Undetectable Transcription of cap in a Clinical AAV Vector: Implications for Preformed Capsid in Immune Responses
Authors: 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
Journal: Molecular Therapy (2008)

In a gene therapy clinical trial for hemophilia B, adeno-associated virus 2 (AAV2) capsid–specific CD8+ T cells were previously implicated in the elimination of vector-transduced hepatocytes, resulting in loss of human factor IX (hFIX) transgene expression. To test the hypothesis that expression of AAV2 cap DNA impurities in the AAV2-hFIX vector was the source of epitopes presented on transduced cells, transcription of cap was assessed by quantitative reverse transcription–PCR (Q-RT-PCR) following transduction of target cells with the vector used in the clinical trial. Transcriptional profiling was also performed for residual AmpR, and adenovirus E2A and E4. Although trace amounts of DNA impurities were present in the clinical vector, transcription of these sequences was not detected after transduction of human hepatocytes, nor in mice administered a dose 26-fold above the highest dose administered in the clinical study. Two methods used to minimize encapsidated DNA impurities in the clinical vector were: (i) a vector (cis) production plasmid with a backbone exceeding the packaging limit of AAV; and (ii) a vector purification step that achieved separation of the vector from vector-related impurities (e.g., empty capsids). In conclusion, residual cap expression was undetectable following transduction with AAV2-hFIX clinical vectors. Preformed capsid protein is implicated as the source of epitopes recognized by CD8+ T cells that eliminated vector-transduced cells in the clinical study. In a gene therapy clinical trial for hemophilia B, adeno-associated virus 2 (AAV2) capsid–specific CD8+ T cells were previously implicated in the elimination of vector-transduced hepatocytes, resulting in loss of human factor IX (hFIX) transgene expression. To test the hypothesis that expression of AAV2 cap DNA impurities in the AAV2-hFIX vector was the source of epitopes presented on transduced cells, transcription of cap was assessed by quantitative reverse transcription–PCR (Q-RT-PCR) following transduction of target cells with the vector used in the clinical trial. Transcriptional profiling was also performed for residual AmpR, and adenovirus E2A and E4. Although trace amounts of DNA impurities were present in the clinical vector, transcription of these sequences was not detected after transduction of human hepatocytes, nor in mice administered a dose 26-fold above the highest dose administered in the clinical study. Two methods used to minimize encapsidated DNA impurities in the clinical vector were: (i) a vector (cis) production plasmid with a backbone exceeding the packaging limit of AAV; and (ii) a vector purification step that achieved separation of the vector from vector-related impurities (e.g., empty capsids). In conclusion, residual cap expression was undetectable following transduction with AAV2-hFIX clinical vectors. Preformed capsid protein is implicated as the source of epitopes recognized by CD8+ T cells that eliminated vector-transduced cells in the clinical study. IntroductionGene transfer vectors based on adeno-associated virus (AAV) have demonstrated significant promise for human gene therapy based on excellent safety and long-term efficacy in animal models, and several clinical studies have been initiated.1Carter BJ Adeno-associated virus vectors in clinical trials.Hum Gene Ther. 2005; 16: 541-550Crossref PubMed Scopus (164) Google Scholar,2Warrington KH Herzog RW Treatment of human disease by adeno-associated viral gene transfer.Hum Genet. 2006; 119: 571-603Crossref PubMed Scopus (116) Google Scholar,3Fiandaca M Forsayeth J Bankiewicz K Current status of gene therapy trials for Parkinson's disease.Exp Neurol. 