André Berger

SAMHD1-Deficient CD14+ Cells from Individuals with Aicardi-Goutieres Syndrome Are Highly Susceptible to HIV-1 Infection

Myeloid blood cells are largely resistant to infection with human immunodeficiency virus type 1 (HIV-1). Recently, it was reported that Vpx from HIV-2/SIVsm facilitates infection of these cells by counteracting the host restriction factor SAMHD1. Here, we independently confirmed that Vpx interacts with SAMHD1 and targets it for ubiquitin-mediated degradation. We found that Vpx-mediated SAMHD1 degradation rendered primary monocytes highly susceptible to HIV-1 infection; Vpx with a T17A mutation, defective for SAMHD1 binding and degradation, did not show this activity. Several single nucleotide polymorphisms in the SAMHD1 gene have been associated with Aicardi-Goutières syndrome (AGS), a very rare and severe autoimmune disease. Primary peripheral blood mononuclear cells (PBMC) from AGS patients homozygous for a nonsense mutation in SAMHD1 (R164X) lacked endogenous SAMHD1 expression and support HIV-1 replication in the absence of exogenous activation. Our results indicate that within PBMC from AGS patients, CD14+ cells were the subpopulation susceptible to HIV-1 infection, whereas cells from healthy donors did not support infection. The monocytic lineage of the infected SAMHD1 -/- cells, in conjunction with mostly undetectable levels of cytokines, chemokines and type I interferon measured prior to infection, indicate that aberrant cellular activation is not the cause for the observed phenotype. Taken together, we propose that SAMHD1 protects primary CD14+ monocytes from HIV-1 infection confirming SAMHD1 as a potent lentiviral restriction factor.

PKCδ-induced PU.1 phosphorylation promotes hematopoietic stem cell differentiation to dendritic cells.

Human CD34+ hematopoietic stem cells (HSCs) exhibit the potential to differentiate into a variety of specialized blood cells. The distinct intracellular mechanisms that control cell fate and lineage commitment of these multipotent cells are not well defined. In this study, we investigate and modulate the signaling processes during HSC differentiation toward myeloid dendritic cells (mDCs). DC differentiation induced by the cytokines Granulocyte macrophage colony‐stimulating factor (GM‐CSF) and Interleukin‐4 (IL‐4) led to activation of the Extracellular‐signal‐regulated kinase (ERK), protein kinase C (PKC), and Janus kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) but not the SAPK/c‐Jun NH2‐terminal kinase and p38 mitogen‐activated protein kinase signaling pathways. From the activated signaling pathways the PKC isoform δ was found to phosphorylate the transcription factor PU.1, which is described as one of the key factors for myeloid HSC differentiation. On molecular level, PKCδ regulated PU.1 activity by affecting its transactivation activity, whereas its DNA binding activity remained unaffected. This was accompanied by PKCδ‐induced phosphorylation of the PU.1 transactivation domain. Furthermore, treatment with PKC‐ and ERK1/2‐specific signaling inhibitors impaired both HSC differentiation toward mDCs as well as phosphorylation‐mediated transactivation activity of PU.1. Taken together, these results provide new insights into the molecular mechanisms promoting the differentiation process of HSCs toward mDCs and introduce the PKC isoform δ as critical mediator. STEM CELLS 2011;29:297–306

Interaction of Vpx and Apolipoprotein B mRNA-editing Catalytic Polypeptide 3 Family Member A (APOBEC3A) Correlates with Efficient Lentivirus Infection of Monocytes

The accessory protein Vpx is encoded by lentiviruses of the human immunodeficiency virus type 2 (HIV-2) and the simian immunodeficiency SIVsm/SIVmac lineage. It is packaged into virions and is indispensable in early steps of monocyte infection. HIV-1, which does not encode Vpx, is not able to infect human monocytes, but Vpx enables infection with HIV-1. The underlying mechanism is not completely understood. In this work, we focus on Vpx-mediated intracellular postentry events as counteraction of host cell proteins. We found that Vpx binds to apolipoprotein B mRNA-editing catalytic polypeptide 3 family member A (APOBEC3A; A3A), a member of the family of cytidine deaminases, present in monocytes. This interaction led to a reduction of the steady-state protein level of A3A. A single-point mutation in Vpx (H82A) abrogated binding to A3A and single-round infection of monocytes by HIV-1. Taken together, our data indicate that lentiviral Vpx counteracts A3A in human monocytes.

