Stochastic packaging of Cas proteins into exosomes

Capa

Citar

Texto integral

Acesso aberto Acesso aberto
Acesso é fechado Acesso está concedido
Acesso é fechado Somente assinantes

Resumo

CRISPR/Cas systems are perspective molecular tools for targeted manipulation with genetic materials, including gene editing, regulation of gene transcription, modification of epigenome etc. While CRISPR/Cas systems proved to be highly effective for correcting genetic disorders and treating infectious diseases and cancers in experimental settings, the clinical translation of these results is hampered by the lack of efficient CRISPR/Cas delivery vehicles. Modern synthetic nanovehicles based on organic and inorganic polymers have many disadvantages, including toxicity issues, the lack of targeted delivery, complex and expensive production pipelines. In turn, exosomes are secreted biological nanoparticles exhibiting high biocompatibility, physico-chemical stability, and ability to cross biological barriers. Early clinical trials found no toxicity associated with exosome injections. In recent years, exosomes have been considered as perspective delivery vehicles for CRISPR/Cas systems in vivo. The aim of this study was to analyze the efficacy of CRISPR/Cas stochastic packaging into exosomes at several human cell lines. Here, we show that Cas9 protein is effectively localized into the compartment of intracellular exosome biogenesis, but stochastic packaging of Cas9 into exosomes turns to be very low (~1%). As such, stochastic packaging of Cas9 protein is very ineffective, and cannot be used for gene editing purposes. Developing novel tools and technologies for loading CRISPR/Cas systems into exosomes is required.

Texto integral

Acesso é fechado

Sobre autores

N. Ponomareva

First Moscow State Medical University (Sechenov University); Sirius University of Science and Technology

Autor responsável pela correspondência
Email: ponomareva.n.i13@yandex.ru

Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases, Department of Pharmaceutical and Toxicological Chemistry

Rússia, Moscow, 119991; Sochi, 354340

S. Brezgin

First Moscow State Medical University (Sechenov University); Sirius University of Science and Technology

Email: ponomareva.n.i13@yandex.ru

Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases

Rússia, Moscow, 119991; Sochi, 354340

A. Kostyusheva

First Moscow State Medical University (Sechenov University)

Email: ponomareva.n.i13@yandex.ru

Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases

Rússia, Moscow, 119991

O. Slatinskaya

Lomonosov Moscow State University

Email: ponomareva.n.i13@yandex.ru

Faculty of Biology

Rússia, Moscow, 119991

E. Bayurova

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products, Russian Academy of Sciences

Email: ponomareva.n.i13@yandex.ru
Rússia, Moscow, 108819

I. Gordeychuk

Chumakov Federal Scientific Center for Research and Development of Immune-and-Biological Products, Russian Academy of Sciences

Email: ponomareva.n.i13@yandex.ru
Rússia, Moscow, 108819

G. Maksimov

Lomonosov Moscow State University

Email: ponomareva.n.i13@yandex.ru

Faculty of Biology

Rússia, Moscow, 119991

D. Sokolova

N.N. Blokhin National Medical Research Center of Oncology

Email: ponomareva.n.i13@yandex.ru
Rússia, Moscow, 115478

G. Babaeva

N.N. Blokhin National Medical Research Center of Oncology

Email: ponomareva.n.i13@yandex.ru
Rússia, Moscow, 115478

I. Khan

N.N. Blokhin National Medical Research Center of Oncology

Email: ponomareva.n.i13@yandex.ru
Rússia, Moscow, 115478

V. Pokrovsky

N.N. Blokhin National Medical Research Center of Oncology

Email: ponomareva.n.i13@yandex.ru
Rússia, Moscow, 115478

A. Lukashev

First Moscow State Medical University (Sechenov University)

Email: ponomareva.n.i13@yandex.ru

Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases

Rússia, Moscow, 11999

V. Chulanov

National Medical Research Center of Tuberculosis and Infectious Diseases, Ministry of Health

Email: ponomareva.n.i13@yandex.ru
Rússia, Moscow, 127994

D. Kostyushev

First Moscow State Medical University (Sechenov University); Sirius University of Science and Technology

Email: ponomareva.n.i13@yandex.ru

Martsinovsky Institute of Medical Parasitology, Tropical and Vector-Borne Diseases

Rússia, Moscow, 119991; Sochi, 354340

Bibliografia

  1. Банников А.В., Лавров А.В. (2017) CRISPR/Cas9 – король геномного редактирования. Молекуляр. биология. 51, 582–594.
  2. Wiedenheft B., Sternberg S.H., Doudna J.A. (2012) RNA-guided genetic silencing systems in bacteria and archaea. Nature. 482, 331–338.
  3. Ran F.A., Hsu P.D., Wright J., Agarwala V., Scott D.A., Zhang F. (2013) Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308.
  4. Kostyushev D., Brezgin S., Kostyusheva A., Zarifyan D., Goptar I., Chulanov V. (2019) Orthologous CRISPR/Cas9 systems for specific and efficient degradation of covalently closed circular DNA of hepatitis B virus. Cell. Mol. Life Sci. 76, 1779–1794.
  5. Brezgin S., Kostyusheva A., Kostyushev D., Chulanov V. (2019) Dead Cas systems: types, principles, and applications. Int. J. Mol. Sci. 20(23), 6041.
  6. Kostyusheva A., Brezgin S., Babin Y., Vasilyeva I., Glebe D., Kostyushev D., Chulanov V. (2022) CRISPR-Cas systems for diagnosing infectious diseases. Methods. 203, 431–446.
  7. Chen S., Lee B., Lee A.Y., Modzelewski A.J., He L. (2016) Highly efficient mouse genome editing by CRISPR ribonucleoprotein electroporation of zygotes. J. Biol. Chem. 291(28), 14457–14467.
  8. Bharathkumar N., Sunil A., Meera P., Aksah S., Kannan M., Saravanan K.M., Anand T. (2022) CRISPR/Cas-based modifications for therapeutic applications: a review. Mol. Biotechnol. 64, 355–372.
  9. Brezgin S., Kostyusheva A., Ponomareva N., Volia V., Goptar I., Nikiforova A., Shilovskiy I., Smirnov V., Kostyushev D., Chulanov V. (2020) Clearing of foreign episomal DNA from human cells by crispra-mediated activation of cytidine deaminases. Int. J. Mol. Sci. 21, 1–17.
  10. Kostyushev D., Brezgin S., Kostyusheva A., Ponomareva N., Bayurova E., Zakirova N., Kondrashova A., Goptar I., Nikiforova A., Sudina A., Babin Y., Gordeychuk I., Lukashev A., Zamyatnin A., Ivanov A., Chulanov V. (2023) Transient and tunable CRISPRa regulation of APOBEC/AID genes for targeting hepatitis B virus. Mol. Ther. Nucl. Acids. 32, 478–493.
  11. Kostyushev D., Kostyusheva A., Brezgin S., Ponomareva N., Zakirova N.F., Egorshina A., Yanvarev D.V., Bayurova E., Sudina A., Goptar I., Nikiforova A., Dunaeva E., Lisitsa T., Abramov I., Frolova A., Lukashev A., Gordeychuk I., Zamyatnin A.A., Ivanov A., Chulanov V. (2023) Depleting hepatitis B virus relaxed circular DNA is necessary for resolution of infection by CRISPR-Cas9. Mol. Ther. Nucl. Acids, 31, 482–493.
  12. Костюшева А.П., Костюшев Д.С., Брезгин С.А., Зарифьян Д.Н., Волчкова Е.В., Чуланов В.П. (2019) Низкомолекулярные ингибиторы путей репарации двухцепочечных разрывов ДНК усиливают противовирусное действие системы CRISPR/Cas9 на моделях вируса гепатита B. Молекуляр. биология. 53, 311–323.
  13. Костюшева А.П., Брезгин С.А., Пономарева Н.И., Гоптарь И.А., Никифорова А.В., Гегечкори В.И., Полуэктова В.Б., Туркадзе К.А., Судина А.Е., Чуланов В.П., Костюшев Д.С. (2022) Противовирусное действие рибонуклеопротеиновых комплексов CRISPR/Cas9 на модели вируса гепатита В in vivo. Молекуляр. биология. 56, 884–891.
  14. Sharma G., Sharma A.R., Bhattacharya M., Lee S., Chakraborty C. (2021) CRISPR-Cas9 : a preclinical and clinical perspective for the treatment of human diseases. Mol. Ther. 29(2), 571–586.
  15. Rafii S., Tashkandi E., Bukhari N., Al-shamsi H.O. (2022) Current status of CRISPR/Cas9 application in clinical cancer research: opportunities and challenges. Cancers (Basel). 14(4), 1–14.
  16. Kostyushev D., Kostyusheva A., Brezgin S., Smirnov V., Volchkova E., Lukashev A., Chulanov V. (2020) Gene editing by extracellular vesicles. Int. J. Mol. Sci. 21, 1–34.
  17. Li A., Lee C.M., Hurley A.E., Jarrett K.E., De Giorgi M., Lu W., Balderrama K.S., Doerfler A.M., Deshmukh H., Ray A., Bao G., Lagor W.R. (2019) A self-deleting AAV-CRISPR system for in vivo genome editing. Mol. Ther. Methods Clin. Dev. 12, 111–122.
  18. Scott T., Moyo B., Nicholson S., Maepa M.B., Watashi K., Ely A., Weinberg M.S., Arbuthnot P. (2017) SsAAVs containing cassettes encoding SaCas9 and guides targeting hepatitis B virus inactivate replication of the virus in cultured cells. Sci. Rep. 7 (1), 7401.
  19. Bulcha J.T., Wang Y., Ma H., Tai P.W.L., Gao G. (2021) Viral vector platforms within the gene therapy landscape. Signal Transduct. Target. Ther. 6(1), 53.
  20. Jiang C., Mei M., Li B., Zhu X., Zu W., Tian Y., Wang Q., Guo Y., Dong Y., Tan X. (2017) A non-viral CRISPR/Cas9 delivery system for therapeutically targeting HBV DNA and pcsk9 in vivo. Cell. Res. 27(3), 440–443.
  21. Vader P., Mol E.A., Pasterkamp G., Schiffelers R.M. (2016) Extracellular vesicles for drug delivery. Adv. Drug Deliv. Rev. 106, 148–156.
  22. Doyle L.M., Wang M.Z. (2019) Overview of extracellular vesicles, their origin, composition, purpose, and methods for exosome isolation and analysis. Cells. 8(7), 727.
  23. Phillips W., Willms E., Hill A.F. (2021) Understanding extracellular vesicle and nanoparticle heterogeneity: novel methods and considerations. Proteomics. 21(13–14), e2000118.
  24. Alvarez-erviti L., Seow Y., Yin H., Betts C., Lakhal S., Wood M.J.A. (2011) Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nat. Biotechnol. 29(4), 341–345.
  25. Yang B., Chen Y., Shi J. (2019) Exosome biochemistry and advanced nanotechnology for next-generation theranostic platforms. Adv. Mater. 31(2), e1802896.
  26. Kim M.S., Haney M.J., Zhao Y., Mahajan V., Deygen I., Klyachko N.L., Inskoe E., Piroyan A., Sokolsky M., Okolie O., Hingtgen S.D., Kabanov A.V., Batrakova E.V. (2016) Development of exosome-encapsulated paclitaxel to overcome MDR in cancer cells. Nanomedicine. 12(3), 655–664.
  27. Yuan D., Zhao Y., Banks W.A., Bullock K.M., Haney M., Batrakova E., Kabanov A.V. (2017) Macrophage exosomes as natural nanocarriers for protein delivery to inflamed brain. Biomaterials. 142, 1–12.
  28. Kim H., Yun N., Mun D., Kang J.Y., Lee S.H., Park H., Park H., Joung B. (2018) Cardiac-specific delivery by cardiac tissue-targeting peptide-expressing exosomes. Biochem. Biophys. Res. Commun. 499(4), 803–808.
  29. Brezgin S., Parodi A., Kostyusheva A., Ponomareva N., Lukashev A., Sokolova D., Pokrovsky V.S., Slatinskaya O., Maksimov G., Zamyatnin A.A. Jr., Chulanov V., Kostyushev D. (2023) Technological aspects of manufacturing and analytical control of biological nanoparticles. Biotechnol. Adv. 64, 108122.
  30. Chen R., Huang H., Liu H., Xi J., Ning J., Zeng W., Shen C., Zhang T., Yu G., Xu Q., Chen X., Wang J., Lu F. (2019) Friend or foe? Evidence indicates endogenous exosomes can deliver functional gRNA and Cas9 protein. Small. 15(38), e1902686.
  31. Dai J., Su Y., Zhong S., Cong L., Liu B., Yang J., Tao Y., He Z., Chen C., Jiang Y. (2020) Exosomes : key players in cancer and potential therapeutic strategy. Signal. Transduct. Target. Ther. 5(1), 145.
