Sleeping Beauty kit sets provide rapid and accessible generation of artificial antigen-presenting cells for natural killer cell expansion.

Artificial antigen-presenting cells (aAPCs) offer a cost effective and convenient tool for the expansion of chimeric antigen receptor (CAR)-bearing T cells and NK cells. aAPCs are particularly useful because of their ability to efficiently expand low-frequency antigen-reactive lymphocytes in bulk cultures. Commonly derived from the leukemic cell line K562, these aAPCs lack most major histocompatibility complex expression and are therefore useful for NK cell expansion without triggering allogeneic T-cell proliferation. To combat difficulties in accessing existing aAPC lines, while circumventing the iterative lentiviral gene transfers with antibody-mediated sorting required for the isolation of stable aAPC clones, we developed a single-step technique using Sleeping Beauty (SB)-based vectors with antibiotic selection options. Our SB vectors contain options of two to three genes encoding costimulatory molecules, membrane-bound cytokines as well as the presence of antibiotic-resistance genes that allow for stable transposition-based transfection of feeder cells. Transfection of K562 with SB vectors described in this study allows for the surface expression of CD86, 4-1BBL, membrane-bound (mb) interleukin (IL)-15 and mbIL-21 after simultaneous transposition and antibiotic selection using only two antibiotics. aAPCs successfully expanded NK cells to high purity (80-95%). Expanded NK cells could be further engineered by lentiviral CAR transduction. The multivector kit set is publicly available and will allow convenient and reproducible in-house production of effective aAPCs for the in vitro expansion of primary cells.

CRISISS: A Novel, Transcriptionally and Post-Translationally Inducible CRISPR/Cas9-Based Cellular Suicide Switch.

With the ever-increasing developing rate of gene and cellular therapy applications and growing accessibility due to products receiving regulatory approval, the need for effective and reliable safety mechanisms to prevent or eliminate potentially fatal side effects is of the utmost importance. In this study, we present the CRISPR-induced suicide switch (CRISISS) as a tool to eliminate genetically modified cells in an inducible and highly efficient manner by targeting Cas9 to highly repetitive Alu retrotransposons in the human genome, causing irreparable genomic fragmentation by the Cas9 nuclease and resulting cell death. The suicide switch components, including expression cassettes for a transcriptionally and post-translationally inducible Cas9 and an Alu-specific single-guide RNA, were integrated into the genome of target cells via Sleeping-Beauty-mediated transposition. The resulting transgenic cells did not show signs of any impact on overall fitness when uninduced, as unintended background expression, background DNA damage response and background cell killing were not observed. When induced, however, a strong expression of Cas9, a strong DNA damage response and a rapid halt of cell proliferation coupled with near complete cell death within four days post-induction were seen. With this proof-of-concept study, we present a novel and promising approach for a robust suicide switch with potential utility for gene and cell therapy in the future.

Latest Advances for the Sleeping Beauty Transposon System: 23 Years of Insomnia but Prettier than Ever: Refinement and Recent Innovations of the Sleeping Beauty Transposon System Enabling Novel, Nonviral Genetic Engineering Applications.

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.

Engineered Sleeping Beauty Transposon as Efficient System to Optimize Chimp Adenoviral Production.

Sleeping Beauty (SB) is the first DNA transposon employed for efficient transposition in vertebrate cells, opening new applications for genetic engineering and gene therapies. A transposon-based gene delivery system holds the favourable features of non-viral vectors and an attractive safety profile. Here, we employed SB to engineer HEK293 cells for optimizing the production of a chimpanzee Adenovector (chAd) belonging to the Human Mastadenovirus C species. To date, chAd vectors are employed in several clinical settings for infectious diseases, last but not least COVID-19. A robust, efficient and quick viral vector production could advance the clinical application of chAd vectors. To this aim, we firstly swapped the hAd5 E1 with chAd-C E1 gene by using the CRISPR/Cas9 system. We demonstrated that in the absence of human Ad5 E1, chimp Ad-C E1 gene did not support HEK293 survival. To improve chAd-C vector production, we engineered HEK293 cells to stably express the chAd-C precursor terminal protein (ch.pTP), which plays a crucial role in chimpanzee Adenoviral DNA replication. The results indicate that exogenous ch.pTP expression significantly ameliorate the packaging and amplification of recombinant chAd-C vectors thus, the engineered HEK293ch.pTP cells could represent a superior packaging cell line for the production of these vectors.

