Integration Site Selection Research Articles

LEDGF/p75 interacts with mRNA splicing factors and targets HIV-1 integration to highly spliced genes.

The host chromatin-binding factor LEDGF/p75 interacts with HIV-1 integrase and directs integration to active transcription units. To understand how LEDGF/p75 recognizes transcription units, we sequenced 1 million HIV-1 integration sites isolated from cultured HEK293T cells. Analysis of integration sites showed that cancer genes were preferentially targeted, raising concerns about using lentivirus vectors for gene therapy. Additional analysis led to the discovery that introns and alternative splicing contributed significantly to integration site selection. These correlations were independent of transcription levels, size of transcription units, and length of the introns. Multivariate analysis with five parameters previously found to predict integration sites showed that intron density is the strongest predictor of integration density in transcription units. Analysis of previously published HIV-1 integration site data showed that integration density in transcription units in mouse embryonic fibroblasts also correlated strongly with intron number, and this correlation was absent in cells lacking LEDGF. Affinity purification showed that LEDGF/p75 is associated with a number of splicing factors, and RNA sequencing (RNA-seq) analysis of HEK293T cells lacking LEDGF/p75 or the LEDGF/p75 integrase-binding domain (IBD) showed that LEDGF/p75 contributes to splicing patterns in half of the transcription units that have alternative isoforms. Thus, LEDGF/p75 interacts with splicing factors, contributes to exon choice, and directs HIV-1 integration to transcription units that are highly spliced.

Host determinants of HTLV-1 integration site preference

Integration of the provirus into the host genome is an essential step in the establishment of a chronic retroviral infection. Retroviruses do not integrate at random. Each genus of retrovirus preferentially integrates in genomic sites associated with certain genomic features. This preference is thought to result from an interaction of the pre-integration complex with a specific host co-factor. Such an interaction has been well documented between HIV-1 integrase and lens epithelium-derived growth factor (LEDGF) and between MLV and members of the Bromoextraterminal Domain Protein (BET) family. HTLV-1 also has a reproducible preference to integrate near various genomic features; however, the putative host co-factor of HTLV-1 integrase remains unknown. We present evidence that demonstrates that the HTLV-1 integrase co-factor may be the protein phosphatase 2A (PP2A) complex. Two independent protein interaction screens (yeast-2-hybrid and co-immunoprecipitation) both revealed an interaction between HTLV-1 integrase and the PP2A holoenzyme. We used co-culture with HTLV-1 producer cell line MT2 to infect rat cell lines containing an inducible knockdown of PP2A. We found that PP2A-knockdown altered the integration profile of these de novo integration sites. In contrast to infection in PP2A-competent cells, integration sites in PP2A-knockdown cells lacked the characteristic proximity to genomic features associated with transcriptional activity (genes, transcription start sites, CpG islands) and to epigenetic marks associated with active chromatin. We are in the process of investigating how PP2A inhibitors and shRNAs targeting specific regulatory subunits of the PP2A holoenzyme influence integration site selection.

Impact of Nucleoporin-Mediated Chromatin Localization and Nuclear Architecture on HIV Integration Site Selection.

It has been known for a number of years that integration sites of human immunodeficiency virus type 1 (HIV-1) DNA show a preference for actively expressed chromosomal locations. A number of viral and cellular proteins are implicated in this process, but the underlying mechanism is not clear. Two recent breakthrough publications advance our understanding of HIV integration site selection by focusing on the localization of the preferred target genes of integration. These studies reveal that knockdown of certain nucleoporins and components of nucleocytoplasmic trafficking alter integration site preference, not by altering the trafficking of the viral genome but by altering the chromatin subtype localization relative to the structure of the nucleus. Here, we describe the link between the nuclear basket nucleoporins (Tpr and Nup153) and chromatin organization and how altering the host environment by manipulating nuclear structure may have important implications for the preferential integration of HIV into actively transcribed genes, facilitating efficient viral replication.

