[Genetically modified regulatory T cells: therapeutic concepts and regulatory aspects]. / Genetisch modifizierte regulatorische T-Zellen: Therapiekonzepte und ihr regulatorischer Rahmen.

Adoptive T­cell therapies are emerging tools to combat various human diseases. CAR­T cells are approved and marketed as last line therapeutics in advanced B­cell lymphomas and leukemias. TCR-engineered T cells are being evaluated in clinical trials for a variety of hematological and solid tumors. Genetically modified regulatory T cells, however, are still in the initial stages of clinical development for the induction of immune tolerance in various indications.Here we outline the general role of regulatory T cells in establishing self-tolerance and the mechanisms by which these suppress the effector immune cells. Further, the role of regulatory T cells in the pathomechanism of certain immune diseases is presented, and the current status of clinical developments of genetically modified Treg cells is discussed. We also present the regulatory framework for genetically modified regulatory T cells as advanced therapy medicinal products, including aspects of manufacture and quality control, as well as nonclinical and clinical development requirements.

Going non-viral: the Sleeping Beauty transposon system breaks on through to the clinical side.

Molecular medicine has entered a high-tech age that provides curative treatments of complex genetic diseases through genetically engineered cellular medicinal products. Their clinical implementation requires the ability to stably integrate genetic information through gene transfer vectors in a safe, effective and economically viable manner. The latest generation of Sleeping Beauty (SB) transposon vectors fulfills these requirements, and may overcome limitations associated with viral gene transfer vectors and transient non-viral gene delivery approaches that are prevalent in ongoing pre-clinical and translational research. The SB system enables high-level stable gene transfer and sustained transgene expression in multiple primary human somatic cell types, thereby representing a highly attractive gene transfer strategy for clinical use. Here we review several recent refinements of the system, including the development of optimized transposons and hyperactive SB variants, the vectorization of transposase and transposon as mRNA and DNA minicircles (MCs) to enhance performance and facilitate vector production, as well as a detailed understanding of SB's genomic integration and biosafety features. This review also provides a perspective on the regulatory framework for clinical trials of gene delivery with SB, and illustrates the path to successful clinical implementation by using, as examples, gene therapy for age-related macular degeneration (AMD) and the engineering of chimeric antigen receptor (CAR)-modified T cells in cancer immunotherapy.

Cytokine-induced interleukin-1 receptor antagonist protein expression in genetically engineered equine mesenchymal stem cells for osteoarthritis treatment.

BACKGROUND: A combination of tissue engineering methods employing mesenchymal stem cells (MSCs) together with gene transfer takes advantage of innovative strategies and highlights a new approach for targeting osteoarthritis (OA) and other cartilage defects. Furthermore, the development of systems allowing tunable transgene expression as regulated by natural disease-induced substances is highly desirable. METHODS: Bone marrow-derived equine MSCs were transduced with a lentiviral vector expressing interleukin-1 receptor antagonist (IL-1Ra) gene under the control of an inducible nuclear factor-kappa B-responsive promoter and IL-1Ra production upon pro-inflammatory cytokine stimulation [tumor necrosis factor (TNF)α, interleukin (IL)-1ß] was analysed. To assess the biological activity of the IL-1Ra protein that was produced and the therapeutic effect of IL-1Ra-expressing MSCs (MSC/IL-1Ra), cytokine-based two- and three-dimensional in vitro models of osteoarthritis using equine chondrocytes were established and quantitative real-time polymerase chain reaction (PCR) analysis was used to measure the gene expression of aggrecan, collagen IIA1, interleukin-1ß, interleukin-6, interleukin-8, matrix metalloproteinase-1 and matrix metalloproteinase-13. RESULTS: A dose-dependent increase in IL-1Ra expression was found in MSC/IL-1Ra cells upon TNFα administration, whereas stimulation using IL-1ß did not lead to IL-1Ra production above the basal level observed in nonstimulated cells as a result of the existing feedback loop. Repeated cycles of induction allowed on/off modulation of transgene expression. In vitro analyses revealed that IL-1Ra protein present in the conditioned medium from MSC/IL-1Ra cells blocks OA onset in cytokine-treated equine chondrocytes and co-cultivation of MSC/IL-1Ra cells with osteoarthritic spheroids alleviates the severity of the osteoarthritic changes. CONCLUSIONS: Thus, pro-inflammatory cytokine induced IL-1Ra protein expression from genetically modified MSCs might represent a promising strategy for osteoarthritis treatment.

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.

