Volume 13, Issue 1, Pages 167-174 (January 2006) Dimerizer regulation of AADC expression and behavioral response in AAV-transduced 6- OHDA lesioned rats  Laura M. Sanftner, Victor M. Rivera, Brian M. Suzuki, Lan Feng, Lori Berk, Shangzhen Zhou, John R. Forsayeth, Tim Clackson, Janet Cunningham  Molecular Therapy  Volume 13, Issue 1, Pages 167-174 (January 2006) DOI: 10.1016/j.ymthe.2005.06.480 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 1 In AAV-CMV-TF, the cytomegalovirus (CMV) enhancer/promoter drives expression of a bicistronic message encoding the activation and DNA binding domain fusions. The activation domain fusion contains the rapamycin binding domain of human FRAP (FRB) fused to the transcriptional activation domain derived from the p65 subunit of NF-κB (p65). The internal ribosome entry sequence (IRES) is derived from encephalomyocarditis virus. The DNA binding domain fusion contains two DNA binding domains from the human transcription factor Zif268, a homeodomain derived from Oct-1 (ZFHD1), and three drug-binding domains from the cytosolic receptor for FK506 (3× FKBP). In AAV-Z12-hAADC, expression of human AADC is controlled by 12 binding sites for the transcription factor fused to a minimal IL-2 promoter. The AAV2 inverted terminal repeat (ITR), hAADC coding sequence (hAADC), and human growth hormone polyadenylation (pA) are indicated. Molecular Therapy 2006 13, 167-174DOI: (10.1016/j.ymthe.2005.06.480) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 2 In vitro transduction of HeLa D7-4 cells. Relative optical density was measured in an AADC expression ELISA. Cells transduced with AAV-CMV-TF + AAV-Z12-hAADC (transcription factor vector plus the regulated AADC vector) at a 1:1 ratio and treated with three different concentrations of the rapamycin analog AP21967 induced gene expression in a dose-responsive manner. The graph also depicts results from various controls including nontransduced cells, transcription factor vector alone (AAV-CMV-TF), constitutively expressed hAADC (AAV-CMV-hAADC2), and regulated AADC alone (AAV-Z12-hAADC). Molecular Therapy 2006 13, 167-174DOI: (10.1016/j.ymthe.2005.06.480) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 3 (A) Experimental time line for monitoring rotational response to l-dopa in 6-OHDA-lesioned rats and (B) rotational response to l-dopa in 6-OHDA-lesioned rats. (A) A baseline rotational test of 5 mg/kg l-dopa was given to all rats prior to treatment. Vector or excipient was infused intrastriatally on day 0. On day 17 rats scheduled to receive rapamycin were induced with rapamycin or diluent. Induction consisted of 4 consecutive days of ip injection of 10 mg/kg/day of rapamycin. On day 21 rats were again tested for a rotational response to 5 mg/kg l-dopa. Rats were allowed to recover from rapamycin for 1 week and then tested for a response on day 28. Rats were induced a second time on day 31 and tested for a rotational response on day 35. After recovery from rapamycin for a week, the rotational test was repeated on day 42. On day 45 rats received the third and final course of induction followed by a final rotational test on day 49. Animals were euthanized and processed for immunohistochemistry at this time. (B) Rotational response to l-dopa in 6-OHDA-lesioned rats. At weeks 3, 5, and 7 the rotational response of the vector-infused (+) rapamycin group was significantly increased above that of both the vector-infused (−) rapamycin group and the excipient control group (P < 0.001). The vector-infused (−) rapamycin and the excipient control groups were not significantly different at any of the time points (P > 0.05). At the preinfusion time point and at time points following withdrawal of rapamycin (weeks 4 and 6) the vector-infused (+) rapamycin group was not significantly different from the two control groups (P > 0.05). Statistical differences were compared by using one-way ANOVA for multiple groups. Rapamycin induction was 10 mg/kg/day for 4 consecutive days. Arrows indicate rapamycin induction. Molecular Therapy 2006 13, 167-174DOI: (10.1016/j.ymthe.2005.06.480) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Immunohistochemistry for AADC within the rat striatum. (A–C) Immunostaining at high magnification is localized to the medium spiny neurons in the rat striatum at 7 weeks postinfusion. Representative sections from (A) the vector-infused (+) rapamycin group, (B) the vector-infused (−) rapamycin group, and (C) the excipient-infused (+) rapamycin group are shown. Scale bar, 75 μm. (D–F) Representative animals from each group are shown in whole-mounted brain sections in cross section through the infusion site at 7 weeks postinfusion. Animals infused with AAV-CMV-TF + AAV-Z12-hAADC (3 × 1010 vg of each vector) (D) with rapamycin induction or (E) without rapamycin are shown in comparison to (F) the excipient control with rapamycin. Left hemisphere: site of both 6-OHDA lesion and intrastriatal vector (or excipient) infusions (D–F). Right hemisphere: endogenous staining is present from intact rat AADC-positive fibers (D–F). Molecular Therapy 2006 13, 167-174DOI: (10.1016/j.ymthe.2005.06.480) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

FIG. 5 Modulation of hAADC expression by rapamycin induction. There was an increase in hAADC expression when the vector-infused rats were induced with rapamycin at several time points and examined at 7 weeks post-vector infusion. Top: Western blot bands from representative vector-infused (+/−) rapamycin and excipient-infused (+) rapamycin unilaterally 6-OHDA-lesioned rats showing changes in protein levels of hAADC (50 kDa) within the striatum. β-Actin is a loading control. Bottom: Analysis of band density of Western blots between vector-infused and excipient-infused rats. Band density was significantly increased in the vector-infused (+) rapamycin group in comparison to both control groups, P < 0.001. Molecular Therapy 2006 13, 167-174DOI: (10.1016/j.ymthe.2005.06.480) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions