144 results on '"Buchholz CJ"'
Search Results
2. P06.13 A novel local treatment approach? Targeted immunotherapy of glioblastoma via AAV-mediated gene transfer of checkpoint inhibitors through locally administered HER2-AAVs in combination with CAR-NK cells
- Author
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Strecker, MI, primary, Strassheimer, F, additional, Reul, J, additional, Harter, PN, additional, Tonn, T, additional, Steinbach, JP, additional, Wels, WS, additional, Buchholz, CJ, additional, and Burger, MC, additional
- Published
- 2020
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3. Holo-APP and G-protein-mediated signaling are required for sAPPa-induced activation of the Akt survival pathway
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Milosch, N, Tanriöver, G, Kundu, A, Rami, A, Francois, J-C, Baumkötter, F, Weyer, SW, Samanta, Ayan, Jäschke, A, Brod, F, Buchholz, CJ, Kins, S, Behl, C, Müller, UC, Kögel, D, Milosch, N, Tanriöver, G, Kundu, A, Rami, A, Francois, J-C, Baumkötter, F, Weyer, SW, Samanta, Ayan, Jäschke, A, Brod, F, Buchholz, CJ, Kins, S, Behl, C, Müller, UC, and Kögel, D
- Abstract
Accumulating evidence indicates that loss of physiologic amyloid precursor protein (APP) function leads to reduced neuronal plasticity, diminished synaptic signaling and enhanced susceptibility of neurons to cellular stress during brain aging. Here we investigated the neuroprotective function of the soluble APP ectodomain sAPPα (soluble APPα), which is generated by cleavage of APP by α-secretase along the non-amyloidogenic pathway. Recombinant sAPPα protected primary hippocampal neurons and SH-SY5Y neuroblastoma cells from cell death induced by trophic factor deprivation. We show that this protective effect is abrogated in neurons from APP-knockout animals and APP-depleted SH-SY5Y cells, but not in APP-like protein 1- and 2- (APLP1 and APLP2) depleted cells, indicating that expression of membrane-bound holo-APP is required for sAPPα-dependent neuroprotection. Trophic factor deprivation diminished the activity of the Akt survival pathway. Strikingly, both recombinant sAPPα and the APP-E1 domain were able to stimulate Akt activity in wild-type (wt) fibroblasts, SH-SY5Y cells and neurons, but failed to rescue in APP-deficient neurons or fibroblasts. The ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10) inhibitor GI254023X exacerbated neuron death in organotypic (hippocampal) slice cultures of wt mice subjected to trophic factor and glucose deprivation. This cell death-enhancing effect of GI254023X could be completely rescued by applying exogenous sAPPα. Interestingly, sAPPα-dependent Akt induction was unaffected in neurons of APP-ΔCT15 mice that lack the C-terminal YENPTY motif of the APP intracellular region. In contrast, sAPPα-dependent rescue of Akt activation was completely abolished in APP mutant cells lacking the G-protein interaction motif located in the APP C-terminus and by blocking G-protein-dependent signaling with pertussis toxin. Collectively, our data provide new mechanistic insights into the physiologic role of APP in antagonizing
- Published
- 2014
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4. Restriction of measles virus RNA synthesis by a mouse host cell. Trans-complementation by polymerase components or human cellular factor(s)
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S Tigaud I Schneider H Buchholz Cj Yanagi Y Gerlier D., Vincent, Virologie et pathogenèse virale (VPV), Centre National de la Recherche Scientifique (CNRS)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon, Laboratoire de Cytogénétique Moléculaire, Hôpital Edouard Herriot [CHU - HCL], Hospices Civils de Lyon (HCL)-Hospices Civils de Lyon (HCL), Institut für Molekularbiologie, Universität Zürich [Zürich] = University of Zurich (UZH), Department of Virology, Faculty of Medicine, Kyushu University, and This work was performed with financial support from the Commission of European Communities (RTD programme 'Quality of Life and Management of Living Resources' [QLK2-CT2001-01225]) and from the Ministère de l'Education Nationale de la Recherche et de la Technologie (PRFMMIP). The content of this publication does not necessarily reflect the views of the Commission of European Communities and in no way anticipates the Commission's future policy in this area.
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Glycoproteins/genetics/*metabolism ,Genetic Complementation Test ,Viral/*biosynthesis/genetics ,Immunoglobulins/genetics/*metabolism ,Epithelial Cells/virology ,Hybrid Cells/virology ,DNA-Directed RNA Polymerases/genetics/*metabolism ,Viral Proteins/genetics/metabolism ,Research Support ,Virus Replication ,Cell Line ,CD/genetics/*metabolism ,Measles virus/genetics/pathogenicity/*physiology ,Mice ,Hela Cells/virology ,[SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology ,Animals ,Humans ,RNA ,Antigens ,Non-U.S. Gov't ,OCIS 000.1430 ,CD46 ,Membrane Glycoproteins/genetics/*metabolism ,Intestines/cytology - Abstract
The mouse epithelial MODE-K cell line expressing human CD46 or CD150 cellular receptors was found to be nonpermissive for measles virus (MV) replication. The virus binding and membrane fusion steps were unimpaired, but only very limited amounts of virus protein and RNA synthesized were detected after the infection. In a minigenome chloramphenicol acetyltransferase assay, MODE-K cells were as able as the permissive HeLa cells in supporting MV polymerase activity. The restriction phenotype of MODE-K cells could be alleviated by providing, in trans, either N-P-L or N-P functional protein complexes but not by P-L complexes or individual N, P, and L proteins. Several human x mouse (HeLa x MODE-K) somatic hybrid clones expressing human CD46 were isolated and found to be either nonpermissive or permissive according to their human chromosomal contents. The MV-restricted phenotype exhibited by the MODE-K cell line suggests that a cellular factor(s) can control MV transcription, possibly by stabilizing the incoming virus polymerase templates.
- Published
- 2006
5. Pseudotyping lentiviral vectors with the wild-type measles virus glycoproteins improves titer and selectivity.
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Funke, S., Schneider, IC, Glaser, S., Mühlebach, MD, Moritz, T., Cattaneo, R., Cichutek, K., and Buchholz, CJ
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MEASLES ,GLYCOPROTEINS ,VIRUS diseases ,PARAMYXOVIRUSES ,LYMPHOCYTE transformation - Abstract
We pseudotyped HIV-1 vectors with cytoplasmic tail-truncated envelope glycoproteins of a wild-type (WT) measles virus (MV). The particles entered the lymphatic cells exclusively through the signaling lymphocyte activation molecule (SLAM, CD150), whereas particles pseudotyped with the MV vaccine strain glycoproteins also recognized the ubiquitous membrane cofactor protein (CD46) as receptor and had less specific cell entry. MV
WT -HIV vectors reached titers of 108 t.u. ml−1 , which were up to 10-fold higher than those of MVVac -HIV vectors, and discriminated between SLAM-positive and SLAM-negative cells, also in mixed cell cultures. As these vectors transduce primary human cells more efficiently than vesicular stomatitis virus-G pseudotyped vectors do, they are promising candidates for gene transfer to human lymphocytes and certain epithelial cells.Gene Therapy (2009) 16, 700–705; doi:10.1038/gt.2009.11; published online 12 February 2009 [ABSTRACT FROM AUTHOR]- Published
- 2009
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6. T-cell specific in vivo gene delivery with DART-AAVs targeted to CD8.
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Demircan MB, Zinser LJ, Michels A, Guaza-Lasheras M, John F, Gorol JM, Theuerkauf SA, Günther DM, Grimm D, Greten FR, Chlanda P, Thalheimer FB, and Buchholz CJ
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- Animals, Mice, Humans, Transduction, Genetic, Capsid Proteins genetics, Dependovirus genetics, CD8-Positive T-Lymphocytes immunology, CD8-Positive T-Lymphocytes metabolism, Genetic Vectors administration & dosage, Genetic Vectors genetics, Gene Transfer Techniques, Genetic Therapy methods
- Abstract
One of the biggest challenges for in vivo gene therapy are vectors mediating highly selective gene transfer into a defined population of therapy-relevant cells. Here we present DARPin-targeted AAVs (DART-AAVs) displaying DARPins specific for human and murine CD8. Insertion of DARPins into the GH2/GH3 loop of the capsid protein 1 (VP1) of AAV2 and AAV6 resulted in high selectivity for CD8-positive T cells with unimpaired gene delivery activity. Remarkably, the capsid core structure was unaltered with protruding DARPins detectable. In complex primary cell mixtures, including donor blood or systemic injections into mice, the CD8-targeted AAVs were by far superior to unmodified AAV2 and AAV6 in terms of selectivity, target cell viability, and gene transfer rates. In vivo, up to 80% of activated CD8+ T cells were hit upon a single vector injection into conditioned humanized or immunocompetent mice. While gene transfer rates decreased significantly under non-activated conditions, genomic modification selectively in CD8+ T cells was still detectable upon Cre delivery into indicator mice. In both mouse models, selectivity for CD8+ T cells was close to absolute with exceptional detargeting from liver. The CD8-AAVs described here expand strategies for immunological research and in vivo gene therapy options., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)
- Published
- 2024
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7. In vivo CAR T cell therapy against angioimmunoblastic T cell lymphoma.
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Krug A, Saidane A, Martinello C, Fusil F, Michels A, Buchholz CJ, Ricci JE, and Verhoeyen E
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- Animals, Mice, Humans, Immunoblastic Lymphadenopathy therapy, Immunoblastic Lymphadenopathy immunology, CD8-Positive T-Lymphocytes immunology, Cell Line, Tumor, Receptors, Chimeric Antigen immunology, Immunotherapy, Adoptive methods, Lymphoma, T-Cell therapy, Lymphoma, T-Cell immunology, Lymphoma, T-Cell pathology, Disease Models, Animal
- Abstract
Background: For angioimmunoblastic T cell lymphoma (AITL), a rare cancer, no specific treatments are available and survival outcome is poor. We previously developed a murine model for AITL that mimics closely human disease and allows to evaluate new treatments. As in human AITL, the murine CD4
+ follicular helper T (Tfh) cells are drivers of the malignancy. Therefore, chimeric antigen receptor (CAR) T cell therapy might represent a new therapeutic option., Methods: To prevent fratricide among CAR T cells when delivering an CD4-specific CAR, we used a lentiviral vector (LV) encoding an anti-CD4 CAR, allowing exclusive entry into CD8 T cells., Results: These anti-CD4CAR CD8-targeted LVs achieved in murine AITL biopsies high CAR-expression levels in CD8 T cells. Malignant CD4 Tfh cells were eliminated from the mAITL lymphoma, while the CAR + CD8 T cells expanded upon encounter with the CD4 receptor and were shaped into functional cytotoxic cells. Finally, in vivo injection of the CAR + CD8-LVs into our preclinical AITL mouse model carrying lymphomas, significantly prolonged mice survival. Moreover, the in vivo generated functional CAR + CD8 T cells efficiently reduced neoplastic T cell numbers in the mAITL tumors., Conclusion: This is the first description of in vivo generated CAR T cells for therapy of a T cell lymphoma. The strategy described offers a new therapeutic concept for patients suffering from CD4-driven T cell lymphomas., (© 2024. The Author(s).)- Published
- 2024
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8. Early induction of cytokine release syndrome by rapidly generated CAR T cells in preclinical models.
