204 results on '"Cathomen T"'
Search Results
2. 803 Editing a missense to a nonsense keratin 9 gene mutation restores intermediate filament integrity
- Author
-
Trafoier, T., primary, Ortner-Tobider, D., additional, Hainzl, S., additional, Kocher, T., additional, Koller, U., additional, Bauer, J., additional, Rhiel, M., additional, Andrieux, G., additional, Cornu, T., additional, Cathomen, T., additional, Reichelt, J., additional, Tiefenthaler, C. Heufler, additional, and Schmuth, M., additional
- Published
- 2023
- Full Text
- View/download PDF
3. Progress and harmonization of gene editing to treat human diseases: Proceeding of COST Action CA21113 GenE-HumDi.
- Author
-
Cavazza, A., Hendel, A., Bak, R.O., Rio, P., Güell, M., Lainšček, D., Arechavala-Gomeza, V., Peng, L., Hapil, F.Z., Harvey, J., Ortega, F.G., Gonzalez-Martinez, C., Lederer, C.W., Mikkelsen, K., Gasiunas, G., Kalter, N., Gonçalves, M.A.F.V., Petersen, J., Garanto, A., Montoliu, L., Maresca, M., Seemann, S.E., Gorodkin, J., Mazini, L., Sanchez, R., Rodriguez-Madoz, J.R., Maldonado-Pérez, N., Laura, T., Schmueck-Henneresse, M., Maccalli, C., Grünewald, J., Carmona, G., Kachamakova-Trojanowska, N., Miccio, A., Martin, F., Turchiano, G., Cathomen, T., Luo, Y, Tsai, S.Q., Benabdellah, K., Cavazza, A., Hendel, A., Bak, R.O., Rio, P., Güell, M., Lainšček, D., Arechavala-Gomeza, V., Peng, L., Hapil, F.Z., Harvey, J., Ortega, F.G., Gonzalez-Martinez, C., Lederer, C.W., Mikkelsen, K., Gasiunas, G., Kalter, N., Gonçalves, M.A.F.V., Petersen, J., Garanto, A., Montoliu, L., Maresca, M., Seemann, S.E., Gorodkin, J., Mazini, L., Sanchez, R., Rodriguez-Madoz, J.R., Maldonado-Pérez, N., Laura, T., Schmueck-Henneresse, M., Maccalli, C., Grünewald, J., Carmona, G., Kachamakova-Trojanowska, N., Miccio, A., Martin, F., Turchiano, G., Cathomen, T., Luo, Y, Tsai, S.Q., and Benabdellah, K.
- Abstract
Item does not contain fulltext, The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi's primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing's potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing.
- Published
- 2023
4. A selection free ex vivo gene therapy approach to congenital neutropenia causing HAX1 mutations
- Author
-
Ritter, MU, additional, Nasri, M, additional, Dannenmann, B, additional, Kaufmann, MM, additional, Zeidler, KA, additional, Zeidler, C, additional, Klimiankou, M, additional, Cathomen, T, additional, Welte, K, additional, and Skokowa, J, additional
- Published
- 2022
- Full Text
- View/download PDF
5. Biodegradable nanocarriers resembling extracellular vesicles deliver genetic material with the highest efficiency to various cell types
- Author
-
Tarakanchikova, Y. (Yana), Alzubi, J. (Jamal), Pennucci, V. (Valentina), Follo, M. (Marie), Kochergin, B. (Boris), Muslimov, A. (Albert), Skovorodkin, I. (Ilya), Vainio, S. (Seppo), Antipina, M. N. (Maria N.), Atkin, V. (Vsevolod), Popov, A. (Alexey), Meglinski, I. (Igor), Cathomen, T. (Toni), Cornu, T. I. (Tatjana I.), Gorin, D. A. (Dmitry A.), Sukhorukov, G. B. (Gleb B.), Nazarenko, I. (Irina), Tarakanchikova, Y. (Yana), Alzubi, J. (Jamal), Pennucci, V. (Valentina), Follo, M. (Marie), Kochergin, B. (Boris), Muslimov, A. (Albert), Skovorodkin, I. (Ilya), Vainio, S. (Seppo), Antipina, M. N. (Maria N.), Atkin, V. (Vsevolod), Popov, A. (Alexey), Meglinski, I. (Igor), Cathomen, T. (Toni), Cornu, T. I. (Tatjana I.), Gorin, D. A. (Dmitry A.), Sukhorukov, G. B. (Gleb B.), and Nazarenko, I. (Irina)
- Abstract
Efficient delivery of genetic material to primary cells remains challenging. Here, efficient transfer of genetic material is presented using synthetic biodegradable nanocarriers, resembling extracellular vesicles in their biomechanical properties. This is based on two main technological achievements: generation of soft biodegradable polyelectrolyte capsules in nanosize and efficient application of the nanocapsules for co‐transfer of different RNAs to tumor cell lines and primary cells, including hematopoietic progenitor cells and primary T cells. Near to 100% efficiency is reached using only 2.5 × 10–4 pmol of siRNA, and 1 × 10–3 nmol of mRNA per cell, which is several magnitude orders below the amounts reported for any of methods published so far. The data show that biodegradable nanocapsules represent a universal and highly efficient biomimetic platform for the transfer of genetic material with the utmost potential to revolutionize gene transfer technology in vitro and in vivo.
- Published
- 2020
6. Zinc positive: tailored genome engineering meets reprogramming
- Author
-
Cathomen, T and Schambach, A
- Published
- 2010
- Full Text
- View/download PDF
7. Gene-modified stem cells: Rational approaches to prevent off-target effects: V943
- Author
-
Baum, C., Modlich, U., Bode, J., Schiedlmeier, B., Meyer, J., Li, Z., Cathomen, T., and Schambach, A.
- Published
- 2010
8. Grundlagen und klinische Anwendung der Genomeditierung
- Author
-
Klermund, J., primary and Cathomen, T., additional
- Published
- 2019
- Full Text
- View/download PDF
9. Pointing the finger at genetic disease
- Author
-
Cathomen, T and Weitzman, M D
- Published
- 2005
10. A novel chimeric antigen receptor CAR-T cell approach eliminates prostate cancer in a mouse tumour model
- Author
-
Alzubi, J., Dettmer, V., Kuehle, J., Seidl, M., Taromi, S., Zeiser, R., Abken, H., Cathomen, T., Wolf, P., Alzubi, J., Dettmer, V., Kuehle, J., Seidl, M., Taromi, S., Zeiser, R., Abken, H., Cathomen, T., and Wolf, P.