Caracterización genómica de la integración simultánea del virus de la inmunodeficiencia humana 1 y el virus linfotrópico humano tipo1

The selection of retroviral cDNA integration sites in the human genome is a critical step to condition the dynamics of infection. The objective was to analyze the combination of genomic characteristics of infected cells that would condition the genomic profiles of HIV-1/HTLV-1 simultaneous integration. We carried out a computer simulation using 203 human genome sequences flanking 3´LTR of both retroviruses previously deposited in the GenBank, and applying several computational tools. The analyses were focused on determining the chromosomal integration, CpG islands, Alu sequences and the expression of integration target class II genes in lymphocytic populations in a 100 kb chromatin structure associated with simultaneous integration. We found 47.3% of simultaneous cDNA integrations localized in regions rich in repetitive elements. The rest of retroviral cDNA integrations occurred in class II gene introns (p<0.05). We determined a differential chromosomal distribution for both types of provirus where HTLV-1 provirus were preferentially placed in pericentromeric and centromeric regions in contrast with HIV-1 distribution, which was registered in telomeric and subtelomeric zones (p<0.001). The genomic environment of integration for both retroviruses was characterized by genes encoding molecular binding and signal transduction, as well as by high density of CpG islands and Alu sequences. The data resulting from the computer simulation did support the hypothesis that a combination of specific chromatin characteristics would determine the dynamics of HIV-1/HTLV-1 simultaneous integration process.

Nuclear architecture dictates HIV-1 integration site selection.

Long-standing evidence indicates that human immunodeficiency virus type 1 (HIV-1) preferentially integrates into a subset of transcriptionally active genes of the host cell genome. However, the reason why the virus selects only certain genes among all transcriptionally active regions in a target cell remains largely unknown. Here we show that HIV-1 integration occurs in the outer shell of the nucleus in close correspondence with the nuclear pore. This region contains a series of cellular genes, which are preferentially targeted by the virus, and characterized by the presence of active transcription chromatin marks before viral infection. In contrast, the virus strongly disfavours the heterochromatic regions in the nuclear lamin-associated domains and other transcriptionally active regions located centrally in the nucleus. Functional viral integrase and the presence of the cellular Nup153 and LEDGF/p75 integration cofactors are indispensable for the peripheral integration of the virus. Once integrated at the nuclear pore, the HIV-1 DNA makes contact with various nucleoporins; this association takes part in the transcriptional regulation of the viral genome. These results indicate that nuclear topography is an essential determinant of the HIV-1 life cycle.

Intasome architecture and chromatin density modulate retroviral integration into nucleosome.

BackgroundRetroviral integration depends on the interaction between intasomes, host chromatin and cellular targeting cofactors as LEDGF/p75 or BET proteins. Previous studies indicated that the retroviral integrase, by itself, may play a role in the local integration site selection within nucleosomal target DNA. We focused our study on this local association by analyzing the intrinsic properties of various retroviral intasomes to functionally accommodate different chromatin structures in the lack of other cofactors.ResultsUsing in vitro conditions allowing the efficient catalysis of full site integration without these cofactors, we show that distinct retroviral integrases are not equally affected by chromatin compactness. Indeed, while PFV and MLV integration reactions are favored into dense and stable nucleosomes, HIV-1 and ASV concerted integration reactions are preferred into poorly dense chromatin regions of our nucleosomal acceptor templates. Predicted nucleosome occupancy around integration sites identified in infected cells suggests the presence of a nucleosome at the MLV and HIV-1 integration sites surrounded by differently dense chromatin. Further analyses of the relationships between the in vitro integration site selectivity and the structure of the inserted DNA indicate that structural constraints within intasomes could account for their ability to accommodate nucleosomal DNA and could dictate their capability to bind nucleosomes functionally in these specific chromatin contexts.ConclusionsThus, both intasome architecture and compactness of the chromatin surrounding the targeted nucleosome appear important determinants of the retroviral integration site selectivity. This supports a mechanism involving a global targeting of the intasomes toward suitable chromatin regions followed by a local integration site selection modulated by the intrinsic structural constraints of the intasomes governing the target DNA bending and dictating their sensitivity toward suitable specific nucleosomal structures and density.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-015-0145-9) contains supplementary material, which is available to authorized users.

Meta-Analysis of DNA Tumor-Viral Integration Site Selection Indicates a Role for Repeats, Gene Expression and Epigenetics

Meta-Analysis of DNA Tumor-Viral Integration Site Selection Indicates a Role for Repeats, Gene Expression and Epigenetics

HIV-1 integrase variants retarget viral integration and are associated with disease progression in a chronic infection cohort.

