Allogeneic CD19-specific chimeric antigen receptor (CAR) T cells with inactivated donor T cell receptor (TCR) expression can be used as an ‘off-the-shelf’ therapeutic modality for lymphoid ...malignancies, thus offering an attractive alternative to autologous, patient-derived T cells. Current approaches for T cell engineering mainly rely on the use of viral vectors. Here, we optimized and validated a non-viral genetic modification platform based on Sleeping Beauty (SB) transposons delivered with minicircles to express CD19-28z.CAR and CRISPR/Cas9 ribonucleoparticles to inactivate allogeneic TCRs. Efficient TCR gene disruption was achieved with minimal cytotoxicity and with attainment of robust and stable CD19-28z.CAR expression. The CAR T cells were responsive to CD19+ tumor cells with antitumor activities that induced complete tumor remission in NALM6 tumor-bearing mice while significantly reducing TCR alloreactivity and GvHD development. Single CAR signaling induced the similar T cell signaling signatures in TCR-disrupted CAR T cells and control CAR T cells. In contrast, TCR disruption inhibited T cell signaling/protein phosphorylation compared to the control CAR T cells during dual CAR/TCR signaling. This non-viral SB transposon-CRISPR/Cas9 combination strategy serves as an alternative for generating next-generation CD19-specific CAR T while reducing GvHD risk and easing potential manufacturing constraints intrinsic to viral vectors.
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Non-viral SB transposon-CRISPR/Cas9 T cell engineering results in efficient CD19-specific CAR/TCR knockout T cell production. These universal CD19-CAR/TCR-negative T cells exhibited potent antitumor effects in vitro and in vivo while reducing TCR alloreactivity and GvHD development. CD19-CAR/TCR-negative T cells exhibit reduced TCR/CD3 signaling upon dual CAR/TCR co-activation.
The relationship between disruption and delayed recognition is a critical research topic, yet the connection between the degree of disruption and delayed acknowledgment remains unclear. This study ...investigates the extent of recognition delay for disruptive papers using the SciSciNet dataset. We conducted a quantitative analysis based on this extensive dataset to examine the relationship between the Disruption Index and the Sleeping Beauty Index, revealing that highly disruptive papers often face a latency period before gaining acknowledgment, with significant variations across disciplines and over time. Our analysis of team dynamics indicates that larger teams, the presence of high-impact authors, fixed teams, and hierarchically structured teams can significantly reduce this delay. These findings provide insights into optimizing team strategies and understanding the complexities of academic recognition. They offer valuable implications for researchers and policymakers aiming to foster and accelerate the acknowledgment of groundbreaking scientific contributions.
•Disruptive papers have a higher probability to have delayed recognition.•We highlight how recognition delays for disruptive research vary significantly across academic disciplines and over time.•Larger teams and pre-established collaborative relationships can reduce delays in recognizing disruptive research.•Highly reputed scientists in research teams accelerate the acknowledgment of groundbreaking findings.
Significance Scientific papers typically have a finite lifetime: their rate to attract citations achieves its maximum a few years after publication, and then steadily declines. Previous studies ...pointed out the existence of a few blatant exceptions: papers whose relevance has not been recognized for decades, but then suddenly become highly influential and cited. The Einstein, Podolsky, and Rosen âparadoxâ paper is an exemplar Sleeping Beauty. We study how common Sleeping Beauties are in science. We introduce a quantity that captures both the recognition intensity and the duration of the âsleepingâ period, and show that Sleeping Beauties are far from exceptional. The distribution of such quantity is continuous and has power-law behavior, suggesting a common mechanism behind delayed but intense recognition at all scales.
A Sleeping Beauty (SB) in science refers to a paper whose importance is not recognized for several years after publication. Its citation history exhibits a long hibernation period followed by a sudden spike of popularity. Previous studies suggest a relative scarcity of SBs. The reliability of this conclusion is, however, heavily dependent on identification methods based on arbitrary threshold parameters for sleeping time and number of citations, applied to small or monodisciplinary bibliographic datasets. Here we present a systematic, large-scale, and multidisciplinary analysis of the SB phenomenon in science. We introduce a parameter-free measure that quantifies the extent to which a specific paper can be considered an SB. We apply our method to 22 million scientific papers published in all disciplines of natural and social sciences over a time span longer than a century. Our results reveal that the SB phenomenon is not exceptional. There is a continuous spectrum of delayed recognition where both the hibernation period and the awakening intensity are taken into account. Although many cases of SBs can be identified by looking at monodisciplinary bibliographic data, the SB phenomenon becomes much more apparent with the analysis of multidisciplinary datasets, where we can observe many examples of papers achieving delayed yet exceptional importance in disciplines different from those where they were originally published. Our analysis emphasizes a complex feature of citation dynamics that so far has received little attention, and also provides empirical evidence against the use of short-term citation metrics in the quantification of scientific impact.
