Hemophilia A is an X-linked genetic disorder caused by mutations in the F8 gene, which encodes the blood coagulation factor VIII. Almost half of all severe hemophilia A cases result from two gross (140-kbp or 600-kbp) chromosomal inversions that involve introns 1 and 22 of the F8 gene, respectively. We derived induced pluripotent stem cells (iPSCs) from patients with these inversion genotypes and used CRISPR-Cas9 nucleases to revert these chromosomal segments back to the WT situation. We isolated inversion-corrected iPSCs with frequencies of up to 6.7% without detectable off-target mutations based on whole-genome sequencing or targeted deep sequencing. Endothelial cells differentiated from corrected iPSCs expressed the F8 gene and functionally rescued factor VIII deficiency in an otherwise lethal mouse model of hemophilia. Our results therefore provide a proof of principle for functional correction of large chromosomal rearrangements in patient-derived iPSCs and suggest potential therapeutic applications.

The other 600-kbp inversion involving three int22h sequences (designated int22h-1, -2, and -3) is eight times more prevalent than is the 140-kbp inversion, but it is technically more challenging to revert partially due to the larger size of the inverted region and the presence of three, rather than two, homologs on chromosome X. Furthermore, because int22h is much larger (10 kbp) than int1h (1 kbp), it is very difficult to genotype the int22h inversion or its reversion using conventional PCR: the entire 10-kbp int22h must be amplified. In fact, to the best of our knowledge, it has never been shown that the 600-kbp chromosomal segment involving int22h can be reverted in human cells, not to mention in patient-derived iPSCs, using programmable nucleases. In this study, we used the type II clustered, regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, a.k.a. RNA-guided engineered nucleases (RGENs) (), to revert these two large inverted regions back to the normal orientation in hemophilia A patient-derived iPSCs. We also showed that endothelial cells differentiated from the inversion-corrected iPSCs expressed the F8 gene in vitro and rescued the F8 deficiency in a hemophilia mouse model, demonstrating a proof of principle for cell-based hemophilia therapy.

Previously, we had used custom-made zinc-finger nucleases (ZFNs) in immortalized WT human cell lines () and transcriptional activator-like effect nucleases (TALENs) in WT induced pluripotent stem cells (iPSCs) () to invert the chromosomal segment between the two identical int1h sequences (designated hereinafter int1h-1 and int1h-2), which are separated by 140 kilobase pairs (kbp), at a frequency of 0.1% and 1.9%, respectively. This process mimicked the erroneous DSB repair, artificially inducing the inversion genotype.

Hemophilia A is one of the most common genetic disorders, with an incidence of 1 in 5,000 male births in the US; it is caused by various mutations in the blood coagulation factor VIII (F8) gene on chromosome X. Depending on mutant genotypes, clinical symptoms range from mild (5%–30% F8 activity, 50% of all hemophilia A patients) to moderate (2%–5% activity, 10%) to severe (<1% activity, 40%) (). Nearly half of all severe hemophilia A patients harbor one of the two different types of gross chromosomal inversions, rather than point mutations, that involve the F8 intron 1 homolog (int1h) (a minor type, ∼5% of severe hemophilia A cases) or the intron 22 homolog (int22h) (a major type, ∼40%). These two large inversions result from erroneous repair of DNA double-strand breaks (DSBs) accidently induced in the homologs via non-allelic homologous recombination (NAHR).

Results

Figure 1 Correction of the Partially Inverted F8 Gene in Hemophilia A Patient-Derived iPSCs Show full caption Bagnall et al., 2006 Bagnall R.D.

Giannelli F.

Green P.M. Int22h-related inversions causing hemophilia A: a novel insight into their origin and a new more discriminant PCR test for their detection. (A) Genomic DNA was isolated from human dermal fibroblasts (WT) and hemophilia A patient-derived urine cells (Pa1, Pa2, and Pa3) and subjected to PCR analysis using appropriate primers shown in Figure S1 A and a previous report () to detect the intron 1 (left) or 22 (right) inversions. (B) Schematic representation of intron 22 inversion and reversion. Three homolog regions are shown as int22h-1, int22h-2, and int22h-3. Blue arrowheads indicate PCR primers. Nuclease target sites near int22h-1 or int22h-3 are shown as black (RGEN 02) or red (RGEN 03) lightning symbols. (C) Mutations at nuclease target sites in HeLa cells were confirmed by the T7E1 assay. (D) PCR products corresponding to the inversion of the 563-kbp chromosomal segment in HeLa cells. (E) PCR analysis to confirm inversion correction in iPSC clones. (F) DNA sequences of breakpoint junctions in the inversion-corrected iPSC clones. Each RGEN target sequence is underlined. The PAM sequence is shown in green. Dashes indicate deleted bases. Lowercase letters indicate inserted bases. Two blue arrows indicate cleavage sites. See also Figures S1 and S2 and Table S1 First, we genotyped 11 unrelated, severe hemophilia A patients and identified one patient with the int1 inversion and three patients with the int22 inversion. We chose the one patient with the int1 inversion (termed Pa1) and two patients with the int22 inversion (termed Pa2 and Pa3) for further study ( Figure 1 A). We established their respective iPSCs by introducing the four Yamanaka factors via an episomal vector or Sendai virus into urinary epithelial cells, avoiding an invasive biopsy to obtain fibroblasts from these patients with this bleeding disorder.