Distinct integration patterns of different retroviruses, including HIV-1, have puzzled virologists for over 20years. A tetramer of the viral integrase (IN) assembles on the two viral cDNA ends, docks onto the target DNA (tDNA), and catalyzes viral genome insertion into the host chromatin. We identified the amino acids in HIV-1 IN that directly contact tDNA bases and affect local integration site sequence selection. These residues also determine the propensity of the virus to integrate into flexible tDNA sequences. Remarkably, natural polymorphisms INS119G and INR231G retarget viral integration away from gene-dense regions. Precisely these variants were associated with rapid disease progression in a chronic HIV-1 subtype C infection cohort. These findings link integration site selection to virulence and viral evolution, but also to the host immune response and antiretroviral therapy, since HIV-1 IN119 is under selection by HLA alleles and integrase inhibitors.

Host Cofactors and Pharmacologic Ligands Share an Essential Interface in HIV-1 Capsid That Is Lost upon Disassembly

The HIV-1 capsid is involved in all infectious steps from reverse transcription to integration site selection, and is the target of multiple host cell and pharmacologic ligands. However, structural studies have been limited to capsid monomers (CA), and the mechanistic basis for how these ligands influence infection is not well understood. Here we show that a multi-subunit interface formed exclusively within CA hexamers mediates binding to linear epitopes within cellular cofactors NUP153 and CPSF6, and is competed for by the antiretroviral compounds PF74 and BI-2. Each ligand is anchored via a shared phenylalanine-glycine (FG) motif to a pocket within the N-terminal domain of one monomer, and all but BI-2 also make essential interactions across the N-terminal domain: C-terminal domain (NTD:CTD) interface to a second monomer. Dissociation of hexamer into CA monomers prevents high affinity interaction with CPSF6 and PF74, and abolishes binding to NUP153. The second interface is conformationally dynamic, but binding of NUP153 or CPSF6 peptides is accommodated by only one conformation. NUP153 and CPSF6 have overlapping binding sites, but each makes unique CA interactions that, when mutated selectively, perturb cofactor dependency. These results reveal that multiple ligands share an overlapping interface in HIV-1 capsid that is lost upon viral disassembly.

Molecular mechanisms of retroviral integration site selection.

Retroviral replication proceeds through an obligate integrated DNA provirus, making retroviral vectors attractive vehicles for human gene-therapy. Though most of the host cell genome is available for integration, the process of integration site selection is not random. Retroviruses differ in their choice of chromatin-associated features and also prefer particular nucleotide sequences at the point of insertion. Lentiviruses including HIV-1 preferentially integrate within the bodies of active genes, whereas the prototypical gammaretrovirus Moloney murine leukemia virus (MoMLV) favors strong enhancers and active gene promoter regions. Integration is catalyzed by the viral integrase protein, and recent research has demonstrated that HIV-1 and MoMLV targeting preferences are in large part guided by integrase-interacting host factors (LEDGF/p75 for HIV-1 and BET proteins for MoMLV) that tether viral intasomes to chromatin. In each case, the selectivity of epigenetic marks on histones recognized by the protein tether helps to determine the integration distribution. In contrast, nucleotide preferences at integration sites seem to be governed by the ability for the integrase protein to locally bend the DNA duplex for pairwise insertion of the viral DNA ends. We discuss approaches to alter integration site selection that could potentially improve the safety of retroviral vectors in the clinic.

Toward a Safer Integration Profile of MLV-based Retroviral Vectors

Toward a Safer Integration Profile of MLV-based Retroviral Vectors

Repair of oxidative DNA base damage in the host genome influences the HIV integration site sequence preference.

