Figure 1.
Purification of murine BChE (mBChE).
A) SDS-PAGE gel with single major protein bands after purification of wild type mouse enzyme and the cocaine hydrolase mutant (mCocH); B) Active-site titration of mCocH with diisopropylfluorophosphate.
Table 1.
Summary of kinetic constants for hydrolysis of cocaine, acetylcholine (ACh), and butyrylthiocholine (BThCh) by wild type mouse BChE as previously determined [14], [15] and mCocH, determined as described in Methods.
Figure 2.
Expression of mouse and human CocH with AAV vector.
A). Mice were treated with either of two different i.v. doses of AAV-CMV-mCocH vector: 7×1010 viral particles (filled circles, n = 4) or 3×1011 viral particles (open circles, n = 12). Other mice were given empty vector or saline treatment. Shown are cocaine hydrolase activities (mU/ml) in plasma samples at the indicated times (data from control mice are not represented because cocaine hydrolysis was barely measurable, less than 0.1 mU/ml). Note duration approaching 2 years. B). AAV-CMV-hCocH vector (1011 viral particles) was administered (n = 10), and cocaine hydrolase activities were monitored during the comparatively brief expression (time scale in weeks).
Figure 3.
Turnover of vector-delivered mCocH and native BChE.
Mice treated 5 months beforehand with 3×1011 particles of AAV vector encoding mCocH, and mice with no prior treatment, received 50 mg/kg of iso-OMPA at time zero, causing ∼95% inhibition of plasma CocH/BChE activities. Time-course of recovery was monitored as an index of replacement by newly synthesized enzyme. Relative rates are shown in activities as percent of pre-treatment levels.
Table 2.
Anti-BChE antibodies detected by immunoprecipitation.
Figure 4.
Effect of vector promoter and vector dose on mCocH expression.
A) Mice were given injections of AAV-CMV-mCocH vector (3×1011 viral particles) or AAV-VIP mCocH vector (1011 viral particles). Plasma samples were collected at the indicated time points and assayed for cocaine hydrolysis activity. Means and standard errors of the means are shown (n = 12). B) Mice received AAV-VIP-mCocH vector in the indicated doses, and cocaine hydrolase activity was measured repeatedly from 2 weeks to 2 months. Data, in mU, are reported on a logarithmic scale. Means and standard errors are shown (n = 10 per group).
Figure 5.
Quantitative PCR evidence for liver as primary site of vector.
Mice were euthanized 2 months after i.v. injection of AAV-VIP vector with mutated BChE at a dose of 1012 viral particles per animal. Vector DNA in the indicated tissues was quantitated by qPCR and expressed as viral genome copies per ng of host DNA.
Figure 6.
Extreme mCocH expression with high-dose hdAD vector.
Mice (n = 10) were given vector injections of 1.7×1012 viral particles. Circulating levels of cocaine hydrolase activity are shown (note ordinate scale in units (U) rather than milliunits).
Figure 7.
Spontaneous and stimulant-induced locomotor activity in control mice and mice transduced with mCocH by high-dose hdAD vector.
Subjects (n = 8 to 12) were tested in locomotor activity chambers 6 weeks after receiving hdAD-mCocH vector. Controls were previously untreated animals. Shown are means and SEM of locomotor activity (cm traveled per 2-min bin) from hour-long sessions on consecutive days beginning immediately after injection of saline (days 1–3), cocaine 40 mg/kg (day 4), and D-amphetamine 5 mg/kg (day 7). One-way analysis of variance indicated a highly significant difference between treatment groups (F = 10.6, p<0.001). Statistical significance of the most relevant significant pairwise multiple comparisons is indicated by asterisks (* t >3.1, p<0.01; ** t >3.8, p<0.001). The complete analysis is available in Supplementary Materials (Table S2).
Table 3.
Biomarker tests of liver, muscle and heart toxicity in vector-treated animals.