dCas9-VPR-mediated transcriptional activation of functionally equivalent genes for gene therapy

Lisa M. Riedmayr, Klara S. Hinrichsmeyer, Nina Karguth, Sybille Böhm, Victoria Splith, Stylianos Michalakis, Elvir Becirovic

Published: 2022-02-06 DOI: 10.1038/s41596-021-00666-3

gene therapy

transcriptional activation

dCas9-VPR

functionally equivalent genes

inherited retinal diseases

AI 解读

The GFP thermal shift assay for screening ligand and lipid interactions to solute carrier transporters

Co-fractionation–mass spectrometry to characterize native mitochondrial protein assemblies in mammalian neurons and brain

Simultaneously measuring the methylation of parent and daughter strands of replicated DNA at the single-molecule level by Hammer-seq

The Nodewalk assay to quantitate chromatin fiber interactomes in very small cell populations

Nondestructive production of exosomes loaded with ultrathin palladium nanosheets for targeted bio-orthogonal catalysis

An optimized culture system for efficient derivation of porcine expanded potential stem cells from preimplantation embryos and by reprogramming somatic cells

Engineering SARS-CoV-2 using a reverse genetic system

cfSNV: a software tool for the sensitive detection of somatic mutations from cell-free DNA

Growing and making nano- and microcrystals

Composable microfluidic spinning platforms for facile production of biomimetic perfusable hydrogel microtubes

查看全部

Sections

Figures

References

Many disease-causing genes possess functionally equivalent counterparts, which are often expressed in distinct cell types. An attractive gene therapy approach for inherited disorders caused by mutations in such genes is to transcriptionally activate the a
Introduction
Materials
Procedure
Troubleshooting
Timing
Anticipated results
- Reporting Summary
- References
References
ACKNOWLEDGMENTS

Dunbar, C. E. et al. Gene therapy comes of age. Science 359, eaan4672 (2018).
Wang, D., Tai, P. W. L. & Gao, G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat. Rev. Drug Discov. 18, 358–378 (2019).
Eid, A., Alshareef, S. & Mahfouz, M. M. CRISPR base editors: genome editing without double-stranded breaks. Biochem. J. 475, 1955–1964 (2018).
Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).
Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).
Wang, M. et al. Targeted base editing in rice with CRISPR/ScCas9 system. Plant Biotechnol. J. 18, 1645–1647 (2020).
Komor, A. C., Kim, Y. B., Packer, M. S., Zuris, J. A. & Liu, D. R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533, 420–424 (2016).
Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).
Maeder, M. L. et al. CRISPR RNA-guided activation of endogenous human genes. Nat. Methods 10, 977–979 (2013).
Mali, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat. Biotechnol. 31, 833–838 (2013).
Perez-Pinera, P. et al. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Nat. Methods 10, 973–976 (2013).
Böhm, S. et al. A gene therapy for inherited blindness using dCas9-VPR-mediated transcriptional activation. Sci. Adv. 6, eaba5614 (2020).
Gilbert, L. A. et al. CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes. Cell 154, 442–451 (2013).
Gilbert, L. A. et al. Genome-scale CRISPR-mediated control of gene repression and activation. Cell 159, 647–661 (2014).
Konermann, S. et al. Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex. Nature 517, 583–588 (2015).
Chavez, A. et al. Comparison of Cas9 activators in multiple species. Nat. Methods 13, 563–567 (2016).
Wu, Z., Yang, H. & Colosi, P. Effect of genome size on AAV vector packaging. Mol. Ther. 18, 80–86 (2010).
Zinn, E. & Vandenberghe, L. H. Adeno-associated virus: fit to serve. Curr. Opin. Virol. 8, 90–97 (2014).
Chew, W. L. et al. A multifunctional AAV-CRISPR-Cas9 and its host response. Nat. Methods 13, 868–874 (2016).
Moreno, A. M. et al. In situ gene therapy via AAV-CRISPR-Cas9-mediated targeted gene regulation. Mol. Ther. 26, 1818–1827 (2018).
Truong, D. J. et al. Development of an intein-mediated split-Cas9 system for gene therapy. Nucleic Acids Res. 43, 6450–6458 (2015).
Ma, D., Peng, S. & Xie, Z. Integration and exchange of split dCas9 domains for transcriptional controls in mammalian cells. Nat. Commun. 7, 13056 (2016).
Lamb, T. D. Evolution of phototransduction, vertebrate photoreceptors and retina. Prog. Retin. Eye Res. 36, 52–119 (2013).
Fu, Y., Kefalov, V., Luo, D. G., Xue, T. & Yau, K. W. Quantal noise from human red cone pigment. Nat. Neurosci. 11, 565–571 (2008).
Kefalov, V. J. Rod and cone visual pigments and phototransduction through pharmacological, genetic, and physiological approaches. J. Biol. Chem. 287, 1635–1641 (2012).
Sakurai, K. et al. Physiological properties of rod photoreceptor cells in green-sensitive cone pigment knock-in mice. J. Gen. Physiol. 130, 21–40 (2007).
Shi, G., Yau, K. W., Chen, J. & Kefalov, V. J. Signaling properties of a short-wave cone visual pigment and its role in phototransduction. J. Neurosci. 27, 10084–10093 (2007).
Liao, H. K. et al. In vivo target gene activation via CRISPR/Cas9-mediated trans-epigenetic modulation. Cell 171, 1495–1507.e15 (2017).
Matharu, N. et al. CRISPR-mediated activation of a promoter or enhancer rescues obesity caused by haploinsufficiency. Science 363, eaau0629 (2019).
Kemaladewi, D. U. et al. A mutation-independent approach for muscular dystrophy via upregulation of a modifier gene. Nature 572, 125–130 (2019).
Boye, S. E., Boye, S. L., Lewin, A. S. & Hauswirth, W. W. A comprehensive review of retinal gene therapy. Mol. Ther. 21, 509–519 (2013).
Keeler, A. M. & Flotte, T. R. Recombinant adeno-associated virus gene therapy in light of Luxturna (and Zolgensma and Glybera): where are we, and how did we get here? Annu. Rev. Virol. 6, 601–621 (2019).
Dahlman, J. E. et al. Orthogonal gene knockout and activation with a catalytically active Cas9 nuclease. Nat. Biotechnol. 33, 1159–1161 (2015).
Kiani, S. et al. Cas9 gRNA engineering for genome editing, activation and repression. Nat. Methods 12, 1051–1054 (2015).
Riedmayr, L. et al. A highly efficient dual AAV technology for therapeutic (epi)genome editing applications [abstract]. ASGCT 23rd Annual Meeting, 2020 May 12–15, abstract no. 1472.
Narasimhan, I., Murali, A., Subramanian, K., Ramalingam, S. & Parameswaran, S. Autosomal dominant retinitis pigmentosa with toxic gain of function: mechanisms and therapeutics. Eur. J. Ophthalmol. 31, 304–320 (2021).
Lugo-Martinez, J. et al. The loss and gain of functional amino acid residues is a common mechanism causing human inherited disease. PLoS Comput. Biol. 12, e1005091 (2016).
Li, Y., Zhang, Y., Li, X., Yi, S. & Xu, J. Gain-of-function mutations: an emerging advantage for cancer biology. Trends Biochem. Sci. 44, 659–674 (2019).
Li, X. H. & Babu, M. M. Human diseases from gain-of-function mutations in disordered protein regions. Cell 175, 40–42 (2018).
Kajiwara, K., Berson, E. L. & Dryja, T. P. Digenic retinitis pigmentosa due to mutations at the unlinked peripherin/RDS and ROM1 loci. Science 264, 1604–1608 (1994).
Burkard, M. et al. Accessory heterozygous mutations in cone photoreceptor CNGA3 exacerbate CNG channel-associated retinopathy. J. Clin. Invest. 128, 5663–5675 (2018).
Das, R. G. et al. Variabilities in retinal function and structure in a canine model of cone-rod dystrophy associated with RPGRIP1 support multigenic etiology. Sci. Rep. 7, 12823 (2017).
Bazaga, A., Leggate, D. & Weisser, H. Genome-wide investigation of gene-cancer associations for the prediction of novel therapeutic targets in oncology. Sci. Rep. 10, 10787 (2020).
Wang, D., Zhang, F. & Gao, G. CRISPR-based therapeutic genome editing: strategies and in vivo delivery by AAV vectors. Cell 181, 136–150 (2020).
Gallego, C., Goncalves, M. & Wijnholds, J. Novel therapeutic approaches for the treatment of retinal degenerative diseases: focus on CRISPR/Cas-based gene editing. Front. Neurosci. 14, 838 (2020).
Seto, M., Weiner, R. L., Dumitrescu, L. & Hohman, T. J. Protective genes and pathways in Alzheimer’s disease: moving towards precision interventions. Mol. Neurodegener. 16, 29 (2021).
Harper, A. R., Nayee, S. & Topol, E. J. Protective alleles and modifier variants in human health and disease. Nat. Rev. Genet. 16, 689–701 (2015).
Levy, J. M. et al. Cytosine and adenine base editing of the brain, liver, retina, heart and skeletal muscle of mice via adeno-associated viruses. Nat. Biomed. Eng. 4, 97–110 (2020).
Tornabene, P. et al. Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina. Sci. Transl. Med. 11, eaav4523 (2019).
Carvalho, L. S. et al. Evaluating efficiencies of dual AAV approaches for retinal targeting. Front. Neurosci. 11, 503 (2017).
Riviere, C., Danos, O. & Douar, A. M. Long-term expression and repeated administration of AAV type 1, 2 and 5 vectors in skeletal muscle of immunocompetent adult mice. Gene Ther. 13, 1300–1308 (2006).
Bennett, J. et al. Safety and durability of effect of contralateral-eye administration of AAV2 gene therapy in patients with childhood-onset blindness caused by RPE65 mutations: a follow-on phase 1 trial. Lancet 388, 661–672 (2016).
Bainbridge, J. W. et al. Long-term effect of gene therapy on Leber’s congenital amaurosis. N. Engl. J. Med. 372, 1887–1897 (2015).
Mitchell, A. L. et al. InterPro in 2019: improving coverage, classification and access to protein sequence annotations. Nucleic Acids Res. 47, D351–D360 (2019).
de Castro, E. et al. ScanProsite: detection of PROSITE signature matches and ProRule-associated functional and structural residues in proteins. Nucleic Acids Res. 34, W362–W365 (2006).
Concordet, J. P. & Haeussler, M. CRISPOR: intuitive guide selection for CRISPR/Cas9 genome editing experiments and screens. Nucleic Acids Res. 46, W242–W245 (2018).
al-Ubaidi, M. R. et al. Bilateral retinal and brain tumors in transgenic mice expressing simian virus 40 large T antigen under control of the human interphotoreceptor retinoid-binding protein promoter. J. Cell Biol. 119, 1681–1687 (1992).
Tan, E. et al. Expression of cone-photoreceptor-specific antigens in a cell line derived from retinal tumors in transgenic mice. Invest. Ophthalmol. Vis. Sci. 45, 764–768 (2004).
Cox, J. et al. A practical guide to the MaxQuant computational platform for SILAC-based quantitative proteomics. Nat. Protoc. 4, 698–705 (2009).
Palmer, A. E. & Tsien, R. Y. Measuring calcium signaling using genetically targetable fluorescent indicators. Nat. Protoc. 1, 1057–1065 (2006).
Modena, M. M., Chawla, K., Misun, P. M. & Hierlemann, A. Smart cell culture systems: integration of sensors and actuators into microphysiological systems. ACS Chem. Biol. 13, 1767–1784 (2018).
Becirovic, E. et al. AAV vectors for FRET-based analysis of protein-protein interactions in photoreceptor outer segments. Front. Neurosci. 10, 356 (2016).
DeWeirdt, P. C. et al. Genetic screens in isogenic mammalian cell lines without single cell cloning. Nat. Commun. 11, 752 (2020).
Fenno, L. E. et al. Targeting cells with single vectors using multiple-feature Boolean logic. Nat. Methods 11, 763–772 (2014).
Petrs-Silva, H. et al. High-efficiency transduction of the mouse retina by tyrosine-mutant AAV serotype vectors. Mol. Ther. 17, 463–471 (2009).
Dalkara, D. et al. In vivo-directed evolution of a new adeno-associated virus for therapeutic outer retinal gene delivery from the vitreous. Sci. Transl. Med. 5, 189ra176 (2013).
Khabou, H. et al. Insight into the mechanisms of enhanced retinal transduction by the engineered AAV2 capsid variant -7m8. Biotechnol. Bioeng. 113, 2712–2724 (2016).
Jat, P. S., Cepko, C. L., Mulligan, R. C. & Sharp, P. A. Recombinant retroviruses encoding simian virus 40 large T antigen and polyomavirus large and middle T antigens. Mol. Cell. Biol. 6, 1204–1217 (1986).
Xu, J. Preparation, culture, and immortalization of mouse embryonic fibroblasts. Curr. Protoc. Mol. Biol. Ch. 28, Unit 28.1 (2005).
Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).
Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 3, 1101–1108 (2008).
Vandenberghe, L. H. et al. Efficient serotype-dependent release of functional vector into the culture medium during adeno-associated virus manufacturing. Hum. Gene Ther. 21, 1251–1257 (2010).
D’Costa, S. et al. Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR. Mol. Ther. Methods Clin. Dev. 5, 16019 (2016).
Becirovic, E. et al. In vivo analysis of disease-associated point mutations unveils profound differences in mRNA splicing of peripherin-2 in rod and cone photoreceptors. PLoS Genet. 12, e1005811 (2016).
Murenu, E. et al. A universal protocol for isolating retinal ON bipolar cells across species via fluorescence-activated cell sorting. Mol. Ther. Methods Clin. Dev. 20, 587–600 (2021).
Jonkman, J., Brown, C. M., Wright, G. D., Anderson, K. I. & North, A. J. Tutorial: guidance for quantitative confocal microscopy. Nat. Protoc. 15, 1585–1611 (2020).
Bohm, S. et al. Peripherin-2 and Rom-1 have opposing effects on rod outer segment targeting of retinitis pigmentosa-linked peripherin-2 mutants. Sci. Rep. 7, 2321 (2017).
Panagiotopoulos, A. L. et al. Antisense oligonucleotide- and CRISPR-Cas9-mediated rescue of mRNA splicing for a deep intronic CLRN1 mutation. Mol. Ther. Nucleic Acids 21, 1050–1061 (2020).
Wang, Z. et al. Adeno-associated virus serotype 8 efficiently delivers genes to muscle and heart. Nat. Biotechnol. 23, 321–328 (2005).
Bish, L. T. et al. Adeno-associated virus (AAV) serotype 9 provides global cardiac gene transfer superior to AAV1, AAV6, AAV7, and AAV8 in the mouse and rat. Hum. Gene Ther. 19, 1359–1368 (2008).
Su, H. et al. AAV serotype 1 mediates more efficient gene transfer to pig myocardium than AAV serotype 2 and plasmid. J. Gene Med. 10, 33–41 (2008).
Xiao, W. et al. Gene therapy vectors based on adeno-associated virus type 1. J. Virol. 73, 3994–4003 (1999).
Gregorevic, P. et al. Systemic delivery of genes to striated muscles using adeno-associated viral vectors. Nat. Med. 10, 828–834 (2004).
Katwal, A. B. et al. Adeno-associated virus serotype 9 efficiently targets ischemic skeletal muscle following systemic delivery. Gene Ther. 20, 930–938 (2013).
Sumner-Jones, S. G., Davies, L. A., Varathalingam, A., Gill, D. R. & Hyde, S. C. Long-term persistence of gene expression from adeno-associated virus serotype 5 in the mouse airways. Gene Ther. 13, 1703–1713 (2006).
Limberis, M. P., Vandenberghe, L. H., Zhang, L., Pickles, R. J. & Wilson, J. M. Transduction efficiencies of novel AAV vectors in mouse airway epithelium in vivo and human ciliated airway epithelium in vitro. Mol. Ther. 17, 294–301 (2009).
Pfeifer, C., Aneja, M. K., Hasenpusch, G. & Rudolph, C. Adeno-associated virus serotype 9-mediated pulmonary transgene expression: effect of mouse strain, animal gender and lung inflammation. Gene Ther. 18, 1034–1042 (2011).
Burger, C. et al. Recombinant AAV viral vectors pseudotyped with viral capsids from serotypes 1, 2, and 5 display differential efficiency and cell tropism after delivery to different regions of the central nervous system. Mol. Ther. 10, 302–317 (2004).
Broekman, M. L., Comer, L. A., Hyman, B. T. & Sena-Esteves, M. Adeno-associated virus vectors serotyped with AAV8 capsid are more efficient than AAV-1 or -2 serotypes for widespread gene delivery to the neonatal mouse brain. Neuroscience 138, 501–510 (2006).
Dayton, R. D., Wang, D. B. & Klein, R. L. The advent of AAV9 expands applications for brain and spinal cord gene delivery. Expert Opin. Biol. Ther. 12, 757–766 (2012).
Gao, G. et al. Biology of AAV serotype vectors in liver-directed gene transfer to nonhuman primates. Mol. Ther. 13, 77–87 (2006).
Auricchio, A. et al. Exchange of surface proteins impacts on viral vector cellular specificity and transduction characteristics: the retina as a model. Hum. Mol. Genet. 10, 3075–3081 (2001).
Wu, H., Hu, Z. & Liu, X. Q. Protein trans-splicing by a split intein encoded in a split DnaE gene of Synechocystis sp. PCC6803. Proc. Natl Acad. Sci. USA 95, 9226–9231 (1998).
Aranko, A. S., Wlodawer, A. & Iwai, H. Nature’s recipe for splitting inteins. Protein Eng. Des. Sel. 27, 263–271 (2014).
Li, J., Sun, W., Wang, B., Xiao, X. & Liu, X. Q. Protein trans-splicing as a means for viral vector-mediated in vivo gene therapy. Hum. Gene Ther. 19, 958–964 (2008).
Stevens, A. J. et al. A promiscuous split intein with expanded protein engineering applications. Proc. Natl Acad. Sci. USA 114, 8538–8543 (2017).
Zetsche, B., Volz, S. E. & Zhang, F. A split-Cas9 architecture for inducible genome editing and transcription modulation. Nat. Biotechnol. 33, 139–142 (2015).
Nihongaki, Y., Kawano, F., Nakajima, T. & Sato, M. Photoactivatable CRISPR-Cas9 for optogenetic genome editing. Nat. Biotechnol. 33, 755–760 (2015).
Li, C. & Samulski, R. J. Engineering adeno-associated virus vectors for gene therapy. Nat. Rev. Genet. 21, 255–272 (2020).

dCas9-VPR-mediated transcriptional activation of functionally equivalent genes for gene therapy

Supplementary information

推荐阅读