- Published
- 2018
11. Gene Repair: Pointing the finger at genetic disease
- Author
-
Cathomen, T and Weitzman, M D
- Published
- 2005
- Full Text
- View/download PDF
12. Therapeutic gene editing in hematopoietic progenitor cells from a mouse model of Fanconi anemia
- Author
-
Pino-Barrio, MJ, primary, Gimenez, Y, additional, Villanueva, M, additional, Hildenbeutel, M, additional, Sánchez-Dominguez, R, additional, Rodriguez-Perales, S, additional, Pujol, R, additional, Surrallés, J, additional, Rio, P, additional, Cathomen, T, additional, Mussolino, C, additional, Bueren, JA, additional, and Navarro, S, additional
- Published
- 2018
- Full Text
- View/download PDF
13. LB795 TALEN-mediated elimination of mutant keratin 14 as a gene therapy for epidermolysis bullosa simplex
- Author
-
Aushev, M., primary, Mussolino, C., additional, Cathomen, T., additional, and Reichelt, J., additional
- Published
- 2016
- Full Text
- View/download PDF
14. Gene editing – From modeling diseases to treating patients
- Author
-
Cathomen, T., primary
- Published
- 2016
- Full Text
- View/download PDF
15. Translating the genomics revolution: the need for an international gene therapy consortium for monogenic diseases
- Author
-
Tremblay, Jp, Xiao, X., Aartsma-Rus, A., Barbas, C., Blau, H.m., Bogdanove, A.j., Boycott, K., Braun, S., Breakefield, Xo, Bueren, J.a., Buschmann, M., Byrne, B.j., Calos, M., Cathomen, T., Chamberlain, J., Chuah, Marinee, Cornetta, K., Davies, K.e., Dickson, J.g., Duchateau, P., Flotte, T.r., Gaudet, D., Gersbach, C.a., Gilbert, R., Glorioso, J., Herzog, R., High, K.a., Huang, W., Joung, J.k., Liu, D., Löchmuller, H., Lustig, L., Martens, J., Massie, B., Mavilio, F., Mendell, J.r., Nathwani, A., Ponder, K., Porteus, M., Puymirat, J., Samulski, J., Takeda, S., Thrasher, A., VandenDriessche, Thierry, Wei, Y., Wilson, J., Wilton, S.d., Wolfe, J.h., Gao, G., Cell Biology and Histology, and Division of Gene Therapy & Regenerative Medicine
- Subjects
genomics revolution ,Gene Therapy ,monogenic diseases ,Consortium - Published
- 2013
16. A novel zinc-finger nuclease platform with a sequence-specific cleavage module
- Author
-
Schierling, B., Dannemann, N., Gabsalilow, L., Wende, W., Cathomen, T., Pingoud, A., and Institute of Biochemistry
- Subjects
restriction endonuclease PvuII ,fusion constructs ,ddc:570 ,Zinc-finger nucleasese (ZFNs) ,dna cleavage ,restriction endonuclease FokI ,Life sciences - Abstract
Zinc-finger nucleases (ZFNs) typically consist of three to four zinc fingers (ZFs) and the non-specific DNA-cleavage domain of the restriction endonuclease FokI. In this configuration, the ZFs constitute the binding module and the FokI domain the cleavage module. Whereas new binding modules, e.g. TALE sequences, have been considered as alternatives to ZFs, no efforts have been undertaken so far to replace the catalytic domain of FokI as the cleavage module in ZFNs. Here, we have fused a three ZF array to the restriction endonuclease PvuII to generate an alternative ZFN. While PvuII adds an extra element of specificity when combined with ZFs, ZF-PvuII constructs must be designed such that only PvuII sites with adjacent ZF-binding sites are cleaved. To achieve this, we introduced amino acid substitutions into PvuII that alter Km and kcat and increase fidelity. The optimized ZF-PvuII fusion constructs cleave DNA at addressed sites with a >1000-fold preference over unaddressed PvuII sites in vitro as well as in cellula. In contrast to the analogous ZF-FokI nucleases, neither excess of enzyme over substrate nor prolonged incubation times induced unaddressed cleavage in vitro. These results present the ZF-PvuII platform as a valid alternative to conventional ZFNs.
- Published
- 2011
17. Molecular surgery for epidermolysis bullosa simplex
- Author
-
Aushev, M., Mussolino, C., Cathomen, T., Törmä, Hans, Reichelt, J., Aushev, M., Mussolino, C., Cathomen, T., Törmä, Hans, and Reichelt, J.
- Published
- 2014
18. Generation of CF-patient derived iPS cells and efficient footprintless designer nuclease-based gene targeting
- Author
-
Merkert, S, primary, Wunderlich, S, additional, Bednarski, C, additional, Haase, A, additional, Dreyer, AK, additional, Schwanke, K, additional, Cathomen, T, additional, and Martin, U, additional
- Published
- 2014
- Full Text
- View/download PDF
19. Transfusion-transmitted hepatitis E in Germany, 2013
- Author
-
Huzly, D, primary, Umhau, M, additional, Bettinger, D, additional, Cathomen, T, additional, Emmerich, F, additional, Hasselblatt, P, additional, Hengel, H, additional, Herzog, R, additional, Kappert, O, additional, Maassen, S, additional, Schorb, E, additional, Schulz-Huotari, C, additional, Thimme, R, additional, Unmüssig, R, additional, Wenzel, J J, additional, and Panning, M, additional
- Published
- 2014
- Full Text
- View/download PDF
20. Translating the genomics revolution: The need for an international gene therapy consortium for monogenic diseases
- Author
-
Tremblay, J.P., Xiao, X., Aartsma-Rus, A., Barbas, C., Blau, H.M., Bogdanove, A.J., Boycott, K., Braun, S., Breakefield, X.O., Bueren, J.A., Buschmann, M., Byrne, B.J., Calos, M., Cathomen, T., Chamberlain, J., Chuah, M., Cornetta, K., Davies, K.E., Dickson, J.G., Duchateau, P., Flotte, T.R., Gaudet, D., Gersbach, C.A., Gilbert, R., Glorioso, J., Herzog, R.W., High, K.A., Huang, W., Huard, J., Joung, J.K., Liu, D., Lochmüller, H., Lustig, L., Martens, J., Massie, B., Mavilio, F., Mendell, J.R., Nathwani, A., Ponder, K., Porteus, M., Puymirat, J., Samulski, J., Takeda, S., Thrasher, A., VandenDriessche, T., Wei, Y., Wilson, J.M., Wilton, S.D., Wolfe, J.H., Gao, G., Tremblay, J.P., Xiao, X., Aartsma-Rus, A., Barbas, C., Blau, H.M., Bogdanove, A.J., Boycott, K., Braun, S., Breakefield, X.O., Bueren, J.A., Buschmann, M., Byrne, B.J., Calos, M., Cathomen, T., Chamberlain, J., Chuah, M., Cornetta, K., Davies, K.E., Dickson, J.G., Duchateau, P., Flotte, T.R., Gaudet, D., Gersbach, C.A., Gilbert, R., Glorioso, J., Herzog, R.W., High, K.A., Huang, W., Huard, J., Joung, J.K., Liu, D., Lochmüller, H., Lustig, L., Martens, J., Massie, B., Mavilio, F., Mendell, J.R., Nathwani, A., Ponder, K., Porteus, M., Puymirat, J., Samulski, J., Takeda, S., Thrasher, A., VandenDriessche, T., Wei, Y., Wilson, J.M., Wilton, S.D., Wolfe, J.H., and Gao, G.
- Abstract
Letter to the Editor
- Published
- 2013
21. 803 Editing a missense to a nonsense keratin 9gene mutation restores intermediate filament integrity
- Author
-
Trafoier, T., Ortner-Tobider, D., Hainzl, S., Kocher, T., Koller, U., Bauer, J., Rhiel, M., Andrieux, G., Cornu, T., Cathomen, T., Reichelt, J., Tiefenthaler, C. Heufler, and Schmuth, M.
- Published
- 2023
- Full Text
- View/download PDF
22. DNA-Binding Activity of Adeno-Associated Virus Rep Is Required for Inverted Terminal Repeat-Dependent Complex Formation with Herpes Simplex Virus ICP8
- Author
-
Alex, M., primary, Weger, S., additional, Mietzsch, M., additional, Slanina, H., additional, Cathomen, T., additional, and Heilbronn, R., additional
- Published
- 2011
- Full Text
- View/download PDF
23. Zinc positive: tailored genome engineering meets reprogramming
- Author
-
Cathomen, T, primary and Schambach, A, additional
- Published
- 2009
- Full Text
- View/download PDF
24. A matrix-less measles virus is infectious and elicits extensive cell fusion: consequences for propagation in the brain.
- Author
-
Cathomen, T, Mrkic, B, Spehner, D, Drillien, R, Naef, R, Pavlovic, J, Aguzzi, A; https://orcid.org/0000-0002-0344-6708, Billeter, M A, Cattaneo, R, Cathomen, T, Mrkic, B, Spehner, D, Drillien, R, Naef, R, Pavlovic, J, Aguzzi, A; https://orcid.org/0000-0002-0344-6708, Billeter, M A, and Cattaneo, R
- Abstract
Measles viruses (MV) can be isolated from the brains of deceased subacute sclerosing panencephalitis patients only in a cell-associated form. These viruses are often defective in the matrix (M) protein and always seem to have an altered fusion protein cytoplasmic tail. We reconstituted a cell-free, infectious M-less MV (MV-DeltaM) from cDNA. In comparison with standard MV, MV-DeltaM was considerably more efficient at inducing cell-to-cell fusion but virus titres were reduced approximately 250-fold. In MV-DeltaM-induced syncytia the ribonucleocapsids and glycoproteins largely lost co-localization, confirming the role of M protein as the virus assembly organizer. Genetically modified mice were inoculated with MV-DeltaM or with another highly fusogenic virus bearing glycoproteins with shortened cytoplasmic tails (MV-Delta(tails)). MV-DeltaM and MV-Delta(tails) lost acute pathogenicity but penetrated more deeply into the brain parenchyma than standard MV. We suggest that enhanced cell fusion may also favour the propagation of mutated, assembly-defective MV in human brains.
- Published
- 1998
25. Influence of N-linked oligosaccharide chains on the processing, cell surface expression and function of the measles virus fusion protein
- Author
-
Hu, A., primary, Cathomen, T., additional, Cattaneo, R., additional, and Norrby, E., additional
- Published
- 1995
- Full Text
- View/download PDF
26. Targeted integration of hFANCA in the mouse Mbs85-locus of Fanca-/- fibroblasts and hematopoietic progenitor cells
- Author
-
Pino-Barrio, M. J., Chinchon, R., Gimenez, Y., Sanchez-Dominguez, R., Rodriguez-Perales, S., Pujol, R. M., Mussolino, C., Cathomen, T., Juan Bueren, and Navarro, S.
