CoChat
Sign Up
Cited by 614RNA Interference and Gene DeliveryCRISPR and Genetic EngineeringNanoparticle-Based Drug Delivery

CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy

Open access
Rosenblum, Daniel, Gutkin, Anna, Kedmi, Ranit, Ramishetti, Srinivas, Veiga, Nuphar, Jacobi, Ashley M., Schubert, Mollie S., Friedmann-Morvinski, Dinorah +4 more·2020·Science Advances
DOI 10.1126/sciadv.abc9450PMC PMC7673804
Open this paper in CoChat — free accountOpen PDF

Abstract

CRISPR lipid nanoparticles promote in vivo therapeutic genome editing in two aggressive cancer mouse models.

AI snapshot

Written from the paper’s full text.

TL;DR

This paper reports lipid nanoparticles (LNPs) co-encapsulating Cas9 mRNA and sgRNA that enable targeted CRISPR-Cas9 genome editing in vitro and in vivo. In mouse models of glioblastoma and metastatic ovarian cancer, PLK1 editing inhibited tumor growth and improved survival after single or double administrations.

Key findings

  • L8-cLNPs co-encapsulated Cas9 mRNA and sgRNA with diameters of 71 to 80 nm, PDI 0.024 to 0.103, and >90% encapsulation efficiency for L6, L8, L10, and MC3 formulations; L1 was ~65%. In HEK293/GFP cells, L8-cLNPs reduced GFP+ cells to 4% at 1.0 μg/ml total RNA, achieved 94% GFP genomic editing by NGS, and showed <0.1% editing at the nontargeted PLK1 locus. In multiple GFP-expressing cancer cell lines, GFP fluorescence remained in only 3 to 18% of cells after sgGFP-cLNPs.
  • sgPLK1-cLNPs caused 98% PLK1 editing in HEK293/GFP cells with <0.1% GFP editing, induced G2-M arrest, and reduced viability fivefold. In GBM 005 and OV8 cells, sgPLK1-cLNPs caused 84 and 91% PLK1 editing, respectively, with fivefold and tenfold viability reductions.
  • In orthotopic GBM, intratumoral sgGFP-cLNPs (0.05 mg/kg) produced ~72% GFP editing, and sgPLK1-cLNPs produced ~68% PLK1 editing; caspase-3 activation was seen only in sgPLK1-treated tumors. A single sgPLK1-cLNP injection increased median survival from 32.5 to >48 days, with 30% surviving to 60 days versus all controls dying by 40 days.
  • In metastatic OV8 ovarian cancer, EGFR-targeted cLNPs accumulated three times more in tumors than IgG controls, T-sgPLK1-cLNPs achieved ~82% PLK1 editing with <1% in controls, and two intraperitoneal doses (0.75 mg/kg) increased overall survival by ~80%. Systemic sgGFP-cLNPs (1 mg/kg) showed no apparent toxicity and no significant differences in liver enzymes, blood counts, or plasma cytokines (IL-1β, IL-2, TNF-α, IFN-γ, IL-10) 24 hours after injection.

Methods

  • LNPs were formulated by microfluidic mixing of ionizable cationic lipids, DSPC, cholesterol, PEG-lipids, and Cas9 mRNA/sgRNA; L8 was selected from a lipid library screen. Cas9 mRNA was 5-methoxyuridine-modified, and highly modified sgRNAs were used. EGFR-targeted cLNPs were assembled using the ASSET antibody-linker system.
  • In vitro studies used HEK293/GFP, GBM 005, OV8, NAR, HCT116, and A549 cells; editing was measured by NGS, GFP disruption by flow cytometry, cell cycle by DAPI flow cytometry, and viability by XTT and annexin V/DAPI.
  • In vivo studies used C57BL/6 mice bearing orthotopic 005 GBM tumors and athymic nude mice bearing intraperitoneal OV8-mCherry tumors. Editing was analyzed by NGS in sorted GFP+ or mCherry+ tumor cells; tumor growth was monitored by bioluminescence or fluorescence imaging, and survival was analyzed by Kaplan-Meier curves.

