Biology subjects

Volk, H.-D.

Publications and source records attributed to Volk, H.-D..

14 recordsLinked to original sources

bioRxiv2025

Clinical development of gene edited tacrolimus-resistant Treg (FKBP12KO-Treg) to enable simultaneous immunosuppression and support of immune regulation

Background: Unwanted immune responses play a central role in the pathogenesis of solid organ allograft rejection. These are managed by life-long immunosuppression with considerable burden for the patient and society. Adoptive therapy with regulatory T-cells (Treg) is a promising approach to restore sustainable immune balance and avoid long-term adverse effects of immunosuppression. While Treg effectively inhibit activation of unwanted immune responses, they are less effective in controlling pre-existing/activated memory effector T-cells (Teff). Thus, co-administration of Treg with immunosuppressants is required to achieve a sustainable organ acceptance. Calcineurin inhibitors (CNI) are powerful in controlling de novo generated and preformed Teff. However, CNI also dampen Treg immunoregulatory function. Thus, we hypothesize improved results of adoptive Treg therapy in immunosuppressed patients applying tacrolimus-resistant Treg. Methods: While retaining CNI modulation of Teff with tacrolimus, we knocked-out FKBP12 in Treg (FKBP12KO-Treg) by gene-editing using ribonucleoprotein-based CRISPR/Cas9 technology to generate tacrolimus-resistant Treg and characterised them using flow cytometry, functional assays and in-depth phenotyping. Results: This detailed in vitro analysis showed FKBP12KO-Treg were comparable to non-gene edited Treg and impervious to tacrolimus while maintaining immunoregulatory function and sensitivity to alternative CNIs raising no safety concerns. Furthermore, we aligned our methodology to achieve GMP compliance laying the basis for a manufacturing license in preparation of a clinical trial. Conclusion: Based on the presented preclinical dataset implying safety and efficacy of FKBP12KO-Treg, we are now seeking to undertake a proof-of-concept clinical trial to evaluate the co-administrationof FKBP12KO-Treg and tacrolimus to enhance the management of living donor kidney transplant recipients.

immunology↗

bioRxiv2025

Repurposing base editors for targeted knock-in and simultaneous knockouts to generate multiplex-edited allogeneic CAR T cells with minimal translocations

The CRISPR-Cas system enables precise genome engineering of cell therapies. For allogeneic applications, multiplex editing is frequently required to improve efficacy, persistence, and safety. However, strategies involving multiple DNA double-strand breaks (DSBs) induce genotoxicity by provoking chromosomal aberrations. Base editors, which enable sequence changes without generating DSBs, are widely used for gene disruption, but their capacity for gene insertion remains unexplored. Here, we developed Base editor-mediated knock-in (BEKI), a non-viral platform that allows targeted transgene insertion in parallel with multiplex gene disruption using a single base editor. Repurposing the Cas9 nickase domain of base editors generates paired nicks, inducing homology-directed repair (HDR). In human T cells, optimized guide RNA orientation and nick distance, together with HDR-enhancing modulators, enabled efficient transgene knock-in at the TRAC, CD3{zeta}, B2M, and CD3{varepsilon} loci. Simultaneous base editing of multiple additional genes produced chimeric antigen receptor (CAR) T cells with increased cytokine secretion, drug resistance, and resistance to allo-rejection. Compared to multiplex editing with Cas9, BEKI markedly reduced chromosomal translocations. BEKI therefore provides a streamlined, scalable strategy for multiplex CAR T-cell engineering with a single enzyme, offering a safer route to clinical-grade manufacturing of off-the-shelf therapies for cancer and autoimmune diseases. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=193 SRC="FIGDIR/small/676172v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@f5f6e5org.highwire.dtl.DTLVardef@246411org.highwire.dtl.DTLVardef@125a478org.highwire.dtl.DTLVardef@510278_HPS_FORMAT_FIGEXP M_FIG C_FIG