Tropism, intracerebral distribution, and transduction efficiency of HIV- and SIV-based lentiviral vectors after injection into the mouse brain: a qualitative and quantitative in vivo study.

Lentiviruses are suitable to transfer potential therapeutic genes into non-replicating cells such as neurons, but systematic in vivo studies on transduction of neural cells within the complete brain are missing. We analysed the distribution of transduced cells with respect to brain structure, virus tropism, numbers of transduced neurons per brain, and influence of the Vpx or Vpr accessory proteins after injection of vectors based on SIVsmmPBj, HIV-2, and HIV-1 lentiviruses into the right striatum of the mouse brain. Transduced cells were found ipsilaterally around the injection canal, in corpus striatum and along corpus callosum, irrespective of the vector type. All vectors except HIV-2SEW transduced also single cells in the olfactory bulb, hippocampus, and cerebellum. Vector HIV-2SEW was the most neuron specific. However, vectors PBjSEW and HIV-1SEW transduced more neurons per brain (means 41,299 and 32,309) than HIV-2SEW (16,102). In the presence of Vpx/Vpr proteins, HIV-2SEW(Vpx) and HIV-1SEW(Vpr) showed higher overall transduction efficiencies (30,696 and 27,947 neurons per brain) than PBjSEW(Vpx) (6636). The distances of transduced cells from the injection canal did not differ among the viruses but correlated positively with the numbers of transduced neurons. The presence of Vpx/Vpr did not increase the numbers of transduced neurons. Parental virus type and the vector equipment seem to influence cellular tropism and transduction efficiency. Thus, precision of injection and choice of virus pseudotype are not sufficient when targeted lentiviral vector transduction of a defined brain cell population is required.

Inflammation-induced transgene expression in genetically engineered equine mesenchymal stem cells.

BACKGROUND: Osteoarthritis, a chronic and progressive degenerative joint disorder, ranks amongst the top five causes of disability. Given the high incidence, associated socioeconomic costs and the absence of effective disease-modifying therapies of osteoarthritis, cell-based treatments offer a promising new approach. Owing to their paracrine, differentiation and self-renewal abilities, mesenchymal stem cells (MSCs) have great potential for regenerative medicine, which might be further enhanced by targeted gene therapy. Hence, the development of systems allowing transgene expression, particularly when regulated by natural disease-dependent occuring substances, is of high interest. METHODS: Bone marrow-isolated equine MSCs were stably transduced with an HIV-1 based lentiviral vector expressing the luciferase gene under control of an inducible nuclear factor κB (NFκB)-responsive promoter. Marker gene expression was analysed by determining luciferase activity in transduced cells stimulated with different concentrations of interleukin (IL)-1ß or tumour necrosis factor (TNF)α. RESULTS: A dose-dependent increase in luciferase expression was observed in transduced MSCs upon cytokine stimulation. The induction effect was more potent in cells treated with TNFα compared to those treated with IL-1ß. Maximum transgene expression was obtained after 48 h of stimulation and the same time was necessary to return to baseline luciferase expression levels after withdrawal of the stimulus. Repeated cycles of induction allowed on-off modulation of transgene expression without becoming refractory to induction. The NFκB-responsive promoter retained its inducibility also in chondrogenically differentiated MSC/Luc cells. CONCLUSIONS: The results of the present study demonstrate that on demand transgene expression from the NFκB-responsive promoter using naturally occurring inflammatory cytokines can be induced in undifferentiated and chondrogenically differentiated equine MSCs. Copyright © 2016 John Wiley & Sons, Ltd.

Report of the International Regulatory Forum on Human Cell Therapy and Gene Therapy Products.

The development of human cell therapy and gene therapy products has progressed internationally. Efforts have been made to address regulatory challenges in the evaluation of quality, efficacy, and safety of the products. In this forum, updates on the specific challenges in quality, efficacy, and safety of products in the view of international development were shared through the exchange of information and opinions among experts from regulatory authorities, academic institutions, and industry practitioners. Sessions identified specific/critical points to consider for the evaluation of human cell therapy and gene therapy products that are different from conventional biological products; common approaches and practices among regulatory regions were also shared. Certain elements of current international guidelines might not be appropriate to be applied to these products. Further, international discussion on the concept of potency and in vivo tumorigenicity studies, among others, is needed. This forum concluded that the continued collective actions are expected to promote international convergence of regulatory approaches of the products. The Pharmaceuticals and Medical Devices Agency and Japanese Society for Regenerative Medicine jointly convened the forum with support from the National Institutes of Biomedical Innovation, Health and Nutrition. Participants at the forum include 300 experts in and outside of Japan.

