Neurological disorders are often debilitating conditions with no cure. The majority of current therapies are palliative rather than disease-modifying; therefore, new strategies for treating neurological disorders are greatly needed. mRNA-based therapeutics have great potential for treating such neurological disorders; however, challenges with delivery have limited their clinical potential. Lipid nanoparticles (LNPs) are a promising delivery vector for the brain, given their safer toxicity profile and higher efficacy. Despite this, very little is known about LNP-mediated delivery of mRNA into the brain. Here, we employ MC3-based LNPs and successfully deliver Cre mRNA and Cas9 mRNA/Ai9 sgRNA to the adult Ai9 mouse brain; greater than half of the entire striatum and hippocampus was found to be penetrated along the rostro-caudal axis by direct intracerebral injections of MC3 LNP mRNAs. MC3 LNP Cre mRNA successfully transfected cells in the striatum (∼52% efficiency) and hippocampus (∼49% efficiency). In addition, we demonstrate that MC3 LNP Cas9 mRNA/Ai9 sgRNA edited cells in the striatum (∼7% efficiency) and hippocampus (∼3% efficiency). Further analysis demonstrates that MC3 LNPs mediate mRNA delivery to multiple cell types including neurons, astrocytes, and microglia in the brain. Overall, LNP-based mRNA delivery is effective in brain tissue and shows great promise for treating complex neurological disorders.

Department of Bioengineering University of California Berkeley California 94720 United States

Department of Pathophysiology Faculty of Medicine in Pilsen Charles University alej Svobody 75 323 00 Plzen Czech Republic

The Department of Cellular and Integrative Physiology The University of Texas Health Science Center at San Antonio San Antonio Texas 78229 United States

The Innovative Genomics Institute 2151 Berkeley Way Berkeley California 94704 United States

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Damase TR; Sukhovershin R; Boada C; Taraballi F; Pettigrew RI.Cooke JP. The Limitless Future of RNA Therapeutics, Front Bioeng Biotechnol 2021, 9, 628137. PubMed PMC

Qin S; Tang X; Chen Y; Chen K; Fan N; Xiao W; Zheng Q; Li G; Teng Y; Wu M.Song X. mRNA-based therapeutics: powerful and versatile tools to combat diseases, Signal Transduct Target Ther 2022, 7, 166. PubMed PMC

Anthony K. RNA-based therapeutics for neurological diseases, RNA Biol. 2022, 19, 176–190. PubMed PMC

Peng H; Guo X; He J; Duan C; Yang M; Zhang X; Zhang L; Fu R; Wang B; Wang D; Chen H; Xie M; Feng P; Dai L; Tang X.Luo J. Intracranial delivery of synthetic mRNA to suppress glioblastoma, Mol Ther Oncolytics 2022, 24, 160–170. PubMed PMC

Pardi N; Tuyishime S; Muramatsu H; Kariko K; Mui BL; Tam YK; Madden TD; Hope MJ.Weissman D. Expression kinetics of nucleoside-modified mRNA delivered in lipid nanoparticles to mice by various routes, J Control Release 2015, 217, 345–351. PubMed PMC

Tanaka H; Nakatani T; Furihata T; Tange K; Nakai Y; Yoshioka H; Harashima H.Akita H. In Vivo Introduction of mRNA Encapsulated in Lipid Nanoparticles to Brain Neuronal Cells and Astrocytes via Intracerebroventricular Administration, Mol Pharm 2018, 15, 2060–2067. PubMed

Li Y; Jarvis R; Zhu K; Glass Z; Ogurlu R; Gao P; Li P; Chen J; Yu Y; Yang Y.Xu Q. Protein and mRNA Delivery Enabled by Cholesteryl-Based Biodegradable Lipidoid Nanoparticles, Angew Chem Int Ed Engl 2020, 59, 14957–14964. PubMed PMC

Hou X; Zaks T; Langer R.Dong Y. Lipid nanoparticles for mRNA delivery, Nat Rev Mater 2021, 6, 1078–1094. PubMed PMC

Di J; Du Z; Wu K; Jin S; Wang X; Li T.Xu Y. Biodistribution and Non-linear Gene Expression of mRNA LNPs Affected by Delivery Route and Particle Size, Pharm Res 2022, 39, 105–114. PubMed PMC

Kariko K; Keller JM; Harris VA; Langer DJ.Welsh FA. In vivo protein expression from mRNA delivered into adult rat brain, J Neurosci Methods 2001, 105, 77–86. PubMed

Mahato RI; Kawabata K; Nomura T; Takakura Y.Hashida M. Physicochemical and pharmacokinetic characteristics of plasmid DNA/cationic liposome complexes, J Pharm Sci 1995, 84, 1267–1271. PubMed

