Engineered <i>Sleeping Beauty</i> transposase redirects transposon integration away from genes

Miskey, Csaba; Kesselring, Lisa; Querques, Irma; Abrusán, György; Barábas, O.; Ivics, Zoltán

doi:10.1093/nar/gkac092

Cited by 22 publications

(10 citation statements)

References 100 publications

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“…Lentiviruses are commonly used in clinical trials today, and recent advances have improved their safety. However, there are still certain safety risks associated with the pathogenic origin of such viruses, and a recent study has suggested a higher preference of lentiviral insertion into active genes compared to Sleeping Beauty-based gene transfer methods 61, 62 .…”

Section: Discussionmentioning

confidence: 99%

Therapeutic immune cell engineering with an mRNA : AAV-Sleeping Beautycomposite system

Ye¹,

Lam²,

Yang³

et al. 2023

Preprint

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Adoptive cell therapy has shown clinical success in patients with hematological malignancies. Immune cell engineering is critical for production, research, and development of cell therapy; however, current approaches for generation of therapeutic immune cells face various limitations. Here, we establish a composite gene delivery system for the highly efficient engineering of therapeutic immune cells. This system, termed MAJESTIC (mRNA AAV-Sleeping-Beauty Joint Engineering of Stable Therapeutic Immune Cells), combines the merits of mRNA, AAV vector, and transposon into one composite system. In MAJESTIC, the transient mRNA component encodes a transposase that mediates permanent genomic integration of the Sleeping Beauty (SB) transposon, which carries the gene-of-interest and is embedded within the AAV vector. This system can transduce diverse immune cell types with low cellular toxicity and achieve highly efficient and stable therapeutic cargo delivery. Compared with conventional gene delivery systems, such as lentiviral vector, DNA transposon plasmid, or minicircle electroporation, MAJESTIC shows higher cell viability, chimeric antigen receptor (CAR) transgene expression, therapeutic cell yield, as well as prolonged transgene expression. CAR-T cells generated by MAJESTIC are functional and have strong anti-tumor activity in vivo. This system also demonstrates versatility for engineering different cell therapy constructs such as canonical CAR, bi-specific CAR, kill switch CAR, and synthetic TCR; and for CAR delivery into various immune cells, including T cells, natural killer cells, myeloid cells, and induced pluripotent stem cells.

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Section: Discussionmentioning

confidence: 99%

Therapeutic immune cell engineering with an mRNA : AAV-Sleeping Beautycomposite system

Ye¹,

Lam²,

Yang³

et al. 2023

Preprint

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“…The conditions and thresholds of the raw read processing and mapping parameters have been specified previously ( 41 ). In short, the raw reads were subjected to quality trimming, and the resulting reads were mapped to the hg38 human genome assembly using bowtie2 ( 42 ).…”

Section: Methodsmentioning

confidence: 99%

“…The HepG2-specific chromatin states based on imputed histone modification (ChIP-Seq) datasets were downloaded from the Roadmap Epigenomics Project ( https://egg2.wustl.edu/roadmap/web_portal/imputed.html#chr_imp ). The insertion site frequencies of PS and SB ( 41 ) within these regions were compared with a set of 100 000 computationally generated random loci in the human genome. The coordinates of PS and SB insertions in the human genome are listed in Supplementary Files S2 and S3 .…”

Section: Methodsmentioning

confidence: 99%

Passer, a highly active transposon from a fish genome, as a potential new robust genetic manipulation tool

Wang

Gao

Miskey

et al. 2023

Nucleic Acids Research

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The discovery of new, active DNA transposons can expand the range of genetic tools and provide more options for genomic manipulation. In this study, a bioinformatics analysis suggested that Passer (PS) transposons, which are members of the pogo superfamily, show signs of recent and current activity in animals and may be active in some species. Cell-based transposition assays revealed that the native PS transposases from Gasterosteus aculeatus and Danio rerio displayed very high activity in human cells relative to the Sleeping Beauty transposon. A typical overproduction inhibition phenomenon was observed for PS, and transposition capacity was decreased by ∼12% with each kilobase increase in the insertion size. Furthermore, PS exhibited a pronounced integration preference for genes and their transcriptional regulatory regions. We further show that two domesticated human proteins derived from PS transposases have lost their transposition activity. Overall, PS may represent an alternative with a potentially efficient genetic manipulation tool for transgenesis and mutagenesis applications.

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“…We used ChIPseeker to annotate the positions of the spliced sequences in the human genome and used clusterProfiler 26 for gene enrichment analysis in the pathways of the Kyoto Encyclopedia of Genes and Genomes 21 . Targeted DNA-sequencing of the insertion sites, their quantification and annotation has been described 23,27 . In addition to the tools described above we used ChIPseeker 28 to annotate the insertion sites in gene related segments of the human genome.…”

Section: Methodsmentioning

confidence: 99%

HTLV-1 Splice Sites in Prevalent Gene Vectors Cause Splicing Perturbations in Transgenic Human Cells

Miskey

Prommersberger

Mestermann

et al. 2023

Preprint

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The use of any semi-randomly integrating gene vector in a therapeutic setting is associated with genotoxic risks. The two major mechanisms of genotoxicity are disruption of a coding sequence (loss-of-function) or transcriptional upregulation of genes (gain-of-function) in the cellular genome where the genetic modifications are executed. A third, less widely recognized genotoxic risk stems from splice sites and polyadenylation sites within the vector sequences. These transcriptional elements may drive aberrant splicing and/or polyadenylation between transgene-contained and genomic sequences. A widely used promoter/enhance element present in gene vectors to ensure high transgene expression levels in mammalian cells is composed of a hybrid EF1a/HTLV-1 LTR, in which the retroviral LTR contains an intron. We assessed aberrant splicing initiated from the splice donor (SD) element present in the HTLV-1 LTR in CAR-T cells that had been engineered by either lentiviral vector (LV) or Sleeping Beauty (SB) transposon-mediated gene transfer. We establish that the vector-contained canonical SD site gives rise to aberrantly spliced RNA species and thereby can cause misexpression of host gene segments that are involved in various host cell functions. This, potentially genotoxic, effect could be abrogated by mutating or completely eliminating the SD (or the entire intron) from the HTLV-1 LTR segment. CAR-T cells generated by the modified vectors are equally potent in efficiency of CAR-T cell manufacturing and in functionality. The simple genetic modifications that we describe here affecting vector design therefore enhance genomic safety while maintaining efficacy of gene-modified therapeutic cells.

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Engineered Sleeping Beauty transposase redirects transposon integration away from genes

Cited by 22 publications

References 100 publications

Therapeutic immune cell engineering with an mRNA : AAV-Sleeping Beautycomposite system

Therapeutic immune cell engineering with an mRNA : AAV-Sleeping Beautycomposite system

Passer, a highly active transposon from a fish genome, as a potential new robust genetic manipulation tool

HTLV-1 Splice Sites in Prevalent Gene Vectors Cause Splicing Perturbations in Transgenic Human Cells

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