Natural killer (NK) cells have high intrinsic cytotoxic capacity, and clinical trials have demonstrated their safety and efficacy for adoptive cancer therapy. Expression of chimeric antigen receptors ...(CARs) enhances NK cell target specificity, with these cells applicable as off-the-shelf products generated from allogeneic donors. Here, we present for the first time an innovative approach for CAR NK cell engineering employing a non-viral Sleeping Beauty (SB) transposon/transposase-based system and minimized DNA vectors termed minicircles. SB-modified peripheral blood-derived primary NK cells displayed high and stable CAR expression and more frequent vector integration into genomic safe harbors than lentiviral vectors. Importantly, SB-generated CAR NK cells demonstrated enhanced cytotoxicity compared with non-transfected NK cells. A strong antileukemic potential was confirmed using established acute lymphocytic leukemia cells and patient-derived primary acute B cell leukemia and lymphoma samples as targets in vitro and in vivo in a xenograft leukemia mouse model. Our data suggest that the SB-transposon system is an efficient, safe, and cost-effective approach to non-viral engineering of highly functional CAR NK cells, which may be suitable for cancer immunotherapy of leukemia as well as many other malignancies.
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Ullrich and colleagues present an innovative non-viral SB transposon/transposase-based system for CAR-NK cell engineering. The SB-transposon system is an efficient, safe, and cost-effective approach to virus-free engineering of highly functional CAR-NK cells, which can be suitable for cancer immunotherapy of leukemia. as well as many other malignancies.
The paper discusses the similarities between Primo Levi's documentary novel Se questo è un uomo (1947) and his short science fiction story La bella addormentata nel frigo from the collection Storie ...naturali (1966), on the basis of the themes of the novel. The concentration camps form an element of continuity between the novel, which bears witness to the horrors Levi experienced, and the seemingly light-hearted story. There are many parallels between human perversion documented in the novel and the grotesque experiment of the ‘Laboratory of evil’ seen in the fictional work. The story is almost a parody of Auschwitz; dreadful seriousness is converted to comedy. Patricia, the protagonist of the short story, is Levi's alter ego and a caricature of the Sleeping Beauty archetype from the traditional fairy tale.
While a large majority of scientific publications get most of their citations within the initial few years after publication, there is an interesting number of papers—termed as
sleeping beauties
...—which do not get much cited for several years after being published, but then suddenly start getting cited heavily. In this work, we focus on sleeping beauties (SBs) in the domain of Computer Science. We identify more than 5,000 sleeping beauties in Computer Science, and characterise them based on their sub-field and their citation profile after awakening. We also reveal some interesting factors which led to their awakening long after publication. Furthermore, we also propose a methodology for early identification of sleeping beauties, and develop a machine learning-based classification approach that attempts to classify publications based on whether they are likely to be SBs. The classifier achieves a precision of 0.73 and a recall of 0.45 in identifying SBs immediately after their year of publications, and the performance significantly improves with time. To our knowledge, this is the first study on sleeping beauties in Computer Science.
Stable gene expression in mammalian cells is a prerequisite for many in vitro and in vivo experiments. However, either the integration of plasmids into mammalian genomes or the use of ...retro‐/lentiviral systems have intrinsic limitations. The use of transposable elements, e.g. the Sleeping Beauty system (SB), circumvents most of these drawbacks (integration sites, size limitations) and allows the quick generation of stable cell lines. The integration process of SB is catalyzed by a transposase and the handling of this gene transfer system is easy, fast and safe. Here, we report our improvements made to the existing SB vector system and present two new vector types for robust constitutive or inducible expression of any gene of interest. Both types are available in 16 variants with different selection marker (puromycin, hygromycin, blasticidin, neomycin) and fluorescent protein expression (GFP, RFP, BFP) to fit most experimental requirements. With this system it is possible to generate cell lines from stable transfected cells quickly and reliably in a medium‐throughput setting (three to five days). Cell lines robustly express any gene‐of‐interest, either constitutively or tightly regulated by doxycycline. This allows many laboratory experiments to speed up generation of data in a rapid and robust manner.
The integration of plasmids into mammalian genomes and the use of retro‐/lentiviral systems have intrinsic limitations, and the use of transposable elements, e.g. the Sleeping Beauty system (SB), circumvents most of these drawbacks and allows the quick generation of stable cell lines. In this manuscript, the SB vector was used to design two new vector families with optimized expression of fluorescent and selective markers, inducibility of transgenes and long‐term gene expression. These vectors should allow the robust expression of any gene of interest, either constitutively or tightly regulated by doxycycline.
The Sleeping Beauty transposon system is a nonviral DNA transfer tool capable of efficiently mediating transposition‐based, stable integration of DNA sequences of choice into eukaryotic genomes. ...Continuous refinements of the system, including the emergence of hyperactive transposase mutants and novel approaches in vectorology, greatly improve upon transposition efficiency rivaling viral‐vector‐based methods for stable gene insertion. Current developments, such as reversible transgenesis and proof‐of‐concept RNA‐guided transposition, further expand on possible applications in the future. In addition, innate advantages such as lack of preferential integration into genes reduce insertional mutagenesis‐related safety concerns while comparably low manufacturing costs enable widespread implementation. Accordingly, the system is recognized as a powerful and versatile tool for genetic engineering and is playing a central role in an ever‐expanding number of gene and cell therapy clinical trials with the potential to become a key technology to meet the growing demand for advanced therapy medicinal products.
The Sleeping Beauty transposon system represents a nonviral DNA transfer tool capable of efficiently mediating stable integration of DNA sequences into the genome of eukaryotic cells. This article reviews its latest developments highlighting clinical applications and discusses potential future directions based on novel additions to the system's versatility.
Standard principles of chance deference face two kinds of problems. In the first place, they face difficulties with a priori knowable contingencies. In the second place, they face difficulties in ...cases where you've lost track of the time. I provide a principle of chance deference which handles those problem cases. This principle has a surprising consequence for Adam Elga's Sleeping Beauty puzzle.
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