Supplementary information: Optimization of design and production strategies for novel adeno-associated viral display peptide libraries

Transcription

1 Supplementary information: Optimization of design and production strategies for novel adeno-associated viral display peptide libraries Jakob Körbelin, Agnes Hunger, Malik Alawi, Timo Sieber, Mascha Binder, Martin Trepel Supplementary Figure 1 Two steps protocol for the generation of random AAV display peptide libraries After cloning the AAV plasmid library by insertion of heptapeptide-encoding oligonucleotides at nucleotide position 3,967 of the AAV2 genome (R588, VP1 numbering), in a first step so-called AAV library transfer shuttles are generated by triple transfection of HEK 293T producer cells with the AAV plasmid library, an adenoviral helper plasmid and an AAV helper plasmid encoding the codon-modified wild type AAV2 cap gene. In a second step HEK 293T producer cells are infected with the library transfer shuttles at a low MOI followed by superinfection with adenovirus. Thereby an AAV display peptide library is generated in which each peptidepresenting capsid carries its own corresponding genome. Figure adapted from Müller et al. 1 and Waterkamp et al. 2

2 Supplementary Figure 2 Clonal distribution measured by repeated Next-Generation Sequencing with independent samples of one individual AAV library. Six samples (5x10 8 viral genomes each) of the same AAV2 two-steps library with random 7-mer peptide insertion were coupled to different NGS barcode adapters in individual PCR reactions and subsequently analyzed by Next- Generation Sequencing with approx. 100,000 or 5 million filter-passing reads per sample. The clonal distribution within the six individual samples of the AAV2 two-steps 7-mer library is displayed as bar graphs. The relative number of NGS reads is given for the most abundant 100 peptide sequences (black bars), 1,000 peptide sequences (black + orange bars) and 5,000 sequences (black + orange + blue bars). The six samples only display minor deviations, showing that a sample size of 100,000 reads is highly representative for the population of the whole library. 2

3 Supplementary Figure 3 Amino acid distribution measured by repeated Next-Generation Sequencing with independent samples of one individual AAV library. Six samples (5x10 8 viral genomes each) of the same AAV2 two-steps library with random 7-mer peptide insertion were coupled to different NGS barcode adapters in individual PCR reactions and subsequently analyzed by Next- Generation Sequencing with approx. 100,000 or 5 million filter-passing reads per sample. The frequency of each of the 19 amino acids is displayed for each position within the library inserts in color code ( heat maps ) for the six samples of the AAV2 two-steps 7-mer library. Codons that are exactly as frequent as expected (1/19 = 5.26%) have a value of 1 (1-fold expected value), displayed without color. Underrepresented codons are displayed in blue and overrepresented codons in red. All six samples yield almost identical values, showing that a sample size of 100,000 reads is highly representative for the population of the whole library. 3

4 A B C Supplementary Figure 4 - Read frequency plots (rarefaction plots) of Next-Generation Sequencing experiments with AAV plasmid libraries AAV2-encoding plasmid libraries with random peptide insertions were analyzed by Next-Generation Sequencing with approx. 100,000 reads passing the quality filters. The number of reads is plotted against the number of different sequences. The absence of a plateau indicates that 100,000 reads do not cover the library s actual diversity. A) 6-mer library. B) 7-mer library. C) 12-mer library 4

5 A B C Supplementary Figure 5 - Read frequency plots (rarefaction plots) of Next-Generation Sequencing experiments with AAV transfer shuttles AAV2 transfer shuttles with random peptide insertions were analyzed by Next-Generation Sequencing with approx. 100,000 reads passing the quality filters. The number of reads is plotted against the number of different sequences. The absence of a plateau indicates that 100,000 reads do not cover the library s actual diversity. A) 6- mer library. B) 7-mer library. C) 12-mer library 5

6 A B C Supplementary Figure 6 - Read frequency plots (rarefaction plots) of Next-Generation Sequencing experiments with AAV one-step libraries AAV2 peptide libraries produced in one step via transfection were analyzed by Next-Generation Sequencing with approx. 100,000 reads passing the quality filters. The number of reads is plotted against the number of different sequences. The absence of a plateau indicates that 100,000 reads do not cover the library s actual diversity. A) 6- mer library. B) 7-mer library. C) 12-mer library 6

7 A B C Supplementary Figure 7- Read frequency plots (rarefaction plots) of Next-Generation Sequencing experiments with AAV two-steps libraries AAV2 peptide libraries produced in two steps via transfer shuttles were analyzed by Next-Generation Sequencing with approx. 100,000 reads passing the quality filters. The number of reads is plotted against the number of different sequences. The absence of a plateau indicates that 100,000 reads do not cover the library s actual diversity. A) 6-mer library. B) 7-mer library. C) 12-mer library 7

8 References: 1. Müller OJ, Kaul F, Weitzman MD, Pasqualini R, Arap W, Kleinschmidt JA et al. Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors. Nat Biotechnol 2003; 21: Waterkamp DA, Muller OJ, Ying Y, Trepel M, Kleinschmidt JA. Isolation of targeted AAV2 vectors from novel virus display libraries. J Gene Med 2006; 8: