Adeno-associated viruses (AAVs) are small (26 nm in diameter) replication-defective, non-enveloped viruses with a linear single-stranded DNA (ssDNA) genome of 4.8 kilobases. They belong to the genus Dependoparvovirus, which in turn belongs to the family Parvoviridae [1]. The genome encodes for viral replication, packaging, and capsid assembly proteins, flanked by two inverted terminal repeats (ITRs) [2]. There are at least 13 different AAV subtypes which have different tissue tropisms due to differing interactions with their receptor [3]. In 2012, the first gene therapy product, Glybera, which is based on an AAV gene delivery approach for the treatment of patients with lipoprotein lipase deficiency was approved in Europe and today, AAV vectors are well established in clinical trials for in vivo gene therapy [4]. In 2019, 288 AAV-based clinical trials were registered with the US FDA database [5]. AAV vectors are commonly used due to high efficacy of transduction and a high safety profile. They are characterized by a broad tropism, low immunogenicity, ease of production, they are non-pathogenic, rarely integrates into the host chromosome and results in long-term expression of the transgene [4].
Naturally occurring AAVs infect both dividing and non-dividing cells in humans and animals and can remain latent in the host in a circular DNA episomal form, although integration in the host could occur, but it is very infrequent. Infection with AAVs could result in the production of neutralizing antibodies (NAbs), and these can interfere with treatment based on recombinant AAVs vectors.
The Göttingen Minipigs is an attractive large animal model in many clinical areas due to the high similarity in anatomy, physiology, and immunology to humans [6-8]. Göttingen Minipigs are smaller in size compared to other pig breeds, making them more manageable in a laboratory setting. Their cardiovascular, respiratory, digestive, and renal systems, as well as metabolic functions, are closer to those of humans compared to other animal models. Anatomical similarities are important in studies that involve surgical procedures, medical device testing, or the development of surgical techniques. Göttingen Minipigs can be used to model human diseases such as cardiovascular diseases, diabetes, obesity, and metabolic disorders. Because of the similarities of the porcine and human cardiovascular system, Göttingen Minipigs are a widely used as large animal model for cardiovascular diseases [9-11]. This allows preclinical testing of new therapies and interventions. Since Göttingen Minipigs are bred under controlled conditions, a high reproducibility of experimental results can be achieved. Knowledge of AAV prevalence in Göttingen Minipigs is thus relevant when considering this species for gene therapy studies. In the best case, animals free of AAVs can be selected and used.
In a study by Rapti et al. [12], the prevalence of antibodies against several AAV serotypes were assessed in a variety of small and large animal models (excluding non-human primates, NHP). The antibody titers varied widely between species and between serotypes. Rats displayed the lowest level of NAbs, whereas porcine sera strongly inhibited all AAV serotypes and displayed the largest variation between individual animals [12]. When Göttingen Minipigs were injected with an AAV1 vector containing the coding sequence of the sarcoplasmatic reticulum ATPase (SERCA2a), the results showed that the vector genome copies in the heart were lower in the animals with pre-existing neutralizing antibodies 2 days post injection, demonstrating that even low NAbs against AAV vectors can have a negative transduction effect in vivo [11]. The wide prevalence of cross-reactive antibodies against AAV in different animal species, including pigs, and AAVs that have been isolated from pigs [12-14] provide a potential explanation for the presence of neutralizing antibodies in the sera of minipigs.
In another study, 3 strains of pigs (Norsvin Topigs-20 strain, Yucatan minipigs, and Göttingen Minipigs) were used to assess pre-existing antibodies against several AAV capsids, including AAV1, AAV2, AAV5, AAV6, AAV8 and AAV9 [16]. Although, the results for the 3 strains cannot be compared because of the intrinsic strain differences, and also the different ages used in this study, overall, the results showed a low prevalence of antibodies against most of the serotypes, except for AAV2. As a matter of fact, in some cases, for example, the Norsvin Topigs-20 strain, the seroprevalence of antibodies against AAV2 was very high. Interestingly, when the titers were defined in those positive animals, the Göttingen Minipigs showed the lower titer (less than 1:20), while Topigs-20 and Yucatan had 1:80-1:320 and 1:160-1:640, respectively. This study demonstrated that the minipigs strains used in this study could be used as animal models for gene therapy studies when using the AAV1, AAV5, AAV8 and the AAV9 serotypes. It was also demonstrated a prevalence of antibodies to multiple serotypes, suggesting that there has been exposure to multiple viruses or that the pre-existing antibodies cross react with multiple serotypes. These results also suggest that prescreening on circulating anti-AAV antibodies could be helpful before inclusion of pigs into gene therapy studies.
In addition to gene therapy, knowledge of AAVs in Göttingen Minipigs may be relevant for xenotransplantation where the patient typically undergoes significant immunosuppression. Göttingen Minipigs were considered to be used as donor animals for islet cells in order to treat diabetic patients. Therefore, these animals were carefully screened for different xenotransplantation-relevant viruses [17-23]. AAV were not screened until now in Göttingen Minipigs, and they are not included into the list of viruses “not permitted in swine with designated pathogen-free status” to be used in xenotransplantation published by Fishman [24]. However, porcine adenovirus (pAdV) is included in this list. Although AAVs are thought to be non-pathogenic, it is unclear whether these viruses may induce diseases in humans [25], and whether they pose a risk for xenotransplantation. It cannot be excluded that transfer of AAVs from the donor pig may result in disease in the recipient. Here we analyzed Göttingen Minipigs breed at Ellegaard Göttingen Minipigs in Denmark and at Marshall BioResources in the USA for neutralizing and binding (total IgG) antibodies against AAV to evaluate the seroprevalence against 4 serotypes.