Targeted Transgene Integration Research Articles

55. AAVHSC Vectors Encoding Zinc-Finger Nucleases Mediate Efficient Targeted Integration at the Human AAVS1 Locus in CD34+ Human Hematopoietic Stem Cells

Zinc-finger nucleases (ZFN) are powerful genome editing tools that may be used to prevent expression of specified genes, correct mutations, or insert transgenes at safe harbor loci. ZFNs create double-stranded breaks at specified locations in the genome which may subsequently be repaired via the non-homologous end joining pathway to interrupt expression of a gene. Targeted integration of genes at pre-determined sites in the genome can be achieved through homology-directed repair of ZFN-mediated targeted double-strand breaks using a co-delivered donor construct. We recently isolated and characterized a panel of novel AAVs (AAVHSC) derived from CD34+ human hematopoietic stem cells (HSC) from healthy donors. We have previously shown that vectors derived from AAVHSCs have the ability to efficiently transduce CD34+ cells. Here we tested the hypothesis that delivery of ZFNs and donor DNA by AAVHSC vectors to CD34+ human cells results in the creation of double stranded site-specific breaks and subsequent efficient targeted transgene integration. A promoterless GFP gene was targeted to Intron 1 of the PPP1R12C gene in the AAVS1 locus such that expression would be driven by the endogenous PPP1R12C promoter following site specific transgene integration. ZFNs specific for PPP1R12C Intron 1 and donor DNA were packaged in AAVHSCs and used to transduce either K562 cells or primary human cytokine-primed peripheral blood CD34+ cells (PBSC). Flow-cytometric analysis at different time points post-transduction revealed specific GFP expression in ZFN + Donor-transduced cells but not in cells transduced with ZFN alone. The multiplicities of infection, ratio of ZFN:donor, and timing of transduction were optimized in K562 cells. Simultaneous transduction of AAVHSC encoding both ZFN and donor DNA at multiplicities of infection of 50,000 and 150,000 respectively was found to be optimal and resulted in GFP expression in up to 50% of transduced cells. Optimized conditions were confirmed in primary CD34+ PBSC from different donors. Targeted integration into AAVS1 was consistently found to be more efficient in CD34+ PBSC as compared with K562 cells, with GFP expression ranging from 10 to 65% of cells. Integrated GFP was expressed long term, with approximately 20% GFP positive cells persisting at 35 days post-transduction in vitro in the absence of selection. Targeted integration into the AAVS1 locus was independently confirmed by amplification using transgene-specific and chromosome-specific primers. Sequencing of the amplified vector chromosome junctions confirmed site-specific integration into AAVS1. No unexpected modifications were found at the junction of the AAVS1 chromosomal sequence and donor homology arms. These results indicate that AAVHSCs function as efficient delivery tools for genome editing in therapeutically relevant cells such as primary PBSC.

TALE nickase mediates high efficient targeted transgene integration at the human multi-copy ribosomal DNA locus

Although targeted gene addition could be stimulated strikingly by a DNA double strand break (DSB) created by either zinc finger nucleases (ZFNs) or TALE nucleases (TALENs), the DSBs are really mutagenic and toxic to human cells. As a compromised solution, DNA single-strand break (SSB) or nick has been reported to mediate high efficient gene addition but with marked reduction of random mutagenesis. We previously demonstrated effective targeted gene addition at the human multicopy ribosomal DNA (rDNA) locus, a genomic safe harbor for the transgene with therapeutic potential. To improve the transgene integration efficiency by using TALENs while lowering the cytotoxicity of DSBs, we created both TALENs and TALE nickases (TALENickases) targeting this multicopy locus. A targeting vector which could integrate a GFP cassette at the rDNA locus was constructed and co-transfected with TALENs or TALENickases. Although the fraction of GFP positive cells using TALENs was greater than that using TALENickases during the first few days after transfection, it reduced to a level less than that using TALENickases after continuous culture. Our findings showed that the TALENickases were more effective than their TALEN counterparts at the multi-copy rDNA locus, though earlier studies using ZFNs and ZFNickases targeting the single-copy loci showed the reverse. Besides, TALENickases mediated the targeted integration of a 5.4kb fragment at a frequency of up to 0.62% in HT1080 cells after drug selection, suggesting their potential application in targeted gene modification not being limited at the rDNA locus.

