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Research Article

Widespread and sustained target engagement in Huntington’s disease minipigs upon intrastriatal microRNA-based gene therapy

Science Translational Medicine
7 Apr 2021
Vol 13, Issue 588

Targeting HTT in pigs

Huntington’s disease (HD) is a genetic neurodegenerative disorder caused by mutated huntingtin (HTT) gene. Reducing the expression of the aberrant HTT has been shown to be effective in preclinical models. Now, Vallès et al. evaluated the effects of an adeno-associated viral vector (AAV)–mediated strategy delivering microRNA (miRNA) targeting human mutant HTT (mHTT) in a pig model of HD that closely resembles the human condition. Intracerebral delivery of the miRNA into the striatum resulted in widespread distribution and reduced mHTT for up to a year after injection. The results suggest that the approach could be effective in patients with HD.


Huntingtin (HTT)–lowering therapies hold promise to slow down neurodegeneration in Huntington’s disease (HD). Here, we assessed the translatability and long-term durability of recombinant adeno-associated viral vector serotype 5 expressing a microRNA targeting human HTT (rAAV5-miHTT) administered by magnetic resonance imaging–guided convention-enhanced delivery in transgenic HD minipigs. rAAV5-miHTT (1.2 × 1013 vector genome (VG) copies per brain) was successfully administered into the striatum (bilaterally in caudate and putamen), using age-matched untreated animals as controls. Widespread brain biodistribution of vector DNA was observed, with the highest concentration in target (striatal) regions, thalamus, and cortical regions. Vector DNA presence and transgene expression were similar at 6 and 12 months after administration. Expression of miHTT strongly correlated with vector DNA, with a corresponding reduction of mutant HTT (mHTT) protein of more than 75% in injected areas, and 30 to 50% lowering in distal regions. Translational pharmacokinetic and pharmacodynamic measures in cerebrospinal fluid (CSF) were largely in line with the effects observed in the brain. CSF miHTT expression was detected up to 12 months, with CSF mHTT protein lowering of 25 to 30% at 6 and 12 months after dosing. This study demonstrates widespread biodistribution, strong and durable efficiency of rAAV5-miHTT in disease-relevant regions in a large brain, and the potential of using CSF analysis to determine vector expression and efficacy in the clinic.

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Supplementary Material


Fig. S1. Design and mechanism of action of rAAV5-miHTT leading to HTT lowering in the cell.
Fig. S2. Brain dissection scheme for bioanalytics.
Fig. S3. VG copies in deep brain regions of tgHD minipigs at 6 and 12 months after rAAV5-miHTT administration in the caudate and putamen.
Fig. S4. Correlation between miHTT expression and mHTT protein lowering (as percentage from control), 12 months after rAAV5-miHTT administration.
Fig. S5. HTT protein concentrations in the CSF of tgHD minipigs increase with age, as determined by two independent immunoassays.
Fig. S6. No differences in CSF NFL between wild-type and tgHD minipigs up to 4 years of age.
Table S1. Statistical analysis of mHTT in the CSF of tgHD minipigs (control versus treated).
Table S2. Experimental approach for intrastriatal miRNA-based gene therapy in tgHD minipigs.
Data file S1. Raw data (provided as supplementary Excel file).


File (abb8920_data_file_s1.xlsx)
File (abb8920_sm.pdf)


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Published In

Science Translational Medicine
Volume 13 | Issue 588
April 2021

Submission history

Received: 25 March 2020
Accepted: 9 January 2021


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We would like to thank O. Lewis, M. Wooley, and D. Johnson from Renishaw for valuable support during the CED surgical procedure. We are grateful to the teams of Process Development and Analytical Development at uniQure for the production and characterization of rAAV5-miHTT and to E. Sawyer and E. Broug for critically reading the manuscript. OCS Life Sciences provided support with statistical analysis of CSF mHTT. We are also grateful for the support from the CHDI Foundation, in particular, D. Macdonald and D. Howland. Funding: Part of this work was supported by PIGMOD Center’s sustainability program: National Sustainability Programme, project number LO1609 (Czech Ministry of Education, Youth and Sports) to J.M. Author contributions: Conceptualization: A.V., M.M.E., B. Blits, J.M., Z.E., H.P., S.v.D., and P.K. Surgery: B. Bohuslavova, R.L., D.U., Z.S., M.C., B. Blits, Z.E., and J.M. Sample collection: A.V., M.S.-G., C.B., L.P., J.K., B. Bohuslavova, and Z.E. Molecular and data analyses: A.V., M.M.E., A.S., M.S.-G., C.B., C.V.-T., S.A.-B., L.P., R.P., V.F., and A.B. Formal analysis: A.V. and M.M.E. Writing: A.V. Funding acquisition: Z.E., J.M., H.P., S.v.D., and P.K. Supervision: A.V., M.M.E., Z.E., J.M., and P.K. Competing interests: A.V., M.M.E., A.S., M.S.-G., C.B., C.V.-T., S.A.-B., L.P., H.P., S.v.D., and P.K. are employees and shareholders at uniQure; Z.E., J.M., and PIGMOD have a collaborative agreement with uniQure. Filed patent applications pertaining to the results presented in this paper include the following: RNAi-induced huntingtin gene suppression (WO2016/102664, resulting in at least US 10,174,321, US 10,767,180, and EP 3237618B1), A companion diagnostic to monitor the effects of gene therapy (PCT/EP2019/081759), Method and means to deliver miRNA to target cells (PCT/EP2019/081822), and Targeting misspliced transcripts in genetic disorders (PCT/EP2020/075871); the latter three have not yet been published. Data and materials availability: All data associated with this study are present in the paper or the Supplementary Materials, and, if required, materials may be available subject to at least a material transfer agreement.



Department of Research and Development, uniQure biopharma B.V., Paasheuvelweg 25a, 1105 BP Amsterdam, Netherlands.
Department of Research and Development, uniQure biopharma B.V., Paasheuvelweg 25a, 1105 BP Amsterdam, Netherlands.
Anouk Stam
Department of Research and Development, uniQure biopharma B.V., Paasheuvelweg 25a, 1105 BP Amsterdam, Netherlands.
Department of Research and Development, uniQure biopharma B.V., Paasheuvelweg 25a, 1105 BP Amsterdam, Netherlands.
Department of Research and Development, uniQure biopharma B.V., Paasheuvelweg 25a, 1105 BP Amsterdam, Netherlands.
Carlos Vendrell-Tornero
Department of Research and Development, uniQure biopharma B.V., Paasheuvelweg 25a, 1105 BP Amsterdam, Netherlands.
Seyda Acar-Broekmans
Department of Research and Development, uniQure biopharma B.V., Paasheuvelweg 25a, 1105 BP Amsterdam, Netherlands.
Department of Research and Development, uniQure biopharma B.V., Paasheuvelweg 25a, 1105 BP Amsterdam, Netherlands.
Institute of Animal Physiology and Genetics, Rumburská 89, 277 21 Libechov, Czech Republic.
Bozena Bohuslavova
Institute of Animal Physiology and Genetics, Rumburská 89, 277 21 Libechov, Czech Republic.
Department of Translational Biology, IRBM Science Park S.p.A., Via Pontina km 30,600, 00071 Pomezia, Italy.
Valentina Fodale
Department of Translational Biology, IRBM Science Park S.p.A., Via Pontina km 30,600, 00071 Pomezia, Italy.
Department of Translational Biology, IRBM Science Park S.p.A., Via Pontina km 30,600, 00071 Pomezia, Italy.
Roman Liscak
Department of Stereotactic Radioneurosurgery, Na Homolce Hospital, Roentgenova 37/2, 150 30, Prague 5, Czech Republic.
Dusan Urgosik
Department of Stereotactic Radioneurosurgery, Na Homolce Hospital, Roentgenova 37/2, 150 30, Prague 5, Czech Republic.
Zdenek Starek
Interventional Cardiac Electrophysiology, St. Anne’s University Hospital, Pekařská 53, 656 91 Brno, Czech Republic.
Michal Crha
Small Animal Clinic, Veterinary and Pharmaceutical University, Palackého třída 1946/1, 612 42 Brno, Czech Republic.
Bas Blits
Department of Research and Development, uniQure biopharma B.V., Paasheuvelweg 25a, 1105 BP Amsterdam, Netherlands.
Harald Petry
Department of Research and Development, uniQure biopharma B.V., Paasheuvelweg 25a, 1105 BP Amsterdam, Netherlands.
Institute of Animal Physiology and Genetics, Rumburská 89, 277 21 Libechov, Czech Republic.
Jan Motlik
Institute of Animal Physiology and Genetics, Rumburská 89, 277 21 Libechov, Czech Republic.
Sander van Deventer
Department of Research and Development, uniQure biopharma B.V., Paasheuvelweg 25a, 1105 BP Amsterdam, Netherlands.
Pavlina Konstantinova
Department of Research and Development, uniQure biopharma B.V., Paasheuvelweg 25a, 1105 BP Amsterdam, Netherlands.

Funding Information

National Sustainability Programme (Czech Ministry of Education, Youth and Sports): project number LO1609


Corresponding author. Email: [email protected] (A.V.); [email protected] (M.M.E.)
These authors contributed equally to this work.

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