Optimization of the protocol for generation of shuffled AAV libraries

As shown in Figure 15 above, a prerequisite for AAV DFS is the initial isolation and then fragmentation of the cap genes of choice. In the original protocol by Grimm and co-workers, 8 different cap sequences were isolated from respective plasmids by restriction digestion, using enzyme binding sites that had been engineered into each of the cap plasmids. While feasible and efficient, a drawback of this strategy is that it may not be applicable to all present or future cap genes because some of them may inherently contain binding sites for the enzymes originally used (HindIII/SpeI). Moreover, since the DNase digestion step may have to be repeated until desired fragments are produced (see below), it can become necessary to digest large amounts of cap plasmid with restriction enzymes which is time-, work- and cost-intensive. Finally, since many labs have set up their own plasmids and restriction strategies, it is very difficult to share and use constructs between individual groups which hampers a broader evaluation and application of the whole technology.

To overcome these problems and to standardize the complete protocol, we decided to refine the original strategy and switched to a PCR-based approach for cap gene amplification rather than enzymatic digestion. Therefore, we generated an entirely new set of AAV donor plasmids in which the cap genes of 12 different AAV serotypes - 1 to 9, rh10, po1 and 12 - are flanked by a collection of PCR primer binding sites (Figure 16). These serotypes were chosen because they have been studied extensively in our and other labs, and because they differ substantially in their tropisms and efficiencies, making them interesting templates for DFS. The primer binding sites were identical to

[71]

those used for shuffled cap gene amplification in the 2008 Grimm study (CUF/R); in addition, we included the SAF/R primer binding sites (see Materials and Methods 2.2.4.2, Figure 11) that another PhD student in our lab (Nina Schürmann) had concurrently found to work well for shuffling of other genes (encoding human Argonaute proteins) [270]. As the plasmids were based on pBlueScript, they additionally contained binding sites for the standard M13 and T3/T7 primers. Finally, in our eventual constructs, each cap fragment is flanked by recognition sites for the restriction enzymes PacI (5’) or AscI (3’) which allowed a) the initial cloning of all cap sequences into the new plasmids, and b) the final subcloning of shuffled sequences into an appropriately modified AAV recipient plasmid, i.e., a replication-competent construct containing the AAV2 ITRs and the AAV2 rep gene followed by PacI/AscI sites. While this strategy again introduces a need for a restriction digestion, it is important to point out that both PacI and AscI recognize an 8 bp target sequence (in contrast to most other enzymes which bind only 6 bp, including HindIII/SpeI) and thus cut very infrequently; indeed, there are no PacI or AscI sites in any of the 12 AAV cap genes used here. Figure 16 schematically depicts the composition of the new modular AAV donor and recipient plasmids described above.

Figure 16: New AAV cap gene donor and recipient plasmids for standardized AAV DNA family shuffling. Top: AAV cap

donor plasmids containing binding sites for 4 different primer pairs on each side of the cap gene. Also shown schematically are binding sites for the capF and capR primers used to initially amplify the capsid genes for subcloning into these plasmids, as well as the location of several restriction sites. Bottom: Similar depiction of the new AAV cap gene recipient plasmids, containing AAV2 ITRs and rep gene, followed by PacI/AscI sites for cap subcloning. Also shown are binding sites for two primers (LseqR/F) that were used for sequencing of the shuffled and cloned cap genes.

[72]

Using these plasmids, we next assessed which primer pairs would give the best results, as measured by the strength of the band after the second PCR (for amplification of shuffled fragments, see Figure 15 above). For this proof-of-concept work, we DNase-fragmented and then re-assembled AAV serotypes 2, 8 and 9. The M13 primers (Figure 16, top) were used to amplify all cap genes prior to DNase digestion. According to the original protocol [212], we first tested the CUF/R primer pair and found that while it worked, the yields of the final cap chimera band were relatively weak which hampered subsequent large-scale cloning (data not shown). The same was observed for a nested PCR with the T7/T3 primers in a first reaction, followed by amplification with CUF/R; moreover, this primer combination produced multiple non-specific bands (Figure 17A MTC). In fact, the only setting which produced satisfactory results were “nested” PCRs in which the T3/T7 primers were re-used (Figure 17A, MTT). Interestingly, a single PCR with the SAF/R primers alone was equally efficient and specific, especially at higher annealing temperatures of up to 67°C (Figure 17B). We therefore continued to use this simple two-step PCR (including the first primerless PCR) with these two primers in the second PCR step in all subsequent shuffling reactions in this thesis.

