RNA interference (RNAi)-based therapeutics hold the potential for dominant genetic disorders, enabling sequence-specific inhibition of pathogenic gene products. We aimed to direct RNAi for the selective suppression of the heterozygous GNAO1 c.607 G > A variant causing GNAO1 encephalopathy. By screening short interfering RNA (siRNA), we showed that GNAO1 c.607G>A is a druggable target for RNAi. The si1488 candidate achieved at least twofold allelic discrimination and downregulated mutant protein to 35%. We created vectorized RNAi by incorporating the si1488 sequence into the short hairpin RNA (shRNA) in the adeno-associated virus (AAV) vector. The shRNA stem and loop were modified to improve the transcription, processing, and guide strand selection. All tested shRNA constructs demonstrated selectivity toward mutant GNAO1, while tweaking hairpin structure only marginally affected the silencing efficiency. The selectivity of shRNA-mediated silencing was confirmed in the context of AAV vector transduction. To conclude, RNAi effectors ranging from siRNA to AAV-RNAi achieve suppression of the pathogenic GNAO1 c.607G>A and discriminate alleles by the single-nucleotide substitution. For gene therapy development, it is crucial to demonstrate the benefit of these RNAi effectors in patient-specific neurons and animal models of the GNAO1 encephalopathy.
Get full access to this article
View all access options for this article.
References
1.
WilsonR and JADoudna. (2013). Molecular mechanisms of RNA interference. Annu Rev Biophys, 42:217–239.
2.
O'CarrollD and ASchaefer. (2013). General principals of miRNA biogenesis and regulation in the brain. Neuropsychopharmacology, 38:39–54.
3.
AngartP, VocelleD, Chan C and SPWalton. (2013). Design of siRNA therapeutics from the molecular scale. Pharmaceuticals, 6:440–468.
4.
SettenRL, RossiJJ and HanS. (2019). The current state and future directions of RNAi-based therapeutics. Nat Rev Drug Discov, 18:421–446.
5.
RamachandranPS, KeiserMS and DavidsonBL. (2013). Recent advances in RNA interference therapeutics for CNS diseases. Neurotherapeutics, 10:473–485.
6.
BorelF and CMueller. (2019). Design of AAV vectors for delivery of RNAi. In: Adeno-Associated Virus Vectors: Design and Delivery. Castle MJ, ed. Springer, New York, NY, pp. 3–18.
7.
HocquemillerM, GierschL, AudrainM, Parker S and NCartier. (2016). Adeno-associated virus-based gene therapy for CNS diseases. Hum Gene Ther, 27:478–496.
8.
MartierR and P.Konstantinova (2020). Gene therapy for neurodegenerative diseases: slowing down the ticking clock. Front Neurosci, 14:580179.
9.
EllisCA, Petrovski S and SFBerkovic. (2020). Epilepsy genetics: clinical impacts and biological insights. Lancet Neurol, 19:93–100.
10.
NakamuraK, KoderaH, AkitaT, ShiinaM, KatoM, HoshinoH, TerashimaH, OsakaH, NakamuraS, et al. (2013). De Novo mutations in GNAO1, encoding a Gαo subunit of heterotrimeric G proteins, cause epileptic encephalopathy. Am J Hum Genet, 93:496–505.
11.
JiangM and NSBajpayee. (2009). Molecular mechanisms of go signaling. Neurosignals, 17:23–41.
12.
NovelliM, GalosiS, ZorziG, MartinelliS, CapuanoA, NardecchiaF, GranataT, PolliniL, Di RoccoM, et al. (2023). GNAO1-related movement disorder: an update on phenomenology, clinical course, and response to treatments. Parkinsonism Relat Disord, 111:105405.
13.
LarasatiYA, SavitskyM, KovalA, SolisGP, Valnohova J and VL.Katanaev (2022). Restoration of the GTPase activity and cellular interactions of Gαo mutants by Zn2+ in GNAO1 encephalopathy models. Sci Adv, 8:eabn9350.
14.
FengH, SjögrenB, KarajB, ShawV, Gezer A and RRNeubig. (2017). Movement disorder in GNAO1 encephalopathy associated with gain-of-function mutations. Neurology, 89:762–770.
15.