Targeted transgene integration overcomes variability of position effects in zebrafish

Zebrafish transgenesis is increasingly popular owing to the optical transparency and external development of embryos, which provide a scalable vertebrate model for in vivo experimentation. The ability to express transgenes in a tightly controlled spatio-temporal pattern is an important prerequisite for exploitation of zebrafish in a wide range of biomedical applications. However, conventional transgenesis methods are plagued by position effects: the regulatory environment of genomic integration sites leads to variation of expression patterns of transgenes driven by engineered cis-regulatory modules. This limitation represents a bottleneck when studying the precise function of cis-regulatory modules and their subtle variants or when various effector proteins are to be expressed for labelling and manipulation of defined sets of cells. Here, we provide evidence for the efficient elimination of variability of position effects by developing a PhiC31 integrase-based targeting method. To detect targeted integration events, a simple phenotype scoring of colour change in the lens of larvae is used. We compared PhiC31-based integration and Tol2 transgenesis in the analysis of the activity of a novel conserved enhancer from the developmentally regulated neural-specific esrrga gene. Reporter expression was highly variable among independent lines generated with Tol2, whereas all lines generated with PhiC31 into a single integration site displayed nearly identical, enhancer-specific reporter expression in brain nuclei. Moreover, we demonstrate that a modified integrase system can also be used for the detection of enhancer activity in transient transgenesis. These results demonstrate the power of the PhiC31-based transgene integration for the annotation and fine analysis of transcriptional regulatory elements and it promises to be a generally desirable tool for a range of applications, which rely on highly reproducible patterns of transgene activity in zebrafish.

Efficient Designer Nuclease-Based Homologous Recombination Enables Direct PCR Screening for Footprintless Targeted Human Pluripotent Stem Cells

SummaryGenetic engineering of human induced pluripotent stem cells (hiPSCs) via customized designer nucleases has been shown to be significantly more efficient than conventional gene targeting, but still typically depends on the introduction of additional genetic selection elements. In our study, we demonstrate the efficient nonviral and selection-independent gene targeting in human pluripotent stem cells (hPSCs). Our high efficiencies of up to 1.6% of gene-targeted hiPSCs, accompanied by a low background of randomly inserted transgenes, eliminated the need for antibiotic or fluorescence-activated cell sorting selection, and allowed the use of short donor oligonucleotides for footprintless gene editing. Gene-targeted hiPSC clones were established simply by direct PCR screening. This optimized approach allows targeted transgene integration into safe harbor sites for more predictable and robust expression and enables the straightforward generation of disease-corrected, patient-derived iPSC lines for research purposes and, ultimately, for future clinical applications.

Production of CMAH Knockout Preimplantation Embryos Derived From Immortalized Porcine Cells Via TALE Nucleases

Although noncancerous immortalized cell lines have been developed by introducing genes into human and murine somatic cells, such cell lines have not been available in large domesticated animals like pigs. For immortalizing porcine cells, primary porcine fetal fibroblasts were isolated and cultured using the human telomerase reverse transcriptase (hTERT) gene. After selecting cells with neomycin for 2 weeks, outgrowing colonized cells were picked up and subcultured for expansion. Immortalized cells were cultured for more than 9 months without changing their doubling time (~24 hours) or their diameter (< 20 µm) while control cells became replicatively senescent during the same period. Even a single cell expanded to confluence in 100 mm dishes. Furthermore, to knockout the CMAH gene, designed plasmids encoding a transcription activator-like effector nuclease (TALENs) pairs were transfected into the immortalized cells. Each single colony was analyzed by the mutation-sensitive T7 endonuclease I assay, fluorescent PCR, and dideoxy sequencing to obtain three independent clonal populations of cells that contained biallelic modifications. One CMAH knockout clone was chosen and used for somatic cell nuclear transfer. Cloned embryos developed to the blastocyst stage. In conclusion, we demonstrated that immortalized porcine fibroblasts were successfully established using the human hTERT gene, and the TALENs enabled biallelic gene disruptions in these immortalized cells.

