C Coutelle

Imperial College London, London, ENG, United Kingdom

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Publications (195)717.72 Total impact

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    ABSTRACT: Genotoxicity models are extremely important to assess retroviral vector biosafety before gene therapy. We have developed an in utero model that demonstrates that hepatocellular carcinoma (HCC) development is restricted to mice receiving nonprimate (np) lentiviral vectors (LV) and does not occur when a primate (p) LV is used regardless of woodchuck post-translation regulatory element (WPRE) mutations to prevent truncated X gene expression. Analysis of 839 npLV and 244 pLV integrations in the liver genomes of vector-treated mice revealed clear differences between vector insertions in gene dense regions and highly expressed genes, suggestive of vector preference for insertion or clonal outgrowth. In npLV-associated clonal tumors, 56% of insertions occurred in oncogenes or genes associated with oncogenesis or tumor suppression and surprisingly, most genes examined (11/12) had reduced expression as compared with control livers and tumors. Two examples of vector-inserted genes were the Park 7 oncogene and Uvrag tumor suppressor gene. Both these genes and their known interactive partners had differential expression profiles. Interactive partners were assigned to networks specific to liver disease and HCC via ingenuity pathway analysis. The fetal mouse model not only exposes the genotoxic potential of vectors intended for gene therapy but can also reveal genes associated with liver oncogenesis.Molecular Therapy (2012); doi:10.1038/mt.2012.224.
    Molecular Therapy 11/2012; · 7.04 Impact Factor
  • Charles Coutelle, Simon N Waddington
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    ABSTRACT: This chapter gives a comparative review of the different vector systems applied to date in prenatal gene therapy experiments highlighting the need for versatility and choice for application in accordance with the actual aim of the study. It reviews the key characteristics of the four main gene therapy vector systems and gives examples for their successful application in prenatal gene therapy experiments.
    Methods in molecular biology (Clifton, N.J.) 01/2012; 891:41-53. · 1.29 Impact Factor
  • Charles Coutelle, Richard Ashcroft
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    ABSTRACT: The still experimental nature of prenatal gene therapy carries a certain degree of risk, both for the pregnant mother as well as for the fetus. Some of the risks are procedural hazards already known from more conventional fetal medicine interventions. Others are more specific to gene therapy such as the potential for interference with normal fetal development, the possibility of inadvertent germ line gene transfer, and the danger of oncogenesis. This chapter reviews the potential risks in relation to the expected benefits of prenatal gene therapy. It discusses the scientific, ethical, legal, and social implications of this novel preventive approach to genetic disease and outlines preconditions to be met in preparation for a potential future clinical application.
    Methods in molecular biology (Clifton, N.J.) 01/2012; 891:371-87. · 1.29 Impact Factor
  • Charles Coutelle, Simon N Waddington
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    ABSTRACT: This introductory chapter provides a short review of the ideas and practical approaches that have led to the present and perceived future development of prenatal gene therapy. It summarizes the advantages and the potential adverse effects of this novel preventive and therapeutic approach to the management of prenatal diseases. It also provides guidance to the range of conditions to which prenatal gene therapy may be applied and to the technical approaches, vectors, and societal/ethical considerations for this newly emerging field of Fetal Medicine.
    Methods in molecular biology (Clifton, N.J.) 01/2012; 891:1-7. · 1.29 Impact Factor
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    ABSTRACT: Safety is an absolute prerequisite for introducing any new therapy, and the need to monitor the consequences of administration of both vector and transgene to the fetus is particularly important. The unique features of fetal development that make it an attractive target for gene therapy, such as its immature immune system and rapidly dividing populations of stem cells, also mean that small perturbations in pregnancy can have significant short- and long-term consequences. Certain features of the viral vectors used, the product of the delivered gene, and sometimes the invasive techniques necessary to deliver the construct to the fetus in utero have the potential to do harm. An important goal of prenatal gene therapy research is to develop clinically relevant techniques that could be applied to cure or ameliorate human disease in utero on large animal models such as sheep or nonhuman primates. Equally important is the use of these models to monitor for potential adverse effects of such interventions. These large animal models provide good representation of individual patient-based investigations. However, analyses that require defined genetic backgrounds, high throughput, defined variability and statistical analyses, e.g. for initial studies on teratogenic and oncogenic effects, are best performed on larger groups of small animals, in particular mice. This chapter gives an overview of the potential adverse effects in relation to prenatal gene therapy and describes the techniques that can be used experimentally in a large animal model to monitor the potential adverse consequences of prenatal gene therapy, with relevance to clinical application. The sheep model is particularly useful to allow serial monitoring of fetal growth and well-being after delivery of prenatal gene therapy. It is also amenable to serially sampling using minimally invasive and clinically relevant techniques such as ultrasound-guided blood sampling. For more invasive long-term monitoring, we describe telemetric techniques to measure the haemodynamics of the mother or fetus, for example, that interferes minimally with normal animal behaviour. Implanted catheters can also be used for serial fetal blood sampling during gestation. Finally, we describe methods to monitor events around birth and long-term neonatal follow-up that are important when considering human translation of this therapy.
