reporting the mutation. Validation of mutations was carried out by Sanger capillary sequencing on parental Fibroblast-B, A1ATD-hIPSC line B, the homozygously targeted B-16 cells and the piggyBac-excised B-16-C2 cells.
Sendaiviral regrogramming, RT-PCR, quantitative RT-PCR, bisulfite sequencing, immunostaining, flow cytometric analysis, ELISA and EndoH analysis
These experiments were performed as described previously see refs 16,24,25,36
20q11.21 Amplification
In the initial analysis of CGH data, we found amplification of 20q11.21 in one homozygous clone, C-B6 (FIG. 7 a). Through analysis of SNP array data, however, this amplification was found in all lines derived from A1ATD-iPSC line C (FIG. 7 b). This region is found to be frequently amplified following prolonged human ESC culture^37,38. We individually analyzed CGH data of the 20q11.21 region in 3 primary A1ATD-iPSC lines and 3 homozygous clones derived from line C. We first compared log2 ratios in the affected region (position 29,297,270-30,638,579; 167 probes) and those in the adjacent normal copy number region (position 30,642,607-32,056,016) that contains the same number of probes (FIG. 7 a). Consistent with the SNP array data, all three homozygous clones showed significantly higher ratios in the affected region than in the adjacent region (FIG. 7 c). However, the ratios in C-C11 and C-G4 were lower than the cut-off used for the initial CGH data analysis. C-B6 showed a much higher ratio than C-C11 and C-G4, suggesting that C-B6 carried more copies of the 20q11.21 region than the others. Surprisingly, the primary hIPSC line C also showed a slightly higher ratio in the affected region than in the adjacent region whereas line A and B, both carrying normal chromosome 20 based on the SNP analysis, had same ratios. This result strongly suggests that line C is heterogeneous with a minor population of cells with amplified 20q11.21. The mean log2 ratio of this region in line C is 0.208, which indicates 2.3 copies per diploid genome (FIG. 7 d). On the assumption that line C consists of 2 cell types (2-copy normal and 3-copy abnormal cells), 30% of the population carries 3 copies of 20q11.21. Since we used genomic DNA from line C as a reference for subsequent CGH analyses of the line C-derived cell lines, the log2 ratio of the 20q11.21 region in C-C11 and C-G4 was lower than a ratio representing single-copy gain (0.58) and thus this region was not called as a “Gain”. We recalculated the copy number for 3 homozygous clones using the corrected copy number for the line C. Normalized copy numbers of 20q11.21 were 3 copies for C-C11 and C-G4, and 3.8 for C-B6 (FIG. 7 d), which is consistent with the SNP array data. Thus, in C-B6, the 20q11.21 region was further amplified. The ZFN-stimulated gene targeting in line C was conducted several passages after CGH analysis. The abnormal cells might become dominant at the time of gene targeting. As a result all derivative lines carried 20q11.21 amplification.
Analysis on One Point Mutation Introduced During ZFN-Stimulated Targeting
As ZFN-induced mutations are primarily InDels, the one observed single base substitution is unlikely to be the result of off-target ZFN cleavage. To support this conclusion we scanned the human genome (hg19) for potential ZFN cleavage sites and calculated the distance between maximum-likelihood off-target sites and the mutation. Both heterodimers and homodimers of either site with 5 or 6bp between individual ZFN sites and up to 6 mismatches from the intended binding sites were allowed; this returned 495 potential cleavage sites. The smallest distance between any of these 495 potential cleavage sites and any of the mutation was 110,407 bp. Thus, it is extremely unlikely that ZFN cleavage at any of these putative off-target sites caused the observed mutation.