Standardization of Good Manufacturing Practice-compliant production of bone marrow-derived human mesenchymal stromal cells for immunotherapeutic applications.

BACKGROUND AIMS: Human mesenchymal stem or stromal cells (MSCs) represent a potential resource not only for regenerative medicine but also for immunomodulatory cell therapies. The application of different MSC culture protocols has significantly hampered the comparability of experimental and clinical data from different laboratories and has posed a major obstacle for multicenter clinical trials. Manufacturing of cell products for clinical application in the European Community must be conducted in compliance with Good Manufacturing Practice and requires a manufacturing license. In Germany, the Paul-Ehrlich-Institut as the Federal Authority for Vaccines and Biomedicines is critically involved in the approval process. METHODS: This report summarizes a consensus meeting between researchers, clinicians and regulatory experts on standard quality requirements for MSC production. RESULTS: The strategy for quality control testing depends on the product's cell composition, the manufacturing process and the indication and target patient population. Important quality criteria in this sense are, among others, the immunophenotype of the cells, composition of the culture medium and the risk for malignant transformation, as well as aging and the immunosuppressive potential of the manufactured MSCs. CONCLUSIONS: This position paper intends to provide relevant information to interested parties regarding these criteria to foster the development of scientifically valid and harmonized quality standards and to support approval of MSC-based investigational medicinal products.

Cell therapy clinical trials in Germany--Critical aspects of quality data content: Summary of meeting presentation.

This article is summarizing a presentation given by the author at the International Alliance for Biological Standardization and Japan Science and Technology Agency (IABS-JST) Joint Workshop on "Challenges towards sound scientific regulation of cell therapy products" held at the Kyoto International Conference Center, Kyoto Japan on March 7-8, 2014. The main topics of the presentation were to give a short overview about the regulatory approval process for clinical trials in Germany and to summarize important manufacturing aspects of cell based medicinal products (CBMPs) which are intended to be studied in clinical trials in Germany.

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.

Marketing Regulatory Oversight of Advanced Therapy Medicinal Products (ATMPs) in Europe: The EMA/CAT Perspective.

With the release of Regulation 1394/2007, a new framework for gene and cell therapy medicinal products and tissue-engineered products was established in the European Union. For all three product classes, called advanced therapy medicinal products, a centralised marketing authorisation became mandatory. The European Medicines Agency (EMA) together with its Committee for Advanced Therapies, Committee for Human Medicinal Products and the network of national agencies is responsible for scientific evaluation of the marketing authorisation applications. For a new application, data and information relating to manufacturing processes and quality control of the active substance and the final product have to be submitted for evaluation together with data from non-clinical and clinical safety and efficacy studies. Technical requirements for ATMPs are defined in the legislation, and guidance for different products is available through several EMA/CAT guidelines. Due to the diversity of ATMPs, a tailored approach for regulating these products is considered necessary. Thus, a risk-based approach has been introduced for ATMPs allowing flexibility for the regulatory requirements. Since the regulatory framework for ATMPs was established, five products have been licenced in the European Union. However, the pipeline of new ATMPs is much bigger, as seen from the significant numbers of different products discussed by the CAT in scientific advice and classification procedures. In 2013, a public consultation on the ATMP Regulation was conducted by the European Commission, and the results were published in 2014. The report proposes several improvements for the current framework and established procedures for the regulation of ATMPs.

[Therapeutic approaches using genetically modified cells]. / Genetisch modifizierte Zellen zur Therapie verschiedener Erkrankungen.

Medicinal products containing genetically modified cells are, in most cases, classified as gene therapy and cell therapy medicinal products. Although no medicinal product containing genetically modified cells has been licensed in Europe yet, a variety of therapeutic strategies using genetically modified cells are in different stages of clinical development for the treatment of acquired and inherited diseases. In this chapter, several examples of promising approaches are presented, with an emphasis on gene therapy for inherited immunodeficiencies and on tumour immunotherapy with genetically modified T-cells expressing a chimeric antigen receptor or a recombinant T-cell receptor.

Regulatory Considerations for Clinical Trial Applications with CRISPR-Based Medicinal Products.