2008; 209: 51-57Crossref PubMed Scopus (47) Google Scholar,4Maguire AM Simonelli F Pierce EA Pugh EN Mingozzi F Bennicelli J et al.Safety and efficacy of gene transfer for Leber's Congenital Amaurosis.N Engl J Med. 2008; 358: 2240-2248Crossref PubMed Scopus (1695) Google Scholar In a recent clinical trial in which recombinant AAV2–expressing human coagulation factor IX (AAV2-hFIX) was administered to the liver and resulted in therapeutic hFIX expression, an AAV capsid–specific CD8+ T-cell response was documented in the setting of loss of transgene expression starting 4 weeks after vector administration.5Manno CS Pierce GF Arruda VR Glader B Ragni M Rasko J et al.Successful transduction of liver in hemophilia by AAV-factor IX and limitations imposed by the host immune response.Nat Med. 2006; 12: 342-347Crossref PubMed Scopus (1535) Google Scholar,6Mingozzi F Maus MV Hui DJ Sabatino DE Murphy SL Rasko JE et al.CD8+ T-cell responses to adeno-associated virus capsid in humans.Nat Med. 2007; 13: 419-422Crossref PubMed Scopus (516) Google Scholar Mingozzi and colleagues characterized this T-cell response in samples of peripheral blood mononuclear cells obtained from clinical trial subjects, and reported that a population of AAV capsid–specific cytotoxic T lymphocytes (CTLs) expanded following vector administration with kinetics that overlapped with the presumptive elimination of the transduced hepatocytes.6Mingozzi F Maus MV Hui DJ Sabatino DE Murphy SL Rasko JE et al.CD8+ T-cell responses to adeno-associated virus capsid in humans.Nat Med. 2007; 13: 419-422Crossref PubMed Scopus (516) Google Scholar In these studies, presentation by major histocompatibility complex class I molecules of epitopes derived from the preformed, input capsid protein component of the vector inoculum was proposed as the pathway that resulted in the elimination of the hFIX-expressing cells. The delayed onset of the immune-mediated elimination of hFIX-transduced cells may be partly attributable to slow intracellular degradation of AAV2 capsids.7Thomas CE Storm TA Huang Z Kay MA Rapid uncoating of vector genomes is the key to efficient liver transduction with pseudotypes of adeno-associated virus vectors.J Virol. 2004; 78: 3110-3122Crossref PubMed Scopus (292) Google Scholar An alternative hypothesis, proposed by others to explain the provenance of capsid epitopes presented on the surface of vector-transduced cells, is that AAV cap DNA impurities present in the lot of vector used in the clinical study were expressed.8NIH Recombinant DNA Advisory Committee, AAV Symposium Immune responses to adeno-associated virus (AAV) vectors.http://www4.od.nih.gov/oba/RAC/meeting.htmlDate: 2007Google Scholar Presentation of peptides derived from de novo synthesized capsid protein through classical major histocompatibility complex class I pathways could account for the loss of vector-transduced hepatocytes observed in the clinical study if cap impurities were present in sufficient quantities in the vector lot used and were expressed in the majority of hFIX-expressing cells. Identifying the major source of the AAV capsid–derived epitopes that sensitized vector-transduced hepatocytes to recognition and elimination by CD8+ T cells in the hemophilia B clinical study is important to enable design of effective strategies to address limitations imposed by human host immune responses.Encapsidated DNA impurities in AAV vector preparations, derived from production plasmids used for transient or stable transfection, and from the producer cell genome, have been reported by several groups.9Allen JM Debeklak DJ Reynolds TC Miller AD Identification and elimination of replication-competent adeno-associated virus (AAV) that can arise by nonhomologous recombination during AAV vector production.J Virol. 1997; 71: 6816-6822Crossref PubMed Google Scholar,10Wang XS Qing K Ponnazhagan S Srivastava A Adeno-associated virus type 2 DNA replication in vivo: mutation analyses of the D sequence in viral inverted terminal repeats.J Virol. 1997; 71: 3077-3082PubMed Google Scholar,11Nony P Chadeuf G Tessier J Moullier P Salvetti A Evidence for packaging of rep-cap sequences into adeno-associated virus (AAV) type 2 capsids in the absence of inverted terminal repeats: a model for generation of rep-positive AAV particles.J Virol. 