Published 2025 | Version v1
Thesis Open

Nanoparticle delivery of CRISPR based gene-therapy into the human brain

Authors/Creators

  • 1. ROR icon Freie Universität Berlin
  • 2. ROR icon University of California, San Francisco

Description

Neurological diseases such as amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), and frontotemporal dementia (FTD) share numerous pathological and molecular characteristics. These attributes include the accumulation of proteins such as TDP-43, amyloid and tau, a gradual acceleration in neuronal cell-death and progressive neurodegeneration. A particularly notable commonality among these pathologies, lies within their genetic signatures. Beyond monogenic and polygenic risk factors, such as the APOE4 allele and MAPT gene-variants, repeat expansions like those found in C9orf72 and HTT, manifest a common feat of neurodegenerative diseases. Unfortunately, in spite of the deep and detailed genetic descriptions of these neurological pathologies, cures do not exist, and effective therapies remain limited.

CRISPR-based gene therapy offers promising solutions to these genetic challenges; but successful delivery of genome-editing tools into target-cells remains a significant challenge in the field of molecular medicine. Numerous modalities are being explored; including lipid nanoparticles (LNPs), virus-like particles (VLPs), and peptide-conjugated ribonucleoprotein complexes (RNPs); each with distinct advantages and limitations. In this study we report the development of a high-throughput screening platform, based on a bi-fluorescent human induced neuron reporter-cell. The system combines both visual and sequencing data, to enable temporal and visual monitoring of genome-editing outcomes over time. Using this platform, we present highly efficient genome editing in human neurons, using engineered nanoparticles on the basis of HIV-1. These results highlight the advantages of image based screening methods for CRISPR-delivery, and underwrite the importance of scalable nanoparticle discovery platforms in the quest for cures.

Files

Nanoparticle delivery of CRISPR based gene therapy into the human brain.pdf

Files (18.8 MB)