Evaluation of a Gene-Directed Enzyme-Product Therapy (GDEPT) in Human Pancreatic Tumor Cells and Their Use as In Vivo Models for Pancreatic Cancer

Background Gene-directed enzyme prodrug therapy (GDEPT) is a two-step treatment protocol for solid tumors that involves the transfer of a gene encoding a prodrug-activating enzyme followed by administration of the inactive prodrug that is subsequently activated by the enzyme to its tumor toxic form. However, the establishment of such novel treatment regimes to combat pancreatic cancer requires defined and robust animal model systems. Methods Here, we comprehensively compared six human pancreatic cancer cell lines (PaCa-44, PANC-1, MIA PaCa-2, Hs-766T, Capan-2, and BxPc-3) in subcutaneous and orthotopical mouse models as well as in their susceptibility to different GDEPTs. Results Tumor uptake was 83% to 100% in the subcutaneous model and 60% to 100% in the orthotopical mouse model, except for Hs-766T cells, which did not grow orthotopically. Pathohistological analyses of the orthotopical models revealed an infiltrative growth of almost all tumors into the pancreas; however, the different cell lines gave rise to tumors with different morphological characteristics. All of the resultant tumors were positive for MUC-1 staining indicating their origin from glandular or ductal epithelium, but revealed scattered pan-cytokeratin staining. Transfer of the cytochrome P450 and cytosine deaminase suicide gene, respectively, into the pancreatic cancer cell lines using retroviral vector technology revealed high level infectibility of these cell lines and allowed the analysis of the sensitivity of these cells to the chemotherapeutic drugs ifosfamide and 5-fluorocytosine, respectively. Conclusion These data qualify the cell lines as part of valuable in vitro and in vivo models for the use in defined preclinical studies for pancreas tumor therapy.

[The certification of advanced therapy medicinal products. A quality label for product development in small and medium-sized enterprises].

Advanced therapy medicinal products (ATMPs) are gene therapy, cell therapy, and tissue engineered products. To gain access to the market within the European Union, ATMPs must be authorized by the European Commission (EC). Especially for small and medium-sized enterprises (SMEs), the European centralized procedure of marketing authorization that is conducted by the European Medicines Agency (EMA) constitutes a major challenge, because SMEs often have little experience with regulatory procedures and many have limited financial possibilities. To tackle these challenges, a certification procedure exclusively for SMEs and their ATMP development was introduced by the EC. Independently from a marketing authorization application, development and/or production processes can be certified. An issued certificate demonstrates that the respective process meets the current regulatory and scientific requirements of the EMA, representing a valuable milestone for putative investors and licensees. This article highlights the background, the detailed procedure, the minimum requirements, as well as the costs of certification, while giving further noteworthy guidance for interested parties.ZusammenfassungArzneimittel für neuartige Therapien (Advanced Therapy Medicinal Products, ATMPs) sind Gentherapeutika, Zelltherapeutika und biotechnologisch bearbeitete Gewebeprodukte. Um ATMPs innerhalb der Europäischen Union (EU) auf den Markt bringen zu können, müssen diese zentralisiert durch die EU-Kommission zugelassen werden. Die zentrale Zulassung über die EMA stellt vor allem für kleine und mittlere Unternehmen (KMUs) eine große Herausforderung dar, da diese oft nur begrenzte Erfahrung mit den regulatorischen Vorgängen haben und über eine geringe Finanzkraft verfügen. Um dieser Problematik Rechnung zu tragen, wurde von der EU-Kommission ein von der zentralisierten Zulassung unabhängiges Zertifizierungsverfahren für KMUs und deren ATMP-Entwicklung eingeführt. Mit einem Zertifikat „ausgezeichnete“ Entwicklungs- und Produktionsprozesse stehen dafür, dass diese den gegenwärtigen regulatorischen und wissenschaftlichen Anforderungen der EMA entsprechen. Auf diese Weise wird die Attraktivität eines Unternehmens für potenzielle oder bestehende Investoren und Lizenznehmer gesteigert. Der vorliegende Beitrag beleuchtet die Hintergründe, den Verfahrensablauf, die Mindestanforderungen und die Kosten der Zertifizierung und gibt weitere bedenkenswerte Hinweise.AbstractAdvanced therapy medicinal products (ATMPs) are gene therapy, cell therapy, and tissue engineered products. To gain access to the market within the European Union, ATMPs must be authorized by the European Commission (EC). Especially for small and medium-sized enterprises (SMEs), the European centralized procedure of marketing authorization that is conducted by the European Medicines Agency (EMA) constitutes a major challenge, because SMEs often have little experience with regulatory procedures and many have limited financial possibilities. To tackle these challenges, a certification procedure exclusively for SMEs and their ATMP development was introduced by the EC. Independently from a marketing authorization application, development and/or production processes can be certified. An issued certificate demonstrates that the respective process meets the current regulatory and scientific requirements of the EMA, representing a valuable milestone for putative investors and licensees. This article highlights the background, the detailed procedure, the minimum requirements, as well as the costs of certification, while giving further noteworthy guidance for interested parties.

