Csaba Miskey

• Paul Ehrlich Institut

Most CAR-T therapies rely on genetic T cell engineering with integrating viral vectors that, although effective, are associated with prohibitive costs. Here we have generated TranspoCART19 cells, a fully functional 4-1BB second-generation CAR-T cell product targeting CD19, fused to a truncated version of the human EGFR (hEGFRt) as reporter gene and...

RNA editing by adenosine deaminases acting on RNA (ADARs) is an evolutionarily conserved posttranscriptional modification essential for organismal development and normal cell function. Three catalytically active ADARs are conserved in mammals: two isoforms of ADAR1 referred to as ADAR1-p150 and ADAR1-p110, as well as ADAR2. All recognize and edit d...

The Sleeping Beauty (SB) transposon system is a useful tool for genetic applications, including gene therapy. We discovered a hyperactive variant of the SB100X transposase, called SB200X. This mutant, resulting from a specific amino acid replacement (Q124C), showed an ∼2-fold increase in transposition activity in various human and murine cells. Oth...

The accessory protease transmembrane protease serine 2 (TMPRSS2) enhances severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) uptake into ACE2-expressing cells, although how increased entry impacts downstream viral and host processes remains unclear. To investigate this in more detail, we performed infection assays in engineered cells prom...

Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture-adapted HCV population (p100pop) which exhibits increased replicative c...

Lung immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is critical for the ability to deal with infection. Using light sheet fluorescence microscopy of hamster lung slices in combination with virological, immunohistochemical, and RNA sequencing analyses, we show a wave of monocyte-derived macrophage (MDM) infiltration...

Hepatitis C virus (HCV) infection progresses to chronicity in the majority of infected individuals. Its high intra-host genetic variability enables HCV to evade the continuous selection pressure exerted by the host, contributing to persistent infection. Utilizing a cell culture adapted HCV population (p100pop) which exhibits increased replicative c...

Transposons are nature’s gene delivery vehicles that can be harnessed for experimental and therapeutic purposes. The Sleeping Beauty (SB) transposon shows efficient transposition and long-term transgene expression in human cells, and is currently under clinical development for gene therapy. SB transposition occurs into the human genome in a random...

[This corrects the article DOI: 10.1016/j.omto.2023.07.003.].

Discrimination between hematopoietic stem cells and leukemic stem cells remains a major challenge for acute myeloid leukemia immunotherapy. CAR T cells specific for the CD117 antigen can deplete malignant and healthy hematopoietic stem cells before consolidation with allogeneic hematopoietic stem cell transplantation in absence of cytotoxic conditi...

Acute Myeloid Leukemia originates from the accumulation of mutations in hematopoietic stem and progenitor cells, leading to the emergence of leukemia-initiating cells, which sustain blast formation. CAR T cells specific for the CD117 antigen can deplete malignant and healthy hematopoietic stem cells. Here we exploit non-viral technology to achieve...

Factors of the innate immune response to SARS-CoV-2 in the lungs are pivotal for the ability of the host to deal with the infection. In humans, excessive macrophage infiltration is associated with disease severity. Using 3D spatiotemporal analysis of optically cleared hamster lung slices in combination with virological, immunohistochemical and RNA...

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, le...

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...

Background Viral safety of blood products in Germany has improved significantly over the last two decades. We describe the second documented transfusion-transmitted (TT) episode for the hepatitis C virus (HCV) in Germany since mandatory nucleic acid amplification techniques (NAT) screening was introduced in 1999. Study Design and Methods When a re...

Both ZeBrafish (ZB), a recently identified DNA transposon in the zebrafish genome, and SB, a reconstructed transposon originally discovered in several fish species, are known to exhibit high transposition activity in vertebrate cells. Although a similar structural organization was observed for ZB and SB transposons, the evolutionary profiles of the...

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 t...

Megakaryopoiesis describes the stepwise differentiation of megakaryocytes (MK) from hematopoietic stem and progenitor cells (HSPC) mostly guided by thrombopoietin. Rapid platelet production in response to thrombocytopenic events during inflammation implies MK generation via a different mechanism, called emergency megakaryopoiesis (EM). In wild-type...

Respiratory tract infections are among the deadliest communicable diseases worldwide. Severe cases of viral lung infections are often associated with a cytokine storm and alternating platelet numbers. We report that hematopoietic stem and progenitor cells (HSPCs) sense a non-systemic influenza A virus (IAV) infection via inflammatory cytokines. Irr...

