The EMEA today released a reflection paper regarding Quality, non-clinical and clinical issues relating specifically to recombinant adeno-associated viral vectors. It is still open for comment until September 2009.
Recombinant adeno-associated viral (rAAV) vectors are derived from the single stranded DNA virus adeno-associated virus which belongs to the genus dependovirus within the Parvoviridae family. As the name suggests the wild type virus is incapable of independent replication and relies on co-infection of a helper virus to enable a lytic replication cycle (Gonclaves, 2005). Adenovirus (Ad), herpes simplex virus (HSV), pseudorabies virus (PrV) and human papilloma virus (HPV) are known to support wild type AAV replication.
Given the basic biology of the ‘parent’ virus, the methods for manufacture and quality control of product are complicated, and the long-term fate of the administered vector is at present unknown. There are a number of manufacturing strategies that can be used to produce rAAV vectors.
The aim of this reflection paper is to discuss quality, non-clinical and clinical issues that are specific only to the development of rAAV vectors as medicinal products. The paper goes on to discuss in some details the different manufacturing methodologies that can be used to generate rAAV including:
- Virus containing production systems (helper virus & hybrid vectors)
- Virus-Free production systems (tri-plasmid transfection & packaging cell lines)
- Self-complementary adeno-associated virus
There are a number of quality considerations that are specific to these systems, from the standard issues of cell banking, seed stock and qualified cell lines, virus origine and stock control/testing. There are also a number of specific issues to be considered:
Virus Containing Production Systems: The main disadvantage of this system is the potential for contamination of the product with the helper/hybrid virus,and strategies for dealing with this are described. “It is recommended that a quality specification for the helper/hybrid virus is set, and the testing strategy detailed in the Ph. Eur. (Monograph 5.14 Gene Transfer Medicinal Products for Human use) can be used for guidance in defining an appropriate testing program. In particular, if the helper/hybrid virus is considered to be replication incompetent, the specification should include a test for replication-competent virus contamination.”
Virus Free Production Systems: Limitations of a manufacturing approach that relies solely on plasmid transfection lie in the difficulties of process scale up and the consistency of manufacture due to the inherent variability of the transfection process itself. However, the advantage of such an approach is that the quality of the final product is improved as there will be no contamination of the product with a helper/hybrid virus. It is recommended that the transfection conditions are thoroughly evaluated and optimized at each scale of manufacture to assure consistency in product quality and yield. Following each manufacturing change product characterization should be undertaken to assure that the introduced changes do not impact on product quality. Furthermore, the purification process should be sufficiently robust to assure removal of excess plasmid from the final product. Quality issues specific to packaging cell lines are identical for those used to manufacture recombinant proteins in that the genetic stability of construct should be shown, at or beyond the expected number of population doublings required for manufacture.
Issues of Non-Clinical Evaluation
Choice of Animal Model – AAV is a species specific virus, therefore it is possible that the biodistribution of a human serotype derived vector in a mouse or rat may not correlate to that when administered to man as cellular/organ uptake may be different as a result of differences in, or differential expression of, the receptor used for entry. A number of animal species have been used in non-clinical evaluation of rAAV vectors (rats, mice, rhesus monkey, non-human primates, dogs, cats and pigs); however it is not clear which is the most appropriate model to use, and it may be necessary for more than one species to be used to complete a full non-clinical development program. Given these difficulties there may be scientific justification for using in pivotal non-clinical studies, a serotype of virus that is specific to the animal model of choice, rather than the human serotype that will be used in clinical studies. Such studies may provide more useful information in relation to biodistribution and the impact of pre-existing immunity to the vector to it.
Vector Persistance – The safety of rAAV in terms of insertional mutagenesis is still under debate following a recent publication where an increased rate of hepatocellular carcinoma was observed in neonatal mice treated with a rAAV (Donsante, 2007). While this study is not definitive in confirming the oncogenic potential of these vectors (Kay, 2007), the implications of the study can not be ignored, and the level of integration of the vector under investigation should be evaluated. Non-clinical studies should be considered which are designed to investigate how long-term gene expression is expected to be achieved i.e. episomal or integration.
