Long-term health sequelae and quality of life at least 6 months after infection with SARS-CoV-2: design and rationale of the COVIDOM-study as part of the NAPKON population-based cohort platform (POP).

PURPOSE: Over the course of COVID-19 pandemic, evidence has accumulated that SARS-CoV-2 infections may affect multiple organs and have serious clinical sequelae, but on-site clinical examinations with non-hospitalized samples are rare. We, therefore, aimed to systematically assess the long-term health status of samples of hospitalized and non-hospitalized SARS-CoV-2 infected individuals from three regions in Germany. METHODS: The present paper describes the COVIDOM-study within the population-based cohort platform (POP) which has been established under the auspices of the NAPKON infrastructure (German National Pandemic Cohort Network) of the national Network University Medicine (NUM). Comprehensive health assessments among SARS-CoV-2 infected individuals are conducted at least 6 months after the acute infection at the study sites Kiel, Würzburg and Berlin. Potential participants were identified and contacted via the local public health authorities, irrespective of the severity of the initial infection. A harmonized examination protocol has been implemented, consisting of detailed assessments of medical history, physical examinations, and the collection of multiple biosamples (e.g., serum, plasma, saliva, urine) for future analyses. In addition, patient-reported perception of the impact of local pandemic-related measures and infection on quality-of-life are obtained. RESULTS: As of July 2021, in total 6813 individuals infected in 2020 have been invited into the COVIDOM-study. Of these, about 36% wished to participate and 1295 have already been examined at least once. CONCLUSION: NAPKON-POP COVIDOM-study complements other Long COVID studies assessing the long-term consequences of an infection with SARS-CoV-2 by providing detailed health data of population-based samples, including individuals with various degrees of disease severity. TRIAL REGISTRATION: Registered at the German registry for clinical studies (DRKS00023742).

Mutant KIT as imatinib-sensitive target in metastatic sinonasal carcinoma.

Background: Sinonasal carcinomas (SNCs) comprise various rare tumor types that are characterized by marked histologic diversity and largely unknown molecular profiles, yet share an overall poor prognosis owing to an aggressive clinical course and frequent late-stage diagnosis. The lack of effective systemic therapies for locally advanced or metastatic SNC poses a major challenge to therapeutic decision making for individual patients. We here aimed to identify actionable genetic alterations in a patient with metastatic SNC whose tumor, despite all diagnostic efforts, could not be assigned to any known SNC category and was refractory to multimodal therapy. Patients and methods: We used whole-exome and transcriptome sequencing to identify a KIT exon 11 mutation (c.1733_1735del, p.D579del) as potentially druggable target in this patient and carried out cancer hotspot panel sequencing to detect secondary resistance-conferring mutations in KIT. Furthermore, as a step towards clinical exploitation of the recently described signatures of mutational processes in cancer genomes, we established and applied a novel bioinformatics algorithm that enables supervised analysis of the mutational catalogs of individual tumors. Results: Molecularly guided treatment with imatinib in analogy to the management of gastrointestinal stromal tumor (GIST) resulted in a dramatic and durable response with remission of nearly all tumor manifestations, indicating a dominant driver function of mutant KIT in this tumor. KIT dependency was further validated by a secondary KIT exon 17 mutation (c.2459_2462delATTCinsG, p.D820_S821delinsG) that was detected upon tumor progression after 10 months of imatinib treatment and provided a rationale for salvage therapy with regorafenib, which has activity against KIT exon 11/17 mutant GIST. Conclusions: These observations highlight the potential of unbiased genomic profiling for uncovering the vulnerabilities of individual malignancies, particularly in rare and unclassifiable tumors, and underscore that KIT exon 11 mutations represent tractable therapeutic targets across different histologies.

Lentivirus-induced 'Smart' dendritic cells: Pharmacodynamics and GMP-compliant production for immunotherapy against TRP2-positive melanoma.

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 (SmartDC-TRP2) 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 self-differentiated 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 a simpler 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.

10-year stability of clinical-grade serum-free γ-retroviral vector-containing medium.

