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Monocyte-derived conventional dendritic cells (ConvDCs) loaded with melanoma antigens showed modest responses in clinical trials. Efficacy studies were hampered by difficulties in ConvDC manufacturing and low potency. Overcoming these issues, we demonstrated higher potency of lentiviral vector (LV)-programmed DCs. Monocytes were directly induced to self-differentiate into DCs (SmartDCTRP2) upon transduction with a tricistronic LV encoding for cytokines (granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4)) and a melanoma antigen (tyrosinase-related protein 2 (TRP2)). Here, SmartDC-TRP2 generated with monocytes from five advanced melanoma patients were tested in autologous DC:T cell stimulation assays, validating the activation of functional TRP2-specific cytotoxic T lymphocytes (CTLs) for all patients. We described methods compliant to good manufacturing practices (GMP) to produce LV and SmartDC-TRP2. Feasibility of monocyte transduction in a bag system and cryopreservation following a 24-h standard operating procedure were achieved. After thawing, 50% of the initial monocyte input was recovered and SmartDC-TRP2 selfdifferentiated in vitro, showing uniform expression of DC markers, detectable LV copies and a polyclonal LV integration pattern not biased to oncogenic loci. GMP-grade SmartDC-TRP2 expanded TRP2-specific autologous CTLs in vitro. These results demonstrated asimpler GMP-compliant method of manufacturing an effective individualized DC vaccine. Such DC vaccine, when in combination with checkpoint inhibition therapies, might provide higher specificity against melanoma.

The clinical application of self-inactivating (SIN) retroviral vectors requires an efficient vector production technology. To enable production of γ-retroviral SIN vectors from stable producer cells, new targetable HEK293-based producer clones were selected, providing amphotropic, GALV, or RD114 pseudotyping. Viral vector expression constructs can reliably be inserted at a predefined genomic locus via Flp-recombinase-mediated cassette exchange. Introduction of a clean-up step, mediated by Cre-recombinase, allows the removal of residual sequences that were required for targeting and selection, but were dispensable for the final producer clones and eliminated homology-driven recombination between the tagging and the therapeutic vector. The system was used to establish GALV and RD114 pseudotyping producer cells (HG- and HR820) for a clinically relevant long terminal repeat-driven therapeutic vector, designed for the transfer of a recombinant TCR that delivered titers in the range of 2×10(7) infectious particles (IP)/ml. Production capacity of the amphotropic producer cell (HA820) was challenged by a therapeutic SIN vector transferring the large COL7A1 cDNA. The final producer clone delivered a titer of 4×10(6) IP/ml and the vector containing supernatant was used directly to functionally restore primary fibroblasts and keratinocytes isolated from recessive dystrophic epidermolysis bullosa patients. Thus, the combinatorial approach (fc-technology) to generate producer cells for therapeutic γ-retroviral (SIN) vectors is feasible, is highly efficient, and allows their safe production and application in clinical trials.

Therapeutic vaccination with tumor antigen-encoding RNAs is being investigated in various clinical trials. Typically, the RNA vaccine is administered intradermally, subcutaneously or intranodally with the intention to get expression of the encoded antigens in local antigen-presenting cells (APCs). We have developed a novel class of RNA-lipoplex (RNA(LIP)) immunotherapeutics for intravenous application, which allow systemic targeting of APCs. RNA(LIP) is a novel nanoparticulate formulation of lipid-complexed mRNA which selectively delivers the functional mRNA to APCs in lymphoid compartments body-wide for efficient mRNA uptake and expression of the encoded antigen by APCs. Moreover, this formulation has intrinsically strong adjuvant activity, mimics a systemic viral infection and induces synchronized activation of potent adaptive as well as type-I-IFN-mediated innate immune responses (Kranz et al., Nature 2016). The first-in-human phase I/II dose escalation Lipo-MERIT trial (NCT02410733) conducted in four German study centers assesses the safety, tolerability, and biological efficacy of RNA(LIP) immunotherapy in patients with stage IIIB/C and IV melanoma. This trial is the first to investigate intravenous administration of a RNA-based cancer vaccine. Following antigen expression stratification on routinely collected tumor samples, eligible patients are treated with repeated dosing of the tetravalent Lipo-MERIT vaccine composed of RNA(LIP) products encoding the shared melanoma-associated antigens NY-ESO-1, tyrosinase, MAGE-A3, and TPTE. Pharmacodynamic activity and immunogenicity of the vaccine is investigated by concerted immune monitoring and correlative biomarker studies. Clinical activity is assessed following imaging according to irRECIST1.1. As of January 2018, >50 patients have been treated with escalating or constant dosing under the guidance of an independent data safety and monitoring board. The Lipo-MERIT vaccine was generally well-tolerated and no dose-limiting toxicities (DLTs) were observed so far. Further patient enrollment is continuing. Preliminary data on the assessment of vaccine-induced immune responses and clinical responses from the first patients treated will be presented. Citation Format: Robert A. Jabulowsky, Carmen Loquai, Heidrun Mitzel-Rink, Jochen Utikal, Christoffer Gebhardt, Jessica C. Hassel, Roland Kaufmann, Andreas Pinter, Evelyna Derhovanessian, Christine Anft, Sebastian Attig, Alexandra Deubel, Mustafa Diken, Maike Gold, Claudia Guertler, Heinrich Haas, Ludwig Heesen, Alexandra Kemmer-Brück, Lena M. Kranz, Klaus Kuehlcke, Andreas Kuhn, Peter Langguth, Ulrich Luxemburger, Daniel Maurus, Martin Meng, Felicitas Müller, Richard Rae, Fatih Sari, Katharina Schreeb, Doreen Schwarck-Kokarakis, Malte Stein, Dirk Jäger, Stephan Grabbe, Sebastian Kreiter, Christoph Huber, Özlem Türeci, Ugur Sahin. A first-in-human phase I/II clinical trial assessing novel mRNA-lipoplex nanoparticles encoding shared tumor antigens for immunotherapy of malignant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr CT156.