Tissue Tropism – Different serotypes of AAV have been associated with specific tissue tropisms, for example AAV 1, 6 and 7 are effective at transducing muscle cells; serotype 9 preferentially transducing the myocardium and AAV 5 is suggested to be more tropic to the airway epithelium and the central nervous system (at least in the mouse model). This preferential transduction activity does not mean however, that the vector is not distributed to other organs. It is possible therefore, that tissue tropism defined non-clinically may not be observed following administration to humans, and it is recommended that a cautious approach is taken when translating non-clinical data to humans.
Reactivation of Productive Infection – When developing rAAV vectors as medicinal products the consequence of long-term episomal maintenance and the potential for re-activation of virus if the subject is infected with both wild-type AAV and a helper virus should be considered. Where possible or relevant, this should be investigated in non-clinical studies such as those described by Afione et al (Afione, 1996). Associated treatment during clinical studies i.e. chemotherapy, immuno-suppression, anti-inflammatory medicines, may also impact on virus biodistribution and maybe even the likelihood of viral reactivation, particularly if immuno-suppression is being given. Where possible these additional treatments should be addressed during non-clinical evaluation of the product.
Germ-line transmission – Biodistribution studies have shown in the mouse and the rat that rAAV DNA can be detected in gonadal DNA (Arruda, 2001) for a variable duration. Furthermore following hepatic artery delivery of a rAAV for the treatment of hemophilia B, transient dissemination to the semen in 1 patient was observed (Schuettrumpt, 2006). The potential for germ-line transmission can not therefore be entirely ruled out (Honaramooz, 2008), as such it is recommended that germ-line transmission studies are undertaken prior to first in man studies.
Environmental risk Considerations
There is a substantial amount of literature available suggesting that shedding of rAAV is dependent on the dose and route of administration, and that vector DNA can be detected for a number of weeks in serum, and early times i.e. day 1 post administration, in saliva, serum, urine and semen (Favre, 2001; Manno, 2006; Provost, 2005). Ideally, if positive DNA signals are observed, the samples should be followed up for infectious virus quantification. The data derived from non-clinical shedding studies and from early phase clinical studies can then be used to assess the likelihood of transmission and to justify the extent of viral shedding evaluation in subsequent trials.
Clinical Considerations
Biodistribution and shedding studies – The extrapolation of biodistribution data from animal models to humans is not straight forward. It is recommended that wherever possible an investigation into the biodistribution of the vector, by screening for DNA sequences in the first instance, should be included within a clinical trial protocol is included.
Immunogenicity – Equally the extrapolation of immunogenicity data for therapeutic applications of AAV vectors from animal models to humans is not simple, and the route of administration may also impact on the immunogenic profile of the product. It is recommended therefore that consideration is given to the potential of subjects having pre-existing antibodies to the serotype of AAV under investigation, and that evaluation of the immunogenicity of both the vector and the transgene is assessed in terms of neutralizing and non-neutralizing antibody formation during clinical trials
Germ-line transmission – The question of germ-line transmission in humans has not been fully resolved and short term DNA persistence has been observed in semen (serotype 2), therefore it is recommended that germline transmission is investigated during clinical studies and that the use of barrier contraception for individuals enrolled in clinical trials is included in study protocols.
Long-term follow up – Non-clinical studies may indicate long-term persistence of the vector, be it due to viral DNA integration or episomal maintenance, in which case long-term follow-up of the patients treated with a rAAV product could be necessary, not only in terms of safety evaluation but also efficacy. It should also be considered that where these vectors are being investigated for preventive vaccination uses, long term expression of the antigenic proteins may be a safety risk rather than a desired outcome.
The paper goes into greater detail on these issues and requires detailed consideration for those working in this field.
If you would like more detail in this area please get in touch with Damien Bové damien.bove@idaconsultants.com
Damien Bové works as a drug development consultant (pharmaceutical or biotechnology) and regulatory consultant, we work with our clients to define a drug development target, define a drug development strategy, define a regulatory strategy or define a commercial strategy. Our clients are generally raising funds or looking to license out their technology and we help them achieve it. If you want to know more don’t hesitate to get in touch.
