Current Gene Therapy - Volume 3, Issue 6, 2003

Biosafety of viral vectors was first conceived as a set of guidelines for the risk assessment and management of viral vectors under various research and development situations. People from academia or industrial area concerned with basic research, gene therapy and vaccine technologies, were demanding ways to improve their daily research practices or production activities, their in vivo experiments and their clinical trials with appropriate safeguards. Such a need was originating either from safety concerns or from the necessity of compliance of pharmaceuticals and therapeutic development to international regulatory standards. Our idea was then not to evaluate conceptual risks -that nobody is able to assess anyway- but to build an coherent scientific image of the current biosafety of each type of viral vectors from the puzzle of published data and spread experiences. The project was set-up by a multidisciplinary group of experts organized by the secretariat of the Belgian Biosafety Advisory Council. Virologists, geneticists, vectors engineers, medical and veterinean users, experts familiar with clinical trials, registration of medicinal products, and post-marketing surveillance could be gathered. Most of the experts were members of safety advisory bodies in the European Union or at the level of the European Commission. “Viral vectors” are mainly engineered derivatives from eukaryotic viruses or attenuated variants thereof such as adenovirus, retrovirus, parvovirus, herpes viruses and poxviridae. Depending on the viewpoint, viral vectors are also conceptually transgenic viruses, chimerical transgenes, gene therapy vectors, vaccine vectors, or seen in innovative biotechnology as a source of future medicinal products. But viral vectors already have a long story. It started with the golden years of bacteriophages and with genetic engineering. How many students and biotechnologists today, still know the brilliant science of phage lambda, a bacterial virus, underlying the commercial packaging kits sold for the building of genomic libraries? As an example, do they know that the phage DNA sequences used in those packaging kits are restricted to the phage terminal repeats as carriers of “insert”, substrates for the encapsidation, and, in certain cases, as promoters for gene expression? Some readers may perhaps recognize or discover the amazing similarities between the classic recombinant lambda phages and some of the viral vectors described here. Whereas gene therapy vectors are intended for use with diseased animals and later on patients, life recombinant vaccines are intended for treatment of healthy people and animals. The corresponding scientific and medical literatures have few overlaps: somehow lessons from the design and use of vaccine vectors could be exploited for the design or assay of gene therapy vectors and vice-versa. Therefore, gathering “vaccine” and “gene therapy” people in a single review of risk assessment should certainly enrich both of them. Assessment of viral vectors is usually carried out after assessment of their components: the biology and ecology of gene donors and acceptors. The components of the expression cassette, the functional genetics of the construct, the natural spectrum of hosts of the construct acceptor are the main elements illustrated by the authors. The present review is the first but modest attempt to globally illustrate the various strategies of assessment at various steps of vector development and testing. One ambition of the review is to show that the risk assessment exercise may lead to unfamiliar conclusions and perhaps also show how to identify key knowledge that is missing and -in fact- should be known or demonstrated to justify claims of vector “safety”. In that sense the authors felt that biosafety assessment is not a way to block research and development but rather a responsible and qualitative way to frame it professionally. We do not know everything about the in vivo biology of viral vectors, these extra-ordinary chimerical genetic entities wherein huge amounts of human smartness and scientific culture are concentrated in an apparently innocent puzzle of DNA sequences. If the DNA sequence is indeed a program, safety can certainly be integrated in the engineered constructs. But this has limitations defined by our lack of knowledge of virus-host(s) interactions in vitro, and most certainly in vivo. However, these tools are the necessary probes to make Knowledge advance. Nowadays, industrial scales of vector production are rather impressive; The last fifteen years, the uses of virus derived genetic constructs, viral vectors, virosomes moved from academia to industries and even to production centers working under good manufacturing practices. The scale of production of these “products” evolved from titers of 10 3-10 5 pfu(particle forming units) up to those titers of 10 10 - 10 13 pfu necessary for relevant in vivo experiments. Such a suspension spread on the floor is not exactly the planned outcome of viral vectors. How do you estimate what to do as a clinician or a lab scientist should such a black day once occur? Containing the design and uses of these vectors in appropriate facilities seems obvious to everybody. Elaborating good laboratory or husbandry practices and quality procedures sounds also as logical. However, what do we do in the real world when designing a new construct, with not fully characterized or understood promoters, spacers, enhancers, terminators? What are the related precautions and decisions, what are the preventive measures, what are the training needs, how is the key art of “educated guessing” applied to viral vectors and transmitted to young scientists? The examples of assessment gathered in the present review might bring some rationale in the handling of such questions. The publication of “biosafety of viral vectors” is not just an academic exercise. Hopefully, it could trigger the appropriate criticisms, amendments and most probably a need for permanent improvements

For people starting off in the field of gene therapy, the encountered terminology is often quite confusing. Moreover, the background on basic virology may be modest. The following introduction provides a head start to any novice willing to gain more in-depth knowledge on the subject. The development of gene therapy is also addressed from a historical perspective.

