DNA Transposons Research Articles

Transcriptome Analysis Reveals Higher Levels of Mobile Element-Associated Abnormal Gene Transcripts in Temporal Lobe Epilepsy Patients.

Mesial temporal lobe epilepsy (MTLE) is the most common form of epilepsy, and temporal lobe epilepsy patients with hippocampal sclerosis (TLE-HS) show worse drug treatment effects and prognosis. TLE has been shown to have a genetic component, but its genetic research has been mostly limited to coding sequences of genes with known association to epilepsy. Representing a major component of the genome, mobile elements (MEs) are believed to contribute to the genetic etiology of epilepsy despite limited research. We analyzed publicly available human RNA-seq-based transcriptome data to determine the role of mobile elements in epilepsy by performing de novo transcriptome assembly, followed by identification of spliced gene transcripts containing mobile element (ME) sequences (ME-transcripts), to compare their frequency across different sample groups. Significantly higher levels of ME-transcripts in hippocampal tissues of epileptic patients, particularly in TLE-HS, were observed. Among ME classes, short interspersed nuclear elements (SINEs) were shown to be the most frequent contributor to ME-transcripts, followed by long interspersed nuclear elements (LINEs) and DNA transposons. These ME sequences almost in all cases represent older MEs normally located in the intron sequences. For protein coding genes, ME sequences were mostly found in the 3′-UTR regions, with a significant portion also in the coding sequences (CDSs), leading to reading frame disruption. Genes associated with ME-transcripts showed enrichment for the mRNA splicing process and an apparent bias in epileptic transcriptomes toward neural- and epilepsy-associated genes. The findings of this study suggest that abnormal splicing involving MEs, leading to loss of functions in critical genes, plays a role in epilepsy, particularly in TLE-HS, thus providing a novel insight into the molecular mechanisms underlying epileptogenesis.

A high-resolution genome of an euryhaline and eurythermal rhinogoby (Rhinogobius similis Gill 1895).

Rhinogobius similis is distributed in East and Southeast Asia. It is an amphidromous species found mostly in freshwater and sometimes brackish waters. We have obtained a high-resolution assembly of the R. similis genome using nanopore sequencing, high-throughput chromosome conformation capture (Hi-C), and transcriptomic data. The assembled genome was 890.10 Mb in size and 40.15% in GC content. Including 1373 contigs with contig N50 is 1.54 Mb, and scaffold N50 is 41.51 Mb. All of the 1373 contigs were anchored on 22 pairs of chromosomes. The BUSCO evaluation score was 93.02% indicating high quality of genome assembly. The repeat sequences accounted for 34.92% of the whole genome, with retroelements (30.13%), DNA transposons (1.64%), simple repeats (2.34%), and so forth. A total of 31,089 protein-coding genes were predicted in the genome and functionally annotated using Maker, of those genes, 26,893 (86.50%) were found in InterProScan5. There were 1910 gene families expanded in R. similis, 1171 gene families contracted and 170 gene families rapidly evolving. We have compared one rapidly change gene family (PF05970) commonly found in four species (Boleophthalmus pectinirostris, Neogobius melanostomus, Periophthalmus magnuspinnatus, and R. similis), which was found probably related to the lifespan of those species. During 400–10 Ka, the period of the Guxiang Ice Age, the population of R. similis decreased drastically, and then increased gradually following the last interglacial period. A high-resolution genome of R. similis should be useful to study taxonomy, biogeography, comparative genomics, and adaptive evolution of the most speciose freshwater goby genus, Rhinogobius.

CRISPR/Cas Technologies and Their Applications in Escherichia coli.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems have revolutionized genome editing and greatly promoted the development of biotechnology. However, these systems unfortunately have not been developed and applied in bacteria as extensively as in eukaryotic organism. Here, the research progress on the most widely used CRISPR/Cas tools and their applications in Escherichia coli is summarized. Genome editing based on homologous recombination, non-homologous DNA end-joining, transposons, and base editors are discussed. Finally, the state of the art of transcriptional regulation using CRISPRi is briefly reviewed. This review provides a useful reference for the application of CRISPR/Cas systems in other bacterial species.