Mutanome engineered RNA immuno-therapy (MERIT) for patients with triple negative breast cancer

Effective immunization of patients shortly after allogeneic hematopoietic stem cell transplantation (allo-HSCT) is an unmet clinical need due to delayed de novo development of T and B cells. We demonstrated a recombinant, donor-derived cell vaccine against cytomegalovirus that can be easily manufactured, cryopreserved and, upon thaw and administration, turn themselves into highly-viable dendritic cells (DCs). The cell source consisted of monocytes from peripheral blood or cord blood (CB), transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the cytomegalovirus antigen pp65). The pharmacodynamics of the frozen/thawed vaccine (“SmyleDCpp65”) was evaluated by immunization of NOD.Rag1–/–.IL2rγ–/– mice transplanted with human CD34+ CB stem cells. SmyleDCpp65 stimulated in vivo thymic and extrathymic expansion of human cytotoxic T cells (CTL) reactive against pp65, and high levels of human immunoglobulins and immune-stimulatory cytokines in plasma. Standardized production of the IDLV and SmyleDCpp65 was established using Good Manufacturing Practices (GMP). Analytical parameters for quality control demonstrated high recovery, uniformity, purity and viability of SmyleDCpp65 after thawing. Stimulation of autologous T cells by GMP-grade SmyleDCpp65 was validated. SmyleDCpp65 showed only residual and polyclonal IDLV integration, unbiased to protooncogenic hot-spots. These results underscore further developments of this individualized cell vaccine to accelerate immune reconstitution against cytomegalovirus after allo-HSCT.

Background Reactivation of latent viruses such as human cytomegalovirus (HCMV) after allogeneic hematopoietic stem cell transplantation (HSCT) results in high morbidity and mortality. Effective immunization against HCMV shortly after allo-HSCT is an unmet clinical need due to delayed adaptive T cell development. Donor-derived dendritic cells (DCs) have a critical participation in stimulation of naïve T cells and immune reconstitution, and therefore adoptive DC therapy could be used to protect patients after HSCT. However, previous methods for ex vivo generation of adoptive donor-derived DCs were complex and inconsistent, particularly regarding cell viability and potency after thawing. We have previously demonstrated in humanized mouse models of HSCT the proof-of-concept of a novel modality of lentivirus-induced DCs (“SmyleDCpp65”) that accelerated antigen-specific T cell development. Methods Here we demonstrate the feasibility of good manufacturing practices (GMP) for production of donor-derived DCs consisting of monocytes from peripheral blood transduced with an integrase-defective lentiviral vector (IDLV, co-expressing GM-CSF, IFN-α and the cytomegalovirus antigen pp65) that were cryopreserved and thawed. Results Upscaling and standardized production of one lot of IDLV and three lots of SmyleDCpp65 under GMP-compliant conditions were feasible. Analytical parameters for quality control of SmyleDCpp65 identity after thawing and potency after culture were defined. Cell recovery, uniformity, efficacy of gene transfer, purity and viability were high and consistent. SmyleDCpp65 showed only residual and polyclonal IDLV integration, unbiased to proto-oncogenic hot-spots. Stimulation of autologous T cells by GMP-grade SmyleDCpp65 was validated. Conclusion These results underscore further developments of this individualized donor-derived cell vaccine to accelerate immune reconstitution against HCMV after HSCT in clinical trials. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0599-5) contains supplementary material, which is available to authorized users.

Graft-versus-host disease (GvHD) is a severe complication in the context of allogeneic stem cell transplantation and adoptive immunotherapy. The transfer of a suicide gene into donor T-lymphocytes (TLCs) allows selective elimination of GvHD-causing cells. As retroviral gene transfer into hematopoietic stem cells can induce leukaemia, there is an urgent need also to analyze retroviral integration sites in TLCs. We examined suicide gene-transduced TLCs in four grafts and from four transplanted patients. One-hundred and fifteen integration sites were detected in vitro. Of these 90 could be mapped to the human genome; 50% (45) were located in genes and 32% (29) were detected 10 kb upstream or downstream of transcription start sites. We found a significant overrepresentation of genes encoding for proteins with receptor activity, signal transducer activity, transcription regulator activity, nucleic acid binding activity and translation regulator activity. Similar data were obtained from patient samples. Our results point to preferred vector integration patterns, which are specific for the target cell population and probably independent of selection processes. Thus, future preclinical analysis of the integration repertoire with abundant amounts of transduced cells could allow a prediction also for the in vivo situation, where target cells are scarce.

Retroviral producer cells are generated by the introduction of a viral genome into "helper" cell lines containing all the necessary components for viral packaging and the release of infectious particles. The selection of high-titer vector producer cells is most efficient if the vector genome encodes a selectable marker, while it is extremely tedious to select high-titer producer clones if the transgene cannot be detected and selected directly. Here we describe the development of a screening system that uses reversible integration of lox-P-flanked eGFP as a qualitative and quantitative marker gene in two different vector systems, greatly facilitating the selection of viral producer cells. After selection and titration of high-titer viral producer cells based on eGFP expression, the eGFP gene could be removed from the provirus by transient introduction of Cre-recombinase into the producer cells, thus allowing the production of therapeutic relevant vectors expressing solely the gene of interest. However, after removal of the marker gene a slight but consistent increase in viral titers compared to the respective control vectors was found, independent of the transgene or backbone used. The single lox-P site retained in the vector backbone does not affect gene expression level or fidelity of RNA processing.

In a clinical trial that we recently reported, a suicide gene transfer in human primary T cells required 12 days of ex vivo culture, including activation of peripheral blood mononuclear cells (PBMC) with CD3 monoclonal antibody (CD3 mAb), retrovirus-mediated transduction, and selection of gene-modified cells (GMC) by G418. The aim of the present study was to determine the impact of the initial T cell activation and of the transduction/selection on T cell receptor beta variable chain (TCRBV) repertoire of GMC by using the spectratyping method. The TCRBV repertoires of nontransduced, nonselected control (Co) cells and of GMC generated after an initial stimulation with CD3 mAb, CD3/CD28 beads, or allogeneic PBMC or Epstein-Barr virus-transformed B (B-EBV) cells were compared to the ones of their corresponding PBMC. The TCRBV repertoires were skewed in Co cells generated after CD3 mAb or after allogeneic stimulation, and even more so in their corresponding GMC, demonstrating that both culture-dependent and transduction/selection-dependent events led to TCRBV repertoire alterations. However, TCRBV repertoires were not altered, or to a lesser extent, in Co cells or GMC produced after CD3/CD28 bead activation, demonstrating a protective effect on both culture-dependent and transduction/selection-dependent repertoire alterations. Thus, we suggest to replace the initial CD3 mAb stimulation by CD3/CD28 beads for the production of clinical-grade GMC in the setting of future gene therapy trials.

