1. Volume 16, Issue 12, Pages 1944-1952 (December 2008)
A Single Intravenous Injection of Adeno-associated Virus Serotype-9 Leads to Whole Body Skeletal Muscle Transduction in Dogs 
Yongping Yue, Arkasubhra Ghosh, Chun Long, Brian Bostick, Bruce F Smith, Joe N Kornegay, Dongsheng Duan 
Molecular Therapy 
Volume 16, Issue 12, Pages (December 2008)
DOI: /mt
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

2. Figure 1
Local adeno-associated virus-9 (AAV-9) injection leads to robust transduction in neonatal, but not adult, canine muscles. (a) Representative photomicrographs of alkaline phosphatase (AP) and hematoxylin & eosin (HE) staining after direct muscle injection in the cranial sartorius muscle. [Adult, 4 × 1011 vector genome (vg) particles, 8 weeks after injection; newborn, 1.6 × 1011 vg particles, 10 weeks after injection. N = 2 for each group]. Arrow, rare AP positive myofibers in AAV-9 infected adult dog muscle. (b) Representative photomicrographs of CD4+ and CD8+ T-cell immunostaining at 8 weeks after local muscle injection in adult dogs (AAV, 4 × 1011 vg particles; mock, HEPES buffer. N = 2 for each group). Arrow, CD4+ and CD8+ T cells.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

3. Figure 2
Schematic outline of systemic AAV-9 AV.RSV.AP infection in newborn dogs. Left column shows dog's name and injection details [adeno-associated virus (AAV) or HEPES buffer, dosage]. Arrow, age at the time of AAV delivery; arrowhead, age at the time of biopsy; asterisk, age at the time of necropsy. vg, vector genome.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

4. Figure 3
Skeletal muscle transduction efficiency correlates with vector dose used in systemic delivery. (a) Representative photomicrographs of alkaline phosphatase (AP) staining from the cranial sartorius muscles at 2 and 6 weeks after intravenous adeno-associated virus-9 (AAV-9) delivery, respectively. Dog's name is marked for each panel. Caleb and Seth received 1 × 1011 vector genome (vg)/g body weight AAV-9 AV.RSV.AP. Miles and Dojo were injected with 2.5 × 1011 and 2 × 1011 vg/g body weight AAV-9 AV.RSV.AP, respectively. (b) Confirmation of histochemical staining with immunofluorescence staining. Serial muscle sections from Dojo's biopsy were stained for AP expression by histochemical (left panel) and immunofluorescence staining (right panel) using a human placental AP specific antibody. p, a myofiber that was transduced by AAV-9; n, a myofiber that was not transduced by AAV-9.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

5. Figure 4
Systemic adeno-associated virus-9 (AAV-9) delivery in newborn dogs shows a fiber type, microvasculature, and receptor-independent muscle preference. (a) Representative photomicrographs of alkaline phosphatase (AP) staining and AP positive cell quantification from the high-dose group (Dojo and Miles) biopsy at 2, 3, and 6 weeks after AAV-9 injection. Biopsy from a mock-infected dog (Frank) served as the negative control. (b) Both slow and fast twitch myofibers were equally transduced by AAV-9. Serial muscle sections were stained for AP expression and fiber type. Cross, a slow fiber transduced by AAV-9; double cross, a slow fiber not transduced by AAV-9; asterisk, a fast fiber transduced by AAV-9; pound sign, a fast fiber not transduced by AAV-9. (c) Quantification of microvasculature density in different canine muscles (left panels) and representative photomicrographs of arteriole staining (red circles in the top right panel) and capillary staining (blue dots in the bottom right panel). Low, muscles that were poorly transduced by AAV-9 at the dose of 1 × 1011 vector genome (vg)/g body weight; medium, muscles that were moderately transduced by AAV-9 at the dose of 1 × 1011 vg/g body weight; high, muscles that were efficiently transduced by AAV-9 at the dose of 1 × 1011 vg/g body weight. Abd, abdominal muscle; CS, cranial sartorius muscle; CT, cranial tibialis muscle; Dia, diaphragm; Gas, gastrocnemius muscle; I-cost, intercostal muscle; LDE, long digital extensor muscle; Tong, tongue; VL, vastus lateralis muscle. Scale bar in each panel applies to all images in the same panel. (d) Representative immunofluorescence staining of AAV-9 receptor laminin receptor (LamR) in the CS, VL, and LDE muscles. LamR is highly enriched in slow-twitch myofibers. Scale bar applies to all the images in the same row.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

