Safe harbours for the integration of new DNA in the human genome

Sadelain, Michel; Papapetrou, Eirini P.; Bushman, Frederic D.

doi:10.1038/nrc3179

Cited by 429 publications

(406 citation statements)

References 81 publications

Supporting

Mentioning

400

Contrasting

Unclassified

Order By: Relevance

“…When comparing the insertion profiles to a control set of 10,000 computationally generated random human genomic loci, no apparent bias was detected for any of the categories analyzed (Figure 7B). Furthermore, SB transposon integrations occur in genomic safe harbors, defined by five criteria32, 33 (Figure 7C), at about 25% (Figure 7D). These results are in good agreement with numerous previous studies, which established a close-to-random insertion profile for the SB system in human cells 34, 35, 36, 37…”

Section: Resultsmentioning

confidence: 99%

Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids

Thumann

Harmening

Prat-Souteyrand

et al. 2017

Molecular Therapy - Nucleic Acids

View full text Add to dashboard Cite

Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal blood vessels growing into the subretinal space, leading to retinal pigment epithelial (RPE) cell degeneration and vision loss. Vessel growth results from an imbalance of pro-angiogenic (e.g., vascular endothelial growth factor [VEGF]) and anti-angiogenic factors (e.g., pigment epithelium-derived factor [PEDF]). Current treatment using intravitreal injections of anti-VEGF antibodies improves vision in about 30% of patients but may be accompanied by side effects and non-compliance. To avoid the difficulties posed by frequent intravitreal injections, we have proposed the transplantation of pigment epithelial cells modified to overexpress human PEDF. Stable transgene integration and expression is ensured by the hyperactive Sleeping Beauty transposon system delivered by pFAR4 miniplasmids, which have a backbone free of antibiotic resistance markers. We demonstrated efficient expression of the PEDF gene and an optimized PEDF cDNA sequence in as few as 5 × 103 primary cells. At 3 weeks post-transfection, PEDF secretion was significantly elevated and long-term follow-up indicated a more stable secretion by cells transfected with the optimized PEDF transgene. Analysis of transgene insertion sites in human RPE cells showed an almost random genomic distribution. The results represent an important contribution toward a clinical trial aiming at a non-viral gene therapy of nvAMD.

show abstract

Section: Resultsmentioning

confidence: 99%

Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids

Thumann

Harmening

Prat-Souteyrand

et al. 2017

Molecular Therapy - Nucleic Acids

View full text Add to dashboard Cite

show abstract

“…Therefore, genome editing techniques have been used to insert transgenes into defined genomic sites. Although no genomic site has been fully validated as a genomic "safe harbor", some candidates from the human genome have been proposed, including the adeno-associated virus site 1 (AAVS1) locus, the chemokine (CC motif) receptor 5 (CCR5) locus, and the human orthologue of the mouse ROSA26 locus [1]. Among these candidates, the AAVS1 locus stands out for the ubiquitous, constitutive, and high-level expression of the integrated transgene [2].…”

Section: Introductionmentioning

confidence: 99%

“…Pluripotent stem cells with exogenous DNA inserted into the AAVS1 locus show normal phenotype and differentiation potential [5,6]. However, misgivings have arisen because the function of MBS85 is not yet completely understood [1].…”

Section: Introductionmentioning

confidence: 99%

Transgene integration into the human AAVS1 locus enhances myosin II-dependent contractile force by reducing expression of myosin binding subunit 85