MunteanBS, MasuhoI, DaoM, SuttonLP, ZuccaS, IwamotoH, PatilDN, WangD, BirnbaumerL, et al. (2021). Gαo is a major determinant of cAMP signaling in the pathophysiology of movement disorders. Cell Rep, 34:108718.
16.
MihalekI, WaughJL, ParkM, KayaniS, Poduri A and O.Bodamer (2020). Molecular map of GNAO1-related disease phenotypes and reactions to therapy. bioRxiv. https://doi.org/10.1101/232058
17.
LunevEA, ShmidtAA, VassilievaSG, SavchenkoIM, LoginovVA, MarinaVI, EgorovaTV and BardinaMV. (2022). Effective viral delivery of genetic constructs to neuronal culture for modeling and gene therapy of GNAO1 encephalopathy. Mol Biol, 56:559–571.
18.
LiuZ, ZhangJ, WuL, Liu J and MZhang. (2014). Overexpression of GNAO1 correlates with poor prognosis in patients with gastric cancer and plays a role in gastric cancer cell proliferation and apoptosis. Int J Mol Med, 33:589–596.
19.
MoffatJ, GruenebergDA, YangX, KimSY, KloepferAM, HinkleG, PiqaniB, EisenhaureTM, LuoB, et al. (2006). A lentiviral RNAi library for human and mouse genes applied to an arrayed viral high-content screen. Cell, 124:1283–1298.
20.
TranNT, HeinerC, WeberK, WeiandM, WilmotD, XieJ, WangD, BrownA, ManokaranS, et al. (2020). AAV-genome population sequencing of vectors packaging CRISPR components reveals design-influenced heterogeneity. Mol Ther Methods Clin Dev, 18:639–651.
21.
WilmottP, LisowskiL, AlexanderIE and LoganGJ. (2019). A user's guide to the inverted terminal repeats of adeno-associated virus. Hum Gene Ther Methods, 30:206–213.
22.
StarikovaAV, SkopenkovaVV, PolikarpovaAV, ReshetovDA, VassilievaSG, VelyaevOA, ShmidtAA, SavchenkoIM, SoldatovVO, et al. (2022). Therapeutic potential of highly functional codon-optimized microutrophin for muscle-specific expression. Sci Rep, 12:848.
23.
BuclezP-O, Dias FlorencioG, RelizaniK, BeleyC, Garcia L and R.Benchaouir (2016). Rapid, scalable, and low-cost purification of recombinant adeno-associated virus produced by baculovirus expression vector system. Mol Ther Methods Clin Dev, 3:16035.
24.
WorleyPF, BarabanJM, Van DopC, NeerEJ and SnyderSH. (1986). Go, a guanine nucleotide-binding protein: immunohistochemical localization in rat brain resembles distribution of second messenger systems. Proc Natl Acad Sci U S A, 83:4561–4565.
25.
PolikarpovaAV, EgorovaTV, LunevEA, TsitrinaAA, VassilievaSG, SavchenkoIM, SilaevaYY, DeykinAV and Bardina MV. (2023). CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics. Front Genome Ed, 5:1034720
26.
MannM, WrightPR and BackofenR. (2017). IntaRNA 2.0: enhanced and customizable prediction of RNA-RNA interactions. Nucleic Acids Res, 45:W435–W439.
27.
HudryE and LHVandenberghe. (2019). Therapeutic AAV gene transfer to the nervous system: a clinical reality. Neuron, 101:839–862.
28.
MartierR, LiefhebberJM, García-OstaA, MiniarikovaJ, Cuadrado-TejedorM, EspelosinM, UrsuaS, PetryH, van DeventerSJ, et al. (2019). Targeting RNA-mediated toxicity in C9orf72 ALS and/or FTD by RNAi-based gene therapy. Mol Ther Nucleic Acids, 16:26–37.
29.
GuS, JinL, ZhangY, HuangY, ZhangF, ValdmanisPN and KayMA. (2012). The loop position of shRNAs and pre-miRNAs is critical for the accuracy of Dicer processing in vivo. Cell, 151:900–911.
30.
Bofill-De Ros X and SGu. (2016). Guidelines for the optimal design of miRNA-based shRNAs. Methods San Diego Calif, 103:157–166.
31.
MaH, WuY, DangY, ChoiJ-G, Zhang J and H.Wu (2014). Pol III promoters to express small RNAs: delineation of transcription initiation. Mol Ther Nucleic Acids, 3:e161.
