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GDNF gene therapy for alcohol use disorder in male non-human primates

Nature Medicine volume 29, pages 2030–2040 (2023)Cite this article

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Abstract

Alcohol use disorder (AUD) exacts enormous personal, social and economic costs globally. Return to alcohol use in treatment-seeking patients with AUD is common, engendered by a cycle of repeated abstinence-relapse episodes even with use of currently available pharmacotherapies. Repeated ethanol use induces dopaminergic signaling neuroadaptations in ventral tegmental area (VTA) neurons of the mesolimbic reward pathway, and sustained dysfunction of reward circuitry is associated with return to drinking behavior. We tested this hypothesis by infusing adeno-associated virus serotype 2 vector encoding human glial-derived neurotrophic factor (AAV2-hGDNF), a growth factor that enhances dopaminergic neuron function, into the VTA of four male rhesus monkeys, with another four receiving vehicle, following induction of chronic alcohol drinking. GDNF expression ablated the return to alcohol drinking behavior over a 12-month period of repeated abstinence–alcohol reintroduction challenges. This behavioral change was accompanied by neurophysiological modulations to dopamine signaling in the nucleus accumbens that countered the hypodopaminergic signaling state associated with chronic alcohol use, indicative of a therapeutic modulation of limbic circuits countering the effects of alcohol. These preclinical findings suggest gene therapy targeting relapse prevention may be a potential therapeutic strategy for AUD.

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Fig. 1: Chronic alcohol self-administration in rhesus macaques recapitulates drinking in humans.
Fig. 2: AAV2-hGDNF delivery to the VTA.
Fig. 3: AAV2-hGDNF treatment curbs excessive drinking over a 1-year period.
Fig. 4: Effects of AAV2-hGDNF on food and water intake, weight, sensorimotor tasks and alternative reward motivation.
Fig. 5: AAV2-hGDNF effects on GDNF expression, DA concentrations, and DA metabolism.
Fig. 6: AAV2-hGDNF treatment alters dopaminergic neuron dynamics and modulation in the NAc.

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Data availability

The data that support the findings of this study, including behavioral, biochemical, and voltammetry analyses, are available on the public data repository Zenodo.org at https://zenodo.org/record/7236274.

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Acknowledgements

We thank N. Newman, K. Diem, H. VanderJagt, C. Rudnicky, J. Schoen and J. Mootz for animal husbandry and data collection of daily drinking sessions; S. Gonzales for technical support with drinking session programming and equipment maintenance; and B. Park for consultation with statistical modeling and experimental design. We also acknowledge the contributions of C. Kroenke and M. Reusz for MRI support; L. Martin and T. Hobbs for surgical services support; A. Lewis, A. Johnson and L. Colgin, as well as W. Price and A. Beckman, for pathology services support; M. Travis and V. Sudhakar for histology support; the Electrophysiology Core overseen by V. Cuzon-Carlson for hosting and supporting the voltammetry experiments; the Molecular Virology Core at the Oregon National Primate Research Center (ONPRC) for performing viral serology assays; and the Clinical Pathology Laboratory (ONPRC) for conducting in-house assays for complete blood count and a comprehensive chemistry panel. This study was funded by National Institute on Alcohol Abuse and Alcoholism (NIAAA) grant R01 AA024757 (to M.M.F.). Additional support was provided by NIH grants U01 AA013510 (K.A.G.), U01 AA014091 (S.R.J.), P60 AA010760 (K.A.G. and M.M.F.), R24 AA019431 (K.A.G.) and P50 AA026117 (S.R.J.).

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Author notes

  1. These authors contributed equally: Matthew M. Ford, Brianna E. George, Victor S. Van Laar, Katherine M. Holleran.

Authors and Affiliations

  1. Division of Neuroscience, Oregon National Primate Research Center, Oregon Health & Science University, Portland, OR, USA

    Matthew M. Ford, Lauren E. Vanderhooft, Jodi L. McBride & Kathleen A. Grant

  2. Department of Psychology, Lewis & Clark College, Portland, OR, USA

    Matthew M. Ford

  3. Department of Physiology and Pharmacology, Wake Forest School of Medicine, Winston Salem, NC, USA

    Brianna E. George, Katherine M. Holleran, Emily G. Peck, Monica H. Dawes & Sara R. Jones

  4. Department of Neurological Surgery, The Ohio State University, Columbus, OH, USA

    Victor S. Van Laar, Jerusha Naidoo, Piotr Hadaczek, Lluis Samaranch & Krystof S. Bankiewicz

  5. Department of Neurological Surgery, University of California, San Francisco, CA, USA

    Jerusha Naidoo, Piotr Hadaczek, Kousaku Ohno, John Bringas, Lluis Samaranch, John R. Forsayeth & Krystof S. Bankiewicz