REFERENCES

- 1. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861-872 (2007).
- 2 Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917-1920 (2007).
- 3 Stadtfeld, M. & Hochedlinger, K. Induced pluripotency: history, mechanisms, and applications. Genes Dev 24, 2239-2263 (2010).
- 4 Hanna, J. et al. Treatment of sickle cell anemia mouse model with iPS cells generated from autologous skin. Science 318, 1920-1923 (2007).
- 5 Fairchild, P. J. The challenge of immunogenicity in the quest for induced pluripotency. Nat Rev Immunol 10, 868-875 (2010).
- 6 Tenzen, T., Zembowicz, F. & Cowan, C. A. Genome modification in human embryonic stem cells. J Cell Physiol 222, 278-281 (2010).
- 7 Urnov, F. D., Rebar, E. J., Holmes, M. C., Zhang, H. S. & Gregory, P. D. Genome editing with engineered zinc finger nucleases. Nat Rev Genet 11, 636-646 (2010).
- 8 Yusa, K., Zhou, L., Li, M. A., Bradley, A. & Craig, N. L. A hyperactive piggyBac transposase for mammalian applications. Proc Natl Acad Sci U S A 108, 1531-1536 (2011). 9 Wang, W. et al. Chromosomal transposition of PiggyBac in mouse embryonic stem cells. Proc Natl Acad Sci U S A 105, 9290-9295 (2008).
- 10 Hockemeyer, D. et al. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat Biotechnol 27, 851-857 (2009).
- 11 van der Weyden, L., Adams, D. J. & Bradley, A. Tools for targeted manipulation of the mouse genome. Physiol Genomics 11, 133-164 (2002).
- 12 Meier, I. D. et al. Short DNA sequences inserted for gene targeting can accidentally interfere with off-target gene expression. FASEB J 24, 1714-1724 (2010).
- 13 Lacoste, A., Berenshteyn, F. & Brivanlou, A. H. An efficient and reversible transposable system for gene delivery and lineage-specific differentiation in human embryonic stem cells. Cell Stem Cell 5, 332-342 (2009).
- 14 Fraser, M. J., Ciszczon, T., Elick, T. & Bauser, C. Precise excision of TTAA-specific lepidopteran transposons piggyBac (IFP2) and tagalong (TFP3) from the baculovirus genome in cell lines from two species of Lepidoptera. Insect Mol Biol 5, 141-151 (1996).
- 15 Yusa, K., Rad, R., Takeda, J. & Bradley, A. Generation of transgene-free induced pluripotent mouse stem cells by the piggyBac transposon. Nat Methods 6, 363-369 (2009).
- 16 Rashid, S. T. et al. Modeling inherited metabolic disorders of the liver using human induced pluripotent stem cells. J Clin Invest 120, 3127-3136 (2010).
- 17 Perlmutter, D. H. Autophagic disposal of the aggregation-prone protein that causes liver inflammation and carcinogenesis in alpha-1-antitrypsin deficiency. Cell Death Differ 16, 39-45 (2009).
- 18 Gooptu, B. & Lomas, D. A. Conformational pathology of the serpins: themes, variations, and therapeutic strategies. Annu Rev Biochem 78, 147-176 (2009).
- 19 Zou, J. et al. Gene targeting of a disease-related gene in human induced pluripotent stem and embryonic stem cells. Cell Stem Cell 5, 97-110 (2009).
- 20 Mitalipova, M. M. et al. Preserving the genetic integrity of human embryonic stem cells. Nat Biotechnol 23, 19-20 (2005).
- 21 Baker, D. E. et al. Adaptation to culture of human embryonic stem cells and oncogenesis in vivo. Nat Biotechnol 25, 207-215 (2007).
- 22 Lefort, N. et al. Human embryonic stem cells reveal recurrent genomic instability at 20q11.21. Nat Biotechnol 26, 1364-1366 (2008).
- 23 Spits, C. et al. Recurrent chromosomal abnormalities in human embryonic stem cells. Nat Biotechnol 26, 1361-1363 (2008).
- 24 Touboul, T. et al. Generation of functional hepatocytes from human embryonic stem cells under chemically defined conditions that recapitulate liver development. Hepatology 51, 1754-1765 (2010).
- 25 Fusaki, N., Ban, H., Nishiyama, A., Saeki, K. & Hasegawa, M. Efficient induction of transgene-free human pluripotent stem cells using a vector based on Sendai virus, an RNA virus that does not integrate into the host genome. Proc Jpn Acad Ser B Phys Biol Sci 85, 348-362 (2009).
- 26 Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol 25, 681-686 (2007).
- 27 Cadinanos, J. & Bradley, A. Generation of an inducible and optimized piggyBac transposon system. Nucleic Acids Res 35, e87 (2007).
- 28 Skarnes, W. C. et al. A conditional knockout resource for the genome-wide study of mouse gene function. Nature 474, 337-342 (2011).
- 29 Liu, P., Jenkins, N. A. & Copeland, N. G. A highly efficient recombineering-based method for generating conditional knockout mutations. Genome Res 13, 476-484 (2003).
- 30 Urnov, F. D. et al. Highly efficient endogenous human gene correction using designed zinc-finger nucleases. Nature 435, 646-651 (2005).
- 31 Beerli, R. R. & Barbas, C. F., 3rd. Engineering polydactyl zinc-finger transcription factors. Nat Biotechnol 20, 135-141 (2002).
- 32 Pavletich, N. P. & Pabo, C. O. Zinc finger-DNA recognition: crystal structure of a Zif268-DNA complex at 2.1 A. Science 252, 809-817 (1991).
- 33 Guschin, D. Y. et al. A rapid and general assay for monitoring endogenous gene modification. Methods Mol Biol 649, 247-256 (2010).
- 34 Varela, I. et al. Exome sequencing identifies frequent mutation of the SWI/SNF complex gene PBRM1 in renal carcinoma. Nature 469, 539-542 (2011).
- 35 Consortium, T. G. P. A map of human genome variation from population-scale sequencing. Nature 467, 1061-1073 (2010).
- 36 Seki, T. et al. Generation of induced pluripotent stem cells from human terminally differentiated circulating T cells. Cell Stem Cell 7, 11-14 (2010).
- 37 Lefort, N. et al. Human embryonic stem cells reveal recurrent genomic instability at 20q11.21. Nat Biotechnol 26, 1364-1366 (2008).
- 38 Spits, C. et al. Recurrent chromosomal abnormalities in human embryonic stem cells. Nat Biotechnol 26, 1361-1363 (2008).

