Lentivirale Vektoren für den stabilen Gentransfer in primäre humane mononukleäre Blutzellen

Für bestimmte Gentherapiestrategien bieten sich retrovirale Vektoren auf Grund der stabilen Integration der von ihnen übertragenen genetische Information an. Mit bisher vorhandenen retro- und lentiviralen Vektorsystemen kann jedoch kein effizienter Gentransfer in ruhende primäre Zellen wie unstimulierte PBMC oder undifferenzierte Monozyten erreicht werden. Ziel dieser Arbeit war es daher, retrovirale Vektorsysteme zu etablieren, die naive primäre Zellen effizient transduzieren können. Zunächst wurden HIV-1-Vektoren mit den Hüllproteinen (Env) des amphotropen MLV, des MLV-Klons 10A1, des felinen endogenen Retrovirus RD114 und des VSV sowie einem modifizierten GaLV-Env pseudotypisiert, um ihre Eignung für die Transduktion von primären T-Lymphozyten zu untersuchen. Mit diesen Vektoren konnten primäre stimulierte humane T-Lymphozyten mit ähnlichen Transduktionsraten von ca. 20% transduziert werden. Im Gegensatz dazu wurden mit MLV-Vektoren, die mit den gleichen Hüllproteinen pseudotypisiert waren, bei gleichem Transduktionsprotokoll untereinander deutlich unterschiedliche Transduktionsraten zwischen 1 - 45% erreicht. Dieser unerwartete Unterschied ist möglicherweise auf eine längere, dabei untereinander ähnliche Halbwertszeit der HIV- 1-abgeleiteten Vektoren zurückzuführen. Unstimulierte PBMC konnten allerdings unabhängig vom verwendeten Hüllprotein weder von den MLV- noch von den HIV-1-abgeleiteten Vektoren transduziert werden. Demgegenüber hatte sich in meiner vorangegangenen Diplomarbeit die Transduzierbarkeit ruhender humaner Zellen durch Vektoren angedeutet, die von dem akut pathogenen simianen Immundefizienzvirus SIVsmmPBj1.9 abgeleitet waren. In der hier vorgestellten Arbeit zeigten die PBj-Vektoren, die nur im env-Gen eine Deletion aufwiesen, dass sie auch in der G0-Phase des Zellzyklus arretierte GHOST-Indikatorzellen oder diploide Fibroblasten effizient und unabhängig von dem zur Pseudotypisierung verwendeten Hüllprotein transduzieren können. Die korrespondierenden HIV-1-Vektoren waren dazu nicht in der Lage. Auch PBj- und HIV-1-Vektoren, die egfp als Markergen transferieren, zeigten das gleiche Transduktionsverhalten. Die egfp-transferierenden SIVsmmPBj-Vektoren konnten schließlich im Unterschied zu den entsprechenden HIV-1-Vektoren auch frisch isolierte primäre humane Monozyten sehr effizient transduzieren. Mit Nef-deletierten Mutanten wurde schließlich gezeigt, dass die beschriebenen Fähigkeiten PBj-abgeleiteter Vektoren nicht vom viralen Nef-Protein abhängen, obwohl dieses regulatorische Gen die pathogenen Eigenschaften und die Replikationsfähigkeit von SIVsmmPBj-Viren bestimmt. Für alle mit SIVsmmPBj-abgeleiteten Vektoren transduzierten Zellen konnte in der vorliegenden Arbeit ferner die chromosomale Integration des Transfervektors gezeigt werden. Schließlich gelang die Konstruktion eines ersten 3-Plasmid-Vektorsystems, mit dem transduktionsfähige Vektorpartikel generiert werden können. Mit SIVsmmPBj-abgeleiteten Vektoren steht somit ein neues retrovirales Vektorsystem zur Verfügung, mit dem auch ruhende primäre Zellen stabil genetisch modifiziert werden können. Durch die Erschließung dieser wichtigen Zielzellen werden die Perspektiven einer dauerhaften Gentherapie in vitro und in vivo beträchtlich erweitert

CD20 and CD19 targeted vectors induce minimal activation of resting B lymphocytes