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Jamali A, Ho N, Braun A, Adabi E, Thalheimer FB, and Buchholz CJ
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- Humans, Animals, Mice, Interleukin-10, Interleukin-6, Tumor Necrosis Factor-alpha, Interleukin-2, Cytokines, Immunotherapy, Adoptive, T-Lymphocytes, Cytokine Release Syndrome, Neoplasms
- Abstract
Cytokine release syndrome (CRS) is a significant side-effect of conventional chimeric antigen receptor (CAR) T-cell therapy. To facilitate patient accessibility, short-term (st) CAR T cells, which are administered to patients only 24 h after vector exposure, are in focus of current investigations. Their impact on the incidence and severity of CRS has been poorly explored. Here, we evaluated CD19-specific stCAR T cells in preclinical models. In co-culture with tumor cells and monocytes, stCAR T cells exhibited anti-tumoral activity and potent release of CRS-related cytokines (IL-6, IFN-γ, TNF-α, GM-CSF, IL-2, IL-10). When administered to NSG-SGM3 mice, stCAR T cells, but not conventional CAR T cells, induced severe acute adverse events within 24 h, including hypothermia and weight loss, as well as high body scores, independent of the presence of tumor target cells. Human (IFN-γ, TNF-α, IL-2, IL-10) and murine (MCP-1, IL-6, G-CSF) cytokines, typical for severe CRS, were systemically elevated. Our data highlight potential safety risks of rapidly manufactured CAR T cells and suggest NSG-SGM3 mice as sensitive model for their preclinical safety evaluation., (© 2024. The Author(s).)
- Published
- 2024
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9. Heterogeneity of layer 4 in visual areas of rhesus macaque cortex.
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Günther DM, Batiuk MY, Petukhov V, De Oliveira R, Wunderle T, Buchholz CJ, Fries P, and Khodosevich K
- Abstract
Recently, single-cell RNA-sequencing (scRNA-seq) has enabled unprecedented insights to the cellular landscape of the brains of many different species, among them the rhesus macaque as a key animal model. Building on previous, broader surveys of the macaque brain, we closely examined five immediately neighboring areas within the visual cortex of the rhesus macaque: V1, V2, V4, MT and TEO. To facilitate this, we first devised a novel pipeline for brain spatial archive - the BrainSPACE - which enabled robust archiving and sampling from the whole unfixed brain. SnRNA-sequencing of ~100,000 nuclei from visual areas V1 and V4 revealed conservation within the GABAergic neuron subtypes, while seven and one distinct principle neuron subtypes were detected in V1 and V4, respectively, all most likely located in layer 4. Moreover, using small molecule fluorescence in situ hybridization, we identified cell type density gradients across V1, V2, V4, MT, and TEO appearing to reflect the visual hierarchy. These findings demonstrate an association between the clear areal specializations among neighboring areas with the hierarchical levels within the visual cortex of the rhesus macaque., Competing Interests: Competing interests: P.F. has a patent on thin-film electrodes and is a member of the Advisory Board of CorTec GmbH (Freiburg, Germany). All other authors declare they have no competing interests.
- Published
- 2024
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10. External validation of novel Revised Intensity Battle Score and comparison of static rib fracture scoring systems.
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Buchholz CJ, D'Aquila ML, and Lollar DI
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- Adult, Humans, Retrospective Studies, Prospective Studies, Injury Severity Score, Length of Stay, Rib Fractures complications, Rib Fractures diagnosis, Rib Fractures therapy, Thoracic Injuries
- Abstract
Background: This study aims to compare and externally validate the previously developed Revised Intensity Battle Score (RIBS) against other proposed scores for predicting poor outcomes after rib fractures., Methods: An external validation set was assembled retrospectively, comprising 1,493 adult patients with one or more rib fractures admitted to a Level 1 trauma center between 2019 and 2022. The following rib fracture scores were calculated for each patient: RIBS, Injury Severity Score, Rib Fracture Score, Chest Trauma Score, and Battle score. Each was investigated to assess utility in predicting mortality, intensive care unit upgrade, unplanned intubation and ventilator days. Performance was measured by area under the receiver operating characteristic curve., Results: Of the 1,493 patients who met inclusion criteria, 239 patients (16%) experienced one of more of the investigated outcomes. Generally, scores performed best at predicting mortality and ventilator days. The RIBS stood out as best predicting "any complication" (AUC = 0.735) and ">7 ventilator days" (AUC = 0.771)., Conclusion: The RIBS represents an externally validated triage score in patients with rib fractures and compares favorably to other static scoring systems. Use of this score as a triage tool may allow stratifying patients who may benefit from direct intensive care unit admission, neuraxial anesthesia and aggressive respiratory care. Next steps include prospective investigation of how pairing these interventions with score directed triage impacts outcomes., Level of Evidence: Prognostic and Epidemiological; Level IV., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)
- Published
- 2024
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11. A new age of precision gene therapy.
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Schambach A, Buchholz CJ, Torres-Ruiz R, Cichutek K, Morgan M, Trapani I, and Büning H
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- Humans, Genetic Therapy, CRISPR-Cas Systems, Gene Editing
- Abstract
Gene therapy has become a clinical reality as market-approved advanced therapy medicinal products for the treatment of distinct monogenetic diseases and B-cell malignancies. This Therapeutic Review aims to explain how progress in genome editing technologies offers the possibility to expand both therapeutic options and the types of diseases that will become treatable. To frame these impressive advances in the context of modern medicine, we incorporate examples from human clinical trials into our discussion on how genome editing will complement currently available strategies in gene therapy, which still mainly rely on gene addition strategies. Furthermore, safety considerations and ethical implications, including the issue of accessibility, are addressed as these crucial parameters will define the impact that gene therapy in general and genome editing in particular will have on how we treat patients in the near future., Competing Interests: Declaration of interests We declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)
- Published
- 2024
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12. AAV vectors displaying bispecific DARPins enable dual-control targeted gene delivery.
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Theuerkauf SA, Herrera-Carrillo E, John F, Zinser LJ, Molina MA, Riechert V, Thalheimer FB, Börner K, Grimm D, Chlanda P, Berkhout B, and Buchholz CJ
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- Mice, Humans, Animals, Transduction, Genetic, Dependovirus genetics, Genetic Vectors genetics, Genetic Therapy, Designed Ankyrin Repeat Proteins, HIV Infections
- Abstract
Precise delivery of genes to therapy-relevant cells is crucial for in vivo gene therapy. Receptor-targeting as prime strategy for this purpose is limited to cell types defined by a single cell-surface marker. Many target cells are characterized by combinations of more than one marker, such as the HIV reservoir cells. Here, we explored the tropism of adeno-associated viral vectors (AAV2) displaying designed ankyrin repeat proteins (DARPins) mono- and bispecific for CD4 and CD32a. Cryo-electron tomography revealed an unaltered capsid structure in the presence of DARPins. Surprisingly, bispecific AAVs transduced CD4/CD32a double-positive cells at much higher efficiencies than single-positive cells, even if present in low amounts in cell mixtures or human blood. This preference was confirmed when vector particles were systemically administered into mice. Cell trafficking studies revealed an increased cell entry rate for bispecific over monospecific AAVs. When equipped with an HIV genome-targeting CRISPR/Cas cassette, the vectors prevented HIV replication in T cell cultures. The data provide proof-of-concept for high-precision gene delivery through tandem-binding regions on AAV. Reminiscent of biological products following Boolean logic AND gating, the data suggest a new option for receptor-targeted vectors to improve the specificity and safety of in vivo gene therapy., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:Christian J Buchholz reports financial support was provided by National Institutes of Health. Christian J Buchholz, Vanessa Riechert has patent pending to Assignee., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)
- Published
- 2023
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13. CAR Gene Delivery by T-cell Targeted Lentiviral Vectors is Enhanced by Rapamycin Induced Reduction of Antiviral Mechanisms.
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Charitidis FT, Adabi E, Ho N, Braun AH, Tierney C, Strasser L, Thalheimer FB, Childs L, Bones J, Clarke C, and Buchholz CJ
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- Humans, Mice, Animals, Lentivirus genetics, Genetic Vectors genetics, Gene Transfer Techniques, Antiviral Agents, Receptors, Chimeric Antigen genetics
- Abstract
Lentiviral vectors (LV) have become the dominant tool for stable gene transfer into lymphocytes including chimeric antigen receptor (CAR) gene delivery to T cells, a major breakthrough in cancer therapy. Yet, room for improvement remains, especially for the latest LV generations delivering genes selectively into T cell subtypes, a key requirement for in vivo CAR T cell generation. Toward improving gene transfer rates with these vectors, whole transcriptome analyses on human T lymphocytes are conducted after exposure to CAR-encoding conventional vectors (VSV-LV) and vectors targeted to CD8+ (CD8-LV) or CD4+ T cells (CD4-LV). Genes related to quiescence and antiviral restriction are found to be upregulated in CAR-negative cells exposed to all types of LVs. Down-modulation of various antiviral restriction factors, including the interferon-induced transmembrane proteins (IFITMs) is achieved with rapamycin as verified by mass spectrometry (LC-MS). Strikingly, rapamycin enhances transduction by up to 7-fold for CD8-LV and CD4-LV without compromising CAR T cell activities but does not improve VSV-LV. When administered to humanized mice, CD8-LV results in higher rates of green fluorescent protein (GFP) gene delivery. Also in vivo CAR T cell generation is improved in kinetics and tumor control, however to a moderate extent, leaving room for improvement by optimizing the rapamycin administration schedule. The data favor multi-omics approaches for improvements in gene delivery., (© 2023 The Authors. Advanced Science published by Wiley-VCH GmbH.)
- Published
- 2023
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14. Revised Intensity Battle Score (RIBS): Development of a Clinical Score for Predicting Poor Outcomes After Rib Fractures.
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Buchholz CJ, Jia L, Manea C, Petersen T, Wang H, Stright A, Young J, and Calland JF
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- Humans, Injury Severity Score, Ribs, Retrospective Studies, Rib Fractures complications, Rib Fractures therapy, Thoracic Injuries, Lung Diseases
- Abstract
Background: Patients with rib fractures have variable clinical courses and it is difficult to predict which patients will do poorly. Ideally this prediction would happen at the time of admission to facilitate effective triage. One scoring system devised to this end, is the Battle score. This study aims to evaluate the efficacy of the Battle score as triage tool, and to re-tool it for performance in an inpatient trauma setting., Methods: A multivariate logistic regression model was trained on patients admitted to a level one trauma center with at least one rib fracture. A composite outcome was used to classify those who had poor outcomes. Eighteen candidate predictors were analyzed in univariate analysis, then the most promising fed into the logistic model until a triage score was built and internally validated by bootstrapping., Results: Of the 838 patients who met the inclusion criteria, 145 (17.3%) patients had a defined poor outcome. The relevant predictors included in the final scoring system were number of ribs fractured, chest tube, pulmonary contusions, chronic obstructive pulmonary disease, and Glasgow coma score. Age was not found to be predictive. This score was found to have higher fidelity in predicting poor outcomes than the original Battle score (AUROC .858 vs .649.)., Discussion: An easy to calculate clinical scoring system was created to triage patients with rib fractures at the time of admission. Age may be of less importance than previously thought, while injury burden and history of lung disease may play a larger role., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
- Published
- 2023
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15. Substantially improved gene transfer to interneurons with second-generation glutamate receptor-targeted DART-AAV vectors.
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Günther DM, Kovacs R, Wildner F, Salivara A, Thalheimer FB, Fries P, Geiger JRP, and Buchholz CJ
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- Rats, Animals, Gene Transfer Techniques, Cell Line, Genetic Therapy methods, Transduction, Genetic, Genetic Vectors, Dependovirus genetics
- Abstract
Background: Adeno-associated viral vectors (AAVs) are a widely used gene transfer platform in neuroscience. Although naturally AAV serotypes can have preferences for certain tissues, selectivity for particular cell types in the CNS does not exist. Towards interneuron targeting, capsid engineering of AAV2 including display of the designed ankyrin repeat protein (DARPin) 2K19 specific for the glutamate receptor subunit 4 (GluA4) at the N-terminus of the VP2 capsid protein has been established. The resulting AAV-VP2N is highly specific for interneurons, but exhibits rather moderate transduction efficiencies., Methods: Two alternative insertion sites for 2K19 in the GH2/GH3 loop of capsid proteins VP1 (AAV-VP1L) or VP2 (AAV-VP2L) were exploited to yield second generation GluA4-AAVs. Having packaged reporter genes under ubiquitous promoters, the vectors were characterized for biochemical properties as well as gene delivery into cell lines and rat hippocampal slice cultures. Electrophysiological recordings monitored the functional properties of transduced cells., Results: Compared to AAV-VP2N, the second-generation vectors, especially AAV-VP1L, achieved about 2-fold higher genomic titers as well as a substantially improved GluA4 binding. Improvements in gene transfer activities were 18-fold on GluA4-overexpressing A549 cells and five-fold on rat hippocampal organotypic slice cultures reaching approximately 60 % of all parvalbumin positive interneurons upon a single administration. The spiking behaviour of transduced cells was unaltered and characteristic for a heterogeneous group of interneurons., Conclusion: The substantially improved gene transfer activity of the second generation GluA4-targeted AAV combined with low toxicity makes this vector an attractive tool for interneuron-directed gene transfer with unrestricted promotor and transgene choice., Competing Interests: Declaration of Competing Interest P.F. has a patent on thin-film electrodes and is a member of the Advisory Board of CorTec GmbH (Freiburg, Germany). All other authors do not have any conflict of interests to declare., (Copyright © 2023. Published by Elsevier B.V.)
- Published
- 2023
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16. CD62L as target receptor for specific gene delivery into less differentiated human T lymphocytes.
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Kapitza L, Ho N, Kerzel T, Frank AM, Thalheimer FB, Jamali A, Schaser T, Buchholz CJ, and Hartmann J
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- Humans, Animals, Mice, Cell Differentiation, Disease Models, Animal, L-Selectin genetics, RNA, Genetic Therapy, CD8-Positive T-Lymphocytes
- Abstract
Chimeric antigen receptor (CAR)-expressing T cells are a complex and heterogeneous gene therapy product with variable phenotype compositions. A higher proportion of less differentiated CAR T cells is usually associated with improved antitumoral function and persistence. We describe in this study a novel receptor-targeted lentiviral vector (LV) named 62L-LV that preferentially transduces less differentiated T cells marked by the L-selectin receptor CD62L, with transduction rates of up to 70% of CD4+ and 50% of CD8+ primary T cells. Remarkably, higher amounts of less differentiated T cells are transduced and preserved upon long-term cultivation using 62L-LV compared to VSV-LV. Interestingly, shed CD62L neither altered the binding of 62L-LV particles to T cells nor impacted their transduction. The incubation of 2 days of activated T lymphocytes with 62L-LV or VSV-LV for only 24 hours was sufficient to generate CAR T cells that controlled tumor growth in a leukemia tumor mouse model. The data proved that potent CAR T cells can be generated by short-term ex vivo exposure of primary cells to LVs. As a first vector type that preferentially transduces less differentiated T lymphocytes, 62L-LV has the potential to circumvent cumbersome selections of T cell subtypes and offers substantial shortening of the CAR T cell manufacturing process., Competing Interests: CB, JH, FT, LK, and TS are listed as inventors on a patent describing 62L-LV. Author TS is a full-time employee of the company Miltenyi Biotec GmbH. 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., (Copyright © 2023 Kapitza, Ho, Kerzel, Frank, Thalheimer, Jamali, Schaser, Buchholz and Hartmann.)
- Published
- 2023
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17. FcγRIIA-specific DARPins as novel tools in blood cell analysis and platelet aggregation.
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Riechert V, Hein S, Visser M, Zimmermann M, Wesche J, Adams PA, Theuerkauf SA, Jamali A, Wangorsch A, Reuter A, Pasternak AO, Hartmann J, Greinacher A, Herrera-Carrillo E, Berkhout B, Cichutek K, and Buchholz CJ
- Subjects
- Humans, Antibodies metabolism, Blood Platelets metabolism, HIV-1, Protein Isoforms metabolism, Virus Latency, T-Lymphocytes virology, Designed Ankyrin Repeat Proteins metabolism, Platelet Aggregation, Receptors, IgG metabolism
- Abstract
Fc receptors are involved in a variety of physiologically and disease-relevant responses. Among them, FcγRIIA (CD32a) is known for its activating functions in pathogen recognition and platelet biology, and, as potential marker of T lymphocytes latently infected with HIV-1. The latter has not been without controversy due to technical challenges complicated by T-B cell conjugates and trogocytosis as well as a lack of antibodies distinguishing between the closely related isoforms of FcγRII. To generate high-affinity binders specific for FcγRIIA, libraries of designed ankyrin repeat proteins (DARPins) were screened for binding to its extracellular domains by ribosomal display. Counterselection against FcγRIIB eliminated binders cross-reacting with both isoforms. The identified DARPins bound FcγRIIA with no detectable binding for FcγRIIB. Their affinities for FcγRIIA were in the low nanomolar range and could be enhanced by cleavage of the His-tag and dimerization. Interestingly, complex formation between DARPin and FcγRIIA followed a two-state reaction model, and discrimination from FcγRIIB was based on a single amino acid residue. In flow cytometry, DARPin F11 detected FcγRIIA
+ cells even when they made up less than 1% of the cell population. Image stream analysis of primary human blood cells confirmed that F11 caused dim but reliable cell surface staining of a small subpopulation of T lymphocytes. When incubated with platelets, F11 inhibited their aggregation equally efficient as antibodies unable to discriminate between both FcγRII isoforms. The selected DARPins are unique novel tools for platelet aggregation studies as well as the role of FcγRIIA for the latent HIV-1 reservoir., Competing Interests: Conflict of interest V. R., J. H., K. C., and C. J. B. are listed as inventors on a filed patent describing FcγRIIA-specific DARPins., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)- Published
- 2023
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18. APPsα rescues CDK5 and GSK3β dysregulation and restores normal spine density in Tau transgenic mice.
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Baltissen D, Bold CS, Rehra L, Banićević M, Fricke J, Just J, Ludewig S, Buchholz CJ, Korte M, and Müller UC
- Abstract
The Tau protein can be phosphorylated by numerous kinases. In Alzheimer's disease (AD) hyperphosphorylated Tau species accumulate as neurofibrillary tangles that constitute a major hallmark of AD. AD is further characterized by extracellular Aβ plaques, derived from the β-amyloid precursor protein APP. Whereas Aβ is produced by amyloidogenic APP processing, APP processing along the competing non-amyloidogenic pathway results in the secretion of neurotrophic and synaptotrophic APPsα. Recently, we demonstrated that APPsα has therapeutic effects in transgenic AD model mice and rescues Aβ-dependent impairments. Here, we examined the potential of APPsα to regulate two major Tau kinases, GSK3β and CDK5 in THY-Tau22 mice, a widely used mouse model of tauopathy. Immunohistochemistry revealed a dramatic increase in pathologically phosphorylated (AT8 and AT180) or misfolded Tau species (MC1) in the hippocampus of THY-Tau22 mice between 3 and 12 months of age. Using a highly sensitive radioactive kinase assay with recombinant human Tau as a substrate and immunoblotting, we demonstrate an increase in GSK3β and CDK5 activity in the hippocampus of THY-Tau22 mice. Interestingly, AAV-mediated intracranial expression of APPsα in THY-Tau22 mice efficiently restored normal GSK3β and CDK5 activity. Western blot analysis revealed upregulation of the CDK5 regulatory proteins p35 and p25, indicating CDK5 hyperactivation in THY-Tau22 mice. Strikingly, AAV-APPsα rescued p25 upregulation to wild-type levels even at stages of advanced Tau pathology. Sarkosyl fractionation used to study the abundance of soluble and insoluble phospho-Tau species revealed increased soluble AT8-Tau and decreased insoluble AT100-Tau species upon AAV-APPsα injection. Moreover, AAV-APPsα reduced misfolded (MC1) Tau species, particularly in somatodendritic compartments of CA1 pyramidal neurons. Finally, we show that AAV-APPsα upregulated PSD95 expression and rescued deficits in spine density of THY-Tau22 mice. Together our findings suggest that APPsα holds therapeutic potential to mitigate Tau-induced pathology., Competing Interests: The 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., (Copyright © 2023 Baltissen, Bold, Rehra, Banićević, Fricke, Just, Ludewig, Buchholz, Korte and Müller.)
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- 2023
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19. Dasatinib is a potent enhancer for CAR T cell generation by CD3-targeted lentiviral vectors.
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Braun AH, Frank AM, Ho N, and Buchholz CJ
- Abstract
CD3-targeted lentiviral vectors (CD3-LVs) mediate selective transduction of human T lymphocytes in vitro and in vivo while simultaneously activating the targeted cells. Previously, we have demonstrated that CD3-LV leads to downmodulation of the CD3:T cell receptor (TCR) complex. We therefore hypothesized that inhibition of CD3 phosphorylation by Src/Abl tyrosine kinase inhibitors such as dasatinib results in enhancement of gene delivery by T cell-targeted LVs. Indeed, dasatinib treatment of T cells prior to incubation with CD3-LV increased reporter gene delivery by 3- to 10-fold. Moreover, the presence of dasatinib enhanced selective transduction into non-activated target cells present in whole blood. When combined with delivery of the CD19-chimeric antigen receptor (CAR) gene, dasatinib increased CAR T cell numbers by close to 10-fold. Importantly, the short-term exposure of T cells to dasatinib during vector incubation did not interfere with tumor cell killing by the resulting CAR T cells and rather came along with less upregulated exhaustion markers and a more naive phenotype. Our data suggest that dasatinib prevents CD3-LV-induced phosphorylation and CD3:TCR intake, thereby increasing the amount of CD3-LV bound to the cell surface. This is the first description of dasatinib as transduction enhancer, an activity particularly relevant for CAR T cell generation with CD3-LV., Competing Interests: C.J.B., A.M.F., and A.H.B. are inventors of a patent describing the use of dasatinib as a transduction enhancer., (© 2022 The Authors.)
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- 2022
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20. Expansion of T memory stem cells with superior anti-tumor immunity by Urolithin A-induced mitophagy.
