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Cited by 65RNA Interference and Gene DeliveryNanoparticle-Based Drug DeliveryBarrier Structure and Function Studies

Lipid Nanoparticles Deliver mRNA to the Brain after an Intracerebral Injection

Open access
Tuma, Jan, Chen, Yu-Ju, Collins, Michael G., Paul, Abhik, Li, Jie, Han, Hesong, Sharma, Rohit, Murthy, Niren +1 more·2023·Biochemistry
DOI 10.1021/acs.biochem.3c00371PMC PMC10760911
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Abstract

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.

AI snapshot

Written from the paper’s full text.

TL;DR

MC3-based lipid nanoparticles delivered Cre mRNA and Cas9 mRNA/Ai9 sgRNA into the adult Ai9 mouse striatum and hippocampus after direct intracerebral injection, producing widespread Cre recombination and detectable Cas9 editing. Cre mRNA reached roughly half of the striatum and hippocampus with ~52% and ~49% local transfection efficiency, while Cas9 mRNA/sgRNA editing was lower at ~7% and ~3%.

Key findings

  • MC3 LNP Cre mRNA penetrated more than half of the striatum and hippocampus along the rostro-caudal axis. Transfection distances for low and high doses were 2,207 ± 254 μm and 2,680 ± 500 μm in striatum, and 2,700 ± 40 μm and 2,770 ± 110 μm in hippocampus. Total tdTomato+;DAPI+ cells for low and high doses were 52,160 ± 17,075 and 107,742 ± 7,050 in striatum, and 53,484 ± 5,158 and 123,210 ± 9,366 in hippocampus. Local recombination efficiency was ~53.0% versus ~28.0% in striatum and ~49.0% versus ~14.9% in hippocampus for high versus low dose.
  • MC3 LNP Cas9 mRNA/Ai9 sgRNA produced gene editing in both regions. Transfection distances for low and high doses were 430 ± 250 μm and 1,360 ± 303 μm in striatum, and 180 ± 180 μm and 2,527 ± 144 μm in hippocampus. Total tdTomato+;DAPI+ cells for low and high doses were 252 ± 240 and 1,524 ± 558 in striatum, and 18 ± 18 and 3,344 ± 1,514 in hippocampus. Local editing efficiency was ~7.0% versus ~0.5% in striatum and ~2.8% versus ~0.3% in hippocampus for high versus low dose.
  • Cell-type analysis after high-dose MC3 LNP Cre mRNA showed that among recombined cells, striatum had ~33.7% neurons, ~47.9% astrocytes, and ~2.5% microglia; hippocampus had ~78.7% neurons, ~6.0% astrocytes, and ~0.4% microglia. After high-dose MC3 LNP Cas9 mRNA/Ai9 sgRNA, edited cells in striatum were ~1.5% neurons, ~79.4% astrocytes, and ~2.9% microglia; in hippocampus, ~45.1% neurons, ~44.5% astrocytes, and ~4.2% microglia.

Methods

  • MC3 LNPs were formulated with MC3, DOPE, cholesterol, and DMG-PEG at 36.8:23.8:38.2:1.2 molar ratios; mRNA in 200 mM citrate buffer (pH 4) was mixed with lipids at a 1:3 volume/volume ratio. Average LNP size with Cre mRNA was 162 nm, with a reported range of approximately 150–170 nm.
  • Male and female Ai9 mice aged 2–3 months received stereotaxic intracerebral injections into striatum or hippocampus. Treatments included saline, Cre mRNA, or Cas9 mRNA/Ai9 sgRNA with or without MC3 LNPs, at low (0.125 μg μL−1) or high (0.250 μg μL−1) dose. Cre mRNA was injected as 1.5 μL per site; Cas9 mRNA/Ai9 sgRNA was injected as 2.1 μL across three sites. Brains were collected 21 days after injection.
  • Coronal brain sections (20 μm) were DAPI-stained and immunostained for RFP/tdTomato, NeuN, GFAP, and Iba1. Every 3rd or 4th section was imaged and analyzed with ImageJ; local efficiency and cell-type counts used defined ROIs and z-stacks. Statistics used two-tailed permutation t-tests with 10,000 permutations, significance at P < 0.05, and data are mean ± SEM.