Additional details

Dates

Submitted
2025-07-01

References

  • [1] Herculano-Houzel S. (2009). The human brain in numbers: a linearly scaled-up primate brain. Frontiers in human neuroscience, 3, 31. https://doi.org/10.3389/neuro.09.031.2009
  • [2] Ensafi, Shahab et al. "3D reconstruction of neurons in electron microscopy images." 2014 36th Annual International Conference of the IEEE Engineering in Medicine and Biology Society (2014): 6732-6735.
  • [3] Jones RA, Harrison C, Eaton SL, Llavero Hurtado M, Graham LC, Alkhammash L, Oladiran OA, Gale A, Lamont DJ, Simpson H, Simmen MW, Soeller C, Wishart TM, Gillingwater TH. Cellular and Molecular Anatomy of the Human Neuromuscular Junction. Cell Rep. 2017 Nov 28;21(9):2348-2356.
  • [4] Harris AL. Electrical coupling and its channels. J Gen Physiol. 2018 Dec 03;150(12):1606-1639.
  • [5] Südhof TC. The presynaptic active zone. Neuron. 2012 Jul 12;75(1):11-25.
  • [6] Purves D, Augustine GJ, Fitzpatrick D, et al., editors. Sunderland (MA): Sinauer Associates; 2001.
  • [7] Jones, B. E. (2003). Arousal systems. Front. Biosci. 8, s438–s451.
  • [8] Kant, I. (2003). Critique of pure reason (N. Kemp Smith, Trans.). Palgrave Macmillan. (Original work published 1781)
  • [9] Crick, F., and Koch, C. (1990). Towards a neurobiological theory of consciousness. Semin. Neurosci. 2, 263–275.
  • [10] Hameroff, S., and Penrose, R. (2014). Consciousness in the universe: a review of the 'Orch OR' theory. Phys. Life Rev. 11, 39–78. doi: 10.1016/j.plrev.2013.08.002
  • [11] Sandra A. Heldstab et al (2022) Current Biology 32, R697–R708, 2022 Elsevier Inc. doi.org/ 10.1016/j.cub.2022.04.096
  • [12] Siegel GJ, Agranoff BW, Albers RW, et al., editors. Philadelphia: Lippincott-Raven; 1999.
  • [13] Eriksson, P., Perfilieva, E., Björk-Eriksson, T. et al. Neurogenesis in the adult human hippocampus. Nat Med 4, 1313–1317 (1998). https://doi.org/10.1038/3305
  • [14] Anda, F., Madabhushi, R., Rei, D. et al. Cortical neurons gradually attain a post-mitotic state. Cell Res 26, 1033–1047 (2016). https://doi.org/10.1038/cr.2016.76
  • [15] Nicholls JG, Adams WB, Eugenin J, Geiser R, Lepre M, Luque JM, Wintzer M. Why does the central nervous system not regenerate after injury? Surv Ophthalmol. 1999 Jun;43 Suppl 1:S136-41. doi: 10.1016/s0039-6257(99)00008-9. PMID: 10416756.
  • [16] Sender R, Milo R. The distribution of cellular turnover in the human body. Nat Med. 2021 Jan;27(1):45-48. doi: 10.1038/s41591-020-01182-9. Epub 2021 Jan 11. PMID: 33432173.
  • [17] Castro-Aldrete, L., Einsiedler, M., Novakova Martinkova, J. et al. Alzheimer disease seen through the lens of sex and gender. Nat Rev Neurol 21, 235–249 (2025). https://doi.org/10.1038/ s41582-025-01071-0
  • [18] Tenner, A.J., Petrisko, T.J. Knowing the enemy: strategic targeting of complement to treat Alzheimer disease. Nat Rev Neurol 21, 250–264 (2025).https://doi.org/10.1038/s41582-025-01073-y
  • [19] Lamptey, Richard N L et al. "A Review of the Common Neurodegenerative Disorders: Current Therapeutic Approaches and the Potential Role of Nanotherapeutics." International journal of molecular sciences vol. 23,3 1851. 6 Feb. 2022, doi:10.3390/ijms23031851
  • [20] Campbell, B.C.V., De Silva, D.A., Macleod, M.R. et al. Ischaemic stroke. Nat Rev Dis Primers 5, 70 (2019). https://doi.org/10.1038/s41572-019-0118-8
  • [21] Tabrizi, S.J., Flower, M.D., Ross, C.A. et al. Huntington disease: new insights into molecular pathogenesis and therapeutic opportunities. Nat Rev Neurol 16, 529–546 (2020). https://doi.org/ 10.1038/s41582-020-0389-4
  • [22] Deuschl G, Beghi E, Fazekas F, Varga T, Christoforidi KA, Sipido E, Bassetti CL, Vos T, Feigin VL. The burden of neurological diseases in Europe: an analysis for the Global Burden of Disease Study 2017. Lancet Public Health. 2020 Oct;5(10):e551-e567. doi: 10.1016/ S2468-2667(20)30190-0. PMID: 33007212
  • [23] Feigin, Valery L et al. "The global burden of neurological disorders: translating evidence into policy." The Lancet. Neurology vol. 19,3 (2020): 255-265. doi:10.1016/S1474-4422(19)30411-9