27. Getting personal: mutation-specific gene editing and addition strategies for beta-thalassemia and beyond
- Author
-
Lederer, C. W., Patsali, P., Papasavva, P., Coralea Stephanou, Turchiano, G., Romito, M., Christou, S., Sitarou, M., Kolnagou, A., Mussolino, C., Cornu, T. I., Antoniou, M. N., Cathomen, T., and Kleanthous, M.
28. Assessing the role of LRRK2 G2019S mutation and the genomic background to the development of PD-related neurodegeneration
- Author
-
Antonella Consiglio, Raya, A., Cathomen, T., Mussolino, C., Tolosa, E., Richaud-Patin, Y., Fernandez, I., Jimenez, S., Faella, A., and Calatayud, C.
29. Gene editing of NCF1 loci is associated with homologous recombination and chromosomal rearrangements.
- Author
-
Raimondi F, Siow KM, Wrona D, Fuster-García C, Pastukhov O, Schmitz M, Bargsten K, Kissling L, Swarts DC, Andrieux G, Cathomen T, Modlich U, Jinek M, Siler U, and Reichenbach J
- Subjects
- Humans, Granulomatous Disease, Chronic genetics, CRISPR-Cas Systems, Chromosome Aberrations, Cell Line, Gene Editing methods, Homologous Recombination, NADPH Oxidases genetics
- Abstract
CRISPR-based genome editing of pseudogene-associated disorders, such as p47
phox -deficient chronic granulomatous disease (p47 CGD), is challenged by chromosomal rearrangements due to presence of multiple targets. We report that interactions between highly homologous sequences that are localized on the same chromosome contribute substantially to post-editing chromosomal rearrangements. We successfully employed editing approaches at the NCF1 gene and its pseudogenes, NCF1B and NCF1C, in a human cell line model of p47 CGD and in patient-derived human hematopoietic stem and progenitor cells. Upon genetic engineering, a droplet digital PCR-based method identified cells with altered copy numbers, spanning megabases from the edited loci. We attributed the high aberration frequency to the interaction between repetitive sequences and their predisposition to recombination events. Our findings emphasize the need for careful evaluation of the target-specific genomic context, such as the presence of homologous regions, whose instability can constitute a risk factor for chromosomal rearrangements upon genome editing., (© 2024. The Author(s).)- Published
- 2024
- Full Text
- View/download PDF
30. CRISPR/Cas9 editing of NKG2A improves the efficacy of primary CD33-directed chimeric antigen receptor natural killer cells.
- Author
-
Bexte T, Albinger N, Al Ajami A, Wendel P, Buchinger L, Gessner A, Alzubi J, Särchen V, Vogler M, Rasheed HM, Jung BA, Wolf S, Bhayadia R, Oellerich T, Klusmann JH, Penack O, Möker N, Cathomen T, Rieger MA, Imkeller K, and Ullrich E
- Subjects
- Humans, Cell Line, Tumor, Animals, Mice, Immunotherapy, Adoptive methods, CRISPR-Cas Systems, NK Cell Lectin-Like Receptor Subfamily C genetics, NK Cell Lectin-Like Receptor Subfamily C metabolism, NK Cell Lectin-Like Receptor Subfamily C immunology, Killer Cells, Natural immunology, Receptors, Chimeric Antigen immunology, Receptors, Chimeric Antigen genetics, Gene Editing methods, Leukemia, Myeloid, Acute immunology, Leukemia, Myeloid, Acute therapy, Leukemia, Myeloid, Acute genetics, Sialic Acid Binding Ig-like Lectin 3 genetics, Sialic Acid Binding Ig-like Lectin 3 immunology
- Abstract
Chimeric antigen receptor (CAR)-modified natural killer (NK) cells show antileukemic activity against acute myeloid leukemia (AML) in vivo. However, NK cell-mediated tumor killing is often impaired by the interaction between human leukocyte antigen (HLA)-E and the inhibitory receptor, NKG2A. Here, we describe a strategy that overcomes CAR-NK cell inhibition mediated by the HLA-E-NKG2A immune checkpoint. We generate CD33-specific, AML-targeted CAR-NK cells (CAR33) combined with CRISPR/Cas9-based gene disruption of the NKG2A-encoding KLRC1 gene. Using single-cell multi-omics analyses, we identified transcriptional features of activation and maturation in CAR33-KLRC1
ko -NK cells, which are preserved following exposure to AML cells. Moreover, CAR33-KLRC1ko -NK cells demonstrate potent antileukemic killing activity against AML cell lines and primary blasts in vitro and in vivo. We thus conclude that NKG2A-deficient CAR-NK cells have the potential to bypass immune suppression in AML., (© 2024. The Author(s).)- Published
- 2024
- Full Text
- View/download PDF
31. Effective genome editing with an enhanced ISDra2 TnpB system and deep learning-predicted ωRNAs.
- Author
-
Marquart KF, Mathis N, Mollaysa A, Müller S, Kissling L, Rothgangl T, Schmidheini L, Kulcsár PI, Allam A, Kaufmann MM, Matsushita M, Haenggi T, Cathomen T, Kopf M, Krauthammer M, and Schwank G
- Abstract
Transposon (IS200/IS605)-encoded TnpB proteins are predecessors of class 2 type V CRISPR effectors and have emerged as one of the most compact genome editors identified thus far. Here, we optimized the design of Deinococcus radiodurans (ISDra2) TnpB for application in mammalian cells (TnpBmax), leading to an average 4.4-fold improvement in editing. In addition, we developed variants mutated at position K76 that recognize alternative target-adjacent motifs (TAMs), expanding the targeting range of ISDra2 TnpB. We further generated an extensive dataset on TnpBmax editing efficiencies at 10,211 target sites. This enabled us to delineate rules for on-target and off-target editing and to devise a deep learning model, termed TnpB editing efficiency predictor (TEEP; https://www.tnpb.app ), capable of predicting ISDra2 TnpB guiding RNA (ωRNA) activity with high performance (r > 0.8). Employing TEEP, we achieved editing efficiencies up to 75.3% in the murine liver and 65.9% in the murine brain after adeno-associated virus (AAV) vector delivery of TnpBmax. Overall, the set of tools presented in this study facilitates the application of TnpB as an ultracompact programmable endonuclease in research and therapeutics., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)
- Published
- 2024
- Full Text
- View/download PDF
32. Improving the Assessment of Risk Factors Relevant to Potential Carcinogenicity of Gene Therapies: A Consensus Article.
- Author
-
Klapwijk JC, Del Rio Espinola A, Libertini S, Collin P, Fellows MD, Jobling S, Lynch AM, Martus H, Vickers C, Zeller A, Biasco L, Brugman MH, Bushmann FD, Cathomen T, Ertl HCJ, Gabriel R, Gao G, Jadlowsky JK, Kimber I, Lanz TA, Levine BL, Micklethwaite KP, Onodera M, Pizzurro DM, Reed S, Rothe M, Sabatino DE, Salk JJ, Schambach A, Themis M, and Yuan J
- Subjects
- Humans, Risk Factors, Animals, Genetic Vectors adverse effects, Consensus, Neoplasms therapy, Neoplasms genetics, Risk Assessment, Genetic Therapy adverse effects, Genetic Therapy methods
- Abstract
Regulators and industry are actively seeking improvements and alternatives to current models and approaches to evaluate potential carcinogenicity of gene therapies (GTs). A meeting of invited experts was organized by NC3Rs/UKEMS (London, March 2023) to discuss this topic. This article describes the consensus reached among delegates on the definition of vector genotoxicity, sources of uncertainty, suitable toxicological endpoints for genotoxic assessment of GTs, and future research needs. The collected recommendations should inform the further development of regulatory guidelines for the nonclinical toxicological assessment of GT products.
- Published
- 2024
- Full Text
- View/download PDF
33. Safe and effective liver-directed AAV-mediated homology-independent targeted integration in mouse models of inherited diseases.