Host base excision repair (BER) proteins that repair oxidative damage enhance HIV infection. These proteins include the oxidative DNA damage glycosylases 8-oxo-guanine DNA glycosylase (OGG1) and mutY homolog (MYH) as well as DNA polymerase beta (Polβ). While deletion of oxidative BER genes leads to decreased HIV infection and integration efficiency, the mechanism remains unknown. One hypothesis is that BER proteins repair the DNA gapped integration intermediate. An alternative hypothesis considers that the most common oxidative DNA base damages occur on guanines. The subtle consensus sequence preference at HIV integration sites includes multiple G:C base pairs surrounding the points of joining. These observations suggest a role for oxidative BER during integration targeting at the nucleotide level. We examined the hypothesis that BER repairs a gapped integration intermediate by measuring HIV infection efficiency in Polβ null cell lines complemented with active site point mutants of Polβ. A DNA synthesis defective mutant, but not a 5′dRP lyase mutant, rescued HIV infection efficiency to wild type levels; this suggeted Polβ DNA synthesis activity is not necessary while 5′dRP lyase activity is required for efficient HIV infection. An alternate hypothesis that BER events in the host genome influence HIV integration site selection was examined by sequencing integration sites in OGG1 and MYH null cells. In the absence of these 8-oxo-guanine specific glycosylases the chromatin elements of HIV integration site selection remain the same as in wild type cells. However, the HIV integration site sequence preference at G:C base pairs is altered at several positions in OGG1 and MYH null cells. Inefficient HIV infection in the absence of oxidative BER proteins does not appear related to repair of the gapped integration intermediate; instead oxidative damage repair may participate in HIV integration site preference at the sequence level.

MLV integration site selection is driven by strong enhancers and active promoters.

Retroviruses integrate into the host genome in patterns specific to each virus. Understanding the causes of these patterns can provide insight into viral integration mechanisms, pathology and genome evolution, and is critical to the development of safe gene therapy vectors. We generated murine leukemia virus integrations in human HepG2 and K562 cells and subjected them to second-generation sequencing, using a DNA barcoding technique that allowed us to quantify independent integration events. We characterized >3 700 000 unique integration events in two ENCODE-characterized cell lines. We find that integrations were most highly enriched in a subset of strong enhancers and active promoters. In both cell types, approximately half the integrations were found in <2% of the genome, demonstrating genomic influences even narrower than previously believed. The integration pattern of murine leukemia virus appears to be largely driven by regions that have high enrichment for multiple marks of active chromatin; the combination of histone marks present was sufficient to explain why some strong enhancers were more prone to integration than others. The approach we used is applicable to analyzing the integration pattern of any exogenous element and could be a valuable preclinical screen to evaluate the safety of gene therapy vectors.

Targeted Gene Addition in Human Epithelial Stem Cells by Zinc-finger Nuclease-mediated Homologous Recombination

Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a "safe harbor" locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of > 20% in a human keratinocyte cell line, > 10% in immortalized keratinocytes, and < 1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs.

HERV-H RNA is abundant in human embryonic stem cells and a precise marker for pluripotency

BackgroundCertain post-translational modifications to histones, including H3K4me3, as well as binding sites for the transcription factor STAT1, predict the site of integration of exogenous gamma-retroviruses with great accuracy and cell-type specificity. Statistical methods that were used to identify chromatin features that predict exogenous gamma-retrovirus integration site selection were exploited here to determine whether cell type-specific chromatin markers are enriched in the vicinity of endogenous retroviruses (ERVs).ResultsAmong retro-elements in the human genome, the gamma-retrovirus HERV-H was highly associated with H3K4me3, though this association was only observed in embryonic stem (ES) cells (p < 10-300) and, to a lesser extent, in induced pluripotent stem (iPS) cells. No significant association was observed in nearly 40 differentiated cell types, nor was any association observed with other retro-elements. Similar strong association was observed between HERV-H and the binding sites within ES cells for the pluripotency transcription factors NANOG, OCT4, and SOX2. NANOG binding sites were located within the HERV-H 5′LTR itself. OCT4 and SOX2 binding sites were within 1 kB and 2 kB of the 5′LTR, respectively. In keeping with these observations, HERV-H RNA constituted 2% of all poly A RNA in ES cells. As ES cells progressed down a differentiation pathway, the levels of HERV-H RNA decreased progressively. RNA-Seq datasets showed HERV-H transcripts to be over 5 kB in length and to have the structure 5′LTR-gag-pro-3′LTR, with no evidence of splicing and no intact open reading frames.ConclusionThe developmental regulation of HERV-H expression, the association of HERV-H with binding sites for pluripotency transcription factors, and the extremely high levels of HERV-H RNA in human ES cells suggest that HERV-H contributes to pluripotency in human cells. Proximity of HERV-H to binding sites for pluripotency transcription factors within ES cells might be due to retention of the same chromatin features that determined the site of integration of the ancestral, exogenous, gamma-retrovirus that gave rise to HERV-H in the distant past. Retention of these markers, or, alternatively, recruitment of them to the site of the established provirus, may have acted post-integration to fix the provirus within the germ-line of the host species. Either way, HERV-H RNA provides a specific marker for pluripotency in human cells.