synthetic biology↗

bioRxiv2025

Revisiting the need for mRNA nucleoside modification in CAR T cell engineering

mRNA-based chimeric antigen receptor (CAR)-T cells offer the promise of enhanced safety and simplified manufacturing. However, in vitro-transcribed (IVT) mRNA is known to trigger antiviral immune responses, inflammatory signaling, and apoptosis in transfected cells. To address these challenges and enable efficient IVT-mRNA expression, modified nucleosides, such as N1-methyl-pseudouridine (m1{Psi}), have become the gold standard for CAR-T cell production, albeit at increased cost. In this study, immune responses to IVT-mRNA were evaluated across five primary human cell types, including T-cells. Unexpectedly, T-cells, unlike other immune and non-immune cell types tested, exhibited no immune activation in response to unmodified mRNA. T-cell viability and cytokine secretion patterns remained unaffected, regardless of whether unmodified mRNA was delivered via lipid nanoparticles (LNPs) or electroporation. Furthermore, CAR expression levels in T-cells were not influenced by mRNA modification with m1{Psi} or 5-methoxy-uridine (5moU) nucleosides. The absence of nucleoside modifications did not compromise CAR-T cell cytotoxic potency, demonstrating that such modifications are not required for producing functional CAR-T cells. These findings eliminate the need for nucleoside modification in T-cell mRNA, simplifying and reducing the cost of CAR-T cell manufacturing while positioning IVT-mRNA as a highly efficient and minimally invasive tool for CAR-T cell engineering.

bioengineering↗

bioRxiv2024

Gene editing of CD3 epsilon gene to redirect regulatory T cells for adoptive T cell transfer

I.Adoptive transfer of regulatory T cells (Tregs) is a promising strategy to combat immunopathologies in transplantation and autoimmune diseases. Antigen-specific Tregs are more effective in modulating undesired immune reactions, but their low frequency in peripheral blood poses challenges for manufacturing and their clinical application. Chimeric antigen receptors (CARs) have been used to redirect the specificity of Tregs, employing retroviral vectors. However, retroviral gene transfer is costly, time consuming, and raises safety issues. Here, we explored non-viral gene editing to redirect Tregs with CARs, using HLA-A2-specific constructs for proof-of-concept studies in transplantation models. We introduce a virus-free CRISPR-Cas12a approach to integrate an antigen-binding domain into the CD3 epsilon (CD3{varepsilon}) gene, generating Tregs expressing a T cell receptor fusion construct (TruC). These CD3{varepsilon}-TruC Tregs exhibit potent antigen-dependent activation while maintaining responsiveness to TCR/CD3 stimulation. This enables preferential enrichment of TruC-redirected Tregs via repetitive CD3/CD28-stimulation in a GMP-compatible expansion system. Non-viral gene edited CD3{varepsilon}-TruC Tregs retained their phenotypic, epigenetic, and functional identity. In a humanized mouse model, HLA-A2-specific CD3{varepsilon}-TruC Tregs demonstrate superior protection of allogeneic HLA-A2+ skin grafts from rejection compared to polyclonal Tregs. This approach provides a pathway for developing clinical-grade CD3{varepsilon}-TruC-based Treg cell products for transplantation immunotherapy and other immunopathologies.

immunology↗

bioRxiv2023

Single cell analysis reveals CD45RClow/-TNFR2+CD29lowCD8+ Tregs with superior activity

Although described in the 70s, CD8+ regulatory T cells (Tregs) remain incompletely understood and to date, although several markers are used to define them, they remain poorly defined. The identification of reliable and consistent markers, as it was done for CD4+ Tregs, remains an urgent task and a challenge to advance our understanding. Herein, we analyzed total CD8+ T cells using single cell CITEseq and VDJ T cell receptor sequencing utilizing markers used previously to identify Tregs, in particular CD45RC described by our team and others to divide pro-inflammatory (CD45RChigh) and pro-regulatory (CD45RClow/-) CD8+ T cells in rat, mice and human. 7000 freshly isolated, non-stimulated CD8+ T lymphocytes of four healthy volunteers were analyzed. Combining at a single cell level transcriptome and protein expression data led for the first time to the characterization and definition of three subsets of regulatory CD8+ T cells. Further in vitro functional analysis based on three markers highlighted the superior suppressive activity of the CD8+CD45RClow/-TNFR2+CD29low Tregs subset. To our knowledge, this is the largest characterization of human CD8+ Tregs to date. This data resource will help improve our understanding of CD8+ T cells heterogeneity and will help to translate CD8+ Tregs to the clinic.

