Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Dual AAV vectors for efficient delivery of large transgenes

Nature Protocols volume 21, pages 1466–1522 (2026)Cite this article

Subjects

Abstract

Despite their limited cargo capacity (<5 kb), adeno-associated viral (AAV) vectors remain the gold standard for in vivo delivery of therapeutic genes. Dual AAV vectors have emerged as a valuable tool for delivering large therapeutic genes and CRISPR tools to overcome this limitation. Here we provide a detailed protocol for the design, production and evaluation of dual AAV vectors. We offer guidelines for selecting a suitable dual AAV strategy, designing and cloning the genes to be delivered, and conducting in vitro evaluations of expression efficiency. In addition, we detail the production of dual AAVs and their assessment in human cellular models, such as induced pluripotent stem cell-derived retinal organoids. Finally, we outline the administration of dual AAVs via different routes in mice and the assessment of transgene-derived RNA and protein expression in various tissues. Overall, the instructions in this Protocol will aid in the efficient in vivo delivery of large DNA fragments using dual AAVs. This Protocol is adaptable to a wide range of model organisms as well as to human organoid cultures and, depending on the application, can be completed in 15–44 weeks.

Key points

  • Adeno-associated viral (AAV) vectors are widely used to deliver therapeutic genes in vivo. Dual AAVs, whereby the gene of interest is delivered by two viral vectors and reconstituted upon delivery, are used to overcome the limited cargo capacity of the AAV genome.

  • This Protocol presents three strategies for dual AAV design and provides guidance for the production and evaluation of dual AAV vectors in wild-type and disease mouse models and in human organoids.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on SpringerLink
  • Instant access to the full article PDF.

USD 39.95

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Dual AAV solutions and their mode of action.
Fig. 2: Dual AAV approaches by comparison.
Fig. 3: Overview of the Protocol.
Fig. 4: Cloning procedure to generate dual vector plasmids required for in vitro and in vivo experiments.
Fig. 5: Anticipated results for reconstitution of dual vectors in vitro.
Fig. 6: Anticipated results for reconstitution of dual AAV vectors after in vivo delivery.

Similar content being viewed by others

Data availability

Figures 5 and 6 show example data that were obtained using this Protocol. Source data are provided with this Protocol. Additional data related to this Protocol can be found in the original study20 or may be requested from the authors. Source data are provided with this paper.

References

  1. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Li, C. & Samulski, R. J. Engineering adeno-associated virus vectors for gene therapy. Nat. Rev. Genet. 21, 255–272 (2020).

    Article  CAS  PubMed  Google Scholar 

  3. Ibraheim, R. et al. Self-inactivating, all-in-one AAV vectors for precision Cas9 genome editing via homology-directed repair in vivo. Nat. Commun. 12, 6267 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Jang, M. J. et al. Spatial transcriptomics for profiling the tropism of viral vectors in tissues. Nat. Biotechnol. 41, 1272–1286 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Rittiner, J. E., Moncalvo, M., Chiba-Falek, O. & Kantor, B. Gene-editing technologies paired with viral vectors for translational research into neurodegenerative diseases. Front. Mol. Neurosci. 13, 148 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Srivastava, A. In vivo tissue-tropism of adeno-associated viral vectors. Curr. Opin. Virol. 21, 75–80 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Heo, Y. A. Etranacogene dezaparvovec: first approval. Drugs 83, 347–352 (2023).

    Article  CAS  PubMed  Google Scholar 

  8. Gao, J., Hussain, R. M. & Weng, C. Y. Voretigene neparvovec in retinal diseases: a review of the current clinical evidence. Clin. Ophthalmol. 14, 3855–3869 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Hoy, S. M. Delandistrogene moxeparvovec: first approval. Drugs 83, 1323–1329 (2023).

    Article  CAS  PubMed  Google Scholar 

  10. Wu, Z., Yang, H. & Colosi, P. Effect of genome size on AAV vector packaging. Mol. Ther. 18, 80–86 (2010).

    Article  CAS  PubMed  Google Scholar 

  11. Naso, M. F., Tomkowicz, B., Perry, W. L. & Strohl, W. R. Adeno-associated virus (AAV) as a vector for gene therapy. BioDrugs 31, 317–334 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Lai, Y. et al. Efficient in vivo gene expression by trans-splicing adeno-associated viral vectors. Nat. Biotechnol. 23, 1435–1439 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Yan, Z., Zhang, Y., Duan, D. & Engelhardt, J. F. Trans-splicing vectors expand the utility of adeno-associated virus for gene therapy. Proc. Natl Acad. Sci. USA 97, 6716–6721 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Lopes, V. S. et al. Retinal gene therapy with a large MYO7A cDNA using adeno-associated virus. Gene Ther. 20, 824–833 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Duan, D., Yue, Y. & Engelhardt, J. F. Expanding AAV packaging capacity with trans-splicing or overlapping vectors: a quantitative comparison. Mol. Ther. 4, 383–391 (2001).

    Article  CAS  PubMed  Google Scholar 

  16. Trapani, I. et al. Improved dual AAV vectors with reduced expression of truncated proteins are safe and effective in the retina of a mouse model of Stargardt disease. Hum. Mol. Genet. 24, 6811–6825 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ghosh, A., Yue, Y., Lai, Y. & Duan, D. A hybrid vector system expands adeno-associated viral vector packaging capacity in a transgene-independent manner. Mol. Ther. 16, 124–130 (2008).

    Article  CAS  PubMed  Google Scholar 

  18. Carvalho, L. S. et al. Evaluating efficiencies of dual AAV approaches for retinal targeting. Front. Neurosci. 11, 503 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  19. Dyka, F. M., Boye, S. L., Chiodo, V. A., Hauswirth, W. W. & Boye, S. E. Dual adeno-associated virus vectors result in efficient in vitro and in vivo expression of an oversized gene, MYO7A. Hum. Gene Ther. Methods 25, 166–177 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Riedmayr, L. M. et al. mRNA trans-splicing dual AAV vectors for (epi)genome editing and gene therapy. Nat. Commun. 14, 6578 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Liu, X. et al. Partial correction of endogenous DeltaF508 CFTR in human cystic fibrosis airway epithelia by spliceosome-mediated RNA trans-splicing. Nat. Biotechnol. 20, 47–52 (2002).

    Article  CAS  PubMed  Google Scholar 

  22. Lindley, S. R. et al. Ribozyme-activated mRNA trans-ligation enables large gene delivery to treat muscular dystrophies. Science 386, 762–767 (2024).

    Article  CAS  PubMed  Google Scholar 

  23. Tornabene, P. et al. Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina. Sci. Transl. Med. 11, eaav4523 (2019).

  24. Blair, H. A. Onasemnogene abeparvovec: a review in spinal muscular atrophy. CNS Drugs 36, 995–1005 (2022).

    Article  PubMed  Google Scholar 

  25. Srivastava, A. Rationale and strategies for the development of safe and effective optimized AAV vectors for human gene therapy. Mol. Ther. Nucleic Acids 32, 949–959 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Martin, F. J. et al. Ensembl 2023. Nucleic Acids Res. 51, D933–D941 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Trapani, I. & Auricchio, A. Seeing the light after 25 years of retinal gene therapy. Trends Mol. Med. 24, 669–681 (2018).

    Article  PubMed  Google Scholar 

  28. Willett, K. & Bennett, J. Immunology of AAV-mediated gene transfer in the eye. Front. Immunol. 4, 261 (2013).

    Article  PubMed  PubMed Central  Google Scholar 

  29. Riedmayr, L. M. et al. dCas9-VPR-mediated transcriptional activation of functionally equivalent genes for gene therapy. Nat. Protoc. 17, 781–818 (2022).

    Article  CAS  PubMed  Google Scholar 

  30. Chavez, A. et al. Highly efficient Cas9-mediated transcriptional programming. Nat. Methods 12, 326–328 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Yeo, N. C. et al. An enhanced CRISPR repressor for targeted mammalian gene regulation. Nat. Methods 15, 611–616 (2018).

  32. Nunez, J. K. et al. Genome-wide programmable transcriptional memory by CRISPR-based epigenome editing. Cell 184, 2503–2519 e17 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Zhang, E., Neugebauer, M. E., Krasnow, N. A. & Liu, D. R. Phage-assisted evolution of highly active cytosine base editors with enhanced selectivity and minimal sequence context preference. Nat. Commun. 15, 1697 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Chen, L. et al. Engineering a precise adenine base editor with minimal bystander editing. Nat. Chem. Biol. 19, 101–110 (2023).

    Article  CAS  PubMed  Google Scholar 

  35. Anzalone, A. V. et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149–157 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Bohm, S. et al. A gene therapy for inherited blindness using dCas9-VPR-mediated transcriptional activation. Sci. Adv. 6, eaba5614 (2020).

    Article  PubMed  PubMed Central  Google Scholar 

  37. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Davis, J. R. et al. Efficient prime editing in mouse brain, liver and heart with dual AAVs. Nat. Biotechnol. 42, 253–264 (2024).

    Article  CAS  PubMed  Google Scholar 

  39. Chen, F. et al. Multiplexed base editing through Cas12a variant-mediated cytosine and adenine base editors. Commun. Biol. 5, 1163 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Schmitt-Ulms, C. et al. Programmable RNA writing with trans-splicing. Preprint at bioRxiv (2024).

  41. An, M. et al. Engineered virus-like particles for transient delivery of prime editor ribonucleoprotein complexes in vivo. Nat. Biotechnol. 42, 1526–1537 (2024).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  42. Banskota, S. et al. Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins. Cell 185, 250–265.e16 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kulkarni, J. A. et al. Design of lipid nanoparticles for in vitro and in vivo delivery of plasmid DNA. Nanomedicine 13, 1377–1387 (2017).

    Article  CAS  PubMed  Google Scholar 

  44. Pozzi, D. & Caracciolo, G. Looking Back, moving forward: lipid nanoparticles as a promising frontier in gene delivery. ACS Pharmacol. Transl. Sci. 6, 1561–1573 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Mendell, J. R. et al. Testing preexisting antibodies prior to AAV gene transfer therapy: rationale, lessons and future considerations. Mol. Ther. Methods Clin. Dev. 25, 74–83 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Duan, D. Lethal immunotoxicity in high-dose systemic AAV therapy. Mol. Ther. 31, 3123–3126 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. van der Loo, J. C. & Wright, J. F. Progress and challenges in viral vector manufacturing. Hum. Mol. Genet. 25, R42–R52 (2016).

    Article  PubMed  Google Scholar 

  48. Han, X. et al. Ligand-tethered lipid nanoparticles for targeted RNA delivery to treat liver fibrosis. Nat. Commun. 14, 75 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Musunuru, K. et al. In vivo CRISPR base editing of PCSK9 durably lowers cholesterol in primates. Nature 593, 429–434 (2021).

    Article  CAS  PubMed  Google Scholar 

  50. Kularatne, R. N., Crist, R. M. & Stern, S. T. The future of tissue-targeted lipid nanoparticle-mediated nucleic acid delivery. Pharmaceuticals 15, 897 (2022).

  51. Kulkarni, J. A. et al. The current landscape of nucleic acid therapeutics. Nat. Nanotechnol. 16, 630–643 (2021).

    Article  CAS  PubMed  Google Scholar 

  52. Charlesworth, C. T. et al. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat. Med. 25, 249–254 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. van Haasteren, J., Li, J., Scheideler, O. J., Murthy, N. & Schaffer, D. V. The delivery challenge: fulfilling the promise of therapeutic genome editing. Nat. Biotechnol. 38, 845–855 (2020).

    Article  PubMed  Google Scholar 

  54. Wagner, D. L. et al. High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population. Nat. Med. 25, 242–248 (2019).

    Article  CAS  PubMed  Google Scholar 

  55. Lee, C. S. et al. Adenovirus-mediated gene delivery: potential applications for gene and cell-based therapies in the new era of personalized medicine. Genes Dis. 4, 43–63 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  56. Counsell, J. R. et al. Lentiviral vectors can be used for full-length dystrophin gene therapy. Sci. Rep. 7, 44775 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Jaffe, H. A. et al. Adenovirus-mediated in vivo gene transfer and expression in normal rat liver. Nat. Genet. 1, 372–378 (1992).

    Article  CAS  PubMed  Google Scholar 

  58. Rosenfeld, M. A. et al. Adenovirus-mediated transfer of a recombinant alpha 1-antitrypsin gene to the lung epithelium in vivo. Science 252, 431–434 (1991).

    Article  CAS  PubMed  Google Scholar 

  59. Zak, D. E. et al. Merck Ad5/HIV induces broad innate immune activation that predicts CD8+ T-cell responses but is attenuated by preexisting Ad5 immunity. Proc. Natl Acad. Sci. USA 109, E3503–E3512 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  60. Khare, R., Hillestad, M. L., Xu, Z., Byrnes, A. P. & Barry, M. A. Circulating antibodies and macrophages as modulators of adenovirus pharmacology. J. Virol. 87, 3678–3686 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Raper, S. E. et al. Fatal systemic inflammatory response syndrome in a ornithine transcarbamylase deficient patient following adenoviral gene transfer. Mol. Genet. Metab. 80, 148–158 (2003).

    Article  CAS  PubMed  Google Scholar 

  62. Shirley, J. L., de Jong, Y. P., Terhorst, C. & Herzog, R. W. Immune responses to viral gene therapy vectors. Mol. Ther. 28, 709–722 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Qian, W. et al. Prolonged integration site selection of a lentiviral vector in the genome of human keratinocytes. Med. Sci. Monit. 23, 1116–1122 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Wu, C. & Dunbar, C. E. Stem cell gene therapy: the risks of insertional mutagenesis and approaches to minimize genotoxicity. Front. Med. 5, 356–371 (2011).

  65. Cesana, D. et al. Whole transcriptome characterization of aberrant splicing events induced by lentiviral vector integrations. J. Clin. Invest. 122, 1667–1676 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Cesana, D. et al. Uncovering and dissecting the genotoxicity of self-inactivating lentiviral vectors in vivo. Mol. Ther. 22, 774–785 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Liu, M. et al. Genomic discovery of potent chromatin insulators for human gene therapy. Nat. Biotechnol. 33, 198–203 (2015).

    Article  PubMed  Google Scholar 

  68. Hargrove, P. W. et al. Globin lentiviral vector insertions can perturb the expression of endogenous genes in beta-thalassemic hematopoietic cells. Mol. Ther. 16, 525–533 (2008).

    Article  CAS  PubMed  Google Scholar 

  69. Duverge, A. & Negroni, M. Pseudotyping lentiviral vectors: when the clothes make the virus. Viruses 12, 1311 (2020).

  70. Hirsch, M. L., Agbandje-McKenna, M. & Samulski, R. J. Little vector, big gene transduction: fragmented genome reassembly of adeno-associated virus. Mol. Ther. 18, 6–8 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Trapani, I. Adeno-associated viral vectors as a tool for large gene delivery to the retina. Genes 10, 287 (2019).

  72. Becirovic, E. et al. AAV vectors for FRET-based analysis of protein–protein interactions in photoreceptor outer segments. Front. Neurosci. 10, 356 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  73. Barbon, E. et al. Development of a dual hybrid AAV vector for endothelial-targeted expression of von Willebrand factor. Gene Ther. 30, 245–254 (2023).

    Article  CAS  PubMed  Google Scholar 

  74. Chen, Z. R. et al. Co-transduction of dual-adeno-associated virus vectors in the neonatal and adult mouse utricles. Front. Mol. Neurosci. 15, 1020803 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Kodippili, K. et al. Dual AAV gene therapy for duchenne muscular dystrophy with a 7-kb mini-dystrophin gene in the canine model. Hum. Gene Ther. 29, 299–311 (2018).

    Article  CAS  PubMed  Google Scholar 

  76. Omichi, R., Yoshimura, H., Shibata, S. B., Vandenberghe, L. H. & Smith, R. J. H. Hair cell transduction efficiency of single- and dual-AAV serotypes in adult murine cochleae. Mol. Ther. Methods Clin. Dev. 17, 1167–1177 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Ghosh, A., Yue, Y. & Duan, D. Efficient transgene reconstitution with hybrid dual AAV vectors carrying the minimized bridging sequences. Hum. Gene Ther. 22, 77–83 (2011).

    Article  CAS  PubMed  Google Scholar 

  78. Trapani, I. et al. Effective delivery of large genes to the retina by dual AAV vectors. EMBO Mol. Med. 6, 194–211 (2014).

    Article  CAS  PubMed  Google Scholar 

  79. Ivanchenko, M. V. et al. PCDH15 dual-AAV gene therapy for deafness and blindness in usher syndrome type 1F models. J. Clin. Invest. (2024).