    Methods in Molecular Biology. 01/2012; 891:291-328.
  • Charles Coutelle, Simon N Waddington, Michael Themis
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    ABSTRACT: So far no systematic studies have been conducted to investigate developmental aberrations after prenatal gene transfer in mice. Here, we suggest procedures for such observations to be applied, tested and improved in further in utero gene therapy experiments. They are based on our own experience in husbandry for transgenic human diseases mouse models and breading, rearing, and observing mice after fetal gene transfer as well as on the systematic screens for monitoring of knock-out mutant mouse phenotypes established in international mutagenesis projects (EUMORPHIA and EUMODIC and subsequently the International Mouse Phenotyping Consortium). We also describe here the analysis procedures for detection of germ line mutations based on quantitative PCR (qPCR) by sperm-DNA analysis and breeding studies.
    Methods in molecular biology (Clifton, N.J.) 01/2012; 891:329-40. · 1.29 Impact Factor
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    ABSTRACT: Fetal gene therapy has the potential to treat inherited genetic diseases in utero before significant organ damage has occurred. Rapidly expanding stem cell populations may be targeted and the introduction of transgenes to the fetus during development of the immune system could result in immune tolerance and facilitate repeat treatment postnatally. Genetic diseases such as cystic fibrosis, which are life-threatening and for which there are no currently acceptable treatments available, are suggested targets for this therapy. Ultrasound may be a safe method of delivering a therapeutic gene into the fetus, although most studies have used more invasive techniques, even in large animal models. Viral vectors currently offer the most potential. Adenovirus-based vectors are stable, independent of host cell replication, efficient at tissue infection and have been used as a ‘pathfinder’ to test routes of administration. Unfortunately, they are also highly immunogenic and other systems based on retrovirus or adeno-associated virus may offer advantages because of their lower immunogenicity and potential for permanent transgene expression. Our group is developing the fetal sheep model for the investigation of ultrasound-guided gene therapy in utero. This model is suitable since the sheep fetus is tolerant to manipulations, has a consistent gestation period and shows many similarities with human pregnancy. We have demonstrated significant transfection of the fetal liver and adrenal cortex after ultrasound-guided percutaneous injection of the umbilical vein with adenoviral vectors in the late gestation sheep. We are investigating and here discuss alternative routes of administration to target the fetus in early gestation via ultrasound-guided minimally invasive techniques.
    The Ultrasound Review of Obstetrics & Gynecology 12/2011; 1(1).
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    ABSTRACT: Development of a human mitochondrial gene delivery vector is a critical step in the ability to treat diseases arising from mutations in mitochondrial DNA. Although we have previously cloned the mouse mitochondrial genome in its entirety and developed it as a mitochondrial gene therapy vector, the human mitochondrial genome has been dubbed unclonable in E. coli, due to regions of instability in the D-loop and tRNA(Thr) gene. We tested multi- and single-copy vector systems for cloning human mitochondrial DNA in E. coli and Saccharomyces cerevisiae, including transformation-associated recombination. Human mitochondrial DNA is unclonable in E. coli and cannot be retained in multi- or single-copy vectors under any conditions. It was, however, possible to clone and stably maintain the entire human mitochondrial genome in yeast as long as a single-copy centromeric plasmid was used. D-loop and tRNA(Thr) were both stable and unmutated. This is the first report of cloning the entire human mitochondrial genome and the first step in developing a gene delivery vehicle for human mitochondrial gene therapy.