SARS-CoV-2 entry is promoted by both cell-surface TMPRSS2 and endolysosomal cathepsins. To investigate the impact of differentially routed virions on host and viral processes, lung epithelial cells expressing distinct combinations of entry factors were infected with authentic viruses. Entry route determined early rates of viral replication and tran...

Vaccine-associated enhanced respiratory disease (VAERD) is a severe complication for some respiratory infections. To investigate the potential for VAERD induction in COVID-19, we evaluate two vaccine leads utilizing a severe hamster infection model: a TH1-biased measles vaccine-derived candidate and a TH2-biased alum-adjuvanted, non-stabilized Spik...

Metastasis, a complex, multistep process, is responsible for the overwhelming majority of cancer-related deaths. Despite its devastating consequences, it is not possible to effectively treat cancer that has spread to vital organs, the mechanisms leading to metastasis are still poorly understood, and the catalog of metastasis promoting genes is stil...

The Sleeping Beauty (SB) transposon system is a popular tool for genome engineering, but random integration into the genome carries a certain genotoxic risk in therapeutic applications. Here we investigate the role of amino acids H187, P247 and K248 in target site selection of the SB transposase. Structural modeling implicates these three amino aci...

Human T lymphocytes that transgenically express a chimeric antigen receptor (CAR) have proven efficacy and safety in gene- and cell-based immunotherapy of certain hematological cancers. Appropriate gene vectors and methods of genetic engineering are required for therapeutic cell products to be biologically potent and their manufacturing to be econo...

Since December 2019, the novel human coronavirus SARS-CoV-2 has spread globally, causing millions of deaths. Unprecedented efforts have enabled development and authorization of a range of vaccines, which reduce transmission rates and confer protection against the associated disease COVID-19. These vaccines are conceptually diverse, including e.g. c...

Background: Natural Killer (NK) cells are known for their high intrinsic cytotoxic capacity. Recently, we and others showed that virally transduced NK cells equipped with a synthetic chimeric antigen receptor (CAR) targeting CD19 induced enhanced killing of acute lymphoblastic leukemia (ALL) cells. Here, we demonstrate for the first time that prima...

We report the sequences of two West Nile virus (WNV) strains (lineages 1 and 2) developed by the Paul-Ehrlich-Institut as reference materials. The materials are calibrated against the 1st World Health Organization WNV RNA International Standard and are intended for use in nucleic acid technology assays supporting transfusion safety.

Background People suffering from COVID‐19 are typically considered non‐infectious 14 days after diagnosis if symptoms have disappeared for at least 48 h. We describe three patients who independently acquired their infection. These three patients experienced mild COVID‐19 and completely recovered symptomatically within 10 days, but remained PCR‐posi...

The stable insertion of the retroviral genome into the host chromosomes requires the association between integration complexes and cellular chromatin via the interaction between retroviral integrase and the nucleosomal target DNA. This final association may involve the chromatin-binding properties of both the retroviral integrase and its cellular c...

TLR5 ligand flagellin-containing fusion proteins are potential vaccine candidates for many diseases. A recombinant fusion protein of flagellin A and the major birch pollen allergen Bet v 1 (rFlaA:Betv1) modulates immune responses in vitro and in vivo. We studied the effects of rFlaA:Betv1 on bone marrow-derived macrophages (BMDMs). BMDMs differenti...

Throughout evolution, DNA transposons provide a recurrent supply of genetic information to give rise to novel gene functions by fusion of their transposase domain to various domains of host-encoded proteins. One of these “domesticated”, transposase-derived factors is SETMAR/Metnase which is a naturally occurring fusion protein that consists of a hi...

Transcriptional profiling provides global snapshots of virus-mediated cellular reprogramming, which can simultaneously encompass pro- and antiviral components. To determine early transcriptional signatures associated with HCV infection of authentic target cells, we performed ex vivo infections of adult primary human hepatocytes (PHHs) from seven do...

Transcriptional profiling provides global snapshots of virus-mediated cellular reprogramming, which can simultaneously encompass pro- and antiviral components. To determine early transcriptional signatures associated with HCV infection of authentic target cells, we performed ex vivo infections of adult primary human hepatocytes (PHHs) from seven do...

New genetic tools and strategies are currently under development to facilitate functional genomics analyses. Here, we describe an active member of the Tc1/mariner transposon superfamily, named ZB, which invaded the zebrafish genome very recently. ZB exhibits high activity in vertebrate cells, in the range of those of the widely used transposons pig...

Background A family of Tc1/mariner transposons with a characteristic DD38E triad of catalytic amino acid residues, named Intruder ( IT ), was previously discovered in sturgeon genomes, but their evolutionary landscapes remain largely unknown. Results Here, we comprehensively investigated the evolutionary profiles of ITs , and evaluated their cut-a...

Hepatitis C virus (HCV) has no animal reservoir, infecting only humans. To investigate species barrier determinants limiting infection of rodents, murine liver complementary DNA library screening was performed, identifying transmembrane proteins Cd302 and Cr1l as potent restrictors of HCV propagation. Combined ectopic expression in human hepatoma c...

A novel parvovirus was identified as a cell culture contaminant by metagenomic analysis. Droplet digital PCR (ddPCR) was used to determine viral loads in the cell culture supernatant and further analysis, by ddPCR and DNA sequencing, demonstrated that fetal bovine serum (FBS) used during cell culture was the source of the parvovirus contamination....

An ideal tool for gene therapy would enable efficient gene integration at predetermined sites in the human genome. Here we demonstrate biased genome-wide integration of the Sleeping Beauty (SB) transposon by combining it with components of the CRISPR/Cas9 system. We provide proof-of-concept that it is possible to influence the target site selection...

[This corrects the article DOI: 10.1371/journal.pone.0075344.].

The Sleeping Beauty (SB) transposon system is an efficient non-viral gene transfer tool in mammalian cells, but its broad use has been hampered by uncontrolled transposase gene activity from DNA vectors, posing a risk of genome instability, and by the inability to use the transposase protein directly. In this study, we used rational protein design...

The Sleeping Beauty (SB) transposon is an advanced tool for genetic engineering and a useful model to investigate cut-and-paste DNA transposition in vertebrate cells. Here, we identify novel SB transposase mutants that display efficient and canonical excision but practically unmeasurable genomic re-integration. Based on phylogenetic analyses, we es...

Two different approaches of genomic modification are currently used for genome engineering and gene therapy: integrating vectors, which can efficiently integrate large transgenes but are unspecific with respect to their integration sites, and nuclease-based approaches, which are highly specific but not efficient at integrating large genetic cargoes...

Age-related macular degeneration (AMD) is a progressive retinal disorder characterized by imbalanced pro- and antiangiogenic signals. The aim of this study was to evaluate the effect of ex vivo cell-based gene therapy with stable expression of human pigment epithelium-derived factor (PEDF) release using the non-viral Sleeping Beauty (SB100X) transp...

Widespread treatment of human diseases with gene therapies necessitates the development of gene transfer vectors that integrate genetic information effectively, safely and economically. Accordingly, significant efforts have been devoted to engineer novel tools that i) achieve high-level stable gene transfer at low toxicity to the host cell; ii) ind...

Background We have recently described a peculiar feature of the promoters in two Drosophila Tc1-like elements, Bari1 and Bari3. The AT-richness and the presence of weak core-promoter motifs make these promoters, that we have defined “blurry”, able to activate transcription of a reporter gene in cellular systems as diverse as fly, human, yeast and b...

Developing safe and efficient non-viral delivery systems remains a major challenge for in vivo applications of gene therapy, especially in cystic fibrosis. Unlike conventional cationic polymers or lipids, the emerging poloxamine-based copolymers display promising in vivo gene delivery capabilities. However, poloxamines are invalid for in vitro appl...

Transposon-based vectors have entered clinical trials as an alternative to viral vectors for genetic engineering of T cells. However, transposon vectors require DNA transfection into T cells which we found to cause adverse effects. T cell viability was decreased in a dose-dependent manner and DNA-transfected T cells showed a delayed response upon T...

The Sleeping Beauty (SB) transposon system is a non-viral gene delivery platform that combines simplicity, inexpensive manufacture, and favorable safety features in the context of human applications. However, efficient correction of hematopoietic stem and progenitor cells (HSPCs) with non-viral vector systems, including SB, demands further refineme...

Background Stable insertion of the retroviral DNA genome into host chromatin requires the functional association between the intasome (integrase·viral DNA complex) and the nucleosome. The data from the literature suggest that direct protein–protein contacts between integrase and histones may be involved in anchoring the intasome to the nucleosome....

The moso bamboo genome contains the two structurally intact and thus potentially functional mariner-like elements Ppmar1 and Ppmar2. Both elements contain perfect terminal inverted repeats (TIRs) and a full-length intact transposase gene. Here we investigated whether Ppmar1 is functional in yeast (Saccharomyces cerevisiae). We have designed a two-c...