More than 10 years ago, we developed an efficient protocol for serum-free retroviral transduction of human hematopoietic stem cells derived from mobilized peripheral blood. After upscaling of the methodology, serum-free retroviral gibbon-ape leukemia virus (GALV) pseudotype PG13/LN vector supernatant produced under strict good manufacturing practice (GMP) conditions was used in the first clinical gene-marking trial in Germany. In this study, we analyzed the titer and transduction efficiency of this serum-free clinical-grade retroviral supernatant 10 years after production to evaluate the long-term stability. Long-term storage and transport on dry ice resulted in modestly decreased titers and levels of transduction efficiency in CD34+ cells ranging from 38.4 to 49.1%. We conclude that the stability of retroviral vectors in serum-free medium allows extended storage and distribution of approved clinical-grade retroviral vector stocks to distant sites in multicenter clinical trials.

Prospective, randomized, double-blind, multi-center, Phase III clinical study on transarterial chemoembolization (TACE) combined with Sorafenib versus TACE plus placebo in patients with hepatocellular cancer before liver transplantation - HeiLivCa [ISRCTN24081794].

BACKGROUND: Disease progression of hepatocellular cancer (HCC) in patients eligible for liver transplantation (LTx) occurs in up to 50% of patients, resulting in withdrawal from the LTx waiting list. Transarterial chemoembolization (TACE) is used as bridging therapy with highly variable response rates. The oral multikinase inhibitor sorafenib significantly increases overall survival and time-to-progression in patients with advanced hepatocellular cancer. DESIGN: The HeiLivCa study is a double-blinded, controlled, prospective, randomized multi-centre phase III trial. Patients in study arm A will be treated with transarterial chemoembolization plus sorafenib 400 mg bid. Patients in study arm B will be treated with transarterial chemoembolization plus placebo. A total of 208 patients with histologically confirmed hepatocellular carcinoma or HCC diagnosed according to EASL criteria will be enrolled. An interim patients' analysis will be performed after 60 events. Evaluation of time-to-progression as primary endpoint (TTP) will be performed at 120 events. Secondary endpoints are number of patients reaching LTx, disease control rates, OS, progression free survival, quality of live, toxicity and safety. DISCUSSION: As TACE is the most widely used primary treatment of HCC before LTx and sorafenib is the only proven effective systemic treatment for advanced HCC there is a strong rational to combine both treatment modalities. This study is designed to reveal potential superiority of the combined TACE plus sorafenib treatment over TACE alone and explore a new neo-adjuvant treatment concept in HCC before LTx.

Molecular Evidence of Genome Editing in a Mouse Model of Immunodeficiency.

Genome editing is the introduction of directed modifications in the genome, a process boosted to therapeutic levels by designer nucleases. Building on the experience of ex vivo gene therapy for severe combined immunodeficiencies, it is likely that genome editing of haematopoietic stem/progenitor cells (HSPC) for correction of inherited blood diseases will be an early clinical application. We show molecular evidence of gene correction in a mouse model of primary immunodeficiency. In vitro experiments in DNA-dependent protein kinase catalytic subunit severe combined immunodeficiency (Prkdc scid) fibroblasts using designed zinc finger nucleases (ZFN) and a repair template demonstrated molecular and functional correction of the defect. Following transplantation of ex vivo gene-edited Prkdc scid HSPC, some of the recipient animals carried the expected genomic signature of ZFN-driven gene correction. In some primary and secondary transplant recipients we detected double-positive CD4/CD8 T-cells in thymus and single-positive T-cells in blood, but no other evidence of immune reconstitution. However, the leakiness of this model is a confounding factor for the interpretation of the possible T-cell reconstitution. Our results provide support for the feasibility of rescuing inherited blood disease by ex vivo genome editing followed by transplantation, and highlight some of the challenges.

Previously undetected human hematopoietic cell populations with short-term repopulating activity selectively engraft NOD/SCID-beta2 microglobulin-null mice.