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Denk D, Petrocelli V, Conche C, Drachsler M, Ziegler PK, Braun A, Kress A, Nicolas AM, Mohs K, Becker C, Neurath MF, Farin HF, Buchholz CJ, Andreux PA, Rinsch C, and Greten FR
- Subjects
- Mice, Animals, Wnt Signaling Pathway, Stem Cells, Immunologic Memory, Mitophagy, Coumarins pharmacology
- Abstract
T memory stem cells (T
SCM ) display increased self-renewal and prolonged survival capabilities, thus preventing T cell exhaustion and promoting effective anti-tumor T cell responses. TSCM cells can be expanded by Urolithin A (UA), which is produced by the commensal gut microbiome from foods rich in ellagitannins and is known to improve mitochondrial health. Oral UA administration to tumor-bearing mice conferred strong anti-tumor CD8+ T cell immunity, whereas ex vivo UA pre-treated T cells displayed improved anti-tumor function upon adoptive cell transfer. UA-induced TSCM formation depended on Pink1-mediated mitophagy triggering cytosolic release of the mitochondrial phosphatase Pgam5. Cytosolic Pgam5 dephosphorylated β-catenin, which drove Wnt signaling and compensatory mitochondrial biogenesis. Collectively, we unravel a critical signaling pathway linking mitophagy to TSCM formation and suggest that the well-tolerated metabolic compound UA represents an attractive option to improve immune therapy., Competing Interests: Declaration of interests The authors have filed patents regarding the use of Urolithin in tumor therapy and CAR T cell generation. M.D. is an employee of AstraZeneca, UK. C.R. is CEO and a board member of Amazentis SA; P.A.A. is an employee of Vandria SA. F.R.G. has received consulting fees from Amazentis., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)- Published
- 2022
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21. AAV-mediated gene transfer of a checkpoint inhibitor in combination with HER2-targeted CAR-NK cells as experimental therapy for glioblastoma.
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Strecker MI, Wlotzka K, Strassheimer F, Roller B, Ludmirski G, König S, Röder J, Opitz C, Alekseeva T, Reul J, Sevenich L, Tonn T, Wels WS, Steinbach JP, Buchholz CJ, and Burger MC
- Subjects
- Adenoviridae genetics, Animals, B7-H1 Antigen genetics, Cell Line, Tumor, Cytokines, Immune Checkpoint Inhibitors pharmacology, Immune Checkpoint Inhibitors therapeutic use, Killer Cells, Natural metabolism, Killer Cells, Natural transplantation, Mice, RNA, Messenger, Receptor, ErbB-2 metabolism, Therapies, Investigational, Tumor Microenvironment, Glioblastoma genetics, Glioblastoma therapy
- Abstract
Glioblastoma (GB) is the most common primary brain tumor, which is characterized by low immunogenicity of tumor cells and prevalent immunosuppression in the tumor microenvironment (TME). Targeted local combination immunotherapy is a promising strategy to overcome these obstacles. Here, we evaluated tumor-cell specific delivery of an anti-PD-1 immunoadhesin (aPD-1) via a targeted adeno-associated viral vector (AAV) as well as HER2-specific NK-92/5.28.z (anti-HER2.CAR/NK-92) cells as components for a combination immunotherapy. In co-culture experiments, target-activated anti-HER2.CAR/NK-92 cells modified surrounding tumor cells and bystander immune cells by triggering the release of inflammatory cytokines and upregulation of PD-L1. Tumor cell-specific delivery of aPD-1 was achieved by displaying a HER2-specific designed ankyrin repeat protein (DARPin) on the AAV surface. HER2-AAV mediated gene transfer into GB cells correlated with HER2 expression levels, without inducing anti-viral responses in transduced cells. Furthermore, AAV-transduction did not interfere with anti-HER2.CAR/NK-92 cell-mediated tumor cell lysis. After selective transduction of HER2
+ cells, aPD-1 expression was detected at the mRNA and protein level. The aPD-1 immunoadhesin was secreted in a time-dependent manner, bound its target on PD-1-expressing cells and was able to re-activate T cells by efficiently disrupting the PD-1/PD-L1 axis. Moreover, high intratumoral and low systemic aPD-1 concentrations were achieved following local injection of HER2-AAV into orthotopic tumor grafts in vivo . aPD-1 was selectively produced in tumor tissue and could be detected up to 10 days after a single HER2-AAV injection. In subcutaneous GL261-HER2 and Tu2449-HER2 immunocompetent mouse models, administration of the combination therapy significantly prolonged survival, including complete tumor control in several animals in the GL261-HER2 model. In summary, local therapy with aPD-1 encoding HER2-AAVs in combination with anti-HER2.CAR/NK-92 cells may be a promising novel strategy for GB immunotherapy with the potential to enhance efficacy and reduce systemic side effects of immune-checkpoint inhibitors., Competing Interests: J.P.S has a consulting or advisory board membership with, or has received honoraria or travel or accommodation expenses from AbbVie, Medac, Novocure, Roche, and UCB. M.C.B has received honoraria for lectures or advisory board participation from Bristol-Myers Squibb and Gilead Sciences. All other authors report no conflict of interest., (© 2022 The Author(s). Published with license by Taylor & Francis Group, LLC.)- Published
- 2022
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22. APPsα Rescues Tau-Induced Synaptic Pathology.
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Bold CS, Baltissen D, Ludewig S, Back MK, Just J, Kilian L, Erdinger S, Banicevic M, Rehra L, Almouhanna F, Nigri M, Wolfer DP, Spilger R, Rohr K, Kann O, Buchholz CJ, von Engelhardt J, Korte M, and Müller UC
- Subjects
- Animals, Female, Hippocampus metabolism, Male, Mice, Mice, Transgenic, Neuronal Plasticity physiology, Alzheimer Disease metabolism, Tauopathies pathology
- Abstract
Alzheimer's disease (AD) is histopathologically characterized by Aβ plaques and the accumulation of hyperphosphorylated Tau species, the latter also constituting key hallmarks of primary tauopathies. Whereas Aβ is produced by amyloidogenic APP processing, APP processing along the competing nonamyloidogenic pathway results in the secretion of neurotrophic and synaptotrophic APPsα. Recently, we demonstrated that APPsα has therapeutic effects in transgenic AD model mice and rescues Aβ-dependent impairments. Here, we examined the potential of APPsα to mitigate Tau-induced synaptic deficits in P301S mice (both sexes), a widely used mouse model of tauopathy. Analysis of synaptic plasticity revealed an aberrantly increased LTP in P301S mice that could be normalized by acute application of nanomolar amounts of APPsα to hippocampal slices, indicating a homeostatic function of APPsα on a rapid time scale. Further, AAV-mediated in vivo expression of APPsα restored normal spine density of CA1 neurons even at stages of advanced Tau pathology not only in P301S mice, but also in independent THY-Tau22 mice. Strikingly, when searching for the mechanism underlying aberrantly increased LTP in P301S mice, we identified an early and progressive loss of major GABAergic interneuron subtypes in the hippocampus of P301S mice, which may lead to reduced GABAergic inhibition of principal cells. Interneuron loss was paralleled by deficits in nest building, an innate behavior highly sensitive to hippocampal impairments. Together, our findings indicate that APPsα has therapeutic potential for Tau-mediated synaptic dysfunction and suggest that loss of interneurons leads to disturbed neuronal circuits that compromise synaptic plasticity as well as behavior. SIGNIFICANCE STATEMENT Our findings indicate, for the first time, that APPsα has the potential to rescue Tau-induced spine loss and abnormal synaptic plasticity. Thus, APPsα might have therapeutic potential not only because of its synaptotrophic functions, but also its homeostatic capacity for neuronal network activity. Hence, APPsα is one of the few molecules which has proven therapeutic effects in mice, both for Aβ- and Tau-dependent synaptic impairments and might therefore have therapeutic potential for patients suffering from AD or primary tauopathies. Furthermore, we found in P301S mice a pronounced reduction of inhibitory interneurons as the earliest pathologic event preceding the accumulation of hyperphosphorylated Tau species. This loss of interneurons most likely disturbs neuronal circuits that are important for synaptic plasticity and behavior., (Copyright © 2022 the authors.)
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- 2022
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23. Precision medicine: In vivo CAR therapy as a showcase for receptor-targeted vector platforms.
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Michels A, Ho N, and Buchholz CJ
- Subjects
- Animals, Liposomes, Mice, Nanoparticles, Receptors, Antigen, T-Cell genetics, T-Lymphocytes, Immunotherapy, Adoptive, Precision Medicine, Receptors, Chimeric Antigen genetics
- Abstract
Chimeric antigen receptor (CAR) T cells are a cancer immunotherapy of extremes. Unprecedentedly effective, but complex and costly to manufacture, they are not yet a therapeutic option for all who would benefit. This disparity has motivated worldwide efforts to simplify treatment. Among the proposed solutions, the generation of CAR T cells directly in the patient, i.e., in vivo, is arguably simultaneously the most technically challenging and clinically useful approach to convert CAR therapy from a cell-based autologous medicinal product into a universally applicable off-the-shelf treatment. Here, we review the current state of the art of in vivo CAR therapy, focusing especially on the vector technologies used. These cover lentiviral vectors and adenovirus-associated vectors as well as synthetic polymer nanocarriers and lipid nanoparticles. Proof of concept, i.e., the generation of CAR cells directly in mouse models, has been demonstrated for all vector platforms. Receptor targeting of vector particles is crucial, as it can prevent CAR gene delivery into off-target cells, thus reducing toxicities. We discuss the properties of the vector platforms, such as their immunogenicity, potency, and modes of CAR delivery (permanent versus transient). Finally, we outline the work required to advance in vivo CAR therapy from proof of concept to a robust, scalable technology for clinical testing., Competing Interests: Declaration of interests The authors declare no conflict of interest., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)
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- 2022
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24. In vivo generation of CAR T cells in the presence of human myeloid cells.
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Ho N, Agarwal S, Milani M, Cantore A, Buchholz CJ, and Thalheimer FB
- Abstract
Pre-clinical humanized mouse models are a powerful tool to evaluate immunotherapies. NSG-SGM3 mice reconstituted with human stem cells (huSGM3) develop pronounced human myeloid cells due to transgenic expression of stem cell factor, granulocyte-macrophage colony-stimulating factor, and interleukin-3 (IL-3) compared with the widely used humanized NSG (huNSG) model. We assessed in vivo generation of CD19-CAR T cells in huSGM3 mice upon single intravenous injection of the T cell-specific lentiviral vectors (LVs) CD4-LV and CD8-LV. While in vivo CAR T cell generation was clearly detectable in individual mice, generation appeared less efficient than previously observed for huNSG mice. Especially for the CD4-LV group, this correlated with increased IL-15 and decreased GM-CSF levels, indicating activation of monocytes and macrophages. Co-culture assays identified macrophages as a potential barrier for gene transfer. Refining CD4-LV and CD8-LV with a less immunogenic surface by using modified packaging cells substantially improved the transduction of lymphocytes in vitro in the presence of macrophages, as well in vivo in huSGM3 mice. Notably, two mice that developed less CAR T cells showed high interferon-α or -β levels before vector injection. Our data emphasize the relevance of innate immune responses for in vivo generation of CAR T cells, which can be overcome by vector surface engineering., Competing Interests: C.J.B. is listed as inventor on patents covering T cell-targeted lentiviral vectors. A.C. is inventor on patents related to MHC-negative lentiviral vectors. All other authors declare no competing interests., (© 2022 The Author(s).)
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- 2022
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25. Time 2EVOLVE: predicting efficacy of engineered T-cells - how far is the bench from the bedside?
