Volume 16, Issue 2, Pages 280-289 (February 2008) Optimization of Self-complementary AAV Vectors for Liver-directed Expression Results in Sustained Correction of Hemophilia B at Low Vector Dose  Zhijian Wu, Junjiang Sun, Taiping Zhang, Chaoying Yin, Fang Yin, Terry Van Dyke, Richard J Samulski, Paul E Monahan  Molecular Therapy  Volume 16, Issue 2, Pages 280-289 (February 2008) DOI: 10.1038/sj.mt.6300355 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Higher human factor IX (hFIX) expression was achieved in male mice rather than in female mice regardless of whether the adeno-associated virus (AAV) vector is single-stranded (ss) or self-complementary (sc). Each vector contains a human β-globin/IgG chimeric intron, human FIX complementary DNA and mouse α-globin polyadenylation signal. hFIX expression in male versus female C57Bl/6 mice following administration of 4 × 1010 vector genomes (a) ssAAV-CBA-hFIX or (b) scAAV-TTR-hFIX via portal vein. CBA, chicken β -actin; IgG, immunoglobulin G; TTR, transthyretin. Molecular Therapy 2008 16, 280-289DOI: (10.1038/sj.mt.6300355) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Selection of transcriptional elements for self-complementary adeno-associated virus 2 (scAAV2)-mediated human factor IX (hFIX) expression in liver. (a, b) Selection of promoters. Human β-globin/IgG chimeric intron, hFIX complementary DNA (cDNA) and mouse α -globin polyadenylation (pA) signal were kept identical in scAAV2 vector. U1a1, U1b2, H2a and transthyretin (TTR) promoters were swapped in to make scAAV2 vectors for uniform in vivo comparison. Vectors were infused into C57Bl/6 male mice via portal vein at a dose of 4 × 1010 vector genomes (v.g.) Blood samples were collected at time-points indicated and serum hFIX was tested by enzyme-linked immunosorbent assay. An ssAAV-CBA-hFIX vector as shown in Figure1a was used as a benchmark. (c, d) Selection of introns. TTR enhancer–promoter, hFIX cDNA and mouse α-globin pA were fixed in the vectors, while minute virus of mice (MVM) intron and chimeric intron were swapped in between promoter and hFIX cDNA. A third scAAV vector incorporated a truncated hFIX intron 1 (m2) maintained in the natural position between exon 1 and exon 2 within the FIX coding sequence. An scAAV vector without an intron and an ssAAV vector with an MVM intron were also included in the study. A dose of 1 × 1011 v.g. AAV/mouse was used. (e, f) Selection of pA signals. TTR enhancer–promoter, MVM intron and hFIX cDNA were fixed in the vectors, while mouse α-globin pA, synthetic pA and BGHpA were swapped in downstream of hFIX cDNA. A dose of 1 × 1011 v.g. AAV/mouse was used. BGH, bovine growth hormone; CBA, chicken β-actin; gbpA, pA signal derived from mouse β -globin; IgG, immunoglobulin G; ITR, inverted terminal repeat; spA, synthetic pA. Molecular Therapy 2008 16, 280-289DOI: (10.1038/sj.mt.6300355) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Codon-optimized human factor IX (hFIXopt) complementary DNA (cDNA) achieves increased hFIX expression compared to wild-type hFIX cDNA. Human FIX cDNA and hFIXopt cDNA were placed into the identical self-complementary adeno-associated virus (scAAv) context including transthyretin (TTR) enhancer–promoter, minute virus of mice (MVM) intron and bovine growth hormone polyadenylation. Wild-type C57Bl/6 male mice were infused with 1 × 1011 v.g. AAV vectors via portal vein. Blood samples were collected at 1 week and 4 weeks post injection. (a) Human FIX levels in mice serum were tested by enzyme-linked immunosorbent assay. (b) Four weeks after vector administration, the mice were sacrificed and total DNA was extracted from the liver. The DNA was digested with XbaI and Bgl II. Southern hybridization was performed to evaluate the vector copy number. A 32P-labled Xba I-Bgl II DNA fragment containing TTR promoter region was used as probe. No difference in the amount of vector DNA persistent in mice liver was observed between the two vectors. Molecular Therapy 2008 16, 280-289DOI: (10.1038/sj.mt.6300355) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Comparison of human factor IX (hFIX) expression from ssAAV2-CM1 (closed diamond), scAAV2-hFIX (closed square) and scAAV2-hFIXopt (closed triangle) vectors. (a) Diagram of ssAAV2-CM1, scAAV2-hFIX and scAAV2-hFIXopt vectors. (b) Dose response of the vectors at 8 weeks postadministration. (c–f), hFIX expression following administration of 1E+11 v.g/mouse (c), 3E+10 v.g./mouse (d), 1E+10 v.g./mouse (e) and 3E+09 v.g./mouse (f). BGHpA, bovine growth hormone polyadenylation; cDNA, complementary DNA; ITR, inverted terminal repeat; MVM, minute virus of mice; scAAV, self-complementary adeno-associated virus; ssAAV, single-stranded AAV; TTR, transthyretin. Molecular Therapy 2008 16, 280-289DOI: (10.1038/sj.mt.6300355) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 Different distribution patterns of transgene expression from scAAV2-hFIX and ssAAV2-CM1. C57Bl/6 mice were infused with 3–10 × 1010 vector genomes (v.g.) of AAV vectors via portal vein. Twelve months later, mice were screened for circulating human factor IX (hFIX) expression and mice with equivalent plasma FIX levels (∼900 ng/ml plasma FIX in each animal) were euthanized and livers collected. (a) Messenger RNA (mRNA) in situ hybridization and immunohistochemistry were performed on these liver samples. For mRNA in situ hybridization, 10 μm sections of liver were probed using 35S labeled, in vitro transcribed antisense RNA of partial hFIX sequence. For immunohistochemistry, the livers were sectioned into 5 μm sections and embedded with paraffin. Polyclonal antibodies against hFIX raised in rabbits (DAKO) were applied to detect hFIX in liver sections. The negative controls were mice liver without vector infusion. (b) mRNA in situ hybridization was also performed on liver from mice that received identical vector doses (3.0 × 1010 v.g.). When contrasted with the small subpopulation of cells strongly transduced by ssAAV2, administration of scAAV2-hFIX vectors resulted in evenly distributed hFIX expression throughout the liver, with moderate hFIX transcription and translation within the individual hepatocytes. (c) In addition, liver from R333Q-hFIX mice, expressing endogenous hFIX from the mouse FIX locus, was stained with the same anti-hFIX antibodies to demonstrate the physiological pattern of hepatic FIX expression. KO, knockout; scAAV, self-complementary adeno-associated virus; ssAAV, single-stranded AAV. Molecular Therapy 2008 16, 280-289DOI: (10.1038/sj.mt.6300355) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 6 Comparison of human factor IX (hFIX) expression and function from ssAAV2-CM1, scAAV2-hFIX, and scAAV2-hFIXopt. FIX knockout mice were infused with 1 × 1010 vector genomes of each vector through portal vein. At the time-points indicated, blood samples were collected. (a) hFIX protein levels were tested using enzyme-linked immunosorbent assay. (b) hFIX function was tested by the hFIX-specific one-stage clotting assay (FIX activated partial thromboplastin time). scAAV, self-complementary adeno-associated virus; ssAAV, single-stranded AAV. Molecular Therapy 2008 16, 280-289DOI: (10.1038/sj.mt.6300355) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions