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Cholesterol-functionalized DNA/RNA heteroduplexes cross the blood–brain barrier and knock down genes in the rodent CNS

Abstract

Achieving regulation of endogenous gene expression in the central nervous system (CNS) with antisense oligonucleotides (ASOs) administered systemically would facilitate the development of ASO-based therapies for neurological diseases. We demonstrate that DNA/RNA heteroduplex oligonucleotides (HDOs) conjugated to cholesterol or α-tocopherol at the 5′ end of the RNA strand reach the CNS after subcutaneous or intravenous administration in mice and rats. The HDOs distribute throughout the brain, spinal cord and peripheral tissues and suppress the expression of four target genes by up to 90% in the CNS, whereas single-stranded ASOs conjugated to cholesterol have limited activity. Gene knockdown was observed in major CNS cell types and was greatest in neurons and microglial cells. Side effects, such as thrombocytopenia and focal brain necrosis, were limited by using subcutaneous delivery or by dividing intravenous injections. By crossing the blood–brain barrier more effectively, cholesterol-conjugated HDOs may overcome the limited efficacy of ASOs targeting the CNS without requiring intrathecal administration.

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Fig. 1: Lipid-conjugated HDOs reduce gene expression in the CNS after systemic administration.
Fig. 2: Chol-HDO reduces Malat1 transcript expression throughout the CNS in mice and rats and reduces the expression of clinically relevant targets expressed by neurons and glia after systemic administration.
Fig. 3: PK/PD characterization of Chol-HDO in CNS tissues of mice after systemic injections.
Fig. 4: Comparison of PD/PK between i.c.v. and i.v. injection.

Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its extended data and supplementary information files. Source data are provided with this paper.

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Acknowledgements

We thank A. Oda, Y. Kakoi and A. Kunugi (Research, Takeda Pharmaceutical Company) for conducting the in vivo studies, and S. Matsumoto and I. Chisaki (Research, Takeda Pharmaceutical Company) for quantifying the ASO and conducting PK analyses. We also thank N. Post (Ionis Pharmaceuticals) for the quantification of the ASO, M. Jackson and M. Afetian (Ionis Pharmaceuticals) for valuable technical assistance, S. Klein (Ionis Pharmaceuticals) and A. Nakamura (TMDU) for histopathological studies and A. Abe and Y. Okamoto (WDB) for their care of the laboratory animals. This research was supported by the Basic Science and Platform Technology Programs for Innovative Biological Medicine (18am0301003h0005) and Advanced Biological Medicine (20am0401006h0002) to T.Y., a Research Project on the Elucidation of Chronic Pain (19ek0610013h0003) to T.Y. and T.N. from the Japan Agency for Medical Research and Development (AMED), JST CREST (JPMJCR12L4 to T.Y.) and a JSPS KAKENHI Grant-in-Aid for Scientific Research (S) (17H06109 to T.Y. and T.N.), (A) (19H01016 to T.N. and T.Y.) and (B) (16H05221 to T.N.) from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan (Tokyo).

Author information

Authors and Affiliations

Authors

Contributions

T.N., C.A.D., F.R., P.P.S., C.F.B., Kenichi Miyata and T.Y. designed the research. T.N., C.A.D., K.Y-T., K.I., M.O., H.K., H.M., T. Ishii, S.E., K.Y., Kanjiro Miyata, Kenichi Miyata, B.P., T. Igari, S.Y., N.A., H.H., T.U., R.I.H. and T.W. performed the experiments and analyzed the results. T.N., C.A.D., P.P.S., C.F.B., F.R. and T.Y. wrote the manuscript. T.Y. and T.N. coordinated and supervised the project. All authors have read and approved the final manuscript.

Corresponding author

Correspondence to Takanori Yokota.