  32. Kornilov R., Puhka M., Mannerström B., Hiidenmaa H., Peltoniemi H., Siljander P., Seppänen-Kaijansinkko R., Kaur S. (2018) Efficient ultrafiltration-based protocol to deplete extracellular vesicles from fetal bovine serum. J. Extracell. Vesicles. 7(1), 1422674.
  33. Hessvik N.P., Llorente A. (2018) Current knowledge on exosome biogenesis and release. Cell. Mol. Life Sci. 75(2), 193–208.
  34. Ostrowski M., Carmo N.B., Krumeich S., Fanget I., Raposo G., Savina A., Moita C.F., Schauer K., Hume A.N., Freitas R.P., Goud B., Benaroch P., Hacohen N., Fukuda M., Desnos C., Seabra M.C., Darchen F., Amigorena S., Moita L.F., Thery C. (2009) Rab27a and Rab27b control different steps of the exosome secretion pathway. Nat. Cell Biol. 12(1), 19–30.
  35. Gurung S., Perocheau D., Touramanidou L., Baruteau J. (2021) The exosome journey : from biogenesis to uptake and intracellular signalling. Cell Commun. Signal. 19(1), 47.
  36. Manders E.M., Verbeek F., Aten J. (1992) Measurement of co-localization of objects in dual colour confocal images. J. Microsc. 169, 375–382.
  37. Luo T., Mitra S., McBride J.W. (2018) Ehrlichia chaffeensis TRP75 interacts with host cell targets. mSphere. 3(2), e00147–18.
  38. Brezgin S., Kostyusheva A., Ponomareva N., Bayurova E., Kondrashova A., Frolova A., Slatinskaya O., Fatkhutdinova L., Maksimov G., Zyuzin M., Gordeychuk I., Lukashev A., Makarov S., Ivanov A., Zamyatnin A.A. Jr., Chulanov V., Parodi A., Kostyushev D. (2023) Hydroxychloroquine enhances cytotoxic properties of extracellular vesicles and extracellular vesicle – mimetic nanovesicles loaded with chemotherapeutics. Pharmaceutics. 15(2), 534.
  39. Théry C., Witwer K.W., Aikawa E., Alcaraz M.J., Anderson J.D., Andriantsitohaina R., Antoniou A., Arab T., Archer F., Atkin‐Smith G.K. (2018) Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for extracellular vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles. 7(1), 1535750.
  40. Witwer K.W., Goberdhan D.C., O’Driscoll L., Théry C., Welsh J.A., Blenkiron C., Buzás E.I., Di Vizio D., Erdbrügger U., Falcón-Pérez J.M., Fu Q.L, Hill A.F., Lenassi M., Lötvall J., Nieuwland R., Ochiya T., Rome S., Sahoo S., Zheng L. (2021) Updating MISEV : Evolving the minimal requirements for studies of extracellular vesicles. J. Extracell. Vesicles. 10(14), e12182.
  41. Gee P., Lung M.S.Y., Okuzaki Y., Sasakawa N., Iguchi T., Makita Y., Hozumi H., Miura Y., Yang L.F., Iwasaki M., Wang X.H., Waller M.A., Shirai N., Abe Y.O., Fujita Y., Watanabe K., Kagita A., Iwabuchi K.A., Yasuda M., Xu H., Noda T., Komano J., Sakurai H., Inukai N., Hotta A. (2020) Extracellular nanovesicles for packaging of CRISPR-Cas9 protein and sgRNA to induce therapeutic exon skipping. Nat. Commun. 11(1), 1334.
  42. Liang X., Gupta D., Xie J., Wonterghem E., Van Hoecke L., Van Hean J., Niu Z., Wiklander O.P.B., Zheng W., Wiklander R.J., , Rui He, Doste R. Mamand, Bost J., Zhou G., Zhou H., Roudi S., Zickler A.M., Görgens A., Hagey D.W., de Jong O.G., Uy A. G., Zong Y., Mäger Imre., Perez C.M., Roberts T.C., Vader P., Vandenbroucke R.E., Nordin J.Z., EL-Andaloussiet S. (2023) Multimodal engineering of extracellular vesicles for efficient intracellular protein delivery. bioRxiv. 2023.04.30.535834. doi: 10.1101/2023.04.30.535834
  43. Li T., Zhang L., Lu T., Zhu T., Feng C., Gao N., Liu F., Yu J., Chen K., Zhong J., Tang Q., Zhang Q., Deng X., Ren J., Zeng J., Zhou H., Zhu J. (2023) Engineered extracellular vesicle-delivered CRISPR / CasRx as a novel RNA editing tool. Adv. Sci. (Weinh). 10(10), e2206517.
  44. Patel S., Kim J., Herrera M., Mukherjee A., Kabanov A.V., Sahay G. (2019) Brief update on endocytosis of nanomedicines. Adv. Drug Deliv. Rev. 144, 90–111.