Trait stacking via targeted genome editing

Modern agriculture demands crops carrying multiple traits. The current paradigm of randomly integrating and sorting independently segregating transgenes creates severe downstream breeding challenges. A versatile, generally applicable solution is hereby provided: the combination of high-efficiency targeted genome editing driven by engineered zinc finger nucleases (ZFNs) with modular 'trait landing pads' (TLPs) that allow 'mix-and-match', on-demand transgene integration and trait stacking in crop plants. We illustrate the utility of nuclease-driven TLP technology by applying it to the stacking of herbicide resistance traits. We first integrated into the maize genome an herbicide resistance gene, pat, flanked with a TLP (ZFN target sites and sequences homologous to incoming DNA) using WHISKERS™-mediated transformation of embryogenic suspension cultures. We established a method for targeted transgene integration based on microparticle bombardment of immature embryos and used it to deliver a second trait precisely into the TLP via cotransformation with a donor DNA containing a second herbicide resistance gene, aad1, flanked by sequences homologous to the integrated TLP along with a corresponding ZFN expression construct. Remarkably, up to 5% of the embryo-derived transgenic events integrated the aad1 transgene precisely at the TLP, that is, directly adjacent to the pat transgene. Importantly and consistent with the juxtaposition achieved via nuclease-driven TLP technology, both herbicide resistance traits cosegregated in subsequent generations, thereby demonstrating linkage of the two independently transformed transgenes. Because ZFN-mediated targeted transgene integration is becoming applicable across an increasing number of crop species, this work exemplifies a simple, facile and rapid approach to trait stacking.

Baculoviral transduction facilitates TALEN-mediated targeted transgene integration and Cre/LoxP cassette exchange in human-induced pluripotent stem cells

Safety and reliability of transgene integration in human genome continue to pose challenges for stem cell-based gene therapy. Here, we report a baculovirus-transcription activator-like effector nuclease system for AAVS1 locus-directed homologous recombination in human induced pluripotent stem cells (iPSCs). This viral system, when optimized in human U87 cells, provided a targeted integration efficiency of 95.21% in incorporating a Neo-eGFP cassette and was able to mediate integration of DNA insert up to 13.5 kb. In iPSCs, targeted integration with persistent transgene expression was achieved without compromising genomic stability. The modified iPSCs continued to express stem cell pluripotency markers and maintained the ability to differentiate into three germ lineages in derived embryoid bodies. Using a baculovirus-Cre/LoxP system in the iPSCs, the Neo-eGFP cassette at the AAVS1 locus could be replaced by a Hygro-mCherry cassette, demonstrating the feasibility of cassette exchange. Moreover, as assessed by measuring γ-H2AX expression levels, genome toxicity associated with chromosomal double-strand breaks was not detectable after transduction with moderate doses of baculoviral vectors expressing transcription activator-like effector nucleases. Given high targeted integration efficiency, flexibility in transgene exchange and low genome toxicity, our baculoviral transduction-based approach offers great potential and attractive option for precise genetic manipulation in human pluripotent stem cells.

A Modular Toolset for Recombination Transgenesis and Neurogenetic Analysis of Drosophila

Transgenic Drosophila have contributed extensively to our understanding of nervous system development, physiology and behavior in addition to being valuable models of human neurological disease. Here, we have generated a novel series of modular transgenic vectors designed to optimize and accelerate the production and analysis of transgenes in Drosophila. We constructed a novel vector backbone, pBID, that allows both phiC31 targeted transgene integration and incorporates insulator sequences to ensure specific and uniform transgene expression. Upon this framework, we have built a series of constructs that are either backwards compatible with existing restriction enzyme based vectors or utilize Gateway recombination technology for high-throughput cloning. These vectors allow for endogenous promoter or Gal4 targeted expression of transgenic proteins with or without fluorescent protein or epitope tags. In addition, we have generated constructs that facilitate transgenic splice isoform specific RNA inhibition of gene expression. We demonstrate the utility of these constructs to analyze proteins involved in nervous system development, physiology and neurodegenerative disease. We expect that these reagents will facilitate the proficiency and sophistication of Drosophila genetic analysis in both the nervous system and other tissues.

Chromatin structure of two genomic sites for targeted transgene integration in induced pluripotent stem cells and hematopoietic stem cells

