Stability of mRNA/Cationic Lipid Lipoplexes in Human and Rat Cerebrospinal Fluid: Methods and Evidence for Nonviral mRNA Gene Delivery to the Central Nervous System
Abstract
Clinical applications of gene therapy require advances in gene delivery systems. Although numerous clinical trials are already underway, the ultimate success of gene therapies will depend on gene transfer vectors that facilitate the expression of a specific gene at therapeutic levels in the desired cell populations without eliciting cytotoxicity. In clinical applications for which transient expression is desirable, mRNA delivery is of particular interest. We have shown cationic lipid-mediated mRNA delivery to be feasible, efficient, and reproducible in vitro. mRNA delivery to the cerebrospinal fluid (CSF) in vivo would provide a means of vector distribution throughout the central nervous system (CNS). This study examined the functional integrity and protection from degradation of mRNA/cationic complexes (lipoplexes) in human cerebrospinal fluid (hCSF) in vitro and expression of these lipoplexes in vivo. Results obtained from gel electrophoresis indicate that cationic lipids protect mRNA transcripts from RNases in hCSF for at least 4 hr. This is in contrast to the total disappearance of nonlipid-complexed mRNA in less than 5 min. We confirmed the importance of RNase activity by incubating mRNA transcripts encoding luciferase or green fluorescent protein (GFP) in hCSF to which RNase inhibitors had been added. After incubation, these solutions were used to transfect Chinese hamster ovary (CHO) cells in vitro. Next, assays for both GFP and luciferase were used to demonstrate functional integrity and translation of the mRNA transcripts. Finally, we delivered in vitro transcribed mRNA vectors encoding for Hsp70 and luciferase to the lateral ventricle of the rat in a series of preliminary in vivo experiments. Initial immunohistochemistry analysis demonstrates that the distribution, uptake, and expression of reporter sequences using lipid-mediated mRNA vector delivery is extensive, as we earlier reported using similar methods with DNA vectors but that the expression may be less intense. Expression was noted in coronal sections throughout the rat brain, confirming the potential for lipid-mediated mRNA delivery to the CNS. These findings confirm that complexing mRNA with cationic lipid before exposure to CSF confers protection against RNase activity, facilitating distribution, cellular uptake, and expression of mRNA delivered into the CNS.
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mRNA-based protein replacement therapies have been applied to haematological diseases, central nervous system disorders, skin diseases and hearing loss in preclinical studies.
“In addition, mRNA-based protein replacement therapies have been applied to haematological diseases (for example, haemophilia A232, haemophilia B91,104,233 and thrombotic thrombocytopaenic purpura234), central nervous system disorders67,152 (for example, Friedreich’s ataxia67), skin diseases235,236 (for example, elastin deficiency235) and hearing loss237 in preclinical”
From Lipid nanoparticles for mRNA delivery (2021) · cites this paper as [152]
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- Authors
- Anderson, D. M., Hall, L. L., Ayyalapu, A. R., Irion, V. R., Nantz, M. H., & Hecker, J. G.
- Affiliations
- University of California, Davis (US)
- University of Pennsylvania (US)
- ORCID
- Nantz, Michael H.
- Venue
- Human Gene Therapy
- Publisher
- SAGE Publications
- Published
- 2003-02-10
- Volume
- 14
- Issue
- 3
- Pages
- 191-202
- ISSN
- 1043-0342 (ISSN-L)
- 1557-7422
- OpenAlex
- W2068047816
- Cited by
- 66
- References
- 39
- Citations by year
- 2026: 1 · 2025: 2 · 2024: 4 · 2023: 5 · 2022: 4 · 2021: 5 · 2020: 3 · 2019: 1 · 2018: 5 · 2017: 1 · 2016: 2 · 2015: 5 · 2014: 3 · 2013: 3 · 2012: 3
- FWCI
- 0.58
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Human Gene Therapy (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Institute of Neurological Disorders and Stroke (K08NS001960, K08 NS01960)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
- Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
- Keywords
- mRNA delivery, cationic lipids, lipoplexes, cerebrospinal fluid, central nervous system gene delivery, RNase protection, mRNA stability, lateral ventricle injection, HSP70, luciferase reporter, green fluorescent protein, in vivo transfection
- MeSH terms
- Animals, Central Nervous System, Cricetinae, Humans, Lipids, RNA, Messenger, Transfection, CHO Cells, Genes, Reporter, Gene Transfer Techniques, Lipid Metabolism, Rats
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