Increasing use of purified or cultured human hematopoietic cells as transplants has revealed an urgent need for better methods to predict the speed and durability of their engraftment potential. We now show that NOD/SCID-beta2 microglobulin-null (NOD/SCID-beta2m-/-) mice are sequentially engrafted by two distinct and previously unrecognized populations of transplantable human short-term repopulating hematopoietic cells (STRCs), neither of which efficiently engraft NOD/SCID mice. One is predominantly CD34+CD38+ and is myeloid-restricted; the other is predominantly CD34+CD38- and has broader lymphomyeloid differentiation potential. In contrast, the long-term repopulating human cells that generate lymphoid and myeloid progeny in NOD/SCID mice engraft and self-renew in NOD/SCID-beta2m-/- mice equally efficiently. In short-term expansion cultures of adult bone marrow cells, myeloid-restricted STRCs were preferentially amplified (greater than tenfold) and, interestingly, both types of STRC were found to be selectively elevated in mobilized peripheral blood harvests. These results suggest an enhanced sensitivity of STRCs to natural killer cell-mediated rejection. They also provide new in vivo assays for different types of human STRC that may help to predict the engraftment potential of clinical transplants and facilitate future investigation of early stages of human hematopoietic stem cell differentiation.

Cooperation of BRAF(F595L) and mutant HRAS in histiocytic sarcoma provides new insights into oncogenic BRAF signaling.

Activating BRAF mutations, in particular V600E/K, drive many cancers and are considered mutually exclusive with mutant RAS, whereas inactivating BRAF mutations in the D(594)F(595)G(596) motif cooperate with RAS via paradoxical MEK/ERK activation. Due to the increasing use of comprehensive tumor genomic profiling, many non-V600 BRAF mutations are being detected whose functional consequences and therapeutic actionability are often unknown. We investigated an atypical BRAF mutation, F595L, which was identified along with mutant HRAS in histiocytic sarcoma and also occurs in epithelial cancers, melanoma and neuroblastoma, and determined its interaction with mutant RAS. Unlike other DFG motif mutants, BRAF(F595L) is a gain-of-function variant with intermediate activity that does not act paradoxically, but nevertheless cooperates with mutant RAS to promote oncogenic signaling, which is efficiently blocked by pan-RAF and MEK inhibitors. Mutation data from patients and cell lines show that BRAF(F595L), as well as other intermediate-activity BRAF mutations, frequently coincide with mutant RAS in various cancers. These data define a distinct class of activating BRAF mutations, extend the spectrum of patients with systemic histiocytoses and other malignancies who are candidates for therapeutic blockade of the RAF-MEK-ERK pathway and underscore the value of comprehensive genomic testing for uncovering the vulnerabilities of individual tumors.

Effect of recombinant canine stem cell factor, a c-kit ligand, on hematopoietic recovery after DLA-identical littermate marrow transplants in dogs.

We studied the effect of recombinant canine stem cell factor (rcSCF) on hematopoietic recovery, incidence of graft failure, graft-vs.-host disease (GVHD), and survival after marrow transplantation from dog leukocyte antigen (DLA)-identical canine littermates. Ten animals received 100 microg rcSCF/kg/day b.i.d. by subcutaneous injection on days 1 through 10 after 920 cGy total body irradiation and transplantation of a mean of 3.7x10(8) marrow cells/kg body weight. None of the dogs received GVHD prophylaxis. All animals showed hematopoietic engraftment. The median number of days to achieve 1000 neutrophils/mm3 was 9; 100 monocytes/mm3 were reached after 15 days, 500 lymphocytes/mm3 after 21 days, and 20,000 platelets/mm3 after 16 days. One animal developed GVHD involving skin, gut, and liver and died of bacterial pneumonia 21 days after transplantation. The remaining nine dogs were observed for a median of 37 days (range 29-84 days) posttransplantation until they were killed. Facial edema was seen in three dogs during the first 2-3 days of rcSCF administration. These results show that within the limits of this study it appears to be safe to administer SCF after DLA-identical littermate marrow transplants in dogs. Comparison with previously published data in the same model showed that neutrophil and monocyte recovery was significantly faster in dogs receiving SCF treatment compared with dogs without growth factor treatment (recovery to achieve 1000 neutrophils/mm3: median 9 days vs. 13 days, p = 0.002; recovery to 100 monocytes/mm3: median 15 days vs. 105 days, p = 0.0002). Otherwise, no significant differences were seen. Results obtained with SCF treatment were similar to those previously obtained in the same model with recombinant human granulocyte colony-stimulating factor (rhG-CSF) treatment except that recovery of lymphocytes to 500/mm3 appeared to be more rapid in G-CSF-treated dogs (median 15 days vs. 21 days, p = 0.03).

Long-term persistence of canine hematopoietic cells genetically marked by retrovirus vectors.

In 1991 we reported gene transduction into autologous long-term repopulating marrow cells in dogs using amphotropic helper-free retrovirus vectors containing the bacterial neomycin phosphotransferase gene (neo) and the human adenosine deaminase gene (ADA). Two of the dogs are still alive and healthy now more than 5 years after transplantation of transduced autologous marrow cells. In one of the surviving dogs, polymerase chain reaction (PCR) analysis showed the neo and ADA genes to be present in peripheral blood granulocytes and lymphocytes up to the present time. The estimated percentage of neo-positive cells ranged from < 0.001% to 0.1%. ADA mRNA expression was detected by reverse transcriptase PCR (RT-PCR) in granulocytes 63 months after transplantation. The other surviving dog failed to show either persistence or expression of the transduced genes after 50 months. Three additional dogs have been transplanted according to the same transduction protocols and with the same retrovirus vectors, and persistence of the transduced neo gene has been documented in peripheral blood myeloid and lymphoid cells along with G418-resistant colony-forming unit-granulocyte/macrophage (CFU-GM) for now more than 2 years. These findings represent the longest follow-up of retrovirus-mediated gene transduction in any animal species. Long-term transduction efficiency, though, has remained low and will need to be improved for therapeutic application to be possible.

Detection and direct genomic sequencing of multiple rare unknown flanking DNA in highly complex samples.

By identifying the sequence of retro- and lentiviral integration sites in peripheral blood leukocytes, the clonal composition and fate of genetically modified hematopoietic progenitor and stem cells could be mapped in vitro and in vivo. Previously available methods have been limited to the analysis of mono- or oligoclonal integration sites present in high copy numbers. Here, we perform characterization of multiple rare retroviral and lentiviral integration sites in highly complex DNA samples. The reliability of this method results from nontarget DNA removal via magnetic extension primer tag selection (EPTS) preceding solid-phase ligation-mediated PCR. EPTS/LM-PCR allowed the simultaneous direct genomic sequencing of multiple proviral LTR-flanking sequences of retro- and lentiviral vectors even if only 1 per 100 to 1000 cells contained the provirus. A primer walking "around" the integration locus demonstrated the adaptability of EPTS/LM-PCR to study unknown flanking DNA regions unrelated to proviruses. The technique is fast, inexpensive, and sensitive in minimal samples. It enables studies of retro- and lentiviral integration, viral vector tracking in gene therapy, insertional mutagenesis, transgene integration, and direct genomic sequencing that until now have been difficult or impossible to perform.

Efficient gene transfer in primitive CD34+/CD38lo human bone marrow cells reselected after long-term exposure to GALV-pseudotyped retroviral vector.

Successful retroviral gene transfer into human hematopoietic stem cells was demonstrated in preliminary clinical trials at low efficiency. We have shown previously that gene transfer into committed hematopoietic progenitor cells is more efficient using a gibbon ape leukemia virus (GALV)-pseudotyped retroviral vector instead of an amphotropic retroviral vector. Here, we have conducted a systematic study of human hematopoietic progenitor cells after extended transduction with a GALV-pseudotyped retroviral vector. CD34+/CD38lo Cells were transduced for 5 days and reselected according to phenotype after culture and analyzed for cell cycle status, long-term culture-initiating cell (LTC-IC) activity, and gene transfer. Reselection of rare, very primitive progenitor cells was successful. Equal to fresh CD34+/CD38lo cells, >90% of reselected CD34+/CD38lo cells were in G0/G1. CD34+/CD38lo reselection enriched for LTC-IC (10-fold), as compared to freshly isolated CD34+/CD38lo cells with excellent specificity (82.7% of total LTC-IC were recovered in the reselected CD34+/CD38lo population) and recovery (62% of initial LTC-IC number in CD34+/CD38lo cells were recovered in the reselected fraction after transduction). Gene transfer into primitive progenitor cells was efficient with 50.5% G418-resistant LTC-IC colonies and more than 40 copies of vector provirus detectable per 100 nuclei of CD34+/CD38lo cells. To our knowledge, this is the first systematic analysis of phenotype, function, and cell cycle demonstrating retroviral gene transfer into rare, very primitive human hematopoietic progenitor cells. The chosen strategy should be of considerable value for analyzing and improving gene therapy of the hematopoietic system.