Limitations

  • Stated: more extensive toxicity evaluation is needed for preclinical development; off-target editing remains a safety concern, and the authors suggest mitigation with tissue-specific miR binding sites. The study used PLK1 as proof of concept and S. pyogenes Cas9, which could be substituted with other nucleases.
  • Evident: off-target assessment was limited to selected nontargeted loci (e.g., <0.1% at PLK1 or GFP), not genome-wide off-target analysis. Toxicity and immunogenicity were assessed after a single intravenous dose at 24 hours, so longer-term or repeated-dose safety is not established.
  • Evident: therapeutic efficacy was shown in two mouse cancer models only, and the ovarian cancer targeting relied on EGFR expression in OV8 cells; generalizability to other tumors or human patients is not demonstrated.

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

Integrity signals

Registry

Crossref · 10.1126/sciadv.abc9450

Cites

50 of 50 references verified0 citation errors · 0 not checkable

Statistics

Nothing to recompute.

Claims the paper makes

0 of 41 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Cy5.5-labeled cLNPs distributed throughout the tumor after intratumoral injection.

    “The Cy5.5-labeled cLNPs distributed throughout the tumor (Fig. 4B).”
    Quote in the paperNot a substantial claim
  • Findingown data

    A plasma cytokine panel showed no significant differences after intravenous injection of sgGFP-cLNPs.

    “A plasma cytokine panel [interleukin-1β (IL-1β), IL-2, tumor necrosis factor–α (TNF-α), interferon-γ (IFN-γ), and IL-10] also showed no significant differences (fig. S5C).”
    Quote in the paperSubstantial claim
  • Findingown data

    After incubation with sgGFP-cLNPs (1 μg/ml; 7 nM total RNA), GFP fluorescence was only detected in 3 to 18% of multiple GFP-expressing aggressive cancer cell lines.

    “After incubation with sgGFP-cLNPs (1 μg/ml; 7 nM of total RNA), GFP fluorescence was only detected in 3 to 18% of these cancer cells (Fig. 1H).”
    Quote in the paperSubstantial claim
  • Findingown data

    sgPLK1-cLNPs facilitated ~68% editing in the PLK1 locus in tumor cells.

    “sgPLK1-cLNPs facilitated ~68% editing in the PLK1 locus in tumor cells (Fig. 4C).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: PLK1 is a kinase required for mitosis; lack of it leads to G2-M phase cell cycle arrest and cell death in dividing cells.
  • Findingown data

    A single intracerebral injection of sgGFP-cLNPs (0.05 mg/kg) facilitated ~72% editing in the GFP locus in tumor cells.

    “A single intracerebral injection facilitated ~72% of editing in the GFP locus in tumor cells (fig. S6A).”
    Quote in the paperSubstantial claim
  • May combine two claims. 34
  • Classified as probably not a substantial claim. 9

  • May combine two claims.

    Findingown data

    PLK1 gene editing caused potent G2-M arrest 48 hours later, while control sgGFP-cLNPs had no effect on cell cycle profile.

    “PLK1 gene editing caused potent G2-M arrest 48 hours later, while control sgGFP-cLNPs had no effect on cell cycle profile (Fig. 2, C and D).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: PLK1 is a kinase required for mitosis; lack of it leads to G2-M phase cell cycle arrest and cell death in dividing cells.

  • May combine two claims.

    Findingown data

    DAPI and/or annexin V staining increased after incubation with sgPLK1, but not sgGFP-cLNPs.

    “Similarly, DAPI and/or annexin V staining increased after incubation with sgPLK1, but not sgGFP-cLNPs (Fig. 3, E and J, and fig. S4, B and D).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: PLK1 is a kinase required for mitosis; lack of it leads to G2-M phase cell cycle arrest and cell death in dividing cells.

  • May combine two claims.

    Findingown data

    There were no apparent clinical signs of toxicity and no significant difference in liver enzyme levels or blood counts 24 hours after intravenous injection of sgGFP-cLNPs (1 mg/kg) into C57BL/6 mice.

    “There were no apparent clinical signs of toxicity and no significant difference in liver enzyme (alanine transaminase, aspartate aminotransferase, and alkaline phosphatase) levels (fig. S5A) or blood counts (fig. S5B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    L1-, L8-, and L10-cLNPs all disrupted GFP in a concentration-dependent manner, and L8 was the most efficient.

    “L1-, L8-, and L10-cLNPs all disrupted GFP in a concentration-dependent manner, and L8 was the most efficient.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Encapsulation efficiency of Cas9 mRNA and sgRNA was similarly high (>90%) in L6, L8, L10, and MC3-LNPs but lower in L1-cLNPs (~65%).