Tc Buster Transposon Engineered CLL-1 CAR-NK Cells Efficiently Target Acute Myeloid Leukemia

Introduction:DNA transposons are efficient, integrating, non-viral vector systems which have been applied to clinical scale CAR-T cell production, aiming to reduce cost and deliver larger genetic cargos relative to viral transduction. CAR-NK cells are a promising cell therapy platform, with a potential for ‘off-the-shelf’, allogeneic application. Inefficient delivery of plasmid DNA and substantial associated toxicity have limited the utility of DNA transposon systems in primary NK cell engineering. Here we describe an efficient process for the production of transposon engineered primary CAR-NK cells, and the in vitro activity of a CAR-NK cell product targeting human C-type lectin-like molecule-1 (CLL-1/ C-type lectin domain family 12 member A, CLEC12A) for acute myeloid leukemia (AML) produced using the Tc Buster (TcB) transposon system. Since CLL-1 is frequently expressed by leukemic stem cells (LSC) but absent on hematopoietic stem cells, it represents a rational antigenic target to enhance the innate activity of allogeneic NK cell therapies in AML.Methods:NK cells were isolated from 30ml of healthy donor peripheral blood by negative immunomagnetic selection (NK Cell Isolation Kit, Miltenyi Biotec), then activated and expanded using a good manufacturing practice (GMP) grade, 100 Gy irradiated, Epstein Barr Virus-transformed lymphoblastoid cell line (EBV-LCL) in the presence of IL-2 and IL-21. On day 4, NK cells were electroporated with a nanoplasmid TcB transposon carrying a second generation CLL-1 CAR (CD28/CD3ζ or 41BB/CD3ζ) and hyperactive TcB transposase mRNA (Maxcyte ATx). Following transposition, the NK cells were stimulated with the feeder line for a second time. Purified CAR-NK cell populations were produced by immunomagnetic selection of CAR expressing cells using anti-biotin beads (Miltenyi Biotec) and biotinylated CLL-1 protein (ACRO Biosystems), which was also used to detect CAR expression by flow cytometry. For CRISPR/Cas9 gene knockout (KO), pooled sgRNAs targeting 3 sites within the target gene were complexed with Cas9 protein prior to co-electroporation with the TcB payload. CLL-1 CAR-NK cell function was evaluated in co-culture with AML cell lines or biobanked AML patient samples and analyzed by flow cytometry.Results:This approach (Fig 1A) produced efficient transposition with retained clinical-scale expansion capacity of primary CAR-NK cells with mean CAR expression of 37% (n=3, range 26-46%) at day 21, without selection (Fig 1B). By extrapolation, a mean 4,275-fold expansion was observed by day 21, increasing to 14,023-fold by day 25, sufficient to support many clinical doses of 1x10 7 cells/Kg (Fig 1C). Mean NK cell purity of the final product was 98%. A further increase in the proportion of CAR-NK cells was achieved by immunomagnetic selection, with mean CAR expression increasing to 89.5% (range 84-97%, n=3). A further feeder cell stimulation of these CLL-1 CAR selected cells produced a similar expansion trajectory, 3 days delayed relative to unselected cells (Fig 1C). CLL-1 CAR-NK cell function was confirmed by an enhanced ability to target CLL-1 positive AML cell lines and primary AML blasts relative to control electroporated NK cells (Fig 1 D,E). Increased elimination of a primary AML population enriched for LSC (CD34+, CD38-, CLL-1+) by CLL-1 CAR-NK cells was confirmed (mean 96% vs. 32%, p=0.0002, n=3 AML, n=2 NK donors) (Fig 1E). Preliminary data shows simultaneous KO of the NK cell checkpoint cytokine-inducible SH2 containing protein (CISH) using CRISPR/Cas9 while maintaining efficient transposition (Fig 1F).Conclusions:We describe a novel, non-viral approach to CAR-NK cell production using the TcB DNA transposon system, supported by a clinically validated, GMP grade, irradiated, EBV-LCL with clinical scale expansion capability. Purified CAR-NK cell populations were achieved by immunomagnetic sorting without requiring a selection marker or cytotoxic exposure. Preliminary data supports the ability to simultaneously perform CRISPR/Cas9 gene editing - in this case applied to KO of the CISH gene, an NK cell checkpoint with multiple established benefits, including enhanced in vivo NK cell persistence and improved metabolic health. Manufactured using this process, we also present the first reported pre-clinical activity of a CLL-1 CAR-NK cell therapy in AML, demonstrating enhanced targeting of populations enriched for LSC. [Display omitted] DisclosuresGurney: ONK Therapeutics: Research Funding. O'Reilly: ONK Therapeutics: Research Funding. Corcoran: ONK Therapeutics: Current Employment. Brophy: ONK Therapeutics: Current Employment; Autolus: Ended employment in the past 24 months. Hardwicke: ONK Therapeutics: Current Employment; Novartis: Ended employment in the past 24 months. Hermanson: Bio-Techne: Current Employment. Szegezdi: ONK Therapeutics: Research Funding. O'Dwyer: Janssen: Consultancy; ONK Therapeutics: Current Employment, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Research Funding.

Promising Preliminary Activity of Optimized Affinity, CD38 CAR NK Cells Generated Using a Non-Viral Engineering Approach in Gene Edited Cord Blood Derived NK Cells for the Treatment of Multiple Myeloma

CD38 is a multifunctional cell surface protein that is frequently overexpressed on malignant plasma cells as well as on immune suppressive cells within the tumor microenvironment and constitutes a validated immunotherapeutic target in the treatment of multiple myeloma (MM). At ONK Therapeutics we are developing a gene edited, cord blood-derived NK (CBNK) cell product targeting CD38 for treatment of patients with relapsed and/or refractory MM.The product will be generated using a workflow shown in Figure 1A. This involves starting with cord blood that is processed for NK expansion using a clinically validated, Epstein Barr Virus-transformed lymphoblastoid cell line (EBV-LCL) feeder layer. The NK cells would undergo genetic engineering that involves gene editing followed by a non-viral chimeric antigen receptor (CAR) introduction process mediated by the TcBuster (TcB) DNA transposon system (Biotechne). This is followed by a second round of expansion on the EBV-LCL feeder layer resulting in a characterized NK cell product that can then be cryopreserved.In order to develop protocols for optimizing the best transfection efficiencies using the Maxcyte ATx instrument, GFP mRNA (TriLink) was used for transfecting CBNK cells using different electroporation programs. High transfection efficiency was obtained using all programs (Figure 1B.), with the best from program NK4.Since the product employs an optimized affinity second generation anti CD38 CAR (Stikvoort et al., Hemasphere 2021) which could also target CD38 expressed on neighbouring activated NK cells, it is imperative to knock out (KO) the cell surface expression of CD38 on the CAR-NK cells. To achieve this we carried out CRISPR Cas9 based KO studies of CD38 (Figure 1C. left top), using guide RNAs targeting CD38 (Synthego) in the form of a ribonucleoprotein (RNP) complex with Cas9. CBNK cells were transfected using the Maxcyte ATx instrument and CD38 cell surface expression monitored. As shown in Figure 1C. (left top), complete CD38 KO was achieved 11 days post transfection.ONK Therapeutics is actively involved in targeting and downregulating the negative regulator of cytokine signalling, cytokine inducible SH2-containing protein (CIS), which is encoded by the CISH gene, as part of their CBNK products. It has been demonstrated that in addition to facilitating greater cytokine signalling, CISH KO also confers greater metabolic capacity to NK cells resulting in their increased persistence (Daher et al., Blood 2021). Therefore, ONK Therapeutics have evaluated CISH KO in CBNK cells (Figure 1C, top right) using the same scheme that was used for the CD38 KO. Guide RNAs in the form of a RNP complex with Cas9 (Synthego) were transfected into CBNK cells and intracellular CIS protein levels monitored over time. Almost complete KO was attained by 9 days post transfection. In order to dial in CISH KO as part of the product, we further carried out a simultaneous KO of CD38 and CISH, in addition to individual KO of CD38 or CISH (Fig 1C, bottom). Simultaneous multiplexing of the CD38 and CISH KOs resulted in efficient double KO (DKO) . The extent of knock down leading to KO in the DKO setting was very similar to that of individual gene KOs.We then introduced the anti CD38 CAR as part of a transposon that could be transposed by TcB transposase in CBNK cells. After DKO of CD38 and CISH in CBNK cells, the transposon DNA and mRNA for transposase were electroporated. CAR expression was detected 4-5 days post transposition (Figure 1D) with more than 50% of cells expressing the anti CD38 CAR. These CAR expressing CBNK cells were then tested for functionality in a co-culture kill assay against the CD38 positive MM cell line, RPMI8226, which was engineered to express firefly luciferase. In a 4 hour killing assay, robust killing of the RPMI8226 cells was achieved by the CAR-CBNK cells with an EC 50 ten times lower (more potent) than that of mock electroporation control CBNK cells.To our knowledge this is the first successful expression of an anti CD38 CAR in cord-derived NK cells, and with a double CD38/CISH KO, using non-viral CAR insertion approaches. Current work is focusing on designing and developing a manufacturing-ready workflow for this potential product and further examining the effects of CAR NK cell activity in a DKO setting where both KOs contribute to improved metabolism and potentially NK cell persistence, as well as exploring the added benefit of a DR5 TRAIL variant to enhance cytotoxicity. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Phenotypically Unstable Mutations as Markers of Chromosomal Rearrangements Involving DNA Transposons

Phenotypically Unstable Mutations as Markers of Chromosomal Rearrangements Involving DNA Transposons

Prokaryotic and Eukaryotic Horizontal Transfer of Sailor (DD82E), a New Superfamily of IS630-Tc1-Mariner DNA Transposons.

Simple SummaryTransposable elements, including DNA transposons, play a significant role in genetic material exchanges between prokaryotes and eukaryotes. Comparative profiling of the evolution pattern of DNA transposons between prokaryotes and eukaryotes may identify potential genetic material exchanges between them and provide insights into the evolutionary history of prokaryotic and eukaryotic genomes. The members of the IS630-Tc1-mariner (ITm) group may represent the most diverse and widely distributed DNA transposons in nature, and the discovery of new members of this group is highly expected based on the increasing availability of genome sequencing data. We discovered a new superfamily (termed Sailor) belonging to the ITm hyperfamily, which differed from the known superfamilies of Tc1/mariner, DDxD/pogo and DD34E/Gambol, regarding phylogenetic position and catalytic domain. Our data revealed that Sailor was distributed in both prokaryotes and eukaryotes and suggested that horizontal transfer (HT) events of Sailor may occur from prokaryotic to eukaryotic genomes. Finally, internal transmissions of Sailor in prokaryotes and eukaryotes were also detected.Here, a new superfamily of IS630-Tc1-mariner (ITm) DNA transposons, termed Sailor, is identified, that is characterized by a DD82E catalytic domain and is distinct from all previously known superfamilies of the ITm group. Phylogenetic analyses revealed that Sailor forms a monophyletic clade with a more intimate link to the clades of Tc1/mariner and DD34E/Gambol. Sailor was detected in both prokaryotes and eukaryotes and invaded a total of 256 species across six kingdoms. Sailor is present in nine species of bacteria, two species of plantae, four species of protozoa, 23 species of Chromista, 12 species of Fungi and 206 species of animals. Moreover, Sailor is extensively distributed in invertebrates (a total of 206 species from six phyla) but is absent in vertebrates. Sailor transposons are 1.38–6.98 kb in total length and encoded transposases of ~676 aa flanked by TIRs with lengths between 18, 1362 and 4 bp (TATA) target-site duplications. Furthermore, our analysis provided strong evidence of Sailor transmissions from prokaryotes to eukaryotes and internal transmissions in both. These data update the classification of the ITm group and will contribute to the understanding of the evolution of ITm transposons and that of their hosts.

DNA Transposon Expansion is Associated with Genome Size Increase in Mudminnows.

Genome sizes of eukaryotic organisms vary substantially, with whole-genome duplications (WGD) and transposable element expansion acting as main drivers for rapid genome size increase. The two North American mudminnows, Umbra limi and Umbra pygmaea, feature genomes about twice the size of their sister lineage Esocidae (e.g., pikes and pickerels). However, it is unknown whether all Umbra species share this genome expansion and which causal mechanisms drive this expansion. Using flow cytometry, we find that the genome of the European mudminnow is expanded similarly to both North American species, ranging between 4.5 and 5.4 pg per diploid nucleus. Observed blocks of interstitially located telomeric repeats in U. limi suggest frequent Robertsonian rearrangements in its history. Comparative analyses of transcriptome and genome assemblies show that the genome expansion in Umbra is driven by the expansion of DNA transposon and unclassified repeat sequences without WGD. Furthermore, we find a substantial ongoing expansion of repeat sequences in the Alaska blackfish Dallia pectoralis, the closest relative to the family Umbridae, which might mark the beginning of a similar genome expansion. Our study suggests that the genome expansion in mudminnows, driven mainly by transposon expansion, but not WGD, occurred before the separation into the American and European lineage.

PiggyBac transposase and transposon derivatives for gene transfer targeting the ribosomal DNA loci of CHO cells

Integrative non-viral vectors such as transposons engineered to mediate targeted gene transfer into safe harbor sites in the genome may be a promising approach for the production of therapeutic proteins or for gene therapy in an efficient and secure way. In this context, we designed and evaluated two strategies for targeting the nuclear ribosomal DNA (rDNA) loci. One approach relied on the co-location of the transposase and transposon near transcriptionally active rDNA copies using a nucleolar localization signal (NoLS). Another one consisted of targeting the 18S-coding region in the rDNA loci using a NoLS-FokI-dCas9 endonuclease to perform targeted transgene knock-in. We show that integration into the rDNA of Chinese hamster ovary (CHO) cells can be achieved at a high frequency using the piggyBac transposon system, indicating that the rDNA is highly accessible for transposition. Consistently, rDNA-targeted transposition events were most frequently obtained when both the piggyBac transposon DNA and the transposase were nucleoli-targeted, yielding cells displaying stable and homogeneous expression of the transgene. This approach thus provides an alternative strategy to improve targeted transgene delivery and protein expression using CHO cells.

Recent amplification of microsatellite-associated miniature inverted-repeat transposable elements in the pineapple genome

BackgroundMiniature inverted-repeat transposable elements (MITEs) are non-autonomous DNA transposable elements that play important roles in genome organization and evolution. Genome-wide identification and characterization of MITEs provide essential information for understanding genome structure and evolution.ResultsWe performed genome-wide identification and characterization of MITEs in the pineapple genome. The top two MITE families, accounting for 29.39% of the total MITEs and 3.86% of the pineapple genome, have insertion preference in (TA) n dinucleotide microsatellite regions. We therefore named these MITEs A. comosus microsatellite-associated MITEs (Ac-mMITEs). The two Ac-mMITE families, Ac-mMITE-1 and Ac-mMITE-2, shared sequence similarity in the terminal inverted repeat (TIR) regions, suggesting that these two Ac-mMITE families might be derived from a common or closely related autonomous elements. The Ac-mMITEs are frequently clustered via adjacent insertions. Among the 21,994 full-length Ac-mMITEs, 46.1% of them were present in clusters. By analyzing the Ac-mMITEs without (TA) n microsatellite flanking sequences, we found that Ac-mMITEs were likely derived from Mutator-like DNA transposon. Ac-MITEs showed highly polymorphic insertion sites between cultivated pineapples and their wild relatives. To better understand the evolutionary history of Ac-mMITEs, we filtered and performed comparative analysis on the two distinct groups of Ac-mMITEs, microsatellite-targeting MITEs (mt-MITEs) that are flanked by dinucleotide microsatellites on both sides and mutator-like MITEs (ml-MITEs) that contain 9/10 bp TSDs. Epigenetic analysis revealed a lower level of host-induced silencing on the mt-MITEs in comparison to the ml-MITEs, which partially explained the significantly higher abundance of mt-MITEs in pineapple genome. The mt-MITEs and ml-MITEs exhibited differential insertion preference to gene-related regions and RNA-seq analysis revealed their differential influences on expression regulation of nearby genes.ConclusionsAc-mMITEs are the most abundant MITEs in the pineapple genome and they were likely derived from Mutator-like DNA transposon. Preferential insertion in (TA) n microsatellite regions of Ac-mMITEs occurred recently and is likely the result of damage-limiting strategy adapted by Ac-mMITEs during co-evolution with their host. Insertion in (TA) n microsatellite regions might also have promoted the amplification of mt-MITEs. In addition, mt-MITEs showed no or negligible impact on nearby gene expression, which may help them escape genome control and lead to their amplification.