Therapeutic vaccination with tumor antigen-encoding RNAs by local administration is currently being successfully employed in various clinical trials. Advancing from local to more efficient systemic targeting of antigen-presenting cells (APCs), we have developed pioneering RNA-lipoplex (RNA(LIP)) immunotherapeutics for intravenous application based on the employment of well-known lipid carriers without the need for functionalization of particles with molecular ligands. The novel RNA(LIP) formulation has been engineered to preserve RNA integrity after intravenous injection and physicochemically optimized for efficient uptake and expression of the encoded antigen by APCs in various lymphoid compartments, resulting in the synchronized induction of both potent adaptive as well as type-I-IFN-mediated innate immune responses. The first-in-human phase I/II dose escalation Lipo-MERIT trial (NCT02410733) assesses the safety, tolerability, and biological efficacy of the innovative RNA(LIP) immunotherapy in four study centers in Germany. This is the first example of a clinically applicable and systemic RNA-based cancer vaccine. Following selective antigen stratification on routinely collected tumor samples, eligible patients with malignant melanoma are treated with increasing doses of the tetravalent Lipo-MERIT vaccine - a fixed set of four RNA(LIP) products, each encoding one shared melanoma-associated antigen, i.e. NY-ESO-1, tyrosinase, MAGE-A3, and TPTE, that are administered successively within one treatment cycle. Accompanying correlative biomarker studies and concerted immunological assessments evaluate the pharmacodynamic activity and immunogenicity upon multiple vaccination cycles with the Lipo-MERIT vaccine. As of January 2017, 15 patients have been treated within five dose escalation cohorts thoroughly guided by an independent data safety and monitoring board. Multiple dosing with the Lipo-MERIT vaccine was generally well-tolerated and no dose-limiting toxicities (DLTs) were observed so far. Further patient enrollment is continuing. Detailed information on the ongoing trial, the recruitment and treatment status as well as preliminary data on the assessment of vaccine-induced immune responses from the first patients treated will be presented. Citation Format: Robert A. Jabulowsky, Carmen Loquai, Jochen Utikal, Jessica Hassel, Roland Kaufmann, Evelyna Derhovanessian, Mustafa Diken, Lena M. Kranz, Heinrich Haas, Sebastian Attig, Christine Anft, Janina Buck, Jan Diekmann, Daniel Fritz, Kerstin Hartmann, Alexandra Kemmer-Brueck, Klaus Kuehlcke, Andreas N. Kuhn, Peter Langguth, Ulrich Luxemburger, Martin Meng, Richard Rae, Fatih Sari, Doreen Schwarck-Kokarakis, Malte Stein, Stephan Grabbe, Sebastian Kreiter, Oezlem Tuereci, Christoph Huber, Ugur Sahin. A first-in-human phase I/II clinical trial assessing novel mRNA-lipoplex nanoparticles for potent melanoma immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT034. doi:10.1158/1538-7445.AM2017-CT034

The majority of metastatic cancers remain incurable since the current methods of treatment often fail to target the heterogeneous nature of each individual patient's tumor. Personalized approaches targeting each individual patient's tumor may therefore bring significant improvements. The Mutanome Engineered RNA Immuno-Therapy (MERIT) consortium will clinically validate a pioneering RNA-based immunotherapy concept for the treatment of triple negative breast cancer (TNBC) by targeting shared tumor antigens and individual neo-antigens in TNBC patients. MERIT combines two personalized treatment concepts: (i) treatment with vaccines containing “off-the-shelf” mRNAs selected from a pre-synthesized mRNA vaccine warehouse (MERIT WAREHOUSE) that encode shared breast cancer tumor antigens expressed in the respective patient's tumor; (ii) treatment with mRNAs engineered on-demand that encode patient-specific mutated neo-antigens identified by next generation sequencing (NGS) and ranked according to the predicted immunogenicity (MERIT MUTANOME). The mRNAs are administered intravenously as a nanoparticulate lipoplex formulation, which specifically targets APCs and consequently induces antigen-specific T cell responses. MERIT is a multi-center phase I trial (NCT02316457) conducted in four European countries to assess the feasibility, safety and biological efficacy of this personalized immunotherapy. TNBC patients (pT1cN0M0 - anyTanyNM0) after surgery and adjuvant chemotherapy will be allocated to one of two study arms. Patients in ARM1 will receive eight vaccination cycles with a personalized set of shared tumor antigens selected from the WAREHOUSE that correspond to the patient tumor's antigen-expression profile. Patients in ARM2 will be first treated with the personalized WAREHOUSE vaccine approach followed by six vaccination cycles of on-demand manufactured MUTANOME vaccine targeting the unique mutation signature of the individual patient. During the clinical trial, patients will receive the individualized combination of the RNAs in parallel to standard radiotherapy. The clinical trial is approved and the study start is planned for Q1 2016. The consortium has built a multi-disciplinary clinical workflow and trial design tailored to this unique therapeutic concept, which covers the whole individualized drug development cycle from target discovery, validation to GMP manufacturing and drug release for each individual patient. We will present the therapeutic concept and study protocol as well as the methodologies required for this highly innovative phase I trial. The personalized immunotherapy overcomes the current limitations of fixed, off-the-shelf therapeutics and thus might increase the clinical benefit for TNBC patients. This project is a collaborative effort of five partners from academia and industry funded by the European Commission's FP7 and led by BioNTech AG. Citation Format: Sandra Heesch, Valesca Bukur, Janina Buck, Jan Diekmann, Mustafa Diken, Kerstin Ewen, Heinrich Haas, Alexandra Kemmer-Brueck, Björn-Philipp Kloke, Sebastian Kreiter, Andreas N. Kuhn, Klaus Kuehlcke, Martin Loewer, Anna Paruzynski, Kathrin Schultheiß, Doreen Schwarck, Marcus Schmidt, Fabrice André, Jacques De Greve, Thomas Kuendig, Henrik Lindman, Steve Pascolo, Tobias Sjöblom, Kris Thielemans, Laurence Zitvogel, Özlem Türeci, Ugur Sahin. MERIT: introducing individualized cancer vaccines for the treatment of TNBC - a phase I trial. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT020.

One of the hallmarks of cancer is the inherent instability of the genome leading to multiple genomic alterations and epigenetic changes that ultimately drive carcinogenesis. These processes lead to a unique molecular profile of every given tumor and to substantial intratumoral heterogeneity of cancer tissues. Recently, a series of independent reports revealed that pre-formed neoantigen specific T-cell responses are of crucial relevance for the clinical efficacy of immune checkpoint inhibitors. However, spontaneous immune recognition of neoantigens seems to be a rare event with only less than 1% of mutations inducing a T-cell response in the tumor-bearing patient. Accordingly, only patients with a high burden of mutations profit from currently approved therapies. To overcome this restriction, the IVAC® MUTANOME-project harnesses the individual patient's mutation profile by manufacturing highly potent neoantigen-coding RNA vaccines. To this end, the individual mutation repertoire is identified by next-generation-sequencing, potentially immunogenic mutations are selected and incorporated into a poly-epitopic RNA vaccine that is tailored to activate and expand the individual patient's neoantigen-specific CD4+ and CD8+ T cells. A phase I study to test this novel concept of an active individualized cancer vaccine for the treatment of malignant melanoma was initiated in 2013 (NCT02035956). Notably, BioNTech RNA Pharmaceutical's IVAC® MUTANOME trial is the first-in-human trial that introduces a fully personalized RNA vaccine for the treatment of malignant melanoma. The objective of this clinical trial is to study the feasibility, safety, tolerability, immunogenicity and the potential clinical activity of the IVAC® MUTANOME approach. The recruitment of a patient into the trial triggers the multi-step IVAC® MUTANOME process covering (i) the receipt and processing of tumor and blood sample specimens, (ii) the identification, prioritization and confirmation of mutations, (iii) testing of pre-existing immunity against identified tumor mutations, (iv) the selection of mutant neoantigen sequences as vaccine targets, (v) design, production of a DNA lead structure, (vi) GMP manufacturing and release of the patient-specific mRNA, (vii) shipment to the clinical trial site and (viii) the administration of the IMP to patients. Detailed information on the trial, the recruitment and treatment status as well as data on the assessment of vaccine induced immune responses will be presented. Citation Format: Matthias Miller, Carmen Loquai, Björn-Philipp Kloke, Sebastian Attig, Nicole Bidmon, Stefanie Bolte, Valesca Bukur, Evelyna Derhovanessian, Jan Diekmann, Angela Filbry, Sandra Heesch, Christoph Hoeller, Klaus Kuehlcke, David Langer, Martin Loewer, Felicitas Mueller, Inga Ortseifer, Burkhard Otte, Anna Paruzynski, Richard Rae, Barbara Schroers, Christine Seck, Kristina Spiess, Arbel D. Tadmor, Isabel Vogler, Mathias Vormehr, Alexandra Kemmer-Brueck, Andreas N. Kuhn, Ulrich Luxemburger, Sebastian Kreiter, Jochen Utikal, Stephan Grabbe, Oezlem Tuereci, Ugur Sahin. IVAC® MUTANOME - A first-in-human phase I clinical trial targeting individual mutant neoantigens for the treatment of melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT022.

Immunotherapeutic approaches have evolved as promising and valid alternatives to available conventional cancer treatments. Amongst others, vaccination with tumor antigen-encoding RNAs by local administration is currently successfully employed in various clinical trials. To allow for a more efficient targeting of antigen-presenting cells (APCs) and to overcome potential technical challenges associated with local administration, we have developed a novel RNA immunotherapeutic for systemic application based on a fixed set of four liposome complexed RNA drug products (RNA(LIP)), each encoding one shared melanoma-associated antigen. The novel RNA(LIP) formulation was engineered (i) to protect RNA from degradation by plasma RNases and (ii) to enable directed in vivo targeting of APCs in lymphoid compartments, thus (iii) allowing for intravenous administration of multiple RNA products advancing from local to systemic targeting of APCs. Here, RNA(LIP) products trigger a Toll-like receptor (TLR)-mediated Interferon-α (IFN-α) release from plasmacytoid dendritic cells (DCs) and macrophages stimulating DC maturation and hence inducing innate immune mechanisms as well as potent vaccine antigen-specific immune responses. Notably, BioNTech RNA Pharmaceuticals′ RNA(LIP) formulation is a universally applicable potent novel vaccine class for intravenous APC targeting and the induction of potent synchronized adaptive and type-I interferon-mediated innate immune responses for cancer immunotherapy. Similar to other liposomal drugs, the ready-to-use RNA(LIP) products are prepared individually in a straight-forward manner directly prior to use from three components, namely solutions containing RNA drug product, NaCl diluent, and liposome excipient, that are provided as a kit. A multi-center phase I/II trial to clinically validate this pioneering RNA(LIP) formulation for the treatment of malignant melanoma was initiated in 2015 (NCT02410733). The objective of the clinical trial is to study the feasibility, safety, tolerability, immunogenicity and evaluate potential clinical activity of the RNA(LIP) immunotherapy concept. Detailed information on the ongoing trial, the recruitment and treatment status as well as data on the assessment of vaccine-induced immune responses will be presented. Citation Format: Robert A. Jabulowsky, Carmen Loquai, Mustafa Diken, Lena M. Kranz, Heinrich Haas, Sebastian Attig, Nicole Bidmon, Janina Buck, Evelyna Derhovanessian, Jan Diekmann, Daniel Fritz, Veronika Jahndel, Alexandra Kemmer-Brueck, Klaus Kuehlcke, Andreas N. Kuhn, Peter Langguth, Ulrich Luxemburger, Martin Meng, Felicitas Mueller, Richard Rae, Fatih Sari, Doreen Schwarck-Kokarakis, Christine Seck, Kristina Spieß, Meike Witt, Jessica C. Hassel, Jochen Utikal, Roland Kaufmann, Sebastian Kreiter, Christoph Huber, Oezlem Tuereci, Ugur Sahin. A first-in-human phase I/II clinical trial assessing novel mRNA-lipoplex nanoparticles for potent cancer immunotherapy in patients with malignant melanoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT032.