Molecular and Functional Characterization of BDNF-Overexpressing Human Retinal Pigment Epithelial Cells Established by Sleeping Beauty Transposon-Mediated Gene Transfer.

More and more patients suffer from multifactorial neurodegenerative diseases, such as age-related macular degeneration (AMD). However, their pathological mechanisms are still poorly understood, which complicates the development of effective therapies. To improve treatment of multifactorial diseases, cell-based gene therapy can be used to increase the expression of therapeutic factors. To date, there is no approved therapy for dry AMD, including late-stage geographic atrophy. We present a treatment option for dry AMD that transfers the brain-derived neurotrophic factor (BDNF) gene into retinal pigment epithelial (RPE) cells by electroporation using the plasmid-based Sleeping Beauty (SB) transposon system. ARPE-19 cells and primary human RPE cells were co-transfected with two plasmids encoding the SB100X transposase and the transposon carrying a BDNF transcription cassette. We demonstrated efficient expression and secretion of BDNF in both RPE cell types, which were further increased in ARPE-19 cell cultures exposed to hydrogen peroxide. BDNF-transfected cells exhibited lower apoptosis rates and stimulated neurite outgrowth in human SH-SY5Y cells. This study is an important step in the development of a cell-based BDNF gene therapy that could be applied as an advanced therapy medicinal product to treat dry AMD or other degenerative retinal diseases.

Identification of cancer driver genes using Sleeping Beauty transposon mutagenesis.

Cancer genome sequencing studies have identified driver genes for a variety of different cancers and helped to understand the genetic landscape of human cancer. It is still challenging, however, to identify cancer driver genes with confidence simply from genetic data alone. In vivo forward genetic screens using Sleeping Beauty (SB) transposon mutagenesis provides another powerful genetic tool for identifying candidate cancer driver genes in wild-type and sensitized mouse tumors. By comparing cancer driver genes identified in human and mouse tumors, cancer driver genes can be identified with additional confidence based upon comparative oncogenomics. This review describes how SB mutagenesis works in mice and focuses on studies that have identified cancer driver genes in the mouse gastrointestinal tract.

Targeting CD33 in Chemoresistant AML Patient-Derived Xenografts by CAR-CIK Cells Modified with an Improved SB Transposon System.

The successful implementation of chimeric antigen receptor (CAR)-T cell therapy in the clinical context of B cell malignancies has paved the way for further development in the more critical setting of acute myeloid leukemia (AML). Among the potentially targetable AML antigens, CD33 is insofar one of the main validated molecules. Here, we describe the feasibility of engineering cytokine-induced killer (CIK) cells with a CD33.CAR by using the latest optimized version of the non-viral Sleeping Beauty (SB) transposon system "SB100X-pT4." This offers the advantage of improving CAR expression on CIK cells, while reducing the amount of DNA transposase as compared to the previously employed "SB11-pT" version. SB-modified CD33.CAR-CIK cells exhibited significant antileukemic activity in vitro and in vivo in patient-derived AML xenograft models, reducing AML development when administered as an "early treatment" and delaying AML progression in mice with established disease. Notably, by exploiting an already optimized xenograft chemotherapy model that mimics human induction therapy in mice, we demonstrated for the first time that CD33.CAR-CIK cells are also effective toward chemotherapy resistant/residual AML cells, further supporting its future clinical development and implementation within the current standard regimens.

Overexpression of lncRNAs with endogenous lengths and functions using a lncRNA delivery system based on transposon.

BACKGROUND: Long noncoding RNAs (lncRNAs) play important roles in many physiological and pathological processes, this indicates that lncRNAs can serve as potential targets for gene therapy. Stable expression is a fundamental technology in the study of lncRNAs. The lentivirus is one of the most widely used delivery systems for stable expression. However, it was initially designed for mRNAs, and the applicability of lentiviral vectors for lncRNAs is largely unknown. RESULTS: We found that the lentiviral vector produces lncRNAs with improper termination, appending an extra fragment of ~ 2 kb to the 3'-end. Consequently, the secondary structures were changed, the RNA-protein interactions were blocked, and the functions were impaired in certain lncRNAs, which indicated that lentiviral vectors are not ideal delivery systems of lncRNAs. Here, we developed a novel lncRNA delivery method called the Expression of LncRNAs with Endogenous Characteristics using the Transposon System (ELECTS). By inserting a termination signal after the lncRNA sequence, ELECTS produces transcripts without 3'-flanking sequences and retains the native features and function of lncRNAs, which cannot be achieved by lentiviral vectors. Moreover, ELECTS presents no potential risk of infection for the operators and it takes much less time. ELECTS provides a reliable, convenient, safe, and efficient delivery method for stable expression of lncRNAs. CONCLUSIONS: Our study demonstrated that improper transcriptional termination from lentiviral vectors have fundamental effects on molecular action and cellular function of lncRNAs. The ELECTS system developed in this study will provide a convenient and reliable method for the lncRNA study.