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Contributions

M.F. supervised the research and oversaw coordination across laboratories, designed and performed experiments, analyzed and interpreted data and wrote and edited the manuscript. B.G. and K.H. performed voltammetry experiments, analyzed and interpreted data, assisted in drafting results section and edited the manuscript. V.V. performed tissue immunohistochemistry and microscopy, assisted on data analysis and interpretation, and assisted with manuscript writing and editing. J.N. assisted with coordination of intra-VTA infusions, analyzed GDNF ELISA and HPLC results, and assisted in drafting the results section. P.H. performed ELISA experiments, assisted with interpretation of viral serology results, and edited the manuscript. L.V. served as project lead of animal cohort, collected and analyzed data, coordinated institutional services associated with husbandry, surgery, MRI, and pathology, and edited the manuscript. E.P. and M.D. performed voltammetry experiments on housing control NHPs and assisted in collecting and interpreting voltammetry data. K.O. performed HPLC experiments and assisted with interpretation of HPLC results. J.B. assisted with surgeries and consulted on tissue collection and handling techniques. L.S. consulted on tissue collection and handling techniques and assisted with manuscript writing and editing. J.M. performed brain tissue processing and edited the manuscript. J.F. participated in project inception, and assisted with manuscript writing and editing. S.J. designed voltammetry experiments, interpreted data, supervised B.G., K.H., E.P., and M.D., and edited the manuscript. K.G. and K.B. conceived the project, provided feedback on data analyses, and edited the manuscript. K.G. supervised M.F. and L.V. whereas K.B. supervised J.N., P.H., J.B., L.S., V.V. and J.F. on their efforts. K.B. performed the MRI-guided infusions targeting the VTA.

Corresponding authors

Correspondence to Kathleen A. Grant or Krystof S. Bankiewicz.

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Competing interests

K.S.B. is a consultant for Asklepios BioPharmaceutical, Scribe Therapeutics and Aviado Bio. The remaining authors declare no competing interests.

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Nature Medicine thanks Miguel Sena-Esteves, Alëna Balasanova and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Anna Maria Ranzoni, in collaboration with the Nature Medicine team.

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Extended data

Extended Data Fig. 1 Chronic alcohol self-administration in rhesus macaques.

a) The relationship between blood ethanol concentrations (BECs) collected during the final week of the OAB (n = 2 per subject) and corresponding alcohol intakes recorded at timing of sampling for each subject as identified by drinking panel each was assigned (Panel 0–7), with the dashed horizontal line demarcating 80 mg/dl (the threshold for legal intoxication in humans). 6 of the 8 subjects exhibited alcohol consumption approaching or surpassing the 80 mg/dl threshold during the final week of OAB. b) The relationship between BECs collected during the final two months of the OAB and corresponding alcohol intakes recorded at timing of sampling divided between subjects destined for the Vehicle-treated control group and the AAV2-hGDNF-treated group (4 subjects per group, n = 20 BEC points per group), demonstrating the similarity of BEC-alcohol intake relationship between the two groups.

Extended Data Fig. 2 Pilot surgeries for AAV2 delivery to the VTA.

Prior to viral drug infusion of the experimental groups, a pilot study was conducted using two macaques to confirm surgical coordinates and trajectory planning, as well as infusate and transgene distribution within the VTA of macaques. AAV2-GFP spiked with gadolinium chelate (2 mM, ProHance, Bracco Diagnostics, Princeton, NJ, USA) was bilaterally infused into the VTA target via magnetic-resonance imaging-guided convection-enhanced delivery (MR-guided CED) followed by post-mortem immunohistochemical assessment of GFP protein expression. Below are representative images of MRI with gadoteridol contrast (a) and post-mortem immunohistochemical staining for GFP (b) from one of two preliminary macaque subjects used to confirm targeting and vector distribution in the VTA. AAV2 vector encoding green fluorescent protein (GFP) was delivered by CED as described in Methods. The white arrow (A) shows the cannula actively delivering the vector with contrast agent and the white arrowhead (A) shows the imaging detection of the contrast in the infusate. One month following infusion, subjects were sacrificed by intracardial perfusion, and brain tissue harvested for immunochemical analyses to address GFP expression and distribution (B). The black arrowhead (B) shows the positive immunochemical staining for the GFP antibody.

Extended Data Fig. 3 Individual Monthly Ethanol intakes and BECs, and Table of Day 1 Reintroduction Statistics.

a) Monthly individual subject means of daily alcohol intakes (g/kg/day) across each of the six alcohol reintroduction periods (R1–R6). b) Monthly individual subject means of BECs (mg/dl) across all six alcohol reintroduction periods (R1–R6). c) Table values and statistics of ethanol drinking patterns during each alcohol reintroduction onset (day 1) as a factor of AAV2-hGDNF treatment. All values represent the mean ± SEM of each treatment (n = 4/group). OAB measures reflect the grand mean from the final 7 days of access to ethanol prior to the first forced abstinence (A1) phase onset while measures for alcohol reintroduction phases (R1–R6) are representative of the initial day of alcohol reintroduction following abstinence. †Significant main effects of AAV2-hGDNF treatment; ‡significant treatment x phase interactions. (Mean ± SEM; n = 4/group are shown; *: p ≤ 0.05 and **: p ≤ 0.001 vs. respective Vehicle-treated control group value. Specific statistical tests and p-values are as provided in Fig. 3).

