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l-Type amino acid transporter 1-targeting nanoparticles for antisense oligonucleotide delivery to the CNS

Lim, Yu Na, Ryu, In Soo, Jung, Yeon-Joo, Helmlinger, Gabriel, Kim, Insun, Park, Hye Won, Kang, Hansol, Lee, Jina, Lee, Hyo Jin, Lee, Kang Seon, Jang, Ha-Na, Ha, Dae-In, Park, Junghyung, Won, Jinyoung, Lim, Kyung Seob, Jeon, Chang-Yeop, Cho, Hyun-Jeong, Min, Hyun Su, Ryu, Jin-Hyeob·2024·Molecular Therapy Nucleic Acids
DOI 10.1016/j.omtn.2024.102340PMC PMC11474373
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Figure 1 Schematic illustration of ASO-loaded Phe-PEG-PLL(NA/MPA) (A) PEG-PLL(NA/MPA) with and without Phe ligand complex with ASO drug cargo. Cationic block and hydrophilic block of polymer chains allow for rapid dissociation and intracellular release of the ASO cargo. (B) Phe-PEG-PLL(NA/MPA) synthesis schematic. Phe was introduced through a click reaction between N3-PEG-PLL(NA/MPA) and the alkyne modified tyrosine.
Figure 1 Schematic illustration of ASO-loaded Phe-PEG-PLL(NA/MPA) (A) PEG-PLL(NA/MPA) with and without Phe ligand complex with ASO drug cargo. Cationic block and hydrophilic block of polymer chains allow for rapid dissociation and intracellular release of the ASO cargo. (B) Phe-PEG-PLL(NA/MPA) synthesis schematic. Phe was introduced through a click reaction between N3-PEG-PLL(NA/MPA) and the alkyne modified tyrosine.CC BY-NC-ND · PubMed Central
Figure 2 Characterization studies of Phe-NPs/ASO (A) Hydrodynamic size distribution of Phe-NPs/ASO. (B) Morphologies of Phe-NPs/ASO observed by a cryo-transmission electron microscope before lyophilization and (C) after lyophilization. Scale bar: 50 nm. (D) Changes in total ASO concentration in synthesized Phe-NPs/ASO under various GSH concentrations (0, 0.1, 1.0, 10, and 100 mM). Data are presented as means ± SDs (one-way ANOVA and Tukey multiple comparisons tests; ∗p < 0.05, ∗∗∗∗p < 0.0001 vs. 0 mM GSH, ####p < 0.001 vs. 1 mM GSH). (E) Total ASO concentration in synthesized Phe-NPs/ASO under varying pH conditions (pH 5.28 and pH 7.10). Data are presented as means ± SDs (unpaired t test, ∗∗∗∗p < 0.001 vs. pH 5.28).
Figure 2 Characterization studies of Phe-NPs/ASO (A) Hydrodynamic size distribution of Phe-NPs/ASO. (B) Morphologies of Phe-NPs/ASO observed by a cryo-transmission electron microscope before lyophilization and (C) after lyophilization. Scale bar: 50 nm. (D) Changes in total ASO concentration in synthesized Phe-NPs/ASO under various GSH concentrations (0, 0.1, 1.0, 10, and 100 mM). Data are presented as means ± SDs (one-way ANOVA and Tukey multiple comparisons tests; ∗p < 0.05, ∗∗∗∗p < 0.0001 vs. 0 mM GSH, ####p < 0.001 vs. 1 mM GSH). (E) Total ASO concentration in synthesized Phe-NPs/ASO under varying pH conditions (pH 5.28 and pH 7.10). Data are presented as means ± SDs (unpaired t test, ∗∗∗∗p < 0.001 vs. pH 5.28).CC BY-NC-ND · PubMed Central
Figure 3 In vitro characterization of Phe-NPs/ASO-Cy5.5 (A) Quantification of Phe-NPs/ASO-Cy5.5 cellular uptake in a GL261-Luc cell line, at varying Phe ligand densities. (B) Changes in Cy5.5 fluorescence intensities following exposure to targeting Phe-NPs/ASO-Cy5.5 or NT-NPS/ASO-Cy5.5 in the absence or presence of a Phe inhibitor; GL261-Luc cell line. Data are presented as means ± SDs (one-way ANOVA and Tukey multiple comparison tests; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001). (C) Confocal microscopy imaging of Phe-NPs/ASO-Cy5.5 cellular uptake in GL261-Luc cells; 0, 1, 2, and 4 h of incubation with Phe-NPs/ASO-Cy5.5. Cell nuclei were stained with DAPI (blue), acidic organelles were labeled using LysoTracker (green), and ASO-Cy5.5 was detected under red fluorescence. Scale bars: 20 μm. (D) Temporal changes in mean fluorescence intensity of Phe-NPs/ASO-Cy5.5 alone and its co-localization with acidic organelles.
Figure 3 In vitro characterization of Phe-NPs/ASO-Cy5.5 (A) Quantification of Phe-NPs/ASO-Cy5.5 cellular uptake in a GL261-Luc cell line, at varying Phe ligand densities. (B) Changes in Cy5.5 fluorescence intensities following exposure to targeting Phe-NPs/ASO-Cy5.5 or NT-NPS/ASO-Cy5.5 in the absence or presence of a Phe inhibitor; GL261-Luc cell line. Data are presented as means ± SDs (one-way ANOVA and Tukey multiple comparison tests; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗∗p < 0.0001). (C) Confocal microscopy imaging of Phe-NPs/ASO-Cy5.5 cellular uptake in GL261-Luc cells; 0, 1, 2, and 4 h of incubation with Phe-NPs/ASO-Cy5.5. Cell nuclei were stained with DAPI (blue), acidic organelles were labeled using LysoTracker (green), and ASO-Cy5.5 was detected under red fluorescence. Scale bars: 20 μm. (D) Temporal changes in mean fluorescence intensity of Phe-NPs/ASO-Cy5.5 alone and its co-localization with acidic organelles.CC BY-NC-ND · PubMed Central