Compact Bidirectional Promoters for Dual-Gene Expression in a Sleeping Beauty Transposon.

Promoter choice is an essential consideration for transgene expression in gene therapy. The expression of multiple genes requires ribosomal entry or skip sites, or the use of multiple promoters. Promoter systems comprised of two separate, divergent promoters may significantly increase the size of genetic cassettes intended for use in gene therapy. However, an alternative approach is to use a single, compact, bidirectional promoter. We identified strong and stable bidirectional activity of the RPBSA synthetic promoter comprised of a fragment of the human Rpl13a promoter, together with additional intron/exon structures. The Rpl13a-based promoter drove long-term bidirectional activity of fluorescent proteins. Similar results were obtained for the EF1-α and LMP2/TAP1 promoters. However, in a lentiviral vector, the divergent bidirectional systems failed to produce sufficient titres to translate into an expression system for dual chimeric antigen receptor (CAR) expression. Although bidirectional promoters show excellent applicability to drive short RNA in Sleeping Beauty transposon systems, their possible use in the lentiviral applications requiring longer and more complex RNA, such as dual-CAR cassettes, is limited.

Long-Term Prevention of Congenital Thrombotic Thrombocytopenic Purpura in ADAMTS13 Knockout Mice by Sleeping Beauty Transposon-Mediated Gene Therapy.

OBJECTIVE: Severe deficiency in the von Willebrand factor-cleaving protease ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) because of mutations in the ADAMTS13 gene can lead to acute episodes of congenital thrombotic thrombocytopenic purpura (TTP), requiring prompt treatment. Current treatment consists of therapeutic or prophylactic infusions of fresh frozen plasma. However, lifelong treatment with plasma products is a stressful therapy for TTP patients. Here, we describe the use of the nonviral sleeping beauty (SB) transposon system as a gene therapeutic approach to realize lifelong expression of ADAMTS13 and subsequent protection against congenital TTP. APPROACH AND RESULTS: We demonstrated that hydrodynamic tail vein injection of the SB100X system expressing murine ADAMTS13 in Adamts13-/- mice resulted in long-term expression of supraphysiological levels of transgene ADAMTS13 over a period of 25 weeks. Stably expressed ADAMTS13 efficiently removed the prothrombotic ultralarge von Willebrand factor multimers present in the circulation of Adamts13-/- mice. Moreover, mice stably expressing ADAMTS13 were protected against TTP. The treated mice did not develop severe thrombocytopenia or did organ damage occur when triggered with recombinant von Willebrand factor, and this up to 20 weeks after gene transfer. CONCLUSIONS: These data demonstrate the feasibility of using SB100X-mediated gene therapy to achieve sustained expression of transgene ADAMTS13 and long-term prophylaxis against TTP in Adamts13-/- mice.

Retrofitting baculoviral vector with Sleeping Beauty transposon system: competent for long-term reporter gene imaging in vivo.

Reporter gene imaging is widely used for non-invasively detecting tumorigenesis, trafficking therapeutic cells, and monitoring treatment effect. Baculoviral vectors (BVs) have been utilized as transgenic vectors in the reporter gene imaging systems in recent years. However, BV-mediated report gene imaging can only provide short-term investigation due to its transient transgene expression, which is incompetent for the long-term applications. In the current study, we reconstructed a series of hybrid BVs with several elements, to investigate the feasibility of this hybrid BV-mediated long-term reporter gene imaging in vivo. We showed that with the indispensable assistance of a positive-selection process, hybrid BV containing Sleeping Beauty 100× (SB) transposon system (BV-SB) could significantly prolong the enhanced green fluorescent protein (eGFP) expression for at least 180 days in vitro at nearly 100% eGFP positive percentage and over 1011 arbitrary unit total fluorescence intensity, whereas other hybrid BV-mediated transgene expression gradually faded in only 20 days. Furthermore, BV-SB-mediated eGFP fluorescent reporter gene imaging monitored tumorigenesis in the nude mice for at least 35 days. In addition, we exploited the glucagon-like peptide 1 receptor (glp-1r) gene as a radionuclide reporter gene for in vivo micro-PET imaging. At 50th day post-tumor transplantation, the micro-PET imaging showed considerable radiotracer-receptor-binding in vivo, resulted by stable high level of BV-SB-mediated GLP-1R expression in tumor. In summary, we retrofitted BV with the SB transposon system to make it competent for the long-term reporter gene imaging in vivo, which might broaden the application scopes of BV in the long-term molecular imaging and other biomedicine research fields.