Palmer LR; Chen T; Lam AM; Fenske DB; Wong KF; MacLachlan I.Cullis PR. Transfection properties of stabilized plasmid-lipid particles containing cationic PEG lipids, Biochim Biophys Acta 2003, 1611, 204–216. PubMed

Han X; Zhang H; Butowska K; Swingle KL; Alameh MG; Weissman D.Mitchell MJ. An ionizable lipid toolbox for RNA delivery, Nat Commun 2021, 12, 7233. PubMed PMC

Tam YY; Chen S.Cullis PR. Advances in Lipid Nanoparticles for siRNA Delivery, Pharmaceutics 2013, 5, 498–507. PubMed PMC

Satapathy MK; Yen TL; Jan JS; Tang RD; Wang JY; Taliyan R.Yang CH. Solid Lipid Nanoparticles (SLNs): An Advanced Drug Delivery System Targeting Brain through BBB, Pharmaceutics 2021, 13, 1183. PubMed PMC

Rungta RL; Choi HB; Lin PJ; Ko RW; Ashby D; Nair J; Manoharan M; Cullis PR.Macvicar BA. Lipid Nanoparticle Delivery of siRNA to Silence Neuronal Gene Expression in the Brain, Mol Ther Nucleic Acids 2013, 2, e136. PubMed PMC

Freed CR; Greene PE; Breeze RE; Tsai WY; DuMouchel W; Kao R; Dillon S; Winfield H; Culver S; Trojanowski JQ; Eidelberg D.Fahn S. Transplantation of embryonic dopamine neurons for severe Parkinson’s disease, N Engl J Med 2001, 344, 710–719. PubMed

Kunwar S; Prados MD; Chang SM; Berger MS; Lang FF; Piepmeier JM; Sampson JH; Ram Z; Gutin PH; Gibbons RD; Aldape KD; Croteau DJ; Sherman JW; Puri RK.Cintredekin Besudotox Intraparenchymal Study, G. Direct intracerebral delivery of cintredekin besudotox (IL13-PE38QQR) in recurrent malignant glioma: a report by the Cintredekin Besudotox Intraparenchymal Study Group, J Clin Oncol 2007, 25, 837–844. PubMed

Ma Y; Tang C; Chaly T; Greene P; Breeze R; Fahn S; Freed C; Dhawan V.Eidelberg D. Dopamine cell implantation in Parkinson’s disease: long-term clinical and (18)F-FDOPA PET outcomes, J Nucl Med 2010, 51, 7–15. PubMed PMC

Chaichana KL; Pinheiro L.Brem H. Delivery of local therapeutics to the brain: working toward advancing treatment for malignant gliomas, Ther Deliv 2015, 6, 353–369. PubMed PMC

Chang Z; Mao G; Sun L; Ao Q; Gu Y.Liu Y. Cell therapy for cerebral hemorrhage: Five year follow-up report, Exp Ther Med 2016, 12, 3535–3540. PubMed PMC

Gernert M; Feja M. Bypassing the Blood-Brain Barrier: Direct Intracranial Drug Delivery in Epilepsies, Pharmaceutics 2020, 12, 1134. PubMed PMC

Duerinck J; Schwarze JK; Awada G; Tijtgat J; Vaeyens F; Bertels C; Geens W; Klein S; Seynaeve L; Cras L; D’Haene N; Michotte A; Caljon B; Salmon I; Bruneau M; Kockx M; Van Dooren S; Vanbinst AM; Everaert H; Forsyth R.Neyns B. Intracerebral administration of CTLA-4 and PD-1 immune checkpoint blocking monoclonal antibodies in patients with recurrent glioblastoma: a phase I clinical trial, J Immunother Cancer 2021, 9, e002296. PubMed PMC

Bilang-Bleuel A; Revah F; Colin P; Locquet I; Robert JJ; Mallet J.Horellou P. Intrastriatal injection of an adenoviral vector expressing glial-cell-line-derived neurotrophic factor prevents dopaminergic neuron degeneration and behavioral impairment in a rat model of Parkinson disease, Proc Natl Acad Sci U S A 1997, 94, 8818–8823. PubMed PMC

Gey L; Gernert M.Loscher W. Continuous bilateral infusion of vigabatrin into the subthalamic nucleus: Effects on seizure threshold and GABA metabolism in two rat models, Neurobiol Dis 2016, 91, 194–208. PubMed

Gao Y; Geng L; Chen VP.Brimijoin S. Therapeutic Delivery of Butyrylcholinesterase by Brain-Wide Viral Gene Transfer to Mice, Molecules 2017, 22, 1145. PubMed PMC

Noh JE; Oh SH; Park IH.Song J. Intracerebral Transplants of GMP-Grade Human Umbilical Cord-Derived Mesenchymal Stromal Cells Effectively Treat Subacute-Phase Ischemic Stroke in a Rodent Model, Front Cell Neurosci 2020, 14, 546659. PubMed PMC