Efficient serum-free retroviral gene transfer into primitive human hematopoietic progenitor cells by a defined, high-titer, nonconcentrated vector-containing medium.

Defined serum-free conditions have great conceptual advantages for the biological safety and standardization of clinical gene transfer into hematopoietic stem cells. In the only study reported to date, Sekhar et al. achieved low serum conditions by a complex concentration procedure of a retroviral supernatant initially containing 10% fetal bovine serum. The high cost, small volume, possible coenrichment of serum-derived pathogens, limited recovery of vector particles, and low titer of the final diluted medium restrict the clinical application of this procedure. Transduction of primitive hematopoietic progenitor cells was not demonstrated. In the present study, a defined serum-free medium containing high titers of the pseudotyped retroviral vector PG13/LN was generated from PG13/LN producer cells without requiring a physical enrichment procedure. The transduction of committed hematopoietic progenitor cells in the serum-free vector-containing medium was efficient, and similar to that occurring under serum-containing control conditions. The number of primitive human hematopoietic long-term culture-initiating cell-derived colonies (LTC-IC-derived colonies) generated from CD34+ and CD34+/HLA-DRlo peripheral blood progenitor "stem" cells (PBSCs) increased during 7 days of treatment in this vector-containing medium in the presence of IL-3, SCF, and flt-3 ligand. The described procedure allowed efficient transduction of LTC-IC-derived colonies generated from CD34+, CD34+/HLA-DRlo, and CD34+/CD38lo PBSCs. This is the first report to demonstrate an increase in primitive peripheral blood LTC-IC-derived colonies in vitro as well as their efficient transduction in a high-titer, serum-free vector-containing medium that can be produced exclusively from defined pharmaceutical-grade components, making it ideally suited for applications in clinical gene therapy.

Stem cell clonality and genotoxicity in hematopoietic cells: gene activation side effects should be avoidable.

Two serious adverse events involving activation of the LMO2 oncogene through retrovirus vector insertion in the otherwise extremely successful first gene therapy trial for X-linked severe combined immunodeficieny type 1 (SCID-X1) had initially caused widespread concern in the patient and research communities. Careful consideration 1 year after diagnosis of the second case still finds 12 of the treated patients clearly benefiting from gene therapy (freedom from treatment failure, 80%; survival 100%), a situation that should not portend the end of gene therapy for this disease, and is, in fact encouraging. While current approaches are justified to treat patients with otherwise life-threatening disorders, a broad consensus has developed that systematic basic research is required to further understand the pathophysiology of these serious adverse events and to provide new insights, enabling safer and more effective gene therapy strategies. With the continued success of SCID-X1 gene therapy in the majority of patients treated, it is of even greater importance to understand exactly which vector element or combination of elements predispose to toxicity. An in-depth study of the mechanisms behind the activation of the LMO2 and gammac genes will be highly instructive for the development of safer procedures and vectors. We summarize the central observations, ongoing experimental approaches, new concepts, and developments relevant to understanding, interpreting, and eventually overcoming the real and perceived obstacles posed by insertional mutagenesis due to gene transfer vectors.

A model for the detection of clonality in marked hematopoietic stem cells.

The semirandom location of retroviral integration in the target cell genome introduces a marker in the form of a fusion sequence composed of a genomic and a proviral part that is unique for each transduced cell and its clonal progeny. High-sensitivity detection of these fusion sequences would allow the tracking of clonal contributions of individual, marked hematopoietic progenitor, and stem cells in vivo. Clone detection by Southern blot has helped to analyze models of oligoclonal repopulation but is limited in sensitivity and specificity. Inverse PCR (Nolta et al., Proc. Natl. Acad. Sci. USA 93: 2414-2419) can demonstrate the clonal identity by sequencing but does not permit simultaneous detection of multiple clones. In an efficiently transduced rhesus macaque model (Tisdale et al., Blood 92: 2681-2687; Wu et al., Mol. Ther. 1: 285-293) Kim et al. (Blood 96: 1-8) have identified more than 40 insertion sequences from marrow CFU by inverse PCR. However, no previous study has been able to directly analyze the number of clones active in vivo. Here we demonstrate that the application of a recently developed PCR technology allows the simultaneous visualization of multiple integration sites from small clonal contributions to hematopoietic cells. By combining solid-phase primer extension with ligation-mediated PCR, direct genomic sequencing of retroviral integration sites was obtained in murine bone marrow samples. Further development of this technology will allow analysis of the clonal composition of marked hematopoiesis in small and large animals as well as in human gene transfer.

