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Cryopyrin-associated periodic syndrome (CAPS) is an autoinflammatory syndrome caused by mutations of NLRP3, which was originally identified as cryopyrin. Familial cold autoinflammatory syndrome (FCAS), the mildest form of CAPS, is characterized by cold-induced inflammation induced by the overproduction of IL-1β. However, the molecular mechanism of how mutated NLRP3 causes inflammasome activation in CAPS remains unclear. Here, we found that CAPS-associated NLRP3 mutants form cryo-sensitive aggregates that function as a scaffold for inflammasome activation. Cold exposure promoted inflammasome assembly and subsequent IL-1β release triggered by mutated NLRP3. While K+ efflux was dispensable, Ca2+ was indispensable for mutated NLRP3-mediated inflammasome assembly. Notably, Ca2+ influx was induced during mutated NLRP3-mediated inflammasome assembly. Furthermore, caspase-1 inhibition prevented Ca2+ influx and inflammasome assembly induced by the mutated NLRP3, suggesting a feed-forward Ca2+ influx loop triggered by mutated NLRP3. Thus, the mutated NLRP3 forms cryo-sensitive aggregates to promote inflammasome assembly distinct from canonical NLRP3 inflammasome activation.

The diverse functions of WASP, the deficiency of which causes Wiskott-Aldrich syndrome (WAS), remain poorly defined. We generated three isogenic WAS models using patient induced pluripotent stem cells and genome editing. These models recapitulated WAS phenotypes and revealed that WASP deficiency causes an upregulation of numerous RNA splicing factors and widespread altered splicing. Loss of WASP binding to splicing factor gene promoters frequently leads to aberrant epigenetic activation. WASP interacts with dozens of nuclear speckle constituents and constrains SRSF2 mobility. Using an optogenetic system, we showed that WASP forms phase-separated condensates that encompasses SRSF2, nascent RNA and active Pol II. The role of WASP in gene body condensates is corroborated by ChIPseq and RIPseq. Together our data reveal that WASP is a nexus regulator of RNA splicing that controls the transcription of splicing factors epigenetically and the dynamics of the splicing machinery through liquid-liquid phase separation.

Inherited glycosylphosphatidylinositol (GPI) deficiency (IGD) is caused by mutations in GPI biosynthesis genes. The mechanisms of its systemic, especially neurological, symptoms are not clarified and fundamental therapy has not been established. Here, we report establishment of mouse models of IGD caused by PIGO mutations as well as development of effective gene therapy. As the clinical manifestations of IGD are systemic and lifelong lasting, we treated the mice with adeno-associated virus for homology-independent knock-in as well as extra-chromosomal expression of Pigo cDNA. Significant amelioration of neuronal phenotypes and growth defect was achieved, opening a new avenue for curing IGDs.

Summary Pyroptosis is a form of regulated cell death that is characterized by gasdermin processing and increased membrane permeability. Caspase-1 and caspase-11 have been considered to be essential for gasdermin D processing associated with inflammasome activation. In the present study, we found that NLRP3 inflammasome activation induces delayed necrotic cell death via ASC in caspase-1/11-deficient macrophages. Furthermore, ASC-mediated caspase-8 activation and subsequent gasdermin E processing are necessary for caspase-1-independent necrotic cell death. We define this necrotic cell death as incomplete pyroptosis because IL-1β release, a key feature of pyroptosis, is absent, whereas IL-1α release is induced. Notably, unprocessed pro-IL-1β forms a molecular complex to be retained inside pyroptotic cells. Moreover, incomplete pyroptosis accompanied by IL-1α release is observed under the pharmacological inhibition of caspase-1 with VX765. These findings suggest that caspase-1 inhibition during NLRP3 inflammasome activation modulates forms of cell death and permits the release of IL-1α from dying cells., Graphical Abstract, Highlights • NLRP3 inflammasome induces necrotic cell death in the absence of caspase-1/11 • ASC initiates GSDME-dependent pyroptosis via caspase-8 • IL-1α, but not IL-1β, is released during Casp1/11-independent pyroptosis • Pharmacological inhibition of caspase-1 permits IL-1α release during pyroptosis, Immunology; Cell Biology; Functional Aspects of Cell Biology