Limitations

  • The paper does not include an explicit limitations section. The authors note that Cas9-mediated editing was considerably lower than Cre-mediated recombination and hypothesize that sgRNA may degrade in the cytoplasm before complexing with Cas9 protein; they suggest increasing sgRNA stability in vivo.
  • Evident from the text: all experiments are in adult Ai9 mice (2–3 months) with direct intracerebral injection, and no behavioral, functional therapeutic, or toxicity/safety outcomes are reported.
  • Evident from the text: the study uses two doses and a 21-day endpoint; transfection distance was similar between low and high doses while cell numbers and local efficiency were dose-dependent, and long-term persistence, repeat dosing, and translation to other brain regions or disease models are not tested.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1021/acs.biochem.3c00371

Cites

44 of 44 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

35 of 44 cited sources checked · 15 supported · 1 flagged · accuracy not yet measured

  • Premisecitation

    After ICV LNP mRNA delivery, transfected cells appeared localized to restricted brain regions, primarily the cells lining the ventricle wall, with small levels of transfection in brain tissue.

    “However, transfected cells appeared to be localized to the restricted brain regions, primarily to the cells lining the ventricle wall, with small levels of transfection in the brain tissue.”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [6] source
    • cites [7] source

      The Results and Discussion report that lateral-ventricle infusion of Cre mRNA/LNPs transfected cells lining the ventricle and cells in the hippocampus, with hippocampal signals concentrated in specific regions. The paper does not establish that transfection occurred primarily at the ventricle wall or quantify the level in brain tissue as small; it says overall transfection efficacy remains to be determined. Page numbers are not available in our copy. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Premisecitation

    Cas9 mRNA has not been tested using LNPs in the brain yet.

    “Notably, there has been an effort of Cas9 mRNA/sgRNA delivery using LNPs in cultured cells by Li et al. (2020), however, Cas9 mRNA has not been tested using LNPs in the brain yet (7).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [7] source

      The Results and Discussion report Cas9 mRNA/sgRNA delivery with LNPs in cultured GFP-HEK cells, while the brain experiments used Cre mRNA/LNPs. This supports the distinction between the paper’s cell and brain tests, but it does not establish the broader claim that Cas9 mRNA had not been tested with LNPs in the brain by anyone. (LLM, conf=0.84)

      Experimental — accuracy not yet measured
  • Premisecitation

    LNPs encapsulating siRNA injected into the brain after direct intracerebral or intracerebroventricular injection efficiently knock down neuronal gene expression.

    “Rungta et al. (2013) demonstrated that LNPs encapsulating siRNA have been injected into the brain after a direct intracerebral or intracerebroventricular (ICV) injection, and were able to efficiently knock down neuronal gene expression (16).”
    Quote in the paperNot a substantial claim1 of 1 sources checked
  • Premisecitation

    mRNA can be delivered using LNPs after ICV injection into mouse brain tissue.

    “Further studies by Tanaka et al. (2018) and Li et al. (2020) demonstrated that mRNA can also be delivered using LNPs after ICV injection into mouse brain tissue (6, 7).”
    Quote in the paperSubstantial claim1 of 2 sources checked
    • cites [6] source
    • cites [7] source

      The Results and Discussion report that Cre mRNA-loaded LNPs delivered mRNA to cells in adult mouse brains via the lateral ventricle. However, the paper used continuous infusion through a cannula over several days, not an ICV injection, so it does not establish the claim for that administration method. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
  • Interpretationcitation

    MC3 LNP Cas9 mRNA/Ai9 sgRNA demonstrates similar or better local transfection efficiency in the striatum and the hippocampus compared to previously demonstrated results using Cas9 RNPs (37, 43).

    “MC3 LNP Cas9 mRNA/Ai9 sgRNA demonstrates similar or better local transfection efficiency in the striatum and the hippocampus compared to previously demonstrated results using Cas9 RNPs (37, 43).”
    Quote in the paperSubstantial claim2 of 2 sources checked
  • May combine two claims. 22
  • Classified as probably not a substantial claim. 10

  • May combine two claims.

