Cure of ADPKD by Selection for Spontaneous Genetic Repair Events in Pkd1-Mutated iPS Cells

Induced pluripotent stem cells (iPSCs) generated by epigenetic reprogramming of personal somatic cells have limited therapeutic capacity for patients suffering from genetic disorders. Here we demonstrate restoration of a genomic mutation heterozygous for Pkd1 (polycystic kidney disease 1) deletion (Pkd1(+/−) to Pkd1(+/R+)) by spontaneous mitotic recombination. Notably, recombination between homologous chromosomes occurred at a frequency of 1∼2 per 10,000 iPSCs. Southern blot hybridization and genomic PCR analyses demonstrated that the genotype of the mutation-restored iPSCs was indistinguishable from that of the wild-type cells. Importantly, the frequency of cyst generation in kidneys of adult chimeric mice containing Pkd1(+/R+) iPSCs was significantly lower than that of adult chimeric mice with parental Pkd1(+/−) iPSCs, and indistinguishable from that of wild-type mice. This repair step could be directly incorporated into iPSC development programmes prior to cell transplantation, offering an invaluable step forward for patients carrying a wide range of genetic disorders.


Introduction
Epigenetic reprogramming of personal somatic cells into iPSCs by forced expression of defined transcription factors confers pluripotency [1][2][3], but does not restore mutations that cause genetic disorders. For therapeutic treatment of genetic disorders, techniques using plasmid, zinc finger nucleases and helperdependent adenoviral vector-mediated homologous recombination have been developed to induce genetic editing in diseasespecific iPSCs and embryonic stem cells (ESCs) [4][5][6][7][8][9]. The requirement for target gene-specific vectors coupled with inherent low efficiency has restricted translation of the theoretical possibilities to practical application of these techniques in iPSCs for clinical therapy. Furthermore, Genetic modification in iPSCs gives an obstacle to safety in clinical applications. Mitotic recombination, which functions in DNA repair [10], occurs at a low frequency (,10 26 ) in somatic cells [11]. However, in experiments to drive targeted chromosome elimination from pluripotent ES-somatic hybrid cells [12], evidence suggested the frequency of genetic repair events through spontaneous mitotic recombination in pluripotent stem cells is higher than that in somatic cells [11]. Furthermore, chromosome-specific loss of heterozygosity was created in a KO allele by high-dose G418 selection to the Neo gene in mouse embryonic stem cells [13]. Thus, we investigated whether it was possible to identify and propagate isogenic clones of iPSCs, which retain the property of infinite cell proliferation, in which spontaneously genetic correc-tion had occurred at disease-related mutation alleles through mitotic recombination ( Figure 1).
Here, to demonstrate proof of principle for this theoretical approach, we have investigated a prevalent inherited disorder, autosomal dominant polycystic kidney disease (ADPKD). ADPKD, which is caused by genetic mutation of the PKD1 and PKD2 gene in 85% and 15% of cases, respectively [14], is clinically diagnosed by intrarenal cystogenesis caused by complex mechanisms. Deregulation of PKD1 or PKD2-coding polycystin protein level results in initiation of cyst formation, followed by several signaling pathways that mediate cyst growth and expansion [15].
To address the mechanism of allelic exchange at the Pkd1 locus, DNA extracted from Pkd1(+/R+) iPSC lines was analyzed by Southern blot hybridization with probe 1, 2, and 3 ( Figure 2e). Fragments for the KO allele, 7.8 kb with probe 1 and 7.0 kb with probe 3, were detected in Pkd1(+/2) iPSCs but not wild-type or Pkd1(+/R+) iPSCs, confirming loss of the Neo containing region. The intensity of the 15.1 kb band for the wild-type allele, detected by all three probes, was similar between wild-type and Pkd1(+/R+) iPSCs, and reduced by approximately 50% in Pkd1(+/2) iPSCs, when normalized against an internal loading control Stella, demonstrating replacement of the KO allele with the wild-type allele through mitotic recombination. To exclude the possibility of wild-type cell contamination as a source of apparent Pkd1(+/R+) iPSCs, Southern blot hybridization was performed with a probe specific to Klf4 integrated as a multi-copy transgene in the iPSC genome but not the wild-type genome ( Figure 2f). Detection of the same pattern of multiple bands in Pkd1(+/2) and Pkd1(+/R+) iPSCs demonstrates that Pkd1(+/R+) iPSCs originated from Pkd1(+/2) iPSCs.
To investigate the influence of mitotic recombination on Pkd1 expression, mRNA levels were analyzed by RT-PCR ( Figure S3). Expression in Pkd1(+/2) iPSCs was approximately half that in WT or Pkd1(+/R+) iPSCs. Thus, expression of Pkd1 was restored to the normal level in Pkd1(+/R+) iPSCs. Next, to examine physiological effects of the restoration of the KO to wild-type allele, chimeric mice were generated with Pkd1(+/2) and Pkd1(+/ R+) iPSCs, both of which exhibit ubiquitous GFP expression (Figure 3a). Chimeras were analyzed at more than 6 months old following established criteria for cystogenesis as reported previously, i.e. cyst diameter .200 mm [17]. Kidneys collected in triplicate from mice generated with wild-type, Pkd1(+/2), and Pkd1(+/R+) chimeric genotypes were sacrificed for histological analyses. Extent of chimerism (22,48%) was comparable between Pkd1(+/2) and Pkd1(+/R+) kidneys we examined (Figure 3b). Cyst formation was frequently detected in the cortical region of the Pkd1(+/2) chimeric kidneys but not the Pkd1(+/R+) and wildtype kidneys ( Figure S4). GFP-positive cells comprising part of the cyst wall were observed in Pkd1(+/2) chimeric kidneys (Figure 3c), indicating that unbalanced expression level of polycystin protein in Pkd1(+/2) kidney cells might induce to initiate the cystogensis. Notably, the frequency of cyst formation was more than ten times higher in Pkd1(+/2), than Pkd1(+/R+) chimeric kidneys (Figure 3d). The number of cysts detected in Pkd1(+/R+) chimeric kidneys was comparable to that in wild-type kidneys. These data clearly demonstrate that the mutation in Pkd1(+/2) iPSCs involved in ADPKD was genetically and functionally restored in Pkd1(+/R+) iPSCs through spontaneous mitotic recombination. It is evident that selection of genetic mutation-restored iPSCs by spontaneous mitotic recombination is a realistic way to isolate isogenic and functionally restored iPSCs safely for using clinical applications.
Our results establish a paradigm for enrichment of iPSC clones, by large-scale screening, in which restoration of genetic disorderinducing mutations has occurred spontaneously through mitotic recombination. The frequency of spontaneous mitotic recombination may not be estimated properly in pluripotent stem cells and embryonic cells in vivo and in vitro, since the event occurring in a tiny number of cells was detected using a G418-resistant selection with the Neo gene in many cases. In fact, mouse ESCs homozygous for the Neo-tagged chromosomes were selected and expanded from ESCs heterozygous for Neo-tagged chromosome by the high dose treatment of G418 at the frequency of approximately 1610 26 [12][13]. The generation frequency of G418-resistant cells may be underestimated to that of homologous mitotic recombination, since some pluripotent stem cells homozygous for Neo, expressing insufficient amount of gene product, could stop growing or be killed. FACS analysis of mouse ESCs heterozygous for the GFP reporter gene without drug selection detected GFP-negative cells at the frequency of about 1610 23 (data not shown), which were generated by homologous mitotic recombination or deletion of the GFP gene. The circumstance evidence supports that mitotic homologous recombination at an allele occurs at the frequency of about 1610 24 ,10 25 , similar to our findings using iPSCs. If so, the genetic information may diverge between somatic cells thorough cell divisions rather than we predicted.
An automatic genome-sorting system could make screening of mitotic recombination-mediated genetic correction feasible. Importantly, this approach dispenses with requirement for gene therapy. As with mutation-dominant diseases such as ADPKD and LEOPARD syndrome [18], the approach is similarly applicable to genetic mutations in recessive diseases [19]. Once generated, the disease-repaired iPSCs may be useful for regenerative medicine as well as other applications. Thus, the mitotic recombinationmediated genetic correction approach will open a new path to clinical application for human iPSCs that is relevant to patient groups, for which the iPSC technological revolution was believed to hold little relevance in the clinical setting.