    “The encapsulation efficiency of Cas9 mRNA and sgRNA in L6, L8, L10, and MC3-LNPs was similarly high (>90%) but lower in L1-cLNPs (~65%) (Fig. 1D).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    Activated caspase-3 was only present in sgPLK1-cLNP-treated tumors, while no apparent staining was visualized in tumors treated with sgGFP-cLNPs, demonstrating PLK1-dependent apoptosis.

    “Activated caspase-3 was only present in sgPLK1-cLNP–treated tumors, while no apparent staining was visualized in tumors treated with sgGFP-cLNPs, demonstrating PLK1-dependent apoptosis (Fig. 4D).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: PLK1 is a kinase required for mitosis; lack of it leads to G2-M phase cell cycle arrest and cell death in dividing cells.

  • May combine two claims.

    Findingown data

    Disruption of PLK1 strongly caused G2-M cell cycle arrest 48 hours after treatment and reduced cell viability 96 hours after treatment by 5-fold in GBM 005 and 10-fold in OV8.

    “Disruption of PLK1 also strongly caused G2-M cell cycle arrest 48 hours after treatment (Fig. 3, B, C, G, and H, and fig. S4, A and C) and reduced cell viability 96 hours after treatment by 5-fold in GBM 005 and 10-fold in OV8, respectively (Fig. 3, D and I).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: PLK1 is a kinase required for mitosis; lack of it leads to G2-M phase cell cycle arrest and cell death in dividing cells.

  • May combine two claims.

    Findingown data

    Ninety-four percent of GFP genomic DNA was modified by sgGFP-cLNPs, but <0.1% was edited at a nontargeted locus (PLK1).

    “Ninety-four percent of GFP genomic DNA was modified, but <0.1% was edited at a nontargeted locus (PLK1) (Fig. 1, F and G).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    In vitro incubation of GBM 005 or OV8 with sgPLK1, but not sgGFP, cLNPs efficiently disrupted the PLK1 gene, causing 84 and 91% genomic editing, respectively.

    “In vitro incubation of GBM 005 or OV8 with sgPLK1, but not sgGFP, cLNPs efficiently disrupted the PLK1 gene, causing 84 and 91% genomic editing, respectively (Fig. 3, A and F, and fig. S3).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: PLK1 is a kinase required for mitosis; lack of it leads to G2-M phase cell cycle arrest and cell death in dividing cells.

  • May combine two claims.

    Findingown data

    Treating HEK293/GFP with sgPLK1-cLNPs (0.5 μg/ml) caused 98% PLK1 gene editing, while <0.1% was edited at the nontargeted GFP locus.

    “Treating HEK293/GFP with sgPLK1-cLNPs (0.5 μg/ml) caused 98% PLK1 gene editing, while <0.1% was edited at the nontargeted GFP locus by NGS (Fig. 2, A and B).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: PLK1 is a kinase required for mitosis; lack of it leads to G2-M phase cell cycle arrest and cell death in dividing cells.

  • May combine two claims.

    Findingown data

    cLNPs were uniform in size with a diameter of 71 to 80 nm, polydispersity index of 0.024 to 0.103, and ζ potential of −3 to 18.6 mV.

    “cLNPs were uniform in size with a diameter of 71 to 80 nm, polydispersity index of 0.024 to 0.103, and ζ potential of −3 to 18.6 mV as measured by dynamic light scattering (Fig. 1C).”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Findingown data

    MC3-cLNPs were taken up more efficiently than L8-cLNPs into HEK293/GFP cells, but MC3-cLNPs did not reduce GFP expression at any concentration (0.1 to 1.0 μg/ml total RNA, 0.7 to 7 nM total RNA).

    “Although Cy5.5-labeled MC3-cLNPs were taken up more efficiently than L8-cLNPs into HEK293/GFP cells as measured by flow cytometry (fig. S1B), MC3-cLNPs did not reduce GFP expression at any concentration (0.1 to 1.0 μg/ml total RNA, 0.7 to 7 nM total RNA).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Lipids 1, 6, 8, and 10 were the top hits of the screen and were chosen for further evaluation for CRISPR-Cas9 gene editing.

    “Lipids 1, 6, 8, and 10 were the top hits of the screen and were chosen for further evaluation for CRISPR-Cas9 gene editing (Fig. 1B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Cy5.5 signal in the tumor was three times higher in T-Cy5.5-cLNP-treated mice than in I-Cy5.5-cLNP-treated mice, demonstrating specific targeting and accumulation of T-cLNPs in the tumor.

    “Cy5.5 signal in the tumor was three times higher in T-Cy5.5-cLNP–treated mice rather than in I-Cy5.5-cLNP–treated mice, demonstrating specific targeting and accumulation in the tumor of T-cLNPs (Fig. 5, B and C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    A single intratumoral injection of sgPLK1-cLNPs significantly reduced tumor growth compared to control groups and increased median survival from 32.5 to >48 days.

    “A single intratumoral injection of sgPLK1-cLNPs significantly reduced tumor growth compared to control groups as quantified by live animal luciferase activity (Fig. 4, F and G) and increased median survival from 32.5 to >48 days (Fig. 4H).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Thirty percent of sgPLK1-cLNP-treated mice survived for 60 days when the experiment was terminated, while all control mice died by 40 days.

    “Thirty percent of sgPLK1-cLNP–treated mice survived for 60 days when the experiment was terminated, while all the control mice died by 40 days.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Tumor growth was strongly inhibited only by T-sgPLK1-cLNPs and overall survival increased by ~80%.

    “Tumor growth, monitored using mCherry live animal fluorescent imaging, was strongly inhibited only by T-sgPLK1-cLNPs (Fig. 5, F and G) and increased overall survival by ~80% (Fig. 5H).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    T-sgPLK1-cLNPs facilitated ~82% editing in the PLK1 locus in tumor cells, but <1% was detected in control groups.

    “T-sgPLK1-cLNPs facilitated ~82% of editing in the PLK1 locus in tumor cells, but <1% was detected in control groups (Fig. 5D).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: PLK1 is a kinase required for mitosis; lack of it leads to G2-M phase cell cycle arrest and cell death in dividing cells.

  • May combine two claims.

    Findingown data

    sgGFP-cLNPs had no significant effect on tumor growth or survival.

    “sgGFP-cLNPs had no significant effect on tumor growth or survival.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    sgPLK1-cLNPs efficiently disrupted the targeted gene and caused cell cycle arrest and death of GBM 005 and OV8 in vitro.

    “Thus, sgPLK1-cLNPs efficiently disrupted the targeted gene and caused cell cycle arrest and death of GBM 005 and OV8 in vitro.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: PLK1 is a kinase required for mitosis; lack of it leads to G2-M phase cell cycle arrest and cell death in dividing cells.

  • May combine two claims.

    Premisecitation

    Chemical modification of Cas9 mRNA with 5-methoxyuridine and use of highly modified sgRNAs enhance RNA stability and minimize immunogenicity.

    “To enhance RNA stability and minimize immunogenicity, Cas9 mRNA was chemically modified with 5-methoxyuridine, and highly modified sgRNAs were used (IDT sgRNA XT) (15, 16).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [15] Kauffman K. J., Mir F. F., Jhunjhunwala S., Kaczmarek J. C., Hurtado J. E., Yang J. H., Webber M. J., Kowalski P. S., Heartlein M. W., DeRosa F., Anderson D. G., Efficacy and immunogenicity of unmodified and pseudouridine-modified mRNA delivered systemically with lipid nanoparticles in vivo. Biomaterials 109, 78– 87 (2016). 27680591 PMC5267554

      Not checked in the free run.

    • cites [16] Granot Y., Peer D., Delivering the right message: Challenges and opportunities in lipid nanoparticles-mediated modified mRNA therapeutics—An innate immune system standpoint. Semin. Immunol. 34, 68– 77 (2017). 28890238

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    L8-cLNPs cause efficient and specific gene editing in vitro with low toxicity in multiple cancer cell lines.

    “Thus, L8-cLNPs cause efficient and specific gene editing in vitro with low toxicity in multiple cancer cell lines.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Premisecitation

    Nonviral delivery vehicles for CRISPR components evaluated for liver-associated genetic diseases demonstrated gene editing of up to 60% in mice and rat livers and almost complete reduction of target protein in blood.

    “These systems were evaluated for liver-associated genetic diseases, demonstrated gene editing of up to 60% in mice and rat livers, and almost a complete reduction of target protein in the blood.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [5] Cheng Q., Wei T., Farbiak L., Johnson L. T., Dilliard S. A., Siegwart D. J., Selective organ targeting (SORT) nanoparticles for tissue-specific mRNA delivery and CRISPR–Cas gene editing. Nat. Nanotechnol. 15, 313– 320 (2020). 32251383 PMC7735425 source

      Not checked in the free run.