Horizontal transfer and subsequent explosive expansion of a DNA transposon in sea kraits (Laticauda).

Transposable elements (TEs) are self-replicating genetic sequences and are often described as important ‘drivers of evolution’. This driving force is because TEs promote genomic novelty by enabling rearrangement, and through exaptation as coding and regulatory elements. However, most TE insertions potentially lead to neutral or harmful outcomes, therefore host genomes have evolved machinery to suppress TE expansion. Through horizontal transposon transfer (HTT) TEs can colonize new genomes, and since new hosts may not be able to regulate subsequent replication, these TEs may proliferate rapidly. Here, we describe HTT of the Harbinger-Snek DNA transposon into sea kraits (Laticauda), and its subsequent explosive expansion within Laticauda genomes. This HTT occurred following the divergence of Laticauda from terrestrial Australian elapids approximately 15–25 Mya. This has resulted in numerous insertions into introns and regulatory regions, with some insertions into exons which appear to have altered UTRs or added sequence to coding exons. Harbinger-Snek has rapidly expanded to make up 8–12% of Laticauda spp. genomes; this is the fastest known expansion of TEs in amniotes following HTT. Genomic changes caused by this rapid expansion may have contributed to adaptation to the amphibious-marine habitat.

Enzymatically produced piggyBac transposon vectors for efficient non-viral manufacturing of CD19-specific CAR T cells

The piggyBac transposon system provides a non-viral alternative for cost-efficient and simple chimeric antigen receptor (CAR) T cell production. The generation of clinical-grade CAR T cells requires strict adherence to current good manufacturing practice (cGMP) standards. Unfortunately, the high costs of commonly used lentiviral or retroviral vectors limit the manufacturing of clinical-grade CAR T cells in many non-commercial academic institutions. Here, we present a manufacturing platform for highly efficient generation of CD19-specific CAR T cells (CAR19 T cells) based on co-electroporation of linear DNA transposon and mRNA encoding the piggyBac transposase. The transposon is prepared enzymatically in vitro by PCR and contains the CAR transgene flanked by piggyBac 3′ and 5′ arms. The mRNA is similarly prepared via in vitro transcription. CAR19 T cells are expanded in the combination of cytokines interleukin (IL)-4, IL-7, and IL-21 to prevent terminal differentiation of CAR T cells. The accurate control of vector copy number (VCN) is achieved by decreasing the concentration of the transposon DNA, and the procedure yields up to 1 × 108 CAR19 T cells per one electroporation of 1 × 107 peripheral blood mononuclear cells (PBMCs) after 21 days of in vitro culture. Produced cells contain >60% CAR+ cells with VCN < 3. In summary, the described manufacturing platform enables a straightforward cGMP certification, since the transposon and transposase are produced abiotically in vitro via enzymatic synthesis. It is suitable for the cost-effective production of highly experimental, early-phase CAR T cell products.

Disintegration promotes protospacer integration by the Cas1-Cas2 complex.

'Disintegration'-the reversal of transposon DNA integration at a target site-is regarded as an abortive off-pathway reaction. Here, we challenge this view with a biochemical investigation of the mechanism of protospacer insertion, which is mechanistically analogous to DNA transposition, by the Streptococcus pyogenes Cas1-Cas2 complex. In supercoiled target sites, the predominant outcome is the disintegration of one-ended insertions that fail to complete the second integration event. In linear target sites, one-ended insertions far outnumber complete protospacer insertions. The second insertion event is most often accompanied by the disintegration of the first, mediated either by the 3'-hydroxyl exposed during integration or by water. One-ended integration intermediates may mature into complete spacer insertions via DNA repair pathways that are also involved in transposon mobility. We propose that disintegration-promoted integration is functionally important in the adaptive phase of CRISPR-mediated bacterial immunity, and perhaps in other analogous transposition reactions.

