comprises first the fragmentation of sufficient amounts of parental genes; second the reassembly of capsid sequences from these fragments in a primerless PCR, based on their partial homology; third, the amplification (‘rescue’) of chimeric full-length capsid genes; and finally, cloning and virus production in order to generate a diverse plasmid or viral library, respectively. Because of the complexity of this workflow, only a few groups in the world have thus far been able to establish this technology, which is unfortunate as it continues to hamper its wider application. Therefore, the first specific aim in this thesis was to develop a robust protocol for AAV cap gene shuffling that only requires standard laboratory expertise and equipment, and that can be easily adapted to custom needs including different types and numbers of parental AAV serotypes. In particular, we attempted to streamline and simplify the following aspects of the general workflow:
cap plasmids: A limitation of the original protocol as first reported by Grimm and colleagues [212] and subsequently later used by others [6], [214]–[217], [219], [297]–[301] is the need to isolate desired capsid genes from plasmids via restriction digests. While relatively simple, there may be a need to amplify and then extensively digest large amounts of plasmid DNA since the subsequent DNase I reaction can consume substantial input DNA (see also next paragraph). Here, we improved this step by introducing a new set of standardized and modular plasmids that contain the cap genes of 12 important AAV serotypes flanked by multiple restriction and primer binding sites. As demonstrated, these plasmids allow to isolate any desired cap gene in sufficient amounts in a very efficient and rapid PCR reaction. Accordingly, they help to save significant material and time, and permit to proceed to the DNA fragmentation step within less than two hours (see Figure 53 below).
DNA fragmentation: This step is highly critical for the success of DFS because the cap DNA fragment
size or rather the range of sizes determines the crossover rate and hence the degree of shuffling. Larger fragments will increase the chances of recombination and thus improve the yields of full- length genes, but this comes at the cost of reduced diversity. Vice versa, shorter fragments pose less opportunity for homologous recombination, but where it occurs, it results in highly diverse progeny. Alas, DNA fragmentation via DNase I is very difficult to control due to the high inherent activity of the enzyme. We therefore studied whether physical shearing (Covaris ultra-sonication) of cap DNA could be more reliable and reproducible, and produce more defined fragment sizes. Interestingly, while we found that the latter is indeed the case, we concomitantly noted a drop in shuffling efficiency, as evidenced by low yields of reassembled full-length capsid genes and fewer crossovers. In fact, even
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the best Covaris condition (Fig. 19, 800 bp fragments) which resulted in yields comparable to those from DNase I-based digestion produced clones that were less diverse. Notably, these findings were confirmed independently by another PhD student in the lab (Stefanie Große) who likewise achieved better results for various AAV cap gene combinations with DNase I digestion rather than with Covaris (Große et al., manuscript in preparation). Curiously though, yet another PhD student in our group, Nina Schürmann, successfully applied the Covaris system to create libraries from the four human Argonaute proteins [270], using conditions comparable to those here. Taken together, these three independent observations suggest that the success of library generation through chemical versus physical fragmentation depends on numerous parameters, especially the primary sequences of the parental genes. We thus refrain from generally recommending one method versus the other, but conclude that at least for AAV library production, cap gene fragmentation via DNase I digestion is the preferred approach.
Reassembly & amplification PCRs: Equally important as optimizing the conditions for cap gene
fragmentation was to streamline the subsequent steps of reassembly and amplification of chimeric full-length sequences. This was a very challenging endeavor for two major reasons: (i) the success of the first reassembly PCR can only be visualized through a robust second amplification PCR, since the first PCR itself does not yield a detectable product; hence the two PCRs are closely interrelated and had to be optimized simultaneously. Moreover, (ii) the goal of the first PCR (and the preceding DNA fragmentation step, see above) is to create maximum diversity, whereas the second PCR rather focuses on producing high yields; it was accordingly important to strike a balance between these two aims when optimizing the individual PCR conditions. In the end, we indeed found conditions that fulfill the last requirement and that not only improve but also simplify the original protocol. This is because we eliminated the need for a nested PCR by establishing an experimental setup that allows for robust production of full-length and diverse cap genes via a simple and rapid two-step PCR. The efficiency of this improved PCR is in fact so high that it also permits to skip the intermediate TOPO cloning step that was necessary in the original protocol to increase the amount of cap genes [212]. This is again an essential advance in the present work as it minimizes the risk of a loss of sequence diversity due to suboptimal TOPO ligation and/or subsequent transformation.
Collectively, the improvements implemented in this work substantially streamline the AAV DNA shuffling protocol and now permit the routine production of highly diverse capsid libraries derived from various serotype combinations within less than one week. Figure 53 on the next page illustrates the entire workflow and the individual reactions required to create a typical small-scale AAV library.
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Figure 53: Schematic depiction of the streamlined protocol for AAV library production established in this work. Shown on
the left are the individual steps with estimated times, while the corresponding numbers of reactions (“rxn/s”) are depicted on the right. Numbers outside the circles indicate options for up-scaling. Center arrows denote the required time in days.
4.1.2 The selection process is equally decisive for the success of the shuffling approach