  • [24] Zhang, J., Zhang, Y., Wang, J. et al. Recent advances in Alzheimer's disease: mechanisms, clinical trials and new drug development strategies. Sig Transduct Target Ther 9, 211 (2024). https:// doi.org/10.1038/s41392-024-01911-3
  • [25] GBD 2017 US Neurological Disorders Collaborators. Burden of Neurological Disorders Across the US From 1990-2017: A Global Burden of Disease Study. JAMA Neurol. 2021;78(2):165– 176. doi:10.1001/jamaneurol.2020.4152
  • [26] Shin JH, Yang HJ, Ahn JH, Jo S, Chung SJ, Lee JY, Kim HS, Kim M; on behalf of the Korean Huntington's Disease Society. Evidence-Based Review on Symptomatic Management of Huntington's Disease. J Mov Disord. 2024;17(4):369-386.
  • [27] Bates, G., Dorsey, R., Gusella, J. et al. Huntington disease. Nat Rev Dis Primers 1, 15005 (2015). https://doi.org/10.1038/nrdp.2015.5
  • [28] Snowden JS. Changing perspectives on frontotemporal dementia: A review. J Neuropsychol. 2023 Jun;17(2):211-234. doi: 10.1111/jnp.12297. Epub 2022 Oct 31. PMID: 36315040.
  • [29] Sachdev A, Gill K, Sckaff M, Birk AM, Aladesuyi Arogundade O, Brown KA, Chouhan RS, Issagholian-Lewin PO, Patel E, Watry HL, Bernardi MT, Keough KC, Tsai YC, Smith AST, Conklin BR, Clelland CD. Reversal of C9orf72 mutation-induced transcriptional dysregulation and pathology in cultured human neurons by allele-specific excision. Proc Natl Acad Sci U S A. 2024 Apr 23;121(17):e2307814121. doi: 10.1073/pnas.2307814121. Epub 2024 Apr 15. PMID: 38621131; PMCID: PMC11047104.
  • [30] Mollah, Sha Abbas et al. "A comprehensive review on frontotemporal dementia: its impact on language, speech and behavior." Dementia & neuropsychologia vol. 18 e20230072. 22 Apr. 2024, doi:10.1590/1980-5764-DN-2023-0072
  • [31] Jankovic, Joseph, and L Giselle Aguilar. "Current approaches to the treatment of Parkinson's disease." Neuropsychiatric disease and treatment vol. 4,4 (2008): 743-57. doi:10.2147/ndt.s2006
  • [32] Tzeplaeff, Laura et al. "Current State and Future Directions in the Therapy of ALS." Cells vol. 12,11 1523. 31 May. 2023, doi:10.3390/cells12111523
  • [33] Dotiwala AK, McCausland C, Samra NS. Anatomy, Head and Neck: Blood Brain Barrier. [Updated 2023 Apr 4]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK519556/
  • [34] Luissint AC, Artus C, Glacial F, Ganeshamoorthy K, Couraud PO. Tight junctions at the blood brain barrier: physiological architecture and disease-associated dysregulation. Fluids Barriers CNS. 2012 Nov 09;9(1):23.
  • [35] Correale J, Villa A. Cellular elements of the blood-brain barrier. Neurochem Res. 2009 Dec;34(12):2067-77. doi: 10.1007/s11064-009-0081-y. Epub 2009 Oct 25. PMID: 19856206.
  • [36] Zhao Z, Nelson AR, Betsholtz C, Zlokovic BV. Establishment and Dysfunction of the Blood- Brain Barrier. Cell. 2015 Nov 19;163(5):1064-1078.
  • [37] Wu, D., Chen, Q., Chen, X. et al. The blood–brain barrier: Structure, regulation and drug delivery. Sig Transduct Target Ther 8, 217 (2023). https://doi.org/10.1038/s41392-023-01481-w
  • [38] Matuszek Z, Brown BL, Yrigollen CM, Keiser MS, Davidson BL. Current trends in gene therapy to treat inherited disorders of the brain. Mol Ther. 2025 May 7;33(5):1988-2014. doi: 10.1016/j.ymthe.2025.03.057. Epub 2025 Apr 2. PMID: 40181540; PMCID: PMC12126828.
  • [39] Martin Jinek et al. ,A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity.Science337,816-821(2012).DOI:10.1126/science.1225829
  • [40] Wang, J.Y., Pausch, P. & Doudna, J.A. Structural biology of CRISPR–Cas immunity and genome editing enzymes. Nat Rev Microbiol 20, 641–656 (2022). https://doi.org/10.1038/ s41579-022-00739-4
  • [41] He, Yuxuan et al. "The CRISPR/Cas System: A Customizable Toolbox for Molecular Detection." Genes vol. 14,4 850. 31 Mar. 2023, doi:10.3390/genes14040850
  • [42] Xu, Yuanyuan, and Zhanjun Li. "CRISPR-Cas systems: Overview, innovations and applications in human disease research and gene therapy." Computational and structural biotechnology journal vol. 18 2401-2415. 8 Sep. 2020, doi:10.1016/j.csbj.2020.08.031
  • [43] Li, T., Yang, Y., Qi, H. et al. CRISPR/Cas9 therapeutics: progress and prospects. Sig Transduct Target Ther 8, 36 (2023). https://doi.org/10.1038/s41392-023-01309-7
