Volume 17, Issue 1, Pages 144-152 (January 2009) Undetectable Transcription of cap in a Clinical AAV Vector: Implications for Preformed Capsid in Immune Responses  Bernd Hauck, Samuel L Murphy, Peter H Smith, Guang Qu, Xingge Liu, Olga Zelenaia, Federico Mingozzi, Jürg M Sommer, Katherine A High, J. Fraser Wright  Molecular Therapy  Volume 17, Issue 1, Pages 144-152 (January 2009) DOI: 10.1038/mt.2008.227 Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 1 Transcriptional profiling of residual adeno-associated virus 2 (AAV2) cap in cell culture (HHL5 cells). (a) Schematic overview of the AAV2 cap gene, showing location of the four amplicons (hatched arrows) used for quantitative reverse transcription–PCR (Q-RT-PCR). Locations of cap sequences corresponding to immunodominant epitopes are indicated by open arrows. The human hepatocyte cell line HHL5 was transduced with AAV2-hFIX vector Lot 1053 (b), or Lot 003A (c), at doses of 1 k, 10 k, or 100 k vector genome (vg)/cell. Total RNA was isolated 48 hours after transduction and assessed by Q-RT-PCR. hFIX and cap expression are presented as fold increase relative to excipient-treated cells. Error bars show ±SD of quadruplicate wells. hFIX, human factor IX. Molecular Therapy 2009 17, 144-152DOI: (10.1038/mt.2008.227) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 2 Transcriptional profiling of residual DNA impurities in C57Bl/6 mice. (a) C57Bl/6 mice injected with AAV2-hFIX Lot 1053 at a dose of 5.6 × 1013 vector genome (vg)/kg. RNA was isolated from livers of three mice harvested at each time point, and subjected to quantitative reverse transcription–PCR (Q-RT-PCR) using AAV2 cap–specific primers and probes. Expression is shown as fold increase relative to excipient (phosphate-buffered saline)-treated cells. (b) C57Bl/6 mice injected with AAV2-hFIX Lot 003A at a dose of 1.1 × 1014 vg/kg were assessed for AAV2 cap expression as described in a. (c) RNA from C57Bl/6 mice described in a was subjected to Q-RT-PCR using AmpR and adenovirus E2A and E4-specific primers and probes. (d) RNA isolated 2 weeks after administration of Lot 1053 or Lot 003A were subjected to Q-RT-PCR using hFIX primers and probes. Error bars show ±SD of triplicate mice. AAV2, adeno-associated virus 2; hFIX, human factor IX. Molecular Therapy 2009 17, 144-152DOI: (10.1038/mt.2008.227) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 3 Density gradient fractionation of adeno-associated virus (AAV) particles. (a) AAV2-hFIX vectors [1 × 1010 vector genome (vg)] purified by the gen1-chromatography (gen1-Chr) or chromatography-gradient (chr-gr) methods were subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (10%) followed by silver staining. (b) AAV2-hFIX vector purified by the gen1-chromatography method was subjected to CsCl density gradient ultracentrifugation. Fractions (0.5 ml) collected from the bottom of the tube were analyzed for AAV capsid protein by enzyme-linked immunosorbent assay (shaded), vector genomes (squares), residual HEK 293 genomic DNA (crossouts), and residual total plasmid DNA (triangles) by quantitative PCR (Q-PCR). The density (diamonds) of individual fractions was determined by refractive index. Error bars show ±SD of triplicate Q-PCR determinations. hFIX, human factor IX. Molecular Therapy 2009 17, 144-152DOI: (10.1038/mt.2008.227) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions

Figure 4 Characterization of DNA impurities. (a) Total DNA was isolated from fractions obtained following gradient ultracentrifugation of AAV2-hFIX vector purified by the gen1-chromatography method. Denatured samples were run on an agarose gel containing SYBR Gold. Single-stranded vector genomes migrate at a position approximately corresponding to the 1,000-bp double-stranded DNA marker. M13 is a single-stranded marker (7,250 nucleotides). Plasmid pladeno 6, lacking AmpR, and HEK 293 genomic DNA, are included as negative and positive control, respectively. Topo3 is a fragment amplified from the single-copy human topoisomerase III gene, included as a single-stranded 600-nucleotide marker. (b) Southern blot of the gel shown in a was probed with 32P-labeled HEK293 genomic DNA. The position corresponding to AAV2-hFIX vector genomes (4,297 nt) is indicated by ‘vg’. (c) As described for a, except that M13, topo3, and genomic DNA were not included, and plasmid pAAV-hFIX16 plasmid, containing AmpR, was digested with SbfI and either ClaI, PshAI, or EarI, and fragments pooled, denatured, and included to provide single-stranded DNA size markers for Southern blot analysis. (d) Southern blot of the gel shown in c probed with a 32P-labeled, 612-bp fragment derived from AmpR. AAV, adeno-associated virus; hFIX, human factor IX. Molecular Therapy 2009 17, 144-152DOI: (10.1038/mt.2008.227) Copyright © 2009 The American Society of Gene Therapy Terms and Conditions