    Pharmaceutical Research 07/2011; 28(11):2863-70. · 4.74 Impact Factor
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    ABSTRACT: Over the first decade of this new millennium gene therapy has demonstrated clear clinical benefits in several diseases for which conventional medicine offers no treatment. Clinical trials of gene therapy for single gene disorders have recruited predominantly young patients since older subjects may have suffered irrevocablepathological changes or may not be available because the disease is lethal relatively early in life. The concept of fetal gene therapy is an extension of this principle in that diseases in which irreversible changes occur at or beforebirth can be prevented by gene supplementation or repair in the fetus or associated maternal tissues. This article ccnsiders the enthusiasm and skepticism held for fetal gene therapy and its potential for clinical application. It coversa spectrum of candidate diseases for fetal gene therapy including Pompe disease, Gaucher disease, thalassemia, congenital protein C deficiency and cystic fibrosis. It outlines successful and not-so-successful examples of fetal gene therapy in animal models. Finally the application and potential of fetal gene transfer as a fundamental research tool for developmental biology and generation of somatic transgenic animals is surveyed.
    Therapeutic delivery 04/2011; 2(4):461-9.
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    ABSTRACT: We have previously described the development of a scaffold/matrix attachment region (S/MAR) episomal vector system for in vivo application and demonstrated its utility to sustain transgene expression in the mouse liver for at least 6 months following a single administration. Subsequently, we observed that transgene expression is sustained for the lifetime of the animal. The level of expression, however, does drop appreciably over time. We hypothesised that by eliminating the bacterial components in our vectors, we could improve their performance since bacterial sequences have been shown to be responsible for the immunotoxicity of the vector and the silencing of its expression when applied in vivo. We describe here the development of a minimally sized S/MAR vector, which is devoid of extraneous bacterial sequences. This minicircle vector comprises an expression cassette and an S/MAR moiety, providing higher and more sustained transgene expression for several months in the absence of selection, both in vitro and in vivo. In contrast to the expression of our original S/MAR plasmid vector, the novel S/MAR minicircle vectors mediate increased transgene expression, which becomes sustained at about twice the levels observed immediately after administration. These promising results demonstrate the utility of minimally sized S/MAR vectors for persistent, atoxic gene expression.
    Journal of Molecular Medicine 02/2011; 89(5):515-29. · 4.77 Impact Factor
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    ABSTRACT: The ideal gene therapy vector should enable persistent expression without the limitations of safety and reproducibility. We previously reported that a prototype plasmid vector, containing a scaffold matrix attachment region (S/MAR) domain and the luciferase reporter gene, showed transgene expression for at least 6 months following a single administration to MF1 mice. Following partial hepatectomy of the animals, however, we found no detectable vector replication and subsequent propagation in vivo. To overcome this drawback, we have now developed an in vivo liver selection strategy by which liver cells transfected with an S/MAR plasmid are provided with a survival advantage over non-transfected cells. This allows an enrichment of vectors that are capable of replicating and establishing themselves as extra-chromosomal entities in the liver. Accordingly, a novel S/MAR plasmid encoding the Bcl-2 gene was constructed; Bcl-2 expression confers resistance against apoptosis-mediated challenges by the Fas-activating antibody Jo2. Following hydrodynamic delivery to the livers of mice and frequent Jo2 administrations, we demonstrate that this Bcl-luciferase S/MAR plasmid is indeed capable of providing sustained luciferase reporter gene expression for over 3 months and that this plasmid replicates as an episomal entity in vivo. These results provide proof-of-principle that S/MAR vectors are capable of preventing transgene silencing, are resistant to integration and are able to confer mitotic stability in vivo when provided with a selective advantage.