MicroRNA-mediated multi-tissue detargeting of oncolytic measles virus.

Precise oncotropism is required for successful systemic administration of next-generation oncolytic measles viruses (MVs). We have previously established a system for efficient post-entry targeting by insertion of synthetic microRNA target sites (miRTS) into the MV genome, thereby repressing replication in the presence of cognate microRNAs. Thus, differential expression of microRNAs, as frequently observed in normal compared with malignant tissues, can be exploited to increase vector specificity and safety. Here we report the combination of miRTS for different microRNAs in a single vector to detarget pivotal organs at risk during systemic administration (liver, brain, gastrointestinal tract). Accordingly, miRTS for miR-122, miR-7 and miR-148a that are enriched in these tissues were inserted to create multi-tissue-detargeted MV (MV-EGFP(mtd)). Replication of MV-EGFP(mtd) is repressed in cell lines as well as in non-transformed primary human hepatocytes and liver slices expressing cognate microRNAs. Oncolytic potency of MV-EGFP(mtd) is retained in a model of pancreatic cancer in vitro and in vivo. This work is a proof-of-concept that favorable expression profiles of multiple microRNAs can be exploited concomitantly to reshape the tropism of MV without compromising oncolytic efficacy. This strategy can be adapted to different vectors and cancer entities for safe and efficient high-dose systemic administration in clinical trials.

[Urology in the concept of comprehensive cancer centers]. / Die Urologie im Konzept der "Comprehensive Cancer Center".

BACKGROUND: Urologic cancers comprise one quarter of all newly diagnosed cancers per year in Germany. In addition to the increasing incidence treatment of solid and hematological tumors has become more differentiated, complex and potentially more effective as well as more expensive. Following the example of the USA multidisciplinary translational comprehensive cancer centers (CCCs) have been established in Germany. The financial support from the government and nonprofit organizations, such as the German Cancer Aid aims to ensure and to optimize treatment of tumor patients now and in the future. Coupled with this development new funding opportunities for translational research are opening up for the participating clinical and scientific partners. DISCUSSION: Just as attractive and coherent integration of urology into the structures of a CCC where available appears to be, just as controversial is the professional modus operandi. Using the example of the National Center for Tumor Diseases in Heidelberg (NCT), the current manuscript discusses the risks and opportunities of this new centralized form of oncological care in urology. Detailed knowledge of organizational structures, clinical operations and funding is a prerequisite for any partner of a CCC to succeed in such a highly demanding environment as a specialty instead of becoming mere surgical proceduralists.

Armed and targeted measles virus for chemovirotherapy of pancreatic cancer.

No curative therapy is currently available for locally advanced or metastatic pancreatic cancer. Therefore, new therapeutic approaches must be considered. Measles virus (MV) vaccine strains have shown promising oncolytic activity against a variety of tumor entities. For specific therapy of pancreatic cancer, we generated a fully retargeted MV that enters cells exclusively through the prostate stem cell antigen (PSCA). Besides a high-membrane frequency on prostate cancer cells, this antigen is expressed on pancreatic adenocarcinoma, but not on non-neoplastic tissue. PSCA expression levels differ within heterogeneous tumor bulks and between human pancreatic cell lines, and we could show specific infection of pancreatic adenocarcinoma cell lines with both high- and low-level PSCA expression. Furthermore, we generated a fully retargeted and armed MV-PNP-anti-PSCA to express the prodrug convertase purine nucleoside phosphorylase (PNP). PNP, which activates the prodrug fludarabine effectively, enhanced the oncolytic efficacy of the virus on infected and bystander cells. Beneficial therapeutic effects were shown in a pancreatic cancer xenograft model. Moreover, in the treatment of gemcitabine-resistant pancreatic adenocarcinoma cells, no cross-resistance to both MV oncolysis and activated prodrug was detected.