    • cites [11] Finn J. D., Smith A. R., Patel M. C., Shaw L., Youniss M. R., van Heteren J., Dirstine T., Ciullo C., Lescarbeau R., Seitzer J., Shah R. R., Shah A., Ling D., Growe J., Pink M., Rohde E., Wood K. M., Salomon W. E., Harrington W. F., Dombrowski C., Strapps W. R., Chang Y., Morrissey D. V., A single administration of CRISPR/Cas9 lipid nanoparticles achieves robust and persistent in vivo genome editing. Cell Rep. 22, 2227– 2235 (2018). 29490262 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Using Cas9 mRNA instead of plasmid DNA reduces long-term exposure to the nuclease and minimizes off-target gene modifications.

    “Cas9 mRNA was chosen, instead of plasmid DNA, to reduce long-term exposure to the nuclease to minimize off-target gene modifications (13, 14).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [13] Liang X., Potter J., Kumar S., Zou Y., Quintanilla R., Sridharan M., Carte J., Chen W., Roark N., Ranganathan S., Ravinder N., Chesnut J. D., Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J. Biotechnol. 208, 44– 53 (2015). 26003884

      Not checked in the free run.

    • cites [14] Kim S., Kim D., Cho S. W., Kim J., Kim J.-S., Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012– 1019 (2014). 24696461 PMC4032847 source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    AAV applications are limited by small carrying capacity, immune responses, hepatotoxicity at high doses, and lack of cellular targeting.

    “Nevertheless, AAV applications are limited by its small carrying capacity, immune responses, hepatoxicity at high doses, and the lack of cellular targeting (9, 10).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [9] Senís E., Fatouros C., Große S., Wiedtke E., Niopek D., Mueller A.-K., Börner K., Grimm D., CRISPR/Cas9-mediated genome engineering: An adeno-associated viral (AAV) vector toolbox. Biotechnol. J. 9, 1402– 1412 (2014). 25186301

      Not checked in the free run.

    • cites [10] Yin H., Xue W., Chen S., Bogorad R. L., Benedetti E., Grompe M., Koteliansky V., Sharp P. A., Jacks T., Anderson D. G., Genome editing with Cas9 in adult mice corrects a disease mutation and phenotype. Nat. Biotechnol. 32, 551– 553 (2014). 24681508 PMC4157757

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    CRISPR-Cas9 gene editing can permanently disrupt tumor survival genes, potentially overcoming the repeated dosing limitations of traditional cancer therapies.

    “CRISPR-Cas9 gene editing has the potential to permanently disrupt tumor survival genes, which could overcome the repeated dosing limitations of traditional cancer therapies, improve treatment efficacy, and require fewer treatments (1, 2).”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [1] Foo J., Michor F., Evolution of acquired resistance to anti-cancer therapy. J. Theor. Biol. 355, 10– 20 (2014). 24681298 PMC4058397

      Not checked in the free run.

    • cites [2] Hamis S., Nithiarasu P., Powathil G. G., What does not kill a tumour may make it stronger: In silico insights into chemotherapeutic drug resistance. J. Theor. Biol. 454, 253– 267 (2018). 29909142

      Not checked in the free run.

    • Rests on an assumption: Most cancer drugs require repeated administration, which increases treatment-related toxicity and cost and severely reduces patient quality of life.

  • May combine two claims.

    Interpretationown data

    L8-cLNPs are not toxic or immunogenic when administered systemically at therapeutically relevant doses.

    “Although more extensive evaluation of potential toxicity is needed for preclinical development, these results suggest that L8-cLNPs are not toxic or immunogenic when administered systemically at therapeutically relevant doses (see below).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Long exposure time to the Cas9 nuclease, as well as repeating dosing, might increase the risk for Cas9-related immune responses followed by immune-related adverse reactions and treatment failure.

    “Long exposure time to the Cas9 nuclease, as well as repeating dosing, might increase the risk for Cas9-related immune responses following by immune-related adverse reactions and treatment failure.”
    Quote in the paperSubstantial claim

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

    Study design and conductown data

    All used samples showed a PDI (polydispersity index) lower than 0.2.