Targeted genome editing via engineered nucleases is an exciting area of biomedical research and holds potential for clinical applications. Despite rapid advances in the field, in vivo targeted transgene integration is still infeasible because current tools are inefficient, especially for non-dividing cells, which compose most adult tissues. This poses a barrier for uncovering fundamental biological principles and developing treatments for a broad range of genetic disorders. Based on clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, we demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.

In vivo genome editing represents a powerful strategy for both understanding basic biology and treating inherited diseases. However, it remains a challenge to develop universal and efficient in vivo genome-editing tools for tissues that comprise diverse cell types in either a dividing or non-dividing state. Here, we describe a versatile in vivo gene knock-in methodology that enables the targeting of a broad range of mutations and cell types through the insertion of a minigene at an intron of the target gene locus using an intracellularly linearized single homology arm donor. Asa proof-of-concept, we focused on a mouse model of premature-aging caused by a dominant point mutation, which is difficult to repair using existing in vivo genome-editing tools. Systemic treatment using our new method ameliorated aging-associated phenotypes and extended animal lifespan, thus highlighting the potential of this methodology for a broad range of in vivo genome-editing applications.

Long-term peritoneal dialysis (PD) therapy leads to peritoneal inflammation and fibrosis. However, the mechanism underlying PD-related peritoneal inflammation and fibrosis remains unclear. NLRP3 inflammasome regulates the caspase-1-dependent release of interleukin-1β and mediates inflammation in various diseases. Here, we investigated the role of NLRP3 inflammasome in a murine model of PD-related peritoneal fibrosis induced by methylglyoxal (MGO). Inflammasome-related proteins were upregulated in the peritoneum of MGO-treated mice. MGO induced parietal and visceral peritoneal fibrosis in wild-type mice, which was significantly reduced in mice deficient in NLRP3, ASC, and interleukin-1β (IL-1β). ASC deficiency reduced the expression of inflammatory cytokines and fibrotic factors, and the infiltration of macrophages. However, myeloid cell-specific ASC deficiency failed to inhibit MGO-induced peritoneal fibrosis. MGO caused hemorrhagic ascites, fibrin deposition, and plasminogen activator inhibitor-1 upregulation, but all of these manifestations were inhibited by ASC deficiency. Furthermore, in vitro experiments showed that MGO induced cell death via the generation of reactive oxygen species in vascular endothelial cells, which was inhibited by ASC deficiency. Our results showed that endothelial NLRP3 inflammasome contributes to PD-related peritoneal inflammation and fibrosis, and provide new insights into the mechanisms underlying the pathogenesis of this disorder.

Summary Human pluripotent stem cell-derived kidney organoids recapitulate developmental processes and tissue architecture, but intrinsic limitations, such as lack of vasculature and functionality, have greatly hampered their application. Here we establish a versatile protocol for generating vascularized three-dimensional (3D) kidney organoids. We employ dynamic modulation of WNT signaling to control the relative proportion of proximal versus distal nephron segments, producing a correlative level of vascular endothelial growth factor A (VEGFA) to define a resident vascular network. Single-cell RNA sequencing identifies a subset of nephron progenitor cells as a potential source of renal vasculature. These kidney organoids undergo further structural and functional maturation upon implantation. Using this kidney organoid platform, we establish an in vitro model of autosomal recessive polycystic kidney disease (ARPKD), the cystic phenotype of which can be effectively prevented by gene correction or drug treatment. Our studies provide new avenues for studying human kidney development, modeling disease pathogenesis, and performing patient-specific drug validation.

