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Enhanced genome editing to ameliorate a genetic metabolic liver disease through co-delivery of adeno-associated virus receptor

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Abstract

Genome editing through adeno-associated viral (AAV) vectors is a promising gene therapy strategy for various diseases, especially genetic disorders. However, homologous recombination (HR) efficiency is extremely low in adult animal models. We assumed that increasing AAV transduction efficiency could increase genome editing activity, especially HR efficiency, for in vivo gene therapy. Firstly, a mouse phenylketonuria (PKU) model carrying a pathogenic R408W mutation in phenylalanine hydroxylase (Pah) was generated. Through co-delivery of the general AAV receptor (AAVR), we found that AAVR could dramatically increase AAV transduction efficiency in vitro and in vivo. Furthermore, co-delivery of SaCas9/sgRNA/donor templates with AAVR via AAV8 vectors increased indel rate over 2-fold and HR rate over 15-fold for the correction of the single mutation in PahR408W mice. Moreover, AAVR co-injection successfully increased the site-specific insertion rate of a 1.4 kb Pah cDNA by 11-fold, bringing the HR rate up to 7.3% without detectable global off-target effects. Insertion of Pah cDNA significantly decreased the Phe level and ameliorated PKU symptoms. This study demonstrates a novel strategy to dramatically increase AAV transduction which substantially enhanced in vivo genome editing efficiency in adult animal models, showing clinical potential for both conventional and genome editing-based gene therapy.

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References

  • Barzel, A., Paulk, N.K., Shi, Y., Huang, Y., Chu, K., Zhang, F., Valdmanis, P.N., Spector, L. P., Porteus, M.H., Gaensler, K.M., et al. (2015). Promoterless gene targeting without nucleases ameliorates haemophilia B in mice. Nature 517, 360–364.

    Article  CAS  PubMed  Google Scholar 

  • Blau, N., Bélanger-Quintana, A., Demirkol, M., Feillet, F., Giovannini, M., MacDonald, A., Trefz, F.K., and van Spronsen, F. (2010a). Management of phenylketonuria in Europe: survey results from 19 countries. Mol Genet Metab 99, 109–115.

    Article  CAS  PubMed  Google Scholar 

  • Blau, N., van Spronsen, F.J., and Levy, H.L. (2010b). Phenylketonuria. Lancet 376, 1417–1427.

    Article  CAS  PubMed  Google Scholar 

  • Chen, X., Liu, J., Janssen, J.M., and Gonçalves, M.A.F.V. (2017). The chromatin structure differentially impacts high-specificity CRISPR-Cas9 nuclease strategies. Mol Ther Nucleic Acids 8, 558–563.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dilella, A.G., Marvi, J., Brayton, K., and Woo, S.L.C. (1987). An ammo-acid substitution involved in phenylketonuria is in linkage disequilibrium with DNA haplotype 2. Nature 327, 333–336.

    Article  CAS  PubMed  Google Scholar 

  • Ding, Z., Georgiev, P., and Thöny, B. (2006). Administration-route and gender-independent long-term therapeutic correction of phenylketonuria (PKU) in a mouse model by recombinant adenoassociated virus 8 pseudotyped vector-mediated gene transfer. Gene Ther 13, 587–593.

    Article  CAS  PubMed  Google Scholar 

  • Ding, Z., Harding, C.O., Rebuffat, A., Elzaouk, L., Wolff, J.A., and Thöny, B. (2008). Correction of murine PKU following AAV-mediated intramuscular expression of a complete phenylalanine hydroxylating system. Mol Ther 16, 673–681.

    Article  CAS  PubMed  Google Scholar 

  • Duan, D. (2016). Systemic delivery of adeno-associated viral vectors. Curr Opin Virol 21, 16–25.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Dunbar, C.E., High, K.A., Joung, J.K., Kohn, D.B., Ozawa, K., and Sadelain, M. (2018). Gene therapy comes of age. Science 359, eaan4672.