Immunotherapeutic approaches have evolved as promising and valid alternatives to available conventional cancer treatments. Amongst others, vaccination with tumor antigen-encoding RNAs by local administration is currently successfully employed in various clinical trials. To allow for a more efficient targeting of antigen-presenting cells (APCs) we have developed a novel RNA immunotherapeutic for systemic application based on a fixed set of four liposome complexed RNA drug products (RNA(LIP)) each encoding one shared melanoma-associated antigen. Similar to other liposomal drugs, the four injectable RNA(LIP) products constituting the investigational medicinal product will be prepared individually in a straight-forward manner directly prior to use from three components, namely solutions containing RNA drug product, NaCl diluent, and liposome excipient, that are provided as a kit. The novel lipoplex formulation was engineered (i) to protect RNA from degradation by plasma RNases and (ii) to enable directed in vivo targeting of APCs in lymphoid compartments, thus (iii) allowing for intravenous administration of multiple RNA products advancing from local to systemic targeting of APCs. The improved selective delivery of the RNA(LIP) products into APCs has further been shown to lead to an enhanced induction of vaccine-induced T-cell responses. Extensive pharmacological characterization of the RNA(LIP) platform revealed that upon cellular uptake the encoded antigens will be translated into proteins that will be rapidly processed into peptide fragments, which after presentation by MHC class I and II molecules on the surface of APCs induce tumor antigen-specific CD8+ and CD4+ T-cell responses that spread systemically. These vaccine-induced T cells have been shown to specifically recognize and kill antigen-positive tumor cells eliciting potent anti-tumoral activity in vivo. The potent vaccination effects are additionally enhanced by further immunomodulatory effects based on the transient release of pro-inflammatory cytokines such as IFN-α, IP-10, and IL-6 due to binding of the administered RNA drug products to Toll-like receptors (TLRs). The clinical translation of this pioneering therapeutic concept is currently being realized in a multi-center, first-in-human phase I trial in patients with malignant melanoma. Main objectives of the clinical trial are to study safety, tolerability, and immunogenicity of this innovative immunotherapy approach. The novel lipoplex formulation, RNA(LIP) mechanism of action, study design and clinical workflow, as well as recruitment and treatment status of the ongoing clinical trial will be presented. Citation Format: Robert A. Jabulowsky, Carmen Loquai, Mustafa Diken, Lena M. Kranz, Heinrich Haas, Sebastian Attig, Cedrik M. Britten, Janina Buck, Evelyna Derhovanessian, Jan Diekmann, Isaac Esparza, Daniel Fritz, Yves Huesemann, Veronika Jahndel, Klaus Kuehlcke, Andreas N. Kuhn, Peter Langguth, Ulrich Luxemburger, Martin Meng, Felicitas Mueller, Kerstin C. Reuter, Doreen Schwarck, Kristina Spiess, Meike Witt, Jessica C. Hassel, Jochen Utikal, Roland Kaufmann, Marc Schrott, Sebastian Kreiter, Oezlem Tuereci, Christoph Huber, Ugur Sahin. A novel nanoparticular formulated tetravalent RNA cancer vaccine for treatment of patients with malignant melanoma. [abstract]. In: Proceedings of the CRI-CIMT-EATI-AACR Inaugural International Cancer Immunotherapy Conference: Translating Science into Survival; September 16-19, 2015; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(1 Suppl):Abstract nr B041.

The Mutanome Engineered RNA Immuno-Therapy (MERIT) consortium will clinically and industrially validate a pioneering RNA-based immunotherapy concept that targets individual tumor antigens and tumor-specific mutations in triple negative breast cancer (TNBC) patients. This biomarker-guided, personalized therapy is a collaborative effort of five partners from academia and industry and is funded by the European Commission's FP7 and led by BioNTech AG. TNBC is an aggressive, molecularly heterogeneous cancer that accounts for 20% of all breast cancer patients. The 5-year survival rate is less than 80%. The molecular heterogeneity across TNBCs results in a lack of common targetable molecular alterations, and thus targeted therapies frequently fail to provide clinical benefit. The MERIT concept attempts to address this unmet medical need. The personalized treatment consists in (i) injecting vaccines containing “off the shelf” mRNAs selected from a pre-synthesized mRNA vaccine warehouse (MERIT WAREHOUSE) that encode tumor specific antigens expressed in the respective patient's tumor; and (ii) thereafter mRNAs engineered on-demand that encode patient-specific sequence stretches incorporating non-synonymous mutations identified by next generation sequencing (NGS) and ranked by predicted immunogenicity (MERIT MUTANOME). The mRNAs are administered intravenously as a nanoparticulate lipoplex formulation and are selectively delivered to splenic APCs. The encoded antigens are translated into proteins that are rapidly processed. Subsequent peptide presentation on the surface of APCs induces antigen-specific T cell responses. The central part of the MERIT project, a multi-center first in human trial, will assess the feasibility, safety and biological efficacy of this innovative personalized immunotherapy in TNBC patients. After discussing the regulatory challenges with the German national regulatory agency (PEI), a phase I study is now in preparation. The trial will start in Q2 2015 in five academic centers in Europe and will recruit thirty TNBC patients. Furthermore, the project includes a comprehensive T-cell immunomonitoring and biomarker program. Moreover, an extensive research program will address the optimization of algorithms for improved prediction of immunogenic mutations. Additionally, compounds to enhance vaccine efficacy will be developed and improved to support further clinical development. We have established a RNA delivery platform as well as a MERIT WAREHOUSE containing mRNAs coding for a selection of TNBC specific antigens. Additionally, we have built a multi-disciplinary clinical workflow and trial design tailored to this unique therapeutic concept. We will describe the therapeutic concept and the critical skills, and methodologies required for this project, including cancer genomics, NGS, bioinformatics, tumor immunomics, industrial drug development, GMP manufacturing, clinical immunotherapy and immunological monitoring. Citation Format: Sandra Heesch, Cedrik M. Britten, Valesca Bukur, Janina Buck, John Castle, Jan Diekmann, Mustafa Diken, Katrin Frenzel, Sebastian Kreiter, Andreas N. Kuhn, Klaus Kuehlcke, Martin Loewer, Heinrich Haas, Alexandra Kemmer-Brueck, Bjoern-Philipp Kloke, Burkhard Otte, Anna Paruzynski, Sebastian Petri, Doreen Schwarck-Kokarakis, Marcus Schmidt, Fabrice André, Jacques De Greve, Thomas Kuendig, Henrik Lindman, Steve Pascolo, Tobias Sjöblom, Kris Thielemans, Laurence Zitvogel, Oezlem Tuereci, Ugur Sahin. The Mutanome Engineered RNA Immuno-Therapy (MERIT) project. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr CT201. doi:10.1158/1538-7445.AM2015-CT201