    Gene therapy 01/2011; 18(1):82-7. · 4.75 Impact Factor
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    ABSTRACT: The cellular and molecular environment present in the fetus and early newborn provides an excellent opportunity for effective gene transfer. Innate and pre-existing anti-vector immunity may be attenuated or absent and the adaptive immune system predisposed to tolerance towards xenoproteins. Stem cell and progenitor cell populations are abundant, active and accessible. In addition, for treatment of early lethal genetic diseases of the nervous system, the overarching advantage may be that early gene supplementation prevents the onset of irreversible pathological changes. Gene transfer to the fetal mouse nervous system was achieved, albeit inefficiently, as far back as the mid-1980s. Recently, improvements in vector design and production have culminated in near-complete correction of a mouse model of spinal muscular atrophy. In the present article, we review perinatal gene transfer from both a therapeutic and technological perspective.
    Biochemical Society Transactions 12/2010; 38(6):1489-93. · 2.59 Impact Factor
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    ABSTRACT: Safely targeting the fetal gastrointestinal tract during early gestation is essential to develop effective prenatal gene therapy for gastrointestinal diseases. In this study, we aimed to characterize the development of the fetal sheep stomach sonographically and to determine the optimum gestational age, as well as the shortterm morbidity and mortality of early-gestation ultrasound-guided intragastric injection. In experiments investigating ultrasound-guided prenatal gene therapy, we studied the size and development of the stomach of 185 sheep fetuses (33-144 days' gestational age [GA]; term is 145 days). Ultrasound-guided intragastric injection was performed in 12 fetuses at 55-62 days' GA and postmortem examinations were performed 48 hours later. The stomach was not visible at or before 40 days' GA, but it was seen in all fetuses at 55 days' GA or more. The anteroposterior, transverse and longitudinal diameters of the stomach increased in a quasi-linear fashion throughout gestation. Intragastric injection was successful in 10 out of the 11 fetuses (91%) injected at 60-62 days' GA, with nine fetuses (91%) surviving this procedure. In the early-gestation sheep fetus, ultrasound-guided intragastric injection has a good success rate with a low short-term mortality and morbidity.
    Taiwanese journal of obstetrics & gynecology 03/2010; 49(1):23-9.
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    ABSTRACT: Coronary artery diseases (CAD) inflict a heavy economical and social burden on most populations and contribute significantly to their morbidity and mortality rates. Low-density lipoprotein receptor (LDLR) associated familial hypercholesterolemia (FH) is the most frequent Mendelian disorder and is a major risk factor for the development of CAD. To date there is no cure for FH. The primary goal of clinical management is to control hypercholesterolaemia in order to decrease the risk of atherosclerosis and to prevent CAD. Permanent phenotypic correction with single administration of a gene therapeutic vector is a goal still needing to be achieved. The first ex vivo clinical trial of gene therapy in FH was conducted nearly 18 years ago. Patients who had inherited LDLR gene mutations were subjected to an aggressive surgical intervention involving partial hepatectomy to obtain the patient's own hepatocytes for ex vivo gene transfer with a replication deficient LDLR-retroviral vector. After successful re-infusion of transduced cells through a catheter placed in the inferior mesenteric vein at the time of liver resection, only low-level expression of the transferred LDLR gene was observed in the five patients enrolled in the trial. In contrast, full reversal of hypercholesterolaemia was later demonstrated in in vivo preclinical studies using LDLR-adenovirus mediated gene transfer. However, the high efficiency of cell division independent gene transfer by adenovirus vectors is limited by their short-term persistence due to episomal maintenance and the cytotoxicity of these highly immunogenic viruses. Novel long-term persisting vectors derived from adeno-associated viruses and lentiviruses, are now available and investigations are underway to determine their safety and efficiency in preparation for clinical application for a variety of diseases. Several novel non-viral based therapies have also been developed recently to lower LDL-C serum levels in FH patients. This article reviews the progress made in the 18 years since the first clinical trial for gene therapy of FH, with emphasis on the development, design, performance and limitations of viral based gene transfer vectors used in studies to ameliorate the effects of LDLR deficiency.
    International Archives of Medicine 01/2010; 3:36. · 1.08 Impact Factor
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    ABSTRACT: The clinical application of gene therapy has become a reality with the treatment of patients with X-linked SCID (SCID-X1) using a modified retrovirus. This success has been tempered by the toxicity of the vector used in this trial, which led to oncogenesis in several of the treated patients. The development of safer, alternative vectors, which remain episomal and are therefore less genotoxic, is currently an area of active research. Notable recent developments include the application of modified lentiviral vectors, which stably express transgenes without the risk of integration; plasmid vectors, which exist episomally and are persistently expressed in the livers of mice; and the generation of replicating artificial chromosomes containing genomic loci. In addition, knowledge of the molecular mechanisms of nuclear retention and replication of the transgene is improving and will facilitate further developments in the use of episomal DNA for the genetic modification of cells. This review describes the development and application of gene therapy vectors, with a focus on those that are specifically designed to avoid integration and exist episomally.