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Guedan S, Luu M, Ammar D, Barbao P, Bonini C, Bousso P, Buchholz CJ, Casucci M, De Angelis B, Donnadieu E, Espie D, Greco B, Groen R, Huppa JB, Kantari-Mimoun C, Laugel B, Mantock M, Markman JL, Morris E, Quintarelli C, Rade M, Reiche K, Rodriguez-Garcia A, Rodriguez-Madoz JR, Ruggiero E, Themeli M, Hudecek M, and Marchiq I
- Subjects
- Humans, Immunotherapy, Immunotherapy, Adoptive, T-Lymphocytes, Neoplasms therapy, Receptors, Chimeric Antigen
- Abstract
Immunotherapy with gene engineered CAR and TCR transgenic T-cells is a transformative treatment in cancer medicine. There is a rich pipeline with target antigens and sophisticated technologies that will enable establishing this novel treatment not only in rare hematological malignancies, but also in common solid tumors. The T2EVOLVE consortium is a public private partnership directed at accelerating the preclinical development of and increasing access to engineered T-cell immunotherapies for cancer patients. A key ambition in T2EVOLVE is to assess the currently available preclinical models for evaluating safety and efficacy of engineered T cell therapy and developing new models and test parameters with higher predictive value for clinical safety and efficacy in order to improve and accelerate the selection of lead T-cell products for clinical translation. Here, we review existing and emerging preclinical models that permit assessing CAR and TCR signaling and antigen binding, the access and function of engineered T-cells to primary and metastatic tumor ligands, as well as the impact of endogenous factors such as the host immune system and microbiome. Collectively, this review article presents a perspective on an accelerated translational development path that is based on innovative standardized preclinical test systems for CAR and TCR transgenic T-cell products., Competing Interests: Competing interests: SG is an inventor on patents related to CAR-T cell therapy, filed by the University of Pennsylvania and licensed to Novartis and Tmunity, and has received commercial research funding from Novartis and Gilead. ML is an inventor on a patent application related to CAR-T cell therapy filed by Philipps-University Marburg and the University of Würzburg. DE PhD is cofunded between the academic lab led by ED as PhD supervisor and the industrial partner Invectys. EM is a Scientific Founder and holds stock options for Quell Therapeutics, consults for Orchard Therapeutics, and is Ad hoc advisor and consultant for Gilead Sciences and GSK. MT is inventor on patent applications related to CAR T cell therapy filed by Memorial Sloan Kettering Cancer Center, New York, NY; and VU Medical Center, Amsterdam, Netherlands and licensed to industry. MH is listed as an inventor on patent applications and granted patents related to CAR T cell therapy that have been filed by the Fred Hutchinson Cancer Research Center, Seattle, WA; and the University of Würzburg, Würzburg, Germany and licensed to industry. MH is a cofounder and equity holder of T-CURX, Würzburg, Germany., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)
- Published
- 2022
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26. Erratum: Monitoring CAR T cell generation with a CD8-targeted lentiviral vector by single-cell transcriptomics.
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Charitidis FT, Adabi E, Thalheimer FB, Clarke C, and Buchholz CJ
- Abstract
[This corrects the article DOI: 10.1016/j.omtm.2021.09.019.]., (© 2022 The Author(s).)
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- 2022
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27. Oncolytic measles vaccines encoding PD-1 and PD-L1 checkpoint blocking antibodies to increase tumor-specific T cell memory.
- Author
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Veinalde R, Pidelaserra-Martí G, Moulin C, Jeworowski LM, Küther L, Buchholz CJ, Jäger D, Ungerechts G, and Engeland CE
- Abstract
PD-1/PD-L1 checkpoint blockade has achieved unprecedented success in cancer immunotherapy. Nevertheless, many immune-excluded tumors are resistant to therapy. Combination with oncolytic virotherapy may overcome resistance by inducing acute inflammation, immune cell recruitment, and remodeling of the tumor immune environment. Here, we assessed the combination of oncolytic measles vaccine (MV) vectors and PD-1/PD-L1 blockade. In the MC38cea model of measles virus oncolysis, MV combined with anti-PD-1 and MV vectors encoding anti-PD-1 or anti-PD-L1 antibodies achieved modest survival benefits compared with control MV or vectors encoding the antibody constant regions only. Analyses of tumor samples and tumor-draining lymph nodes revealed slight increases in intratumoral T cell effector cytokines as well as a shift toward an effector memory phenotype in the T cell compartment. Importantly, increased IFN-γ recall responses were observed in tumor rechallenge experiments with mice in complete tumor remission after treatment with MV encoding anti-PD-1 or anti-PD-L1 compared with control MV. These results prompted us to generate MV encoding the clinically approved agents pembrolizumab and nivolumab. Previously, we have generated MV encoding atezolizumab. We demonstrated the functionality of the novel vectors in vitro . We envision these vectors as therapeutics that induce and support durable anti-tumor immune memory., Competing Interests: G.U. is a co-founder, stakeholder, and chief medical officer (CMO)/chief scientific officer (CSO) of CanVirex AG, a company investigating oncolytic viruses as cancer immunotherapeutics., (© 2021 The Authors.)
- Published
- 2021
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28. Monitoring CAR T cell generation with a CD8-targeted lentiviral vector by single-cell transcriptomics.
- Author
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Charitidis FT, Adabi E, Thalheimer FB, Clarke C, and Buchholz CJ
- Abstract
Quantifying gene expression in individual cells can substantially improve our understanding about complex genetically engineered cell products such as chimeric antigen receptor (CAR) T cells. Here we designed a single-cell RNA sequencing (scRNA-seq) approach to monitor the delivery of a CD19-CAR gene via lentiviral vectors (LVs), i.e., the conventional vesicular stomatitis virus (VSV)-LV and the CD8-targeted CD8-LV. LV-exposed human donor peripheral blood mononuclear cells (PBMCs) were evaluated for a panel of 400 immune response-related genes including LV-specific probes. The resulting data revealed a trimodal expression for the CAR and CD8A , demanding a careful distribution-based identification of CAR T cells and CD8+ lymphocytes in scRNA-seq analysis. The fraction of T cells expressing high CAR levels was in concordance with flow cytometry results. More than 97% of the cells hit by CD8-LV expressed the CD8A gene. Remarkably, the majority of the potential off-target cells were in fact on-target cells, resulting in a target cell selectivity of more than 99%. Beyond that, differential gene expression analysis revealed the upregulation of restriction factors in CAR -negative cells, thus explaining their protection from CAR gene transfer. In summary, we provide a workflow and subsetting approach for scRNA-seq enabling reliable distinction between transduced and untransduced cells during CAR T cell generation., Competing Interests: C.J.B. is listed as co-inventor of the patent for the CD8-targeted lentiviral vector. All the other authors declare no competing interests., (© 2021 The Author(s).)
- Published
- 2021
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29. Lentiviral and adeno-associated vectors efficiently transduce mouse T lymphocytes when targeted to murine CD8.
- Author
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Michels A, Frank AM, Günther DM, Mataei M, Börner K, Grimm D, Hartmann J, and Buchholz CJ
- Abstract
Preclinical studies on gene delivery into mouse lymphocytes are often hampered by insufficient activity of lentiviral (LV) and adeno-associated vectors (AAVs) as well as missing tools for cell type selectivity when considering in vivo gene therapy. Here, we selected designed ankyrin repeat proteins (DARPins) binding to murine CD8. The top-performing DARPin was displayed as targeting ligand on both vector systems. When used on engineered measles virus (MV) glycoproteins, the resulting mCD8-LV transduced CD8+ mouse lymphocytes with near-absolute (>99%) selectivity. Despite its lower functional titer, mCD8-LV achieved 4-fold higher gene delivery to CD8+ cells than conventional VSV-LV when added to whole mouse blood. Addition of mCD8-LV encoding a chimeric antigen receptor (CAR) specific for mouse CD19 to splenocytes resulted in elimination of B lymphocytes and lymphoma cells. For display on AAV, the DARPin was inserted into the GH2-GH3 loop of the AAV2 capsid protein VP1, resulting in a DARPin-targeted AAV we termed DART-AAV. Stocks of mCD8-AAV contained similar genome copies as AAV2 but were >20-fold more active in gene delivery in mouse splenocytes, while exhibiting >99% specificity for CD8+ cells. These results suggest that receptor targeting can overcome blocks in transduction of mouse splenocytes., Competing Interests: C.J.B. is listed as inventor on patents covering T cell targeted lentiviral vectors. D.G. is a co-founder, shareholder, and Chief Scientific Officer of AaviGen GmbH. All the other authors declare no competing interests., (© 2021 The Author(s).)
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- 2021
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30. Genetic in vivo engineering of human T lymphocytes in mouse models.
- Author
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Weidner T, Agarwal S, Perian S, Fusil F, Braun G, Hartmann J, Verhoeyen E, and Buchholz CJ
- Subjects
- Animals, Antigens, CD metabolism, HEK293 Cells, Humans, Lentivirus genetics, Mice, Models, Animal, Receptors, Chimeric Antigen genetics, Genetic Engineering methods, T-Lymphocytes metabolism
- Abstract
Receptor targeting of vector particles is a key technology to enable cell type-specific in vivo gene delivery. For example, T cells in humanized mouse models can be modified by lentiviral vectors (LVs) targeted to human T-cell markers to enable them to express chimeric antigen receptors (CARs). Here, we provide detailed protocols for the generation of CD4- and CD8-targeted LVs (which takes ~9 d in total). We also describe how to humanize immunodeficient mice with hematopoietic stem cells (which takes 12-16 weeks) and precondition (over 5 d) and administer the vector stocks. Conversion of the targeted cell type is monitored by PCR and flow cytometry of blood samples. A few weeks after administration, ~10% of the targeted T-cell subtype can be expected to have converted to CAR T cells. By closely following the protocol, sufficient vector stock for the genetic manipulation of 10-15 humanized mice is obtained. We also discuss how the protocol can be easily adapted to use LVs targeted to other types of receptors and/or for delivery of other genes of interest.
- Published
- 2021
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31. Erratum: A Library-Based Screening Strategy for the Identification of DARPins as Ligands for Receptor-Targeted AAV and Lentiviral Vectors.
- Author
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Hartmann J, Münch RC, Freiling RT, Schneider IC, Dreier B, Samukang W, Koch J, Seeger MA, Plückthun A, and Buchholz CJ
- Abstract
[This corrects the article DOI: 10.1016/j.omtm.2018.07.001.]., (© 2021 The Author(s).)
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- 2021
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32. Quantitative assays reveal cell fusion at minimal levels of SARS-CoV-2 spike protein and fusion from without.
- Author
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Theuerkauf SA, Michels A, Riechert V, Maier TJ, Flory E, Cichutek K, and Buchholz CJ
- Abstract
Cell entry of the pandemic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is mediated by its spike protein S. As a main antigenic determinant, S protein is in focus of various therapeutic strategies. Besides particle-cell fusion, S mediates fusion between infected and uninfected cells resulting in syncytia formation. Here, we present sensitive assay systems with a high dynamic range and high signal-to-noise ratios covering not only particle-cell and cell-cell fusion but also fusion from without (FFWO). In FFWO, S-containing viral particles induce syncytia independently of de novo synthesis of S. Neutralizing antibodies, as well as sera from convalescent patients, inhibited particle-cell fusion with high efficiency. Cell-cell fusion, in contrast, was only moderately inhibited despite requiring levels of S protein below the detection limit of flow cytometry and Western blot. The data indicate that syncytia formation as pathological consequence during coronavirus disease 2019 (COVID-19) can proceed at low levels of S protein and may not be effectively prevented by antibodies., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)
- Published
- 2021
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33. Proceedings From the First International Workshop at Sidra Medicine: "Engineered Immune Cells in Cancer Immunotherapy (EICCI): From Discovery to Off-the-Shelf Development", 15 th -16 th February 2019, Doha, Qatar.