Understanding lentiviral vector chromatin targeting: working to reduce insertional mutagenic potential for gene therapy

Replication-deficient retroviruses have been successfully utilized as vectors, offering an efficient, stable method of therapeutic gene delivery. Many examples exist proving this mode of integrative gene transfer is both effective and safe in cultured systems and clinical trials. Along with their success, severe side effects have occurred with early retroviral vectors causing a shift in the approach to vector design before further clinical testing. Several alternative delivery methods are available but lentiviral vectors (LV) are among the most favorable as they are already well understood. LV offer safer integration site selection profiles and a lower degree of genotoxicity, compared with γ-retroviral vectors. Following their introduction, development of the self-inactivating vector configuration was a huge step to this mode of therapy but did not confer full protection against insertional mutagenesis. As a result integration, modeling must be improved to eventually avoid this possibility. The cellular factor LEDGF/p75 seems to play an essential role in the process of LV site selection and its interactions with chromatin are being quickly resolved. LEDGF/p75 is at the center of one example directed integration effort where recombinant products bias the integration event, a step toward fully directed integration into pre-determined benign loci. A more accurate picture of the details of LEDGF/p75 in the natural integration process is emerging, including new binding specificities, chromatin interaction kinetics and additional cellular factors. Together with next-generation sequencing technology and bio-informatics to analyze integration patterns, these advancements will lead to highly focused directed integration, accelerating wide-spread acceptance of LV for gene therapy.

HRP-2 determines HIV-1 integration site selection in LEDGF/p75 depleted cells

BackgroundLens epithelium–derived growth factor (LEDGF/p75) is a cellular co-factor of HIV-1 integrase (IN) that tethers the viral pre-integration complex to the host cell chromatin and determines the genome wide integration site distribution pattern of HIV-1. Recently, we demonstrated that HIV-1 replication was reduced in LEDGF/p75 knockout (KO) cells. LEDGF/p75 KO significantly altered the integration site preference of HIV-1, but the pattern remained distinct from a computationally generated matched random control set (MRC), suggesting the presence of an alternative tethering factor. We previously identified Hepatoma-derived growth factor related protein 2 (HRP-2) as a factor mediating LEDGF/p75-independent HIV-1 replication. However, the role of HRP-2 in HIV-1 integration site selection was not addressed.FindingsWe studied the HIV-1 integration site distribution in the presence and absence of LEDGF/p75 and/or HRP-2, and in LEDGF/p75-depleted cells that overexpress HRP-2. We show that HRP-2 functions as a co-factor of HIV-1 IN in LEDGF/p75-depleted cells. Endogenous HRP-2 only weakly supported HIV-1 replication in LEDGF/p75 depleted cells. However, HRP-2 overexpression rescued HIV-1 replication and restored integration in RefSeq genes to wild-type levels. Additional HRP-2 KD in LEDGF/p75-depleted cells reduces integration frequency in transcription units and shifts the integration distribution towards random.ConclusionsWe demonstrate that HRP-2 overexpression can compensate for the absence of LEDGF/p75 and indicate that the residual bias in integration targeting observed in the absence of LEDGF/p75 can be ascribed to HRP-2. Knockdown of HRP-2 upon LEDGF/p75 depletion results in a more random HIV-1 integration pattern. These data therefore reinforce the understanding that LEDGF/p75 is the dominant HIV-1 IN co-factor.

Investigation of retroviral integration preference via high-throughput sequencing

The integration of retroviral DNA into the host genome is a key step of the retrovirus lifecycle. The positions of such integrations are non-random and are characteristic to the virus in question. Retroviral vectors have been useful in gene therapy, but the approach carries with it the danger of host gene misregulation by viral integration, potentially contributing to cancer. An understanding of how integration sites are determined is critical for any approach seeking to avoid such consequences. To better understand the factors influencing retroviral integration site selection, we are generating integrations of murine leukemia virus (MLV) in human K562 cells. We use massively parallel Illumina technology to sequence the cellular genome and have developed a bioinformatic pipeline to efficiently identify sites of integration. Our aim is to create a high-resolution, high-density integration map with 1 million unique integrations mapped to single-nucleotide resolution. This map will identify regions prone or refractory to MLV integration. We plan to compare this map with data from the ENCODE Consortium, looking for significant associations with characteristics of the local genomic environment, to enhance our understanding of MLV integration site selection.

