AAV-Mediated Expression of Methamphetamine Monoclonal Antibody Attenuates Methamphetamine Behaviour Sensitization in Mice

Abstract

Methamphetamine (Meth) is a psychoactive and neurotoxic chemical. Selective antibodies against Meth molecules have been examined for the treatment of Meth abuse through immunization. Antibodies with high affinity for Meth can capture Meth molecules and reduce Meth response. We previously reported that intraperitoneal administration of adeno-associated virus serotype vector serotype 8 carrying Meth-specific monoclonal antibody transgene (AAV8-MethAb, 2.5 × 10¹⁰ VGC per mouse) induced long-term and stable expression of Meth-antibody in the peripheral. Mice receiving AAV8-MethAb had a lower Meth level in the blood and brain and attenuated Meth-induced locomotor activity after an acute dose of Meth. The effect of AAV-MethAb in animals receiving repeated Meth administration was still not known. In this study, we first investigated the tropism of AAV serotypes in rat primary dopaminergic (DA) neuronal culture. We found that AAV6 is an optimal gene carrier for MethAb. AAV6-MethAb or AAV6-mCherry was used in cellular and animal models of chronic Meth use. In primary DA neuronal culture, repeated Meth administration increased the dendritic branching of DA neurons, which was antagonized by AAV6-MethAb. AAV6-MethAb or AAV6-mCherry was stereotaxically administered to the nucleus accumbens (NAc) of adult CD1 mice. Two weeks after the viral injection, animals were stimulated with a daily dose of Meth for 7 days. Repeat Meth administrations led to a progressive increase in locomotor activity or behaviour sensitization. This response was significantly attenuated in mice receiving AAV6-MethAb. Using qRTPCR and Western analysis, we demonstrated that MethAb mRNA and protein were expressed in the NAc. Previous reports indicated that Meth sensitization was associated with upregulation of tyrosine hydroxylase (TH) in the NAc. Using Western blot analysis, we found that AAV6-MethAb significantly reduced TH protein levels in Meth-sensitized mice. Taken together, our data support that intracerebral administration of AAV6-MethAb reduced Meth sensitization. Our data support a novel antibody gene therapy for Meth abuse.

Keywords: adeno‐associated virus; antibody therapy; methamphetamine; sensitization.

FIGURE 1

Diagram to illustrate the genetic construct and synthesis of MethAb from AAV‐MethAb. Both heavy‐chain and light‐chain genes of the Meth‐specific antibody (MethAb) were linked by the coding sequence of a 2A self‐cleaving peptide and driven by a cytomegalovirus promoter (CMV) in the expression vector AAV‐MethAb. The 2A peptide can cause ribosome skipping at its C‐terminal, leading to co‐translation of both heavy‐chain and light‐chain of MethAb. MethAb comprises two copies of heavy‐chain and two copies of light‐chain proteins and can be detected by anti‐V5 antibodies in the western blot analysis. ITR: inverted‐terminal repeat of AAV2; SP: signal peptide; V5: V5 tag; WPRE: woodchuck hepatitis B virus post‐transcriptional regulation element.

FIGURE 2

Effects of viral serotypes on the expression of MethAb in primary VM cell culture and in vivo. On day 5 of in vitro, rat VM cells were transduced with various serotypes of AAV‐MethAb viral vectors (AAV1, AAV2, AAV5, AAV6, AAV7 and AAV8). On day 15 of in vitro, cells were subjected to immunofluorescence staining to measure the expression of MethAb. (A) The integrated fluorescence density (IF) was calculated from images captured from five microscopic fields and normalized to the AAV1‐transduced group. (B, C) Mice were intracerebral (NAc) injected with AAV6‐MethAb or AAV6‐mCherry. NAc tissues were collected to quantify (B) MethAb mRNA and (C) mCherry mRNA by qRT‐PCR. (B) MethAb mRNA was detected only in the animals receiving AAV6‐MethAb. (C) The expression of mCherry mRNA was significantly increased in mice injected with AAV6‐mCherry. The mRNA levels were normalized to GAPDH and expressed as fold changes relative to the control (naive animal) using the 2‐∆∆Ct method. (D) NAc tissues were collected for Western blot analysis to detect the heavy chain of MethAb using anti‐V5 tag antibodies. Equal sample loading was confirmed by probing β‐actin. MethAb was only detected in mice injected with AAV6‐MethAb. Naive: rats were not injected with any viral vector. ****p < 0.0001, *p < 0.05; ns: not significant.

FIGURE 3

AAV6‐MethAb reduced Meth‐mediated dendritic branching of cultured dopaminergic neurons. (D) Timeline. Primary dopaminergic cells were transfected with AAV6‐mCherry or AAV6‐MethAb on DIV5 and were treated with Meth or vehicle on DIV 10, 12 and 14. TH immunoreactivity was examined on DIV15. (A) Confocal photomicrographs showed that low dose (1 or 10 μM) Meth administration increased the dendritic branching of the dopaminergic neurons in the control cells transducing with AAV6‐mCherry (upper panels). AAV6‐MethAb antagonized this response (lower panels). (B) The number of dendritic branches was measured from AAV6‐mCherry (veh, n = 12; 1 μM, n = 11; 10 μM, n = 12) or AAV6‐MethAb‐treated groups (n = 10, each). AAV6‐MethAb significantly reduced primary (p < 0.001, two‐way ANOVA) and secondary branch numbers (p < 0.001, two‐way ANOVA). Scale: 50 μm. (+)AO: culture media with antioxidants. (−)AO: culture media without antioxidants.

FIGURE 4

Intracerebral administration of AAV6‐Meth Ab reduced Meth‐mediated behavioural sensitization. (A) Timeline. Adult mice received bilateral NAc administration of AAV6‐mCherry (2.5 × 10⁸ VGC/μL × 2 sides; VGC: viral genome copy) or AAV6‐Meth Ab (2.5 × 10⁸ VGC/μL × 2 sides) 14 days (D‐14) before the induction of Meth sensitization. A daily dose of Meth (2.5 mg/kg/day) was given to these animals from day 0 to day 6. Control animals (saline/saline and saline/Meth) received daily saline injection (7 days, D0–D6). Meth (1 mg/kg, s.c.) induced locomotor activity was recorded on days 7 and 21 in animals receiving AAV6‐mCherry or AAV6‐MethAb, and saline/Meth. Saline/saline group animals were given saline on the test days (D7 and 21). (B–E) Repeated Meth treatment (AAV6‐mCherry) induced a significant increase in locomotor and stereotype behaviours as compared to a single Meth injection (saline/Meth), representing Meth behaviour sensitization. This sensitization response was significantly reduced in mice receiving AAV6‐Meth Ab (AAV6‐MethAb vs. AAV6‐mCherry). *Significant difference, two‐way ANOVA.

FIGURE 5

Effects of AAV6‐MethAb on TH expression in Meth‐sensitized mice. Mice were injected with AAV6‐mCherry (n = 5) or AAV‐MethAb (n = 5) in the NAc on day −14, followed by Meth sensitization from days 0 to 6. Animals were subjected to the Meth‐induced behaviour test on days 7 and 22 and sacrificed on day 23. Naive animals (n = 9) were not treated with AAV or Meth. NAc tissues were collected for Western blot analysis. (A) Representative blot demonstrated the expression of TH and β‐actin. (B) The protein levels of TH were quantified and normalized to β‐actin by densitometric analysis. *p < 0.05, **p < 0.01, ns: not significant.