Mutation of a diacidic motif in SIV-PBj Nef impairs T-cell activation and enteropathic disease

BackgroundThe non-pathogenic course of SIV infection in its natural host is characterized by robust viral replication in the absence of chronic immune activation and T cell proliferation. In contrast, acutely lethal enteropathic SIVsmm strain PBj induces a strong immune activation and causes a severe acute and lethal disease in pig-tailed macaques after cross-species transmission. One important pathogenicity factor of the PBj virus is the PBj-Nef protein, which contains a conserved diacidic motif and, unusually, an immunoreceptor tyrosine-based activation motif (ITAM).ResultsMutation of the diacidic motif in the Nef protein of the SIVsmmPBj abolishes the acute phenotype of this virus. In vitro, wild-type and mutant PBj (PBj-Nef202/203GG) viruses replicated to similar levels in macaque PBMCs, but PBj-Nef202/203GG no longer triggers ERK mitogen-activated protein (MAP) kinase pathway including an alteration of a Nef-associated Raf-1/ERK-2 multiprotein signaling complex. Moreover, stimulation of IL-2 and down-modulation of CD4 and CD28 were impaired in the mutant virus. Pig-tailed macaques infected with PBj-Nef202/203GG did not show enteropathic complications and lethality as observed with wild-type PBj virus, despite efficient replication of both viruses in vivo. Furthermore, PBj-Nef202/203GG infected animals revealed reduced T-cell activation in periphery lymphoid organs and no detectable induction of IL-2 and IL-6.ConclusionsIn sum, we report here that mutation of the diacidic motif in the PBj-Nef protein abolishes disease progression in pig-tailed macaques despite efficient replication. These data suggest that alterations in the ability of a lentivirus to promote T cell activation and proliferation can have a dramatic impact on its pathogenic potential.

Die Zertifizierung neuartiger Therapien

Advanced therapy medicinal products (ATMPs) are gene therapy, cell therapy, and tissue engineered products. To gain access to the market within the European Union, ATMPs must be authorized by the European Commission (EC). Especially for small and medium-sized enterprises (SMEs), the European centralized procedure of marketing authorization that is conducted by the European Medicines Agency (EMA) constitutes a major challenge, because SMEs often have little experience with regulatory procedures and many have limited financial possibilities. To tackle these challenges, a certification procedure exclusively for SMEs and their ATMP development was introduced by the EC. Independently from a marketing authorization application, development and/or production processes can be certified. An issued certificate demonstrates that the respective process meets the current regulatory and scientific requirements of the EMA, representing a valuable milestone for putative investors and licensees. This article highlights the background, the detailed procedure, the minimum requirements, as well as the costs of certification, while giving further noteworthy guidance for interested parties.ZusammenfassungArzneimittel für neuartige Therapien (Advanced Therapy Medicinal Products, ATMPs) sind Gentherapeutika, Zelltherapeutika und biotechnologisch bearbeitete Gewebeprodukte. Um ATMPs innerhalb der Europäischen Union (EU) auf den Markt bringen zu können, müssen diese zentralisiert durch die EU-Kommission zugelassen werden. Die zentrale Zulassung über die EMA stellt vor allem für kleine und mittlere Unternehmen (KMUs) eine große Herausforderung dar, da diese oft nur begrenzte Erfahrung mit den regulatorischen Vorgängen haben und über eine geringe Finanzkraft verfügen. Um dieser Problematik Rechnung zu tragen, wurde von der EU-Kommission ein von der zentralisierten Zulassung unabhängiges Zertifizierungsverfahren für KMUs und deren ATMP-Entwicklung eingeführt. Mit einem Zertifikat „ausgezeichnete“ Entwicklungs- und Produktionsprozesse stehen dafür, dass diese den gegenwärtigen regulatorischen und wissenschaftlichen Anforderungen der EMA entsprechen. Auf diese Weise wird die Attraktivität eines Unternehmens für potenzielle oder bestehende Investoren und Lizenznehmer gesteigert. Der vorliegende Beitrag beleuchtet die Hintergründe, den Verfahrensablauf, die Mindestanforderungen und die Kosten der Zertifizierung und gibt weitere bedenkenswerte Hinweise.AbstractAdvanced therapy medicinal products (ATMPs) are gene therapy, cell therapy, and tissue engineered products. To gain access to the market within the European Union, ATMPs must be authorized by the European Commission (EC). Especially for small and medium-sized enterprises (SMEs), the European centralized procedure of marketing authorization that is conducted by the European Medicines Agency (EMA) constitutes a major challenge, because SMEs often have little experience with regulatory procedures and many have limited financial possibilities. To tackle these challenges, a certification procedure exclusively for SMEs and their ATMP development was introduced by the EC. Independently from a marketing authorization application, development and/or production processes can be certified. An issued certificate demonstrates that the respective process meets the current regulatory and scientific requirements of the EMA, representing a valuable milestone for putative investors and licensees. This article highlights the background, the detailed procedure, the minimum requirements, as well as the costs of certification, while giving further noteworthy guidance for interested parties.