Since first proposed as a new tool for gene targeting and genome editing, CRISPR technology has quickly advanced into the clinical stage. Initial studies highlight the potential for CRISPR-Cas9-mediated therapeutic approaches in human medicine to correct incurable genetic diseases and enhance cell-based therapeutic approaches. While acknowledging the opportunities this technology brings for the treatment of patients with severe diseases, timely development of these innovative medicinal products requires regulatory oversight and adaptation of regulatory requirements to ensure the safety and efficacy of medicinal products based on CRISPR technology. We briefly present the current regulatory framework applicable for CRISPR-Cas-based developments as advanced therapy medicinal products. Moreover, scientific- and regulatory-driven considerations relevant for advancing product development toward clinical trial applications in Germany are highlighted by discussing the key aspects of quality and nonclinical and clinical development requirements.

Time to evolve: predicting engineered T cell-associated toxicity with next-generation models.

Despite promising clinical results in a small subset of malignancies, therapies based on engineered chimeric antigen receptor and T-cell receptor T cells are associated with serious adverse events, including cytokine release syndrome and neurotoxicity. These toxicities are sometimes so severe that they significantly hinder the implementation of this therapeutic strategy. For a long time, existing preclinical models failed to predict severe toxicities seen in human clinical trials after engineered T-cell infusion. However, in recent years, there has been a concerted effort to develop models, including humanized mouse models, which can better recapitulate toxicities observed in patients. The Accelerating Development and Improving Access to CAR and TCR-engineered T cell therapy (T2EVOLVE) consortium is a public-private partnership directed at accelerating the preclinical development and increasing access to engineered T-cell therapy for patients with cancer. A key ambition in T2EVOLVE is to design new models and tools with higher predictive value for clinical safety and efficacy, in order to improve and accelerate the selection of lead T-cell products for clinical translation. Herein, we review existing preclinical models that are used to test the safety of engineered T cells. We will also highlight limitations of these models and propose potential measures to improve them.

Comparative evaluation of preclinical in vivo models for the assessment of replicating retroviral vectors for the treatment of glioblastoma.

Despite impressive improvements in neurosurgical techniques, radiation and chemotherapy during the past few years, little progress has been made in the treatment of malignant gliomas. Recently, the efficacy of suicide gene therapy based on replication-competent retroviral (RCR) vectors as delivery vehicles for the therapeutic gene has been described in the treatment of experimental cancer, including gliomas. In this study, we have thus critically evaluated a panel of human and rodent glioma/glioblastoma cell lines (U-87MG, U-118MG, LN-18, LN-229, 8-MG-BA, 42-MG-BA, A-172, T-98G, UVW, C6, 9L, G-26, GL-261, Tu-2449, Tu-9648) with respect to RCR virus vector spread, sensitivity towards the cytosine deaminase (CD)/5-flurocytosine (5-FC)/5-flurouracil (5-FU) suicide system, and orthotopic growth characteristics in mice to identify suitable preclinical animal models for the development of a glioblastoma gene therapy. Rapid virus spread was observed in eight out of nine human cell lines tested in vitro. As expected, only CD-expressing cells became sensitive to 5-FC, due to their ability to convert the prodrug in its toxic form, 5-FU. All LD(50) values were within the range of concentrations obtained in human body fluids after conventional antifungal 5-FC administration. In addition, a significant bystander effect was observed in all human glioma cell lines tested. Injection of the RCR vector into pre-established orthotopic mouse tumor xenografts revealed substantial infection and virus spread of tumor tissue from most cell types.

Influence of vector design and host cell on the mechanism of recombination and emergence of mutant subpopulations of replicating retroviral vectors.

BACKGROUND: The recent advent of murine leukaemia virus (MLV)-based replication-competent retroviral (RCR) vector technology has provided exciting new tools for gene delivery, albeit the advances in vector efficiency which have been realized are also accompanied by a set of fresh challenges. The expression of additional transgene sequences, for example, increases the length of the viral genome, which can lead to reductions in replication efficiency and in turn to vector genome instability. This necessitates efforts to analyse the rate and mechanism of recombinant emergence during the replication of such vectors to provide data which should contribute to improvements in RCR vector design. RESULTS: In this study, we have performed detailed molecular analyses on packaged vector genomes and proviral DNA following propagation of MLV-based RCR vectors both in cell culture and in pre-formed subcutaneous tumours in vivo. The effects of strain of MLV, transgene position and host cell type on the rate of emergence of vector recombinants were quantitatively analysed by applying real-time PCR and real-time RT-PCR assays. Individual mutants were further characterized by PCR, and nucleotide sequence and structural motifs associated with these mutants were determined by sequencing. Our data indicate that virus strain, vector design and host cell influence the rate of emergence of predominating vector mutants, but not the underlying recombination mechanisms in vitro. In contrast, however, differences in the RNA secondary structural motifs associated with sequenced mutants emerging in cell culture and in solid tumours in vivo were observed. CONCLUSION: Our data provide further evidence that MLV-based RCR vectors based on the Moloney strain of MLV and containing the transgene cassette in the 3' UTR region are superior to those based on Akv-MLV and/or containing the transgene cassette in the U3 region of the LTR. The observed discrepancies between the data obtained in solid tumours in vivo and our own and previously published data from infected cells in vitro demonstrates the importance of evaluating vectors designed for use in cancer gene therapy in vivo as well as in vitro.

Transgene expression facilitated by the v-src splice acceptor can impair replication kinetics and lead to genomic instability of Rous sarcoma virus-based vectors.

Rous sarcoma virus (RSV) can be used for the simple generation of high-titer replication-competent retroviral (RCR) vectors. Retroviruses undergo frequent genomic recombination, however, and vectors with reduced replication kinetics are rapidly overgrown by mutant forms. Vector design is hence critical to vector efficacy. In this study, two different designs of RSV-based RCR vectors were evaluated. Vectors in which transgene expression was facilitated by the v-src splice acceptor were revealed to have greatly reduced replication kinetics and genomic stability in comparison to vectors in which transgene expression was mediated by an internal ribosome entry site in the 3' untranslated region.

Suicide genes for cancer therapy.

The principle of using suicide genes for gene directed enzyme prodrug therapy (GDEPT) of cancer has gained increasing significance during the 20 years since its inception. The astute application of suitable GDEPT systems should permit tumour ablation in the absence of off-target toxicity commonly associated with classical chemotherapy, a hypothesis which is supported by encouraging results in a multitude of pre-clinical animal models. This review provides a clear explanation of the rationale behind the GDEPT principle, outlining the advantages and limitations of different GDEPT strategies with respect to the roles of the bystander effect, the immune system and the selectivity of the activated prodrug in contributing to their therapeutic efficacy. An in-depth analysis of the most widely used suicide gene/prodrug combinations is presented, including details of the latest advances in enzyme and prodrug optimisation and results from the most recent clinical trials.

Human Genome Editing in the Clinic: New Challenges in Regulatory Benefit-Risk Assessment.

As genome editing rapidly progresses toward the realization of its clinical promise, assessing the suitability of current tools and processes used for its benefit-risk assessment is critical. Although current regulations may initially provide an adequate regulatory framework, improvements are recommended to overcome several existing technology-based safety and efficacy issues.

Cytochrome P450 reductase dependent inhibition of cytochrome P450 2B1 activity: Implications for gene directed enzyme prodrug therapy.

Cytochrome P450 (P450) enzymes are often used in suicide gene cancer therapy strategies to convert an inactive prodrug into its therapeutic active metabolites. However, P450 activity is dependent on electrons supplied by cytochrome P450 reductase (CPR). Since endogenous CPR activity may not be sufficient for optimal P450 activity, the overexpression of additional CPR has been considered to be a valuable approach in gene directed enzyme prodrug therapy (GDEPT). We have analysed a set of cell lines for the effects of CPR on cytochrome P450 isoform 2B1 (CYP2B1) activity. CPR transfected human embryonic kidney 293 (HEK293) cells showed both strong CPR expression in Western blot analysis and 30-fold higher activity in cytochrome c assays as compared to parental HEK293 cells. In contrast, resorufin and 4-hydroxy-ifosfamide assays revealed that CYP2B1 activity was up to 10-fold reduced in CPR/CYP2B1 cotransfected HEK293 cells as compared to cells transfected with the CYP2B1 expression plasmid alone. Determination of ifosfamide-mediated effects on cell viability allowed independent confirmation of the reduction in CYP2B1 activity upon CPR coexpression. Inhibition of CYP2B1 activity by CPR was also observed in CYP2B1/CPR transfected or infected pancreatic tumour cell lines Panc-1 and Pan02, the human breast tumour cell line T47D and the murine embryo fibroblast cell line NIH3T3. A CPR mediated increase in CYP2B1 activity was only observed in the human breast tumour cell line Hs578T. Thus, our data reveal an effect of CPR on CYP2B1 activity dependent on the cell type used and therefore demand a careful evaluation of the therapeutic benefit of combining cytochrome P450 and CPR in respective in vivo models in each individual target tissue to be treated.