Brigitte Anliker

Cortical dysplasia resembling human type 2 lissencephaly in mice lacking all three APP family members

The Alzheimers disease β‐amyloid precursor protein (APP) is a member of a larger gene family that includes the amyloid precursor‐like proteins, termed APLP1 and APLP2. We previously documented that APLP2−/−APLP1−/− and APLP2−/−APP−/− mice die postnatally, while APLP1−/−APP−/− mice and single mutants were viable. We now report that mice lacking all three APP/APLP family members survive through embryonic development, and die shortly after birth. In contrast to double‐mutant animals with perinatal lethality, 81% of triple mutants showed cranial abnormalities. In 68% of triple mutants, we observed cortical dysplasias characterized by focal ectopic neuroblasts that had migrated through the basal lamina and pial membrane, a phenotype that resembles human type II lissencephaly. Moreover, at E18.5 triple mutants showed a partial loss of cortical Cajal Retzius (CR) cells, suggesting that APP/APLPs play a crucial role in the survival of CR cells and neuronal adhesion. Collectively, our data reveal an essential role for APP family members in normal brain development and early postnatal survival.

A physiologic signaling role for the γ-secretase-derived intracellular fragment of APP

Presenilins mediate an unusual intramembranous proteolytic activity known as γ-secretase, two substrates of which are the Notch receptor (Notch) and the β-amyloid precursor protein (APP). γ-Secretase-mediated cleavage of APP, like that of Notch, yields an intracellular fragment [APP intracellular domain (AICD)] that forms a transcriptively active complex. We now demonstrate a functional role for AICD in regulating phosphoinositide-mediated calcium signaling. Genetic ablation of the presenilins or pharmacological inhibition of γ-secretase activity (and thereby AICD production) attenuated calcium signaling in a dose-dependent and reversible manner through a mechanism involving the modulation of endoplasmic reticulum calcium stores. Cells lacking APP (and hence AICD) exhibited similar calcium signaling deficits, and—notably—these disturbances could be reversed by transfection with APP constructs containing an intact AICD, but not by constructs lacking this domain. Our findings indicate that the AICD regulates phosphoinositide-mediated calcium signaling through a γ-secretase-dependent signaling pathway, suggesting that the intramembranous proteolysis of APP may play a signaling role analogous to that of Notch.

Specific gene transfer to neurons, endothelial cells and hematopoietic progenitors with lentiviral vectors

We present a flexible and highly specific targeting method for lentiviral vectors based on single-chain antibodies recognizing cell-surface antigens. We generated lentiviral vectors specific for human CD105+ endothelial cells, human CD133+ hematopoietic progenitors and mouse GluA-expressing neurons. Lentiviral vectors specific for CD105 or for CD20 transduced their target cells as efficiently as VSV-G pseudotyped vectors but discriminated between endothelial cells and lymphocytes in mixed cultures. CD133-targeted vectors transduced CD133+ cultured hematopoietic progenitor cells more efficiently than VSV-G pseudotyped vectors, resulting in stable long-term transduction. Lentiviral vectors targeted to the glutamate receptor subunits GluA2 and GluA4 exhibited more than 94% specificity for neurons in cerebellar cultures and when injected into the adult mouse brain. We observed neuron-specific gene modification upon transfer of the Cre recombinase gene into the hippocampus of reporter mice. This approach allowed targeted gene transfer to many cell types of interest with an unprecedented degree of specificity.

The Functions of Mammalian Amyloid Precursor Protein and Related Amyloid Precursor-Like Proteins

It is well established that proteolytic processing of the β-amyloid precursor protein (APP) generates β-amyloid which plays a central role in the pathogenesis of Alzheimer’s disease. In contrast, the physiological role of APP and the question of whether a loss of these functions contributes to Alzheimer’s disease are still unclear. For a long time, the characterization of APP functions was markedly hampered by the high redundancy between APP and the related APP family members amyloid precursor-like proteins 1 and 2. The generation and analyses of combined gene deficiencies for APP and amyloid precursor-like proteins in mice finally marked the beginning of uncovering the in vivo roles of these proteins in mammals. In the current review, we summarize recent insights into the functions of the APP gene family from mice lacking one, two or all three family members.

Characterization of zetin 1/rBSPRY, a novel binding partner of 14-3-3 proteins.

14-3-3 proteins are ubiquitously expressed proteins which serve as central adaptors in different signal transduction cascades. In this study, yeast two-hybrid screening of a rat brain cDNA library identified a novel gene product termed zetin 1/rBSPRY that interacts with 14-3-3 zeta. The zetin 1/rBSPRY gene is ubiquitously expressed in a variety of rat tissues, with highest expression being found in testis. In adult brain, high levels of zetin 1/rBSPRY mRNA were observed in the hippocampus, cerebral cortex, and piriform cortex. Biochemical studies confirmed zetin 1/rBSPRY to interact with 14-3-3 zeta. Transient co-transfection in COS 7 cells caused a partial redistribution of zetin 1/rBSPRY into 14-3-3 zeta enriched submembranous foci at leading edges. Our results suggest a role for zetin 1/rBSPRY-14-3-3 interactions at specialized submembrane domains.

Environmental risk assessment for medicinal products containing genetically modified organisms

Many gene therapy medicinal products and also some vaccines consist of, or contain, genetically modified organisms (GMOs), which require specific consideration in the environmental risk assessment (ERA) before marketing authorisation or clinical trial applications. The ERA is performed in order to identify the potential risks for public health and the environment, which may arise due to the clinical use of these medicinal products. If such environmental risks are identified and considered as not acceptable, the ERA should go on to propose appropriate risk management strategies capable to reduce these risks. This article will provide an overview of the legal basis and requirements for the ERA of GMO-containing medicinal products in the context of marketing authorisation in the EU and clinical trials in Germany. Furthermore, the scientific principles and methodology that generally need to be followed when preparing an ERA for GMOs are discussed.ZusammenfassungViele Gentherapeutika und Impfstoffe bestehen aus oder enthalten gentechnisch veränderte Organismen, was bei der Bewertung zur Umweltsicherheit dieser Arzneimittel speziell zu berücksichtigen ist. Sowohl vor Marktzulassung wie auch vor Durchführung klinischer Studien wird daher eine Umweltrisikobewertung durchgeführt. Im Vordergrund steht dabei die Frage, ob durch die klinische Anwendung dieser GVO-haltigen Arzneimittel Risiken für die Gesundheit Dritter oder für die Umwelt entstehen. Werden im Rahmen der Umweltrisikobewertung nicht vertretbare Risiken identifiziert, sind Vorkehrungen und Vorsichtsmaßnahmen zu definieren, welche diese Risiken auf ein akzeptables Niveau senken. Dieser Artikel gibt einen Überblick über die Rechtsgrundlagen und Anforderungen bei der Umweltrisikobewertung GVO-haltiger Arzneimittel bei Marktzulassung in der EU sowie bei klinischer Prüfung in Deutschland. Das Konzept und die Vorgehensweise der Risikobewertung werden dabei im Detail vorgestellt.

Umweltrisikobewertung GVO-haltiger Arzneimittel

Many gene therapy medicinal products and also some vaccines consist of, or contain, genetically modified organisms (GMOs), which require specific consideration in the environmental risk assessment (ERA) before marketing authorisation or clinical trial applications. The ERA is performed in order to identify the potential risks for public health and the environment, which may arise due to the clinical use of these medicinal products. If such environmental risks are identified and considered as not acceptable, the ERA should go on to propose appropriate risk management strategies capable to reduce these risks. This article will provide an overview of the legal basis and requirements for the ERA of GMO-containing medicinal products in the context of marketing authorisation in the EU and clinical trials in Germany. Furthermore, the scientific principles and methodology that generally need to be followed when preparing an ERA for GMOs are discussed.ZusammenfassungViele Gentherapeutika und Impfstoffe bestehen aus oder enthalten gentechnisch veränderte Organismen, was bei der Bewertung zur Umweltsicherheit dieser Arzneimittel speziell zu berücksichtigen ist. Sowohl vor Marktzulassung wie auch vor Durchführung klinischer Studien wird daher eine Umweltrisikobewertung durchgeführt. Im Vordergrund steht dabei die Frage, ob durch die klinische Anwendung dieser GVO-haltigen Arzneimittel Risiken für die Gesundheit Dritter oder für die Umwelt entstehen. Werden im Rahmen der Umweltrisikobewertung nicht vertretbare Risiken identifiziert, sind Vorkehrungen und Vorsichtsmaßnahmen zu definieren, welche diese Risiken auf ein akzeptables Niveau senken. Dieser Artikel gibt einen Überblick über die Rechtsgrundlagen und Anforderungen bei der Umweltrisikobewertung GVO-haltiger Arzneimittel bei Marktzulassung in der EU sowie bei klinischer Prüfung in Deutschland. Das Konzept und die Vorgehensweise der Risikobewertung werden dabei im Detail vorgestellt.