32.
DingH, LiaoG, Wang H and YZhou. (2007). Asymmetrically designed siRNAs and shRNAs enhance the strand specificity and efficacy in RNAi. J RNAi Gene Silenc Int J RNA Gene Target Res, 4:269–280.
33.
XieJ, MaoQ, TaiPWL, HeR, AiJ, SuQ, ZhuY, MaH, LiJ, et al. (2017). Short DNA hairpins compromise recombinant adeno-associated virus genome homogeneity. Mol Ther J Am Soc Gene Ther, 25:1363–1374.
34.
SibleyCR and Wood MJA. (2011). Identification of allele-specific RNAi effectors targeting genetic forms of Parkinson's disease. PLoS One, 6:e26194.
35.
MiniarikovaJ, ZanellaI, HuseinovicA, van der ZonT, HanemaaijerE, MartierR, KoornneefA, SouthwellAL, HaydenMR, et al. (2016). Design, characterization, and lead selection of therapeutic miRNAs targeting Huntingtin for development of gene therapy for Huntington's disease. Mol Ther Nucleic Acids, 5:e297.
36.
JiangJ, WakimotoH, SeidmanJG and SeidmanCE. (2013). Allele-specific silencing of mutant Myh6 transcripts in mice suppresses hypertrophic cardiomyopathy. Science, 342:111–114.
37.
BongianinoR, DenegriM, MazzantiA, LodolaF, VolleroA, BoncompagniS, FascianoS, RizzoG, MangioneD, et al. (2017). Allele-specific silencing of mutant mRNA rescues ultrastructural and arrhythmic phenotype in mice carriers of the R4496C mutation in the ryanodine receptor gene (RYR2). Circ Res, 121:525–536.
38.
KimuraT, FerranB, TsukaharaY, ShangQ, DesaiS, FedoceA, PimentelDR, LuptakI, AdachiT, et al. (2019). Production of adeno-associated virus vectors for in vitro and in vivo applications. Sci Rep, 9:13601.
39.
JudgeAD, SoodV, ShawJR, FangD, McClintock K and IMacLachlan. (2005). Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat Biotechnol, 23:457–462.
40.
GuS, ZhangY, JinL, HuangY, ZhangF, BassikMC, Kampmann M and MAKay. (2014). Weak base pairing in both seed and 3’ regions reduces RNAi off-targets and enhances si/shRNA designs. Nucleic Acids Res, 42:12169–12176.
41.
AkamineS, OkuzonoS, YamamotoH, SetoyamaD, SagataN, OhgidaniM, KatoTA, IshitaniT, KatoH, et al. (2020). GNAO1 organizes the cytoskeletal remodeling and firing of developing neurons. FASEB J, 34:16601–16621.
42.
WallaceLM, SaadNY, PyneNK, FowlerAM, EidahlJO, DomireJS, GriffinDA, HermanAC, SahenkZ, et al. (2018). Pre-clinical safety and off-target studies to support translation of AAV-mediated RNAi therapy for FSHD. Mol Ther Methods Clin Dev, 8:121–130.
43.
McBrideJL, BoudreauRL, HarperSQ, StaberPD, MonteysAM, MartinsI, GilmoreBL, BursteinH, PelusoRW, et al. (2008). Artificial miRNAs mitigate shRNA-mediated toxicity in the brain: implications for the therapeutic development of RNAi. Proc Natl Acad Sci, 105:5868–5873.
44.
HaeryL, DevermanBE, MathoKS, CetinA, WoodardK, CepkoC, GuerinKI, RegoMA, ErsingI, et al. (2019). Adeno-associated virus technologies and methods for targeted neuronal manipulation. Front Neuroanat, 13:93.
45.
SilachevD, KovalA, SavitskyM, PadmasolaG, QuairiauxC, Thorel F and VL.Katanaev (2022). Mouse models characterize GNAO1 encephalopathy as a neurodevelopmental disorder leading to motor anomalies: from a severe G203R to a milder C215Y mutation. Acta Neuropathol Commun, 10:9.
46.
FengH, YuanY, WilliamsMR, RoyAJ, LeipprandtJR and NeubigRR. (2022). Mice with monoallelic GNAO1 loss exhibit reduced inhibitory synaptic input to cerebellar Purkinje cells. J Neurophysiol, 127:607–622.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