    Premisecitation

    In mice, nanoparticles densely coated with PEG measuring up to 200 nm in diameter were able to spread within brain tissue, a size range achievable for LNPs.

    “In mice, nanoparticles densely coated with PEG measuring up to 200 nm in diameter were able to spread within brain tissue (28), a size range achievable for LNPs (9, 29).”
    Quote in the paperSubstantial claim2 of 3 sources checked
    • cites [28] source

      The Results section (‘Particle tracking in live mouse brain’; page not provided) reports that densely PEG-coated 40- and 100-nm particles penetrated mouse brain tissue, but 200-nm particles did not. The separate claim about sizes achievable for LNPs is attributed to other citations and is not judged here. (LLM, conf=0.94)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured
    • cites [9] source

      The Results (Physical and Biological Evaluations of Fluc-mRNA LNPs; page unavailable) report that the authors prepared LNPs in small, medium, and large size categories, supporting the point that LNP size can vary. The passages do not give diameters establishing that LNPs can reach 200 nm. Brain-tissue spread is attached to citation 28, not this source. (LLM, conf=0.76)

      Experimental — accuracy not yet measured
    • cites [29] source

  • May combine two claims.

    Premisecitation

    LNP mRNA complexes have excellent biocompatibility and can transfect a variety of tissues and organs.

    “LNP mRNA complexes have excellent biocompatibility and can transfect a variety of tissues and organs (5–9).”
    Quote in the paperSubstantial claim4 of 5 sources checked
    • cites [5] source

      The Results (§3.2; page unavailable) report mRNA-LNP-driven protein production in mouse liver, lung, and muscle, supporting delivery to multiple tissues and organs. The paper does not establish that the mRNA-LNP complexes have “excellent biocompatibility”; its Introduction describes safety evidence for similarly composed LNPs used to deliver siRNA, not the mRNA-LNP complexes studied here. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [6] source
    • cites [7] source

      The Results and Discussion report negligible cytotoxicity for mRNA-loaded OCholB LNPs in HeLa cells and functional mRNA delivery in mouse skeletal muscle, spleen, and brain tissue (page unavailable). The claim is broader than these findings: it describes LNP–mRNA complexes generally as having excellent biocompatibility, whereas the paper's toxicity evidence for loaded complexes is in vitro and it cautions that cargo may change in vivo toxicity. (LLM, conf=0.83)

      Experimental — accuracy not yet measured
    • cites [8] source

      The Overcoming physiological barriers section describes LNP–mRNA delivery to different cells and organs, including the spleen and lung. But the Safety profiles section does not establish excellent biocompatibility as a general property: it discusses immune activation, cytotoxicity and reported liver and lung injuries in rodents, as well as ways to improve biocompatibility. Page numbers are not available in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured
    • cites [9] source

      The Results section reports mRNA-LNP transgene expression in mouse liver, muscle, spleen, and lymph nodes, supporting transfection in multiple tissues and organs. The paper does not establish excellent biocompatibility; its Discussion instead notes potential adverse reactions from systemic exposure. (LLM, conf=0.87)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Direct injection of 4xNLS-Cas9-2xNLS RNPs into the striatum and hippocampus of Ai14 mice edited genes in brain tissue efficiently.

    “Specifically, Staahl et al. (2017) demonstrated that direct injection of the Cas9 protein fused to 6 SV40 nuclear localization signals (4xNLS-Cas9–2xNLS), complexed to sgRNA (4xNLS-Cas9–2xNLS RNPs) into the striatum and hippocampus of Ai14 mice was able to edit genes in brain tissue efficiently (37).”
    Quote in the paperNot a substantial claim1 of 1 sources checked

  • May combine two claims.

    Premisecitation

    Li et al. (2020) infused LNPs containing Cre mRNA into the lateral ventricle of adult Ai14 mice continuously from day 1 to day 4 using an osmotic pump connected to a brain cannula, resulting in tdTomato expression in cells lining the injected lateral ventricle and in the hippocampus.

