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Fig. 3. Normal levels of GAA expression are achieved in Gaa^-/- diaphragm after in utero rAAV2-hGAA transduction. 15 day pc Gaa^-/- fetuses were injected with saline (n=4), 3x10⁷ infectious particles (i.p.) of rAAV2-CBA-Luc (n=4), 2x10⁸ i.p. rAAV2-CBA-hGAA (n=8) or 1x10⁹ i.p. rAAV2-CMV-hGAA (n=4). (A) GAA expression was assayed from 1-month-old diaphragms of normal C57B6/129-SvJ mice and in utero-treated Gaa^-/- mice. Black circles represent individual values and bars show averaged values within experimental groups (C57B6/129-SvJ, white bar; rAAV2-CBA-hGAA, light gray bar; rAAV2-CMV-hGAA, dark gray bar). Animals within treated groups are assigned numbers to simplify explanations in the text. (B,C) Protein extracts from same animals as shown in A were separated by SDS-PAGE and the blots probed with a polyclonal antibody against human placental GAA. Normal C57B6/129-SvJ (B,C; lane 2), untreated Gaa^-/- (B,C; lane 3), rAAV2-CBA-hGAA-treated Gaa^-/- (B, lanes 4-11) and rAAV2-CMV-hGAA-treated Gaa^-/- (C, lanes 4-7) diaphragms are shown. GAA purified from human placenta was included as a positive control showing predominant isoforms of 95 kDa precursor, and 76 and 67 kDa processed forms (B,C; lane 1). Arrows indicate the 76 kDa mature form. The asterisk designates a cross-reacting protein of unknown origin.

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