  80. Ferla, R. et al. Efficacy, pharmacokinetics, and safety in the mouse and primate retina of dual AAV vectors for Usher syndrome type 1B. Mol. Ther. Methods Clin. Dev. 28, 396–411 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  81. Occelli, L. M. et al. Development of a translatable gene augmentation therapy for CNGB1-retinitis pigmentosa. Mol. Ther. 31, 2028–2041 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  82. Kügler, S., Kilic, E. & Bähr, M. Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression from an adenoviral vector in the adult rat brain depending on the transduced area. Gene Ther. 10, 337–347 (2003).

    Article  PubMed  Google Scholar 

  83. Kuhn, B., Ozden, I., Lampi, Y., Hasan, M. T. & Wang, S. S. An amplified promoter system for targeted expression of calcium indicator proteins in the cerebellar cortex. Front. Neural. Circuits 6, 49 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Prasad, K. M., Xu, Y., Yang, Z., Acton, S. T. & French, B. A. Robust cardiomyocyte-specific gene expression following systemic injection of AAV: in vivo gene delivery follows a Poisson distribution. Gene Ther. 18, 43–52 (2011).

    Article  CAS  PubMed  Google Scholar 

  85. Schröder, L. C., Frank, D. & Müller, O. J. Transcriptional targeting approaches in cardiac gene transfer using AAV vectors. Pathogens 12, 1301 (2023).

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

    Article  CAS  PubMed  Google Scholar 

  87. Xu, J. Preparation, culture, and immortalization of mouse embryonic fibroblasts. Curr. Protoc. Mol. Biol. 28, 28.1 (2005).

  88. Cowan, C. S. et al. Cell types of the human retina and its organoids at single-cell resolution. Cell 182, 1623–1640.e34 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Muralidharan, V. & Muir, T. W. Protein ligation: an enabling technology for the biophysical analysis of proteins. Nat. Methods 3, 429–438 (2006).

    Article  CAS  PubMed  Google Scholar 

  90. Shah, N. H. & Muir, T. W. Inteins: nature’s gift to protein chemists. Chem. Sci. 5, 446–461 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  91. Stevens, A. J. et al. A promiscuous split intein with expanded protein engineering applications. Proc. Natl Acad. Sci. USA 114, 8538–8543 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Schmittgen, T. D. & Livak, K. J. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 3, 1101–1108 (2008).

    Article  CAS  PubMed  Google Scholar 

  93. Goldman, A., Harper, S. & Speicher, D. W. Detection of proteins on blot membranes. Curr. Protoc. Protein Sci. 86, 10.8.1–10.8.11 (2016).

    PubMed  PubMed Central  Google Scholar 

  94. Ni, D., Xu, P. & Gallagher, S. Immunoblotting and immunodetection. Curr. Protoc. Immunol. 114, 8.10.1–8.10.36 (2016).

    Article  PubMed  Google Scholar 

  95. 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).

    Article  PubMed  PubMed Central  Google Scholar 

  96. Kim, S. et al. Generation, transcriptome profiling, and functional validation of cone-rich human retinal organoids. Proc. Natl Acad. Sci. USA 116, 10824–10833 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  97. Völkner, M., Pavlou, M., Büning, H., Michalakis, S. & Karl, M. O. Optimized adeno-associated virus vectors for efficient transduction of human retinal organoids. Hum. Gene Ther. 32, 694–706 (2021).

    Article  PubMed  Google Scholar 

  98. Weinmann, J. et al. Identification of broadly applicable adeno-associated virus vectors by systematic comparison of commonly used capsid variants in vitro. Hum. Gene Ther. 33, 1197–1212 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  99. Tessmer, K., Borsch, O., Ader, M. & Gasparini, S. J. in Brain Organoid Research (ed. Gopalakrishnan, J.) 81–98 (Springer US, 2023).

  100. Collins, A. L. et al. Mild overexpression of MeCP2 causes a progressive neurological disorder in mice. Hum. Mol. Genet. 13, 2679–2689 (2004).