Kim et al., 2010 Kim S.

Lee H.J.

Kim E.

Kim J.S. Analysis of targeted chromosomal deletions induced by zinc finger nucleases. Lee et al., 2010 Lee H.J.

Kim E.

Kim J.S. Targeted chromosomal deletions in human cells using zinc finger nucleases. In parallel, we tested RGENs, which consisted of the Cas9 protein and a small guide RNA (sgRNA), for their ability to induce or revert these two inversions in WT HeLa cells and patient iPSCs. RGEN 01 was designed to target a site in int1h ( Figure S1 A). This RGEN can cleave two identical sites, one in int1h-1 and the other in its homolog, int1h-2, that is located 140 kbp upstream. Inversions can occur via NAHR or non-homologous end joining (NHEJ). RGEN 01 was highly active, inducing small insertions and deletions (indels) at a frequency of 34% at the target site in int1h ( Figure S1 B). In addition, RGEN 01 induced the inversion of the 140-kbp chromosomal segment in HeLa cells, as shown by inversion-specific PCR ( Figure S1 C). The frequency of this inversion ranged from 2.2% to 3.1%, as measured by digital PCR ( Table S1 ) (). We determined the DNA sequences of the inversion-specific PCR amplicons and found that indels were induced at the two inversion breakpoint junctions ( Figure S1 D). Encouraged by this high frequency, we co-transfected plasmids encoding the Cas9 protein and the sgRNA into iPSCs derived from Pa1 (Pa1-iPSCs) and analyzed iPSC colonies using PCR. Eight colonies (not necessarily derived from single cells) out of 120 colonies (6.7%) produced positive PCR bands on an agarose gel. Four colonies were then further cultured to obtain single-cell-derived clones. These clones produced PCR amplicons corresponding to the int1h-1 and int1h-2 regions, indicating that the inverted 140-kbp chromosomal segment in Pa1 cells was reverted ( Figure S1 E). In contrast, no such PCR amplicons were produced from Pa1 iPSCs or urinary cells. We sequenced the PCR amplicons and found that no indels were induced at the target site in three clones. In the other clone, there was a 13-bp deletion at the target site ( Figure S1 F).

We then focused on the other larger and more prevalent int22h inversion. To rule out the possibility that unwanted deletions or inversions involving any two of the three int22 homologs, rather than the desired reversion of the inverted 600-kbp segment, were induced by cutting a site within int22h, we used two RGENs that target sites outside of the homologs ( Figure 1 B). This strategy also facilitated detection of the reversion event using appropriate PCR primers: amplification of two ∼1-kbp segments outside of int22h rather than chromosomal segments that spanned the entire 10-kbp int22h allowed genotyping.

We designed two RGENs (termed RGEN 02 and RGEN 03) to target sites near int22h-1 and int22h-3 and tested their nuclease activity in HeLa cells using the T7 endonuclease I (T7E1) assay. These RGENs were highly active, inducing indels at each target site with a frequency of 44% or 32% ( Figure 1 C). Next, we used PCR to detect the inversion of the 563-kbp chromosomal segment between the two target sites. Transfection of either RGEN 02 or RGEN 03 alone into HeLa cells did not produce inversion-specific PCR amplicons. In contrast, co-transfection of these RGEN plasmids gave rise to two inversion-specific PCR amplicons ( Figure 1 D). The inversion frequency was in the range of 1.5% to 2.2% ( Table S1 ). We determined the DNA sequences of the PCR amplicons and found that indels accompanied most of the two inversion breakpoint junctions, supporting the idea that two DSBs induced by the two RGENs were repaired by error-prone NHEJ ( Figure S2 A). Note that HeLa cells are WT with respect to the F8 exon orientation. In Pa2 and Pa3 cells, F8 exons 1 to 22 are inverted. But still, the two RGEN target sites are conserved, enabling the reversion of the large chromosomal segment.

Next, we transfected RGEN 02 and 03 into Pa2 iPSCs using an electroporator, and we isolated 135 colonies whose genomic DNA samples were subjected to PCR analysis. Five colonies (3.7%) yielded PCR amplicons corresponding to the inversion-correction (namely reversion) event. No such PCR products were obtained using genomic DNA isolated from Pa2 iPSCs or WT iPSCs ( Figure 1 E). These colonies were further expanded to enable isolation of three independent single-cell-derived clones. We then determined the DNA sequences at the two inversion breakpoint junctions, which confirmed the reversion of the 563-kbp chromosomal segment between the two RGEN sites ( Figure 1 F). As in HeLa cells, indels, characteristic of error-prone NHEJ, were observed at the two breakpoint junctions in these inversion-corrected iPSCs.

Figure 2 Characterization of Inversion-Corrected iPSC Clones Show full caption (A) Quantitative real-time PCR (qPCR) was carried out to detect endogenous OCT4, SOX2, LIN28, and NANOG mRNAs from parental and corrected cell lines. The expression level of each gene was normalized to that of GAPDH. (B) In vitro differentiation of inversion-corrected lines. The expression of marker proteins representing ectoderm (Nestin), mesoderm (α-smooth muscle actin [α-SMA]), and endoderm (α-fetoprotein [AFP]) in corrected lines is shown. Scale bar, 50 μm. (C) The expression of OCT4 and SSEA4, human-ESC-specific markers, was detected by immunocytochemistry. Scale bar, 100 μm. Karyotypes of the indicated iPSC lines are shown. (D) F8 gene expression in cells differentiated from intron 1 and 22 inversion-corrected iPSC lines. RT-PCR (upper) and qPCR (lower) was used to detect expression of F8 and a mesoderm marker gene (Brachyury) in cells derived from WT iPSCs, patient iPSCs (Pa1, Pa2 and Pa3), and inversion-corrected Pa1- (Co-1 and Co-2) or Pa2-iPSCs (Co-1, Co-2 and Co-3). GAPDH expression was used as a loading control. ND, not detected. (E) Chromatograms showing correct splicing between exons 1 and 2 or exons 22 and 23 in inversion-corrected iPSC lines (related to Figure 2 D). See also Figure S3 Table S2 , and Table S3 We then investigated whether the inversion-corrected iPSCs remained pluripotent. First, we checked the expression of stem cell marker genes in inversion-corrected Pa1 (int1h inversion) and Pa2 (int22h inversion) iPSCs and found that four marker genes, namely, OCT4, SOX2, LIN28, and NANOG, were transcribed actively in these cells ( Figure 2 A). Second, these inversion-corrected iPSCs were successfully differentiated into three primary germ layers ( Figure 2 B). Furthermore, they showed a normal karyotype ( Figure 2 C). Taken together, these results show that gross chromosomal reversions induced by RGENs do not negatively affect the pluripotency of patient-derived iPSCs.

Shahani et al., 2010 Shahani T.

Lavend’homme R.

Luttun A.

Saint-Remy J.M.

Peerlinck K.

Jacquemin M. Activation of human endothelial cells from specific vascular beds induces the release of a FVIII storage pool. Endothelial cells derived from mesoderm are a major source of F8 gene expression (). We differentiated patient iPSCs and inversion-corrected patient iPSCs into mesoderm and measured the levels of F8 mRNA using RT-PCR. As expected, no PCR bands corresponding to F8 exons 1 and 2 were detected in cells differentiated from Pa1-iPSCs, indicating that F8 was not expressed in patient-derived cells ( Figure 2 D). In contrast, PCR bands corresponding to these exons were detected in cells differentiated from the WT iPSCs or the two inversion-corrected Pa1-iPSCs (termed Co-1 and Co-2). Likewise, PCR amplicons corresponding to F8 exons 22 and 23 were not detected in cells differentiated from Pa2- and Pa3-iPSCs, but were detected in cells differentiated from the three inversion-corrected iPSCs ( Figure 2 D). We also performed Sanger sequencing to confirm that exons 1 and 2 or exons 22 and 23 were spliced correctly in cells differentiated from the inversion-corrected iPSCs ( Figure 2 E). These results prove that the F8 gene was repaired in patient iPSCs that had harbored intron 1 and 22 inversions, supporting the expression of the F8 gene in mesoderm cells.

Merkely et al., 2015 Merkely B.

Gara E.

Lendvai Z.

Skopál J.

Leja T.

Zhou W.

Kosztin A.

Várady G.

Mioulane M.

Bagyura Z.

et al. Signaling via PI3K/FOXO1A pathway modulates formation and survival of human embryonic stem cell-derived endothelial cells. Pandey et al., 2013 Pandey G.S.