Mesenchymale Stromazellen bei der Therapie der Graft-versus-Host-Erkrankung: Wo stehen wir?@@@Mesenchymal stromal cells in the treatment of graft-versus-host disease: where do we stand?

Medicinal products based on mesenchymal stromal cells (MSC) are expected to have a therapeutic benefit in a variety of conditions and, accordingly, are being tested in many clinical studies. The treatment and prevention of graft-versus-host disease (GVHD) is one of the worlds most widely studied MSC therapy concepts. So far, one MSC medicinal product has been approved for the treatment of GvHD. This article gives an overview of the particular features related to the production of MSC-based medicinal products, the state of non-clinical research, and the clinical development status of MSCs and the associated challenges, especially in the context of GvHD.Arzneimittel auf der Basis mesenchymaler Stromazellen (MSCs) lassen einen therapeutischen Nutzen bei einer Vielzahl von Erkrankungen erwarten und werden daher in vielen klinischen Studien untersucht. Die Behandlung und Prävention der Graft-versus-Host-Reaktion (GvHD) ist eine der weltweit am häufigsten untersuchten MSC-Anwendungskonzepte. Bisher ist ein auf MSCs basierendes Arzneimittel zur Behandlung von GvHD zugelassen worden. Der vorliegende Beitrag gibt einen Überblick über die Besonderheiten bei der Herstellung von MSC-basierten Arzneimitteln, über den Stand der nichtklinischen Forschung mit und der klinischen Entwicklung von MSCs und über die damit verbundenen Herausforderungen speziell bei ihrer Anwendung bei GvHD. Medicinal products based on mesenchymal stromal cells (MSC) are expected to have a therapeutic benefit in a variety of conditions and, accordingly, are being tested in many clinical studies. The treatment and prevention of graft-versus-host disease (GVHD) is one of the world’s most widely studied MSC therapy concepts. So far, one MSC medicinal product has been approved for the treatment of GvHD. This article gives an overview of the particular features related to the production of MSC-based medicinal products, the state of non-clinical research, and the clinical development status of MSCs and the associated challenges, especially in the context of GvHD.

Mesenchymale Stromazellen bei der Therapie der Graft-versus-Host-Erkrankung: Wo stehen wir?

