In this study, we carried out DNA shuffling of AAV capsid genomes corresponding to AAV serotypes 1–9 to generate a chimeric AAV library. Until recently, attempts to elucidate structure–function attributes of the AAV capsid have primarily focused on site-directed and insertional mutagenesis, including epitope-tag or ligand-insertion mutants and epitope substitution mutants.14
However, such strategies are often biased toward regions previously identified as potential sites for manipulation. Starting with no prior knowledge of the best serotype to choose or the number of mutations needed to generate new phenotypes, random mutagenesis of any single parent would require libraries that are orders of magnitude larger. Thus, we rationalized that the application of molecular breeding to the AAV family in conjunction with rational mutagenesis should enable high-throughput characterization of critical domains on AAV serotype capsids as well as the accelerated evolution of novel tissue-specific AAV vectors.
As proof of principle, a chimeric virus composed of capsid sequences from AAV1, 8, 2, and 9 was obtained through selection in a hamster melanoma cell line (). Selection of chimeric library on CS1 cell has preferentially selected a chimeric particle with enhanced tropism for CS1 cell line compared to parental donors (). However, this tropism is not unique to CS1 integrin minus cells but extends to all rodent melanoma cell lines tested regardless of absence or presence of integrin (see , ). Although chimeric-1829 transduced these cells readily, none of the parental donor capsids was able to infect the rodent melanoma cells demonstrating that in the case of this chimeric capsid, the whole is the best of the parts. We then expanded our study to characterize the transduction profile of chimeric-1829 in a range of human melanoma cell lines and cultures. Interestingly, chimeric-1829 displayed transduction efficiencies comparable to parental AAV2 vectors in all human cells, while parental serotypes 8 and 9 were negative and type 1 restrictive to transducing one of nine cell lines (, ). It is tempting to speculate that such selective tropism for hamster/mouse melanoma cells displayed by chimeric-1829 could arise from the ability of the capsid to exploit a species-specific cellular factor (such as a cell surface coreceptor) for rodent melanoma cell entry. Although this critical finding highlights a potential caveat associated with application of this technique in developing AAV vectors for human gene therapy, it is important to note that the strategy allows elucidation of the determinants of species-specific differences in AAV serotypes. Obvious solutions to this problem are the use of human cell lines and “humanized” mouse models for directed evolution.
Following the isolation of such cell type–specific mutant(s), we utilized a battery of structural and molecular cloning tools to further investigate specific domains and residues that might potentially constitute determinants for melanoma cell–specific tropism in chimeric-1829. Structural analysis of a 3D model generated for the chimeric-1829 vector suggests that the heparin-binding regions are derived from AAV2, while antigenic regions were derived from more than one serotype capsid (). These results were further corroborated through heparin-inhibition studies () and characterization of the immunological profile of chimeric-1829 (). In particular, the unique immunological profile of AAV1829 in comparison with its parental serotypes is an important finding reported in this study (). We observed low-to-modest crossreactivity of NAbs against chimeric-1829 with AAV2 capsids (25-fold) and no crossreactivity to parental capsids 1, 8, or 9. The primary outcome of this analysis suggests that antigenic regions on the AAV capsid can be manipulated by grafting domains derived from other serotype capsids. This approach could aid in the generation of NAb escape mutants relevant for vector readministration in a clinical setting. The generation of chimeric mutants through rational mutagenesis was a critical component in establishing the molecular determinants for melanoma tropism displayed by chimeric-1829. A cumulative assessment of mutants generated through domain swapping and site-directed mutagenesis suggests that the C-terminal residues derived from AAV9 likely play a necessary, but not sufficient, role in conferring melanoma cell tropism to the chimeric-1829 capsid. In particular, we demonstrated that residues derived from AAV9, which form the wall of the twofold dimple, could play an important role in transducing CS1 hamster melanoma cells. This suggests that this region likely interacts with a yet unidentified cellular factor, or potentially involved in capsid structural/conformational transitions that are important for efficient rodent melanoma cell entry and transduction. It is worth noting that we recently generated a chimeric AAV capsid currently being used in a phase I clinical trial for Duchene muscular dystrophy. In this vector, three of the five amino acids that were altered to enhance skeletal muscle transduction were also located in the 705–735 amino acid region, independently confirming the importance of this domain in AAV infectivity (Bowles and R.J.S., unpublished results).
In addition to displaying selective tropism for melanoma cells in vitro
, chimeric-1829 displays tropism for CS1 cells in severe combined immunodeficiency model (Supplementary Materials and Methods
) and altered tropism for murine liver and skeletal muscle in vivo
(). Although modest, such “detargeting” of capsids vectors, in conjunction with tropism for specific cell types, could afford greater control over the biodistribution of viral vectors, thereby avoiding potential side effects due to transduction of nontarget tissues in vivo
. It is further corroborated through our vector-infusion studies in the rat brain (). Previous studies have shown that the parental serotypes of chimeric-1829 exhibit a preferential tropism for neurons.32-34
In contrast, chimeric-1829 appears to exhibit reduced tropism for neurons in both rat and monkey brains and potentially, preferential tropism for glial fibrillary acidic protein/nestin positive non-neuronal cells of rodent origin. Besides the fact that most melanoma metastasizes to the brain (54%),24
neuronal precursors (nestin positive cells) in the central nervous system and melanocytes are derived from a common linage (neural crest). As a number of brain tumors demonstrate surface markers similar to progenitor cells (nestin positive), which suggests reversion back to an earlier precursor cell type, we realized that the ability of chimeric-1829 to transduce a selected cell population may suggest a common link between these central nervous system cell types and the fact that they may share a similar lineage pathway with melanocytes. Thus, DNA shuffling/directed evolution proves capable of dramatically shifting capsid in vivo
In summary, we have described the utilization of DNA shuffling of a family of AAV capsid genes and directed evolution to generate a chimeric capsid library and a cell type–specific chimeric particle isolate (i.e., 1829), respectively. Further exploitation of the capsid library for isolation of novel chimeric mutants in vitro and in vivo is currently in progress (airway, heart, and brain; W.L., unpublished results) and likely to yield new capsid vectors with altered tropisms and antigenic profiles. More important, we anticipate that the mutants generated through shuffling of AAV serotype–specific capsid motifs or capsid sequences from similar (B19) or divergent (SV40, Ad, etc.) viruses will lay the foundation for designing the next generation of novel vectors for human gene therapy applications namely “biological nanoparticles” that process unique tissue targeting, immune profiles, and biodistribution.