2003; 77: 776-781Crossref PubMed Scopus (41) Google Scholar,12Smith PH Wright JF Qu G Patarroyo-White S Parker A Sommer JM Packaging of host cell and plasmid DNA into recombinant adeno-associated virus particles produced by triple transfection.Mol Ther. 2003; 7: S348Google Scholar,13Chadeuf G Ciron C Moullier P Salvetti A Evidence for encapsidation of prokaryotic sequences during recombinant adeno-associated virus production and their in vivo persistence after vector delivery.Mol Ther. 2005; 12: 744-753Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar,14Report from the CHMP Gene Therapy Expert Group Meeting European Medicines Agency 2005. EMEA/CHMP/183989/2004.www.emea.europa.eu/pdfs/human/genetherapy/18398904en.pdfDate: 2005Google Scholar,15Gao G Wang Q Wang L Vandenberghe L Lock M Grant R et al.Inadvertent gene transfer of co-packaged Rep and Cap sequences during the production of AAV vector and its potential impact on vector performance.Mol Ther. 2008; 16: S105Google Scholar Nony and colleagues reported packaged rep-cap sequences in the absence of inverted terminal repeats (ITRs) at levels up to 2% of vector genomes in AAV2 vectors, and implicated a role for a Rep-binding motif (CARE) in generation of this impurity.11Nony P Chadeuf G Tessier J Moullier P Salvetti A Evidence for packaging of rep-cap sequences into adeno-associated virus (AAV) type 2 capsids in the absence of inverted terminal repeats: a model for generation of rep-positive AAV particles.J Virol. 2003; 77: 776-781Crossref PubMed Scopus (41) Google Scholar Chadeuf and colleagues reported that encapsidated prokaryotic DNA impurities derived from production plasmids were present at levels ranging from 1.2 to 6.3% in recombinant AAV vectors generated by transfection of HEK293 cells or by helper-virus infection of stable producer cell lines.13Chadeuf G Ciron C Moullier P Salvetti A Evidence for encapsidation of prokaryotic sequences during recombinant adeno-associated virus production and their in vivo persistence after vector delivery.Mol Ther. 2005; 12: 744-753Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar Gao and colleagues reported residual cap at levels ranging from 0.4 to 1.0% in 17 lots of recombinant AAV 2, 7, and 8, and that cap was transcriptionally active.15Gao G Wang Q Wang L Vandenberghe L Lock M Grant R et al.Inadvertent gene transfer of co-packaged Rep and Cap sequences during the production of AAV vector and its potential impact on vector performance.Mol Ther. 2008; 16: S105Google Scholar Wang and colleagues reported that while cross-presentation of AAV2 capsid protein could activate CTLs, vector-transduced hepatocytes were not targets for capsid-specific CTLs in mice.16Wang L Figueredo J Calcedo R Lin J Wilson JM Cross-presentation of adeno-associated virus serotype 2 capsids activates cytotoxic T-cells but does not render hepatocytes effective cytolytic targets.Hum Gene Ther. 2007; 18: 185-194Crossref PubMed Scopus (92) Google Scholar Li and colleagues reported that capsid-specific CTLs eliminated liver and muscle cells that endogenously expressed cap in cell culture and in vivo, but that cells transduced by AAV2 vectors were rarely eliminated by capsid-specific CTLs in a mouse model.17Li C Hirch M Asokan A Zeithaml B Ma H Kafri T et al.Adeno-associated virus type 2 (AAV2) capsid-specific cytotoxic T lymphocytes eliminate only vector-transduced cells coexpressing the AAV2 capsid in vivo.J Virol. 2007; 81: 7540-7547Crossref PubMed Scopus (80) Google Scholar These findings document the presence and transcriptional potential of cap and other DNA impurities in recombinant AAV preparations, and the inefficiency of preformed capsid protein to sensitize vector-transduced hepatocytes to CTL effector functions in animal models. We tested the hypothesis that expression of AAV cap impurities was the source of epitopes presented on vector-transduced cells that sensitized them to elimination by CTLs in the clinical trial. Vector-manufacturing steps that were used to minimize levels and transcriptional potential of cap and other DNA impurities in the clinical vector are described.ResultsUndetectable transcription of residual cap in the human hepatocyte cell line HHL5Primer-probe sets corresponding to four independent amplicons distributed from 5′ to 3′ in the VP3 region of the cap sequence were used to quantify residual cap in AAV2-hFIX vectors by quantitative PCR (Q-PCR), and to quantify cap transcripts following transduction of HHL5 cells and mice by Q-RT-PCR (Figure 1a). Amplicons 1–3 overlap with four known immunodominant epitopes: VPQYGYLTL and SADNNNSEY were identified as a CTL target in a human subject treated with AAV-hFIX;5Manno CS Pierce GF Arruda VR Glader B Ragni M Rasko J et al.Successful transduction of liver in hemophilia by AAV-factor IX and limitations imposed by the host immune response.Nat Med. 2006; 12: 342-347Crossref PubMed Scopus (1535) Google Scholar and TTSTRTWAL and YHLNGRDSL were identified in screening of human peripheral blood mononuclear cell samples.6Mingozzi F