e13669 Background: Recently, several cancer gene panel tests have been developed to determine many driver mutations including lung cancer simultaneously. However, the current next-sequencing based platform is not ideal testing method in the clinical practice due to the high examination cost and the time required for the result to reach the field. By refining recently reported CRISPR-Cas-based nucleic acid detection methods, we developed a novel CRISPR-Cas12 family based rapid-detection kit for targetable EGFR mutations in non-small lung cancer (NSCLC) patients. Methods: To develop a powerful CRISPR-based testing method, we focused on unique Cas12 family proteins which contain a single RuvC nuclease domain that cleave target double-stranded DNA adjacent to protospacer adjacent motif (PAM) sequences as well as non-target single-stranded DNA (ssDNA) collaterally. We purified 6 Cas12 family proteins (Cas12a, Cas12c1, Cas12c2, Cas12g, Cas12i1, and Cas12i2) as well as two types of target-defined guide RNA for EGFR Ex.19 del(E746-A750) and L858R mutations. The target sequences including EGFR Ex.19(E746-A750) and L858R mutations were amplified from EGFR mutated cell lines, FFPE lung cancer tissues or cell free DNA (cfDNA) of NSCLC patients using PCR with PrimeSTAR GXL DNA polymerase (Takara, Shiga, Japan) or isothermal recombinase polymerase amplification (RPA) with TwistAmp Basic (TwistDx, Cambridge, UK). CRISPR-Cas12-based cleavage assay was conducted in a reaction buffer consisting of 10 nM Cas12 proteins, 10 nM purified guide RNA, 0.4nM target DNA, and 50 nM collateral ssDNA (quenched fluorescent DNA reporter) in a 20 μl volume at 37 °C for 1 h. The excited fluorescence, which is an indicator of the presence of target cancer mutation, was measured by Synergy H1 hybrid Multi-Mode Reader (BioTek, Vermont, USA). For simpler instrument-free and portable detection lateral flow readouts were also developed and tested using Milenia HybriDetect1 kit (TwistDx). Results: Cas12a, Cas12i1, Cas12i2-based assays successfully detected the EGFR Ex.19 del(E746-A750) and L858R mutations from PCR and RPA products. Lateral flow readout kit could also detect EGFR Ex.19 del (E746-A750) mutation from cfDNA derived from NSCLC patients. It took about 3 hours from plasma collection to DNA extraction, RPA, and measurement with the kit. Conclusions: CRISPR-Cas based lateral flow readout kit can detect EGFR mutation at an unprecedented pace with low cost. Our developed system will be an innovative testing tool for lung cancer patients with low cost, rapid, multiplexable, and noninvasive procedure.

Wiskott-Aldrich syndrome (WAS), caused by mutations in the WASP protein, displaysimmunological dysfunctions and predisposition to cancer. Despite studies in cell linesand mouse models the molecular mechanisms of WAS remain obscure. We generatedinduced pluripotent stem cells (iPSCs) from patients with WAS (WAS-iPSCs) andisogenic gene-corrected iPSCs by genome editing. Immune cells derived from WASiPSCs,genetically engineered B lymphoblastoid cell lines, and patient primarylymphocytes were subjected to imaging, proteomic and transcriptomic analyses. TheWAS-iPSC model not only recapitulated known disease phenotypes but also revealed,for the first time, roles of WASP in the organization of the nucleolus and nuclearspeckles and PML nuclear bodies. WASP interacts with components of the nucleolusand nuclear speckles, including chromatin modifiers and splicing factors. Innate andadaptive immune cells from WAS patients display global dysregulation of cell cycleregulation and alternative splicing. WASP mutation is sufficient to drive an acceleratedcell cycle and tumor-promoting splicing changes. Our data show that WASP acts as atumor suppressor and specific WASP mutants behave as oncogenes and cause cellintrinsicalterations that predispose patients to cancer.