Ethics declarations

Competing interests

T.Y. is collaborating with Daiichi Sankyo Co., Ltd. (Tokyo, Japan), Mitsubishi Tanabe Pharma Corporation (Osaka, Japan), NanoCarrier Co., Ltd. (Chiba, Japan), Ono Pharmaceutical Co., Ltd. (Osaka, Japan), Rena Therapeutics Inc. (Tokyo, Japan), Takeda Pharmaceutical Co., Ltd. (Tokyo, Japan) and Toray Industries, Inc. (Tokyo, Japan) and Ionis Pharmaceuticals (CA, USA). T.Y. also serves as academic adviser for Rena Therapeutics Inc. C.A.D., B.P., C.F.B., P.P.S. and F.R. are paid employees of Ionis Pharmaceuticals, Inc. Kenichi Miyata, T. Igari, S.Y., N.A. and H.H. are paid employees of Takeda Pharmaceutical Company Limited. T.Y. and T.N. are inventors on patents on BBB-HDO filed by Tokyo Medical and Dental University and have benefits from licensing of the associated intellectual property.

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Peer review information Nature Biotechnology thanks Arthur Krieg and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Processing mechanism of HDO.

a, Schematic illustration of presumed processing mechanism of HDO in the cell. HDO is composed of a gapmer ASO, duplexed with a cRNA conjugated to delivery ligands. Gapmer ASOs have a central gap-region of DNA flanked by modified nucleotides that enhance affinity for cRNA, such as LNA, cEt or MOE, and whole nucleotides of ASO strand are protected from nucleases by PS modification. In the cRNA strand, the center portion hybridized to the gap portion of ASO remains unmodified for recognition of RNase H, and terminal nucleotides hybridized to both wing portions of ASO are protected from exonucleases by both PS modification and OMe. b, The effect of the cholesterol position and number in cRNA strand of Chol-HDO with LNA wings on Malat1 RNA levels. Cortex was harvested 3 days after one IV dose per week for 4 weeks of PBS or 50 mg/kg Chol-HDO (3–4/group; *P < 0.05; ** P < 0.001). c, Evaluation of Hdac2 expression in the cortex after co-administration of ASO targeting Hdac2 and Chol-HDO containing ASO with cEt wings targeting Malat1 after a single IV injection. Tissues were collected 1 week after dosing. Results show Chol-HDO does not facilitate Hdac2 ASO activity in the CNS indicating the absence of BBB damage by Chol-HDO (3/group). d, ICV administration of ASO targeting Hdac2 with cEt wings shows gene knockdown in the cerebral cortex at 30 or 100 µg doses (2/group), indicating that ASO targeting Hdac2 is effective in the CNS. Abbreviations (for all figures): ASO, antisense oligonucleotide; cEt, 2′, 4′- constrained Ethyl BNA; Chol, cholesterol; Chol-HDO, heteroduplex oligonucleotide conjugated with cholesterol; cRNA, complementary ribonucleic acid; DNA, deoxyribonucleic acid; DHA, Docosahexaenoic acid; Dmpk, dystrophia myotonica protein kinase; Gfap, glial fibrillary acidic protein; Hdac2, histone deacetylase 2; HDO, heteroduplex oligonucleotide; LNA, locked nucleic acid; Malat1, metastasis associated lung adenocarcinoma 1; MOE, 2′-O-Methoxyethyl ribonucleic acid; mRNA, messenger ribonucleic acid; OMe, 2′-O-methyl RNA ; PBS, phosphate-buffered saline; PO phosphodiester; PS phosphorothioate; RNA, ribonucleic acid; s.e.m., standard error of the mean; SOD1, superoxide dismutase 1; Tmax, time to maximal plasma concentration (Tmax); Toc, tocopherol; Toc-HDO, heteroduplex oligonucleotide conjugated with tocopherol.