Neovascular age-related macular degeneration (nvAMD) is characterized by choroidal blood vessels growing into the subretinal space, leading to retinal pigment epithelial (RPE) cell degeneration and vision loss. Vessel growth results from an imbalance of pro-angiogenic (e.g., vascular endothelial growth factor (VEGF)) and anti-angiogenic factors (e....

Plasmid DNA is being used as a pharmaceutical agent in vaccination, as well as a basic substance and starting material in gene and cell therapy, and viral vector production. Since the uncontrolled expression of backbone sequences present in such plasmids and the dissemination of antibiotic resistance genes may have profound detrimental effects, an...

Immunotherapy with T cell modified with gamma-retroviral or lentiviral (LV) vectors to express a chimeric antigen receptor (CAR) has shown remarkable efficacy in clinical trials. However, the potential for insertional mutagenesis and genotoxicity of viral vectors is a safety concern, and their cost and regulatory demands a roadblock for rapid and b...

Helitron transposons capture and mobilize gene fragments in eukaryotes, but experimental evidence for their transposition is lacking in the absence of an isolated active element. Here we reconstruct Helraiser, an ancient element from the bat genome, and use this transposon as an experimental tool to unravel the mechanism of Helitron transposition....

Supplementary Figures 1-5, Supplementary Table 1-4, Supplementary Notes 1-18 and Supplementary References

The inherent risks associated with vector insertion in gene therapy need to be carefully assessed. We analyzed the genome-wide distributions of Sleeping Beauty (SB) and piggyBac (PB) transposon insertions as well as MLV retrovirus and HIV lentivirus insertions in human CD4+ T cells cells with respect to a panel of 40 chromatin states. The distribut...

We recently developed adenovirus/transposase hybrid-vectors utilizing the previously described hyperactive Sleeping Beauty (SB) transposase HSB5 for somatic integration and we could show stabilized transgene expression in mice and a canine model for hemophilia B. However, the safety profile of these hybrid-vectors with respect to vector dose and ge...

Background: Transposon-mediated transgenesis is an excellent method for creating stable transgenic lines and insertional mutants. In the chordate Ciona intestinalis, Minos is the only transposon that has been used as the tool for germline transformation. Adding another transposon system in this organism enables us to conduct genetic techniques whi...

The Sleeping Beauty (SB) transposon is a nonviral, integrating vector system with proven efficacy in preclinical animal models, and thus holds promise for future clinical applications. However, SB has a close-to-random insertion profile that could lead to genotoxic effects, thereby presenting a potential safety issue. We evaluated zinc finger (ZF)...

The Sleeping Beauty (SB), piggyBac (PB) and Tol2 transposons are promising instruments for genome engineering. Integration site profiling of SB, PB and Tol2 in human cells showed that PB and Tol2 insertions were enriched in genes, whereas SB insertions were randomly distributed. We aimed to introduce a bias into the target site selection properties...

It has been previously shown that integrase-defective HIV-1-based gene vectors can serve, with moderate efficiency, as substrate for DNA transposition by a transiently expressed Sleeping Beauty (SB) transposase. Here, we describe the enhanced gene transfer properties of a HIV-1/SB hybrid vector that allows efficient DNA transposition, facilitated b...

The Sleeping Beauty (SB) transposon is a promising technology platform for gene transfer in vertebrates; however, its efficiency of gene insertion can be a bottleneck in primary cell types. A large-scale genetic screen in mammalian cells yielded a hyperactive transposase (SB100X) with approximately 100-fold enhancement in efficiency when compared t...

Human embryonic stem (HuES) cells represent a new potential tool for cell-therapy and gene-therapy applications. However, these approaches require the development of efficient, stable gene delivery, and proper progenitor cell and tissue separation methods. In HuES cell lines, we have generated stable, enhanced green fluorescent protein (EGFP)-expre...

The Sleeping Beauty (SB) transposable element shows efficient transposition in human cells, and provides long-term transgene expression in preclinical animal models. Random chromosomal insertion of SB vectors represents a safety issue in human gene therapeutic applications, due to potential genotoxic effects associated with transposon integration....

Hsmar1, one of the two subfamilies of mariner transposons in humans, is an ancient element that entered the primate genome lineage ∼50 million years ago. Although Hsmar1 elements are inactive due to mutational damage, one particular copy of the transposase gene has apparently been under selection. This transposase coding region is part of the SETMA...

Genome sequences of many model organisms of developmental or agricultural importance are becoming available. The tremendous amount of sequence data is fuelling the next phases of challenging research: annotating all genes with functional information, and devising new ways for the experimental manipulation of vertebrate genomes. Transposable element...