B lymphocytes are an important cell population of the immune system. However, until recently it was not possible to transduce resting B lymphocytes with retro- or lentiviral vectors, making them unsusceptible for genetic manipulations by these vectors. Lately, we demonstrated that lentiviral vectors pseudotyped with modified measles virus (MV) glycoproteins hemagglutinin, responsible for receptor recognition, and fusion protein were able to overcome this transduction block. They use either the natural MV receptors, CD46 and signaling lymphocyte activation molecule (SLAM), for cell entry (MV-LV) or the vector particles were further modified to selectively enter via the CD20 molecule, which is exclusively expressed on B lymphocytes (CD20-LV). It has been shown previously that transduction by MV-LV does not induce B lymphocyte activation. However, if this is also true for CD20-LV is still unknown. Here, we generated a vector specific for another B lymphocyte marker, CD19, and compared its ability to transduce resting B lymphocytes with CD20-LV. The vector (CD19ds-LV) was able to stably transduce unstimulated B lymphocytes, albeit with a reduced efficiency of about 10% compared to CD20-LV, which transduced about 30% of the cells. Since CD20 as well as CD19 are closely linked to the B lymphocyte activation pathway, we investigated if engagement of CD20 or CD19 molecules by the vector particles induces activating stimuli in resting B lymphocytes. Although, activation of B lymphocytes often involves calcium influx, we did not detect elevated calcium levels. However, the activation marker CD71 was substantially up-regulated upon CD20-LV transduction and most importantly, B lymphocytes transduced with CD20-LV or CD19ds-LV entered the G1b phase of cell cycle, whereas untransduced or MV-LV transduced B lymphocytes remained in G0. Hence, CD20 and CD19 targeting vectors induce activating stimuli in resting B lymphocytes, which most likely renders them susceptible for lentiviral vector transduction

Nutrition issues in Codex: health claims, nutrient reference values and WTO agreements: a conference report

BACKGROUND: Codex documents may be used as educational and consensus materials for member governments. Also, the WTO SPS Agreement recognizes Codex as the presumptive international authority on food issues. Nutrient bioavailability is a critical factor in determining the ability of nutrients to provide beneficial effects. Bioavailability also influences the quantitative dietary requirements that are the basis of nutrient intake recommendations and NRVs. HEALTH CLAIMS: Codex, EFSA and some national regulatory authorities have established guidelines or regulations that will permit several types of health claims. The scientific basis for claims has been established by the US FDA and EFSA, but not yet by Codex. Evidence-based nutrition differs from evidence-based medicine, but the differences are only recently gaining recognition. Health claims on foods may provide useful information to consumers, but many will interpret the information to mean that they can rely upon the food or nutrient to eliminate a disease risk. NUTRIENT REFERENCE VALUES: NRVs are designed to provide a quantitative basis for comparing the nutritive values of foods, helping to illustrate how specific foods fit into the overall diet. The INL-98 and the mean of adult male and female values provide NRVs that are sufficient when used as targets for individual intakes by most adults. WORLD TRADE ORGANIZATION AGREEMENTS: WTO recognizes Codex as the primary international authority on food issues. Current regulatory schemes based on recommended dietary allowances are trade restrictive. A substantial number of decisions by the EFSA could lead to violation of WTO agreements

The Measles Virus Fusion Protein Transmembrane Region Modulates Availability of an Active Glycoprotein Complex and Fusion Efficiency▿

The glycoprotein complex of paramyxoviruses mediates receptor binding and membrane fusion. In particular, the measles virus (MV) fusion (F) protein executes membrane fusion, after receptor binding by the hemagglutinin (H) protein. Structures and single amino acids influencing fusion function have been identified in the F-protein ectodomain and cytoplasmic tail, but not in its transmembrane (TM) region. Since this region influences function of the envelope proteins of other viruses, we examined its role in the MV F protein. Alanine-scanning mutagenesis revealed that an F protein with a single mutation of a central TM region leucine (L507A) was more fusogenic than the unmodified F protein while retaining similar kinetics of proteolytic processing. In contrast, substitution of residues located near the edges of the lipid bilayer reduced fusion activity. This was true not only when the mutated F proteins were coexpressed with H but also in the context of infections with recombinant viruses. Analysis of the H-F complexes with reduced fusion activities revealed that more precursor (F0) than activated (F1+2) protein coprecipitated with H. In contrast, in complexes with enhanced fusion activity, including H-FL507A, the F0/F1+2 ratio shifted toward F1+2. Thus, fusion activity correlated with an active F-H protein complex, and the MV F protein TM region modulated availability of this complex