Drug toxicity and viral resistance limit the long-term efficacy of antiviral drug treatment for human immunodeficiency virus (HIV) infection. Thus, alternative therapies need to be explored. We tested the infusion of T lymphocytes transduced with a retroviral vector (M87o) that expresses an HIV entry-inhibitory peptide (maC46). Gene-modified autologous T cells were infused into ten HIV-infected patients with advanced disease and multidrug-resistant virus during anti-retroviral combination therapy. T-cell infusions were tolerated well, with no severe side effects. A significant increase of CD4 counts was observed after infusion. At the end of the 1-year follow-up, the CD4 counts of all patients were still around or above baseline. Gene-modified cells could be detected in peripheral blood, lymph nodes, and bone marrow throughout the 1-year follow-up, and marking levels correlated with the cell dose. No significant changes of viral load were observed during the first 4 months. Four of the seven patients who changed their antiviral drug regimen thereafter responded with a significant decline in plasma viral load. In conclusion, the transfer of gene-modified cells was safe, led to sustained levels of gene marking, and may improve immune competence in HIV-infected patients with advanced disease and multidrug-resistant virus.

Despite significant improvements of the survival of HIV-infected patients during HAART, treatment limitations such as the development of drug-resistant HIV strains and long-term toxicities call for innovative treatment strategies. In this study, a novel antiviral gene that effectively inhibits virus entry was tested in a phase I/II clinical trial. The antiviral gene is expressed from a retroviral vector (M87o) and encodes for the membrane-anchored antiviral peptide C46. C46 comprises 46 amino acids, is derived from the second heptad repeat of the HIV-1 envelope glycoprotein gp41 and effectively inhibits fusion of the viral and cellular membranes during virus entry. The membrane-anchored form (maC46) was shown to effectively block entry of a broad range of HIV isolates, including viruses resistant to the fusion inhibitory peptide C36 (T20). C36 (T20) corresponds to the 36 C-terminal amino acids of C46. The gene was introduced into autolous CD4 T lymphocytes from 10 patients with severe immunodeficiency and HAART failure. Cells were expanded by co-stimulation with anti-CD3 and anti-CD28 immobilized on beads. Between 2 and 15 |[times]| 10E9 cells were infused. No major toxicities were observed and neither cellular nor humoral immune responses to the transgene product were detected. While a significant rise in T helper cell counts was seen, viral load was not affected. Gene marking could be detected throughout the one-year follow-up, but the levels were too low to account for the marked rise in CD4 counts.

Previous studies have demonstrated leukemic complications in mice after high-copy retroviral gene transfer of the multidrug resistance 1 (MDR1) cDNA, encoding a membrane-located efflux pump expressed in hematopoietic stem cells. In contrast, no such complications or MDR1-associated alterations of hematopoiesis were observed in numerous other studies exploring MDR1 gene transfer into cell lines, mice, dogs, nonhuman primates, and human subjects. Here, we show that leukemias associated with retroviral expression of MDR1 depend on high vector dose, and involve the selection of clones with combinatorial insertional mutagenesis of proto-oncogenes or other signaling genes. Compared with insertion patterns in normal long-term repopulating hematopoietic cells, such hits were overrepresented in leukemic clones, pointing to a causal role. A similar constellation of insertion sites was also observed in a leukemia arising after high-copy retroviral gene transfer of a fluorescent protein. Spectral karyotyping demonstrated additional chromosomal translocations in a subset of cases, indicative of secondary genetic instability. We also show that insertional mutants can be amplified in vitro prior to transplantation. On the basis of these findings, we suggest the use of preclinical dose-escalation studies to define a therapeutic index for retroviral transgene delivery.

Methods to analyze the clonality of an adverse event in preclinical or clinical retroviral stem cell gene therapy protocols are needed. We analyzed the progeny of retrovirally transduced human peripheral blood progenitor cells (PBPCs) after transplantation and engraftment in immune-deficient mice. The integration site of the provirus serves as a unique tag of the individual transduced PBPC. A plasmid library of junctions between proviral and human genomic DNA was generated. We were able to detect individual transduced cell clones that amounted to 0.14%–0.0001% of chimeric bone marrow cells. This is the first report in which the contribution of individual marrow-repopulating cells to human hematopoiesis is directly quantified.

Objective. Hematopoietic progenitor cells are a promising source for generation of genetically modified dendritic cells. A prerequisite for using these cells in therapeutic approaches is stable vector-mediated transgene expression during and after cell maturation. We investigated the expression of enhanced green fluorescence protein (EGFP) mediated by retroviral vectors in dendritic cells and other hematopoietic cells differentiated in vitro. Material and Methods. CD34 + cells were efficiently transduced with retroviral vector constructs known to mediate different expression levels due to distinct cis -acting elements. EGFP + cells were purified by cell sorting and differentiated to monocytes, granulocytes, dendritic cells, and erythrocytes. Coexpression of EGFP and cell type-specific markers was analyzed by flow cytometry. Results. Transgene expression from various retroviral vectors was silenced exclusively in dendritic cells, but not in other mature myeloid cells. Loss of EGFP was most pronounced in cells initially displaying low expression levels. This was confirmed by using a retroviral vector coding for a variant of EGFP with significantly reduced half-life. In contrast, a majority of dendritic cells showed stable expression when a self-inactivating retroviral construct using an internal cytomegalovirus promotor was used. Conclusions. We suggest that expression from the retroviral long terminal repeat is silenced during dendritic cell differentiation in vitro. High levels of stable transgene product in progenitor cells may mask a loss of expression. An improvement of retroviral vectors mediating stable transgenic expression is necessary for therapeutic approaches using gene-modified dendritic cells.