Mizutani

Haga

et al. 2015

Biochemical and Biophysical Research Communications

View full text Add to dashboard Cite

The adeno-associated virus site 1 (AAVS1) locus in the human genome is a strong candidate for gene therapy by insertion of an exogenous gene into the locus. The AAVS1 locus includes the coding region for myosin binding subunit 85 (MBS85). Although the function of MBS85 is not well understood, myosin II-dependent contractile force may be affected by altered expression of MBS85. The effect of altered expression of MBS85 on cellular contractile force should be examined prior to the application of gene therapy. In this study, we show that transgene integration into AAVS1 and consequent reduction of MBS85 expression changes myosin II-dependent cellular contractile force. We established a human fibroblast cell line with exogenous DNA knocked-in to AAVS1 (KI cells) using the CRISPR/Cas9 genome editing system. Western blotting analysis showed that KI cells had significantly reduced MBS85 expression. KI cells also showed greater cellular contractile force than control cells. The increased contractile force was associated with phosphorylation of the myosin II regulatory light chain (MRLC). Transfection of KI cells with an MBS85 expression plasmid restored cellular contractile force and phosphorylation of MRLC to the levels in control cells. These data suggest that transgene integration into the human AAVS1 locus induces an increase in cellular contractile force and thus should be considered as a gene therapy to effect changes in cellular contractile force. 3 Highlights•Transgene integration to human AAVS1 locus decreases MBS85 expression.•Decrease in MBS85 expression increases cellular contractile force.•MBS85 is involved in myosin regulatory light chain phosphorylation.

show abstract

“…The NCI cancer gene index project assigned a number for each gene as the count of sentences from MEDLINE abstracts in which the gene name and a cancer term cooccurred (26). We also included databases with annotations of cancer-related genes, including the Bushman Laboratory cancer driver gene list (27,28), the COSMIC somatic mutation catalog (29), and the CCGD mouse cancer driver genes (30). We found TFs with higher percentage of tumors showing target differential expression are associated with the cancer gene annotations in all databases ( Fig.…”

Section: Significancementioning

confidence: 99%

Inference of transcriptional regulation in cancers

Jiang

Freedman

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Despite the rapid accumulation of tumor-profiling data and transcription factor (TF) ChIP-seq profiles, efforts integrating TF binding with the tumor-profiling data to understand how TFs regulate tumor gene expression are still limited. To systematically search for cancerassociated TFs, we comprehensively integrated 686 ENCODE ChIPseq profiles representing 150 TFs with 7484 TCGA tumor data in 18 cancer types. For efficient and accurate inference on gene regulatory rules across a large number and variety of datasets, we developed an algorithm, RABIT (regression analysis with background integration). In each tumor sample, RABIT tests whether the TF target genes from ChIP-seq show strong differential regulation after controlling for background effect from copy number alteration and DNA methylation. When multiple ChIP-seq profiles are available for a TF, RABIT prioritizes the most relevant ChIP-seq profile in each tumor. In each cancer type, RABIT further tests whether the TF expression and somatic mutation variations are correlated with differential expression patterns of its target genes across tumors. Our predicted TF impact on tumor gene expression is highly consistent with the knowledge from cancer-related gene databases and reveals many previously unidentified aspects of transcriptional regulation in tumor progression. We also applied RABIT on RNAbinding protein motifs and found that some alternative splicing factors could affect tumor-specific gene expression by binding to target gene 3′UTR regions. Thus, RABIT (rabit.dfci.harvard.edu) is a general platform for predicting the oncogenic role of gene expression regulators.regulatory inference | tumor profiling | transcription factor | RNA-binding proteinT umorigenesis is a multistep process requiring alterations in gene expression programs (1, 2). Transcription factors (TFs) are instrumental in driving these gene expression programs, and misregulation of these TFs can result in the acquisition of tumorrelated properties (3). For example, E2F1 is overexpressed in many cancer types and promotes tumor proliferation by regulating expression of genes involved in cell differentiation, metabolism, and development (4). As another example, FOXM1 plays an important role in promoting cell proliferation and cell cycle progression through transcriptional activation of many G2/M-specific genes. Increased FOXM1 gene expression was detected in numerous cancer types, and FOXM1 is a promising therapeutic target for cancer treatment (5). TFs also play critical roles in inducing the tumor microenvironment for metastasis. For example, SNAI1/2, TWIST, and ZEB1/2 orchestrate the expression of genes involved in cell polarity, cell-cell contact, cytoskeleton structure, and extracellular matrix degradation. The joint effect of these TFs promotes cancer cell motility and invasion in the metastatic process (2, 6).With the rapid development of high-throughput technologies, large amounts of datasets have been generated for regulatory proteins. For example, the ENCODE project generated 689 C...

show abstract

Safe harbours for the integration of new DNA in the human genome

Cited by 429 publications

References 81 publications

Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids

Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids

Transgene integration into the human AAVS1 locus enhances myosin II-dependent contractile force by reducing expression of myosin binding subunit 85

Inference of transcriptional regulation in cancers

Contact Info

Product

Resources

About