    Current opinion in molecular therapeutics 09/2009; 11(4):433-41. · 3.42 Impact Factor
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    ABSTRACT: Fetal gene therapy for cystic fibrosis may have advantages over adult treatment. We aimed (1) to develop a percutaneous ultrasound guided technique to inject adenoviral vectors into the fetal sheep trachea and (2) to improve transgene expression with transduction enhancing agents. Adenoviral vectors containing the β-galactosidase gene (2.0×1011–8.3×1011 particles/kg) and transduction enhancing agents were delivered to the trachea via a needle inserted through the thorax of late (n = 3) or mid gestation (n = 15) fetal sheep using ultrasound guidance. Tissues were analysed 48 hours after injection (n = 17) or 12 hours after birth (n = 1) for transgene expression. Transthoracic injection of the trachea was successful in 16 fetuses with 100% survival. Expression of β-galactosidase, as measured by ELISA, was low after delivery of adenoviral vector alone, but increased 10-fold when the vector was complexed with the polycation DEAE dextran. Pretreatment of the fetal airways with sodium caprate, which opens tight junctions to reach the basolateral surface of lung epithelia, resulted in a 90-fold increase in expression. A synergistic effect of the two agents resulted in widespread staining of the trachea, main bronchi and all airways, confirmed by immunohistochemistry for β-galactosidase. Instillation of perflubron following sodium caprate and vector/DEAE dextran complex injection increased transduction of the peripheral small airways at the expense of tracheal and large airways transduction.
    Journal of Obstetrics & Gynaecology. 07/2009; 23(s1).
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    ABSTRACT: An ideal gene therapy vector should enable persistent transgene expression without limitations of safety and reproducibility. Here we report the development of a non-viral episomal plasmid DNA (pDNA) vector that appears to fulfil these criteria. This pDNA vector combines a scaffold/matrix attachment region (S/MAR) with a human liver-specific promoter (alpha1-antitrypsin (AAT)) in such a way that long-term expression is enabled in murine liver following hydrodynamic injection. Long-term expression is demonstrated by monitoring the longitudinal luciferase expression profile for up to 6 months by means of in situ bioluminescent imaging. All relevant control pDNA constructs expressing luciferase are unable to sustain significant transgene expression beyond 1 week post-administration. We establish that this shutdown of expression is due to promoter methylation. In contrast, the S/MAR element appears to inhibit methylation of the AAT promoter thereby preventing transgene silencing. Although this vector appears to be maintained as an episome throughout, we have no evidence for its establishment as a replicating entity. We conclude that the combination of a mammalian, tissue-specific promoter with the S/MAR element is sufficient to drive long-term episomal pDNA expression of genes in vivo.
    Gene therapy 12/2008; 15(24):1593-605. · 4.75 Impact Factor
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    ABSTRACT: In vivo bioimaging of transgenic luciferase in the lung and nose is an expedient method by which to continually measure expression of this marker gene after gene transduction. Its substrate, luciferin, is typically injected into the peritoneal cavity before bioimaging. Here we demonstrate that, compared with intraperitoneal injection, intranasal instillation of luciferin confers approximately an order of magnitude increase in luciferase bioluminescence detection in both lung and nose. This effect was observed after administration of viral vectors based on adenovirus type 5, adeno-associated virus type 8, and gp64-pseudotyped HIV lentivirus and, to a lesser extent, after nonviral polyethylenimine (PEI)-DNA delivery. Detection increased relative to the concentration of luciferin; however, a standard concentration of 15 mg/ml was well beyond the saturation point. Compared with intraperitoneal injection, intranasal instillation yields about a 10-fold increase in sensitivity with an approximate 30-fold reduction in luciferin usage when bioimaging in the nasal and pulmonary airways of mice.
    Human gene therapy 11/2008; 19(10):1050-6. · 4.20 Impact Factor
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    ABSTRACT: In vivo bioimaging of transgenic luciferase in the lung and nose is an expedient method of continually measuring expression of this marker gene after gene transduction. Standardly, its substrate, luciferin, is injected into the peritoneal cavity prior to bioimaging. Here we demonstrate that, compared with intra-peritoneal injection, intra-nasal instillation of luciferin confers approximately an order of magnitude increase in luciferase bioluminescence detection in both lung and nose. This effect was observed following administration of viral vectors based on adenovirus-5, AAV8 and gp64-pseudotyped HIV lentivirus and, to a lesser extent, after non-viral PEI DNA delivery. Detection increased relative to the concentration of luciferin however a standard concentration of 15 mg/ml was well beyond the saturation point. Compared with intra-peritoneal injection, intra-nasal instillation yields around a ten-fold increase in sensitivity with an approximate thirty-fold reduction in the luciferin usage when bioimaging in the nasal and pulmonary airways of mice.
    Human gene therapy 09/2008; · 4.20 Impact Factor
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    ABSTRACT: Due to its early onset and severe prognosis, cystic fibrosis (CF) has been suggested as a candidate disease for in utero gene therapy. In 1997, a study was published claiming that to how transient prenatal expression of CF transmembrane conductance regulator (CFTR) from an in utero-injected adenovirus vector could achieve permanent reversal of the CF intestinal pathology in adult CF knockout mice, despite the loss of CFTR transgene expression by birth. This would imply that the underlying cause of CF is a prenatal defect for which lifelong cure can be achieved by transient prenatal expression of CFTR. Despite criticism at the time of publication, no independent verification of this contentious finding has been published so far. This is vital for the development of future therapeutic strategies as it may determine whether CF gene therapy should be performed prenatally or postnatally. We therefore reinvestigated this finding with an identical adenoviral vector and a knockout CF mouse line (Cftr(tmlCam)) with a completely inbred genetic background to eliminate any effects due to genetic variation. After delivery of the CFTR-expressing adenovirus to the fetal mouse, both vector DNA and transgenic CFTR expression were detected in treated animals postpartum but statistically no significant difference in survival was observed between the Cftr(-/-) mice treated with the CFTR-adenovirus and those treated with the control vector.
    Molecular Therapy 06/2008; 16(5):819-24. · 7.04 Impact Factor

Publication Stats

3k Citations
717.72 Total Impact Points

Institutions

  • 1993–2012
    • Imperial College London
      • • Section of Computational and Systems Medicine (CSM)
      • • Molecular Medicine
      • • Division of Cell and Molecular Biology
      • • Faculty of Medicine
      • • Section of Molecular Genetics & Genomics
      London, ENG, United Kingdom
  • 2011
    • The University of Manchester
      • Faculty of Medical and Human Sciences
      Manchester, ENG, United Kingdom
  • 2006–2008
    • University College London
      • Department of Haematology
      London, ENG, United Kingdom
  • 1978–2005
    • University of London
      Londinium, England, United Kingdom
  • 1998–2000
    • French Institute of Health and Medical Research
      Lutetia Parisorum, Île-de-France, France
  • 1996–1998
    • University of Barcelona
      • Departament d'Anatomia Patològica, Farmacologia i Microbiologia
      Barcelona, Catalonia, Spain
  • 1994–1996
    • St. Mary’s Hospital for Children
      New York City, New York, United States
    • Imperial College Healthcare NHS Trust
      Londinium, England, United Kingdom
  • 1992–1994
    • Max-Delbrück-Centrum für Molekulare Medizin
      Berlín, Berlin, Germany
  • 1981–1991
    • Institute of Molecular Biology
      Mayence, Rheinland-Pfalz, Germany
  • 1989
    • Oxford University Hospitals NHS Trust
      Oxford, England, United Kingdom
    • Humboldt State University
      Arcata, California, United States
  • 1987–1989
    • Humboldt-Universität zu Berlin
      • Microbiology, Molecular Biology, and Biochemistry Section
      Berlin, Land Berlin, Germany
  • 1971
    • Russian Academy of Sciences
      • Institute of Molecular Biology
      Moscow, Moscow, Russia