Biology subjects

Reinke, P.

Publications and source records attributed to Reinke, P..

13 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↗

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

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↗

bioRxiv2019

Sex-associated differences in cytomegalovirus prevention: Prophylactic strategy is associated with a strong kidney function impairment in female renal transplant patients

Post-transplantation cytomegalovirus (CMV) syndrome can be prevented using the antiviral drug (val)ganciclovir. (Val)ganciclovir is typically administered following a prophylactic or a pre-emptive strategy. The prophylactic strategy entails early universal administration, the pre-emptive strategy, early treatment in case of infection. However, it is not clear which strategy is superior with respect to transplantation outcome; sex-specific effects of these prevention strategies are not known. We have retrospectively analysed 540 patients from the multi-centre Harmony study along eight pre-defined visits: 308 were treated according to a prophylactic, 232 according to a pre-emptive strategy. As expected, we observed an association of prophylactic strategy with lower incidence of CMV syndrome, delayed onset and lower viral loads compared to the pre-emptive strategy. However, in female patients, the prophylactic strategy was associated with a strong impairment of glomerular filtration rate one year post-transplant (difference: -12.0{+/-}4.2 mL{middle dot}min-1{middle dot}1.73m-2, P=0.005). Additionally, we observed a tendency of higher incidence of acute rejection and severe BK virus reactivation in the prophylactic strategy group. While the prophylactic strategy was more effective for preventing CMV syndrome, our results suggest for the first time that the prophylactic strategy might lead to inferior transplantation outcomes in female patients, providing evidence for a strong association with sex.

pharmacology and toxicology↗

bioRxiv2019

BKV clearance time correlates with exhaustion state and T-cell receptor repertoire shape of BKV-specific T-cells in renal transplant patients

Reactivation of the BK polyomavirus is known to lead to severe complications in kidney transplant patients. The current treatment strategy relies on decreasing the immunosuppression to allow the immune system to clear the virus. Recently, we demonstrated a clear association between the resolution of BKV reactivation and reconstitution of BKV-specific CD4+ T-cells. However, which factors determine the duration of viral infection clearance remains so far unclear. Here we apply a combination of in-depth multi-parametric flow cytometry and NGS-based CDR3 beta chain receptor repertoire analysis of BKV-specific T-cells to a cohort of 7 kidney transplant patients during the clinical course of BKV reactivation. This way we followed TCR repertoires at single clone levels and functional activity of BKV-specific T-cells during the resolution of BKV infection. The duration of BKV clearance did not depend on the number of peripheral blood BKV-specific T-cells nor on a few immunodominant BKV-specific T-cell clones. Rather, the T-cell receptor repertoire diversity and exhaustion status of BKV-specific T-cells affected the duration of viral clearance: high clonotype diversity and lack of PD1 and TIM3 exhaustion markers on BKV-specific T-cells was associated with short clearance time. Our data thus demonstrate how the diversity and the exhaustion state of the T-cells can determine the clinical course of BKV infection.

immunology↗

bioRxiv2018

A novel approach reveals that HLA class 1 single antigen bead-signatures provide a means of high-accuracy pre-transplant risk assessment of acute cellular rejection

Acute cellular rejection (ACR) is associated with complications after kidney transplantation, such as graft dysfunction and graft loss. Early risk assessment is therefore critical for the improvement of transplantation outcomes. In this work, we retrospectively analyzed a pre-transplant HLA antigen bead assay data set that was acquired by the e:KID consortium as part of a systems medicine approach. The data set included single antigen bead (SAB) reactivity profiles of 52 low-risk graft recipients (negative complement dependent cytotoxicity crossmatch, PRA<30%) who showed detectable pre-transplant anti-HLA 1 antibodies. To assess whether the reactivity profiles provide a means for ACR risk assessment, we established a novel approach which differs from standard approaches in two aspects: the use of quantitative continuous data and the use of a multiparameter classification method. Remarkably, it achieved significant prediction of the 38 graft recipients who experienced ACR with a balanced accuracy of 82.7% (sensitivity=76.5%, specificity= 88.9%). The resultant classifier achieved one of the highest prediction accuracies in the literature for pre-transplant risk assessment of ACR. Importantly, it can facilitate risk assessment in non-sensitized patients who lack donor-specific antibodies. As the classifier is based on continuous data and includes weak signals, our results emphasize that not only strong but also weak binding interactions of antibodies and HLA 1 antigens contain predictive information.

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↗