Medicinal products based on mesenchymal stromal cells (MSC) are expected to have a therapeutic benefit in a variety of conditions and, accordingly, are being tested in many clinical studies. The treatment and prevention of graft-versus-host disease (GVHD) is one of the worlds most widely studied MSC therapy concepts. So far, one MSC medicinal product has been approved for the treatment of GvHD. This article gives an overview of the particular features related to the production of MSC-based medicinal products, the state of non-clinical research, and the clinical development status of MSCs and the associated challenges, especially in the context of GvHD.Arzneimittel auf der Basis mesenchymaler Stromazellen (MSCs) lassen einen therapeutischen Nutzen bei einer Vielzahl von Erkrankungen erwarten und werden daher in vielen klinischen Studien untersucht. Die Behandlung und Prävention der Graft-versus-Host-Reaktion (GvHD) ist eine der weltweit am häufigsten untersuchten MSC-Anwendungskonzepte. Bisher ist ein auf MSCs basierendes Arzneimittel zur Behandlung von GvHD zugelassen worden. Der vorliegende Beitrag gibt einen Überblick über die Besonderheiten bei der Herstellung von MSC-basierten Arzneimitteln, über den Stand der nichtklinischen Forschung mit und der klinischen Entwicklung von MSCs und über die damit verbundenen Herausforderungen speziell bei ihrer Anwendung bei GvHD. Medicinal products based on mesenchymal stromal cells (MSC) are expected to have a therapeutic benefit in a variety of conditions and, accordingly, are being tested in many clinical studies. The treatment and prevention of graft-versus-host disease (GVHD) is one of the world’s most widely studied MSC therapy concepts. So far, one MSC medicinal product has been approved for the treatment of GvHD. This article gives an overview of the particular features related to the production of MSC-based medicinal products, the state of non-clinical research, and the clinical development status of MSCs and the associated challenges, especially in the context of GvHD.

Die Zertifizierung neuartiger Therapien@@@The certification of advanced therapy medicinal products: Ein Gütesiegel für die Produktentwicklung in kleinen und mittelständischen Unternehmen@@@A quality label for product development in small and medium-sized enterprises

Advanced therapy medicinal products (ATMPs) are gene therapy, cell therapy, and tissue engineered products. To gain access to the market within the European Union, ATMPs must be authorized by the European Commission (EC). Especially for small and medium-sized enterprises (SMEs), the European centralized procedure of marketing authorization that is conducted by the European Medicines Agency (EMA) constitutes a major challenge, because SMEs often have little experience with regulatory procedures and many have limited financial possibilities. To tackle these challenges, a certification procedure exclusively for SMEs and their ATMP development was introduced by the EC. Independently from a marketing authorization application, development and/or production processes can be certified. An issued certificate demonstrates that the respective process meets the current regulatory and scientific requirements of the EMA, representing a valuable milestone for putative investors and licensees. This article highlights the background, the detailed procedure, the minimum requirements, as well as the costs of certification, while giving further noteworthy guidance for interested parties.ZusammenfassungArzneimittel für neuartige Therapien (Advanced Therapy Medicinal Products, ATMPs) sind Gentherapeutika, Zelltherapeutika und biotechnologisch bearbeitete Gewebeprodukte. Um ATMPs innerhalb der Europäischen Union (EU) auf den Markt bringen zu können, müssen diese zentralisiert durch die EU-Kommission zugelassen werden. Die zentrale Zulassung über die EMA stellt vor allem für kleine und mittlere Unternehmen (KMUs) eine große Herausforderung dar, da diese oft nur begrenzte Erfahrung mit den regulatorischen Vorgängen haben und über eine geringe Finanzkraft verfügen. Um dieser Problematik Rechnung zu tragen, wurde von der EU-Kommission ein von der zentralisierten Zulassung unabhängiges Zertifizierungsverfahren für KMUs und deren ATMP-Entwicklung eingeführt. Mit einem Zertifikat „ausgezeichnete“ Entwicklungs- und Produktionsprozesse stehen dafür, dass diese den gegenwärtigen regulatorischen und wissenschaftlichen Anforderungen der EMA entsprechen. Auf diese Weise wird die Attraktivität eines Unternehmens für potenzielle oder bestehende Investoren und Lizenznehmer gesteigert. Der vorliegende Beitrag beleuchtet die Hintergründe, den Verfahrensablauf, die Mindestanforderungen und die Kosten der Zertifizierung und gibt weitere bedenkenswerte Hinweise.AbstractAdvanced therapy medicinal products (ATMPs) are gene therapy, cell therapy, and tissue engineered products. To gain access to the market within the European Union, ATMPs must be authorized by the European Commission (EC). Especially for small and medium-sized enterprises (SMEs), the European centralized procedure of marketing authorization that is conducted by the European Medicines Agency (EMA) constitutes a major challenge, because SMEs often have little experience with regulatory procedures and many have limited financial possibilities. To tackle these challenges, a certification procedure exclusively for SMEs and their ATMP development was introduced by the EC. Independently from a marketing authorization application, development and/or production processes can be certified. An issued certificate demonstrates that the respective process meets the current regulatory and scientific requirements of the EMA, representing a valuable milestone for putative investors and licensees. This article highlights the background, the detailed procedure, the minimum requirements, as well as the costs of certification, while giving further noteworthy guidance for interested parties.