Source data

Extended Data Fig. 2 The distribution and gene knockdown effect of Chol-HDO in multiple CNS regions.

a, Dose response curve showing Malat1 RNA knockdown in the spinal cord after a single IV injection of PBS or Chol-HDO with LNA or cEt wings (Dose for LNA wings, 12.5, 25, 50 or 75 mg/kg, for cEt wings 7, 20 or 50 mg/kg) (4/group). Spinal cord was harvested 3 days after dosing. Malat1 RNA levels in the cortex were normalized to Actin mRNA. Dose dependency was also observed in other brain regions with the cerebellum being the least sensitive to the HDO (data not shown). Time course of Malat1 RNA reductions in the spinal cord on 3, 7, 14, 28, 56, 168 or 252 days after a single IV injection of 50 mg/kg Chol-HDO with LNA wings (4/group; *P < 0.05; ** P < 0.001). b, The effect of 50 mg/kg Toc-HDO and Chol-HDO with LNA wings after a single IV injection on Malat1 RNA expression in various brain regions and spinal cord compared to 50 mg/kg ASO or PBS. Tissues were harvested 3 days after injection (4/group; *P < 0.05; ** P < 0.001). (c/d) In situ hybridization with a Malat1 probe in the cortex (layers I/II–VI) (c) and the anterior horn of spinal cord (d), dosing paradigm as described in Fig. 1c. Each panel (c) shows all cortical layers from the pial surface/layer I (left) to layer VI (right). The dashed lines in (d) indicate the border between white and gray matter, and the area within the dashed line is the anterior horn of spinal cord. The scale bar is 100 μm. Since Malat1 is ubiquitously expressed15, the near complete inhibition of probe signal suggests that RNA expression was reduced in both neurons and glia after Chol-HDO administration. Similar Malat1 RNA reduction was also confirmed in cerebellum, striatum, hippocampus, and brain stem (data not shown). e, Time courses of Malat1 RNA levels in the cortex and spinal cord on 7, 14 or 28 days after single SC injection of 50 mg/kg Chol-HDO with LNA wings (4–6/group; *P < 0.05; ** P < 0.001). f, Dose response curves of Malat1 gene knockdown effect comparing IV and SC injection with dosing of 50 mg/kg Chol-HDO with cEt wings once a week for four weeks (2–3/group). g, The effect of 50 mg/kg Chol-HDO with MOE gapmer ASOs on Malat1 RNA expression levels in different brain regions after a single IV injection of 50 mg/kg. Tissues were harvested 3 days after the last injection (4/group; *P < 0.05). h, The effect of Chol-HDO with cEt gapmer ASOs on Malat1 RNA in brain regions and spinal cord after IV administration of 50 mg/kg Chol-HDO once a week for four weeks. Tissues were harvested 3 days after the last injection (4/group; *P < 0.05; ** P < 0.001). i, Chol-HDO with MOE wings reduces the expression of Gfap, causative gene of Alexander disease, in various brain regions and spinal cord after IV administration of 50 mg/kg Chol-HDO once a week for four weeks. The brain and spinal cords were harvested 3 days after the last injection (4/group; *P < 0.05; ** P < 0.001). There is no clear reduction in GFAP protein in the CNS of mice treated with Chol-HDO by a Quanterix ELISA assay. While this experimental design described above was sufficient for detecting mRNA knockdown, it was too short to evaluate GFAP protein reduction as this protein is known to have a long half-life (~27 days for cortex and ~9 weeks for spinal cord). j, Chol-HDO with LNA wings reduces the expression of SOD1, the causative gene of familial amyotrophic lateral sclerosis, in various brain regions and spinal cord after SC administration of 50 mg/kg Chol-HDO once a week for four weeks (upper panel). The brain and spinal cords were harvested 7 days after the last injection (4–6/group; *P < 0.05). Human SOD1 protein expression from the brain treated with Chol-HDO was also reduced as compared to that from the brain treated with PBS on immunoblotting (lower panel). Immunoblotting and densitometry analysis (3/group; *P < 0.05) showing that Chol-HDO significantly reduced the levels of SOD1 protein applied by 10 μg total protein in brain as compared to the brain treated with PBS (lower panel).