Maus MV Hui DJ Sabatino DE Murphy SL Rasko JE et al.CD8+ T-cell responses to adeno-associated virus capsid in humans.Nat Med. 2007; 13: 419-422Crossref PubMed Scopus (516) Google Scholar Residual cap DNA was detected in AAV2-hFIX Clinical Lot 1053 at a level of 0.00018 (±0.00006) copies per vector genome (cap/vg), an average of the values obtained using the four amplicons: 0.00021 cap/vg (amplicon 1), 0.00016 cap/vg (amplicon 2), 0.00020 cap/vg (amplicon 3), and 0.00017 cap/vg (amplicon 4). Transcription of hFIX in human hepatocyte cell line HHL5 transduced with AAV2-hFIX, Lot 1053 resulted in a dose-dependent increase in hFIX mRNA, ranging from a 14-fold increase relative to the excipient control at a dose of 1,000 vg/cell, to a 920-fold increase at 100,000 vg/cell (Figure 1b). A similar dose–response was observed using AAV2-hFIX reference vector Lot 003A (Figure 1c). In contrast, for both the clinical Lot 1053 (Figure 1b) and reference Lot 003A (Figure 1c), no significant expression of cap was detectable following transduction of HHL5 cells at any dose.Undetectable transcription of residual DNA impurities in C57Bl/6 miceTo investigate the transcriptional potential of cap and other DNA impurities in vivo, mice were injected via tail vein with AAV2-hFIX vectors at doses of 1.3 × 1012 vg/mouse (5.2 × 1013 vg/kg) (Lot 1053), or 2.7 × 1012 vg/mouse (1.1 × 1014 vg/kg) (Lot 003A). These doses were 26-fold (Lot 1053) and 56-fold (Lot 003A) higher than the highest dose (2.0 × 1012 vg/kg) that was administered in the hemophilia B clinical trial.5Manno CS Pierce GF Arruda VR Glader B Ragni M Rasko J et al.Successful transduction of liver in hemophilia by AAV-factor IX and limitations imposed by the host immune response.Nat Med. 2006; 12: 342-347Crossref PubMed Scopus (1535) Google Scholar As measured by Q-RT-PCR, hFIX mRNA levels were 161,550-fold (Lot 1053) and 337,731-fold (Lot 003A) at week 2 following vector administration elevated relative to excipient-treated mice (Figure 2d). Supraphysiological levels of hIX protein were measured in blood samples, 32.5 ± 6.4 µg/ml for Lot 1053 (n = 3) and 70.0 ± 6.0 µg/ml for Lot 003A (n = 3) at week 6 following vector administration. The levels of hFIX mRNA and protein measured after vector administration confirmed efficient transduction by both vectors. Mice injected with Ad-cap at a dose of 4 × 1012 vg/kg resulted in readily detectable cap mRNA at 1 week measured using each of the four amplicons. In contrast, AAV2 cap mRNA levels following administration of AAV2-hFIX, Lot 1053 (Figure 2a), or Lot 003A (Figure 2b) were not significantly different from those measured in excipient-treated animals. The limit of sensitivity of the Q-RT-PCR assay for cap expression, measured as the lowest copy number of cap plasmid DNA spiked into RNA isolated from excipient-treated mouse liver that gave a significant positive signal, was determined to be 20 copies per 500 ng RNA input, corresponding to ~1 copy per 2,500 cells (based on 10 pg total RNA per cell). Transcriptional profiling of mRNA for AmpR, and Ad E2A and E4 was also performed using RNA extracted from the mice that received AAV2-hFIX, Lot 1053, and no transcription of these genes was observed (Figure 2c).Figure 2Transcriptional 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Hence, using our AAV2-hFIX vectors that contained low amounts of cap impurities, cap expression was undetectable following transduction in a human hepatocyte cell line and after high-dose administration in mice. However, others have reported transcription of residual cap and other DNA impurities following transduction with AAV vectors, and higher levels of residual DNA in AAV vector preparations.11Nony P Chadeuf G Tessier J Moullier P Salvetti A Evidence for packaging of rep-cap sequences into adeno-associated virus (AAV) type 2 capsids in the absence of inverted terminal repeats: a model for generation of rep-positive AAV particles.J Virol. 