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et al. PATH (Personalized Alternative Therapies for Hemophilia) Study Investigators

Endogenous factor VIII synthesis from the intron 22-inverted F8 locus may modulate the immunogenicity of replacement therapy for hemophilia A. Figure 3 Functional Rescue of the Factor VIII Deficiency in Hemophilia Mice Using Inversion-Corrected iPSCs Show full caption (A) The F8 protein in endothelial cells differentiated from WT, patient iPSCs (Pa1 and Pa2), and inversion-corrected iPSCs (Pa1 Co-1 and Pa2 Co-1) was detected by immunocytochemistry. DAPI signals (blue) indicate the total cell presence in the image. FVIII, F8 protein; vWF, von Willebrand factor (a marker protein for mature endothelial cells). Scale bar, 100 μm. (B) Proportions of surviving mice after the tail-clip challenge. HA, hemophilia mice (n = 5); HAT, hemophilia mice transplanted with cells derived from Pa2 iPSCs (n = 9) or Pa2 Co-1 iPSCs (n = 9). n.s., not significant compared with HA; ∗p < 0.01 compared with the Pa2 group (log-rank test). (C) Average survival time of mice that died after the tail-clip challenge. Note that the three out of nine hemophilia mice transplanted with Pa2 Co-1 cells that survived the challenge were excluded in this analysis. n.s., not significant compared with HA; ∗p < 0.05 compared with the Pa2 group (Student’s t test). (D) Relative factor VIII activities were determined in plasma obtained from non-transplanted hemophilia A mice (HA, n = 5) or transplanted hemophilia A mice (HAT, n = 9 each). Data are presented as the percent of the F8 activity in WT mice (100%, n = 4). n.s., not significant compared with HA; ∗p < 0.05 compared with the Pa2 group (Student’s t test). See also Figure S3 Next, we further differentiated WT, patient-derived, and inversion-corrected iPSCs into mature endothelial cells () and checked the expression of the F8 protein by immunocytochemistry. As expected, the endothelial cells differentiated from the WT and the three inversion-corrected Pa1-iPSC clones (termed Pa1 Co-1 to Pa1 Co-3) expressed the F8 protein ( Figures 3 A and S3 ). In contrast, no signals corresponding to the F8 protein were detected in cells differentiated from Pa1-iPSCs, although these cells differentiated into mature endothelial cells, as shown by the expression of von Willebrand factor, a marker protein of mature endothelial cells ( Figure 3 A). Because the entire amino acid sequence of the F8 protein is still expressed as two inactive polypeptide chains in cells with the int22h inversion (), positive signals were detected in mature endothelial cells derived from both Pa2 iPSCs (int22h inversion) and inversion-corrected Pa2 iPSCs (termed Pa2 Co-1 to Pa2 Co-3). This implies that no neutralizing antibodies against the F8 protein will be produced in patients transplanted with the int22h inversion-corrected cells.

To test whether endothelial cells differentiated from inversion-corrected iPSCs could functionally rescue the F8 deficiency in an animal model, we transplanted endothelial cells generated from patient iPSCs (Pa2) and inversion-corrected iPSCs (Pa2 Co-1) into the hind-limb of hemophilia A mice whose F8 gene was disrupted. Two weeks after transplantation, these mice were subjected to a tail-clip challenge. All hemophilia mice (n = 5) that had not received a transplant, as well as those (n = 9) transplanted with Pa2-derived cells, soon died; the average survival time was 71 min and 65 min, respectively ( Figure 3 B). Notably, three out of nine mice transplanted with cells derived from the Pa2 Co-1 iPSCs were alive 2 days after the tail-clip challenge, which was the end point of this experiment. Furthermore, the other six mice that did not survive the challenge also showed a significant increase in the survival time (111 min on average) compared to non-transplanted mice or mice transplanted with Pa2-derived cells ( Figure 3 C). We then measured the F8 enzymatic activity in plasma samples obtained from these hemophilia mice and WT mice using a chromogenic assay. The relative F8 activity in mice transplanted with Pa2 Co-1 iPSC-derived cells was 10% of that in WT mice, significantly higher than that in non-transplanted mice (3.3%) and those transplanted with Pa2 iPSC-derived cells (4.3%) ( Figure 3 D). Taken together, these results indicate that the F8 deficiency in hemophilia mice can be functionally rescued by transplantation of endothelial cells derived from inversion-corrected iPSCs.

Kim et al., 2014 Kim S.

Kim D.

Cho S.W.

Kim J.

Kim J.S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. To prevent unwanted insertions of plasmid fragments at RGEN on-target and off-target sites, we transfected recombinant Cas9 protein purified after expression in E. coli and in vitro transcribed sgRNAs (termed RGEN ribonucleoproteins, or RNPs), rather than plasmids encoding these components, into two patient (Pa1 and Pa3) iPSCs, each harboring an intron 1 or 22 inversion. We used PCR and Sanger sequencing to confirm the reversion of the 140-kbp or 563-kbp chromosomal segments in these iPSCs, restoring the genetic integrity of the F8 gene ( Figures S2 B and S2C). RGEN RNP delivery can also reduce off-target effects without sacrificing genome editing activity at on-target sites, because RNPs, unlike plasmids, cleave chromosomal target DNA immediately after transfection and are rapidly degraded in cells ().