- Author
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Guerrouahen B, Elnaggar M, Al-Mohannadi A, Kizhakayil D, Bonini C, Benjamin R, Brentjens R, Buchholz CJ, Casorati G, Ferrone S, Locke FL, Martin F, Schambach A, Turtle C, Veys P, van der Vliet HJ, and Maccalli C
- Subjects
- Animals, Genetic Engineering, Humans, Immunotherapy, Adoptive, Qatar, Receptors, Chimeric Antigen immunology, T-Lymphocytes immunology, Immunotherapy, Neoplasms therapy
- Abstract
The progress in the isolation and characterization of tumor antigen (TA)-specific T lymphocytes and in the genetic modification of immune cells allowed the clinical development of adoptive cell therapy (ACT). Several clinical studies highlighted the striking clinical activity of T cells engineered to express either Chimeric Antigen (CAR) or T Cell (TCR) Receptors to target molecularly defined antigens expressed on tumor cells. The breakthrough of immunotherapy is represented by the approval of CAR-T cells specific for advanced or refractory CD19
+ B cell malignancies by both the Food and Drug Administration (FDA) and the European Medicinal Agency (EMA). Moreover, advances in the manufacturing and gene editing of engineered immune cells contributed to the selection of drug products with desired phenotype, refined specificity and decreased toxicity. An important step toward the optimization of CAR-T cell therapy is the development of "off-the shelf" T cell products that allow to reduce the complexity and the costs of the manufacturing and to render these drugs available for a broad number of cancer patients. The Engineered Immune Cells in Cancer Immunotherapy (EICCI) workshop hosted in Doha, Qatar, renowned experts, from both academia and industry, to present and discuss the progress on both pre-clinical and clinical development of genetically modified immune cells, including advances in the "off-the-shelf" manufacturing. These experts have addressed also organizational needs and hurdles for the clinical grade production and application of these biological drugs., Competing Interests: CBo received a research contract from Intellia Therapeutics and participated to the advisory boards of Molmed, Intellia Therapeutics, TxCell, Novartis, GSK, Allogene, Kiadis. CBu is the inventor of patents in the field of adoptive T cell therapy. RB is a recipient of research funding from Servier. IJ is employed at Miltenyi Biotec. FL has scientific advisory role for Kite, a Gilead Company, Novartis, Celgene/Bristol-Myers Squibb, GammaDelta Therapeutics, Wugen, Amgen, Calibr, Amgen, and Allogene; is a consultant with grant options for Cellular Biomedicine Group, Inc.; and has research support from Kite, a Gilead Company. AM is employed at Immatics. CT received research funding from Juno Therapeutics/BMS, Nektar Therapeutics, Minerva, AstraZeneca, and TCR2 Therapeutics. He is a member of Scientific Advisory Boards of Precision Biosciences, Eureka Therapeutics, Caribou Biosciences, T-CURX, Myeloid Therapeutics, ArsenalBio, and Century Therapeutics, and ad hoc advisory boards (last 12 months) of Nektar Therapeutics, Allogene, PACT Pharma, Astra Zeneca, and Amgen. He has stock/options of Precision Biosciences, Eureka Therapeutics, Caribou Biosciences, Myeloid Therapeutics, and ArsenalBio. CT is the inventor of a patent licensed to Juno Therapeutics. PV is a recipient of a grant from SERVIER to investigate Universal chimeric antigen receptor T cells for ALL (UCAR19-PALL). HV is employed as a chief scientific officer (CSO) of Lava Therapeutics. 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., (Copyright © 2021 Guerrouahen, Elnaggar, Al-Mohannadi, Kizhakayil, Bonini, Benjamin, Brentjens, Buchholz, Casorati, Ferrone, Locke, Martin, Schambach, Turtle, Veys, van der Vliet, Maccalli and The EICCI Faculty Group.)- Published
- 2021
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34. Designed Ankyrin Repeat Protein (DARPin) to target chimeric antigen receptor (CAR)-redirected T cells towards CD4 + T cells to reduce the latent HIV + cell reservoir.
- Author
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Patasic L, Seifried J, Bezler V, Kaljanac M, Schneider IC, Schmitz H, Tondera C, Hartmann J, Hombach A, Buchholz CJ, Abken H, König R, and Cichutek K
- Subjects
- CD4-Positive T-Lymphocytes drug effects, CD4-Positive T-Lymphocytes immunology, Dose-Response Relationship, Immunologic, Drug Evaluation, Preclinical, Gammaretrovirus genetics, Genetic Vectors genetics, HEK293 Cells, HIV Infections virology, Humans, Lymphocyte Activation, Peptides chemistry, Single-Chain Antibodies immunology, Transduction, Genetic, Ankyrin Repeat, CD4-Positive T-Lymphocytes virology, HIV isolation & purification, HIV Infections immunology, Immunotherapy, Adoptive, Lymphocyte Depletion methods, Peptides pharmacology
- Abstract
Chimeric Antigen Receptor (CAR)-redirected T cells show great efficacy in the patient-specific therapy of hematologic malignancies. Here, we demonstrate that a DARPin with specificity for CD4 specifically redirects and triggers the activation of CAR engineered T cells resulting in the depletion of CD4
+ target cells aiming for elimination of the human immunodeficiency virus (HIV) reservoir.- Published
- 2020
- Full Text
- View/download PDF
35. Combining T-cell-specific activation and in vivo gene delivery through CD3-targeted lentiviral vectors.
- Author
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Frank AM, Braun AH, Scheib L, Agarwal S, Schneider IC, Fusil F, Perian S, Sahin U, Thalheimer FB, Verhoeyen E, and Buchholz CJ
- Subjects
- Animals, Leukocytes, Mononuclear, Mice, T-Lymphocytes, Transduction, Genetic, Genetic Vectors, Lentivirus genetics
- Abstract
Genetic modification of T lymphocytes is a key issue in research and therapy. Conventional lentiviral vectors (LVs) are neither selective for T cells nor do they modify resting or minimally stimulated cells, which is crucial for applications, such as efficient in vivo modification of T lymphocytes. Here, we introduce novel CD3-targeted LVs (CD3-LVs) capable of genetically modifying human T lymphocytes without prior activation. For CD3 attachment, agonistic CD3-specific single-chain variable fragments were chosen. Activation, proliferation, and expansion mediated by CD3-LVs were less rapid compared with conventional antibody-mediated activation owing to lack of T-cell receptor costimulation. CD3-LVs delivered genes not only selectively into T cells but also under nonactivating conditions, clearly outperforming the benchmark vector vesicular stomatitis-LV glycoproteins under these conditions. Remarkably, CD3-LVs were properly active in gene delivery even when added to whole human blood in absence of any further stimuli. Upon administration of CD3-LV into NSG mice transplanted with human peripheral blood mononuclear cells, efficient and exclusive transduction of CD3+ T cells in all analyzed organs was achieved. Finally, the most promising CD3-LV successfully delivered a CD19-specific chimeric antigen receptor (CAR) into T lymphocytes in vivo in humanized NSG mice. Generation of CAR T cells was accompanied by elimination of human CD19+ cells from blood. Taken together, the data strongly support implementation of T-cell-activating properties within T-cell-targeted vector particles. These particles may be ideally suited for T-cell-specific in vivo gene delivery., (© 2020 by The American Society of Hematology.)
- Published
- 2020
- Full Text
- View/download PDF
36. [Chimeric antigen receptors in oncology: clinical applications and new developments].
- Author
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Michels A, Hartmann J, and Buchholz CJ
- Subjects
- Animals, Germany, Humans, Immunotherapy, Adoptive, Medical Oncology, Receptors, Antigen, T-Cell genetics, T-Lymphocytes, Receptors, Chimeric Antigen
- Abstract
In 2018, two novel cancer therapies based on chimeric antigen receptors (CARs) were granted marketing authorization in the European Union. Authorized for use against advanced lymphoma and/or leukemia, the products were at the center of international attention, not only due to their novel mode of action and their encouraging efficacy but also because of their sometimes severe side effects and the economic and logistic challenges posed by their manufacture. Now, almost two years later, hundreds of active clinical trials emphasize the global drive to harness the full potential of CAR technology.In this article, we describe the mode of action of CAR T and CAR NK cells and review the clinical testing situation as well as early real-world data. In recent years, preclinical studies using advanced animal models have provided first insights into the mechanisms underlying the severe side effects of CAR T therapy. We summarize their results and describe the available models. Additionally, we discuss potential solutions to the hurdles currently limiting CAR technology. So far used as last-line treatment for patients with aggressive disease, CAR technology has the potential to become a new, broadly effective standard for tumor therapy.
- Published
- 2020
- Full Text
- View/download PDF
37. In Vivo Generation of CAR T Cells Selectively in Human CD4 + Lymphocytes.
- Author
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Agarwal S, Hanauer JDS, Frank AM, Riechert V, Thalheimer FB, and Buchholz CJ
- Subjects
- Animals, Antigens, CD19 immunology, Antigens, Neoplasm immunology, CD8-Positive T-Lymphocytes immunology, Disease Models, Animal, Humans, Mice, Receptors, Antigen, T-Cell genetics, Receptors, Antigen, T-Cell metabolism, Receptors, Chimeric Antigen genetics, Receptors, Chimeric Antigen metabolism, T-Lymphocyte Subsets immunology, T-Lymphocyte Subsets metabolism, CD4-Positive T-Lymphocytes immunology, CD4-Positive T-Lymphocytes metabolism, Immunotherapy, Adoptive adverse effects, Immunotherapy, Adoptive methods, Receptors, Antigen, T-Cell immunology, Receptors, Chimeric Antigen immunology
- Abstract
T cells modified with CD19-specific chimeric antigen receptors (CARs) result in significant clinical benefit for leukemia patients but constitute a challenge for manufacturing. We have recently demonstrated the in vivo generation of CD19-CAR T cells using the CD8-targeted lentiviral vector (CD8-LV). In this study, we investigated the in vivo generation of CD4
+ CAR T cells using CD4-targeted LV (CD4-LV). Administration of CD4-LV into NSG mice transplanted with human peripheral blood mononuclear cells (PBMCs) led to 40%-60% of human CD4+ lymphocytes being CAR positive while CD8+ cells remained CAR negative. CAR+ T cells displayed a T helper 1 (Th1)/Th2 phenotype, which was accompanied by CD19+ B cell elimination. Intravenous administration of CD4-LV into NSG mice reconstituted with human CD34+ cells induced CAR expression and B cell elimination within 2-3 weeks post-injection. Preclinical analysis in a tumor mouse model revealed that mice administered CD4-LV exhibited faster and superior tumor cell killing compared to mice injected with CD8-LV alone or as a mixture with CD4-LV. Further analysis suggests that CD4+ CAR+ cells may outperform CD8+ CAR+ cells, especially at a high burden of target antigen, mainly since CD8 cells are more prone to exhaustion. This first description of in vivo-generated CD4+ CAR T cells supports their importance for cellular therapy., (Copyright © 2020 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)- Published