- Author
-
Esposito F, Dell'Aquila F, Rhiel M, Auricchio S, Chmielewski KO, Andrieux G, Ferla R, Horrach PS, Padmanabhan A, Di Cunto R, Notaro S, Santeularia ML, Boerries M, Dell'Anno M, Nusco E, Padula A, Nutarelli S, Cornu TI, Sorrentino NC, Piccolo P, Trapani I, Cathomen T, and Auricchio A
- Subjects
- Animals, Mice, Hepatocytes metabolism, Humans, Virus Integration genetics, CRISPR-Cas Systems genetics, Transgenes, Genetic Diseases, Inborn genetics, Genetic Diseases, Inborn therapy, Genetic Therapy methods, Mice, Inbred C57BL, Albumins genetics, Albumins metabolism, Dependovirus genetics, Liver metabolism, Liver pathology, Disease Models, Animal, Genetic Vectors genetics
- Abstract
Liver-directed adeno-associated viral (AAV) vector-mediated homology-independent targeted integration (AAV-HITI) by CRISPR-Cas9 at the highly transcribed albumin locus is under investigation to provide sustained transgene expression following neonatal treatment. We show that targeting the 3' end of the albumin locus results in productive integration in about 15% of mouse hepatocytes achieving therapeutic levels of systemic proteins in two mouse models of inherited diseases. We demonstrate that full-length HITI donor DNA is preferentially integrated upon nuclease cleavage and that, despite partial AAV genome integrations in the target locus, no gross chromosomal rearrangements or insertions/deletions at off-target sites are found. In line with this, no evidence of hepatocellular carcinoma is observed within the 1-year follow-up. Finally, AAV-HITI is effective at vector doses considered safe if directly translated to humans providing therapeutic efficacy in the adult liver in addition to newborn. Overall, our data support the development of this liver-directed AAV-based knockin strategy., Competing Interests: Declaration of interests F.E., F.D.A., R.F., M.L.S., and A.A. are listed as inventors on the patent WO2023213831 “Homology independent targeted integration for gene editing” related to this work. A.A. is founder, shareholder, and consultant of InnovaVector s.r.l. and of AAVantgarde Bio s.r.l. R.F. is currently an employee of AAVantgarde Bio s.r.l. T.C., M.B., and G.A. are listed as inventors of CAST-Seq (EP3856928B1)., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
- Full Text
- View/download PDF
34. Hypoxic extracellular vesicles from hiPSCs protect cardiomyocytes from oxidative damage by transferring antioxidant proteins and enhancing Akt/Erk/NRF2 signaling.
- Author
-
Bobis-Wozowicz S, Paw M, Sarna M, Kędracka-Krok S, Nit K, Błażowska N, Dobosz A, Hammad R, Cathomen T, Zuba-Surma E, Tyszka-Czochara M, and Madeja Z
- Subjects
- Humans, Oxidative Stress drug effects, Cell Hypoxia drug effects, Apoptosis drug effects, Extracellular Signal-Regulated MAP Kinases metabolism, Animals, Myocytes, Cardiac metabolism, Myocytes, Cardiac drug effects, Extracellular Vesicles metabolism, NF-E2-Related Factor 2 metabolism, Proto-Oncogene Proteins c-akt metabolism, Induced Pluripotent Stem Cells metabolism, Induced Pluripotent Stem Cells cytology, Signal Transduction drug effects, Antioxidants pharmacology
- Abstract
Background: Stem cell-derived extracellular vesicles (EVs) are an emerging class of therapeutics with excellent biocompatibility, bioactivity and pro-regenerative capacity. One of the potential targets for EV-based medicines are cardiovascular diseases (CVD). In this work we used EVs derived from human induced pluripotent stem cells (hiPSCs; hiPS-EVs) cultured under different oxygen concentrations (21, 5 and 3% O
2 ) to dissect the molecular mechanisms responsible for cardioprotection., Methods: EVs were isolated by ultrafiltration combined with size exclusion chromatography (UF + SEC), followed by characterization by nanoparticle tracking analysis, atomic force microscopy (AFM) and Western blot methods. Liquid chromatography and tandem mass spectrometry coupled with bioinformatic analyses were used to identify differentially enriched proteins in various oxygen conditions. We directly compared the cardioprotective effects of these EVs in an oxygen-glucose deprivation/reoxygenation (OGD/R) model of cardiomyocyte (CM) injury. Using advanced molecular biology, fluorescence microscopy, atomic force spectroscopy and bioinformatics techniques, we investigated intracellular signaling pathways involved in the regulation of cell survival, apoptosis and antioxidant response. The direct effect of EVs on NRF2-regulated signaling was evaluated in CMs following NRF2 inhibition with ML385., Results: We demonstrate that hiPS-EVs derived from physiological hypoxia at 5% O2 (EV-H5) exert enhanced cytoprotective function towards damaged CMs compared to EVs derived from other tested oxygen conditions (normoxia; EV-N and hypoxia 3% O2 ; EV-H3). This resulted from higher phosphorylation rates of Akt kinase in the recipient cells after transfer, modulation of AMPK activity and reduced apoptosis. Furthermore, we provide direct evidence for improved calcium signaling and sustained contractility in CMs treated with EV-H5 using AFM measurements. Mechanistically, our mass spectrometry and bioinformatics analyses revealed differentially enriched proteins in EV-H5 associated with the antioxidant pathway regulated by NRF2. In this regard, EV-H5 increased the nuclear translocation of NRF2 protein and enhanced its transcription in CMs upon OGD/R. In contrast, inhibition of NRF2 with ML385 abolished the protective effect of EVs on CMs., Conclusions: In this work, we demonstrate a superior cardioprotective function of EV-H5 compared to EV-N and EV-H3. Such EVs were most effective in restoring redox balance in stressed CMs, preserving their contractile function and preventing cell death. Our data support the potential use of hiPS-EVs derived from physiological hypoxia, as cell-free therapeutics with regenerative properties for the treatment of cardiac diseases., (© 2024. The Author(s).)- Published
- 2024
- Full Text
- View/download PDF
35. Engineering of potent CAR NK cells using non-viral Sleeping Beauty transposition from minimalistic DNA vectors.
- Author
-
Bexte T, Botezatu L, Miskey C, Gierschek F, Moter A, Wendel P, Reindl LM, Campe J, Villena-Ossa JF, Gebel V, Stein K, Cathomen T, Cremer A, Wels WS, Hudecek M, Ivics Z, and Ullrich E
- Subjects
- Humans, Animals, Mice, Xenograft Model Antitumor Assays, Transposases genetics, Transposases metabolism, Cell Line, Tumor, DNA Transposable Elements, Cytotoxicity, Immunologic, Precursor Cell Lymphoblastic Leukemia-Lymphoma therapy, Precursor Cell Lymphoblastic Leukemia-Lymphoma genetics, Precursor Cell Lymphoblastic Leukemia-Lymphoma immunology, Cell Engineering methods, Killer Cells, Natural immunology, Killer Cells, Natural metabolism, Genetic Vectors genetics, Receptors, Chimeric Antigen genetics, Receptors, Chimeric Antigen immunology, Receptors, Chimeric Antigen metabolism, Immunotherapy, Adoptive methods
- Abstract
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., Competing Interests: Declaration of interests E.U. is an Advisory Board member for Phialogics and has sponsored research projects with Gilead and BMS. Z.I. is an inventor on patents related to Sleeping Beauty and MC technology. M.H. is listed as inventor on patent applications and granted patents related to CAR technologies and transposon-based gene transfer that are, in part licensed to industry. M.H. is a co-founder and equity owner of T-CURX GmbH, Würzburg, Germany. M.H. and E.U. are inventors on patents related to CAR and MC technology. T.B., P.W., W.S.W., and E.U. are inventors on patents related to optimized CAR designs. T.B., P.W., and E.U. are inventors on non-viral gene-editing technologies of NK cells., (Copyright © 2024. Published by Elsevier Inc.)
