Design of AAV virus for the targeting of ATXN3 gene in human lt-NES cells

The general composition of an AAV targeting vector is illustrated in Fig. 3.1. Two homology arms (HAs) flanking the selection cassette are necessary for site-specific targeting and were amplified from genomic DNA of the same cell line that was later targeted. For primer design and in-silico genome analysis, the Ensembl databases (http://www.ensembl.org) and Primer3 (http://frodo.wi.mit.edu/) were used. For selection, a promoterless cassette bearing neomycin resistance was chosen and cloned into the commercially pAAV-MCS vector (Stratagene).

3.6.1 Generation of homology arms

The ATXN3 gene consists of 12 exons, of which exon 10 contains the polyglutamine encoding CAG repeat motif. Based on an in silico analysis with RepeatMasker, exon 10 and the surrounding intronic sequences did not contain any repetitive motifs beside the CAG repeat and thus was found to be suitable for our targeting strategy. The AAV vector system allows the incorporation of around 2 kb for HA usage, which should be evenly distributed between the two HAs lying upstream and downstream of the selection cassette. With a size of 405 bp, Exon 10 of the ATXN3 gene is small enough for the complete incorporation into the downstream HA. Primers were picked using the Primer3 algorithm (Rozen and Skaletsky, 2000) on the intronic sequences surrounding the exon, generating a downstream HA of 1165 bp length (Fig. 3.1). The upstream HA was chosen to contain only intronic sequences found between exons 9 and 10 with a length of 814 bp.

For the amplification of the two homology arms, genomic DNA was isolated from MJD-lt-NES cells using the Qiagen DNeasy Blood and Tissue DNA isolation kit according to the manufacturer’s instructions. The homology arms were amplified from genomic DNA by PCR using primers with overhanging ends containing restriction sites for NotI, SpeI or NdeI (Tab.

2.14). Primer sequences encompassed 26-27 nucleotides of target binding, the restriction enzyme recognition site and additional nucleotides homologous to the selection cassettes resulting in an overall-length of 41-43 nucleotides.

2 µM of each forward and reverse primers, 0.025 U/µl Taq-Polymerase, 2 mM of Mg²⁺ and 0.2 mM dNTPS were added to 1 µg of DNA in a total volume of 50 µl PCR buffer. The cycling conditions were the following: 1 cycle of 94°C for 5 min; 20 cycles of 94°C for 30 s, 53°C for 30 s and 72°C for 90 s; 1 cycle of 72°C for 10 min. The primers were removed using the peqLab PCR cycle pure kit. The extracted DNA was digested with NotI (20 units) and SpeI (10 units) in 20 µl NEB buffer 2 or with NotI (10 units) and NdeI (15 units) in 20 µl NEB buffer

sizes, in case of the downstream HA products of both alleles were gained, of which the shorter variant was excised and gel-extracted using the peqLab gel extraction kit.

3.6.2 Cloning of targeting vector

The contents of the targeting vector are depicted in Fig. 3.1. Besides a classical backbone with Amp resistance for bacterial amplification it contains the targeting cassette flanked on both sides by the ITRs that give rise to the hairpin shaped termini of the viral genome.

Between the ITRs the homology arms are situated and in between them, lays the selection cassette containing a synthetic exon promoter trap (SEPT) (Topaloglu et al., 2005) that allows neomycin resistance only if integrated into an actively transcribed gene locus, thus massively increasing the amount of positive resistant clones. The ITRs and the backbone are derived from the pAAV-MCS vector, which is part of the commercial helper-free AAV-system from Stratagene.

The creation of the targeting vector is a multi-step-process: first the upstream HA was ligated into a pWpXL-backbone containing the SEPT cassette, then this larger fragment was excised and, together with the downstream HA, ligated into the pAAV-MCS backbone. The excision and ligation process is shown in Fig. 3.1.