2003; 77: 776-781Crossref PubMed Scopus (41) Google Scholar,13Chadeuf G Ciron C Moullier P Salvetti A Evidence for encapsidation of prokaryotic sequences during recombinant adeno-associated virus production and their in vivo persistence after vector delivery.Mol Ther. 2005; 12: 744-753Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar,15Gao G Wang Q Wang L Vandenberghe L Lock M Grant R et al.Inadvertent gene transfer of co-packaged Rep and Cap sequences during the production of AAV vector and its potential impact on vector performance.Mol Ther. 2008; 16: S105Google Scholar These data support that minimizing cap and other DNA impurities is important to reduce the potential for expression of immunogenic sequences. We identified and further characterized the steps of the vector-manufacturing process that were responsible for reducing cap and other encapsidated DNA impurities in our AAV2-hFIX vectors.Influence of vector plasmid backbone size on levels of DNA impuritiesPrevious studies reported that encapsidated plasmid DNA impurities are primarily derived from the backbone of the ITR-containing vector (cis) production plasmid.12Smith PH Wright JF Qu G Patarroyo-White S Parker A Sommer JM Packaging of host cell and plasmid DNA into recombinant adeno-associated virus particles produced by triple transfection.Mol Ther. 2003; 7: S348Google Scholar,13Chadeuf G Ciron C Moullier P Salvetti A Evidence for encapsidation of prokaryotic sequences during recombinant adeno-associated virus production and their in vivo persistence after vector delivery.Mol Ther. 2005; 12: 744-753Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar The influence of vector plasmid backbone size on the amount of residual plasmid DNA impurities in recombinant AAV2 and AAV6 was assessed. Residual plasmid DNA levels were measured by Q-PCR using primers and probes specific for AmpR, a sequence common to all three production plasmids used for vector generation (Table 1). The vectors compared in this experiment were each generated and purified using a common method (chromatography-gradient) that resulted in high vector purity, removal of empty capsids, and which included an efficient nuclease digestion step that removed nonencapsidated nucleic acid impurities.18Wright JF Le T Prado J Bahr-Davidson J Smith PH Zhen Z et al.Identification of factors that contribute to recombinant AAV2 particle aggregation and methods to prevent its occurrence during vector purification and formulation.Mol Ther. 2005; 12: 171-178Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar The average residual plasmid DNA level measured in five lots generated using a vector plasmid with an oversized (6,980 bp) backbone (Lots 06002, 003A, NHP, 0802, 0803) was 14.2 ± 2.6 pg/109 vg, 7.6-fold lower (P < 0.001) than the average value of 107.6 ± 27.6 pg/109 vg measured in five lots produced with vector plasmids with smaller (2,620 or 2,638 bp) backbones (Lots N0701, 0701, 0702, 0703, 0801). Therefore, the use of the oversized backbone in the vector production plasmid achieved a significant reduction in plasmid-derived DNA impurities.Table 1Levels of DNA impurities in purified AAV vectorsVector Lot #Purification methodSerotypeTransgene/size (nt)Backbone/size (nt)aVector (cis) production plasmid backbone size.293 DNA (pg/109 vg)Plasmid DNA (pg/109 vg)1053grad-onlybgradient-only purification method.2hFIX/42976,980—8.6DCL 1gen1-Chrcgen1-chromatography purification method.2hFIX/42976,98020844.8DCL 2gen1-Chr2hFIX/42976,98012724.2DCL 3gen1-Chr2hFIX/42976,98012230.1DCL 4gen1-Chr2hFIX/42976,9808937.906002chr-graddchromatography-gradient purification method.2hFIX/42976,9802711003Achr-grad2hFIX/42976,98020.913.0NHPchr-grad2hFIX/42976,98038.513.30802chr-grad6hFIX/42976,9807.515.80803chr-grad6hFIX/42976,9806.317.7N0701chr-grad6U/46792,63816.41220701chr-grad6U/46792,63810.177.60702chr-grad6D/48112,62012.81030703chr-grad6D/48112,6208.71470801chr-grad2cFIX/44062,62016.988.3Abbreviations: AAV, adeno-associated virus; vg, vector genome.a Vector (cis) production plasmid backbone size.a gradient-only purification method.c gen1-chromatography purification method.d chromatography-gradient purification method. Open table in a new tab Vector purification methods that minimize encapsidated DNA impuritiesWe next evaluated the influence of vector purification on the levels of DNA impurities. The levels of residual plasmid-derived and host-cell DNA impurities in eight AAV2-hFIX lots are provided in Table 1. Clinical AAV2-hFIX vector Lot 1053 was prepared by a double cesium chloride gradient ultracentrifugation method. This method included an incubation step with nuclease (Benzonase) to remove nonencapsidated DNA impurities. The amount of residual plasmid DNA impurity measured in the clinical vector in this study (8.6 pg/109 vg) was not significantly different than the value originally measured