immunology↗

bioRxiv2023

Integration of ζ-deficient CARs into the CD3-zeta gene conveys potent cytotoxicity in T and NK cells

I.Chimeric antigen receptor (CAR)-reprogrammed immune cells hold significant therapeutic potential for oncology, autoimmune diseases, transplant medicine, and infections. All approved CAR-T therapies rely on personalized manufacturing using undirected viral gene transfer, which results in non-physiological regulation of CAR-signaling and limits their accessibility due to logistical challenges, high costs and biosafety requirements. Here, we propose a novel approach utilizing CRISPR-Cas gene editing to redirect T cells and natural killer (NK) cells with CARs. By transferring shorter, truncated CAR-transgenes lacking a main activation domain into the human CD3{zeta} (CD247) gene, functional CAR fusion-genes are generated that exploit the endogenous CD3{zeta} gene as the CARs activation domain. Repurposing this T/NK-cell lineage gene facilitated physiological regulation of CAR-expression and reprogramming of various immune cell types, including conventional T cells, TCR{gamma}/{delta} T cells, regulatory T cells, and NK cells. In T cells, CD3{zeta} in-frame fusion eliminated TCR surface expression, reducing the risk of graft-versus-host disease in allogeneic off-the-shelf settings. CD3{zeta}-CD19-CAR-T cells exhibited comparable leukemia control to T cell receptor alpha constant (TRAC)-replaced and lentivirus-transduced CAR-T cells in vivo. Tuning of CD3{zeta}-CAR-expression levels significantly improved the in vivo efficacy. Compared to TRAC-edited CAR-T cells, integration of a Her2-CAR into CD3{zeta} conveyed similar in vitro tumor lysis but reduced susceptibility to activation-induced cell death and differentiation, presumably due to lower CAR-expression levels. Notably, CD3{zeta} gene editing enabled reprogramming of NK cells without impairing their canonical functions. Thus, CD3{zeta} gene editing is a promising platform for the development of allogeneic off-the-shelf cell therapies using redirected killer lymphocytes. Key pointsO_LIIntegration of {zeta}-deficient CARs into CD3{zeta} gene allows generation of functional TCR-ablated CAR-T cells for allogeneic off-the-shelf use C_LIO_LICD3{zeta}-editing platform allows CAR reprogramming of NK cells without affecting their canonical functions C_LI

immunology↗

bioRxiv2023

Matching or genetic engineering of HLA Class I and II facilitates successful allogeneic 'off-the-shelf' regulatory T cell therapy

The potential to harness regulatory T cells (Tregs) for the treatment of autoimmune diseases and transplant rejection has been restricted by several barriers: donor variability, manufacturing complications, and time-consuming expansion processes. These issues further complicate the use of autologous Tregs during acute disease phases or when Tregs are low in number or dysfunctional. Here we explore the potential of off-the-shelf allogeneic Tregs, from healthy donors or universal sources, to provide a more practical solution. We discover that the efficacy of these cells is undermined by the recipients immune response, and that that rigorous matching of HLA classes I and II overcomes this barrier. Importantly, genetically manipulating HLA expression enables the use of unmatched allogeneic Tregs with in vivo efficacy. Our findings underscore the transformative potential of HLA-engineered Tregs, offering a novel, ready-to-use therapeutic avenue for treating a wide array of inflammatory diseases. One-Sentence SummaryMatching or engineering of HLA-I and HLA-II facilitates allogeneic off-the-shelf regulatory T cells for immunoregulation.

immunology↗

bioRxiv2022

Combining different CRISPR nucleases for simultaneous knock-in and base editing prevents translocations in multiplex-edited CAR T cells

