Development and testing of AAV-delivered single-chain variable fragments for the treatment of methamphetamine abuse

<div><p>Methamphetamine (METH) substance abuse disorders have major impact on society, yet no medications have proven successful at preventing METH relapse or cravings. Anti-METH monoclonal antibodies can reduce METH brain concentrations; however, this therapy has limitations, including the need for repeated dosing throughout the course of addiction recovery. An adeno-associated viral (AAV)-delivered DNA sequence for a single-chain variable fragment could offer long-term, continuous expression of anti-METH antibody fragments. For these studies, we injected mice via tail vein with 1 x 10¹² vector genomes of two AAV8 scFv constructs and measured long-term expression of the antibody fragments. Mice expressed each scFv for at least 212 days, achieving micromolar scFv concentrations in serum. In separate experiments 21 days and 50 days after injecting mice with AAV-scFvs mice were challenged with METH <i>in vivo</i>. The circulating scFvs were capable of decreasing brain METH concentrations by up to 60% and sequestering METH in serum for 2 to 3 hrs. These results suggest that AAV-delivered scFv could be a promising therapy to treat methamphetamine abuse.</p></div

A comparison of METH or AMP brain and serum concentrations over time, after a 0.56 mg/kg <i>ip</i> injection of METH, between AAV-scFv6H4, AAV-scFv7F9, and a saline control at day 21 post AAV8 administration.

<p>Mice treated with either AAV-scFv6H4 or AAV-scFv7F9 showed significantly lower brain METH concentrations (a) and significantly higher serum concentrations of METH (b) than the saline-treated mice (*, p < 0.05; #, p < 0.001). There was also a significant decrease in AMP brain concentrations (c) in the AAV-scFv treated groups compared to control mice but no difference in serum AMP concentrations (d). Points are shown as mean ± SEM (n = 3–4 per group).</p

Comparison of METH and scFv molar concentrations over time.

<p>On day 0, mice were injected with PBS (control treatment), AAV-scFv6H4, or AAV-scFv7F9. Serum scFv6H4 and 7F9 concentrations were measured via ELISA on days 8 and 15. On day 22, mice were treated with METH as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0200060#pone.0200060.g006" target="_blank">Fig 6</a>. Both serum scFv and METH concentrations were measured. Mice treated with either AAV-scFv6H4 or AAV-scFv7F9 showed significantly higher serum concentrations of METH than the control mice (*, p < 0.05; #, p < 0.001). Data points are mean ± SEM (n = 3–4 per group).</p

Schematic of the prototype scFv design.

<p>V_H, variable heavy region; V_L, variable light region; Linker, 15 amino acid linker; His6, 6-histidine tag for purification and identification; FLAG, FLAG tag for identification; HMM38, a secretory signal sequence. The HMM38 at the 5’ end of the sequences is cleaved during secretion at the site indicated (triangle).</p

Expression levels of AAV-scFv constructs over time.

<p>AAVscFv-6H4 and AAV-scFv7F9 serum relative expression-time profile in AAV-scFv injected mice as measured by functional ELISA. Serum concentrations showed expression of scFv through day 212. Points are shown as mean ± SEM (n = 10 per group).</p

Comparison of IC₅₀ values for METH between culture-produced scFv and expressed AAV-scFv.

<p>A competitive binding assay was performed with each of the variants. The IC₅₀ (in nM) for both scFv and AAV-scFv variants was estimated at 50% ³H-METH bound (dotted line). Individual data points are shown as the mean ± SEM (n = 8 per group).</p