Evaluation of Residual Promoter Activity in γ-Retroviral Self-inactivating (SIN) Vectors

Therapeutic gene delivery mediated by retroviral vectors has the advantage of stable integration into the host genome. A major safety concern for gene delivery achieved by murine leukemia virus (MLV)-based retroviral vectors is the activation of adjacent cellular genes including oncogenes following integration into the host genome. Self-inactivating (SIN) vectors lacking viral enhancers/promoters in their 3' long terminal repeat (LTR) have been proposed as a means of overcoming this safety concern. However the MLV-based SIN vectors currently used by laboratories to assess insertional mutagenesis, integration site selection, and the potency of transgene expression are not uniform in the composition of their 3' LTRs. We constructed a series of SIN vectors representative of the currently employed vectors, but lacking an internal promoter. Green fluorescent protein (GFP) was used as a reporter gene. Target cells exposed to these vectors were evaluated for number of integrants and GFP expression at the messenger RNA (mRNA) level and protein level. We found that viral promoter activity in the 3' LTR is not attenuated in many currently employed SIN vectors. These results suggest that the influence of strong residual promoter activity should be taken into consideration when interpreting experimental results obtained using SIN vectors in gene therapy research.

Genomic Safe Harbors in Human iPS Cells

Abstract SCI-47Current hematopoietic stem cell (HSC) gene therapy relies on randomly integrated retroviral vectors and is hampered by the risk of insertional oncogenesis often leading to leukemia. This risk would be minimized if therapeutic transgenes could be inserted in selected sites of the genome that permit appropriate function without disruption or dysregulation of endogenous genes — referred to as “genomic safe harbors.” The advent of induced pluripotent stem (iPS) cell technology offered unprecedented opportunities for the genetic engineering of human cells. iPS cells, unlike HSC, can be extensively cultured in vitro, enabling the selection and study of unique sites of transgene integration for the first time in a relevant setting. We proposed a definition of safe harbor sites, based on their topology in the genome with relation to coding genes and other genomic landmarks, using five criteria: (i) distance of at least 50 kb from the 5' end of any gene, (ii) distance of at least 300 kb from any cancer-related gene, (iii) distance of at least 300 kb from any microRNA, (iv) location outside a transcription unit, and (v) location outside ultraconserved regions of the human genome (1). To test them, we developed a strategy to select iPS cell clones harboring a single copy of a randomly integrating vector at sites that meet our safe harbor criteria. In a recent proof-of-principle study, using a model for genetic correction of β-thalassemia major, we demonstrated that erythroid progeny of patient-specific iPS cell clones harboring a lentivirally encoded β-globin transgene in a safe harbor site express therapeutic levels of β-globin without perturbing neighboring genes. This approach, entailing the prospective screening and selection of integration sites, based on combined bioinformatics and functional analyses, provides a robust and dependable strategy for the genetic engineering of human iPS cells. iPS cell-derived cell products used in regenerative medicine will need to be genetically engineered to correct a genetic disease or permit in vivo cell tracking or the elimination of residual undifferentiated cells or progeny gone astray. Our strategy should be broadly applicable to introducing reporter, suicide, or therapeutic genes in a clinically relevant manner. We are currently exploiting this strategy to express a conditional HSV-tk suicide gene for purging of iPS cell progeny from teratoma-initiating cells. With the emergence of improved technologies for homologous recombination into human cells, targeted gene addition may soon become a realistic option if predefined validated safe harbor sites in the human genome are identified. We are utilizing our selection strategy in iPS cells (using lentiviral vectors with reporter cassettes that can be exchanged using Cre recombinase-mediated cassette exchange) as a platform for the de novo discovery and characterization of putative universal safe harbor sites that can be broadly used for the genetic engineering of multiple human cell types. Disclosures:No relevant conflicts of interest to declare.