    “All used samples showed a PDI (polydispersity index) lower than 0.2.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Suppression of mRNAs in hepatocytes and Kupffer cells can be achieved by inserting miR122 and miR142 binding sites, respectively.

    “The main off-target site of LNP-based platforms is the liver, more specifically hepatocytes and Kupffer cells (49, 50), and suppression of mRNAs in these cell types can be achieved by inserting miR122 and miR142 binding sites, respectively.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [49] Shi B., Keough E., Matter A., Leander K., Young S., Carlini E., Sachs A. B., Tao W., Abrams M., Howell B., Sepp-Lorenzino L., Biodistribution of small interfering RNA at the organ and cellular levels after lipid nanoparticle-mediated delivery. J. Histochem. Cytochem. 59, 727– 740 (2011). 21804077 PMC3261601

      Not checked in the free run.

    • cites [50] Woitok M. M., Zoubek M. E., Doleschel D., Bartneck M., Mohamed M. R., Kießling F., Lederle W., Trautwein C., Cubero F. J., Lipid-encapsulated siRNA for hepatocyte-directed treatment of advanced liver disease. Cell Death Dis. 11, 343 (2020). 32393755 PMC7214425

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Intraperitoneally injected chemotherapy improves progression-free survival and overall survival.

    “Recent clinical studies have demonstrated an improved pharmacokinetic profile of intraperitoneally injected chemotherapy resulting in higher drug concentration in the abdominal cavity and improved progression-free survival and overall survival.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [44] Markman M., Reichman B., Hakes T., Curtin J., Jones W., Lewis J. L. Jr., Barakat R., Rubin S., Mychalczak B., Saigo P., Almadrones L., Hoskins W., Intraperitoneal chemotherapy in the management of ovarian cancer. Cancer 71, 1565– 1570 (1993). 8431894

      Not checked in the free run.

    • cites [45] Jewell A., McMahon M., Khabele D., Heated intraperitoneal chemotherapy in the management of advanced ovarian cancer. Cancers 10, 296 (2018). PMC6162496 30200420

      Not checked in the free run.

    • cites [46] Skaznik-Wikiel M. E., Lesnock J. L., McBee W. C., Beriwal S., Zorn K. K., Richard S. D., Krivak T. C., Edwards R. P., Intraperitoneal chemotherapy for recurrent epithelial ovarian cancer is feasible with high completion rates, low complications, and acceptable patient outcomes. Int. J. Gynecol. Cancer 22, 232– 237 (2012). 22080886

      Not checked in the free run.

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

    Study design and conductnone given

    Mice were randomly divided in a blinded fashion at the beginning of each experiment.

    “Mice were randomly divided in a blinded fashion at the beginning of each experiment.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All cells were routinely checked every 2 months for mycoplasma contamination using the EZ-PCR Mycoplasma Test Kit.

    “All cells were routinely checked every 2 months for mycoplasma contamination using the EZ-PCR Mycoplasma Test Kit (Biological Industries, Israel).”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    The majority of ovarian cancers are diagnosed at late stages when tumor metastasizes throughout the peritoneal cavity.

    “The majority of ovarian cancers are diagnosed at late stages when tumor metastasizes throughout the peritoneal cavity (42, 43).”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [42] Stewart C., Ralyea C., Lockwood S., Ovarian cancer: An integrated review. Semin. Oncol. Nurs. 35, 151– 156 (2019). 30867104

      Not checked in the free run.

    • cites [43] Lheureux S., Braunstein M., Oza A. M., Epithelial ovarian cancer: Evolution of management in the era of precision medicine. CA Cancer J. Clin. 69, 280– 304 (2019). 31099893

      Not checked in the free run.

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

    Study design and conductnone given

    Preestablished criteria for the removal of animals from the experiment were based on animal health, behavior, and well-being as required by ethical guidelines.

    “Preestablished criteria for the removal of animals from the experiment were based on animal health, behavior, and well-being as required by ethical guidelines.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S4 (based). May combine two claims.

    Interpretationreasoning

    The permanent nature of genome editing should have a therapeutic impact even after one or a few doses, which could strongly affect toxicity, development of adverse reactions, compliance, and cost.

    “The permanent nature of genome editing should have a therapeutic impact even after one or a few doses, which could strongly affect toxicity, development of adverse reactions, compliance, and cost.”
    Quote in the paperNot a substantial claim

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

    Study design and conductnone given

    All animal protocols were approved by the Tel Aviv University Institutional Animal Care and Usage Committee and in accordance with current regulations and standards of the Israel Ministry of Health.