APOBEC4 Enhances the Replication of HIV-1

APOBEC4 (A4) is a member of the AID/APOBEC family of cytidine deaminases. In this study we found a high mRNA expression of A4 in human testis. In contrast, there were only low levels of A4 mRNA detectable in 293T, HeLa, Jurkat or A3.01 cells. Ectopic expression of A4 in HeLa cells resulted in mostly cytoplasmic localization of the protein. To test whether A4 has antiviral activity similar to that of proteins of the APOBEC3 (A3) subfamily, A4 was co-expressed in 293T cells with wild type HIV-1 and HIV-1 luciferase reporter viruses. We found that A4 did not inhibit the replication of HIV-1 but instead enhanced the production of HIV-1 in a dose-dependent manner and seemed to act on the viral LTR. A4 did not show detectable cytidine deamination activity in vitro and weakly interacted with single-stranded DNA. The presence of A4 in virus producer cells enhanced HIV-1 replication by transiently transfected A4 or stably expressed A4 in HIV-susceptible cells. APOBEC4 was capable of similarly enhancing transcription from a broad spectrum of promoters, regardless of whether they were viral or mammalian. We hypothesize that A4 may have a natural role in modulating host promoters or endogenous LTR promoters

Enhanced lysis by bispecific oncolytic measles viruses simultaneously using HER2/neu or EpCAM as target receptors

To target oncolytic measles viruses (MV) to tumors, we exploit the binding specificity of designed ankyrin repeat proteins (DARPins). These DARPin-MVs have high tumor selectivity while maintaining excellent oncolytic potency. Stability, small size, and efficacy of DARPins allowed the generation of MVs simultaneously targeted to tumor marker HER2/neu and cancer stem cell (CSC) marker EpCAM. For optimization, the linker connecting both DARPins was varied in flexibility and length. Flexibility had no impact on fusion helper activity whereas length had. MVs with bispecific MV-H are genetically stable and revealed the desired double-target specificity. In vitro, the cytolytic activity of bispecific MVs was superior or comparable to mono-targeted viruses depending on the target cells. In vivo, therapeutic efficacy of the bispecific viruses was validated in an orthotopic ovarian carcinoma model revealing an effective reduction of tumor mass. Finally, the power of bispecific targeting was demonstrated on cocultures of different tumor cells thereby mimicking tumor heterogeneity in vitro, more closely reflecting real tumors. Here, bispecific excelled monospecific viruses in efficacy. DARPin-based targeting domains thus allow the generation of efficacious oncolytic viruses with double specificity, with the potential to handle intratumoral variation of antigen expression and to simultaneously target CSCs and the bulk tumor mass

Measles Virus-Based Treatments Trigger a Pro-inflammatory Cascade and a Distinctive Immunopeptidome in Glioblastoma

Glioblastoma is an aggressive primary brain tumor with bad prognosis. On the other hand, oncolytic measles virus (MeV) therapy is an experimental glioma treatment strategy with clinical safety and first evidence of anti-tumoral efficacy. Therefore, we investigated the combination of MeV with conventional therapies by cytotoxic survival assays in long-term glioma cell lines LN229, LNZ308, and glioma stem-like GS8 cells, as well as the basal viral infectivity in primary glioblastoma cultures T81/16, T1094/17, and T708/16. We employed Chou-Talalay analysis to identify the synergistic treatment sequence chemotherapy, virotherapy, and finally radiotherapy (CT-VT-RT). RNA sequencing and immunopeptidome analyses were used to delineate treatment-induced molecular and immunological profiles. CT-VT-RT displayed synergistic anti-glioma activity and initiated a type 1 interferon response, along with canonical Janus kinase-signal transducers and activators of transcription (JAK-STAT) signaling, and downstream interferon-stimulated genes were induced, resulting in apoptotic cascades. Furthermore, antigen presentation along with immunostimulatory chemokines was increased in CT-VT-RT-treated glioma cells, indicating a treatment-induced pro-inflammatory phenotype. We identified novel treatment-induced viral and tumor-associated peptides through HLA ligandome analysis. Our data delineate an actionable treatment-induced molecular and immunological signature of CT-VT-RT, and they could be exploited for the design of novel tailored treatment strategies involving virotherapy and immunotherapy. Keywords: glioblastoma, immunopeptidome, oncolytic measles virus, personalized medicine, BATF2, SAMD