Current Gene Therapy (v.13, #6)

General Considerations on the Biosafety of Virus-derived Vectors Used in Gene Therapy and Vaccination by Aline Baldo, Eric van den Akker, Hans E. Bergmans, Filip Lim, Katia Pauwels (385-394).
This introductory paper gathers general considerations on the biosafety of virus-derived vectors that are used inhuman gene therapy and/or vaccination. The importance to assess the potential risks for human health and the environmentrelated to the use of genetically modified organisms (GMO) in this case genetically modified viral vectors is highlightedby several examples. This environmental risk assessment is one of the requirements within the European regulatoryframework covering the conduct of clinical trials using GMO. Risk assessment methodologies for the environmentalrisk assessment of genetically modified virus-derived vectors have been developed.

Environmental Risk Assessment of Replication Competent Viral Vectors Applied in Clinical Trials: Potential Effects of Inserted Sequences by Eric van den Akker, Cecile J.B. van der Vlugt, Diederik A. Bleijs, Hans E. Bergmans (395-412).
Risk assessments of clinical applications involving genetically modified viral vectors are carried out accordingto general principles that are implemented in many national and regional legislations, e.g., in Directive 2001/18/EC of theEuropean Union. Recent developments in vector design have a large impact on the concepts that underpin the risk assessmentsof viral vectors that are used in clinical trials. The use of (conditionally) replication competent viral vectors(RCVVs) may increase the likelihood of the exposure of the environment around the patient, compared to replication defectiveviral vectors. Based on this assumption we have developed a methodology for the environmental risk assessmentof replication competent viral vectors, which is presented in this review. Furthermore, the increased likelihood of exposureleads to a reevaluation of what would constitute a hazardous gene product in viral vector therapies, and a keen interestin new developments in the inserts used. One of the trends is the use of inserts produced by synthetic biology. In thisreview the implications of these developments for the environmental risk assessment of RCVVs are highlighted, with examplesfrom current clinical trials. The conclusion is drawn that RCVVs, notwithstanding their replication competency,can be applied in an environmentally safe way, in particular if adequate built-in safeties are incorporated, like conditionalreplication competency, as mitigating factors to reduce adverse environmental effects that could occur.

Environmental Risk Assessment of Clinical Trials Involving Modified Vaccinia Virus Ankara (MVA)-Based Vectors by Martine Goossens, Katia Pauwels, Nicolas Willemarck, Didier Breyer (413-420).
The modified vaccinia virus Ankara (MVA) strain, which has been developed as a vaccine against smallpox, issince the nineties widely tested in clinical trials as recombinant vector for vaccination or gene therapy applications. AlthoughMVA is renowned for its safety, several biosafety aspects need to be considered when performing the risk assessmentof a recombinant MVA (rMVA). This paper presents the biosafety issues and the main lessons learned from theevaluation of the clinical trials with rMVA performed in Belgium. Factors such as the specific characteristics of therMVA, the inserted foreign sequences/transgene, its ability for reconversion, recombination and dissemination in thepopulation and the environment are the main points of attention. Measures to prevent or manage identified risks are alsodiscussed.

Adenovirus vectors are the most commonly employed vector for cancer gene therapy. They are also used forgene therapy and as vaccines to express foreign antigens. Adenovirus vectors can be replication-defective; certain essentialviral genes are deleted and replaced by a cassette that expresses a foreign therapeutic gene. Such vectors are used forgene therapy, as vaccines, and for cancer therapy. Replication-competent (oncolytic) vectors are employed for cancer genetherapy. Oncolytic vectors are engineered to replicate preferentially in cancer cells and to destroy cancer cells through thenatural process of lytic virus replication. Many clinical trials indicate that replication-defective and replication-competentadenovirus vectors are safe and have therapeutic activity.

Biosafety of Recombinant Adeno-associated Virus Vectors by David J. Dismuke, Liliane Tenenbaum, R. Jude Samulski (434-452).
It is hoped that the use of gene transfer technology to treat both monogenetic and acquired diseases may soonbecome a common therapy option in medicine. For gene therapy to achieve this objective, any gene delivery method willhave to meet several criteria, including ease of manufacturing, efficient gene transfer to target tissue, long-term gene expressionto alleviate the disease, and most importantly safety in patients. Viral vectors are an attractive choice for use ingene therapy protocols due to their relative efficiency in gene delivery. Since there is inherent risk in using viruses, investigatorsin the gene therapy community have devoted extensive efforts toward reengineering viral vectors for enhancesafety. Here we review the approaches and technologies that are being evaluated for the use of recombinant vectors basedupon adeno-associated virus (AAV) in the treatment of a variety of human diseases. AAV is currently the only knownhuman DNA virus that is non-pathogenic and AAV-based vectors are classified as Risk Group 1 agents for all laboratoryand animal studies carried out in the US. Although its apparent safety in natural infection and animals appears well documented,we examine the accumulated knowledge on the biology and vectorology of AAV, lessons learned from gene therapyclinical trials, and how this information is impacting current vector design and manufacturing with an overall emphasison biosafety.