6. Figure 5
Systemic adeno-associated virus-9 delivery in neonatal puppies does not induce a cellular immune response. Representative photomicrographs of hematoxylin & eosin (HE), alkaline phosphatase (AP), CD4+, and CD8+ T-cell staining from 2-week [Miles, infected at 2.5 × 1011 vector genome (vg)/g body weight; top panels] and 6-week (Dojo, infected at 2 × 1011 vg/g body weight; bottom panels) muscle biopsy. Scale bar applies to all images.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

7. Figure 6
Evaluation of low-dose (1 × 1011 vector genome/g body weight) adeno-associated virus-9 transduction at 10 weeks after intravenous delivery. (a) Representative photomicrographs of alkaline phosphatase (AP) staining of different skeletal muscles obtained at necropsy from Caleb and Seth. Negative control is from the cranial sartorius muscle of a mock-infected dog (Frank). BB, biceps brachii muscle; CS, cranial sartorius muscle; CT, cranial tibialis muscle; ECR, extensor carpi radialis muscle. (b) Representative photomicrographs of AP staining from selective internal organs. Arrow, rare occurring AP positive cells. Enlarged images are shown in the boxes affiliated with each respective panel.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

8. Figure 7
Evaluation of high-dose [2× 1011 and 2.5 × 1011 vector genome (vg)/g body weight for Dojo and Miles, respectively] adeno-associated virus-9 (AAV-9) transduction at 6 months after intravenous delivery. (a) Representative photomicrographs of alkaline phosphatase (AP) staining from two representative head muscles, the tongue, and the diaphragm. (b) Representative photomicrographs of AP staining from four representative neck muscles. (c) Representative photomicrographs of AP staining from four representative chest muscles. (d) Representative photomicrographs of AP staining from four representative abdominal muscles. (e) Representative photomicrographs of AP staining from four representative limb muscles. (f) Quantification of AP activity in muscle lysates. N, sample size for each muscle. AM, abdominal muscles (including rectus, transverse, external and internal muscles); BB, biceps brachii muscle; BF, biceps femoris muscle; BT, brachial triceps muscle; CS, cranial sartorius muscle; CT, cranial tibialis muscle; Dia, diaphragm; EC, extensor carpi muscle; Fro, frontoscutularis muscle; Gas, gastrocnemius muscle; IM, intercostal muscles (including both external and internal muscles); LAL, long auricular levator muscle; LD, latissimus dorsi muscle; Neg, 27 skeletal muscles from Frank; NM, neck muscles (including sternohyoideus muscle, sternocephalicus muscle, cleidocervicalis muscle, and trapezius muscle); Occ, occipitalis muscle; Pec, pectineus muscle; PM, pectoral muscles (including both superficial and deep pectoral muscles); Spi, spinate muscles (including both supra and infra muscles); Ton, tongue. (g) Quantification of AAV genome in selected muscles and internal organs. AM, abdominal muscles; BB, biceps brachii muscle; BF, biceps femoris muscle; BT, brachial triceps muscle; CS, cranial sartorius muscle; CT, cranial tibialis muscle; Dia, diaphragm; DPM, deep pectoral muscle; EC, extensor carpi muscle; Fro, frontoscutularis muscle; Gas, gastrocnemius muscle; LAL, long auricle levator muscle; LDE, long digital extensor muscle; SPM, superficial pectoral muscle. (h) Representative photomicrographs of AP staining and AP activity quantification from the heart. LA, left atrium; LV, left ventricle; RA, right atrium; RV, right ventricle. HEPES, heart sample from Frank (N = 5 samples from different locations of the same heart); AAV-9, heart sample from Dojo and Miles (N = 7 samples from different locations of the heart). (i) Representative photomicrographs of AP staining from some internal organs and smooth muscles. Arrow, positive AP staining in the glomerulus in the kidney; Arrow head, positive AP staining in the skeletal muscle layer of the esophagus. Scale bars in e apply to all images in the respective rows in a to e. Scale bars in g and h only apply to images in that panel.
Molecular Therapy  , DOI: ( /mt )
Copyright © 2008 The American Society of Gene Therapy Terms and Conditions