I.Multiple genetic modifications may be required to develop potent off-the-shelf chimeric antigen receptor (CAR) T cell therapies. Conventional CRISPR-Cas nucleases install sequence-specific DNA double-strand breaks (DSBs), enabling gene knock-out (KO) or targeted transgene knock-in (KI). However, simultaneous DSBs provoke a high rate of genomic rearrangements which may impede the safety of the edited cells. Here, we combine a non-viral CRISPR-Cas9 nuclease-assisted KI and Cas9-derived base editing technology for DSB free KOs within a single intervention. We demonstrate efficient insertion of a CAR into the T cell receptor alpha constant (TRAC) gene, along with two KOs that silence major histocompatibility complexes (MHC) class I and II expression. This approach reduced translocations to 1.5% of edited cells. Small insertions and deletion at the base editing target sites indicated guide RNA exchange between the editors. This was overcome by using CRISPR enzymes of distinct evolutionary origins. Combining Cas12a Ultra for CAR KI and a Cas9-derived base editor enabled the efficient generation of triple-edited CAR T cells with a translocation frequency comparable to unedited T cells. Resulting T cell receptor- (TCR-) and MHC-negative CAR T cells resisted allogeneic T cell targeting in vitro. Thus, we demonstrate a solution for safer multiplex-edited cell products and a path towards off-the-shelf CAR therapeutics.

bioengineering↗

bioRxiv2021

Human iPSC-derived renal cells change their immunogenic properties during maturation: Implications for regenerative therapies

Therapeutic success of human induced pluripotent stem cell (hiPSC)-based therapies critically depends on immunological compatibility of the hiPSC-derived transplant. As grafted hiPSC-derived cells are often immature, we hypothesized that their immunologic properties may change due to post-grafting maturation. Subsequently, this will affect their interaction with the host immune system and may compromise graft tolerance. In the present study allogeneic and autologous cellular immunity of primary cells, therof reprogrammed hiPSC, hiPSC-derived progenitor and terminally differentiated cells was investigated in vitro by using renal cells as a model system. In contrast to allogeneic primary cells, hiPSC-derived early renal progenitors and mature renal epithelial cells were both tolerated not only by autologous but also by allogeneic T cells. These immune-privileged properties resulted from active immune-modulation and low immune visibility, which declined during the process of cell maturation. However, autologous and allogeneic natural killer (NK) cell responses were not suppressed by hiPSC-derived renal cells and efficiently changed NK cell activation status. These findings clearly show a dynamic stage-specific dependency of autologous and allogeneic T- and NK cell responses to the hiPSC-derived renal cell lineage with consequences for effective cell therapies. The study suggests that hiPSC-derived early progenitors may provide advantageous immune suppressive properties when applied in cell therapy. The data furthermore indicate a need to suppress NK cell activation in allogeneic as well as autologous settings.

cell biology↗

bioRxiv2021

Fast, efficient and virus-free generation of TRAC-replaced CAR T cells

Chimeric Antigen Receptor (CAR) redirected T cells are a potent treatment option for certain hematological malignancies. Recently, site-specific insertion of CARs into the T cell receptor (TCR) alpha constant (TRAC) locus using gene editing and adeno-associated viruses was shown to generate CAR T cells with improved functionality over their retrovirally transduced counterparts. However, the development of viruses for gene transfer is complex and associated with extensive costs at early clinical stages. Here, we provide an economical and virus-free method for efficient CAR insertion into the TRAC locus of primary human T cells via CRISPR-Cas mediated homology-directed repair (HDR). While the toxicity induced by transfected double-stranded template (donor) DNA was not fully prevented by pharmacological means, the combination of DNA-sensor inhibitors and HDR enhancers resulted in highly efficient gene editing with TCR-to-CAR replacement rates reaching up to 68%. The resulting TCR-deficient CAR T cells show antigen-specific cytotoxicity and cytokine production in vitro. Our GMP-compatible non-viral platform technology lays the foundation for clinical trials and fast-track generation of novel CAR T cells applicable for autologous or allogeneic off-the-shelf use.

immunology↗

bioRxiv2020

Extra-hematopoietic immunomodulatory role of the SCID-susceptibility gene DOCK-2 identified by stepwise maturation of human iPSCs into clonogenic mesodermal stromal progenitors