  • [44] Huai, C., Li, G., Yao, R. et al. Structural insights into DNA cleavage activation of CRISPR- Cas9 system. Nat Commun 8, 1375 (2017). https://doi.org/10.1038/s41467-017-01496-2
  • [45] Hillary, V Edwin, and S Antony Ceasar. "A Review on the Mechanism and Applications of CRISPR/Cas9/Cas12/Cas13/Cas14 Proteins Utilized for Genome Engineering." Molecular biotechnology vol. 65,3 (2023): 311-325. doi:10.1007/s12033-022-00567-0
  • [46] Gao, J., Gunasekar, S., Xia, Z.(. et al. Gene therapy for CNS disorders: modalities, delivery and translational challenges. Nat. Rev. Neurosci. 25, 553–572 (2024). https://doi.org/10.1038/ s41583-024-00829-7
  • [47] Alessia Cavazza, Francisco J. Molina-Estévez et al. Advanced delivery systems for gene editing: A comprehensive review from the GenE-HumDi COST Action Working Group, Molecular Therapy Nucleic Acids, Volume 36, Issue 1, 2025, 102457, ISSN 2162-2531, https://doi.org/ 10.1016/j.omtn.2025.102457.
  • [48] Mali, Shrikant. "Delivery systems for gene therapy." Indian journal of human genetics vol. 19,1 (2013): 3-8. doi:10.4103/0971-6866.112870
  • [49] Bulcha, J.T., Wang, Y., Ma, H. et al. Viral vector platforms within the gene therapy landscape. Sig Transduct Target Ther 6, 53 (2021). https://doi.org/10.1038/s41392-021-00487-6
  • [50] Hou, X., Zaks, T., Langer, R. et al. Lipid nanoparticles for mRNA delivery. Nat Rev Mater 6, 1078–1094 (2021). https://doi.org/10.1038/s41578-021-00358-0
  • [51] Keeler, Allison M, and Terence R Flotte. "Recombinant Adeno-Associated Virus Gene Therapy in Light of Luxturna (and Zolgensma and Glybera): Where Are We, and How Did We Get Here?." Annual review of virology vol. 6,1 (2019): 601-621. doi:10.1146/annurev- virology-092818-015530
  • [52] Wang, JH., Gessler, D.J., Zhan, W. et al. Adeno-associated virus as a delivery vector for gene therapy of human diseases. Sig Transduct Target Ther 9, 78 (2024). https://doi.org/10.1038/ s41392-024-01780-w
  • [53] An, M., Raguram, A., Du, S.W. et al. Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes in vivo. Nat Biotechnol 42, 1526–1537 (2024). https:// doi.org/10.1038/s41587-023-02078-y
  • [54] Foss, D.V., Muldoon, J.J., Nguyen, D.N. et al. Peptide-mediated delivery of CRISPR enzymes for the efficient editing of primary human lymphocytes. Nat. Biomed. Eng 7, 647–660 (2023). https:// doi.org/10.1038/s41551-023-01032-2
  • [55] Chen, K., Stahl, E.C., Kang, M.H. et al. Engineering self-deliverable ribonucleoproteins for genome editing in the brain. Nat Commun 15, 1727 (2024). https://doi.org/10.1038/ s41467-024-45998-2
  • [56] Akcakaya, P., Bobbin, M. L., Guo, J. A., Malagon-Lopez, J., Clement, K., Garcia, S. P., et al. (2018). In vivo CRISPR editing with no detectable genome-wide off-target mutations. Nature 561, 416–419. doi:10.1038/s41586-018-0500-9
  • [57] J.E. Dahlman, K.J. Kauffman, Y. Xing, T.E. Shaw, F.F. Mir, C.C. Dlott, R. Langer, D.G. Anderson, & E.T. Wang, Barcoded nanoparticles for high throughput in vivo discovery of targeted 59 therapeutics, Proc. Natl. Acad. Sci. U.S.A. 114 (8) 2060-2065, https://doi.org/10.1073/ pnas.1620874114 (2017).
  • [58] Davidsson, M., Diaz-Fernandez, P., Schwich, O. et al. A novel process of viral vector barcoding and library preparation enables high-diversity library generation and recombination-free paired-end sequencing. Sci Rep 6, 37563 (2016). https://doi.org/10.1038/srep37563
  • [59] Ngo, Wayne et al. "Mechanism-guided engineering of a minimal biological particle for genome editing." bioRxiv : the preprint server for biology 2024.07.23.604809. 24 Jul. 2024, doi:10.1101/2024.07.23.604809. Preprint.
  • [60] Hamilton, J.R., Chen, E., Perez, B.S. et al. In vivo human T cell engineering with enveloped delivery vehicles. Nat Biotechnol 42, 1684–1692 (2024). https://doi.org/10.1038/s41587-023-02085- z
  • [61] Josh Timmons: HiFi DNA Assembly (NEB). protocols.io https://dx.doi.org/10.17504/ protocols.io.dn85hv
  • [62] Cerbini, Trevor et al. "Transcription activator-like effector nuclease (TALEN)-mediated CLYBL targeting enables enhanced transgene expression and one-step generation of dual reporter human induced pluripotent stem cell (iPSC) and neural stem cell (NSC) lines." PloS one vol. 10,1 e0116032. 14 Jan. 2015, doi:10.1371/journal.pone.0116032