Heterochromatin in aging stem cells Analysis of human aging syndromes, such as Werner syndrome (WS), may lead to greater understanding of both premature and normal aging. Zhang et al. generated isogenic WS-specific human embryonic stem cell lines (see the Perspective by Brunauer and Kennedy). WS-mesenchymal stem cells displayed features characteristic of premature aging, including heterochromatin disorganization. WRN protein thus functions in the maintenance of heterochromatin, and heterochromatin alterations may represent a driving force of human aging. Science , this issue p. 1160 ; see also p. 1093

Pluripotency, the ability to generate any cell type of the body, is an evanescent attribute of embryonic cells. Transitory pluripotent cells can be captured at different time points during embryogenesis and maintained as embryonic stem cells or epiblast stem cells in culture. Since ontogenesis is a dynamic process in both space and time, it seems counterintuitive that these two temporal states represent the full spectrum of organismal pluripotency. Here we show that by modulating culture parameters, a stem-cell type with unique spatial characteristics and distinct molecular and functional features, designated as region-selective pluripotent stem cells (rsPSCs), can be efficiently obtained from mouse embryos and primate pluripotent stem cells, including humans. The ease of culturing and editing the genome of human rsPSCs offers advantages for regenerative medicine applications. The unique ability of human rsPSCs to generate post-implantation interspecies chimaeric embryos may facilitate our understanding of early human development and evolution.

Interspecies blastocyst complementation enables organ-specific enrichment of xenogenic pluripotent stem cell (PSC) derivatives. Here, we establish a versatile blastocyst complementation platform based on CRISPR-Cas9-mediated zygote genome editing and show enrichment of rat PSC-derivatives in several tissues of gene-edited organogenesis-disabled mice. Besides gaining insights into species evolution, embryogenesis, and human disease, interspecies blastocyst complementation might allow human organ generation in animals whose organ size, anatomy, and physiology are closer to humans. To date, however, whether human PSCs (hPSCs) can contribute to chimera formation in non-rodent species remains unknown. We systematically evaluate the chimeric competency of several types of hPSCs using a more diversified clade of mammals, the ungulates. We find that naive hPSCs robustly engraft in both pig and cattle pre-implantation blastocysts but show limited contribution to post-implantation pig embryos. Instead, an intermediate hPSC type exhibits higher degree of chimerism and is able to generate differentiated progenies in post-implantation pig embryos.

Low efficiencies of gene targeting via homologous recombination (HR) have limited basic research and applications using human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs). Here, we show highly and equally efficient gene knockout and knock-in at both transcriptionally active (HPRT1, KU80, LIG1, LIG3) and inactive (HB9) loci in these cells using high-capacity helper-dependent adenoviral vectors (HDAdVs). Without the necessity of introducing artificial DNA double-strand breaks, 7–81% of drug-resistant colonies were gene-targeted by accurate HR, which were not accompanied with additional ectopic integrations. Even at the motor neuron-specific HB9 locus, the enhanced green fluorescent protein (EGFP) gene was accurately knocked in in 23–57% of drug-resistant colonies. In these clones, induced differentiation into the HB9-positive motor neuron correlated with EGFP expression. Furthermore, HDAdV infection had no detectable adverse effects on the undifferentiated state and pluripotency of hESCs and hiPSCs. These results suggest that HDAdV is one of the best methods for efficient and accurate gene targeting in hESCs and hiPSCs and might be especially useful for therapeutic applications.

Human embryonic stem (hES) cells are regarded as a potentially unlimited source of cellular materials for regenerative medicine. For biological studies and clinical applications using primate ES cells, the development of a general strategy to obtain efficient gene delivery and genetic manipulation, especially gene targeting via homologous recombination (HR), would be of paramount importance. However, unlike mouse ES (mES) cells, efficient strategies for transient gene delivery and HR in hES cells have not been established. Here, we report that helper-dependent adenoviral vectors (HDAdVs) were able to transfer genes in hES and cynomolgus monkey ( Macaca fasicularis ) ES (cES) cells efficiently. Without losing the undifferentiated state of the ES cells, transient gene transfer efficiency was ≈100%. Using HDAdVs with homology arms, approximately one out of 10 chromosomal integrations of the vector was via HR, whereas the rate was only ≈1% with other gene delivery methods. Furthermore, in combination with negative selection, ≈45% of chromosomal integrations of the vector were targeted integrations, indicating that HDAdVs would be a powerful tool for genetic manipulation in hES cells and potentially in other types of human stem cells, such as induced pluripotent stem (iPS) cells.

For gene therapy of inherited diseases, targeted integration/gene repair through homologous recombination (HR) between exogenous and chromosomal DNA would be an ideal strategy to avoid potentially serious problems of random integration such as cellular transformation and gene silencing. Efficient sequence-specific modification of chromosomes by HR would also advance both biological studies and therapeutic applications of a variety of stem cells. Toward these goals, we developed an improved strategy of adenoviral vector (AdV)-mediated HR and examined its ability to correct an insertional mutation in the hypoxanthine phosphoribosyl transferase ( Hprt ) locus in male mouse ES cells. The efficiency of HR was compared between four types of AdVs that contained various lengths of homologies at the Hprt locus and with various multiplicities of infections. The frequency of HR with helper-dependent AdVs (HD AdVs) with an 18.6-kb homology reached 0.2% per transduced cell at a multiplicity of infection of 10 genomes per cell. Detection of random integration at DNA levels by PCR revealed extremely high efficiency of 5% per cell. We also isolated and characterized chromosomal sites where HD AdVs integrated in a random manner. In contrast to retroviral, lentiviral, and adeno-associated viral vectors, which tend to integrate into genes, the integration sites of AdV was distributed randomly inside and outside genes. These findings suggest that HR mediated by HD AdVs is efficient and relatively safe and might be a new viable option for ex vivo gene therapy as well as a tool for chromosomal manipulation of a variety of stem cells.

The ortho position of the aromatic ring in 2-aryloxazolines and 2-arylimidazolines is selectively arylated and alkenylated with organic halides in the presence of a ruthenium(II)-phosphine complex. In the case of unsubstituted and para-substituted phenyloxazolines, 1:2 coupled products were obtained preferentially, while 1:1 coupled products were obtained in the case of meta-substituted phenyloxazolines and N-acylarylimidazolines. The reaction is proposed to proceed via the generation of an organoruthenium intermediate, formed by oxidative addition of the organic halide, and ortho-ruthenation directed by the coordination of the 2-oxazolinyl or 2-imidazolinyl group to the ruthenium center.

Werner syndrome (WS) is a premature aging disorder caused by WRN protein deficiency. Here, we report on the generation of a human WS model in human embryonic stem cells (ESCs). Differentiation of WRN-null ESCs to mesenchymal stem cells (MSCs) recapitulates features of premature cellular aging, a global loss of H3K9me3, and changes in heterochromatin architecture. We show that WRN associates with heterochromatin proteins SUV39H1 and HP1α and nuclear lamina-heterochromatin anchoring protein LAP2β. Targeted knock-in of catalytically inactive SUV39H1 in wild-type MSCs recapitulates accelerated cellular senescence, resembling WRN-deficient MSCs. Moreover, decrease in WRN and heterochromatin marks are detected in MSCs from older individuals. Our observations uncover a role for WRN in maintaining heterochromatin stability and highlight heterochromatin disorganization as a potential determinant of human aging.

Fanconi anaemia (FA) is a recessive disorder characterized by genomic instability, congenital abnormalities, cancer predisposition and bone marrow (BM) failure. However, the pathogenesis of FA is not fully understood partly due to the limitations of current disease models. Here, we derive integration free-induced pluripotent stem cells (iPSCs) from an FA patient without genetic complementation and report in situ gene correction in FA-iPSCs as well as the generation of isogenic FANCA-deficient human embryonic stem cell (ESC) lines. FA cellular phenotypes are recapitulated in iPSCs/ESCs and their adult stem/progenitor cell derivatives. By using isogenic pathogenic mutation-free controls as well as cellular and genomic tools, our model serves to facilitate the discovery of novel disease features. We validate our model as a drug-screening platform by identifying several compounds that improve hematopoietic differentiation of FA-iPSCs. These compounds are also able to rescue the hematopoietic phenotype of FA patient BM cells. Altres ajuts: Strategic Priority Research Program of the Chinese Academy of Sciences (XDA01020312), National Basic Research Program of China (973 Program,2014CB964600;2014CB910500), NSFC (81271266, 31222039, 81330008, 31201111, 81371342, 81300261, 81300677), Key Research Program of the Chinese Academy of Sciences (KJZD-EW-TZ-L05), Beijing Natural Science Foundation (7141005; 5142016), the Thousand Young Talents program of China, National Laboratory of Biomacromolecules (012kf02, 2013kf05;2013kf11;2014kf02), and State Key Laboratory of Drug Research (SIMM1302KF-17). M.L. and K.S. are supported by CIRM fellowship. N.M was partially supported by La Fundació Privada La Marató de TV3, 121430/31/32. Y.T. was partially supported by an Uehara Memorial Foundation research fellowship. E.N. was partially supported by an F.M. Kirby Foundation postdoctoral fellowship. J.S. was supported by Fundació Marató TV3 (464/C/2012). J.A.B. was supported by grants from La Fundació Privada La Marató de TV3, 121430/31/32. J.C.I.B. was supported by grants from the G. Harold and Leila Y. Mathers Charitable Foundation, The California Institute of Regenerative Medicine, Ellison Medical Foundation, and The Leona M. and Harry B. Helmsley Charitable Trust grant #2012-PG-MED002.

SummaryThe utility of genome editing technologies for disease modeling and developing cellular therapies has been extensively documented, but the impact of these technologies on mutational load at the whole-genome level remains unclear. We performed whole-genome sequencing to evaluate the mutational load at single-base resolution in individual gene-corrected human induced pluripotent stem cell (hiPSC) clones in three different disease models. In single-cell clones, gene correction by helper-dependent adenoviral vector (HDAdV) or Transcription Activator-Like Effector Nuclease (TALEN) exhibited few off-target effects and a low level of sequence variation, comparable to that accumulated in routine hiPSC culture. The sequence variants were randomly distributed and unique to individual clones. We also combined both technologies and developed a TALEN-HDAdV hybrid vector, which significantly increased gene-correction efficiency in hiPSCs. Therefore, with careful monitoring via whole-genome sequencing it is possible to apply genome editing to human pluripotent cells with minimal impact on genomic mutational load.

Human pluripotent stem cells, such as embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs), have the ability to differentiate into various cell types, and will become a potential source of cellular materials for regenerative medicine. To make full use of hESCs or hiPSCs for both basic and clinical research, genetic modification, especially gene targeting via homologous recombination (HR), would be an essential technique. This report describes the successful gene targeting of the hypoxanthine phosphoribosyl transferase 1 (HPRT1) and the NANOG loci in human pluripotent stem cells with adeno-associated virus (AAV) vectors. At the HPRT1 locus, up to 1% of stable transformants were targeted via HR with an AAV-HPRT1 targeting vector, without loss of pluripotency. On the other hand, 20–87% of stable transformants were targeted using an AAV-NANOG-targeting vector designed for the promoter-trap strategy. In the KhES-3 cell line, which shows particularly high fragility to experimental manipulation, gene targeting was successful only by using an AAV vector but not by electroporation. In addition to hESC, gene targeting was achieved in hiPSC lines at similar frequencies. These data indicate that AAV vectors may therefore be a useful tool to introduce genetic modifications in hESCs and hiPSCs.

