High Capsid–Genome Correlation Facilitates Creation of AAV Libraries for Directed Evolution  Mathieu Nonnenmacher, Harm van Bakel, Roger J Hajjar, Thomas Weber  Molecular Therapy  Volume 23, Issue 4, Pages 675-682 (April 2015) DOI: 10.1038/mt.2015.3 Copyright © 2015 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 1 Low mosaicism in natural adeno-associated virus (AAV) capsid formation. (a) Recombinant viruses were produced with the indicated ratios of Rep-Cap plasmids encoding either wild-type AAV2 or R585/588A mutant (HBDm) capsid, together with an inverted terminal repeat (ITR)-Luciferase-ITR plasmid. Transduction was measured by a luciferase assay. (b) Natural AAV was produced with the indicated ratios of wild-type and HBDm ITR-Rep-Cap-ITR plasmids. Transduction was measured by a DNA replication assay. (c) Recombinant or natural AAV was produced with plasmids encoding a HBD 30-aa insertion mutant (HBDi), wild-type AAV2, or with a 1:1 ratio of these plasmids. Progeny virus was incubated with heparin beads and total or heparin-bound capsids were analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. (d) Recombinant or natural AAV2 and AAV9 virions were produced alone or in a 1:1 ratio and immunoprecipitated with the AAV2-specific antibody A20. Viral DNA was extracted from the beads or the supernatant and quantified by quantitative polymerase chain reaction (qPCR) with rep primers. (e) Recombinant or natural AAV2 and AAV9 virions were produced alone or in a 1:1 ratio and bound to heparin beads. Viral DNA extracted from the beads or the supernatant was quantified by qPCR with rep primers. Molecular Therapy 2015 23, 675-682DOI: (10.1038/mt.2015.3) Copyright © 2015 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 2 High capsid–genome correlation in cells transfected with multiple natural adeno-associated virus (AAV)-genome–encoding plasmids. (a) Genotyping of A20 beads and supernatant fractions described in Figure 1d using primers specific for AAV2 or AAV9. (b) Genotyping of heparin beads and supernatant fractions described in Figure 1e using primers specific for AAV2 or AAV9. (c) Inverted terminal repeat–flanked wild-type and HBDm AAV2 DNA were cotransfected in 293T cells at 1:0 to 1:1,000 ratio. Progeny virions were genotyped (left panel) and an equal amount of virus was added to HeLa cells. Cell-bound virions were analyzed by quantitative polymerase chain reaction with primers specific for wild-type or HBDm AAV2. Absolute genomes copies (middle panel) or percentage of total (right panel) are shown. Molecular Therapy 2015 23, 675-682DOI: (10.1038/mt.2015.3) Copyright © 2015 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 3 Adeno-associated virus (AAV) library generation and diversity. (a) Amino acid sequence alignment of hypervariable loop 4 from common human and non-human primate AAV serotypes. The semi-degenerate amino acid sequence used to synthesize the loop IV library is indicated on bottom. (b) Localization of hypervariable loop IV 13-mer on the AAV9 capsid surface. Amino acids submitted to saturating mutagenesis are depicted in red, semiconserved and conserved positions are shown in yellow. The model was generated with MacPymol version 0.99, Schrödinger LLC, http://www.pymol.org (from the file 3ux1_full.vdb downloaded from the Viper database http://viperdb.scripps.edu/). (c) Schematic of the insertion of the loop IV library into an AAV9 plasmid backbone. (d) Analysis of loop IV plasmid library by next-generation sequencing. Nucleotides (top panel) and amino acid (bottom panel) logos are depicted. Degenerate consensus sequences are indicated. Sequence logos were created by weblogo (http://weblogo.berkeley.edu/).22 (e) Titration of viral libraries by quantitative polymerase chain reaction. Values indicate the total viral genomes produced by transfection of one 15-cm plate with various amounts of plasmid library DNA. (f) Measurement of library diversity by next-generation sequencing. The total read counts for each library was ~3.106, minimal diversity was calculated using Poisson distribution estimates. Molecular Therapy 2015 23, 675-682DOI: (10.1038/mt.2015.3) Copyright © 2015 The American Society of Gene & Cell Therapy Terms and Conditions

Figure 4 Stop codon bias in adeno-associated virus (AAV) libraries. (a) Color plot indicating the bias in amino acid frequency at each position of the loop IV 13-mer. Numbers indicate the ratio of AA frequency in viral libraries versus parent plasmid library. Semiconserved and conserved positions 6 and 11 were excluded from the analysis. (b) Scatter plot of total amino acid frequency in the parent plasmid library (x-axis) versus viral libraries (y-axis). Stop codons are indicated in red. (c) Total stop codon frequency in parent and viral libraries. (d) Fitness analysis of variants containing stop codons. Frequency of variants containing at least one stop codon is represented as a function of variant read count quantile. Molecular Therapy 2015 23, 675-682DOI: (10.1038/mt.2015.3) Copyright © 2015 The American Society of Gene & Cell Therapy Terms and Conditions