  • [63] Zufferey, R et al. "Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors." Journal of virology vol. 73,4 (1999): 2886-92. doi:10.1128/JVI.73.4.2886-2892.1999
  • [64] Pañeda, Astrid et al. 1079. Effect of WPRE on Transgene Expression Using Different Promoters in the Context of Hydrodynamically Delivered Plasmid Vectors - Molecular Therapy Volume 13, Supplement 1, May 2006
  • [65] Gao, C., Jiang, J., Tan, Y. et al. Microglia in neurodegenerative diseases: mechanism and potential therapeutic targets. Sig Transduct Target Ther 8, 359 (2023). https://doi.org/10.1038/ s41392-023-01588-0
  • [66] Fortea, J., Pegueroles, J., Alcolea, D. et al. APOE4 homozygosity represents a distinct genetic form of Alzheimer's disease. Nat Med 30, 1284–1291 (2024). https://doi.org/10.1038/ s41591-024-02931-w
  • [67] Milone, M.C., O'Doherty, U. Clinical use of lentiviral vectors. Leukemia 32, 1529–1541 (2018). https://doi.org/10.1038/s41375-018-0106-0
  • [68] Lin HC, He Z, Ebert S, Schörnig M, Santel M, Nikolova MT, Weigert A, Hevers W, Kasri NN, Taverna E, Camp JG, Treutlein B. NGN2 induces diverse neuron types from human pluripotency. Stem Cell Reports. 2021 Sep 14;16(9):2118-2127. doi: 10.1016/j.stemcr.2021.07.006. Epub 2021 Aug 5. PMID: 34358451; PMCID: PMC8452516.
  • [69] Erika Laraflores, Andy Qi, Luke Reilly, Marianita Santiana, caroline.pantazis, Michael Ward, Mark Cookson 2022. iNDI Transcription Factor-NGN2 differentiation of human iPSC into cortical neurons Version 1. protocols.io https://dx.doi.org/10.17504/protocols.io.8epv5969ng1b/v1
  • [70] Takeshi Uenaka, Alan Bortoncello Napole et al. 2025 Prevention of Transgene Silencing During Human Pluripotent Stem Cell Differentiation. bioRxiv doi: https://doi.org/ 10.1101/2025.04.07.647695
  • [71] Seczynska, M., Bloor, S., Cuesta, S.M. et al. Genome surveillance by HUSH-mediated silencing of intronless mobile elements. Nature 601, 440–445 (2022). https://doi.org/10.1038/ s41586-021-04228-1
  • [72] Forrest, Marc P et al. "Open Chromatin Profiling in hiPSC-Derived Neurons Prioritizes Functional Noncoding Psychiatric Risk Variants and Highlights Neurodevelopmental Loci." Cell stem cell vol. 21,3 (2017): 305-318.e8. doi:10.1016/j.stem.2017.07.008
  • [73] Lee GB, Mazli WNAB, Hao L. Multiomics Evaluation of Human iPSCs and iPSC-Derived Neurons. J Proteome Res. 2024 Aug 2;23(8):3149-3160. doi: 10.1021/acs.jproteome.3c00790. Epub 2024 Feb 28. PMID: 38415376; PMCID: PMC11799864.
  • [74] Vishnu Dileep, Carles A. Boix et al. Neuronal DNA double-strand breaks lead to genome structural variations and 3D genome disruption in neurodegeneration, Cell, Volume 186, Issue 20, 2023, Pages 4404-4421.e20, ISSN 0092-8674, https://doi.org/10.1016/j.cell.2023.08.038.
  • [75] Ramadoss, Gokul N et al. "Neuronal DNA repair reveals strategies to influence CRISPR editing outcomes." bioRxiv : the preprint server for biology 2024.06.25.600517. 17 Mar. 2025, doi:10.1101/2024.06.25.600517. Preprint.
  • [76] Shadfar, Sina et al. "The Complex Mechanisms by Which Neurons Die Following DNA Damage in Neurodegenerative Diseases." International journal of molecular sciences vol. 23,5 2484. 24 Feb. 2022, doi:10.3390/ijms23052484
  • [77] Corish P, Tyler-Smith C. Attenuation of green fluorescent protein half-life in mammalian cells. Protein Eng. 1999 Dec;12(12):1035-40. doi: 10.1093/protein/12.12.1035. PMID: 10611396.
  • [78] Zink, D., Fischer, A. & Nickerson, J. Nuclear structure in cancer cells. Nat Rev Cancer 4, 677–687 (2004). https://doi.org/10.1038/nrc1430
  • [79] Webster M, Witkin KL, Cohen-Fix O. Sizing up the nucleus: nuclear shape, size and nuclear- envelope assembly. J Cell Sci. 2009 May 15;122(Pt 10):1477-86. doi: 10.1242/jcs.037333. PMID: 19420234; PMCID: PMC2680097.
  • [80] Conant D, Hsiau T, Rossi N, Oki J, Maures T, Waite K, Yang J, Joshi S, Kelso R, Holden K, Enzmann BL, Stoner R. Inference of CRISPR Edits from Sanger Trace Data. CRISPR J. 2022 Feb;5(1):123-130. doi: 10.1089/crispr.2021.0113. Epub 2022 Feb 2. PMID: 35119294.