Figure 17: Comparison of experimental setups for the AAV DNA shuffling re-assembly PCR. A) Nested PCR with different

primer combinations at increasing annealing temperatures. M refers to the M13 primer pair that was used in the first PCR from the donor plasmids and is hence part of all PCRs. T, C and S refer to the T3/T7 primers, the CUF/CUR primers and SAF and SAR, respectively. B) Single PCR to re-assemble capsid genes with the newly introduced primer pair SAF and SAR. Yellow arrows mark the desired bands, while yellow brackets indicate the area with unspecific bands.

Having optimized the PCR conditions, we next aimed to also improve the initial step of cap gene fragmentation via DNase I digestion. An inherent problem with this step is that the reaction is hard to standardize, due to the fact that DNase I is an extremely potent enzyme that rapidly degrades input DNA. The representative gel in Figure 18 demonstrates that even small variations in the length of the

61°C – 63°C 61°C – 63°C 61°C – 63°C MTS MTT MTC 61°C 67°C MS 1.6kb 2kb 1kb 4kb 3kb Nested PCR SAF/SAR PCR 1.6kb 2kb 1kb 4kb 3kb

A)

B)

[73]

reaction can already induce substantial differences regarding the size range of the fragments that are produced. The same variation was found when slightly different pre-dilutions of DNase I enzyme were used (data not shown).

Figure 18: Example for DNase I digestion of cap genes (AAV2, 8 and 9). Shown

is an agarose gel electrophoresis analysis of the products of cap gene digestion using the indicated different incubation times (in minutes:seconds). Lane U shows the undigested input cap fragment pool as control. In this example, ideal digests were obtained with incubation times of 1:45 or 2:00 min, which yielded the preferred predominant peak of fragments around 100 to 500 bp (yellow box).

As a result of this high and hard-to-control activity, it can be difficult to avoid complete DNA degradation and to instead generate sufficient cap fragments of a desired size range. In addition, the latter is typically rather broad (roughly 100 to 500 bp gives good results) since a large gel piece has to be isolated (yellow square in Figure 18), which further complicates standardization between different labs. We thus asked whether a more controlled fragmentation by physical means rather than enzymatic digestion could improve this essential step. Specifically, we tested DNA ultrasonification via “Adaptive Focused Acoustic” technology (Covaris, Woburn, USA) and compared it with conventional DNase I digestion. The Covaris system offers the possibility to generate very defined fragment sizes, allowing us to create sheared AAV cap fragments with a peak size of either 150, 300 or 800 bp. Their correct length distribution was confirmed by analysis on an Agilent Bioanalyzer 2100 (Figure 19A). As expected, the fragments generated through DNase I digestion showed a much broader distribution over the entire size range, with a slight accumulation of fragments of around 100 bp (Figure 19B).

For further analysis cap genes from AAV serotypes 1,7-9 and rh10 were fragmented, using either the DNase digestion technique or the previously determined Covaris protocols for 150bp, 300bp and 800bp fragments, respectively. The serotypes were chosen because they are representative for libraries made of several AAV parents (five in this case) with roughly 85% homology to each other. Covaris- and DNase-digested fragments were used in parallel to produce the respective shuffled

1.6kb 2kb 1kb 4kb 3kb 0.5kb U DNase digestion 1:00 1:15 1:30 1:45 2:00 2:15 0.1kb

[74]

libraries. If successful, the first and second PCRs of the shuffling protocol should result in a distinct gel band of around 2.2 kb, corresponding to full-length chimeric cap genes. Notably, as depicted in a representative gel in Figure 19C, the re-assembly reaction did not work for Covaris fragments with an average size of 150 bp, and the PCRs with the 300 bp fragments gave only a weak band of the correct size. In fact, the Covaris conditions that produced a defined fragment size of 800 bp were the only ones that resulted in a full-length cap gene band that was comparable in intensity to that from the conventional DNase I digestion.