Extensive gene therapy studies in preclinical models and in clinical trials underscore the relative safety of onco-retroviral vectors. Up until recently, no adverse effects have been reported in nearly 2000 patients that were enrolled in gene therapy clinical trials involving oncoretroviral vectors. However, the main safety concern of using onco-retroviral vectors is related to the risk of malignant transformation following oncogene activation due to random onco-retroviral genomic integration. Based on primate studies, there is an apparent low risk of malignancy that is predominately associated with the occurrence of chronic retroviremia resulting from replicationcompetent retroviruses (RCR), particularly in immunosuppressed recipient hosts. However, in the latest packaging cell lines and vectors, the risk of RCR-generation has been drastically reduced, primarily by minimizing the homologous overlap between vector and helper sequences. Nevertheless, results from a recent preclinical study in mice and a clinical trial in patients suffering from SCID-X1 strongly suggest that onco-retroviral vectors devoid of RCR can contribute to lymphomagenesis by insertional activation of cellular oncogenes. The risk of inadvertent germline transmission of oncoretroviral vectors appears to be low, especially relative to the endogenous rate of germline insertion, which is known to occur naturally in the human population via transmission of endogenous retro-transposons. The strict dependency of onco-retroviral gene transfer on cell division is an important safety advantage that significantly limits the risks of horizontal transmission. Since improved onco-retroviral vectors or transduction protocols may result in an increased number of retroviral integrations per cell, this may concomitantly increase the risk of malignant transformation. The use of suicide genes, self-inactivating vectors and / or chromosomal insulators is, therefore, warranted to further enhance the safety features of onco-retroviral vectors. Detailed analyses of insertion sites combined with long-term clinical follow-up may contribute to a more accurate risk assessment.

The characteristics of lentiviral vectors (stable integration in non-dividing and dividing cells, long-term expression of the transgene, absence of immune response) make them ideal gene transfer vehicula for future gene therapy. However, the most potent lentiviral vectors are derived from highly pathogenic human viruses, such as HIV. We describe how the field has engineered lentivectors with increasing biosafety both for the lab worker and for the patient. The risk associated with state-of-the-art lentivectors is therefore minimal, although a psychological barrier to use these vectors in the clinic may still have to be overcome. Due to their increased performance, care should be taken to avoid accidental transduction of the lab worker with potential hazardous genes. The precautions which have to be taken are described in detail.

Adenoviral vectors can efficiently transduce a broad variety of different cell types and have been used extensively in preclinical and clinical studies. However, early generation of adenoviral vectors retained residual adenoviral genes that contribute to inflammatory immune responses and toxicity. In addition, these vectors often result in transient expression of the potentially therapeutic transgene. Some clinical trials based on early generation adenoviral vectors have been discontinued because of acute inflammatory responses and toxicity and even one patient has died as a direct consequence of adenoviral toxicity. The latest generation of highcapacity adenoviral vectors is devoid of viral genes, and is having a significantly improved safety profile and yielding more prolonged transgene expression compared to early generation vectors. Nevertheless, transgene expression gradually declines even when high-capacity adenoviral vectors are used, possibly due to the gradual loss of vector genomes. Despite their improved safety, high-capacity adenoviral vectors can still trigger transient toxic effects in animals and patients. Restricting the tropism of adenoviral vectors by immunologic or genetic retargeting may further improve their therapeutic window. The safety of adenoviral vectors has been improved further through the development of safer packaging systems that eliminate the homologous overlap between vector and helper sequences and therefore prevent formation of replication-competent adenoviruses (RCA). RCA could exacerbate inflammatory responses and act as a helper to rescue adenoviral vectors, potentially increasing the effective vector dose. Conditionally replicating adenoviruses (CRAds) have been developed for cancer gene therapy, which replicate selectively in some cancer cells. The use of CRAds in combination with chemotherapy yielded therapeutic effects in patients suffering from cancer but dose-limiting toxicity was apparent. Although there appears to be a very low theoretical risk of malignancy that is predominately associated with the occurrence of E1-positive recombinants, no malignancies have been reported that were associated with adenoviral vectors. Nevertheless, integrating adenoviral vectors carry a greater malignancy risk due to their ability to integrate randomly into the target genomes.

