1. Volume 13, Issue 4, Pages 683-693 (April 2006)
Vascular bed-targeted in vivo gene delivery using tropism-modified adeno-associated viruses 
Lorraine M. Work, Hildegard Büning, Ela Hunt, Stuart A. Nicklin, Laura Denby, Nicola Britton, Kristen Leike, Margarete Odenthal, Uta Drebber, Michael Hallek, Andrew H. Baker 
Molecular Therapy 
Volume 13, Issue 4, Pages (April 2006)
DOI: /j.ymthe
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
In vivo biopanning in the WKY rat. (A) In vivo biopanning was performed as shown. Animals were injected with phage library (round 1) followed by targeted phage populations for (B) the brain and (C) the lung (rounds 2 and 3). Phage recovery from organs for each round was determined and the fold change from round 1 calculated (n = 2 for each organ and each round). (D) A number of peptide inserts from independent phage clones were sequenced (shown in parentheses) and the number of repeated consensus sequences identified at the levels of both the amino acid and its side chain classification (x/y, respectively) was determined. The number in each box represents the number of overlapping peptides between the two organs and highlights the marked level of heterogeneity of the endothelium within different organs.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Candidate targeting peptide characterization. (A) Log10 (observed) occurrences of 6/7 amino acids compared to predicted occurrences, based on amino acid frequencies in rat, human, and mouse. Peptide ID numbers are given, corresponding sequences are shown in Online Supplementary Table 1. (B) Molecular function categories for 438 of 814 genes annotated with GO categories in UniProt for the lung peptide population and for 176 of 355 genes annotated with GO categories in UniProt for the brain peptide population, showing “binding” function for 45 and 46% of genes, respectively. (C) Optimum predose KM13 concentration was determined in 12-week-old WKY rats infused with increasing doses (7 × 1010–7 × 1011 pfu) of insertless KM13 phage and recovery was determined for a number of organs. (D) Animals (n = 3) were infused with homogeneous phage populations displaying QPEHSST, VNTANST, or no peptide (control) following the KM13 predose protocol (C) and the amount of target or control phage recovered from the lung or brain (D) was determined. *P < 0.05 vs control insertless phage.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
Heparin sensitivity of the peptide-modified AAV-2. (A) Ribbon diagram of AAV-2 demonstrating the site of peptide insertion. (B) Peptide-modified AAV-2 was loaded onto a heparin-affinity column and the flowthrough, two washes, and two high-salt elution fractions were collected. These were quantified by PCR and the percentage of the total amount recovered was determined. (C) Rat Arl-6 hepatocyte cells were infected with 20,000 gp of peptide-modified AAV-2 in the presence or absence of competing heparin, and the amount of β-galactosidase activity was determined. *P < 0.05 vs transduction in the absence of heparin.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Biodistribution profiles of the peptide-modified AAV-2 in WKY rats 1 h after intravenous administration. 12-week-old, male WKY rats were infused with 8 × 1010 gp of AAV-2 and sacrificed 1 h later. (A) DNA was extracted from organs and the amount of viral DNA present determined by TaqMan real-time PCR for AAV CTL. (B) Fold changes in viral DNA accumulation between PHK-, HGP-, QPE-, and VNT-modified AAV compared to AAV CTL were determined. Representative TaqMan traces from (C) AAV QPE- or (D) AAV VNT-infused animals showing the shift in amounts of viral DNA present in the liver and lung or brain compared to AAV CTL are presented.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Biodistribution profiles of the peptide-modified AAV-2 in WKY rats 28 days after intravenous administration. Twelve-week-old, male WKY rats were infused with 4.6 × 1011 gp of AAV-2 and sacrificed 28 days later. (A) DNA was extracted from organs (n = 5 per virus group) and the amount of viral DNA present determined by TaqMan real-time PCR for AAV CTL, AAV QPE, and AAV VNT compared to a virus standard curve. *P < 0.05 vs AAV CTL in the corresponding organ; #P < 0.005 (after Bonferroni correction) vs levels in the liver of AAV CTL-infused animals. Representative TaqMan traces from (B) AAV QPE- or (C) AAV VNT-infused animals showing the shift in amounts of viral DNA present in the liver and lung or brain compared to AAV CTL are presented.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
Transgene expression localized by immunohistochemistry 28 days after intravenous infusion. Immunohistochemical detection of β-galactosidase expression in (A) liver, (B) lung, (C) brain, and (D) heart sections 28 days after virus or vehicle administration. Representative sections from seven animals/group. Arrows indicate examples of positively stained cells. Scale bar, 50 μm in (A, C, D) and 20 μm in (B).
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2005 The American Society of Gene Therapy Terms and Conditions