Biosafety Challenges for Use of Lentiviral Vectors in Gene Therapy by Michael Rothe, Ute Modlich, Axel Schambach (453-468).
Lentiviral vectors are promising tools for the genetic modification of cells in biomedical research and genetherapy. Their use in recent clinical trials for the treatment of adrenoleukodystrophy, β -thalassemia, Wiskott-Aldrich-Syndrome and metachromatic leukodystrophy underlined their efficacy for therapies especially in case of hereditary diseases.In comparison to gammaretroviral LTR-driven vectors, which were employed in the first clinical trials, lentiviralvectors present with some favorable features like the ability to transduce also non-dividing cells and a potentially safer insertionprofile. However, genetic modification with viral vectors in general and stable integration of the therapeutic geneinto the host cell genome bear concerns with respect to different levels of personal or environmental safety. Among them,insertional mutagenesis by enhancer mediated dysregulation of neighboring genes or aberrant splicing is still the biggestconcern. However, also risks like immunogenicity of vector particles, the phenotoxicity of the transgene and potential verticalor horizontal transmission by replication competent retroviruses need to be taken into account. This review will givean overview on biosafety aspects that are relevant to the use of lentiviral vectors for genetic modification and gene therapy.Furthermore, assay systems aiming at evaluating biosafety in preclinical settings and recent promising clinical trialsincluding efforts of monitoring of patients after gene therapy will be discussed.

Biosafety Considerations Using Gamma-Retroviral Vectors in Gene Therapy by Annette Deichmann, Manfred Schmidt (469-477).
Gene therapy has become a feasible and efficient strategy for the treatment of human genetic diseases. Themain principle of a gene therapeutic regimen relies on the delivery of a corrected gene of interest in human cells. In aboutone fifth of the clinical trials, gamma-retroviral vectors are used as gene-transfer vehicle. However, previous successfulgene therapy trials revealed gamma-retroviral vector-mediated severe adverse events: Upregulation of proto-oncogenesled to malignant transformation of the affected cells and tumor progression. These severe adverse events enhanced the developmentof new 'safer' gamma-retroviral vectors and comprehensive biosafety studies. This review highlights all possiblesafety and biosafety risks of gamma-retroviral vectors.

Biosafety of Gene Therapy Vectors Derived From Herpes Simplex Virus Type 1 by Filip Lim, Hena Khalique, Maria Ventosa, Aline Baldo (478-491).
The majority of humans have been infected with Herpes Simplex Virus Type 1 (HSV-1) and harbor its viralDNA in the latent form within neurons for lifetime. This, combined with the absence of serious adverse effects due toHSV-1 derived vectors in clinical trials so far, highlight the potential to use this virus to develop neuronal gene transfervectors which are transparent to the host, allowing the effects of the transgene to act without interference from the transfersystem eg., for functional genomics in basic neuroscience or gene therapy of neurological disorders. On the other hand,other HSV-1 derived vectors which also have a promising perspective in the clinic, are designed to have enhanced cytotoxicityin certain cell types, as in the case of oncolytic vectors. Understanding virus-host interactions is fundamental notonly to the success of these gene therapy vectors but also with respect to identifying and minimizing biohazards associatedwith their use. In this review we discuss characteristics of HSV-1 and gene therapy vectors derived from this virus whichare useful to consider in the context of biosafety risk assessment and risk management.

Biosafety of Non-Human Therapeutic Viruses in Clinical Gene Therapy by Rob C. Hoeben, Derrick Louz, Danijela Koppers-Lalic (492-499).
The concept of using viruses as oncolytic agents is not a new one. In an effort to improve the applicability ofviral anti-cancer agents various non-human viruses are being evaluated preclinically and clinically. The application of replication-competent non-human viruses poses new potential hazards, i.e. those associated with the possible adaptation ofthe therapeutic viruses to the human hosts. Therefore it is essential to weigh the potential benefits for the patients againstthe risk for the patients, their close contacts, and the greater public. Many aspects of such assessment parallel with therisks and dilemmas associated with the use of live porcine cells, tissues and organs in a clinical xenotransplantation setting.In this review we will summarize the potential biological hazards and list the points that need to be considered in aformal biosafety risk evaluation. The risk evaluation should include the possible environmental aspects of the non-humanviruses used, also in case the non-human viruses are not formally designated as genetically modified organisms.