The pWpXL vector contained the whole SEPT cassette directly downstream of its multiple cloning site, bearing restriction sites for NotI and SpeI. To open the plasmid and insert the upstream HA in front of the SEPT cassette, both were digested with NotI (20 units) and SpeI (10 units) in 20 µl NEB buffer 2 for 90 min at 37°C, and purified from a 0.8 % agarose gel using the peqLab gel extraction kit according to the manufacturer’s instructions. The linearized backbone (50 ng) was then combined with the digested homology arm (150 ng) so that the insert was in 20 fold molar excess of backbone. Ligation was performed using T4 ligase (400 units) in 20 µl NEB T4 ligation buffer for 30 min at 22°C. The ligated vector was transformed into competent E. coli cells. 100 µl of frozen competent bacteria were thawed on ice, all 20 µl of the DNA solution were added, and the reaction tube chilled on ice for 30 min.

Then, heat shock was performed for 90 sec at 42°C and the sample again chilled for 5 min.

To allow the cells to gain antibiotic resistance, 500 µl SOB medium was added and the tube shaken in an incubator at 37°C for 90 min. Cells were then plated on LB agar plates containing 100 mg/l ampicillin and incubated over night (o/n) at 37°C.

The next morning, plates were put into the fridge to pick colonies in the evening. Each colony was grown o/n at 37°C on a shaker in 3 ml LB medium containing ampicillin. The DNA was isolated using the peqLab DNA mini prep kit according to the manufacturer’s instructions. 1 µl of the preparations were further analyzed by electrophoresis on a 1 % agarose gel after

digestion with NotI (10 units) and NdeI (15 units) in 20 µl NEB buffer 3 for 90 min at 37°C.

Clones showing two bands (backbone and fusion of upper HA with selection cassette) of the correct sizes were considered positive. One of these clones was selected and grown o/n in shaking incubator at 37°C in 200 ml LB medium containing 100 mg/l ampicillin. DNA purification was performed using the Promega PureYield Plasmid Maxiprep System kit according to the manufacturer’s instructions.

The next ligation step combined to downstream HA with adjacent SEPT cassette with the PCR-amplified upstream HA into the pAAV-MCS backbone to gain the functional targeting vector. The pAAV-MCS plasmid was digested with 10 units of NotI in 20 µl of NEB buffer 3 for 90 min. at 37°C and the larger fragment (2887 bp) was purified from a 0.8 % agarose gel using the PeqLab gel extraction kit. To prevent unwanted circularization, the linearized fragment was dephosphorylated using 10 units of alkaline phosphatase in 10 µl of dephosphorylation buffer. After an incubation of 30 min. at 37°C the alkaline phosphatase was inactivated for 15 min. at 65°C. The downstream HA–PCR product and the fusion-construct of upstream HA with selection cassette was digested with NotI and NdeI as described above. Ligation was performed with 50 ng of pAAV-MCS-backbone, 350 ng of downstream HA and 50 ng of upper-HA-selection cassette-fusion-construct in 20 µl NEB T4 ligation buffer with 400 units of T4 ligase for 30 min at 22°C. This time, the HA was used in 200 fold excess of the other segments to ensure efficient incorporation. Transformation of competent cells and clone picking were performed as described above. Positive clones were chosen on the results of digestions with either NotI or SpeI or the combination of both (each 10 units in 20 µl NEB buffer 3) following agarose gel electrophoresis. Again, PCR was conducted to verify integration of the homology arms.

3.6.3 Mutation of targeting vector

To easily discriminate between the original wild type allele and the gene-corrected allele, a 1-bp-mutation was inserted in the targeting vector. This mutation is a silent one, which would not affect the translated protein. For modification of the plasmid, the QuikChange Lightning Site-Directed Mutagenesis Kit (Stratagene) was used following the manufacturers’

instructions. The used primers P11 and P12 were designed with Stratagene’ webpage tool.

Figure 3.1: Cloning strategy for ATXN3-AAV targeting vector for site-specific integration.

Upstream and downstream homology arms (HAs) were PCR-amplified from the shorter allele of exon 10 of the ATXN3 gene of MJD-patient derived lt-NES cells. Primers used also carried restriction nuclease recognition sequences for cloning purposes. The upstream HA was first ligated directly in front of the SEPT cassette, which was already present in a pWPXL vector, using its multiple cloning site (MCS). The new construct was expanded and used to excise an upstream HA+ SEPT fragment for the next ligation step. Together with the downstream HA; it was ligated in a dephosphorylated pAAV-MCS backbone to gain the complete targeting vector composed of a SEPT cassette flanked by homology arms for site directed integration, nested between inverted tandem repeat (ITR) sequences of the viral backbone.