    “All animal protocols were approved by the Tel Aviv University Institutional Animal Care and Usage Committee and in accordance with current regulations and standards of the Israel Ministry of Health.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Interpretationown data

    cLNPs targeting PLK1 inhibit tumor growth and improve survival in two aggressive cancer models in mice following single or double cLNP administrations.

    “cLNPs targeting PLK1 were able to inhibit tumor growth and improve survival in two aggressive cancer models in mice following single or double cLNP administrations.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Intraperitoneally injected chemotherapy has an improved pharmacokinetic profile resulting in higher drug concentration in the abdominal cavity.

    “Recent clinical studies have demonstrated an improved pharmacokinetic profile of intraperitoneally injected chemotherapy resulting in higher drug concentration in the abdominal cavity and improved progression-free survival and overall survival.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [44] Markman M., Reichman B., Hakes T., Curtin J., Jones W., Lewis J. L. Jr., Barakat R., Rubin S., Mychalczak B., Saigo P., Almadrones L., Hoskins W., Intraperitoneal chemotherapy in the management of ovarian cancer. Cancer 71, 1565– 1570 (1993). 8431894

      Not checked in the free run.

    • cites [45] Jewell A., McMahon M., Khabele D., Heated intraperitoneal chemotherapy in the management of advanced ovarian cancer. Cancers 10, 296 (2018). PMC6162496 30200420

      Not checked in the free run.

    • cites [46] Skaznik-Wikiel M. E., Lesnock J. L., McBee W. C., Beriwal S., Zorn K. K., Richard S. D., Krivak T. C., Edwards R. P., Intraperitoneal chemotherapy for recurrent epithelial ovarian cancer is feasible with high completion rates, low complications, and acceptable patient outcomes. Int. J. Gynecol. Cancer 22, 232– 237 (2012). 22080886

      Not checked in the free run.

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

    Study design and conductnone given

    All animal experiments were conducted in a double-blinded fashion; the researchers were blinded to group allocation and administered treatments.

    “All animal experiments were conducted in a double-blinded fashion; the researchers were blinded to group allocation and administered treatments.”
    Quote in the paperNot a substantial claim
40 of 40

  • The paper relies on this without support.

    LNPs are clinically approved nonviral nucleic acid delivery systems capable of delivering large payloads.

    “Lipid nanoparticles (LNPs) are clinically approved nonviral nucleic acid delivery systems capable of delivering potentially such large payloads.”

  • The paper relies on this without support.

    Cationic ionizable lipids are the key component of LNPs that enables efficient nucleic acid encapsulation, cellular delivery, and endosomal release.

    “Cationic ionizable lipids are the key component of LNPs that enables efficient nucleic acid encapsulation, cellular delivery, and endosomal release.”

  • The paper relies on this without support.

    The large size of Cas9 and sgRNA is an obstacle for conventional viral and nonviral delivery systems.

    “However, the large size of Cas9 (160 kDa, 4300 bases) and sgRNA (~31 kDa, 130 bases) is an obstacle for conventional viral and nonviral delivery systems.”

  • The paper relies on this without support.

    Double-strand breaks are predominantly resolved via error-prone nonhomologous end-joining, which can induce insertions or deletions resulting in gene disruption.

    “DSBs are predominantly resolved via the error-prone nonhomologous end-joining repair mechanism, which can induce insertions or deletions that result in gene disruption.”

  • The paper relies on this without support.

    PLK1 is a kinase required for mitosis; lack of it leads to G2-M phase cell cycle arrest and cell death in dividing cells.

    “PLK1 is a kinase required for mitosis; lack of it leads to G2-M phase cell cycle arrest and cell death in dividing cells.”

  • The paper relies on this without support.

    Neurons are terminally differentiated nondividing cells, normal brain tissue has minimal expression of PLK1, and therefore normal brain tissue is not expected to undergo apoptosis.

    “Because neurons are terminally differentiated nondividing cells, normal brain tissue has minimal expression of PLK1; therefore, it is not expected to undergo apoptosis.”

  • The paper relies on this without support.

    Most cancer drugs require repeated administration, which increases treatment-related toxicity and cost and severely reduces patient quality of life.