- Published
- 2024
- Full Text
- View/download PDF
36. Integration of ζ-deficient CARs into the CD3ζ gene conveys potent cytotoxicity in T and NK cells.
- Author
-
Kath J, Franke C, Drosdek V, Du W, Glaser V, Fuster-Garcia C, Stein M, Zittel T, Schulenberg S, Porter CE, Andersch L, Künkele A, Alcaniz J, Hoffmann J, Abken H, Abou-El-Enein M, Pruß A, Suzuki M, Cathomen T, Stripecke R, Volk HD, Reinke P, Schmueck-Henneresse M, and Wagner DL
- Subjects
- Humans, Animals, Mice, T-Lymphocytes immunology, T-Lymphocytes metabolism, Cytotoxicity, Immunologic, Immunotherapy, Adoptive methods, Gene Editing methods, CRISPR-Cas Systems, Mice, Inbred NOD, Killer Cells, Natural immunology, Killer Cells, Natural metabolism, CD3 Complex genetics, Receptors, Chimeric Antigen genetics, Receptors, Chimeric Antigen immunology
- Abstract
Abstract: Chimeric antigen receptor (CAR)-redirected immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in nonphysiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Random gene transfer modalities pose a risk of malignant transformation by insertional mutagenesis. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3ζ (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3ζ gene as the CAR's activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR expression and redirection of various immune cell types, including conventional T cells, TCRγ/δ T cells, regulatory T cells, and NK cells. In T cells, CD3ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3ζ-CD19-CAR-T cells exhibited comparable leukemia control to TCRα chain constant (TRAC)-replaced and lentivirus-transduced CAR-T cells in vivo. Tuning of CD3ζ-CAR-expression levels significantly improved the in vivo efficacy. Notably, CD3ζ gene editing enabled redirection of NK cells without impairing their canonical functions. Thus, CD3ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes., (© 2024 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)
- Published
- 2024
- Full Text
- View/download PDF
37. CRISPR-Cas9n-mediated ELANE promoter editing for gene therapy of severe congenital neutropenia.
- Author
-
Nasri M, Ritter MU, Mir P, Dannenmann B, Kaufmann MM, Arreba-Tutusaus P, Xu Y, Borbaran-Bravo N, Klimiankou M, Lengerke C, Zeidler C, Cathomen T, Welte K, and Skokowa J
- Subjects
- Humans, Animals, Mice, Neutrophils metabolism, Hematopoietic Stem Cells metabolism, Mutation, Disease Models, Animal, Granulocyte Colony-Stimulating Factor genetics, Genetic Diseases, X-Linked therapy, Genetic Diseases, X-Linked genetics, Gene Editing methods, Neutropenia congenital, Neutropenia therapy, Neutropenia genetics, Genetic Therapy methods, Congenital Bone Marrow Failure Syndromes therapy, Congenital Bone Marrow Failure Syndromes genetics, CRISPR-Cas Systems, Promoter Regions, Genetic, Leukocyte Elastase genetics, Leukocyte Elastase metabolism
- Abstract
Severe congenital neutropenia (CN) is an inherited pre-leukemia bone marrow failure syndrome commonly caused by autosomal-dominant ELANE mutations (ELANE-CN). ELANE-CN patients are treated with daily injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, some patients do not respond to rhG-CSF, and approximately 15% of ELANE-CN patients develop myelodysplasia or acute myeloid leukemia. Here, we report the development of a curative therapy for ELANE-CN through inhibition of ELANE mRNA expression by introducing two single-strand DNA breaks at the opposing DNA strands of the ELANE promoter TATA box using CRISPR-Cas9D10A nickases-termed MILESTONE. This editing effectively restored defective neutrophil differentiation of ELANE-CN CD34
+ hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, without affecting the functions of the edited neutrophils. CRISPResso analysis of the edited ELANE-CN CD34+ HSPCs revealed on-target efficiencies of over 90%. Simultaneously, GUIDE-seq, CAST-Seq, and rhAmpSeq indicated a safe off-target profile with no off-target sites or chromosomal translocations. Taken together, ex vivo gene editing of ELANE-CN HSPCs using MILESTONE in the setting of autologous stem cell transplantation could be a universal, safe, and efficient gene therapy approach for ELANE-CN patients., Competing Interests: Declaration of interests M.N., P.M., and J.S. have filed a patent on the invention described in this study. T.C. is an advisor to Cimeio Therapeutics and Excision BioTherapeutics and holds a patent on CAST-Seq., (Copyright © 2024 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)- Published
- 2024
- Full Text
- View/download PDF
38. Generating universal anti-CD19 CAR T cells with a defined memory phenotype by CRISPR/Cas9 editing and safety evaluation of the transcriptome.
- Author
-
Pavlovic K, Carmona-Luque M, Corsi GI, Maldonado-Pérez N, Molina-Estevez FJ, Peralbo-Santaella E, Cortijo-Gutiérrez M, Justicia-Lirio P, Tristán-Manzano M, Ronco-Díaz V, Ballesteros-Ribelles A, Millán-López A, Heredia-Velázquez P, Fuster-García C, Cathomen T, Seemann SE, Gorodkin J, Martin F, Herrera C, and Benabdellah K
- Subjects
- Humans, T-Lymphocytes immunology, T-Lymphocytes metabolism, Phenotype, Cell Line, Tumor, CRISPR-Cas Systems, Immunotherapy, Adoptive methods, Immunotherapy, Adoptive adverse effects, Antigens, CD19 immunology, Antigens, CD19 genetics, Gene Editing methods, Transcriptome, Receptors, Chimeric Antigen genetics, Receptors, Chimeric Antigen immunology, Immunologic Memory
- Abstract
Introduction: Chimeric antigen receptor-expressing T cells (CAR T cells) have revolutionized cancer treatment, particularly in B cell malignancies. However, the use of autologous T cells for CAR T therapy presents several limitations, including high costs, variable efficacy, and adverse effects linked to cell phenotype., Methods: To overcome these challenges, we developed a strategy to generate universal and safe anti-CD19 CAR T cells with a defined memory phenotype. Our approach utilizes CRISPR/Cas9 technology to target and eliminate the B2M and TRAC genes, reducing graft-versus-host and host-versus-graft responses. Additionally, we selected less differentiated T cells to improve the stability and persistence of the universal CAR T cells. The safety of this method was assessed using our CRISPRroots transcriptome analysis pipeline, which ensures successful gene knockout and the absence of unintended off-target effects on gene expression or transcriptome sequence., Results: In vitro experiments demonstrated the successful generation of functional universal CAR T cells. These cells exhibited potent lytic activity against tumor cells and a reduced cytokine secretion profile. The CRISPRroots analysis confirmed effective gene knockout and no unintended off-target effects, validating it as a pioneering tool for on/off-target and transcriptome analysis in genome editing experiments., Discussion: Our findings establish a robust pipeline for manufacturing safe, universal CAR T cells with a favorable memory phenotype. This approach has the potential to address the current limitations of autologous CAR T cell therapy, offering a more stable and persistent treatment option with reduced adverse effects. The use of CRISPRroots enhances the reliability and safety of gene editing in the development of CAR T cell therapies., Conclusion: We have developed a potent and reliable method for producing universal CAR T cells with a defined memory phenotype, demonstrating both efficacy and safety in vitro . This innovative approach could significantly improve the therapeutic landscape for patients with B cell malignancies., Competing Interests: Authors MTM and PJL, were employed by LentiStem Biotech. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Pavlovic, Carmona-Luque, Corsi, Maldonado-Pérez, Molina-Estevez, Peralbo-Santaella, Cortijo-Gutiérrez, Justicia-Lirio, Tristán-Manzano, Ronco-Díaz, Ballesteros-Ribelles, Millán-López, Heredia-Velázquez, Fuster-García, Cathomen, Seemann, Gorodkin, Martin, Herrera and Benabdellah.)
- Published
- 2024
- Full Text
- View/download PDF
39. Deciphering bat influenza H18N11 infection dynamics in male Jamaican fruit bats on a single-cell level.
- Author
-
Kessler S, Burke B, Andrieux G, Schinköthe J, Hamberger L, Kacza J, Zhan S, Reasoner C, Dutt TS, Kaukab Osman M, Henao-Tamayo M, Staniek J, Villena Ossa JF, Frank DT, Ma W, Ulrich R, Cathomen T, Boerries M, Rizzi M, Beer M, Schwemmle M, Reuther P, Schountz T, and Ciminski K
- Subjects
- Animals, Male, Humans, Macrophages immunology, Macrophages virology, Influenza A virus genetics, Influenza A virus immunology, Gene Expression Profiling, Chiroptera virology, Chiroptera immunology, Chiroptera genetics, Single-Cell Analysis, Orthomyxoviridae Infections virology, Orthomyxoviridae Infections immunology, Orthomyxoviridae Infections veterinary
- Abstract
Jamaican fruit bats (Artibeus jamaicensis) naturally harbor a wide range of viruses of human relevance. These infections are typically mild in bats, suggesting unique features of their immune system. To better understand the immune response to viral infections in bats, we infected male Jamaican fruit bats with the bat-derived influenza A virus (IAV) H18N11. Using comparative single-cell RNA sequencing, we generated single-cell atlases of the Jamaican fruit bat intestine and mesentery. Gene expression profiling showed that H18N11 infection resulted in a moderate induction of interferon-stimulated genes and transcriptional activation of immune cells. H18N11 infection was predominant in various leukocytes, including macrophages, B cells, and NK/T cells. Confirming these findings, human leukocytes, particularly macrophages, were also susceptible to H18N11, highlighting the zoonotic potential of this bat-derived IAV. Our study provides insight into a natural virus-host relationship and thus serves as a fundamental resource for future in-depth characterization of bat immunology., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