before the clinical study (6 pg/109 vg). Four other AAV2-hFIX lots were purified by the gen1-chromatography method (Lots DCL1, DCL2, DCL3, and DCL4), and three additional lots by the chromatography-gradient method (Lots 06002, 003A, and NHP). The AAV2-hFIX vector lots purified by the gen1-chromatography method, a first-generation chromatography method, resulted in the co-purification of empty capsids at levels corresponding to 89–95% of total AAV particles.19Sommer JM Smith PH Parthasarathy S Isaacs J Vijay S Kieran J et al.Quantification of adeno-associated virus particles and empty capsids by optical density measurement.Mol Ther. 2003; 7: 122-128Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar This chromatography-only method, which may be comparable to some scalable AAV vector purification processes, results in vectors that are pure based on protein staining, but which contain vector-related impurities such as empty capsids. In contrast, the vectors purified by the chromatography-gradient method, were essentially empty capsid-free as assessed by spectrophotometry.19Sommer JM Smith PH Parthasarathy S Isaacs J Vijay S Kieran J et al.Quantification of adeno-associated virus particles and empty capsids by optical density measurement.Mol Ther. 2003; 7: 122-128Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar Protein purity as assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and silver staining of AAV2-hFIX vectors purified by the gen1-chromatography and chromatography-gradient methods (Figure 3a) showed that while both methods removed non-AAV proteins, the gen1-chromatography method gave more intense capsid protein staining at equal vector genome loading, consistent with the presence of excess capsid protein. The average levels of residual mammalian and plasmid (AmpR) DNA in the four lots purified by the gen1-chromatography method were 136.5 ± 50.6 pg/109 vg and 34.3 ± 9.0 pg/109 vg, respectively. In contrast, the three lots purified by the chromatography-gradient method demonstrated 4.7-fold reduced (P = 0.016) residual mammalian DNA (28.8 ± 8.9 pg/109 vg) and 2.8-fold reduced (P = 0.010) residual plasmid DNA (12.4 ± 1.3 pg/109 vg). To understand how this reduction occurred, an aliquot of vector purified by the gen1-chromatography method, containing 2.5 × 1013 vg and 11-fold excess empty capsids, was subjected to cesium chloride gradient ultracentrifugation, and fractions were then analyzed in detail. As shown in Figure 3b, vector genome–containing particles were detected in fractions 23–33, with the highest concentration in fraction 29 (density 1.38 g/cm3), which coincided with an AAV2 capsid peak detected by enzyme-linked immunosorbent assay (shaded). A larger AAV2 capsid peak detected in fractions 45–57 (densities from 1.30 to 1.33 g/cm3) corresponded to empty capsids. Residual HEK293 and plasmid (AmpR) DNA were primarily distributed in fractions ranging from the vector genome peak to the empty capsid peak (densities from 1.30 to 1.40 g/cm3). The residual plasmid and mammalian DNA were nuclease resistant. The known higher density of DNA (>1.5 g/cm3) when not associated with capsids or other proteins, the absence of detectable noncapsid protein impurities in the gradient starting material, and the observation that the residual DNA impurities detected in the gradient fractions were nuclease-resistant, together support that the DNA impurities were associated and contained within AAV capsids, i.e., encapsidated. Southern blot analysis using a 32P-labeled HEK293 genomic probe revealed that the maximum size of residual mammalian DNA was ~4,300 nt in fraction 29, coincident with the vector genome peak, and decreased progressively in lower density fractions (Figure 4b). Southern blot analysis using an AmpR probe showed that residual plasmid DNA was similarly distributed (Figure 4d). The higher molecular weight bands visible in fractions 29–35 in Figure 4d may correspond to double-stranded DNA resulting from re-annealing of complementary plasmid sequences during sample preparation. The size and density distribution, and nuclease resistance of the DNA impurities in the gradient fractions further support that they are single-stranded DNA impurities contained within AAV2 capsid particles.Figure 3Density 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Characterization 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.View Large Image