    “Most cancer drugs require repeated administration, which increases treatment-related toxicity and treatment cost and severely reduces patient quality of life.”

  • The paper relies on this without support.

    Other genetic therapies based on RNA interference are transient and would require repeated dosing, especially for rapidly dividing cancer cells.

    “Other genetic therapies, such as those based on RNA interference (RNAi), are transient and, therefore, would require repeated dosing, especially for rapidly dividing cancer cells.”

  • The paper relies on this without support.

    The blood-brain barrier is a highly restrictive barrier for most therapeutic modalities.

    “The blood-brain barrier (BBB) is a highly restrictive barrier for most therapeutic modalities.”

  • The paper relies on this without support.

    The clinical course of this devastating disease has not changed for over a decade, partly due to the limitation presented by the low BBB permeability to standard chemo- and immunotherapies.

    “The clinical course of this devastating disease has not changed for over a decade, partly due to the limitation presented by the low BBB permeability to standard chemo- and immunotherapies.”

  • The paper relies on this without support.

    Most LNP and nanoparticle delivery systems have therapeutic effect largely limited to the liver and other central organs, where particles get trapped.

    “Targeting provides a way of overcoming the limitations of most LNP and nanoparticle delivery systems, whose therapeutic effect is largely limited to the liver and other central organs, where particles get trapped.”

  • The paper relies on this without support.

    Off-target gene editing of bystander cells is an additional safety concern for translating CRISPR technologies to the clinic.

    “Additional safety concern of translating CRISPR technologies to the clinic resides in off-target gene editing of bystander cells.”
12 of 12

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Regulation of other tumour-associated genes, such as PLK1, Bax, Maspin, PUMA and PTEN, can delay tumour growth in vivo.

    “Similarly, the regulation of other tumour-associated genes, such as PLK1 (ref.133), Bax219, Maspin37, PUMA70 and PTEN220, can delay tumour growth in vivo.”

    From Lipid nanoparticles for mRNA delivery (2021) · cites this paper as [133]

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Cytokine sample is called both plasma and serum

    Minor

    The results identify the cytokine panel as a plasma assay, but the methods say serum was separated and stored for cytokine analysis.

  • Brain distribution assay has conflicting collection times

    Minor

    The results say mice were euthanized six hours after injection to assess cLNP distribution, whereas the corresponding method says brains were harvested after four hours.

2 of 2

Cites

Flagged first, then by citations

Cited by

Sorted by citations

Related

Authors
Rosenblum, D., Gutkin, A., Kedmi, R., Ramishetti, S., Veiga, N., Jacobi, A. M., Schubert, M. S., Friedmann-Morvinski, D., Cohen, Z. R., Behlke, M. A., Lieberman, J., & Peer, D.
Affiliations
  • Tel Aviv University (IL)
  • Integrated DNA Technologies (United States) (US)
  • Sagol School of Neuroscience, Department of Biochemistry and Molecular Biology, The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel Aviv, Israel.
  • Boston Children's Hospital (US)
Corresponding author
  • Rosenblum, Daniel
  • Gutkin, Anna
  • Peer, Dan
ORCID
Venue
Science Advances
Publisher
American Association for the Advancement of Science (AAAS)
Published
2020-11-20
Volume
6
Issue
47
ISSN
2375-2548 (ISSN-L)
DOI
10.1126/sciadv.abc9450
PMC
PMC7673804
OpenAlex
W3098190681
Cited by
614
References
50
Citations by year
2026: 89 · 2025: 141 · 2024: 123 · 2023: 115 · 2022: 94 · 2021: 50 · 2020: 1
FWCI
21.07
Type
journal article
Language
en
Open access
Licence
Locations
  • Science Advances (publishedVersion, cc-by-nc)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
Funders
ICRF (2016/iL)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
  • Physical Sciences › Materials Science › Biomaterials › Nanoparticle-Based Drug Delivery
Keywords
CRISPR/Cas9 genome editing, lipid nanoparticles, targeted gene delivery, glioblastoma, ovarian cancer, PLK1, orthotopic tumor model, disseminated cancer, tumor cell apoptosis, cancer therapy
MeSH terms
Gene Editing, Liposomes, Neoplasms, Gene Transfer Techniques, Nanoparticles, CRISPR-Cas Systems

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 2026-10-10.

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

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 ›

Are you an author, or do you see a problem with this page? Request a review