40. On- and off-target effects of paired CRISPR-Cas nickase in primary human cells.
- Author
-
Klermund J, Rhiel M, Kocher T, Chmielewski KO, Bischof J, Andrieux G, El Gaz M, Hainzl S, Boerries M, Cornu TI, Koller U, and Cathomen T
- Subjects
- Humans, DNA Breaks, Double-Stranded, Chromosome Aberrations, Collagen Type VII genetics, Collagen Type VII metabolism, Cells, Cultured, CRISPR-Cas Systems, Gene Editing methods, Deoxyribonuclease I metabolism, Deoxyribonuclease I genetics, Keratinocytes metabolism
- Abstract
Undesired on- and off-target effects of CRISPR-Cas nucleases remain a challenge in genome editing. While the use of Cas9 nickases has been shown to minimize off-target mutagenesis, their use in therapeutic genome editing has been hampered by a lack of efficacy. To overcome this limitation, we and others have developed double-nickase-based strategies to generate staggered DNA double-strand breaks to mediate gene disruption or gene correction with high efficiency. However, the impact of paired single-strand nicks on genome integrity has remained largely unexplored. Here, we developed a novel CAST-seq pipeline, dual CAST, to characterize chromosomal aberrations induced by paired CRISPR-Cas9 nickases at three different loci in primary keratinocytes derived from patients with epidermolysis bullosa. While targeting COL7A1, COL17A1, or LAMA3 with Cas9 nucleases caused previously undescribed chromosomal rearrangements, no chromosomal translocations were detected following paired-nickase editing. While the double-nicking strategy induced large deletions/inversions within a 10 kb region surrounding the target sites at all three loci, similar to the nucleases, the chromosomal on-target aberrations were qualitatively different and included a high proportion of insertions. Taken together, our data indicate that double-nickase approaches combine efficient editing with greatly reduced off-target effects but still leave substantial chromosomal aberrations at on-target sites., Competing Interests: Declaration of interests T.C. is an advisor to Cimeio Therapeutics, Excision BioTherapeutics, GenCC, and Novo Nordisk. T.C. and T.I.C. have sponsored research collaborations with Cellectis and Cimeio Therapeutics, respectively. T.C., M.B., and G.A. hold a patent on CAST-seq (US11319580B2)., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
- Full Text
- View/download PDF
41. Artificial Targets: a versatile cell-free platform to characterize CAR T cell function in vitro .
- Author
-
Wang X, Tokarew NJA, Borgelt N, Siemer R, Melo CC, Langer C, Kasampalidis I, Ogusuku IEY, Cathomen T, Gessner I, Dose C, Fauerbach JA, Richter A, and Evaristo C
- Subjects
- Cell Line, Cell Proliferation, Coculture Techniques, Cytokines, Cell-Derived Microparticles
- Abstract
Cancer immunotherapies using chimeric antigen receptor (CAR) T cells have tremendous potential and proven clinical efficacy against a number of malignancies. Research and development are emerging to deepen the knowledge of CAR T cell efficacy and extend the therapeutic potential of this novel therapy. To this end, functional characterization of CAR T cells plays a central role in consecutive phases across fundamental research and therapeutic development, with increasing needs for standardization. The functional characterization of CAR T cells is typically achieved by assessing critical effector functions, following co-culture with cell lines expressing the target antigen. However, the use of target cell lines poses several limitations, including alterations in cell fitness, metabolic state or genetic drift due to handling and culturing of the cells, which would increase variabilities and could lead to inconsistent results. Moreover, the use of target cell lines can be work and time intensive, and introduce significant background due to the allogenic responses of T cells. To overcome these limitations, we developed a synthetic bead-based platform ("Artificial Targets") to characterize CAR T cell function in vitro . These synthetic microparticles could specifically induce CAR T cell activation, as measured by CD69 and CD137 (4-1BB) upregulation. In addition, engagement with Artificial Targets resulted in induction of multiple effector functions of CAR T cells mimicking the response triggered by target cell lines including cytotoxic activity, as assessed by exposure of CD107a (LAMP-1), expression and secretion of cytokines, as well as cell proliferation. Importantly, in contrast to target cells, stimulation with Artificial Targets showed limited unspecific CAR T cell proliferation. Finally, Artificial Targets demonstrated flexibility to engage multiple costimulatory molecules that can synergistically enhance the CAR T cell function and represented a powerful tool for modulating CAR T cell responses. Collectively, our results show that Artificial Targets can specifically activate CAR T cells for essential effector functions that could significantly advance standardization of functional assessment of CAR T cells, from early development to clinical applications., Competing Interests: XW, NT, NB, RS, CM, CL, IK, IO, IG, CD, JAF, AR and CE are employees of Miltenyi Biotec B.V. & Co. KG. The remaining authors declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Wang, Tokarew, Borgelt, Siemer, Melo, Langer, Kasampalidis, Ogusuku, Cathomen, Gessner, Dose, Fauerbach, Richter and Evaristo.)
- Published
- 2024
- Full Text
- View/download PDF
42. CRISPR-Cas12a for Highly Efficient and Marker-Free Targeted Integration in Human Pluripotent Stem Cells.
- Author
-
Hammad R, Alzubi J, Rhiel M, Chmielewski KO, Mosti L, Rositzka J, Heugel M, Lawrenz J, Pennucci V, Gläser B, Fischer J, Schambach A, Moritz T, Lachmann N, Cornu TI, Mussolino C, Schäfer R, and Cathomen T
- Subjects
- Humans, CRISPR-Cas Systems, Hematopoietic Stem Cells, Alleles, Pluripotent Stem Cells, Induced Pluripotent Stem Cells
- Abstract
The CRISPR-Cas12a platform has attracted interest in the genome editing community because the prototypical Acidaminococcus Cas12a generates a staggered DNA double-strand break upon binding to an AT-rich protospacer-adjacent motif (PAM, 5'-TTTV). The broad application of the platform in primary human cells was enabled by the development of an engineered version of the natural Cas12a protein, called Cas12a Ultra. In this study, we confirmed that CRISPR-Cas12a Ultra ribonucleoprotein complexes enabled allelic gene disruption frequencies of over 90% at multiple target sites in human T cells, hematopoietic stem and progenitor cells (HSPCs), and induced pluripotent stem cells (iPSCs). In addition, we demonstrated, for the first time, the efficient knock-in potential of the platform in human iPSCs and achieved targeted integration of a GFP marker gene into the AAVS1 safe harbor site and a CSF2RA super-exon into CSF2RA in up to 90% of alleles without selection. Clonal analysis revealed bi-allelic integration in >50% of the screened iPSC clones without compromising their pluripotency and genomic integrity. Thus, in combination with the adeno-associated virus vector system, CRISPR-Cas12a Ultra provides a highly efficient genome editing platform for performing targeted knock-ins in human iPSCs.
- Published
- 2024
- Full Text
- View/download PDF
43. Transient inhibition of 53BP1 increases the frequency of targeted integration in human hematopoietic stem and progenitor cells.
- Author
-
Baik R, Cromer MK, Glenn SE, Vakulskas CA, Chmielewski KO, Dudek AM, Feist WN, Klermund J, Shipp S, Cathomen T, Dever DP, and Porteus MH
- Subjects
- Humans, Animals, Mice, Hematopoietic Stem Cells metabolism, Recombinant Proteins metabolism, Peptides metabolism, CRISPR-Cas Systems, Gene Editing methods, Hematopoietic Stem Cell Transplantation
- Abstract
Genome editing by homology directed repair (HDR) is leveraged to precisely modify the genome of therapeutically relevant hematopoietic stem and progenitor cells (HSPCs). Here, we present a new approach to increasing the frequency of HDR in human HSPCs by the delivery of an inhibitor of 53BP1 (named "i53") as a recombinant peptide. We show that the use of i53 peptide effectively increases the frequency of HDR-mediated genome editing at a variety of therapeutically relevant loci in HSPCs as well as other primary human cell types. We show that incorporating the use of i53 recombinant protein allows high frequencies of HDR while lowering the amounts of AAV6 needed by 8-fold. HDR edited HSPCs were capable of long-term and bi-lineage hematopoietic reconstitution in NSG mice, suggesting that i53 recombinant protein might be safely integrated into the standard CRISPR/AAV6-mediated genome editing protocol to gain greater numbers of edited cells for transplantation of clinically meaningful cell populations., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