Graft-versus-host-disease (GvHD) is a severe complication in the context of allogeneic stem cell transplantation and adoptive immunotherapy. The transfer of a suicide gene into donor T-lymphocytes (TLCs) allows to selectively eliminate GvHD-causing cells. Since retroviral gene transfer into hematopoietic stem cells can induce leukemia there is an urgent need to analyze retroviral integration sites also in TLCs. We examined suicide gene transduced TLCs in four grafts and from four transplanted patients. Additionally, we generated a random integration pattern which was used for comparison of integration site data.

Chronic Granulomatous Disease (CGD) is a primary immunodeficiency characterized by recurrent and life-threatening bacterial and fungal infections. CGD is caused by mutations in any one of four genes encoding for subunits of the phagocytic NADPH oxidase complex. Most patients contain mutations or deletions within the gene coding for gp91phox, the larger subunit of the complex. Based on our preclinical work, two X-CGD patients, 26 and 25 years old, were treated with gene modified cells. G-CSF mobilized CD34+ cells were transduced with a monocistronic gp91phox retroviral vector. The transduction efficiency was 45% for Pat.1 and 40% for Pat. 2. The number of CD34+ cells reinfused was 1.2|[times]|10e7 per kg for Pat.1 and 0.9|[times]|10e7 per kg for Pat. 2. Before reinfusion, patients were treated with liposomal busulphan given i.v. at a dose of 4 mg/kg at two consecutive days, starting at day -3. The treatment was well tolerated and no adverse effects have been observed. Neutrophil counts declined to less than 100 cells per |[mu]|l at day 15 post reinfusion for both patients and recovered to more than 500 cells per |[mu]|l at day 30 for Pat. 1 and day 20 for Pat. 2. A significant fraction of gene marked cells has been detected in peripheral blood of both patients since day +21. Similarly, therapeutic relevant levels of NADPH oxidase activity have been observed since day +21. Both patients are well and have been free of severe bacterial and fungal infections since transplantation. Our data suggests that gene therapy is an option for the treatment of CGD, despite the fact that gp91phox corrected cells will not have per se a proliferative advantage over non-transduced cells.

Gene transfer into hematopoietic stem cells has been envisaged as an alternative to stem cell transplantation for the treatment of many genetic diseases of the blood system. In 2004 we initiated a gene therapy trial aimed at the correction of Chronic Granulomatous Disease (CGD), a rare inherited immunodeficiency caused by a functional defect in the microbial killing activity of phagocytes. Gene marking and functional correction of phagocytes were high shortly after transplantation of gene modified cells, leading to the eradication of therapy resistant infections from which patients had suffered for many years. However, one of our patients died 27 months after treatment due to a severe sepsis with multiorgan dysfunction. Although gene marking was still high at this time point, expression of the therapeutic gene, gp91phox, was minimal. This down regulation of transgene expression was due to CpG methylation within the viral LTR. Similar effects were observed in a second patient treated more than 3 years ago. In both patients CpG methylation was restricted to the promoter region of the viral LTR, while CpG dinucleotides within the enhancer region of the viral LTRs were not methylated. As a consequence, gp91phox gene expression was suppressed but the capacity of the viral LTRs to transactivate nearby sequences is still intact. Indeed we were able to detect cellular transcripts at predominant retroviral integration sites leading to an unbalanced clonal distribution of gene marked cells in peripheral blood and bone marrow. A third patient, a 5 years old child was treated in a similar way in Zurich. In this case only low levels of engraftment and gene correction were achieved. Although the treatment has been beneficial for all treated patients, the side effects observed in the two adults demand modifications in vector design for sustained gene expression and long term correction of the disease without side effects.

Gene transfer into hematopoietic stem cells has been successfully used to correct immunodeficiencies affecting the lymphoid compartment. However, similar results have not been reported for diseases affecting myeloid cells, mainly due to low engraftment levels of gene-modified cells observed in unconditioned patients. Here we report on two adult patients (P1 and P2, follow up >24 months) and one child (P3, 6 years, follow up 15 months) who received gene-transduced hematopoietic stem cells in combination with nonmyeloablative bone marrow conditioning for the treatment of X-linked Chronic Granulomatous Disease (X-CGD), a primary immunodeficiency caused by a defect in the oxidative antimicrobial activity of phagocytes. Therapeutically significant gene marking was detected in neutrophils of both adult patients (P1 and P2) leading to large numbers (up to 60%) of functionally corrected phagocytes 24 months after gene therapy. This high correction resulted from an unexpected but temporarily restricted expansion of gene transduced myeloid cells in vivo. In contrast gene marking and functionally reconstitution levels in P3 have been low (1–2%). Both adult patients suffered from active infections prior to gene therapy (P1 of bacterial liver abscesses and P2 of lung aspergillosis) and were free of severe bacterial and fungal infections until 24 months after transplantation. P3 suffered from an Aspergillus infection of the spinal cord with paraparesis before transplantation and recovered after gene therapy despite low numbers of functionally corrected cells in the peripheral blood. Large-scale mapping of retroviral integration site distribution revealed that activating insertions in the zinc finger transcription factor homologs MDS1/EVI1, PRDM16, or in SETBP1 have expanded gene-corrected long term myelopoiesis 3- to 4-fold in both adults, providing direct evidence in humans that these genes may influence regulation of normal long-term hematopoiesis. The hematopoietic repopulation in P1 was polyclonal until 18 months after therapy. P1 died of a severe bacterial sepsis after colon perforation 27 months after gene therapy. No evidence of malignant transformation was found in peripheral blood or bone marrow aspirates from this patient. Gene marking at death was still 60%; however the function of gene transduced cells, the number of corrected cell clones and the activity of a predominant clone was greatly decreased. P2 has been free of infections since transplantation (last monitoring: month 26). Hematopoietic repopulation was polyclonal in P2 until day 560. In conclusion, gene therapy in combination with bone marrow conditioning has provided a transitory therapeutic benefit for all 3 patients. Further improvements in vector design and conditioning regimes are under investigation to provide a stable and long term correction of the disease.

Chronic granulomatous disease (CGD) is a primary immunodeficiency in which phagocytic cells of affected patients have impaired antimicrobial activity due to a defect in the production of reactive oxygen species (ROS). CGD is caused by mutations in any one of four genes encoding for the subunits of the NADPH oxidase complex. Although curable by HSC transplantation, this strategy is usually limited only to patients with HLA-matched sibling or unrelated donors, as mismatched transplantation is associated with high morbidity and mortality due to graft failure and slow immune reconstitution. A therapeutic alternative for CGD patients is the genetic modification of autologous HSC. In January 2004 we initiated a Phase I/II clinical trial for X-CGD patients including conditioning with busulfan (8 mg/kg/total dose) prior to infusion of genetically modified HSC. G-CSF mobilized CD34+ cells from 2 adult patients (25 and 26 years) were transduced ex-vivo with a monocistronic gp91phox retroviral vector. Therapeutically significant gene marking levels were detected in neutrophils of both patients with up to 60% functionally corrected phagocytes 14 months after gene therapy. This high correction resulted from an unexpected but temporarily restricted expansion of gene transduced myeloid cells in vivo. In contrast gene marking levels in B-cells has remained constant at a level of 20%, while gene marking in T-cells is below 5%. Gene marking in bone marrow was detected at levels between 30% and 40% one year after transplantation of gene modified cells. Killing assays in vitro have demonstrated antibacterial and antifungal activity in gene transduced phagocytes and both patients recovered of Staph. aureus and Aspergillus fumigatus infections after gene therapy. Our results suggest that gene therapy in combination with bone marrow conditioning is a therapeutic option for inherited diseases affecting the myeloid compartment and can be successfully used to treat CGD.

Genetically modified T-Lymphocytes (TLCs) have been used for adoptive immunotherapy in the context of allogeneic hematopoietic stem cell transplantation (SCT). Infused TLCs have been shown to be susceptible to elimination through exposure to ganciclovir in the event of graft-versus-host disease (GvHD). Yet, reports on insertional mutagenesis in a mouse gene marking study and a clinical gene therapy trial for X-chromosomal severe combined immunodeficiency (X-SCID) reminded us the actual risk of insertional oncogene activation and subsequent leukemia development. We investigated retroviral integration sites in vitro and in vivo. Therefore, donor TLCs transduced with the MoLV-based TK/neoR vector Mo3TIN for a clinical HSV-Tk study were examined. TLCs of four different donors as well as whole blood samples of two patients transplanted with donor TLCs were analyzed either using highly sensitive and specific ligation-mediated PCR (LM-PCR). A total of 114 retroviral integration sites were detected in vitro. 41.2% of all integrations appeared near the transcription start regions (+/−10kb) of genes. Further analysis showed that 57 (50%) of all integrations targeted RefSeq genes. 24 of those appeared in intron 1 (42% of all integrations into genes) while 18% (10/57) of all integrations into genes landed in exon sequences whereas 6 hit the first exon. 18 of the targeted genes (15.8% of all integrations) could be at last assigned to signal transduction pathways, whereas the transcription factor family was afflicted 13 times (11.4% of all integrations). Among the targeted genes we found integrations into the CD8, CD100, CD44, CX3CR1, HLA-DMP and IL10-receptor genes. Within at a range of 5kb up- and 5kb downstream of vector integrations 15 genes were located that were not hit. 5 are known as transcription factors, whereas two of those are involved in leukemia, namely the homo sapiens myeloid/lymphoid or mixed-lineage leukemia 5 gene (MLL5) and the homo sapiens ALL1 fused gene from 5q31 (AF5Q31). Current analyses are focusing at the in vivo pattern of retroviral integration in DNA of TLCs obtained from transplanted patient’s TLCs. Therefore we developed a new high sensitive PCR method (HS-PCR), an improved LM-PCR to even detect minimal quantities of transduced DNA.

High titer retroviral vector supernatant, produced in compliance with GMP, has always been a prerequisite for gene therapy purposes in clinical trials. But most vector production procedures have their limitations: the vector often leads only to low titer supernatant, only a few harvest cycles can be done and harvest volumes are often small.

Retroviral vectors encoding the herpes simplex thymidine kinase gene (HSV-Tk) have been employed to render T-lymphocytes (TLCs) sensitive to the prodrug ganciclovir. Such genetically modified T cells have been used for adoptive immunotherapy in the context of allogeneic hematopoietic stem cell transplantation (SCT). Infused T cells have been shown to be susceptible to elimination through exposure to ganciclovir in the event of graft-versus-host disease (GvHD). Recent reports on insertional “genotoxicity” in a mouse gene marking study and a clinical gene therapy trial for X-chromosomal severe combined immunodeficiency (X-SCID) reminded us the actual risk of insertional oncogene activation and subsequent leukemia development. Here we investigated retroviral integration sites in donor TLCs transduced with the MoLV-based TK/neoR vector Mo3TIN of four donors in a clinical HSV-Tk study. A total of 114 retroviral integration sites were detected using highly sensitive and specific ligation-mediated PCR (LM-PCR). 41.2% of all integrations appeared near the transcription start regions (+/−10kb) of genes. Further analysis showed that 57 (50%) of all integrations targeted RefSeq genes. 24 of those appeared in intron 1 (42% of all integrations into genes) while 18% (10/57) of all integrations into genes landed in exon sequences whereas 6 hit the first exon. 18 of the targeted genes (15.8% of all integrations) could be at last assigned to signal transduction pathways, whereas the transcription factor family was afflicted 13 times (11.4% of all integrations). The zinc ion binding group makes up 4 (resp. 6, minding that GTF2HII contains a C2H2 type and KIAA0427 a C-x8-C-x5-C-x3-H-type zinc finger) of them. Among the targeted genes we found integrations into the CD8, CD100, CD44, CX3CR1, HLA-DMP and IL10-receptor genes. Within at a range of 5kb up- and 5kb downstream of vector integrations 15 genes were located that were not hit. 5 are known as transcription factors, whereas two of those are involved in leukemia, namely the homo sapiens myeloid/lymphoid or mixed-lineage leukemia 5 gene (MLL5) and the homo sapiens ALL1 fused gene from 5q31 (AF5Q31). Hit genes are currently examined more systematically in terms of function, e.g. involvement in signal transduction and transcription promoting processes. RISC (Retroviral Insertion estimate of Chromosomal Integration) scores and integration specific data will be submitted to a data warehouse, the collaborative RISC score database (CRSD). Such a systematic data collection similar to the IBMTR or EBMT databases will allow to recognize factors contributing to the safety, optimal transgene expression and persistence of transduced cells in the setting of allogenic matched donor transplantation.