Claims

1. A method .of modifying the genome of a cell, the method comprising recombining into the genome of the cell a nucleic acid, the exogenous nucleic acid comprising

(i) a modifying sequence and

(ii) a selection element flanked by transposon inverted repeats,

the method further comprising the steps of:

(a) selection of insertion of the nucleic acid into the genome using the selection element, and

(b) use of a transposase to excise the selection element, such that after transposase mediated excision the only exogenous nucleic acid remaining in the genome is the modifying sequence

2. A method of modifying according to claim 1 wherein the modification is correcting or introducing a mutation in a genome of a cell, wherein the modifying sequence comprises nucleic acid which corrects or introduces the mutation, respectively, and wherein after the transposon mediated excision process the genome of the corrected cell is identical to the genome of the original cell with the only exceptions being the mono or biallelic correction of the mutation or mono or biallelic introduction of the mutation.

3. A method according to claim 1 or 2 comprising an additional step of providing to the cell a Zinc Finger Nucleases (ZFNs) or Transcription activator like effector nuclease (TALES) to increase efficiency of the homologous recombination of the nucleic acid into the cell genome.

4. A method according to any preceding claim wherein the transposon sequences are from the piggyback transposon.

5. A method according to any preceding claim 1 wherein the transposase used to excise the nucleic acid comprising the selectable element is a piggyBac transposase.

6. A method according to any preceding claim wherein the cell is an iPS cell from an individual having a genomic mutation associated with disease.

7. A method according to any preceding claim wherein the modification is the correction of a point mutation.

8. A method according to any preceding claim wherein modification is of one allele of a gene.

9. A method according to any preceding claim wherein modification is simultaneous modification of both alleles of a gene.

10. A method according to any preceding claim wherein the method provides at least 40% efficiency in targeting on one allele, determined as number of single allele modifications for cells having selected insertion of a selectable marker.

11. A method according to any preceding claim wherein the method provides an efficiency of at least 3% biallelic targeting, determined as number of biallelic modifications for cells having selected insertion of a selectable marker.

12. A method according to any preceding claim which is a method of modification of the genome of an iPS cell, the genome of which has a mutation associated with a disease phenotype, the method comprising delivering to the cell:

(a) an exogenous nucleic acid comprising:

(i) a modifying sequence and

(ii) a selection element flanked by piggyBac inverted repeats; and

(b) a zinc finger nuclease or TALES to enhance the efficiency of homologous recombination of the nucleic acid into the genome of the cell;

wherein delivery is followed by selection for the insertion of the nucleic acid into the genome using the selection element; and then

providing expression of a piggyBac transposase within the cell to remove the selection element, optionally followed by differentiation of the modified cell.

13. A cell comprising a genome which has been modified by insertion of a modifying sequence using a method according to claim 1-12.

14. A collection of cells, such as an organ or tissue, wherein the genomes of all, or a proportion, of the cells has been modified by insertion of a modifying sequence using a method according to claim 1-12.

15. A pair of cells, or cell lines or tissues differentiated therefrom, the genome of one of the pair having been modified from the other according to the method of any of claims 1-12.

16. A cell, or cell line or tissues differentiated therefrom, made according to any of claims 1-12, optionally in combination with the original unmodified cell, for use in the assessment of any of the following:

the effect of a therapeutic agent (e.g.drug screening);

the identification of targets for drug treatment;

the toxicological effect of a therapeutic agent;

the safety of a therapeutic agent;

the effect of an individual modification or modifications on drug activity;

the effect of an individual modification or modifications on likely patient responsiveness the effect of an individual modification or modifications on likely patient resistance;

in the assessment of which patient sub-groups will respond to drug treatments.

17. A method for treating a disease in an individual, the method comprising modifying the genome of a cell using the method of claims 1-12 to correct a mutation associated with the disease in the cell, and using the cell, or cells derived or differentiated therefrom, in disease treatment.