Stromal cells contribute to organ integrity as fibroblasts and to vascular stability as pericytes, in addition to their enigmatic niche function in many tissues. Their inherent immunomodulatory capacity attracted particular attention, initiating numerous clinical trials, particularly testing trophic regeneration and immunomodulation. Key stromal immune functions are still enigmatic. Here we show that dedicator of cytokinesis (DOCK-2) previously described for causing immune cell dysfunction plays a role in extra-hematopoietic immunity by regulating stromal/fibroblast immunomodulatory function. We used three independent strategies including iPSC-derived mesodermal stromal cell (MSC) lineage maturation, severe combined immunodeficiency (SCID) patient-derived cells and CRISPR/Cas9 knockout to support our findings. Human induced pluripotent stem cells (iPSCs) were generated from healthy bone marrow and umbilical cord blood-derived fibroblasts by Sendai virus-mediated transient expression of Yamanaka factors. After mesoderm induction, stromal differentiation was induced by platelet-derived growth factors under animal serum-free conditions. Under feeder-free defined conditions, iPSCs differentiated into expandable and cryo-preservable CD73+/CD105+/Tra-1-81- early iPS-MSCs lacking immunosuppressive potential. Successive maturation was required for reaching the canonical MSC phenotype and immunomodulatory competence over time, while maintaining clonogenicity, comparable to parental MSCs. Sequential RNAseq revealed acquisition of a spectrum of immune-related genes significantly expressed in mature iPS-MSCs and resembling parental MSCs immune gene expression. The DOCK-2 gene attracted our attention because mutations can cause SCID. Interestingly, SCID patient-derived fibroblast lines harboring bi-allelic DOCK-2 mutations showed significantly reduced immunomodulatory capacity compared to non-mutated control fibroblasts. CRISPR/Cas9-mediated DOCK-2 knockout in healthy iPSCs resulted in iPS-MSCs that also displayed reduced immunomodulatory capacity, thus confirming a role of DOCK-2 in stromal immune function. At a mechanistic level, DOCK-2 deficiency resulted in disturbed subcellular localization of CDC42. This provides first evidence for an extra-hematopoietic immunomodulatory role of DOCK-2 in stromal cells, previously considered restricted to hampering immune cell migration/function resulting in SCID. We may speculate that some of the signs and symptoms of persisting immune disease after successful hematopoietic stem cell transplantation in SCID patients could at least in part be due to mutations, like DOCK-2-/-, permissive outside the hematopoietic immune system, as evidenced also by the increased virus infection susceptibility of DOCK-2 deficient fibroblasts.

immunology↗

bioRxiv2018

Placental-expanded, mesenchymal cells improve muscle function following hip arthroplasty

BackgroundNo regenerative approach has thus far been shown to be effective in skeletal muscle injuries, despite high frequency and associated functional deficits. We sought to address surgical trauma related muscle injuries using local intraoperative application of allogeneic placenta-derived, mesenchymal-like adherent cells (PLX-PAD), using hip arthroplasty as a standardized injury model, because of the high regenerative and immunomodulatory potency of this cell type.\n\nMethodsOur pilot phase I/IIa study was prospective, randomized, double blind and placebo-controlled. Twenty patients undergoing hip arthroplasty via a direct lateral approach were injected with 3.0x108 or 1.5x108 PLX-PAD or a placebo into the gluteus medius muscle.\n\nResultsWe did not observe any relevant PLX-PAD-related adverse events at the 2-year follow-up. Improved gluteus medius strength was noted as early as week 6 in the treatment-groups. Surprisingly, until week 26 the low-dose outperformed the high-dose group and reached significantly improved strength compared to placebo, mirrored by an increase in muscle volume. Histology indicated accelerated healing after cell therapy. Biomarker studies revealed that low-dose treatment reduced the surgery-related immunological stress reaction more than high-dose. Signs of late-onset immune reactivity after high-dose treatment corresponded to reduced functional improvement.\n\nConclusionAllogeneic PLX-PAD therapy improved strength and volume of injured skeletal muscle with a reasonable safety profile. Outcomes could be positively correlated with the modulation of early postoperative stress-related immunological reactions.\n\nTrial RegistrationClinicalTrials.gov (number NCT01525667) and EudraCT (number 2011-003934-16)\n\nFundingThe study was funded by the Sponsor, Pluristem Therapeutics, the Israeli innovation authority and the German Federal Ministry of Education and Research.\n\nConflict of interestT. Winkler, C. Perka and G.N. Duda are members of a clinical advisory board of Pluristem Ltd for future indications. T. Winkler, C. Perka, G.N. Duda, P. von Roth filed a patent together with Pluristem Ltd. E. Lukasiewicz Hagai, R. Ofir, L. Pinzur and E. Eyal are current or former employees of Pluristem Ltd. T. Winkler, P. Reinke and H.-D. Volk received in the past consulting fees from Pluristem Ltd. but not for this project.

