Current Gene Therapy, Vol. 3, No. 6, 2003
Contents
Biosafety
of Virus-Derived Vectors
Guest Editor: William Moens
A
Short Course on Virology / Vectorology / Gene Therapy Pp. 495-499
Zeger Debyser
Biosafety
of Onco-Retroviral Vectors Pp. 501-515
Thierry VandenDriessche, Desire Collen and Marinee K.L.
Chuah
Biosafety
of Lentiviral Vectors Pp. 517-525
Zeger Debyser
Biosafety
of Adenoviral Vectors Pp. 527-543
Marinee K.L. Chuah, Desire Collen and Thierry
VandenDriessche
Evaluation
of Risks Related to the Use of Adeno-Associated Virus-Based Vectors Pp.
545-565
L. Tenenbaum, E. Lehtonen and P.E. Monahan
Risk
Assessment of the Use of Autonomous Parvovirus-Based Vectors Pp. 567-582
Francis Dupont
The
Uses of Poxviruses as Vectors Pp. 583-595
A. Vanderplasschen and P.-P. Pastoret
Biosafety
of Herpesvirus Vectors Pp. 597-611
S. Gogev, F. Schynts, F. Meurens, I. Bourgot and E. Thiry
Abstracts
[Back to top] A Short Course
on Virology / Vectorology / Gene Therapy
Zeger Debyser
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.
[Back to top] Biosafety of
Onco-Retroviral Vectors
Thierry VandenDriessche, Desire Collen and Marinee K.L. Chuah
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 high-
capacity 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.
[Back to top] Biosafety of
Lentiviral Vectors
Zeger Debyser
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.
[Back
to top] Biosafety of Adenoviral Vectors
Marinee K.L. Chuah, Desire
Collen and Thierry VandenDriessche
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 high- capacity 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.
[Back to top] Evaluation of Risks Related to the Use of
Adeno-Associated Virus-Based Vectors
L. Tenenbaum, E. Lehtonen and
P.E. Monahan
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.
[Back to top] Risk Assessment of the Use of Autonomous Parvovirus-Based
Vectors
Francis Dupont
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.
[Back to top] The Uses of Poxviruses
as Vectors
A. Vanderplasschen and P.-P. Pastoret
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.
[Back to top] Biosafety of Herpesvirus Vectors
S. Gogev, F. Schynts, F. Meurens, I. Bourgot and E. Thiry
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.