Changes in Skeletal Muscle and Body Weight on Sleeping Beauty Transposon-Mediated Transgenic Mice Overexpressing Pig mIGF-1.

Insulin-like growth factor (IGF-I) is an important growth factor in mammals, but the functions of the local muscle-specific isoform of insulin-like growth factor 1 (mIGF-1) to skeletal muscle development have rarely been reported. To determine the effect of pig mIGF-1 on body development and muscle deposition in vivo and to investigate the molecular mechanisms, the transgenic mouse model was generated which can also provide experimental data for making transgenic pigs with pig endogenous IGF1 gene. We constructed a skeletal muscle-specific expression vector using 5'- and 3'-regulatory regions of porcine skeletal α-actin gene. The expression cassette was flanked with Sleeping Beauty transposon (SB)-inverted terminal repeats. The recombinant vector could strongly drive enhanced green fluorescence protein (EGFP) reporter gene expression specifically in mouse myoblast cells and porcine fetal fibroblast cells, but not in porcine kidney cells. The EGFP level driven by α-actin regulators was significantly stronger than that driven by cytomegalovirus promoters. These results indicated that the cloned α-actin regulators could effectively drive specific expression of foreign genes in myoblasts, and the skeletal muscle-specific expression vector mediated with SB transposon was successfully constructed. To validate the effect of pig mIGF-1 on skeletal muscle growth, transgenic mice were generated by pronuclear microinjection of SB-mediated mIGF-1 skeletal expression vector and SB transposase-expressing plasmid. The transgene-positive rates of founder mice and the next-generation F1 mice were 30% (54/180) and 90.1% (64/71), respectively. The mIGF-1 gene could be expressed in skeletal muscle specifically. The levels of mRNA and protein in transgenic mice were 15 and 3.5 times higher, respectively, than in wild-type mice. The body weights of F1 transgenic mice were significantly heavier than wild-type mice from the age of 8 weeks onwards. The paraffin-embedded sections of gastrocnemius from 16-week-old transgenic male mice showed that the numbers of myofibers per unit were increased in comparison with those in the wild-type mice. mIGF-1 overexpression in mice skeletal muscle may promote myofibers hypertrophy and muscle production, and increased the average body weight of adult mice. Transgenic mice models can be generated by the mediation of SB transposon with high transgene efficiency.

An in vivo transfection system for inducible gene expression and gene silencing in murine hepatocytes.

BACKGROUND: Hydrodynamic tail vein injection (HTVI) of transposon-based integration vectors is an established system for stably transfecting mouse hepatocytes in vivo that has been successfully employed to study key questions in liver biology and cancer. Refining the vectors for transposon-mediated hepatocyte transfection will further expand the range of applications of this technique in liver research. In the present study, we report an advanced transposon-based system for manipulating gene expression in hepatocytes in vivo. METHODS: Transposon-based vector constructs were generated to enable the constitutive expression of inducible Cre recombinase (CreER) together with tetracycline-inducible transgene or miR-small hairpin RNA (shRNA) expression (Tet-ON system). Transposon and transposase expression vectors were co-injected into R26R-mTmG reporter mice by HTVI. Cre-mediated gene recombination was induced by tamoxifen, followed by the administration of doxycycline to drive tetracycline-inducible gene or shRNA expression. Expression was visualized by immunofluorescence staining in livers of injected mice. RESULTS: After HTVI, Cre recombination by tamoxifen led to the expression of membrane-bound green fluorescent protein in transfected hepatocytes. Activation of inducible gene or shRNA expression was detected by immunostaining in up to one-third of transfected hepatocytes, with an efficiency dependent on the promoter driving the Tet-ON system. CONCLUSIONS: Our vector system combines Cre-lox mediated gene mutation with inducible gene expression or gene knockdown, respectively. It provides the opportunity for rapid and specific modification of hepatocyte gene expression and can be a useful tool for genetic screening approaches and analysis of target genes specifically in genetically engineered mouse models.