Achieving transgene integration into preselected genomic sites is currently one of the central tasks in stem cell gene therapy. A strategy to mediate such targeted integration involves site specific endonucleases. Two genomic sites within the MBS85 and CCR5 genes [AAVS1 and CCR5 zinc finger nuclease (CCR5-ZFN) site, respectively] have recently been suggested as potential target regions for integration as their disruption has no functional consequence. We hypothesized that efficient transgene integration maybe affected by DNA accessibility of endonucleases and therefore studied the transcriptional and chromatin status of the AAVS1 and CCR5 sites in eight human induced pluripotent stem (iPS) cell lines and pooled CD34+ hematopoietic stem cells. Matrixchromatin immunoprecipitation (ChIP) assays demonstrated that the CCR5 site and surrounding regions possessed a predominantly closed chromatin configuration consistent with its transcriptionally inactivity in these cell types. In contrast, the AAVS1 site was located within a transcriptionally active region and exhibited an open chromatin configuration in both iPS cells and hematopoietic stem cells. To show that the AAVS1 site is readily amendable to genome modification, we expressed Rep78, an AAV2-derived protein with AAVS1-specific endonuclease activity, in iPS cells after adenoviral gene transfer. We showed that Rep78 efficiently associated with the AAVS1 site and triggered genome modifications within this site. On the other hand, binding to and modification of the CCR5-ZFN site by a zinc-finger nuclease was relatively inefficient. Our data suggest a critical influence of chromatin structure on efficacy of site-specific endonucleases used for genome editing.

Adeno-associated virus Rep-mediated targeting of integrase-defective retroviral vector DNA circles into human chromosome 19

Retroviral vectors have been employed in clinical trials for gene therapy owing to their relative large packaging capacity, alterable cell tropism, and chromosomal integration for stable transgene expression. However, uncontrollable integrations of transgenes are likely to cause safety issues, such as insertional mutagenesis. A targeted transgene integration system for retroviral vectors, therefore, is a straightforward way to address the insertional mutagenesis issue. Adeno-associated virus (AAV) is the only known virus capable of targeted integration in human cells. In the presence of AAV Rep proteins, plasmids possessing the p5 integration efficiency element (p5IEE) can be integrated into the AAV integration site (AAVS1) in the human genome. In this report, we describe a system that can target the circular DNA derived from non-integrating retroviral vectors to the AAVS1 site by utilizing the Rep/p5IEE integration mechanism. Our results showed that after G418 selection 30% of collected clones had retroviral DNA targeted at the AAVS1 site.

578. Towards Targeted Transgene Integration in Induced Pluripotent Stem Cells

578. Towards Targeted Transgene Integration in Induced Pluripotent Stem Cells

689. Helper-Dependent Adenovirus with Doxycyclin-Inducible Expression of Zinc-Finger Nucleases for Targeted Transgene Integration in Hematopoietic Stem Cells

689. Helper-Dependent Adenovirus with Doxycyclin-Inducible Expression of Zinc-Finger Nucleases for Targeted Transgene Integration in Hematopoietic Stem Cells

Crafting rat genomes with zinc fingers

Expressing zinc-finger nucleases in zygotes enables targeted transgene integration in the mouse and rat genomes.

10. Targeted Transgene Integration in Epidermal Stem Cells by Zinc Finger Nucleases and Integrase-Defective Lentiviral Vectors

10. Targeted Transgene Integration in Epidermal Stem Cells by Zinc Finger Nucleases and Integrase-Defective Lentiviral Vectors

415. Towards Targeted Transgene Integration in Human Hematopoietic and Induced Pluripotent Stem Cells

415. Towards Targeted Transgene Integration in Human Hematopoietic and Induced Pluripotent Stem Cells

93. Chromatin Structure of Two Genomic Sites for Targeted Transgene Integration in Induced Pluripotent Stem Cells

93. Chromatin Structure of Two Genomic Sites for Targeted Transgene Integration in Induced Pluripotent Stem Cells

TRANSLATIONAL PERSPECTIVES — TRANSIENT EPIGENETIC GENE THERAPY: HAZARD-FREE CELL REPROGRAMMING APPROACH AND RISING ARM OF A UNIVERSAL STEM CELL GENE THERAPY PLATFORM