clinical trials↗

bioRxiv2018

High prevalence of S. pyogenes Cas9-specific T cell sensitization within the adult human population - A balanced effector/regulatory T cell response

Summary ParagraphThe field of gene therapy has been galvanized by the discovery of the highly efficient and adaptable site-specific nuclease system CRISPR/Cas9 from bacteria.1,2 Immunity against therapeutic gene vectors or gene-modifying cargo nullifies the effect of a possible curative treatment and may pose significant safety issues.3-5 Immunocompetent mice treated with CRISPR/Cas9-encoding vectors exhibit humoral and cellular immune responses against the Cas9 protein, that impact the efficacy of treatment and can cause tissue damage.5,6 Most applications aim to temporarily express the Cas9 nuclease in or deliver the protein directly into the target cell. Thus, a putative humoral antibody response may be negligible.5 However, intracellular protein degradation processes lead to peptide presentation of Cas9 fragments on the cellular surface of gene-edited cells that may be recognized by T cells. While a primary T cell response could be prevented or delayed, a pre-existing memory would have major impact. Here, we show the presence of a ubiquitous memory/effector T cell response directed towards the most popular Cas9 homolog from Streptococcus pyogenes (SpCas9) within healthy human subjects. We have characterized SpCas9-reactive memory/effector T cells (TEFF) within the CD4/CD8 compartments for multi-effector potency and lineage determination. Intriguingly, SpCas9-specific regulatory T cells (TREG) profoundly contribute to the pre-existing SpCas9-directed T cell immunity. The frequency of SpCas9-reactive TREG cells inversely correlates with the magnitude of the respective TEFF response. SpCas9-specific TREG may be harnessed to ensure the success of SpCas9-mediated gene therapy by combating undesired TEFF response in vivo. Furthermore, the equilibrium of Cas9-specific TEFF and TREG cells may have greater importance in Streptococcus pyogenes-associated diseases. Our results shed light on the T cell mediated immunity towards the much-praised gene scissor SpCas9 and offer a possible solution to overcome the problem of pre-existing immunity.

immunology↗

bioRxiv2018

Distinct housing conditions reveal a major impact of adaptive immunity on the course of obesity-induced type 2 diabetes

Obesity is associated with adipose tissue inflammation, insulin resistance and the development of type 2 diabetes. However, our knowledge is mostly based on conventional murine models and promising pre-clinical studies rarely translated into successful therapies.\n\nThere is a growing awareness of the limitations of studies in laboratory mice, housed in abnormally hygienic specific pathogen-free (SPF) conditions, as relevant aspects of the human immune system remain unappreciated. Here, we assessed the impact of housing conditions on adaptive immunity and metabolic disease processes during high-fat diet. We therefore compared diet-induced obesity in SPF mice with those housed in non-SPF, so called \"antigen exposed\" (AE) conditions. Surprisingly, AE mice fed a high-fat diet maintained increased insulin levels to compensate for insulin resistance, which was reflected in islet hyperplasia and improved glucose tolerance compared to SPF mice. In contrast, we observed higher proportions of effector/memory T cell subsets in blood and liver of high-fat diet AE mice accompanied by the development of nonalcoholic steatohepatitis-like liver pathology. Thus, our data demonstrate the impact of housing conditions on metabolic alterations. Studies in AE mice, in which physiological microbial exposure was restored, could provide a tool for revealing therapeutic targets for immune-based interventions for type 2 diabetes patients.

immunology↗