    “More recently, Li et al. (2020) infused their LNPs, which contained Cre mRNA, into the lateral ventricle of adult Ai14 mice continuously from day 1 to day 4 using an osmotic pump connected to a brain cannula. As a result of Cre recombination, tdTomato expression was observed in the cells lining the injected lateral ventricle (hypothesized to be ependymal cells) and in the hippocampus, specifically”
    Quote in the paperSubstantial claim1 of 1 sources checked

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    Intracerebral injections are clinically feasible and are performed in a variety of clinical scenarios and preclinical settings.

    “Intracerebral injections are clinically feasible, and are performed in a variety of clinical scenarios (17–23), as well as in various preclinical settings (4, 24–27).”
    Quote in the paperNot a substantial claim7 of 10 sources checked
    • cites [17] source
    • cites [18] source
    • cites [19] source
    • cites [20] source
    • cites [21] source
    • cites [22] source
    • cites [23] source
    • cites [4] source
    • cites [24] source
    • cites [25] source

  • May combine two claims.

    Premisecitation

    Human brain extracellular space has pores even larger than 200 nm in diameter, although more than 95% of all pores have diameters of 175 nm or smaller.

    “In addition, a previous study estimated that human brain extracellular space has pores even larger than 200 nm in diameter, although more than 95% of all pores have diameters of 175 nm or smaller (28).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [28] source

      The Results section on particle tracking in human brain tissue ex vivo reports estimated ECS pores as large as 225 nm, supporting the claim that some exceed 200 nm. It reports approximately 28% larger than 100 nm, but does not state that more than 95% are 175 nm or smaller. No page number is available in our copy. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    CRISPR-Gold was able to edit a target gene and rescue increased repetitive behaviors in a mouse model for fragile X syndrome by injecting directly into the striatum.

    “Lee et al. (2018) further demonstrated that CRISPR-Gold which contains gold nanoparticles and Cas9 RNPs was able to successfully edit target gene and rescue increased repetitive behaviors in a mouse model for fragile X syndrome, a neurodevelopmental disorder (33). This was achieved by injecting directly into the striatum (33).”
    Quote in the paperNot a substantial claim1 of 1 sources checked

  • Classified as probably not a substantial claim.

    Study design and conductcitation

    The total number of cells counted from every 4th DAPI stained brain slice was multiplied by 12 to obtain the estimate of the total number of tdTomato+; DAPI+ cells in the striatum or hippocampus, as similar to previously reported methods.

    “Then, the total number of cells counted from every 4th DAPI stained brain slice was multiplied by 12 to obtain the estimate of the total number of tdTomato+; DAPI+ cells in the striatum or hippocampus, as similar to previously reported methods (37).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [37] source

      The Additional information cell-counting passage describes staining sampled brain sections with DAPI, counting tdTomato/DAPI double-positive cells, and scaling the count by section periodicity. That supports the claimed similarity of method, but this paper sampled every sixth dorsal-striatum section and multiplied by 6—not every fourth section and multiplied by 12. The cited passage does not describe this counting procedure for the hippocampus. No page number is provided in our copy. (LLM, conf=0.90)

      Experimental — accuracy not yet measured
    • Rests on an assumption: The tdTomato+; DAPI+ cells counted in sampled sections represent all Cre-recombined cells in the striatum or hippocampus.

  • May combine two claims.

    Premisecitation

    An intracerebral injection avoids the tissue barrier that the blood-CSF barrier or blood-brain-barrier represents and consequently should lead to significantly higher brain transfection levels by specifically targeting local regions of the brain.

    “An intracerebral injection avoids the tissue barrier that blood-cerebrospinal fluid (CSF) barrier or blood-brain-barrier (BBB) represents, and consequently should lead to significantly higher brain transfection levels by specifically targeting local regions of the brain (4, 20, 22, 26, 27).”
    Quote in the paperSubstantial claim5 of 5 sources checked
    • cites [4] source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [20] source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [22] source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [26] source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [27] source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    LNPs have an ionizable lipid that is predominantly neutral at physiological pH, unlike the quaternary lipid DOTMA, allowing an excellent combination of in vivo efficacy and low toxicity.

    “In addition, LNPs have an ionizable lipid, which is predominantly neutral at physiological pH, as opposed to the quaternary lipid DOTMA, and this allows them to have an excellent combination of in vivo efficacy and low toxicity (8, 13).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [8] source
    • cites [13] source

      The Biodegradable ionizable lipids section (page unavailable) describes an ionizable lipid with robust in vivo gene silencing and lower toxicity, and another that maintained potency with improved tolerability. Our copy does not establish the comparison with DOTMA or say that being predominantly neutral at physiological pH causes the claimed combination of efficacy and low toxicity. (LLM, conf=0.82)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    PEGylated polymer nanoparticles similar in size and surface chemistry to LNPs were previously able to transfect DNA into brain tissue after an intracerebral injection, suggesting that LNPs will be able to transfect brain tissue after intracerebral injection.

    “Notably, PEGylated polymer nanoparticles have previously been able to transfect DNA into brain tissue after an intracerebral injection; these nanoparticles were similar in size and surface chemistry as LNPs, suggesting that LNPs will be able to transfect brain tissue after intracerebral injection (28, 30).”
    Quote in the paperSubstantial claim2 of 2 sources checked
    • cites [28] source

      Split into 2 atoms — 0 confirmed, 2 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured
    • cites [30] source

      Split into 2 atoms — 1 confirmed, 1 partially supported, 0 disputed, 0 misattributed, 0 unverifiable. Expand the claim to see per-atom verdicts.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Interpretationcitation

    The concentrations of LNP mRNA complexes used were significantly lower than previously reported doses used for ICV injections while the transfected brain regions were significantly larger than that observed with an ICV injection (7).

    “Notably, the concentration of LNP mRNA complexes that we used in our study were significantly lower than the previously reported doses used for ICV injections while the transfected brain regions were significantly larger than that observed with an ICV injection (7).”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [7] source

      The Results and Discussion (page unavailable) describe Cre mRNA/LNP infusion into the lateral ventricle of mice and report transfected cells along the ventricle and in the hippocampus. This supports citing the paper as a prior ventricular-delivery study with a reported transfection pattern. Our copy does not give a ventricular-infusion dose or establish the citing study’s lower-dose and larger-region comparisons. (LLM, conf=0.78)

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    The sgRNA used targets the STOP sequence upstream of the tdTomato gene and enables tdTomato gene expression.

    “The sgRNA used in these experiments targets the STOP sequence upstream of the fluorescent tdTomato gene and enables tdTomato gene expression, resulting in fluorescence (Figure 1C) as we previously reported (43).”
    Quote in the paperSubstantial claim1 of 1 sources checked

  • May combine two claims.

    Findingown data

    MC3-based lipid nanoparticles successfully deliver Cre mRNA and Cas9 mRNA/Ai9 sgRNA to the adult Ai9 mouse brain.

    “Here, we employ MC3-based LNPs and successfully deliver Cre mRNA and Cas9 mRNA/Ai9 sgRNA to the adult Ai9 mouse brain”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MC3 containing LNPs were stable and able to transfect brain cells for at least 2 days after synthesis if stored at 4°C.

    “Our pilot in vivo experiment demonstrated that MC3 containing LNPs were stable and were able to transfect brain cells for at least 2 days after synthesis if stored at 4°C (Figure 2), therefore our experiments were performed within 2 days after LNP formulation.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Findingown data

    MC3 LNP Cre mRNA generated by hand pipetting had an approximate diameter of 150-170 nm.

    “The mixing was performed by hand pipetting and generated MC3 LNP Cre mRNA of approximately 150–170 nm in diameter (Figure S1).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    High dose MC3 LNP Cas9 mRNA/Ai9 sgRNA elevated transfection distance and tdTomato+;DAPI+ cells in the striatum and more noticeably in the hippocampus.

    “Notably, high dose of MC3 LNP Cas9 mRNA/Ai9 sgRNA elevated transfection distance and tdTomato+; DAPI+ cells in the striatum and more noticeably in the hippocampus.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In striatum, the number of edited cells (tdTomato+) in mice injected with low dose MC3 LNP Cas9 mRNA/Ai9 sgRNA was not significantly different to control (saline or same dose without MC3 LNPs), demonstrating no significant gene-editing occurred.

    “In the striatum, the number of edited cells (tdTomato+) in mice injected with the low dose of MC3 LNP Cas9 mRNA/Ai9 sgRNA was not significantly different to the control composed of mice injected with saline or injected with the same dose of Cas9 mRNA/Ai9 sgRNA without MC3 LNPs (Figure 7A Left panel and Figure 7B), demonstrating that no significant gene-editing occurred.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Approximately a 10-fold increase in transfection distance and approximately a 200-fold increase in total tdTomato+;DAPI+ cells were observed in high dose group of MC3 LNP Cas9 mRNA/Ai9 sgRNA compared to low dose group.

    “Approximately a 10-fold increase in transfection distance and approximately a 200-fold increase in total tdTomato+; DAPI+ cells in high dose group of MC3 LNP Cas9 mRNA/Ai9 sgRNA compared to low dose group of MC3 LNP Cas9 mRNA/Ai9 sgRNA were observed.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    LNP-based mRNA delivery is effective in brain tissue and shows great promise for treating complex neurological disorders.

    “Overall, LNP-based mRNA delivery is effective in brain tissue and shows great promise for treating complex neurological disorders.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    All contrast/brightness parameters were kept identical between ROI images and the counting was performed as a blind experiment.

    “All contrast/brightness parameters were kept identical between ROI images and the counting was performed as a blind experiment.”
    Quote in the paperNot a substantial claim

  • Classified as probably not a substantial claim.

    Study design and conductnone given

    Cells were quantified along the z-stack using the optical dissector to avoid over/under-counting of superimposed cells.

    “The cells were quantified along the z-stack using the optical dissector to avoid the over/under-counting of superimposed cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    MC3 LNPs hold great potential for mRNA delivery in the brain while having both the transfection efficiency and tissue penetration properties needed for a variety of therapeutic applications.

    “Collectively, our results demonstrate that MC3 LNPs hold great potential for mRNA delivery in the brain while having both the transfection efficiency and tissue penetration properties needed for a variety of therapeutic applications.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim.

    Study design and conductreasoning

    The whole brain area except the inner wall of ventricle was analyzed to avoid the possibility that vectors were transported by ventricular CSF.

    “We analyzed whole brain area except the inner wall of ventricle to avoid the possibility that vectors were transported by ventricular CSF.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Study design and conductnone given

    Apotome.2 was utilized to prevent scattered out-of-focus light and to improve resolution.

    “Apotome.2 (Carl Zeiss Microscopy GmbH) was utilized to prevent scattered out-of-focus light and to improve resolution.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    For each experiment, the exposure time was kept consistent to allow a comparative analysis between images within the experiment.

    “For each experiment, the exposure time was kept consistent to allow a comparative analysis between images within the experiment.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Study design and conductreasoning

    All counted Cas9-edited cells were normalized by the total number of DAPI+ cells and therefore the results were not affected by the number of cells.

    “Nevertheless, all counted Cas9-edited cells were normalized by the total number of DAPI+ cells and therefore the results were not affected by the number of cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    MC3 LNP Cre mRNA is capable to induce Cre-mediated recombination in various brain cell types including neurons, astrocytes, and microglia with different transfection efficiencies between striatum and hippocampus.

    “Taken together, MC3 LNP Cre mRNA is capable to induce Cre-mediated recombination in various brain cell types including neurons, astrocytes, and microglia with different transfection efficiencies between striatum and hippocampus.”
    Quote in the paperSubstantial claim
28 of 28

  • The paper relies on this without support.

    The ROI of the heaviest transfected area provides a valid measure of local transfection efficiency for the entire striatum or hippocampus.

    “Quantifications of local transfection efficiency and cell type-specific analyses were performed in defined Region of Interests (ROIs) of the heaviest transfected area of striatum or hippocampus, which was the identical size (219.74 × 165.71 μm) between all animals.”

  • The paper relies on this without support.

    The antibodies used (NeuN, GFAP, Iba1) specifically identify neurons, astrocytes, and microglia in the Ai9 mouse brain.