The large bat Helitron DNA transposase forms a compact monomeric assembly that buries and protects its covalently bound 5′-transposon end

Helitrons are widespread eukaryotic DNA transposons that have significantly contributed to genome variability and evolution, in part because of their distinctive, replicative rolling-circle mechanism, which often mobilizes adjacent genes. Although most eukaryotic transposases form oligomers and use RNase H-like domains to break and rejoin double-stranded DNA (dsDNA), Helitron transposases contain a single-stranded DNA (ssDNA)-specific HUH endonuclease domain. Here, we report the cryo-electron microscopy structure of a Helitron transposase bound to the 5'-transposon end, providing insight into its multidomain architecture and function. The monomeric transposase forms a tightly packed assembly that buries the covalently attached cleaved end, protecting it until the second end becomes available. The structure reveals unexpected architectural similarity to TraI, a bacterial relaxase that also catalyzes ssDNA movement. The HUH active site suggests how two juxtaposed tyrosines, a feature of many replication initiators that use HUH nucleases, couple the conformational shift of an α-helix to control strand cleavage and ligation reactions.

DNA transposon expansion is associated with genome size increase in mudminnows

DNA transposon expansion is associated with genome size increase in mudminnows

Genome of the pincer wasp Gonatopus flavifemur reveals unique venom evolution and a dual adaptation to parasitism and predation

BackgroundHymenoptera comprise extremely diverse insect species with extensive variation in their life histories. The Dryinidae, a family of solitary wasps of Hymenoptera, have evolved innovations that allow them to hunt using venom and a pair of chelae developed from the fore legs that can grasp prey. Dryinidae larvae are also parasitoids of Auchenorrhyncha, a group including common pests such as planthoppers and leafhoppers. Both of these traits make them effective and valuable for pest control, but little is yet known about the genetic basis of its dual adaptation to parasitism and predation.ResultsWe sequenced and assembled a high-quality genome of the dryinid wasp Gonatopus flavifemur, which at 636.5 Mb is larger than most hymenopterans. The expansion of transposable elements, especially DNA transposons, is a major contributor to the genome size enlargement. Our genome-wide screens reveal a number of positively selected genes and rapidly evolving proteins involved in energy production and motor activity, which may contribute to the predatory adaptation of dryinid wasp. We further show that three female-biased, reproductive-associated yellow genes, in response to the prey feeding behavior, are significantly elevated in adult females, which may facilitate the egg production. Venom is a powerful weapon for dryinid wasp during parasitism and predation. We therefore analyze the transcriptomes of venom glands and describe specific expansions in venom Idgf-like genes and neprilysin-like genes. Furthermore, we find the LWS2-opsin gene is exclusively expressed in male G. flavifemur, which may contribute to partner searching and mating.ConclusionsOur results provide new insights into the genome evolution, predatory adaptation, venom evolution, and sex-biased genes in G. flavifemur, and present genomic resources for future in-depth comparative analyses of hymenopterans that may benefit pest control.

Draft Genome of the Mirrorwing Flyingfish (Hirundichthys speculiger).

Draft Genome of the Mirrorwing Flyingfish (Hirundichthys speculiger).

B-Cell Maturation Antigen (BCMA)-Specific, CentyrinTM-Based, PiggyBacTM-Transposed CAR-T Memory Stem Cells Are Effective Against p53-/- and Patient-Derived Multiple Myeloma Tumors

B-Cell Maturation Antigen (BCMA)-Specific, CentyrinTM-Based, PiggyBacTM-Transposed CAR-T Memory Stem Cells Are Effective Against p53-/- and Patient-Derived Multiple Myeloma Tumors

Long Term Follow up after Adoptive Transfer of CD19-Specific CAR+ T Cells Genetically Modified Via Non-Viral Sleeping Beauty S ystem Following Hematopoietic Stem Cell Transplantation (HSCT)

