1. Volume 10, Issue 4, Pages 671-678 (October 2004)
Efficient transduction of skeletal muscle using vectors based on adeno-associated virus serotype 6 
Michael J. Blankinship, Paul Gregorevic, James M. Allen, Scott Q. Harper, Hollie Harper, Christine L. Halbert, Dusty A. Miller, Jeffrey S. Chamberlain 
Molecular Therapy 
Volume 10, Issue 4, Pages (October 2004)
DOI: /j.ymthe
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

2. FIG. 1
Purification and concentration of rAAV6 vectors. (A) A representative tracing from an rAAV heparin-affinity purification with OD280 and conductivity in microsiemens (μs) shown. The loading, wash, and elution stages are denoted L, W, and E, respectively. The detergent eluted peak is noted with an asterisk. (B) An SDS–PAGE gel of samples of rAAV6 vectors collected after heparin column purification stained with Coomassie blue for viral capsid protein detection (VP1, VP2, and VP3). The gel was loaded with either 5 × 109 or 4 × 1010 vector genomes (vg) of material from rAAV2 and rAAV6 vectors expressing either microdystrophin or β galactosidase.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

3. FIG. 2
Persistent transduction of murine skeletal muscles by rAAV6-pseudotyped vectors compared with rAAV2 vectors. Histochemical reaction for β-galactosidase activity (blue) of transverse cryosections obtained from the extensor digitorum longus (EDL) and soleus muscles of mice injected with 2 × 109 vg of rAAV2:CMV-lacZ or rAAV6:CMV-lacZ vectors 2, 4, 8, or 12 weeks before examination. Sections are counterstained with eosin. Representative examples from n = 4 to 6 individual muscles. Scale bar, 500 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

4. FIG. 3
rAAV6-pseudotyped vectors demonstrate increased expression in mouse limb muscles compared with rAAV2 vectors. Mean β-galactosidase activity of (a) extensor digitorum longus (EDL, a) and soleus (b) hindlimb muscles of untreated mice (white bars) and mice administered intramuscular injections of 2 × 105 (light grey), 2 × 107 (grey), or 2 × 109 (black) vg of rAAV2:CMV-lacZ (columns labled 2) or rAAV6:CMV-lacZ (column labled 6) vectors examined 2 or 4 weeks after injection. Mean β-galactosidase activity of EDL (c) and soleus (d) muscles of mice examined 2, 4, or 8 weeks after intramuscular injections of 2 × 109 vg of rAAV2:CMV-lacZ (grey) or rAAV6:CMV-lacZ (black) vectors. Mean activity at time 0 represents samples of respective uninjected muscles.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

5. FIG. 4
Mosaic reporter gene expression in murine muscle fibers administered rAAV vectors is not associated with phenotypic aspects related to distinct myosin heavy chain isoforms. Transverse cryosections of extensor digitorum longus (EDL; predominantly fast fiber type) and soleus (mixed fiber type) muscles of mice examined 8 weeks after intramuscular injection of 2 × 109 vg of rAAV2:CMV-lacZ or rAAV6:CMV-lacZ vectors. Serial sections of each muscle were processed with immunofluorescence detection techniques specific to β-galactosidase (β-Gal) or myosin heavy chain isoform I, IIa, or IIb. Symbols identify a cell on serial sections within each muscle for orientation and comparison of serial sections. Scale bar, 200 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

6. FIG. 5
Intrathoracic administration of rAAV6 vectors to adult mice yields dramatic transduction of skeletal muscles compared with rAAV2 vectors. Gross anatomy of the forelimb and ribcage of an untreated adult mouse and mice examined 4 weeks after intrathoracic injection of 5 × 1011 vg of rAAV2:CMV-lacZ or rAAV6:CMV-lacZ vector that have been histochemically reacted for β-galactosidase activity (blue). Transverse cryosections of the diaphragm muscle were also examined for β-galactosidase activity using histochemical methods (blue, counterstained with eosin) and immunofluorescent detection (green). Examples shown are representative of n = 5 for each group. Scale bar, 500 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

7. FIG. 6
rAAV6-pseudotyped vectors achieve body-wide transgene expression after intraperitoneal injection in newborn (∼3 days of age) mice. Medial aspects of the fore- and hindlimbs and inferior surface of the diaphragm muscles of an uninjected mouse and a mouse examined 2 weeks after intraperitoneal injection of ∼1 × 1011 vg rAAV6:CMV-lacZ at 3 days of age. Images in the right column show immunofluorescent signal detection of β-galactosidase activity (green) in transverse cryosections of the diaphragm muscles of additional, similarly treated mice. Examples are representative of levels of transduction from ≥12 animals per individual serotype. Scale bar, 500 μm.
Molecular Therapy  , DOI: ( /j.ymthe )
Copyright © 2004 The American Society of Gene Therapy Terms and Conditions