Nance EA; Woodworth GF; Sailor KA; Shih TY; Xu Q; Swaminathan G; Xiang D; Eberhart C.Hanes J. A dense poly(ethylene glycol) coating improves penetration of large polymeric nanoparticles within brain tissue, Sci Transl Med 2012, 4, 149ra119. PubMed PMC

Chen S; Tam YY; Lin PJ; Leung AK; Tam YK.Cullis PR. Development of lipid nanoparticle formulations of siRNA for hepatocyte gene silencing following subcutaneous administration, J Control Release 2014, 196, 106–112. PubMed

Mastorakos P; Zhang C; Berry S; Oh Y; Lee S; Eberhart CG; Woodworth GF; Suk JS.Hanes J. Highly PEGylated DNA Nanoparticles Provide Uniform and Widespread Gene Transfer in the Brain, Adv Healthc Mater 2015, 4, 1023–1033. PubMed PMC

Gaj T; Ojala DS; Ekman FK; Byrne LC; Limsirichai P.Schaffer DV. In vivo genome editing improves motor function and extends survival in a mouse model of ALS, Sci Adv 2017, 3, eaar3952. PubMed PMC

Yang S; Chang R; Yang H; Zhao T; Hong Y; Kong HE; Sun X; Qin Z; Jin P; Li S.Li XJ. CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of Huntington’s disease, J Clin Invest 2017, 127, 2719–2724. PubMed PMC

Lee B; Lee K; Panda S; Gonzales-Rojas R; Chong A; Bugay V; Park HM; Brenner R; Murthy N.Lee HY. Nanoparticle delivery of CRISPR into the brain rescues a mouse model of fragile X syndrome from exaggerated repetitive behaviours, Nat Biomed Eng 2018, 2, 497–507. PubMed PMC

Gyorgy B; Loov C; Zaborowski MP; Takeda S; Kleinstiver BP; Commins C; Kastanenka K; Mu D; Volak A; Giedraitis V; Lannfelt L; Maguire CA; Joung JK; Hyman BT; Breakefield XO.Ingelsson M. CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer’s Disease, Mol Ther Nucleic Acids 2018, 11, 429–440. PubMed PMC

Ekman FK; Ojala DS; Adil MM; Lopez PA; Schaffer DV.Gaj T. CRISPR-Cas9-Mediated Genome Editing Increases Lifespan and Improves Motor Deficits in a Huntington’s Disease Mouse Model, Mol Ther Nucleic Acids 2019, 17, 829–839. PubMed PMC

Ricci R; Colasante G. CRISPR/dCas9 as a Therapeutic Approach for Neurodevelopmental Disorders: Innovations and Limitations Compared to Traditional Strategies, Dev Neurosci 2021, 43, 253–261. PubMed

Staahl BT; Benekareddy M; Coulon-Bainier C; Banfal AA; Floor SN; Sabo JK; Urnes C; Munares GA; Ghosh A.Doudna JA. Efficient genome editing in the mouse brain by local delivery of engineered Cas9 ribonucleoprotein complexes, Nat Biotechnol 2017, 35, 431–434. PubMed PMC

Abbasi S; Uchida S; Toh K; Tockary TA; Dirisala A; Hayashi K; Fukushima S.Kataoka, K. Co-encapsulation of Cas9 mRNA and guide RNA in polyplex micelles enables genome editing in mouse brain, J Control Release 2021, 332, 260–268. PubMed

Kulkarni JA; Myhre JL; Chen S; Tam YYC; Danescu A; Richman JM.Cullis PR. Design of lipid nanoparticles for in vitro and in vivo delivery of plasmid DNA, Nanomedicine 2017, 13, 1377–1387. PubMed

Cullis PR; Hope MJ. Lipid Nanoparticle Systems for Enabling Gene Therapies, Mol Ther 2017, 25, 1467–1475. PubMed PMC

Long J; Yu C; Zhang H; Cao Y; Sang Y; Lu H; Zhang Z; Wang X; Wang H; Song G; Yang J.Wang S. Novel Ionizable Lipid Nanoparticles for SARS-CoV-2 Omicron mRNA Delivery, Adv Healthc Mater 2023, e2202590. PubMed PMC

Madisen L; Zwingman TA; Sunkin SM; Oh SW; Zariwala HA; Gu H; Ng LL; Palmiter RD; Hawrylycz MJ; Jones AR; Lein ES.Zeng HA robust and high-throughput Cre reporting and characterization system for the whole mouse brain, Nat. Neurosci. 2010, 13, 133–140. PubMed PMC

Li J; Tuma J; Han H; Kim H; Wilson RC; Lee HY.Murthy N. The Coiled-Coil Forming Peptide (KVSALKE)(5) Is a Cell Penetrating Peptide that Enhances the Intracellular Delivery of Proteins, Adv Healthc Mater 2022, 11, e2102118. PubMed PMC