44. Efficient and safe therapeutic use of paired Cas9-nickases for primary hyperoxaluria type 1.
- Author
-
Torella L, Klermund J, Bilbao-Arribas M, Tamayo I, Andrieux G, Chmielewski KO, Vales A, Olagüe C, Moreno-Luqui D, Raimondi I, Abad A, Torrens-Baile J, Salido E, Huarte M, Hernaez M, Boerries M, Cathomen T, Zabaleta N, and Gonzalez-Aseguinolaza G
- Subjects
- Humans, Animals, Mice, Deoxyribonuclease I genetics, Deoxyribonuclease I metabolism, Gene Editing, CRISPR-Cas Systems, Hyperoxaluria, Primary genetics, Hyperoxaluria, Primary therapy
- Abstract
The therapeutic use of adeno-associated viral vector (AAV)-mediated gene disruption using CRISPR-Cas9 is limited by potential off-target modifications and the risk of uncontrolled integration of vector genomes into CRISPR-mediated double-strand breaks. To address these concerns, we explored the use of AAV-delivered paired Staphylococcus aureus nickases (D10ASaCas9) to target the Hao1 gene for the treatment of primary hyperoxaluria type 1 (PH1). Our study demonstrated effective Hao1 gene disruption, a significant decrease in glycolate oxidase expression, and a therapeutic effect in PH1 mice. The assessment of undesired genetic modifications through CIRCLE-seq and CAST-Seq analyses revealed neither off-target activity nor chromosomal translocations. Importantly, the use of paired-D10ASaCas9 resulted in a significant reduction in AAV integration at the target site compared to SaCas9 nuclease. In addition, our study highlights the limitations of current analytical tools in characterizing modifications introduced by paired D10ASaCas9, necessitating the development of a custom pipeline for more accurate characterization. These results describe a positive advance towards a safe and effective potential long-term treatment for PH1 patients., (© 2024. The Author(s).)
- Published
- 2024
- Full Text
- View/download PDF
45. Gene editing of hematopoietic stem cells restores T-cell response in familial hemophagocytic lymphohistiocytosis.
- Author
-
Dettmer-Monaco V, Weißert K, Ammann S, Monaco G, Lei L, Gräßel L, Rhiel M, Rositzka J, Kaufmann MM, Geiger K, Andrieux G, Lao J, Thoulass G, Schell C, Boerries M, Illert AL, Cornu TI, Ehl S, Aichele P, and Cathomen T
- Subjects
- Humans, Mice, Animals, T-Lymphocytes, Gene Editing, Mutation, Lymphocytic choriomeningitis virus, Hematopoietic Stem Cells, Membrane Proteins genetics, Lymphohistiocytosis, Hemophagocytic genetics, Lymphohistiocytosis, Hemophagocytic therapy, Lymphohistiocytosis, Hemophagocytic diagnosis
- Abstract
Background: Hemophagocytic lymphohistiocytosis (HLH) is a hyperinflammatory disorder characterized by a life-threatening cytokine storm and immunopathology. Familial HLH type 3 (FHL3) accounts for approximately 30% of all inborn HLH cases worldwide. It is caused by mutations in the UNC13D gene that result in impaired degranulation of cytotoxic vesicles and hence compromised T-cell- and natural killer-cell-mediated killing. Current treatment protocols, including allogeneic hematopoietic stem cell (HSC) transplantation, still show high mortality., Objective: We sought to develop and evaluate a curative genome editing strategy in the preclinical FHL3 Jinx mouse model. Jinx mice harbor a cryptic splice donor site in Unc13d intron 26 and develop clinical symptoms of human FHL3 upon infection with lymphocytic choriomeningitis virus (LCMV)., Methods: We employed clustered regularly interspaced short palindromic repeats (CRISPR)-Cas technology to delete the disease-causing mutation in HSCs and transplanted Unc13d-edited stem cells into busulfan-conditioned Jinx recipient mice. Safety studies included extensive genotyping and chromosomal aberrations analysis by single targeted linker-mediated PCR sequencing (CAST-Seq)-based off-target analyses. Cure from HLH predisposition was assessed by LCMV infection., Results: Hematopoietic cells isolated from transplanted mice revealed efficient gene editing (>95%), polyclonality of the T-cell receptor repertoire, and neither signs of off-target effects nor leukemogenesis. Unc13d transcription levels of edited and wild-type cells were comparable. While LCMV challenge resulted in acute HLH in Jinx mice transplanted with mock-edited HSCs, Jinx mice grafted with Unc13d-edited cells showed rapid virus clearance and protection from HLH., Conclusions: Our study demonstrates that transplantation of CRISPR-Cas edited HSCs supports the development of a functional polyclonal T-cell response in the absence of genotoxicity-associated clonal outgrowth., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
- Full Text
- View/download PDF
46. Epitope-engineered human hematopoietic stem cells are shielded from CD123-targeted immunotherapy.
- Author
-
Marone R, Landmann E, Devaux A, Lepore R, Seyres D, Zuin J, Burgold T, Engdahl C, Capoferri G, Dell'Aglio A, Larrue C, Simonetta F, Rositzka J, Rhiel M, Andrieux G, Gallagher DN, Schröder MS, Wiederkehr A, Sinopoli A, Do Sacramento V, Haydn A, Garcia-Prat L, Divsalar C, Camus A, Xu L, Bordoli L, Schwede T, Porteus M, Tamburini J, Corn JE, Cathomen T, Cornu TI, Urlinger S, and Jeker LT
- Subjects
- Humans, Epitopes, Immunotherapy, Hematopoietic Stem Cells metabolism, Immunotherapy, Adoptive, Interleukin-3 Receptor alpha Subunit metabolism, Leukemia, Myeloid, Acute genetics, Leukemia, Myeloid, Acute therapy
- Abstract
Targeted eradication of transformed or otherwise dysregulated cells using monoclonal antibodies (mAb), antibody-drug conjugates (ADC), T cell engagers (TCE), or chimeric antigen receptor (CAR) cells is very effective for hematologic diseases. Unlike the breakthrough progress achieved for B cell malignancies, there is a pressing need to find suitable antigens for myeloid malignancies. CD123, the interleukin-3 (IL-3) receptor alpha-chain, is highly expressed in various hematological malignancies, including acute myeloid leukemia (AML). However, shared CD123 expression on healthy hematopoietic stem and progenitor cells (HSPCs) bears the risk for myelotoxicity. We demonstrate that epitope-engineered HSPCs were shielded from CD123-targeted immunotherapy but remained functional, while CD123-deficient HSPCs displayed a competitive disadvantage. Transplantation of genome-edited HSPCs could enable tumor-selective targeted immunotherapy while rebuilding a fully functional hematopoietic system. We envision that this approach is broadly applicable to other targets and cells, could render hitherto undruggable targets accessible to immunotherapy, and will allow continued posttransplant therapy, for instance, to treat minimal residual disease (MRD)., (© 2023 Marone et al.)
- Published
- 2023
- Full Text
- View/download PDF
47. Integration of ζ-deficient CARs into the CD3-zeta gene conveys potent cytotoxicity in T and NK cells.
- Author
-
Kath J, Franke C, Drosdek V, Du W, Glaser V, Fuster-Garcia C, Stein M, Zittel T, Schulenberg S, Porter CE, Andersch L, Künkele A, Alcaniz J, Hoffmann J, Abken H, Abou-El-Enein M, Pruß A, Suzuki M, Cathomen T, Stripecke R, Volk HD, Reinke P, Schmueck-Henneresse M, and Wagner DL
- Abstract
Chimeric antigen receptor (CAR)-reprogrammed immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in non-physiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3 ζ (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3 ζ gene as the CAR's activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR-expression and reprogramming of various immune cell types, including conventional T cells, TCRγ/δ T cells, regulatory T cells, and NK cells. In T cells, CD3 ζ in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3 ζ-CD19-CAR-T cells exhibited comparable leukemia control to T cell receptor alpha constant ( TRAC )-replaced and lentivirus-transduced CAR-T cells in vivo . Tuning of CD3 ζ-CAR-expression levels significantly improved the in vivo efficacy. Compared to TRAC -edited CAR-T cells, integration of a Her2-CAR into CD3 ζ conveyed similar in vitro tumor lysis but reduced susceptibility to activation-induced cell death and differentiation, presumably due to lower CAR-expression levels. Notably, CD3 ζ gene editing enabled reprogramming of NK cells without impairing their canonical functions. Thus, CD3 ζ gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes., Key Points: Integration of ζ-deficient CARs into CD3 ζ gene allows generation of functional TCR-ablated CAR-T cells for allogeneic off-the-shelf use CD3 ζ-editing platform allows CAR reprogramming of NK cells without affecting their canonical functions.