Figure 19: Fragmentation of AAV capsid genes. A) A pool of different capsid genes

was fragmented to different average fragment lengths by ultrasonification and analyzed on a Bioanalyzer. Plotted are fragment lengths (in bp, X axis) versus fluorescence units (FU, Y axis) from which the amount of DNA is calculated, compared to standard peaks, visible at 15 and 1500bp. The small peak at 2kb in the 800bp fragmentation indicates remaining full-length cap genes (yellow arrow). B) DNA fragmented with DNase I and purified on an agarose gel was analyzed the same way. C) Agarose gel of PCR products from the re-assembly PCR that amplifies full-length genes. Using the Covaris fragmentation, only fragments with an average length of 800 bp resulted in comparable amounts of PCR product. Desired band are indicated by the yellow arrow. D) Average length of fragments (in bp) in assembled capsid genes from a 1,7- 9,rh10 library. In sequences that were generated with DNase I, digested DNA fragments are shorter and therefore the crossover frequency is higher. The 1,7- 9,rh10 library was produced, sequenced and analyzed with Salanto by Stefanie Große. (C) and D) courtesy of Stefanie Große.) (Indet= indetermined; sequence parts for which an exact allocation to a certain serotype is not possible.)

From both reactions, 800 bp Covaris and DNase I digestion, Stefanie Große (PhD student in the lab) produced a plasmid library, sequenced 20 clones and used the Salanto program that Christian Bender had developed together with our lab (chapter 2.1.12) to determine the average length of fragments according to the parental serotypes. Interestingly, with the exception of AAVrh10 (for reasons unknown), the average fragment length in the DNase I-derived library was about half the size of the

A) 150bp fragmentation 800bp fragmentation 300bp fragmentation Covaris DNase digestion DNase 1.6kb2kb 1kb 4kb 2nd_PCR DNase 800 300 150 B) C) 0,00 100,00 200,00 300,00 400,00 [bp ]

Average length of fragments

0,00 100,00 200,00 300,00 400,00 [bp ]

Average length of fragments

Covaris DNase

[75]

800 bp Covaris library (Figure 19D). The fragment length directly correlates with the number of crossovers per clone, with shorter fragments meaning more crossovers, which is beneficial for the library diversity. We therefore decided to continue using the conventional DNase I digestion rather than physical fragmentation for the remaining work in this thesis.

We finally also analyzed whether we could further improve the efficiency of the two PCRs by altering the annealing temperature in the first PCR where the partially homologous fragments are supposed to self-prime. However, we found that 42°C as used in the original Grimm et al. paper [212] as well as in the experiments above gave best results that were not enhanced by lowering or raising the temperature over a 20 degree range (data not shown). We accordingly concluded that 42°C is already an ideal temperature to allow annealing of different cap gene fragments and thus efficient shuffling. In summary, the final optimized protocol for AAV capsid gene shuffling consists of:

1. PCR amplification of the desired cap genes from our new donor plasmids using M13 primers 2. DNase I-based fragmentation (aiming for fragments of 100-500 bp)

3. a primer-less recombination (shuffling) and re-assembly PCR at 42°C annealing temperature 4. a second single-step PCR (i.e., combined annealing/extension at 68°C) using SAF/R primers Important to note, this protocol has not only been used successfully for all further library productions in this thesis, but also independently by Stefanie Große in the lab for generation of >10 additional libraries based on various AAV serotype combinations (Große et al., manuscript in preparation).