BACKGROUND. We evaluated a human application of T cells genetically modified using the Sleeping Beauty (SB) transposon/transposase system to express a CD19-specific chimeric antigen receptor (CAR, designated CD19RCD28). The SB system uses a synthetic DNA transposon for nonviral somatic gene transfer. The initial data were reported (J Clin Invest. 2016 Sep 1;126(9):3363-76) and we now present long-term follow-up (LTFU).METHODS. T cells were genetically modified using DNA plasmids from the SB platform to stably express CD19RCD28 and selectively propagated ex vivo with activating and propagating cells (AaPC) with cytokines. Twenty-six patients with advanced non-Hodgkin lymphoma (n=9) or acute lymphoblastic leukemia (n=17) underwent autologous (n=7) or allogeneic (n=19) HSCT followed by a T-cell infusion as adjuvant therapy on two clinical trials NCT00968760 and NCT01497184, respectively. Patients were subsequently enrolled onto a LTFU study, NCT01492036, to be followed for up to 15 years. During this time, patients could be tested annually for the persistence of genetically modified T cells using PCR and flow cytometry.RESULTS. The persistence of SB-modified T cells was measured in two of the seven autologous HSCT/CAR+T patients as long as 4-years post infusion and both remain progression-free. Similarly, CAR expression was detected in three allogeneic HSCT recipients one-year post T-cell infusion with no disease progression. Upon LTFU of the autologous HSCT group, six of seven patients continue in complete remission (CR) at a median of 3.4 years (range: 1.7 to 4.2 years). The 3-year progression-free (PFS) and overall survival (OS) rates are 86% and 100%, respectively (Figure). After allogeneic HSCT, recipients received donor-derived CAR+ T cells (8 haploidentical, 10 matched-sibling, one 1Ag-mismatched sibling). For this allogeneic group, six patients remain alive and in CR following haplo (n=4) or matched sibling donor (n=2) HSCT with a median follow-up of 1.5 years (range: 0.003 to 2.9 years). The 2-year PFS and OS rates are 45% and 49%, respectively (Figure). Additionally, the 2-year PFS and OS rates for haplo-identical transplants were 57% and 71%, respectively. No long-term safety issues were identified; specifically, no graft failure or excess graft-versus-host-disease.CONCLUSIONS. CD19-specific CAR+ T cells could be detected and were well-tolerated years after adoptive transfer and appear to provide long-term cancer control when infused after HSCT for patients with advanced disease. These encouraging results support further clinical development of this non-viral gene therapy approach.Figure: Overall and progression-free survivals of recipients of SB-modified CAR+ T cells after autologous and allogeneic HSCT. [Display omitted] DisclosuresHuls:Former employee of Intrexon: Employment. De Groot:Ziopharm: Employment. Smith:Ziopharm: Employment. Yang:Ziopharm: Employment. Kantarjian:Bristol-Meyers Squibb: Research Funding; Novartis: Research Funding; Delta-Fly Pharma: Research Funding; Amgen: Research Funding; ARIAD: Research Funding; Pfizer: Research Funding. Cooper:Immatics: Equity Ownership, Patents & Royalties, Research Funding; Intrexon Corporation: Equity Ownership, Patents & Royalties; Sangamo Biosciences: Patents & Royalties; Organovo Holdings: Equity Ownership; Ziopharm: Employment, Equity Ownership, Research Funding; Procter & Gamble: Equity Ownership; Miltenyi Biotec: Honoraria; Ampliphi Biosciences: Equity Ownership; Argos Therapeutics: Equity Ownership; Ferring: Consultancy; Targazyme, Inc.: Equity Ownership.

Satellitome Analysis of the Pacific Oyster Crassostrea gigas Reveals New Pattern of Satellite DNA Organization, Highly Scattered across the Genome.

Several features already qualified the invasive bivalve species Crassostrea gigas as a valuable non-standard model organism in genome research. C. gigas is characterized by the low contribution of satellite DNAs (satDNAs) vs. mobile elements and has an extremely low amount of heterochromatin, predominantly built of DNA transposons. In this work, we have identified 52 satDNAs composing the satellitome of C. gigas and constituting about 6.33% of the genome. Satellitome analysis reveals unusual, highly scattered organization of relatively short satDNA arrays across the whole genome. However, peculiar chromosomal distribution and densities are specific for each satDNA. The inspection of the organizational forms of the 11 most abundant satDNAs shows association with constitutive parts of Helitron mobile elements. Nine of the inspected satDNAs are dominantly found in mobile element-associated form, two mostly appear standalone, and only one is present exclusively as Helitron-associated sequence. The Helitron-related satDNAs appear in more chromosomes than other satDNAs, indicating that these mobile elements could be leading satDNA propagation in C. gigas. No significant accumulation of satDNAs on certain chromosomal positions was detected in C. gigas, thus establishing a novel pattern of satDNA organization on the genome level.