EMA, the European Drug Regulator Publishes Reflection paper on Quality, Non-Clinical and Clinical Issues related to the Development of Recombinant Adeno-Associated Viral Vectors

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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.
Infection with wild-type AAV is not associated with any pathogenic disease, and in the absence of a helper virus co-infection, the virus may integrate into the host cell genome or remain as an extrachromosomal form (Schnepp, 2005). In both situations the virus appears to remain latent. In-vitro studies suggest that wild-type viral DNA integration can occur occasionally in a site specific manner (19q13.3) (Kotin, 1990 and 1991 and 1992), but only at very high multiplicities of infection (Hüser, 2002), and this was originally considered to be a safety feature of vectors derived from this virus. However, it has been subsequently shown that site specific integration is dependent on the presence of both the inverted terminal repeats (ITR) and the Rep gene products (Weitzman, 1994; Linden, 1996), the latter of which is not present in rAAV; as such the site specific integration feature of these vectors is lost. The level of integration of DNA into the cellular chromosome in in-vivo models, however remains contentious. Nonetheless, long term protein expression (in-vivo) from the gene of interest inserted into rAAV vectors has been observed (Flotte, 1993; Kaplitt, 1994; Conrad, 1996; Monahan, 1998; Donahue, 1999; Stieger, 2006), even in the absence of identifiable genetic integration (Miller, 2004; Song, 2004; Flotte, 1994). This persistence is thought to be derived from stable concatemerized duplex genome forms (circular or linear molecules) that are transcriptionally active (Duan, 1998; Yang, 1999; Fisher, 1997).
Examples of diseases studied include haemophilia B (Manno, 2006 and 2003), cystic fibrosis (Flotte, 2003), Parkinson’s disease (Kaplitt, 2007), rheumatoid arthritis (www.targen.com [tgAAC94]), Leber’s congenital amaurosis (Bainbridge, 2008; Maguire, 2008; Jacobson, 2006), infantile neuronal ceroid lipofuscinosis (Worgall, 2008) and muscular dystrophy (Xiao, 2000). Furthermore non-clinical studies indicate rAAV expressing heterologous antigenic sequences (HPV16 – Kuck, 2006; HIV – Xin, 2001 and 2002; SIV – Johnson, 2005; malaria – Logan, 2007) can illicit both humoral and cellular immune responses, and modest immunogenicity has been reported in a phase I/II study using rAAV2 encoding HIV antigens (Mehendal, 2008). However, it has been suggested that cellular responses to the transgene products of rAAV vectors may be impaired (Lin, 2007), as such the utility of these vectors when used for prophylactic purposes needs further investigation.
There are currently 6 confirmed serotypes of adeno-associated virus (AAV-1 to -6) and 2 tentative species (AAV-7 and (source: International Committee on Taxonomy of Viruses [ICTV]). However there are a number of publications describing additional serotypes (i.e. 9 and 10) which are currently not recognized by the ICTV. It is likely therefore, that there are significantly more serotypes circulating that have currently not been formally identified or recognized (Pacak, 2006; Limberis, 2006; Gao, 2004). Nonetheless, the majority of the 67 clinical trials undertaken to date using rAAV for gene delivery have used serotype 2 (Gene Therapy Clinical Trials Worldwide. J. Gene Med. March 2009 Update, http://www.wiley.co.uk/genmed/clinical ).
Evidence is accumulating which suggests that different AAV serotypes may have different tissue tropisms, for example AAV-8 is suggested to have a preferred tropism to the liver (Davidoff, 2005), while for AAV-1, -6 and -7 the preferred tropism is to skeletal muscle (Duan, 2001; Chao, 2000), AAV-4 is highly specific to the retinal pigmented epithelial cells in several animal species (Weber, 2003) and the ependymal cells (Zabner, 2000) and AAV-9 is described as being tropic to cardiacmuscle (Pacak, 2006), thought it also tranduces liver (Van den Driessche, 2007) and brain (Foust, 2009). Vectors based on these serotypes, in-vitro selected AAV with altered tropisms and hybrid vectors (i.e. ITR and Rep from AAV-2, Cap (protein coat) from another serotype i.e. are being investigated (in-vitro and in animal models) to evaluate further the utility of the preferred tropisms and their potential for avoiding pre-existing immunity to AAV-2.
A new development in the field of AAV vectors is the use of self complementary (sc) AAV. Conventional rAAV vectors require 2nd strand synthesis before genes can be expressed, and it is theorized that scAAV bypass this step by delivering a duplex genome. This is achieved by deleting the nicking site of one ITR so that it no longer serves as a replication origin but still forms an AAV hairpin structure. The result is a single stranded, dimeric inverted repeat genome with the altered ITR sequence situated in the middle of the molecule and a wild-type ITR at each end. Following infection and uncoating, the DNA is folded to form a double stranded molecule. A closed hairpin end is formed from the altered ITR, and an open end formed from the two wild-type ITR’s, thus mimicking the structure of a single stranded rAAV after 2nd strand synthesis (McCarty, 2003). It is anticipated that such vectors will improve transduction efficiency and improve the level of protein expression from the transgene. The coding capacity of these vectors, however, is reduced by a factor of two.
Given the basic biology of the ‘parent’ virus as described above, 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 and these are discussed further below, however the basic functional requirements for manufacture are:
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The AAV ITR’s flanking the ‘gene of interest’ (this construct contains the cis elements necessary for packaging and replication of its single stranded DNA genome).
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Genetic sequences (Rep and Cap) necessary for AAV replication and viral capsid proteins (generally provided in trans within a plasmid or in a packaging cell line).
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Helper virus functions: either co-infection of the helper virus or co-transfection/infection of a plasmid/chimeric virus encoding the helper genes (adenovirus: E1a/1b, E2a, E4orf6, VA1 RNA; herpes simplex virus: UL5, UL8, UL52 and UL29).
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A cell line capable of supporting helper virus and AAV replication.
The aim of this paper is to discuss quality, non-clinical and clinical issues that should be considered during the development of medicinal products derived from AAV, and to indicate requirements that might be expected the time of a market authorisation application (MAA). The issues raised are specific only to the development of rAAV vectors as medicinal products; general requirements for MAA are not within the scope of this paper. It is recommended that this paper is read in conjunction with the guidance documents referenced in section 4.2.

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