Ethics statement
Experiments with mice were performed according to the institutional guideline of Kyoto University, Japan. Our animal experiments (W-3-6) are reviewed and permitted by the animal research committee of Kyoto University, JAPAN.

Chromosome
Cells were treated with 0.075 M KCl for 8 min at room temperature and then fixed in 3:1 methanol:acetic acid. Slides of chromosomes prepared by an air-drying method were stained with Hoechst 33258 (10 ng/ml) for G-banding analysis.
PCR primers used in this study are listed in Table S1. For Genomic PCR, products were amplified with GoTaq (Promega) by 35 cycles of reactions. For RT-PCR, total RNA of cultured cells was extracted with TRIzol reagent (Invitrogen, USA). cDNA was synthesized from 500 ng of total RNA with Superscript III (Invitrogen, USA) using random hexamers following manufacturers instructions. Band intensity was measured using Image J (NIH).

Southern blot hybridization
Genomic DNAs was digested with restriction enzyme(s), separated by electrophoresis on a 1.0% agarose gel, and transferred on a Hybond N + filter by alkali blotting. The membrane was hybridized with probes labeled with 32 P using the Megaprime DNA labeling system (Amersham) overnight at 42uC following pre-hybridization treatment. The membrane was washed twice in 26SSC/0.1% SDS at 65uC for 30 minutes and twice in 0.16SSC/0.1% SDS at 65uC for 15 minutes. Band intensity was measured using Image J.