Mapping the Shh long-range regulatory domain.

Coordinated gene expression controlled by long-distance enhancers is orchestrated by DNA regulatory sequences involving transcription factors and layers of control mechanisms. The Shh gene and well-established regulators are an example of genomic composition in which enhancers reside in a large desert extending into neighbouring genes to control the spatiotemporal pattern of expression. Exploiting the local hopping activity of the Sleeping Beauty transposon, the lacZ reporter gene was dispersed throughout the Shh region to systematically map the genomic features responsible for expression activity. We found that enhancer activities are retained inside a genomic region that corresponds to the topological associated domain (TAD) defined by Hi-C. This domain of approximately 900 kb is in an open conformation over its length and is generally susceptible to all Shh enhancers. Similar to the distal enhancers, an enhancer residing within the Shh second intron activates the reporter gene located at distances of hundreds of kilobases away, suggesting that both proximal and distal enhancers have the capacity to survey the Shh topological domain to recognise potential promoters. The widely expressed Rnf32 gene lying within the Shh domain evades enhancer activities by a process that may be common among other housekeeping genes that reside in large regulatory domains. Finally, the boundaries of the Shh TAD do not represent the absolute expression limits of enhancer activity, as expression activity is lost stepwise at a number of genomic positions at the verges of these domains.

Lysosomal storage disease: gene therapy on both sides of the blood-brain barrier.

Most lysosomal storage disorders affect the nervous system as well as other tissues and organs of the body. Previously, the complexities of these diseases, particularly in treating neurologic abnormalities, were too great to surmount. However, based on recent developments there are realistic expectations that effective therapies are coming soon. Gene therapy offers the possibility of affordable, comprehensive treatment associated with these diseases currently not provided by standards of care. With a focus on correction of neurologic disease by systemic gene therapy of mucopolysaccharidoses types I and IIIA, we review some of the major recent advances in viral and non-viral vectors, methods of their delivery and strategies leading to correction of both the nervous and somatic tissues as well as evaluation of functional correction of neurologic manifestations in animal models. We discuss two questions: what systemic gene therapy strategies work best for correction of both somatic and neurologic abnormalities in a lysosomal storage disorder and is there evidence that targeting peripheral tissues (e.g., in the liver) has a future for ameliorating neurologic disease in patients?

Multiplexed gene transfer to a human T-cell line by combining Sleeping Beauty transposon system with methotrexate selection.

Engineered human T-cells are a promising therapeutic modality for cancer immunotherapy. T-cells expressing chimeric antigen receptors combined with additional genes to enhance T-cell proliferation, survival, or tumor targeting may further improve efficacy but require multiple stable gene transfer events. Methods are therefore needed to increase production efficiency for multiplexed engineered cells. In this work, we demonstrate multiplexed, non-viral gene transfer to a human T-cell line with efficient selection (∼ 50%) of cells expressing up to three recombinant open reading frames. The efficient introduction of multiple genes to T-cells was achieved using the Sleeping Beauty transposon system delivered in minicircles by nucleofection. We demonstrate rapid selection for engineered cells using methotrexate (MTX) and a mutant human dihydrofolate reductase resistant to methotrexate-induced metabolic inhibition. Preferential amplification of cells expressing multiple transgenes was achieved by two successive rounds of increasing MTX concentration. This non-viral gene transfer method with MTX step selection can potentially be used in the generation of clinical-grade T-cells housing multiplexed genetic modifications.

A novel method for somatic transgenesis of the mouse prostate using the Sleeping Beauty transposon system.