Regulation of Clinical Trials with Advanced Therapy Medicinal Products in Germany

In the European Union, clinical trials for Advanced Therapy Medicinal Products are regulated at the national level, in contrast to the situation for a Marketing Authorisation Application, in which a centralised procedure is foreseen for these medicinal products. Although based on a common understanding regarding the regulatory requirement to be fulfilled before conduct of a clinical trial with an Advanced Therapy Investigational Medicinal Product, the procedures and partly the scientific requirements for approval of a clinical trial application differ between the European Union Member States. This chapter will thus give an overview about the path to be followed for a clinical trial application and the subsequent approval process for an Advanced Therapy Investigational Medicinal Product in Germany and will describe the role of the stakeholders that are involved. In addition, important aspects of manufacturing, quality control and non-clinical testing of Advanced Therapy Medicinal Products in the clinical development phase are discussed. Finally, current and future approaches for harmonisation of clinical trial authorisation between European Union Member States are summarised.

Clinical translation and regulatory aspects of CAR/TCR-based adoptive cell therapies—the German Cancer Consortium approach

Adoptive transfer of T cells genetically modified by TCRs or CARs represents a highly attractive novel therapeutic strategy to treat malignant diseases. Various approaches for the development of such gene therapy medicinal products (GTMPs) have been initiated by scientists in recent years. To date, however, the number of clinical trials commenced in Germany and Europe is still low. Several hurdles may contribute to the delay in clinical translation of these therapeutic innovations including the significant complexity of manufacture and non-clinical testing of these novel medicinal products, the limited knowledge about the intricate regulatory requirements of the academic developers as well as limitations of funds for clinical testing. A suitable good manufacturing practice (GMP) environment is a key prerequisite and platform for the development, validation, and manufacture of such cell-based therapies, but may also represent a bottleneck for clinical translation. The German Cancer Consortium (DKTK) and the Paul-Ehrlich-Institut (PEI) have initiated joint efforts of researchers and regulators to facilitate and advance early phase, academia-driven clinical trials. Starting with a workshop held in 2016, stakeholders from academia and regulatory authorities in Germany have entered into continuing discussions on a diversity of scientific, manufacturing, and regulatory aspects, as well as the benefits and risks of clinical application of CAR/TCR-based cell therapies. This review summarizes the current state of discussions of this cooperative approach providing a basis for further policy-making and suitable modification of processes.

Regulation for Gene and Cell Therapy Medicinal Products in Europe

An important step in the regulation of cell and gene therapy medicinal products, which are classified as advanced therapy medicinal products (ATMPs) in the European Union, has been made with Regulation 1394/2007/EC. By this regulation a new committee, the Committee for Advanced Therapies, has been established to ensure appropriate coverage of scientific and regulatory aspects of ATMPs. In addition, novel regulatory tools specific for ATMPs such as the classification, certification, and hospital exemption were introduced to support the development of this product class. By nature, ATMPs are a special class of medicinal products with characteristics different to conventional drugs and even other biologicals. Hence, regulatory requirements for manufacturing and clinical evaluation need to be tailored to the type and design of the ATMP, as well as to its manufacturing process and clinical indication. This chapter summarizes the general regulatory pathway for ATMPs as well as the currently applicable ATMP-specific procedures. In addition, the regulatory requirements regarding manufacturing, quality, and nonclinical and clinical testing for cell and gene therapy medicinal products are discussed. Important aspects of the environmental risk assessment, a provision for ATMPs containing genetically modified organisms, are also reviewed.