Arquivos suplementares

Arquivos suplementares
Ação
1. JATS XML
Baixar
2. Fig. 1. Transfection of transformed and non-transformed human cells with CD63-EGFP and dCas9-BFP plasmids. a – Graph of the efficiency of cotransfection of the studied cell lines. b – Microphotographs of cell lines transfected with the CD63-EGFP plasmid. EGFP – green fluorescent protein. BFP – blue fluorescent protein.

Baixar (416KB)
Metadados
3. Fig. 2. Assessment of the levels of colocalization of the dCas9-BFP protein with exosome biogenesis factors in the HEK293T and HepG2 cell lines. a – Representative confocal images of cells transfected with dCas9-BFP with CD63-EGFP or Rab27A-EGFP. The far right row shows luminosity cytograms of dCas9-BFP and CD63-GFP or Rab27A-EGFP. b – Semiquantitative data on the colocalization of dCas9-BFP with the CD63-EGFP and Rab27A-EGFP proteins, calculated using the M1 coefficient. Error bars correspond to standard deviations.

Baixar (440KB)
Metadados
4. Fig. 3. Characteristics of nanoparticles. a – Analysis of exosome size measurements using dynamic light scattering. d – Diameter, nm. b – Particle size distribution. c – Analysis of the ζ-potential of nanoparticles. d – Western blot analysis of the expression of proteins CD63, CD81, Hsp70 in isolates of biological nanoparticles.

Baixar (153KB)
Metadados
5. Fig. 4. Assessment of the levels of stochastic packaging of the Cas9 protein into exosomes. a – Assessment of the proportion of EGFP-positive cells using flow cytometry. b – Histograms of EGFP signal distribution in control cells and in cells 2 and 6 hours after the addition of exosomes.

Baixar (207KB)
Metadados

Declaração de direitos autorais © Russian Academy of Sciences, 2024