BACKGROUND: In vivo ectopic gene expression is a common approach for prostate research through the use of transgenes in germline transgenic mice. For some other organs, somatic transgenesis with the Sleeping Beauty transposon system has allowed in vivo ectopic gene expression with higher throughput and lower cost than germline transgenic approaches. METHODS: Mouse e16 urogenital sinuses (UGSs) were co-injected with plasmids expressing the Sleeping Beauty transposase and plasmids with control or activated BRAF expressing transposons. Following electroporation, the transduced UGSs were grown as allografts in mouse hosts for 8 weeks, and the resulting allografts were evaluated for several endpoints. RESULTS: Transposon-transduced UGS allografts developed into prostatic tissue with normal tissue structure and cellular differentiation. Integration of transposon vectors into the genomes of transduced allografts was confirmed using linker-mediated PCR, sequencing, and in situ PCR. Transduction of UGS allografts with transposons expressing activated BRAF resulted in ectopic BRAF expression that was detectable at both the mRNA and protein levels. Prostatic ducts over-expressing activated BRAF also had ectopic activation of the ERK1/2 mitogen activated kinases and increased epithelial cell proliferation. CONCLUSIONS: The Sleeping Beauty transposon system can be used to achieve somatic transgenesis of prostatic allografts. This new method for achieving ectopic gene expression in the prostate will complement other existing approaches such as ectopic gene expression in cell lines and in germline transgenic mice. Advantages of this new approach include preservation of stromal-epithelial interactions not possible with cell lines, and higher throughput and lower cost than traditional germline transgenic approaches.

Generation of induced pluripotent stem cells from human foetal fibroblasts using the Sleeping Beauty transposon gene delivery system.

Transposon gene delivery systems offer an alternative, non-viral-based approach to generate induced pluripotent stem cells (iPSCs). Here we used the Sleeping Beauty (SB) transposon to generate four human iPSC lines from foetal fibroblasts. In contrast to other gene delivery systems, the SB transposon does not exhibit an integration bias towards particular genetic elements, thereby reducing the risk of insertional mutagenesis. Furthermore, unlike the alternative transposon piggyBac, SB has no SB-like elements within the human genome, minimising the possibility of mobilising endogenous transposon elements. All iPSC lines exhibited the expected characteristics of pluripotent human cells, including the ability to differentiate to derivatives of all three germ layers in vitro. Re-expression of the SB transposase in the iPSCs after reprogramming resulted in the mobilisation of some of the transposons. These results indicate that the SB transposon system is a useful addition to methods for generating human iPSCs, both for basic and applied biomedical research, and in the context of future therapeutic application.

NANO-SBT-PEDF delivery system: A promising approach against ovarian cancer?

Pigment epithelium-derived factor (PEDF) is a secreted glycoprotein involved in various biological processes. Its expression declines during ovarian carcinogenesis where it could decrease macrophages polarization, inhibit angiogenesis and induce apoptosis. Altogether, PEDF represents an ideal anti-cancer agent against ovarian cancer. We previously proposed the non-viral Sleeping Beauty transposon (SBT) system to stably integrate the PEDF transgene into ovarian cancer cells. Here, we report the development of liposomes and lipid nanoparticles for SBT-PEDF gene therapy. We determined that the SBT-PEDF nanolipid delivery system was the best system to increase the expression of PEDF in ovarian cancer spheroids. We also developed an ex vivo model of ovarian tumors which allowed us to show that nanolipoplexe in combination to paclitaxel exhibits synergistic and effective anti-tumor efficacy on ovarian tumors. These findings demonstrate that lipid nanoparticle for SBT-PEDF gene therapy may be a promising therapeutic approach for ovarian cancer.

SB Digestor: a tailored driver gene identification tool for dissecting heterogeneous Sleeping Beauty transposon-induced tumors.

Sleeping Beauty (SB) insertional mutagenesis has been widely used for genome-wide functional screening in mouse models of human cancers, however, intertumor heterogeneity can be a major obstacle in identifying common insertion sites (CISs). Although previous algorithms have been successful in defining some CISs, they also miss CISs in certain situations. A major common characteristic of these previous methods is that they do not take tumor heterogeneity into account. However, intertumoral heterogeneity directly influences the sequence read number for different tumor samples and then affects CIS identification. To precisely detect and define cancer driver genes, we developed SB Digestor, a computational algorithm that overcomes biological heterogeneity to identify more potential driver genes. Specifically, we define the relationship between the sequenced read number and putative gene number to deduce the depth cutoff for each tumor, which can reduce tumor complexity and precisely reflect intertumoral heterogeneity. Using this new tool, we re-analyzed our previously published SB-based screening dataset and identified many additional potent drivers involved in Brca1-related tumorigenesis, including Arhgap42, Tcf12, and Fgfr2. SB Digestor not only greatly enhances our ability to identify and prioritize cancer drivers from SB tumors but also substantially deepens our understanding of the intrinsic genetic basis of cancer.