3.1.1.1 Combination of AAV capsid shuffling and peptide display

As will be described in more detail in a later chapter (3.2), we also engineered all 12 cap genes to contain unique restriction enzyme sites for cloning of short oligonucleotides encoding re-targeting peptides for display on the viral surface. Because the combination of this viral peptide display with DFS should theoretically create the largest possible diversity (of all currently available molecular AAV evolution techniques), we tested whether the two methods were truly compatible. Therefore, all 12 insertion site-modified cap genes were transferred into the new donor plasmid described above and then used in shuffling reactions, using the optimized protocol. The shuffled reaction products were then used as templates for cloning of a randomized peptide library on top. Two factors were particularly important to guarantee the success of this strategy: first, the insertion sites for peptide display were located at comparable positions across all the different serotypes, reducing the risk that one is lost upon recombination. Second, because the peptide insertion site initially creates a frameshift (see chapter 3.2.), VP protein expression and virus production are only possible with a successfully inserted oligonucleotide that corrects this frameshift.

[76]

We then produced eight different shuffled AAV libraries, with or without additional peptide library insertion, as listed in the following Table 13. As can be seen, all eight libraries were produced to decent titers, albeit we noted a 5- to 50-fold reduction in the titers of the peptide-containing libraries as compared to their direct counterparts without peptide insertion. Most likely, this difference is due to the fact that a proportion of the plasmid clones in the shuffling-display libraries has not taken up the oligonucleotide which corrects the frameshift and allows viral library production. Moreover, a fraction of the peptide-encoding oligonucleotides will comprise stop codons, which will further reduce the number of viable sequences in such a complex library. These assumptions are in fact supported by the observation that library diversities on the plasmid level were comparable between the shuffled and corresponding shuffling-display libraries (with minor variations in both directions).

Notably, sequencing of 5 clones from each of the four different shuffling-peptide libraries confirmed that all clones had indeed taken up peptide-encoding oligonucleotides (representative examples in Figure 20A-C). While these libraries were not further used for selection schemes in the context of this thesis, they represent an essential proof-of-concept that our new protocol and plasmids allow for the juxtaposition of two powerful AAV evolution methods and thus provide a novel avenue that can be exploited in the future (Discussed in more detail in chapter 4.3)

Library Diversity [whole library]

Viral titer [vg/ml]

Table 13: Comparison of AAV libraries.

Libraries based on the indicated serotypes were either generated by DNA family shuffling alone, or by combining shuffling with insertion of a peptide library (+PL). The diversity of the respective library was estimated by the number of bacterial colonies after transformation, assuming that each colony carries a distinct clone. Viral libraries were produced in Hek293T cells and purified by Iodixanol density gradient centrifugation. Viral titers were determined by RT-PCR. 289 3.5x106 2.00x1012 289+PL 1.3x106 4.40x1011 789 2.2x106 1.78x1013 789+PL 8.9x105 1.54x1012 1689 1.1x106 1.60x1013 1689+PL 1.1x106 2.50x1012 15689 1.5x105 6.00x1012 15689+PL 5.0x105 1.20x1011

[77]

Figure 20: Comparison of chimeric sequences from different shuffled AAV libraries that harbor additional peptide insertions. A) Clones from the library 289 plus peptide insertion. B) Clones from the library 789 plus peptide insertion. C)

Clones from the libraries 15689 and 1689 plus peptide insertion. Chimeric sequences are shown in alignment to their parental serotypes and compared to a modeled AAV2 sequence, where peptide insertion is indicated by 21 ‘N’ (AAV2mod.). Chimeric sequences are coded with colors that refer to the parental sequences. Note that the chimeric character of the shuffled clones is not always visible within the short area shown around the peptide insertion site, but was always confirmed by further sequencing. Modified sequences flanking the peptide insertion are boxed as triplets. Additional nucleotides are shown in bold, peptide sequences are bold underlined. For details about peptide insertions please refer to chapter 3.2.1, Figure 37 and Figure 39.