Source data

Extended Data Fig. 3 Pharmacokinetics of Chol-HDO in CNS and plasma.

a, Time course of ASO distribution detected by anti-PS antibody staining in the cortex at 1, 24, and 48 hours after a single IV injection of 50 mg/kg Chol-HDO with LNA wings. The scale bar is 20 μm. b, Immunohistochemistry staining of anti-PS antibody in cerebral cortex, the choroid plexus and ependymal cells that line the lateral ventricle in the mouse brain 3 days after a single IV injection of 50 mg/kg single-stranded ASO or Chol-HDO with LNA wings. The ASO signals were observed in the choroid plexus (arrow) and ependymal cells (arrowhead) from mice treated with both ASO and Chol-HDO (upper panel). In contrast, the ASO signals were detected in neuron and glia in cortex from mouse treated with Chol-HDO, but not with ASO (lower panel). The scale bar is 50 μm. Lower panel show the higher magnification image of inset in upper panel. c, Ratiometric FRET imaging to localize separated cRNA and intact Chol-HDO with a Cy5-labeled ASO and a Cy3-labeled cRNA sense strand in layer I and II of the somatosensory cortex (upper panels) and cerebellum (lower panels). When the duplex is intact, the Cy3 transfers energy to Cy5 resulting in the gain of a unique FRET signal (ex540/em680). Far left heat map shows relative distribution of separated cRNA (red) and intact Chol-HDO (purple/blue/green). The majority of Chol-HDO localized at the pial basement membrane is intact and local separation is observed inside of neural cells (inset shows the neuron with close-colocalization of intact duplex and separated cRNA indicating separation of the duplex), vascular endothelial cells and ganglionic (Purkinje cell) layer of the cerebellum. The scale bar is 50 μm in the upper panel or 100 μm in the lower panel. d, Time course of ASO concentration in the plasma and cortex after a single IV injection of 50 mg/kg Chol-HDO containing ASO with LNA wings (3–4/group) on 1, 3, 7, 14, 21, 28, 42 or 56 days. As for plasma, the ASO concentration was also measured at 1, 3, 7, 14, 21, or 28 days after a single IV injection of 50 mg/kg ASO with LNA wings alone (3–4/groups). e, Comparison of Malat1 RNA levels after IV injection of 4 total dosing of 6.25 mg/kg (twice a week) and a single doing of 25 mg/kg Chol-HDO with LNA wings in different brain regions. (4/group; *P < 0.05; ** P < 0.001). f, Correlation of Malat1 RNA levels and ASO concentration in kidney after 1, 2, or 4 total IV dosing (1 dose/week) of 50 mg/kg of ASO or Chol-HDO with LNA wings (2–4/group).

Source data

Extended Data Fig. 4 Comparison of dose response between ASO and Chol-HDO by ICV injection.

a, Comparison of dose response curves generated for ASO or Chol-HDO with cEt wings targeting Malat1 by ICV bolus injection in the cortex and spinal cord (4/group). Tissues were collected 2 weeks after dosing.

Source data

Extended Data Fig. 5 Adverse effects of Chol-HDO and their mitigation by divided administrations or SC administration.

a, Platelet number and Malat1 RNA level after a single IV injection of 50 mg/kg Chol-HDO with LNA wings compared to two divided dosing of 25 mg/kg Chol-HDO with LNA wings in a week (3–12/group; *P < 0.05; ** P < 0.001). b, Platelet number and Malat1 RNA knockdown after a single IV injection or SC injection of 50 mg/kg Chol-HDO with LNA wings (10–12/group; *P < 0.05; ** P < 0.001). In all cases platelet counts and Malat1 knockdown were evaluated in the cortex at 3 days after IV injection and 7 days after SC injection. Divided or SC administration rescued thrombocytopenia observed by IV single administration of Chol-HDO. c, Histology with hematoxylin and eosin staining of the brain after once a week injection of 50 mg/kg Chol-HDO with LNA wings for four weeks. SC administration rescued focal necrosis (arrowhead) observed by IV administration of Chol-HDO (4/group). The scale bar is 300 μm. Lower panels show higher magnification images of the inset in upper panels. The arrowhead indicates necrotic neurons in the brain from intravenously repeated injected mouse. The scale bar is 50 μm.

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Nagata, T., Dwyer, C.A., Yoshida-Tanaka, K. et al. Cholesterol-functionalized DNA/RNA heteroduplexes cross the blood–brain barrier and knock down genes in the rodent CNS. Nat Biotechnol 39, 1529–1536 (2021). https://doi.org/10.1038/s41587-021-00972-x

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