    “the mouse IgG1 monoclonal NeuN antibody (cat. #: MAB377) from Millipore, the rabbit polyclonal GFAP antibody (cat. #: AB5804) from Sigma Aldrich, and the rabbit polyclonal Iba1 antibody (cat. #: 019-19741) from WAKO Chemicals.”

  • The paper relies on this without support.

    The tdTomato+; DAPI+ cells counted in sampled sections represent all Cre-recombined cells in the striatum or hippocampus.

    “For determining the number of total tdTomato+; DAPI+ cells in the injected area, every 4th of DAPI-stained brain slice (every 12th brain slice per mouse) was imaged for analysis using the 20x objective, and then were stitched together.”

  • The paper relies on this without support.

    The transfection distance measured by tdTomato+; DAPI+ cells along the rostro-caudal axis reflects the penetration of MC3 LNP mRNA complexes.

    “For the transfection distance analysis, every 3rd brain slice was stained with DAPI and the transfection distance was defined by identifying the brain slices with the presence of tdTomato and DAPI copositive (tdTomato+; DAPI+) cells.”

  • The paper relies on this without support.

    Cas9 mRNA and sgRNA have never been tested in brain tissue using LNPs.

    “Cas9 mRNA and sgRNA have never been tested in brain tissue using LNPs”

  • The paper relies on this without support.

    Very little is known about LNPs-mediated mRNA delivery in the brain.

    “very little is known about LNPs-mediated mRNA delivery in the brain”

  • The paper relies on this without support.

    The transfection area of the highest transfected brain section is representative of the extent of penetration into the striatum or hippocampus.

    “based on the transfection area of the highest transfected brain section”
7 of 7

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Authors
Tuma, J., Chen, Y.-J., Collins, M. G., Paul, A., Li, J., Han, H., Sharma, R., Murthy, N., & Lee, H. Y.
Affiliations
  • The University of Texas at San Antonio Health Science Center (US)
  • Innovative Genomics Institute (US)
Corresponding author
Lee, Hye Young
ORCID
Venue
Biochemistry
Publisher
American Chemical Society (ACS)
Published
2023-09-20
Volume
62
Issue
24
Pages
3533-3547
ISSN
  • 0006-2960 (ISSN-L)
  • 1520-4995
DOI
10.1021/acs.biochem.3c00371
PMC
PMC10760911
OpenAlex
W4386884157
Cited by
65
References
44
Citations by year
2026: 19 · 2025: 29 · 2024: 16 · 2023: 1
FWCI
4.84
Type
journal article
Language
en
Open access
Licence
Locations
  • Biochemistry (publishedVersion)
  • PubMed (publishedVersion)
  • eScholarship (California Digital Library) (submittedVersion)
  • PubMed Central (submittedVersion)
Funders
  • National Institute of Neurological Disorders and Stroke (UG3NS115599, U19NS132303)
  • University of California Berkeley
  • Innovative Genomics Institute
  • National Institute of Allergy and Infectious Diseases (1UM1AI164559, R33AI119115)
  • National Institute of Biomedical Imaging and Bioengineering (R01EB029320)
  • Simons Foundation Autism Research Initiative (574967)
  • California Institute for Regenerative Medicine (DISC2-14097-0)
  • National Institute on Aging (R21AG072423)
  • National Institute on Drug Abuse (R33DA048444)
  • University of Texas at San Antonio
  • Cystic Fibrosis Foundation Therapeutics
  • National Institute of Mental Health (R01MH125979)
  • CRISPR-Cures grant
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
lipid nanoparticles, mRNA delivery, brain drug delivery, intracerebral injection, CRISPR/Cas9, genome editing, Cre recombinase, striatum, hippocampus, neurological disorders, astrocytes, microglia, neurons, transfection efficiency
MeSH terms
RNA, Guide, CRISPR-Cas Systems, Animals, Brain, Liposomes, Nervous System Diseases, RNA, Messenger, RNA, Small Interfering, Mice, Nanoparticles

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-10-20.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-10-20.

Cited sources are checked by gpt-6-sol.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

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