Background Adeno-associated virus (AAV) vectors have been shown to correct a variety of mutations in human cells by homologous recombination (HR) at high rates, which can overcome insertional mutagenesis and transgene silencing, two of the major hurdles in conventional gene addition therapy of inherited diseases. We examined an ability of AAV vectors to repair a mutation in human hematopoietic cells by HR. Methods We infected a human B-lymphoblastoid cell line (BCL) derived from a normal subject with an AAV, which disrupts the hypoxanthine phosphoribosyl transferase1 (HPRT1) locus, to measure the frequency of AAV-mediated HR in BCL cells. We subsequently constructed an AAV vector encoding the normal sequences from the Fanconi anemia group A (FANCA) locus to correct a mutation in the gene in BCL derived from a FANCA patient. Results Under optimal conditions, approximately 50% of BCL cells were transduced with an AAV serotype 2 (AAV-2) vector. In FANCA BCL cells, up to 0.016% of infected cells were gene-corrected by HR. AAV-mediated restoration of normal genotypic and phenotypic characteristics in FANCA-mutant cells was confirmed at the DNA, protein and functional levels. Conclusions The results obtained in the present study indicate that AAV vectors may be applicable for gene correction therapy of inherited hematopoietic disorders. Copyright © 2009 John Wiley & Sons, Ltd.

For gene therapy of inherited diseases, integration of a therapeutic gene into random sites on chromosomes could potentially lead to serious problems such as cellular transformation and gene silencing. Homologous recombination (HR) between exogenous and chromosomal DNA would be an ideal strategy to obtain safe and stable gene expression stably. We constructed helper-dependent adenoviral vectors (HD AdVs) and examined the ability of AdVs to correct an insertional mutation in exon 3 of the hypoxanthine phosphoribosyl transferase (Hprt) locus in male mouse embryonic stem (ES) cells. These HD AdV contained the wild-type exon 3 sequence of the mouse Hprt gene with 6.7 kb (HD AdHprt6.7) or 18.6 kb (HD AdHprt18.6) of flanking intronic sequences, as well as the |[beta]|-geo marker gene. HR and random integration (RI) frequencies were calculated by dividing the total number of HAT- or neomycin-resistant colonies by the total number of cells plated. HR was only obtained by using the HD AdHprt18.6 vector, and the frequency was nearly 0.0002 per infected cell at an MOI of 1000. In the case of the HD AdHprt6.7 vector, no HR events were detected. The HR frequency by electroporation of a plasmid construct identical to the HD AdHprt18.6 vector was 23-fold less than that by vector infection. The structure of the exon 3 region of Hprt locus was analyzed in HAT-resistant ES clones by Southern analysis, and all the clones showed a pattern of faithful HR. Since the number of target cells might be limited in ex vivo gene therapy, such as stem cell therapy, we next investigated whether HD AdV-mediated HR can be efficiently achieved even in smaller cell populations. We successfully obtained HAT-resistant colonies at the frequency of 2.1 |[times]| 10|[minus]|4 per infected cell in a 24-well dish (25 HAT-resistant colonies out of 1.2 |[times]| 10|[minus]|5 cells infected, on average). These results clearly indicate gene correction can be achieved relatively efficiently even in smaller cell populations using AdVs. Both HD AdVs achieved similar RI frequencies of 10|[minus]|4 |[minus]| 10|[minus]|3 per cell, which was measured as a frequency to produce G418-resistant colonies. In contrast to the HR frequency, which showed an MOI-dependent increase, the RI frequency reached a plateau at an MOI of 100. In order to directly detect integrated vectors at DNA levels, we next performed PCR on ES clones infected with HD AdHprt18.6 and formed in the absence of G418 selection. Surprisingly, vector DNA was detected in 4 of the 74 samples (5 % of colonies). These results indicate that the actual RI frequency of HD AdVs is much higher than previously believed. Next, we determined the integration sites of the HD AdHprt18.6 vector by adaptor-ligated PCR. In contrast to retroviral, lentiviral and adeno-associated viral vectors, which tend to integrate near or within active genes, HD AdV had a tendency to integrate in intergenic regions (12 of 17 integrants, 71%). Small deletions of integrated vector DNA were found at the end of ITRs (16 of 17 integrations, 94%), ranging from 1 to 30 bp in size. These findings suggest that HR mediated by HD AdV is efficient and safe, and might be a viable option for ex vivo gene therapy of stem cells.