The Development and Characterization of an scFv-Fc Fusion-Based Gene Therapy to Reduce the Psychostimulant Effects of Methamphetamine Abuse

Methamphetamine (METH) continues to be among the most addictive and abused drugs in the United States. Unfortunately, there are currently no Food and Drug Administration-approved pharmacological treatments for METH-use disorder. We have previously explored the use of adeno-associated viral (AAV)-mediated gene transfer of an anti-METH monoclonal antibody. Here, we advance our approach by generating a novel anti-METH single-chain variable fragment (scFv)-Fc fusion construct (termed 7F9-Fc) packaged into AAV serotype 8 vector (called AAV-scFv-Fc) and tested in vivo and ex vivo. A range of doses [1 × 10 , 1 × 10 , and 1 × 10 vector copies (vcs)/mouse] were administered to mice, eliciting a dose-dependent expression of 7F9-Fc in serum with peak circulating concentrations of 48, 1785, and 3831 µg/ml, respectively. Expressed 7F9-Fc exhibited high-affinity METH binding, IC = 17 nM. Between days 21 and 35 after vector administration, at both 1 × 10 vc/mouse and 1 × 10 vc/mouse doses, the AAV-7F9-Fc gene therapy significantly decreased the potency of METH in locomotor assays. On day 116 post-AAV administration, mice expressing 7F9-Fc sequestered over 2.5 times more METH in the serum than vehicle-treated mice, and METH concentrations in the brain were reduced by 1.2 times the value for vehicle mice. These data suggest that an AAV-delivered anti-METH Fc fusion antibody could be used to persistently reduce concentrations of METH in the central nervous system. SIGNIFICANCE STATEMENT: In this manuscript, we describe the testing of a novel antimethamphetamine (METH) single-chain variable fragment-Fc fusion protein delivered in mice using gene therapy. The results suggest that the gene therapy delivery system can lead to the production of significant antibody concentrations that mitigate METH's psychostimulant effects in mice over an extended time period

The development and characterization of an scFv-Fc fusion based gene therapy to reduce the psychostimulant effects of methamphetamine abuse

Abstract Methamphetamine (METH) continues to be amongst the most addictive and abused drugs in the US. Unfortunately, there are currently no FDA approved pharmacological treatments for METH substance abuse disorder. As an alternative approach, we have previously explored the use of Adeno-associated viral (AAV) mediated gene transfer of an anti-METH monoclonal antibody. Here, we advance our approach by generating a novel anti-METH scFv-Fc fusion construct (7F9-Fc), packaged into AAV serotype 8 vector (called AAV-scFv-Fc), and tested in vivo and ex vivo. A range of doses (1 × 10 10 . 1 × 10 11 , and 1 × 10 12 vector copies(vc)/mouse) were administered to mice, which exhibited a dose-dependent expression of 7F9-Fc in serum with peak circulating concentrations of 48, 1785, and 3,831 μg/ml. The dose of 1 × 10 12 vc/mouse was further tested in METH locomotor and biodistribution studies to determine the efficacy of the antibody protection. Expressed 7F9-Fc exhibited high affinity binding, 17 nM, to METH. Between days 21 and 35 after vector administration, the 7F9-Fc gene therapy significantly reduced the effects of METH in locomotor assays following administration of moderate and high doses of subcutaneous METH, 3.1 and 9.4 mg/kg respectively. On day 116 post-AAV administration, mice expressing 7F9-Fc sequestered over 2.5 times more METH into the serum than vehicle mice, and METH concentrations in the brain were reduced by 1.2 times compared to vehicle mice. Taken together, these data suggest that a AAV-delivered anti-METH Fc fusion antibody could be a design for persistently reducing concentrations of METH in the CNS. Significance Statement In this manuscript, we describe the use of a novel anti-METH scFv-Fc fusion protein delivered in mice using gene therapy. The results suggest that the gene therapy delivery system can lead to the production of enough antibody to mitigate METH’s psychostimulant effects in mice over an extended time period

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

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 10(12) 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 in vivo. 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

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

Hydrodynamic AAV-scFv plasmid transfected mice expressed functional anti-METH scFv-6H4 and scFv-7F9.

<p>³H-METH binding was determined by equilibrium dialysis. Purified scFv7F9 (4 μg/ml) was used as a positive control. ScFv6H4 transfected serum samples bound ³H-METH significantly higher than GFP (negative control, nonspecific serum binding) [#, p < 0.001; *, p < 0.01] at 24 and 48 hrs post-AAV8 treatment. ScFv7F9 also bound more, but was not statistically significant. (b). Points are shown as mean ± SEM (n = 3 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

Pharmacokinetic study.

<p>Fifty days after AAV-scFv treatment, mice were injected <i>sc</i> with 1 mg/kg METH. Blood samples were collected at 30 and 120 or 60 and 180 min after METH injection. LC-MS/MS was used to determine serum METH concentrations. Because samples were not collected from every mouse at every time point, a non-repeated measures two-way ANOVA was used to analyze the METH concentration-time data. There were significant differences between control and AAV-scFv7F9 treated mice at 60 min post injection and between sham and both AAV-scFv groups at 180 min (* p < 0.05). Data points are shown as mean ± SEM (n = 5–6 per group).</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