    Article  PubMed  CAS  Google Scholar 

  • Graham, T., McIntosh, J., Work, L.M., Nathwani, A., and Baker, A.H. (2008). Performance of AAV8 vectors expressing human factor IX from a hepatic-selective promoter following intravenous injection into rats. Genet Vaccines Ther 6, 9.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Grisch-Chan, H.M., Schlegel, A., Scherer, T., Allegri, G., Heidelberger, R., Tsikrika, P., Schmeer, M., Schleef, M., Harding, C.O., Haberle, J., et al. (2017). Low-dose gene therapy for murine PKU using episomal naked DNA vectors expressing PAH from its endogenous liver promoter. Mol Ther Nucleic Acids 7, 339–349.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Grünewald, J., Zhou, R., Garcia, S.P., Iyer, S., Lareau, C.A., Aryee, M.J., and Joung, J.K. (2019). Transcriptome-wide off-target RNA editing induced by CRISPR-guided DNA base editors. Nature 569, 433–437.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Guan, Y., Ma, Y., Li, Q., Sun, Z., Ma, L., Wu, L., Wang, L., Zeng, L., Shao, Y., Chen, Y., et al. (2016). CRISPR/Cas9-mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse. EMBO Mol Med 8, 477–488.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Hamman, K., Clark, H., Montini, E., Al-Dhalimy, M., Grompe, M., Finegold, M., and Harding, C.O. (2005). Low therapeutic threshold for hepatocyte replacement in murine phenylketonuria. Mol Ther 12, 337–344.

    Article  CAS  PubMed  Google Scholar 

  • Harding, C.O., Gillingham, M.B., Hamman, K., Clark, H., Goebel-Daghighi, E., Bird, A., and Koeberl, D.D. (2006). Complete correction of hyperphenylalaninemia following liver-directed, recombinant AAV2/8 vector-mediated gene therapy in murine phenylketonuria. Gene Ther 13, 457–462.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Jin, S., Zong, Y., Gao, Q., Zhu, Z., Wang, Y., Qin, P., Liang, C., Wang, D., Qiu, J.L., Zhang, F., et al. (2019). Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice. Science 364, 292–295.

    Article  CAS  PubMed  Google Scholar 

  • Komor, A.C., Kim, Y.B., Packer, M.S., Zuris, J.A., and Liu, D.R. (2016). Programmable editing of a target base in genomic DNAwithout double-stranded DNA cleavage. Nature 533, 420–424.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Li, H., Haurigot, V., Doyon, Y., Li, T., Wong, S.Y., Bhagwat, A.S., Malani, N., Anguela, X.M., Sharma, R., Ivanciu, L., et al. (2011). In vivo genome editing restores haemostasis in a mouse model of haemophilia. Nature 475, 217–221.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Manno, C.S., Pierce, G.F., Arruda, V.R., Glader, B., Ragni, M., Rasko, J.J. E., Ozelo, M.C., Hoots, K., Blatt, P., Konkle, B., et al. (2006). Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med 12, 342–347.

    Article  CAS  PubMed  Google Scholar 

  • Mendell, J.R., Al-Zaidy, S., Shell, R., Arnold, W.D., Rodino-Klapac, L.R., Prior, T.W., Lowes, L., Alfano, L., Berry, K., Church, K., et al. (2017). Single-dose gene-replacement therapy for spinal muscular atrophy. N Engl J Med 377, 1713–1722.

    Article  CAS  PubMed  Google Scholar 

  • Mingozzi, F., and High, K.A. (2011). Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nat Rev Genet 12, 341–355.

    Article  CAS  PubMed  Google Scholar 

  • Mochizuki, S., Mizukami, H., Ogura, T., Kure, S., Ichinohe, A., Kojima, K., Matsubara, Y., Kobayahi, E., Okada, T., Hoshika, A., et al. (2004). Long-term correction ofhyperphenylalaninemia by AAV-mediated gene transfer leads to behavioral recovery in phenylketonuria mice. Gene Ther 11, 1081–1086.

    Article  CAS  PubMed  Google Scholar 

  • Nathwani, A.C., Gray, J.T., Ng, C.Y.C., Zhou, J., Spence, Y., Waddington, S.N., Tuddenham, E.G.D., Kemball-Cook, G., McIntosh, J., Boon-Spijker, M., et al. (2006). Self-complementary adeno-associated virus vectors containing a novel liver-specific human factor IX expression cassette enable highly efficient transduction of murine and nonhuman primate liver. Blood 107, 2653–2661.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nathwani, A.C., Reiss, U.M., Tuddenham, E.G.D., Rosales, C., Chowdary, P., McIntosh, J., Della Peruta, M., Lheriteau, E., Patel, N., Raj, D., et al. (2014). Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N Engl J Med 371, 1994–2004.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Nygaard, S., Barzel, A., Haft, A., Major, A., Finegold, M., Kay, M.A., and Grompe, M. (2016). A universal system to select gene-modified hepatocytes in vivo. Sci Transl Med 8, 342ra79.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Pillay, S., Meyer, N.L., Puschnik, A.S., Davulcu, O., Diep, J., Ishikawa, Y., Jae, L.T., Wosen, J.E., Nagamine, C.M., Chapman, M.S., et al. (2016). An essential receptor for adeno-associated virus infection. Nature 530, 108–112.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ran, F.A., Cong, L., Yan, W.X., Scott, D.A., Gootenberg, J.S., Kriz, A.J., Zetsche, B., Shalem, O., Wu, X., Makarova, K.S., et al. (2015). In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rangarajan, S., Walsh, L., Lester, W., Perry, D., Madan, B., Laffan, M., Yu, H., Vettermann, C., Pierce, G.F., Wong, W.Y., et al. (2017). AAV5-factor VIII gene transfer in severe hemophilia A. N Engl J Med 377, 2519–2530.

    Article  CAS  PubMed  Google Scholar 

  • Rees, H.A., Wilson, C., Doman, J.L., and Liu, D.R. (2019). Analysis and minimization of cellular RNA editing by DNA adenine base editors. Sci Adv 5, eaax5717.

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  • Russell, S., Bennett, J., Wellman, J.A., Chung, D.C., Yu, Z.F., Tillman, A., Wittes, J., Pappas, J., Elci, O., McCague, S., et al. (2017). Efficacy and safety of voretigene neparvovec (AAV2-hRPE65v2) in patients with RPE65-mediated inherited retinal dystrophy: a randomised, controlled, open-label, phase 3 trial. Lancet 390, 849–860.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Scriver, C.R., and Clow, C.L. (1980). Phenylketonuria: epitome of human biochemical genetics. N Engl J Med 303, 1336–1342.

    Article  CAS  PubMed  Google Scholar 

  • Shao, Y., Guan, Y., Wang, L., Qiu, Z., Liu, M., Chen, Y., Wu, L., Li, Y., Ma, X., Liu, M., et al. (2014). CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos. Nat Protoc 9, 2493–2512.

    Article  CAS  PubMed  Google Scholar 

  • Shao, Y., Wang, L., Guo, N., Wang, S., Yang, L., Li, Y., Wang, M., Yin, S., Han, H., Zeng, L., et al. (2018). Cas9-nickase-mediated genome editing corrects hereditary tyrosinemia in rats. J Biol Chem 293, 6883–6892.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • van Spronsen, F.J., Ahring, K.K., and Gizewska M. (2009). PKU—What is daily practice in various centres in Europe? J Inherit Metab Dis 32, 58–64.

    Article  CAS  PubMed  Google Scholar 

  • van Wegberg, A.M.J., MacDonald, A., Ahring, K., Bélanger-Quintana, A., Blau, N., Bosch, A.M., Burlina, A., Campistol, J., Feillet, F., Gizewska, M., et al. (2017). The complete European guidelines on phenylketonuria: diagnosis and treatment. Orphanet J Rare Dis 12, 162.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Viecelli, H.M., Harbottle, R.P., Wong, S.P., Schlegel, A., Chuah, M.K., VandenDriessche, T., Harding, C.O., and Thöny, B. (2014). Treatment of phenylketonuria using minicircle-based naked-DNA gene transfer to murine liver. Hepatology 60, 1035–1043.

    Article  CAS  PubMed  Google Scholar 

  • Villiger, L., Grisch-Chan, H.M., Lindsay, H., Ringnalda, F., Pogliano, C.B., Allegri, G., Fingerhut, R., Haberle, J., Matos, J., Robinson, M.D., et al. (2018). Treatment of a metabolic liver disease by in vivo genome base editing in adult mice. Nat Med 24, 1519–1525.

    Article  CAS  PubMed  Google Scholar 

  • Wang, D., Tai, P.W.L., and Gao, G. (2019). Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov 18, 358–378.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang, L., Shao, Y., Guan, Y., Li, L., Wu, L., Chen, F., Liu, M., Chen, H., Ma, Y., Ma, X., et al. (2015). Large genomic fragment deletion and functional gene cassette knock-in via Cas9 protein mediated genome editing in one-cell rodent embryos. Sci Rep 5, 17517.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Wang, L., Wang, H., Bell, P., McMenamin, D., and Wilson, J.M. (2012). Hepatic gene transfer in neonatal mice by adeno-associated virus serotype 8 vector. Hum Gene Ther 23, 533–539.

    Article  CAS  PubMed  Google Scholar 

  • Yagi, H., Ogura, T., Mizukami, H., Urabe, M., Hamada, H., Yoshikawa, H., Ozawa, K., and Kume, A. (2011). Complete restoration of phenylalanine oxidation in phenylketonuria mouse by a self-complementary adeno-associated virus vector. J Gene Med 13, 114–122.

    Article  CAS  PubMed  Google Scholar 

  • Yang, Y., Wang, L., Bell, P., McMenamin, D., He, Z., White, J., Yu, H., Xu, C., Morizono, H., Musunuru, K., et al. (2016). A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice. Nat Biotechnol 34, 334–338.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yarrington, R.M., Verma, S., Schwartz, S., Trautman, J.K., and Carroll, D. (2018). Nucleosomes inhibit target cleavage by CRISPR-Cas9 in vivo. Proc Natl Acad Sci USA 115, 9351–9358.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yin, H., Song, C.Q., Dorkin, J.R., Zhu, L.J., Li, Y., Wu, Q., Park, A., Yang, J., Suresh, S., Bizhanova, A., et al. (2016). Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat Biotechnol 34, 328–333.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yuen, G., Khan, F.J., Gao, S., Stommel, J.M., Batchelor, E., Wu, X., and Luo, J. (2017). CRISPR/Cas9-mediated gene knockout is insensitive to target copy number but is dependent on guide RNA potency and Cas9/sgRNA threshold expression level. Nucleic Acids Res 45, 12039–12053.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zhong, L., Li, B., Mah, C.S., Govindasamy, L., Agbandje-McKenna, M., Cooper, M., Herzog, R.W., Zolotukhin, I., Warrington Kenneth H. J., Weigel-Van Aken, K.A., et al. (2008). Next generation of adeno-associated virus 2 vectors: point mutations in tyrosines lead to high-efficiency transduction at lower doses. Proc Natl Acad Sci USA 105, 7827–7832.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zincarelli, C., Soltys, S., Rengo, G., and Rabinowitz, J.E. (2008). Analysis of AAV serotypes 1–9 mediated gene expression and tropism in mice after systemic injection. Mol Ther 16, 1073–1080.

    Article  CAS  PubMed  Google Scholar 

  • Zuo, E., Sun, Y., Wei, W., Yuan, T., Ying, W., Sun, H., Yuan, L., Steinmetz, L.M., Li, Y., and Yang, H. (2019). Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos. Science 364, 289–292.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

We thank Meizhen Liu from East China Normal University for microinjection and generation of the PKU mouse model, the ECNU Public Platform for innovation(011) for its support, and Dr Stefan Siwko for proof reading of this manuscript. This work was partially supported by grants from the National Key R&D Program of China (2019YFA0110802), the National Natural Science Foundation of China (81670470 and 81873685), grants from the Shanghai Municipal Commission for Science and Technology (18411953500 and 20140900201), a grant from the Innovation Program of Shanghai Municipal Education Commission (2019-01-07-00-05-E00054), and the Fundamental Research Funds for the Central Universities.

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  1. Contributed equally to this work

Authors and Affiliations

  1. Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal University, Shanghai, 200241, China

    Shuming Yin, Lie Ma, Tingting Shao, Mei Zhang, Yuting Guan, Liren Wang, Yaqiang Hu, Xi Chen, Weishi Yu, Mingyao Liu & Dali Li

  2. Bioray Laboratories Inc., Shanghai, 200241, China

    Honghui Han

  3. Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200092, China

    Nan Shen, Wenjuan Qiu, Hongquan Geng & Yongguo Yu

  4. College of Physical Education and Health, East China Normal University, Shanghai, 200241, China

    Shichang Li

  5. Key Laboratory of Adolescent Health Assessment and Exercise Intervention, Ministry of Education, College of Physical Education and Health, East China Normal University, Shanghai, 200241, China

    Shichang Li

  6. CIPHER GENE LLC, Beijing, 100089, China

    Weishi Yu

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  1. Shuming Yin
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  2. Lie Ma
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Correspondence to Dali Li.

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The author(s) declare that they have no conflict of interest. All animal work conformed to the regulations of the animal ethics committee and was approved by the East China Normal University Center for Animal Research.

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Enhanced genome editing to ameliorate a genetic metabolic liver disease through co-delivery of adeno-associated virus receptor

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Yin, S., Ma, L., Shao, T. et al. Enhanced genome editing to ameliorate a genetic metabolic liver disease through co-delivery of adeno-associated virus receptor. Sci. China Life Sci. 65, 718–730 (2022). https://doi.org/10.1007/s11427-020-1744-6

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