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Chimeric piggyBac transposases for genomic targeting in human cells.

ABSTRACT: Integrating vectors such as viruses and transposons insert transgenes semi-randomly and can potentially disrupt or deregulate genes. For these techniques to be of therapeutic value, a method for controlling the precise location of insertion is required. The piggyBac (PB) transposase is an efficient gene transfer vector active in a variety of cell types and proven to be amenable to modification. Here we present the design and validation of chimeric PB proteins fused to the Gal4 DNA binding domain with the ability to target transgenes to pre-determined sites. Upstream activating sequence (UAS) Gal4 recognition sites harbored on recipient plasmids were preferentially targeted by the chimeric Gal4-PB transposase in human cells. To analyze the ability of these PB fusion proteins to target chromosomal locations, UAS sites were randomly integrated throughout the genome using the Sleeping Beauty transposon. Both N- and C-terminal Gal4-PB fusion proteins but not native PB were capable of targeting transposition nearby these introduced sites. A genome-wide integration analysis revealed the ability of our fusion constructs to bias 24% of integrations near endogenous Gal4 recognition sequences. This work provides a powerful approach to enhance the properties of the PB system for applications such as genetic engineering and gene therapy.

SUBMITTER: Owens JB

PROVIDER: S-EPMC3413120 | biostudies-literature | 2012 Aug

REPOSITORIES: biostudies-literature

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Chimeric piggyBac transposases for genomic targeting in human cells.

Owens Jesse B JB Urschitz Johann J Stoytchev Ilko I Dang Nong C NC Stoytcheva Zoia Z Belcaid Mahdi M Maragathavally Kommineni J KJ Coates Craig J CJ Segal David J DJ Moisyadi Stefan S

Nucleic acids research 20120409 14

Integrating vectors such as viruses and transposons insert transgenes semi-randomly and can potentially disrupt or deregulate genes. For these techniques to be of therapeutic value, a method for controlling the precise location of insertion is required. The piggyBac (PB) transposase is an efficient gene transfer vector active in a variety of cell types and proven to be amenable to modification. Here we present the design and validation of chimeric PB proteins fused to the Gal4 DNA binding domain ...[more]

PMID: 22492708

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Project description:BACKGROUND:The anti-tumor immunity of natural killer (NK) cells can be paralyzed by the CD73-induced generation of immunosuppressive adenosine from precursor ATP within the hypoxic microenvironment of solid tumors. In an effort to redirect purinergic immunosuppression of NK cell anti-tumor function, we showed, for the first time, that immunometabolic combination treatment with NKG2D-engineered CAR-NK cells alongside blockade of CD73 ectonucleotidase activity can result in significant anti-tumor responses in vivo. METHODS:NK cells were engineered non-virally with NKG2D.CAR-presenting vectors based on the piggyBac transposon system with DAP10 and CD3? co-signaling domains. The anti-tumor immunity of NKG2D.CAR.NK cells in combination with CD73 targeting was evaluated against multiple solid tumor targets in vitro and humanized mouse xenografts in immunodeficient tumor-bearing mice in vivo. Intratumoral migration was evaluated via immunohistochemical staining, while degranulation capacity and IFN-? production of NK cells were measured in response to solid tumor targets. RESULTS:Our results showed that CD73 blockade can mediate effective purinergic reprogramming and enhance anti-tumor cytotoxicity both in vitro and in vivo by enhancing the killing ability of CAR-engineered NK cells against CD73+ solid tumor targets via mechanisms that might imply alleviation from adenosinergic immunometabolic suppression. CD73 blockade improved the intratumoral homing of CD56+ CAR-NK cells in vivo. These engineered NK cells showed synergistic therapeutic efficacy in combination with CD73 targeting against CD73+ human lung cancer xenograft models. Interestingly, CD73 blockade could inhibit tumor growth in vivo independently of adaptive immune cells, innate immunity or NK cell-mediated ADCC. CONCLUSIONS:Immunotherapies targeting the adenosinergic signaling cascade, which act by neutralizing CD73 ectoenzymatic activity, had thus far not been evaluated in humanized tumor models, nor had the implication of innate immunity been investigated. Taken together, our pre-clinical efficacy data demonstrate, for the first time, the potential of targeting CD73 to modulate purinergic signaling and enhance adoptive NK cell immunotherapy via mechanisms that could implicate autocrine tumor control as well as by mediating adenosinergic signaling.

Project description:BackgroundTransposons are mobile genetic elements that colonize genomes and drive their plasticity in all organisms. DNA transposon-encoded transposases bind to the ends of their cognate transposons and catalyze their movement. In some cases, exaptation of transposon genes has allowed novel cellular functions to emerge. The PiggyMac (Pgm) endonuclease of the ciliate Paramecium tetraurelia is a domesticated transposase from the PiggyBac family. It carries a core catalytic domain typical of PiggyBac-related transposases and a short cysteine-rich domain (CRD), flanked by N- and C-terminal extensions. During sexual processes Pgm catalyzes programmed genome rearrangements (PGR) that eliminate ~ 30% of germline DNA from the somatic genome at each generation. How Pgm recognizes its DNA cleavage sites in chromatin is unclear and the structure-function relationships of its different domains have remained elusive.ResultsWe provide insight into Pgm structure by determining the fold adopted by its CRD, an essential domain required for PGR. Using Nuclear Magnetic Resonance, we show that the Pgm CRD binds two Zn2+ ions and forms an unusual binuclear cross-brace zinc finger, with a circularly permutated treble-clef fold flanked by two flexible arms. The Pgm CRD structure clearly differs from that of several other PiggyBac-related transposases, among which is the well-studied PB transposase from Trichoplusia ni. Instead, the arrangement of cysteines and histidines in the primary sequence of the Pgm CRD resembles that of active transposases from piggyBac-like elements found in other species and of human PiggyBac-derived domesticated transposases. We show that, unlike the PB CRD, the Pgm CRD does not bind DNA. Instead, it interacts weakly with the N-terminus of histone H3, whatever its lysine methylation state.ConclusionsThe present study points to the structural diversity of the CRD among transposases from the PiggyBac family and their domesticated derivatives, and highlights the diverse interactions this domain may establish with chromatin, from sequence-specific DNA binding to contacts with histone tails. Our data suggest that the Pgm CRD fold, whose unusual arrangement of cysteines and histidines is found in all PiggyBac-related domesticated transposases from Paramecium and Tetrahymena, was already present in the ancestral active transposase that gave rise to ciliate domesticated proteins.

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Chimeric piggyBac transposases for genomic targeting in human cells.

Publications

Chimeric piggyBac transposases for genomic targeting in human cells.

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