- 2020
- Full Text
- View/download PDF
38. CD8-Specific Designed Ankyrin Repeat Proteins Improve Selective Gene Delivery into Human and Primate T Lymphocytes.
- Author
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Frank AM, Weidner T, Brynza J, Uckert W, Buchholz CJ, and Hartmann J
- Subjects
- Animals, Cell Line, Chronic Disease therapy, Gene Transfer Techniques, Genetic Therapy methods, HEK293 Cells, Humans, Lentivirus, Leukocytes, Mononuclear, Macaca mulatta, Macaca nemestrina, Neoplasms therapy, Receptors, Antigen, T-Cell genetics, Recombinant Fusion Proteins genetics, T-Lymphocytes, Cytotoxic metabolism, Transduction, Genetic, Ankyrin Repeat, CD8-Positive T-Lymphocytes metabolism, Genetic Vectors, Receptors, Antigen, T-Cell metabolism, Single-Chain Antibodies genetics
- Abstract
Adoptive T cell immunotherapy in combination with gene therapy is a promising treatment concept for chronic infections and cancer. Recently, receptor-targeted lentiviral vectors (LVs) were shown to enable selective gene transfer into particular types of lymphocytes both in vitro and in vivo . This approach might facilitate the genetic engineering of a patient's own T lymphocytes, possibly even shifting this concept from personalized medicine to an off-the shelf therapy in future. Here, we describe novel high-affinity binders for CD8 consisting of designed ankyrin repeat proteins (DARPins), which were selected to bind to the CD8 receptor of human and nonhuman primate (NHP) cells. These binders were identified by ribosome display screening of DARPin libraries using recombinant human CD8 followed by receptor binding analysis on primary lymphocytes. CD8-targeted LVs (CD8-LVs) were then generated that delivered genes exclusively and specifically to human and NHP T lymphocytes by using the same targeting domain. These CD8-LVs were as specific for human T lymphocytes as their single-chain variable fragment-based counterpart, but they could be produced to higher titers. Moreover, they were superior in transducing cytotoxic T cells both in vitro and in vivo when equal particle numbers were applied. Since the here described CD8-LVs transduced primary T lymphocytes from NHP and human donors equally well, they offer the opportunity for preclinical studies in different animal models including large animals such as NHPs without the need for modifications in vector design.
- Published
- 2020
- Full Text
- View/download PDF
39. In vivo generated human CAR T cells eradicate tumor cells.
- Author
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Agarwal S, Weidner T, Thalheimer FB, and Buchholz CJ
- Abstract
Chimeric antigen receptor (CAR) T cells are in prime focus of current research in cancer immunotherapy. Facilitating CAR T cell generation is among the top goals. We have recently demonstrated direct in vivo generation of human CD19-CAR T cells by targeting CD8
+ cells using lentiviral vectors (LVs). The anti-tumor potency of in vivo generated CAR T cells was assessed in human PBMC-transplanted NSG mice carrying i.v. injected CD19+ Nalm-6 tumor cells. A single injection of CD8-targeted LV delivering CD19-CAR was sufficient to completely eliminate the tumor cells from bone marrow and spleen, whereas control animals contained high levels of CD19+ cells. Tumor elimination was due to in vivo generated CAR+ cells. Notably, these were not only composed of T lymphocytes but also included CAR+ natural killer cells (NK and NKT). This is the first demonstration of tumor elimination by in vivo generated human CAR T cells., (© 2019 The Author(s). Published with license by Taylor & Francis Group, LLC.)- Published
- 2019
- Full Text
- View/download PDF
40. GluA4-Targeted AAV Vectors Deliver Genes Selectively to Interneurons while Relying on the AAV Receptor for Entry.
- Author
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Hartmann J, Thalheimer FB, Höpfner F, Kerzel T, Khodosevich K, García-González D, Monyer H, Diester I, Büning H, Carette JE, Fries P, and Buchholz CJ
- Abstract
Selective gene delivery into subtypes of interneurons remains an important challenge in vector development. Adeno-associated virus (AAV) vector particles are especially promising for intracerebral injections. For cell entry, AAV2 particles are supposed to attach to heparan-sulfate proteoglycans (HSPGs) followed by endocytosis via the AAV receptor (AAVR). Here, we assessed engineered AAV particles deficient in HSPG attachment but competent in recognizing the glutamate receptor 4 (GluA4, also known as GluRD or GRIA4) through a displayed GluA4-specific DARPin (designed ankyrin repeat protein). When injected into the mouse brain, histological evaluation revealed that in various regions, more than 90% of the transduced cells were interneurons, mainly of the parvalbumin-positive subtype. Although part of the selectivity was mediated by the DARPin, the chosen spleen focus-forming virus (SFFV) promoter had contributed as well. Further analysis revealed that the DARPin mediated selective attachment to GluA4-positive cells, whereas gene delivery required expression of AAVR. Our data suggest that cell selectivity of AAV particles can be modified rationally and efficiently through DARPins, but expression of the AAV entry receptor remains essential.
- Published
- 2019
- Full Text
- View/download PDF
41. Highly Efficient and Selective CAR-Gene Transfer Using CD4- and CD8-Targeted Lentiviral Vectors.
- Author
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Jamali A, Kapitza L, Schaser T, Johnston ICD, Buchholz CJ, and Hartmann J
- Abstract
Chimeric antigen receptor (CAR)-modified T cells have revealed promising results in the treatment of cancer, but they still need to overcome various hurdles, including a complicated manufacturing process. Receptor-targeted lentiviral vectors (LVs) delivering genes selectively to T cell subtypes may facilitate and improve CAR T cell generation, but so far they have resulted in lower gene delivery rates than conventional LVs (vesicular stomatitis virus [VSV]-LV). To overcome this limitation, we studied the effect of the transduction enhancer Vectofusin-1 on gene delivery to human T cells with CD4- and CD8-targeted LVs, respectively, encoding a second-generation CD19-CAR in conjunction with a truncated version of the low-affinity nerve growth factor receptor (ΔLNGFR) as reporter. Vectofusin-1 significantly enhanced the gene delivery of CD4- and CD8-LVs without a loss in target cell selectivity and killing capability of the generated CAR T cells. Notably, delivery rates mediated by VSV-LV were substantially reduced by Vectofusin-1. Interestingly, a transient off-target signal in samples treated with Vectofusin-1 was observed early after transduction. However, this effect was not caused by uptake and expression of the transgene in off-target cells, but rather it resulted from cell-bound LV particles having ΔLNGFR incorporated into their surface. The data demonstrate that gene transfer rates in the range of those mediated by VSV-LVs can be achieved with receptor-targeted LVs.
- Published
- 2019
- Full Text
- View/download PDF
42. Tumor-Specific Delivery of Immune Checkpoint Inhibitors by Engineered AAV Vectors.
- Author
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Reul J, Frisch J, Engeland CE, Thalheimer FB, Hartmann J, Ungerechts G, and Buchholz CJ
- Abstract
Immune checkpoint inhibitors (ICIs) can block distinct receptors on T cells or tumor cells thus preventing T cell inactivation and tumor immune escape. While the clinical response to treatment with ICIs in cancer patients is impressive, this therapy is often associated with a number of immune-related adverse events. There is therefore a need to explore innovative strategies of tumor-specific delivery of ICIs. Delivery of therapeutic proteins on a genetic level can be accomplished with viral vectors including those derived from adeno-associated virus (AAV). Here, we assessed the tumor-targeted Her2-AAV, a receptor-targeted AAV vector binding to the tumor antigen Her2/neu for cell entry, as vehicle for ICI gene delivery. Initially, we packaged the coding sequence of a scFv-Fc fusion protein directed against mouse programmed cell death protein-1 (PD-1) into Her2-AAV. Upon transduction of Her2/neu
+ RENCA cells, AAV-encoded αPD-1 was readily detectable in the cell culture supernatant and revealed specific binding to its target antigen. In vivo , in BALB/c mice bearing subcutaneous RENCA-Her2/neu tumors, Her2-AAV mediated specific gene delivery into tumor tissue upon intravenous administration as verified by luciferase gene transfer and in vivo imaging thus demonstrating unimpaired tumor-targeting by Her2-AAV vectors in immunocompetent animals. When delivering the αPD-1 gene, levels of ICI were similar in tumor tissue for Her2-AAV and AAV2 but substantially reduced in liver for Her2-AAV. When combined with chemotherapy a tendency for reduced progression of tumor growth was documented for Her2-AAV treated mice. To get closer to the clinical situation, AAV constructs that deliver the complete coding sequence of the therapeutic antibody nivolumab which is directed against human PD-1 were generated next. The AAV-Nivolumab constructs were expressed and released from transduced MDA-MB-453 cells in vitro and from RENCA-Her2/neu cells upon intratumoral as well as intravenous administration in vivo . Antibody processing and expression levels were further improved through optimization of construct design. In conclusion, we provide proof-of-principle for redirecting the biodistribution of ICIs from liver and serum to tumor tissue by the use of engineered AAV vectors. This strategy can be easily combined with other types of immunotherapeutic concepts.- Published
- 2019
- Full Text
- View/download PDF
43. Ligand Coupling to the AAV Capsid for Cell-Specific Gene Transfer.
- Author
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Reul J, Muik A, and Buchholz CJ
- Subjects
- Animals, Biological Products isolation & purification, Cell Line, Chromatography, Affinity, Chromatography, Gel, Dependovirus isolation & purification, Genetic Vectors isolation & purification, Humans, Organ Specificity, Protein Binding, Transduction, Genetic, Capsid Proteins genetics, Capsid Proteins metabolism, Dependovirus genetics, Gene Transfer Techniques, Genetic Vectors genetics, Ligands
- Abstract
Cell entry of AAV vectors is initiated by contacting the cell surface attachment receptor. This process can be rationally engineered through mutating the contact residues on the AAV capsid and covalently coupling targeting ligands to the capsid surface that exhibit high affinity for a cell surface protein of choice. This way, selective gene delivery to target-receptor positive cell types has been achieved. Two methods for coupling targeting ligands to the AAV capsid can be distinguished. Genetic coupling is achieved through expressing fusion proteins composed of the capsid protein VP2 and the targeting ligand in packaging cells. Biochemical coupling involves split-intein-mediated protein trans-splicing between the mutated AAV capsid and the targeting ligand. While genetic coupling is restricted to designed ankyrin repeat proteins as targeting ligand, biochemical coupling tolerates single-chain antibody fragments as well.