Extended Data Fig. 4 Effects of AAV2-hGDNF on food and water intake, weight, and sensorimotor tasks in macaques.

a) Monthly averages of subject weight (kg) shown by group (AAV2-hGDNF in orange; Vehicle in blue) from the last month of OAB and during alcohol reintroduction phases R1 through R6 (Mean ± SEM; n = 4/group; †: significant from within-group OAB value; *: significant from respective Vehicle group in phase [pairwise comparison using Students two-sided T-test]; R1 †, p = 0.0230, *, p = 0.0231; R2 †, p = 0.00938, *, p = 0.00334; R3 †, p = 0.00703, *, p = 0.00066; R4 †, p = 0.0103, *, p = 0.000103; R5 †, p = 0.0109, *, p = 0.00005; and R6 †, p = 0.0103, *, p = 0.000369). b) Monthly averages of subject water intake (ml/kg/day) shown by group (AAV2-hGDNF in orange; Vehicle in blue) from the last month of OAB and during alcohol reintroduction phases R1 through R6 (mean ± s.e.m.; n = 4/group; *: significant from respective Vehicle group in phase [pairwise comparison using Students two-sided T-test]; R2 *, p = 0.0303). c) The average daily caloric intake (kcal/kg/day) from ethanol (7 kcal/g) during the reintroduction phases for the Vehicle-treated (blue) and the AAV2-hGDNF-treated (orange) macaque groups (Mean ± SEM; n = 4/group; *: significant from respective Vehicle group in phase [pairwise comparison using Students two-sided T-test]; R1 *, p = 0.00968; R4 *, p = 0.00249; R5 *, p = 0.00806; and R6 *, p = 0.0182). d) The average group body weight in the final week of each phase in Phases A3-R6 for the Vehicle-treated and AAV2-hGDNF-treated groups. Linear regression lines represent the calculated rate-of-gain during this 8mo time period (0.100 kg/mo for Vehicle, 0.082 kg/mo for AAV2-hGDNF; (Mean ± SEM; n = 4/group). E-L) Panel Reversal Test in R3. Starting on day 12 of experimental phase R2, one GDNF-treated subject (panel 7) inexplicably developed an absolute side preference for the spout designated for alcohol access that was unrelated to any malfunction in apparatus. During the first week of phase R2 this subject consumed 0.94 g/kg/day of alcohol and exhibited an alcohol preference ratio (a measure of choosing alcohol versus water intake) of 0.13, but by the final week of phase R2 this drinking profile switched to 2.71 g/kg/day and a ratio of 0.98. To determine whether this shift in behavior was due to a side bias versus a change in motivation to consume alcohol, the spout designated for alcohol delivery for all subjects was reversed for the first 2 weeks of phase R3 and then returned to original position for the second 2 weeks of this alcohol reintroduction period (see ‘R’ in Fig. 1a). This evaluation clearly demonstrated that each subject, with the exception of the GDNF-treated subject on panel 7 (L), could track the location of the alcohol spout while maintaining a comparable preference ratio. Starting with phase R4, the alcohol spout for GDNF-treated subject on panel 7 was kept in the reversed position so that the subject had daily access to water and could still access ethanol for evaluation.

Extended Data Fig. 5 AAV2-hGDNF effects on 5HT concentrations and metabolism.

a) Concentrations of 5HT (VTA: **, p = 0.0359 vs. respective Vehicle-treated control group value via two-tailed Student’s T-test), and (b) its metabolite 5HIAA (SN: *, p = 0.0220; VTA: *, p = 0.0179 vs. respective Vehicle-treated control group value via two-tailed Student’s T-test) in ng/mg protein as determined by HPLC from tissue homogenates. c) Table of values detected for GDNF protein via ELISA (ng/mg total protein) and monoamine amounts via HPLC (ng/mg total protein). These values were used to generate the graphs shown in Fig. 5 and Extended Fig. 5. (Mean ± SEM; n = 4/group are shown; *: p ≤ 0.05 and **: p ≤ 0.001 vs. respective Vehicle-treated control group value. Specific statistical tests and p-values are as provided in Fig. 5 and above.).

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Ford, M.M., George, B.E., Van Laar, V.S. et al. GDNF gene therapy for alcohol use disorder in male non-human primates. Nat Med 29, 2030–2040 (2023). https://doi.org/10.1038/s41591-023-02463-9

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