Recombinant AAV efficacy has been demonstrated in numerous gene therapy preclinical studies. As this vector is increasingly applied to human clinical trials, it is a priority to evaluate the risks of its use for workers involved in research and clinical trials as well as for the patients and their descendants. At high multiplicity of infection, wild-type AAV integrates into human chromosome 19 in ∼60% of latently infected cell lines. However, it has been recently demonstrated that only approximately 1 out of 1000 infectious units can integrate. The mechanism of this site-specific integration involves AAV Rep proteins which are absent in vectors. Accordingly, recombinant AAV (rAAV) do not integrate site-specifically. Random integration of vector sequences has been demonstrated in established cell lines but only in some cases and at low frequency in primary cultures and in vivo. In contrast, episomal concatemers predominate.Therefore, the risks of insertional mutagenesis and activation of oncogenes are considered low. Biodistribution studies in non-human primates after intramuscular, intrabronchial, hepatic artery and subretinal administration showed low and transient levels of vector DNA in body fluids and distal organs. Analysis of patients body fluids revealed rAAV sequences in urine, saliva and serum at short-term. Transient shedding into the semen has been observed after delivery to the hepatic artery. However, motile germ cells seemed refractory to rAAV infection even when directly exposed to the viral particles, suggesting that the risk of insertion of new genetic material into the germ line is absent or extremely low. Risks related to viral capsid-induced inflammation also seem to be absent since immune response is restricted to generation of antibodies. In contrast, transgene products can elicit both cellular and humoral immune responses, depending on the nature of the expressed protein and of the route of vector administration. Finally, a correlation between early abortion as well as male infertility and the presence of wt AAV DNA in the genital tract has been suggested. Although no causal relationship has been established, this issue stresses the importance of using rAAV stocks devoid of contaminating replication-competent AAV. This review comprehensively examines virus integration, biodistribution, immune interactions, and other safety concerns regarding the wild-type AAV and recombinant AAV vectors.

Autonomous parvoviruses are small, non-enveloped, lytic DNA viruses replicating in the nucleus of actively dividing mammalian cells of appropriate species and tissue origins. In contrast to AAV, the other main subgroup of parvoviruses, autonomous parvoviruses do not require the assistance of an auxiliary virus for productive infection and do not stably integrate in the cellular DNA. Therefore, autonomous parvoviruses are suitable vectors for mediating transient gene transduction in dividing target cells. Interestingly, some of these viruses possess a striking inherent oncotropism, which may render them particularly suitable as selective vehicles in the clinical context of cancer gene therapy. In this chapter, we will present a brief overview of the biology of autonomous parvoviruses. This topic will be followed by a description of the design and recent developments in the production and use of parvoviral vectors, with a particular emphasis on biosafety aspects. Finally, the risk assessment related to the production and use of parvoviral vectors will be discussed in last part of the chapter.

Poxviruses have played an amazing role in the development of virology, immunology and vaccinology. In 1796, deliberate inoculation of cowpox virus to humans was proved by Dr. Edward Jenner to protect against the antigenically related smallpox virus (variola). This discovery founded the science of immunology and eventually led to smallpox eradication from the earth in 1980 after a world wide vaccination campaign with vaccinia virus (another poxvirus). Paradoxically, despite the eradication of smallpox, there has been an explosion of interest in vaccinia virus in the eighties. This interest has stemmed in part from the application of molecular genetics to clone and express foreign genes from recombinant vaccinia virus. The use of these recombinant vaccinia viruses as efficacious in vitro expression system and live vaccine has raised concerns about their safety. The work of the scientific community of the last 20 years has contributed to improve drastically the safety of poxvirus derived vectors. Firstly, the safety of vaccinia virus has been enhanced by production of genetically attenuated strains. Secondly, alternative poxvirus vectors, such as avipoxviruses, were proved to be extremely safe and efficacious non-replicating vectors when used in non avian species. In the present chapter, the basic concepts of poxvirus biology required to assess the safety of a poxvirus derived vector are provided. The principal poxvirus vectors available to date are described in regards to their biosafety.

Herpesviruses are large DNA viruses, which possess a number of advantages as gene delivery vectors. These relate to an ability to package large DNA insertions and establish lifelong latent infections in which the viral genome exists as a stable episome in the nucleus. For gene therapy to become a potential future treatment option, biosafe therapeutically efficient gene transfer is a central, but more and more stringent requirement. This review highlights the progress in development of herpesvirus based vectors, describes their properties as wall as discusses the biosafety concerns that are associated with their use in gene therapy. Thought was also given to biosafety issues pertaining to design and production of herpesvirus vector systems in therapeutic gene delivery.