Figure 4 Pharmacokinetics, biodistribution, and BBB crossing of Phe-NPs/ASO-Cy5.5 in mouse (A) In vivo fluorescence imaging of Hsd:ICR (CD-1) mice after 1.0 mg/kg IV administration of ASO-Cy5.5, Phe-NPs/ASO-Cy5.5, or NT-NPS/ASO-Cy5.5, for up to 24 h. (B) Pharmacokinetics of IV-administered ASO-Cy5.5 or Phe-NPs/ASO-Cy5.5 in blood. (C) Accumulated ASO in mouse brain, expressed as %ID/g, 90 min post-IV administration of 1.0 mg/kg of ASO-Cy5.5 or Phe-NPs/ASO-Cy5.5. Data are presented as means ± SDs (unpaired t test, ∗∗∗∗p < 0.001 vs. naked ASO-Cy5.5). (D and E) IV-CLSM imaging, in vivo BBB crossing: ASO-Cy5.5 (D) and Phe-NPs/ASO-Cy5.5 in mouse brain (E). Scale bar: 100 μm.
Figure 4 Pharmacokinetics, biodistribution, and BBB crossing of Phe-NPs/ASO-Cy5.5 in mouse (A) In vivo fluorescence imaging of Hsd:ICR (CD-1) mice after 1.0 mg/kg IV administration of ASO-Cy5.5, Phe-NPs/ASO-Cy5.5, or NT-NPS/ASO-Cy5.5, for up to 24 h. (B) Pharmacokinetics of IV-administered ASO-Cy5.5 or Phe-NPs/ASO-Cy5.5 in blood. (C) Accumulated ASO in mouse brain, expressed as %ID/g, 90 min post-IV administration of 1.0 mg/kg of ASO-Cy5.5 or Phe-NPs/ASO-Cy5.5. Data are presented as means ± SDs (unpaired t test, ∗∗∗∗p < 0.001 vs. naked ASO-Cy5.5). (D and E) IV-CLSM imaging, in vivo BBB crossing: ASO-Cy5.5 (D) and Phe-NPs/ASO-Cy5.5 in mouse brain (E). Scale bar: 100 μm.CC BY-NC-ND · PubMed Central
Figure 5 Biodistribution and pharmacokinetics of Phe-NPs/ASO-Cy5.5 in NHP (A) Temporal changes in Cy5.5 fluorescence in marmoset, post-IV administration of Phe-NPs/ASO-Cy5.5. (B) Pharmacokinetics of IV-administered Phe-NPs/ASO-Cy5.5 in the CSF of cynomolgus monkeys.
Figure 5 Biodistribution and pharmacokinetics of Phe-NPs/ASO-Cy5.5 in NHP (A) Temporal changes in Cy5.5 fluorescence in marmoset, post-IV administration of Phe-NPs/ASO-Cy5.5. (B) Pharmacokinetics of IV-administered Phe-NPs/ASO-Cy5.5 in the CSF of cynomolgus monkeys.CC BY-NC-ND · PubMed Central
Figure 6 Phe-NPs/ASO target knockdown in sub-regions of mouse brain (A) Target gene knockdown in sub-regions of mouse brain, 1 h post-ICV injection of 1.5 or 10 μg Phe-NPs/ASO. (B) Target gene knockdown in sub-regions of mouse brain, 30 min post-IV injection of 10 or 20 mg/kg of Phe-NPs/ASO. Data are presented as means ± SDs (one-way ANOVA and Tukey multiple comparison tests; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001). OB, olfactory bulb; CB, cerebellum; CTX, cortex; HP, hippocampus; HTH, hypothalamus; ME, medulla; MID, midbrain; PO, pons; STR, striatum; TH, thalamus; n.s., not significant.
Figure 6 Phe-NPs/ASO target knockdown in sub-regions of mouse brain (A) Target gene knockdown in sub-regions of mouse brain, 1 h post-ICV injection of 1.5 or 10 μg Phe-NPs/ASO. (B) Target gene knockdown in sub-regions of mouse brain, 30 min post-IV injection of 10 or 20 mg/kg of Phe-NPs/ASO. Data are presented as means ± SDs (one-way ANOVA and Tukey multiple comparison tests; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001). OB, olfactory bulb; CB, cerebellum; CTX, cortex; HP, hippocampus; HTH, hypothalamus; ME, medulla; MID, midbrain; PO, pons; STR, striatum; TH, thalamus; n.s., not significant.CC BY-NC-ND · PubMed Central

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Authors
Lim, Y. N., Ryu, I. S., Jung, Y.-J., Helmlinger, G., Kim, I., Park, H. W., Kang, H., Lee, J., Lee, H. J., Lee, K. S., Jang, H.-N., Ha, D.-I., Park, J., Won, J., Lim, K. S., Jeon, C.-Y., Cho, H.-J., Min, H. S., & Ryu, J.-H.
ORCID
Ryu, Jin-Hyeob
Venue
Molecular Therapy Nucleic Acids
Publisher
Elsevier BV
Published
2024-12
Volume
35
Issue
4
Pages
102340
ISSN
2162-2531
DOI
10.1016/j.omtn.2024.102340
PMC
PMC11474373
Cited by
17
References
4
Language
en
Licence
Funders
  • Korea Research Institute of Bioscience and Biotechnology
  • Korea Ministry of Small and Medium Enterprises and Startups (RS-2023-00283779)

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