- Published
- 2023
- Full Text
- View/download PDF
48. Cell-Based Models of 'Cytokine Release Syndrome' Endorse CD40L and Granulocyte-Macrophage Colony-Stimulating Factor Knockout in Chimeric Antigen Receptor T Cells as Mitigation Strategy.
- Author
-
Dibas A, Rhiel M, Patel VB, Andrieux G, Boerries M, Cornu TI, Alzubi J, and Cathomen T
- Subjects
- Humans, Animals, Mice, CD40 Ligand, Cytokine Release Syndrome, Interleukin-6, Mice, Knockout, T-Lymphocytes, Granulocyte-Macrophage Colony-Stimulating Factor genetics, Receptors, Chimeric Antigen genetics
- Abstract
While chimeric antigen receptor (CAR) T cell therapy has shown promising outcomes among patients with hematologic malignancies, it has also been associated with undesirable side-effects such as cytokine release syndrome (CRS). CRS is triggered by CAR T-cell-based activation of monocytes, which are stimulated via the CD40L-CD40R axis or via uptake of GM-CSF to secrete proinflammatory cytokines. Mouse models have been used to model CRS, but working with them is labor-intensive and they are not amenable to screening approaches. To overcome this challenge, we established two simple cell-based CRS in vitro models that entail the co-culturing of leukemic B cells with CD19-targeting CAR T cells and primary monocytes from the same donor. Upon antigen encounter, CAR T cells upregulated CD40L and released GM-CSF which in turn stimulated the monocytes to secrete IL-6. To endorse these models, we demonstrated that neutralizing antibodies or genetic disruption of the CD40L and/or CSF2 loci in CAR T cells using CRISPR-Cas technology significantly reduced IL-6 secretion by bystander monocytes without affecting the cytolytic activity of the engineered lymphocytes in vitro. Overall, our cell-based models were able to recapitulate CRS in vitro, allowing us to validate mitigation strategies based on antibodies or genome editing.
- Published
- 2023
- Full Text
- View/download PDF
49. Hypoxia enhances anti-fibrotic properties of extracellular vesicles derived from hiPSCs via the miR302b-3p/TGFβ/SMAD2 axis.
- Author
-
Paw M, Kusiak AA, Nit K, Litewka JJ, Piejko M, Wnuk D, Sarna M, Fic K, Stopa KB, Hammad R, Barczyk-Woznicka O, Cathomen T, Zuba-Surma E, Madeja Z, Ferdek PE, and Bobis-Wozowicz S
- Subjects
- Animals, Humans, Mice, Disease Models, Animal, Fibrosis, Hypoxia, Oxygen, Smad2 Protein genetics, Smad2 Protein metabolism, Transforming Growth Factor beta metabolism, Extracellular Vesicles metabolism, Induced Pluripotent Stem Cells metabolism, MicroRNAs genetics, MicroRNAs metabolism
- Abstract
Background: Cardiac fibrosis is one of the top killers among fibrotic diseases and continues to be a global unaddressed health problem. The lack of effective treatment combined with the considerable socioeconomic burden highlights the urgent need for innovative therapeutic options. Here, we evaluated the anti-fibrotic properties of extracellular vesicles (EVs) derived from human induced pluripotent stem cells (hiPSCs) that were cultured under various oxygen concentrations., Methods: EVs were isolated from three hiPSC lines cultured under normoxia (21% O
2 ; EV-N) or reduced oxygen concentration (hypoxia): 3% O2 (EV-H3) or 5% O2 (EV-H5). The anti-fibrotic activity of EVs was tested in an in vitro model of cardiac fibrosis, followed by a detailed investigation of the underlying molecular mechanisms. Sequencing of EV miRNAs combined with bioinformatics analysis was conducted and a selected miRNA was validated using a miRNA mimic and inhibitor. Finally, EVs were tested in a mouse model of angiotensin II-induced cardiac fibrosis., Results: We provide evidence that an oxygen concentration of 5% enhances the anti-fibrotic effects of hiPS-EVs. These EVs were more effective in reducing pro-fibrotic markers in activated human cardiac fibroblasts, when compared to EV-N or EV-H3. We show that EV-H5 act through the canonical TGFβ/SMAD pathway, primarily via miR-302b-3p, which is the most abundant miRNA in EV-H5. Our results show that EV-H5 not only target transcripts of several profibrotic genes, including SMAD2 and TGFBR2, but also reduce the stiffness of activated fibroblasts. In a mouse model of heart fibrosis, EV-H5 outperformed EV-N in suppressing the inflammatory response in the host and by attenuating collagen deposition and reducing pro-fibrotic markers in cardiac tissue., Conclusions: In this work, we provide evidence of superior anti-fibrotic properties of EV-H5 over EV-N or EV-H3. Our study uncovers that fine regulation of oxygen concentration in the cellular environment may enhance the anti-fibrotic effects of hiPS-EVs, which has great potential to be applied for heart regeneration., (© 2023. The Author(s).)- Published
- 2023
- Full Text
- View/download PDF
50. Progress and harmonization of gene editing to treat human diseases: Proceeding of COST Action CA21113 GenE-HumDi.
- Author
-
Cavazza A, Hendel A, Bak RO, Rio P, Güell M, Lainšček D, Arechavala-Gomeza V, Peng L, Hapil FZ, Harvey J, Ortega FG, Gonzalez-Martinez C, Lederer CW, Mikkelsen K, Gasiunas G, Kalter N, Gonçalves MAFV, Petersen J, Garanto A, Montoliu L, Maresca M, Seemann SE, Gorodkin J, Mazini L, Sanchez R, Rodriguez-Madoz JR, Maldonado-Pérez N, Laura T, Schmueck-Henneresse M, Maccalli C, Grünewald J, Carmona G, Kachamakova-Trojanowska N, Miccio A, Martin F, Turchiano G, Cathomen T, Luo Y, Tsai SQ, and Benabdellah K
- Abstract
The European Cooperation in Science and Technology (COST) is an intergovernmental organization dedicated to funding and coordinating scientific and technological research in Europe, fostering collaboration among researchers and institutions across countries. Recently, COST Action funded the "Genome Editing to treat Human Diseases" (GenE-HumDi) network, uniting various stakeholders such as pharmaceutical companies, academic institutions, regulatory agencies, biotech firms, and patient advocacy groups. GenE-HumDi's primary objective is to expedite the application of genome editing for therapeutic purposes in treating human diseases. To achieve this goal, GenE-HumDi is organized in several working groups, each focusing on specific aspects. These groups aim to enhance genome editing technologies, assess delivery systems, address safety concerns, promote clinical translation, and develop regulatory guidelines. The network seeks to establish standard procedures and guidelines for these areas to standardize scientific practices and facilitate knowledge sharing. Furthermore, GenE-HumDi aims to communicate its findings to the public in accessible yet rigorous language, emphasizing genome editing's potential to revolutionize the treatment of many human diseases. The inaugural GenE-HumDi meeting, held in Granada, Spain, in March 2023, featured presentations from experts in the field, discussing recent breakthroughs in delivery methods, safety measures, clinical translation, and regulatory aspects related to gene editing., Competing Interests: P.R. has licensed the PGK:FANCAWpre∗ LV medicinal product and receives funding and equity from Rocket Pharmaceuticals, Inc., patents and royalties, research & consulting funding. D.L. is an inventor on a patent National Institute of Chemistry filed (WO/2021/032759 patent application, European patent application EP 3783104, China patent application CN 114269930 with National Phase entry EP2020756868). R.O.B. holds patents related to CRISPR-Cas genome editing and has equity in Graphite Bio and is consultant for UNIKUM Tx. G.G. holds patents related to CRISPR-Cas genome editing, is an employee of CasZyme, and has equity in CasZyme. S.Q.T. is a co-inventor on patents for GUIDE-seq, CHANGE-seq, and other genome editing technologies and a member of the scientific advisory boards of Prime Medicine and Ensoma. T.C. is a co-inventor on patents for CAST-seq, Abnoba-Seq, and other genome editing technologies, and a member of the scientific advisory boards of Cimeo Therapeutics, Excision BioTherapeutics, and GenCC. A.C. and G.T. are inventors on a patent for MEGA (WO/2023/079285), G.T. is also co-inventor on a patent for CAST-seq., (© 2023 The Author(s).)
- Published
- 2023
- Full Text
- View/download PDF
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.