- Published
- 2019
- Full Text
- View/download PDF
44. In vivo generation of human CD19-CAR T cells results in B-cell depletion and signs of cytokine release syndrome.
- Author
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Pfeiffer A, Thalheimer FB, Hartmann S, Frank AM, Bender RR, Danisch S, Costa C, Wels WS, Modlich U, Stripecke R, Verhoeyen E, and Buchholz CJ
- Subjects
- Animals, Graft vs Host Disease immunology, HEK293 Cells, Humans, Leukocytes, Mononuclear transplantation, Mice, Syndrome, Antigens, CD19 metabolism, B-Lymphocytes immunology, Cytokines metabolism, Lymphocyte Depletion, Receptors, Chimeric Antigen immunology, T-Lymphocytes immunology
- Abstract
Chimeric antigen receptor (CAR) T cells brought substantial benefit to patients with B-cell malignancies. Notwithstanding, CAR T-cell manufacturing requires complex procedures impeding the broad supply chain. Here, we provide evidence that human CD19-CAR T cells can be generated directly in vivo using the lentiviral vector CD8-LV specifically targeting human CD8
+ cells. Administration into mice xenografted with Raji lymphoma cells and human peripheral blood mononuclear cells led to CAR expression solely in CD8+ T cells and efficacious elimination of CD19+ B cells. Further, upon injection of CD8-LV into mice transplanted with human CD34+ cells, induction of CAR T cells and CD19+ B-cell depletion was observed in 7 out of 10 treated animals. Notably, three mice showed elevated levels of human cytokines in plasma. Tissue-invading CAR T cells and complete elimination of the B-lymphocyte-rich zones in spleen were indicative of a cytokine release syndrome. Our data demonstrate the feasibility of in vivo reprogramming of human CD8+ CAR T cells active against CD19+ cells, yet with similar adverse effects currently notorious in the clinical practice., (© 2018 The Authors. Published under the terms of the CC BY 4.0 license.)- Published
- 2018
- Full Text
- View/download PDF
45. Surface-Engineered Lentiviral Vectors for Selective Gene Transfer into Subtypes of Lymphocytes.
- Author
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Frank AM and Buchholz CJ
- Abstract
Lymphocytes have always been among the prime targets in gene therapy, even more so since chimeric antigen receptor (CAR) T cells have reached the clinic. However, other gene therapeutic approaches hold great promise as well. The first part of this review provides an overview of current strategies in lymphocyte gene therapy. The second part highlights the importance of precise gene delivery into B and T cells as well as distinct subtypes of lymphocytes. This can be achieved with lentiviral vectors (LVs) pseudotyped with engineered glycoproteins recognizing lymphocyte surface markers as entry receptors. Different strategies for envelope glycoprotein engineering and selection of the targeting ligand are discussed. With a CD8-targeted LV that was recently used to achieve proof of principle for the in vivo reprogramming of CAR T cells, these vectors are becoming a key tool to genetically engineer lymphocytes directly in vivo .
- Published
- 2018
- Full Text
- View/download PDF
46. A Library-Based Screening Strategy for the Identification of DARPins as Ligands for Receptor-Targeted AAV and Lentiviral Vectors.
- Author
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Hartmann J, Münch RC, Freiling RT, Schneider IC, Dreier B, Samukange W, Koch J, Seeger MA, Plückthun A, and Buchholz CJ
- Abstract
Delivering genes selectively to the therapeutically relevant cell type is among the prime goals of vector development. Here, we present a high-throughput selection and screening process that identifies designed ankyrin repeat proteins (DARPins) optimally suited for receptor-targeted gene delivery using adeno-associated viral (AAV) and lentiviral (LV) vectors. In particular, the process includes expression, purification, and in situ biotinylation of the extracellular domains of target receptors as Fc fusion proteins in mammalian cells and the selection of high-affinity binders by ribosome display from DARPin libraries each covering more than 10
12 variants. This way, DARPins specific for the glutamate receptor subunit GluA4, the endothelial surface marker CD105, and the natural killer cell marker NKp46 were generated. The identification of DARPins best suited for gene delivery was achieved by screening small-scale vector productions. Both LV and AAV particles displaying the selected DARPins transduced only cells expressing the corresponding target receptor. The data confirm that a straightforward process for the generation of receptor-targeted viral vectors has been established. Moreover, biochemical analysis of a panel of DARPins revealed that their functional cell-surface expression as fusion proteins is more relevant for efficient gene delivery by LV particles than functional binding affinity.- Published
- 2018
- Full Text
- View/download PDF
47. Distinct in vivo roles of secreted APP ectodomain variants APPsα and APPsβ in regulation of spine density, synaptic plasticity, and cognition.
- Author
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Richter MC, Ludewig S, Winschel A, Abel T, Bold C, Salzburger LR, Klein S, Han K, Weyer SW, Fritz AK, Laube B, Wolfer DP, Buchholz CJ, Korte M, and Müller UC
- Subjects
- Alzheimer Disease metabolism, Alzheimer Disease pathology, Animals, Brain metabolism, Brain pathology, Hippocampus metabolism, Hippocampus pathology, Humans, Mice, Mice, Knockout, Neurons metabolism, Neurons pathology, Spine metabolism, Spine pathology, Synaptic Transmission genetics, alpha7 Nicotinic Acetylcholine Receptor genetics, Alzheimer Disease genetics, Amyloid beta-Protein Precursor genetics, Cognition physiology, Neuronal Plasticity genetics
- Abstract
Increasing evidence suggests that synaptic functions of the amyloid precursor protein (APP), which is key to Alzheimer pathogenesis, may be carried out by its secreted ectodomain (APPs). The specific roles of APPsα and APPsβ fragments, generated by non-amyloidogenic or amyloidogenic APP processing, respectively, remain however unclear. Here, we expressed APPsα or APPsβ in the adult brain of conditional double knockout mice (cDKO) lacking APP and the related APLP2. APPsα efficiently rescued deficits in spine density, synaptic plasticity (LTP and PPF), and spatial reference memory of cDKO mice. In contrast, APPsβ failed to show any detectable effects on synaptic plasticity and spine density. The C-terminal 16 amino acids of APPsα (lacking in APPsβ) proved sufficient to facilitate LTP in a mechanism that depends on functional nicotinic α7-nAChRs. Further, APPsα showed high-affinity, allosteric potentiation of heterologously expressed α7-nAChRs in oocytes. Collectively, we identified α7-nAChRs as a crucial physiological receptor specific for APPsα and show distinct in vivo roles for APPsα versus APPsβ. This implies that reduced levels of APPsα that might occur during Alzheimer pathogenesis cannot be compensated by APPsβ., (© 2018 The Authors.)
- Published
- 2018
- Full Text
- View/download PDF
48. Displaying Tetra-Membrane Spanning Claudins on Enveloped Virus-Like Particles for Cancer Immunotherapy.
- Author
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Schneider IC, Hartmann J, Braun G, Stitz J, Klamp T, Bihi M, Sahin U, and Buchholz CJ
- Subjects
- Animals, Claudins genetics, Claudins therapeutic use, Humans, Mice, Neoplasms prevention & control, Neoplasms therapy, Vaccines, Virus-Like Particle immunology, Vaccines, Virus-Like Particle therapeutic use, Viral Envelope Proteins genetics, Claudins immunology, Immunotherapy, Neoplasms immunology, Viral Envelope Proteins immunology
- Abstract
Virus-like particles (VLPs) displaying foreign antigens have become an important tool in vaccination including the induction of immune responses against self-antigens. Claudin 6 (CLDN6) has been identified as tumor-associated antigen and is therefore a potential target for tumor vaccination strategies. However, as tetra-membrane spanning protein its incorporation into VLPs while preserving a native fold is challenging. Here, we attempted the incorporation of a panel of engineered CLDN6 variants into the membrane of retrovirus-derived VLPs. Interestingly, wild-type CLDN6 revealed the most efficient display. VLPs presenting CLDN6 or CLDN9 derived from different donor species were produced and preservation of their native confirmation was demonstrated by antibody binding assays. VLPs displaying murine CLDN6 were used to immunize mice. Antibodies recognizing native CLDN6 as displayed on cell surfaces and mediating complement-dependent cytotoxicity were elicited in vaccinated animals. The data suggest applications of CLDN6 displaying VLPs in cancer immunotherapy., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2018
- Full Text
- View/download PDF
49. CD30-targeted oncolytic viruses as novel therapeutic approach against classical Hodgkin lymphoma.
- Author
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Hanauer JDS, Rengstl B, Kleinlützum D, Reul J, Pfeiffer A, Friedel T, Schneider IC, Newrzela S, Hansmann ML, Buchholz CJ, and Muik A
- Abstract
Classical Hodgkin lymphoma (cHL) is a hematopoietic malignancy with a characteristic cellular composition. The tumor mass is made up of infiltrated lymphocytes and other cells of hematologic origin but only very few neoplastic cells that are mainly identified by the diagnostic marker CD30. While most patients with early stage cHL can be cured by standard therapy, treatment options for relapsed or refractory cHL are still not sufficient, although immunotherapy-based approaches for the treatment of cHL patients have gained ground in the last decade. Here, we suggest a novel therapeutic concept based on oncolytic viruses selectively destroying the CD30
+ -positive cHL tumor cells. Relying on a recently described CD30-specific scFv we have generated CD30-targeted measles virus (MV-CD30) and vesicular stomatitis virus (VSV-CD30). For VSV-CD30 the VSV glycoprotein G reading frame was replaced by those of the CD30-targeted MV glycoproteins. Both viruses were found to be highly selective for CD30-positive cells as demonstrated by infection of co-cultures of target and non-target cells as well as through blocking infection by soluble CD30. Notably, VSV-CD30 yielded much higher titers than MV-CD30 and resulted in a more rapid and efficient killing of cultivated cHL-derived cell lines. Mouse tumor models revealed that intratumorally, as well as systemically injected VSV-CD30, infected cHL xenografts and significantly slowed down tumor growth resulting in a substantially prolonged survival of tumor-bearing mice. Taken together, the data support further preclinical testing of VSV-CD30 as novel therapeutic agent for the treatment of cHL and other CD30+ -positive malignancies., Competing Interests: CONFLICTS OF INTEREST None.- Published
- 2018
- Full Text
- View/download PDF
50. Covalent coupling of high-affinity ligands to the surface of viral vector particles by protein trans-splicing mediates cell type-specific gene transfer.
- Author
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Muik A, Reul J, Friedel T, Muth A, Hartmann KP, Schneider IC, Münch RC, and Buchholz CJ
- Subjects
- Animals, CHO Cells, Cell Line, Tumor, Cricetulus, Genetic Vectors, HEK293 Cells, Humans, Inteins, Ligands, Trans-Splicing, Transduction, Genetic, Dependovirus genetics, Gene Transfer Techniques, Virion genetics
- Abstract
We have established a novel approach for the covalent coupling of large polypeptides to the surface of fully assembled adeno-associated viral gene transfer vector (AAV) particles via split-intein mediated protein-trans-splicing (PTS). This way, we achieved selective gene transfer to distinct cell types. Single-chain variable fragments (scFvs) or designed ankyrin repeat proteins (DARPins), exhibiting high-affinity binding to cell surface receptors selectively expressed on the surface of target cells, were coupled to AAV particles harboring mutations in the capsid proteins which ablate natural receptor usage. Both, the AAV capsid protein VP2 and multiple separately produced targeting ligands recognizing Her2/neu, EpCAM, CD133 or CD30 were genetically fused with complementary split-intein domains. Optimized coupling conditions led to an effective conjugation of each targeting ligand to the universal AAV capsid and translated into specific gene transfer into target receptor-positive cell types in vitro and in vivo. Interestingly, PTS-based AAVs exhibited significantly less gene transfer into target receptor-negative cells than AAVs displaying the same targeting ligand but coupled genetically. Another important consequence of the PTS technology is the possibility to now display scFvs or other antibody-derived domain formats harboring disulfide-bonds in a functionally active form on the surface of AAV particles. Hence, the custom combination of a universal AAV vector particle and targeting ligands of various formats allows for an unprecedented flexibility in the generation of gene transfer vectors targeted to distinct cell types., (Copyright © 2017 Elsevier Ltd. All rights reserved.)
- Published
- 2017
- Full Text
- View/download PDF
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