Random integration of retroviral transgenes is a major oncogenic hazard that hampers both current clinical gene therapy trials and the genesis of clinical-grade induced pluripotent stem (iPS) cells. Noticeably, we have devised a universal stem cell gene therapy platform aimed at eliminating random integration of therapeutic DNA, at reversing inherited diseases including mitochondrial (mt)DNA ones by reestablishing wild-type genomic homeostasis, and at tackling both inherited and acquired/aging disorders through long-term or transient gene therapy protocols culminating in synergistic combinations. Our platform has a transient epigenetic arm directed at long-term transcriptional inactivation/activation of endogenous genes through the transient action of promoter-specific siRNAs or sense/antisense oligonucleotides, thereby opening an attractive avenue for hazard-free reprogramming of therapeutic iPS cells. Therefore, in addition to its obvious original applications such as long-term transcriptional silencing of autosomal-dominant mutant alleles, the transient epigenetic gene therapy arm could initiate the clinical iPS cell era whereby epigenetic reprogramming of patient somatic cells will not only drive regenerative medicine sensu stricto but also the other arms of our universal stem cell gene therapy platform. Both product and drive of our platform, such hazard-free iPS cells and their derivatives should accommodate all kinds of protocols including 1) long-term gene therapy mediated by endonuclease-boosted gene targeting (gene repair/alteration or targeted transgene integration) or cybridization (mtDNA transfer), and 2) transient regenerative gene therapy aimed at synergizing both regenerative cell therapy sensu stricto and long-term/epigenetic gene therapy through the magnification of the homing/regenerative/differentiative potential of therapeutic stem cells. Our approach is thus discussed 1) in light of emerging concurrent transgene-free reprogramming protocols that rely on protein transduction, small molecules or unintegrating vectors and 2) in terms of somatic cell genetic-like breakthrough in which somatic cells can be safely reprogrammed ex vivo into patient-specific pluripotent stem cells, amplified, engineered through standard/personalized protocols, subjected to potential clonal selections and lineage commitments, and then returned to the patient, thereby driving effective therapeutic repopulative/regenerative dynamics into relevant niches (long-term engraftment) and tissues (basic and regenerative cell turn-over).

Elimination of marker genes and targeted integration via FLP/FRT recombination system from yeast in hybrid aspen (Populus tremula L. × P. tremuloides Michx.)

Marker gene elimination was investigated in hybrid aspen (Populus tremula L. × Populus tremuloides Michx.) using the FLP/FRT recombination system. The construct contained the FLP recombinase under control of a heat inducible promoter, the antibiotic resistance gene nptII driven by the CaMV 35S promoter, and a promoterless uidA gene. The construct was integrated into poplar via Agrobacterium-mediated transformation. The active FLP recombinase excised the nptII marker gene and combined the promoterless uidA gene with the CaMV 35S promoter to form an active uidA gene. For targeted transgene integration, two constructs were used. The first one carried FLP under control of the heat-inducible Gmhsp17.5-E promoter from soybean as well as an active nptII gene flanked by two FRT sites; the second contained the promoterless bar selection marker gene also flanked by two FRT sites. Following transformation and induction of FLP, the enzyme mediated a site-specific recombination at the FRT sites of both constructs. This recombination leads to an excision of the FLP and nptII gene from the first as well as an excision of the promoterless bar gene from the second construct. The promoterless bar gene reintegrated exactly at the former position of the FLP and nptII genes in the first construct to form an active bar gene. The FLP/FRT recombination system from yeast forms a promising basis for the production of antibiotic-free transgenic plants and a useful tool for directed integration of transgenes into plant genomes.

Targeted transgene integration in plant cells using designed zinc finger nucleases

Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted, homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait stacking in plants.

A sequence-anchored genetic linkage map for the moss, Physcomitrella patens.

The moss Physcomitrella patens is a model for the study of plant cell biology and, by virtue of its basal position in land plant phylogeny, for comparative analysis of the evolution of plant gene function and development. It is ideally suited for ‘reverse genetic’ analysis by virtue of its outstanding ability to undertake targeted transgene integration by homologous recombination. However, gene identification through mutagenesis and map-based cloning has hitherto not been possible, due to the lack of a genetic linkage map. Using molecular markers [amplified fragment length polymorphisms (AFLP) and simple sequence repeats (SSR)] we have generated genetic linkage maps for Physcomitrella. One hundred and seventy-nine gene-specific SSR markers were mapped in 46 linkage groups, and 1574 polymorphic AFLP markers were identified. Integrating the SSR- and AFLP-based maps generated 31 linkage groups comprising 1420 markers. Anchorage of the integrated linkage map with gene-specific SSR markers coupled with computational prediction of AFLP loci has enabled its correspondence with the newly sequenced Physcomitrella genome. The generation of a linkage map densely populated with molecular markers and anchored to the genome sequence now provides a resource for forward genetic interrogation of the organism and for the development of a pipeline for the map-based cloning of Physcomitrella genes. This will radically enhance the potential of Physcomitrella for determining how gene function has evolved for the acquisition of complex developmental strategies within the plant kingdom.