    Article  CAS  PubMed  Google Scholar 

  101. Xu, Z. et al. Trans-splicing adeno-associated viral vector-mediated gene therapy is limited by the accumulation of spliced mRNA but not by dual vector coinfection efficiency. Hum. Gene Ther. 15, 896–905 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Lek, A. et al. Death after high-dose rAAV9 gene therapy in a patient with duchenne’s muscular dystrophy. N. Engl. J. Med. 389, 1203–1210 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  103. Hinderer, C. et al. Severe toxicity in nonhuman primates and piglets following high-dose intravenous administration of an adeno-associated virus vector expressing human SMN. Hum. Gene Ther. 29, 285–298 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  104. Chew, W. L. et al. A multifunctional AAV–CRISPR–Cas9 and its host response. Nat. Methods 13, 868–874 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Lim, C. K. W. et al. Treatment of a mouse model of ALS by in vivo base editing. Mol. Ther. 28, 1177–1189 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  106. Riedmayr, L. M., Böhm, S., Biel, M. & Becirovic, E. Enigmatic rhodopsin mutation creates an exceptionally strong splice acceptor site. Hum. Mol. Genet. 29, 295–304 (2020).

    Article  CAS  PubMed  Google Scholar 

  107. Stevens, A. J. et al. Design of a split intein with exceptional protein splicing activity. J. Am. Chem. Soc. 138, 2162–2165 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  108. Tornabene, P. et al. Inclusion of a degron reduces levelsof undesired inteins after AAV-mediated proteintrans-splicing in the retina. Mol. Ther. Methods Clin. Dev. 23, 448–459 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  109. Klauer, A. A. & van Hoof, A. Degradation of mRNAs that lack a stop codon: a decade of nonstop progress. Wiley Interdiscip. Rev. RNA 3, 649–660 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  110. Becher, B., Waisman, A. & Lu, L. F. Conditional gene-targeting in mice: problems and solutions. Immunity 48, 835–836 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  111. Lancaster, M. A. & Knoblich, J. A. Generation of cerebral organoids from human pluripotent stem cells. Nat. Protoc. 9, 2329–2340 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  112. McCracken, K. W., Howell, J. C., Wells, J. M. & Spence, J. R. Generating human intestinal tissue from pluripotent stem cells in vitro. Nat. Protoc. 6, 1920–1928 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Miller, A. J. et al. Generation of lung organoids from human pluripotent stem cells in vitro. Nat. Protoc. 14, 518–540 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  114. Takasato, M., Er, P. X., Chiu, H. S. & Little, M. H. Generation of kidney organoids from human pluripotent stem cells. Nat. Protoc. 11, 1681–1692 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work was supported by the Swiss National Science Foundation (grant nos. 310030_212190 and 320030E_221942 to E.B.) and the German Research Foundation (DFG) (project nos. 513025799/FOR5621 to E.B. and M.B.). This work has also been supported by the University Research Priority Program of the University of Zurich (URPP) ITINERARE—Innovative Therapies in Rare Diseases (to E.B.).

Author information

Author notes

  1. Lisa M. Riedmayr

    Present address: Department of Biotechnology and Biomedicine, Technical University of Denmark, Kongens Lyngby, Denmark

  2. These authors contributed equally: David M. Mittas, Lisa M. Riedmayr.

Authors and Affiliations

  1. Department of Pharmacy—Center for Drug Research, Ludwig-Maximilians-Universität, Munich, Germany

    David M. Mittas, Dina Y. Otify, Verena Mehlfeld & Martin Biel

  2. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Lisa M. Riedmayr

  3. Laboratory for Retinal Gene Therapy, Department of Ophthalmology, University Hospital Zurich, University of Zurich, Zurich, Switzerland

    Zoran Gavrilov, Valentin J. Weber, Balint Szalontai, Emina Ucambarlic, Catharina Gandor, Thomas Heigl & Elvir Becirovic

Authors
  1. David M. Mittas
    View author publications

    Search author on:PubMed Google Scholar

  2. Lisa M. Riedmayr
    View author publications

    Search author on:PubMed Google Scholar

  3. Zoran Gavrilov
    View author publications

    Search author on:PubMed Google Scholar

  4. Valentin J. Weber
    View author publications

    Search author on:PubMed Google Scholar

  5. Dina Y. Otify
    View author publications

    Search author on:PubMed Google Scholar

  6. Verena Mehlfeld
    View author publications

    Search author on:PubMed Google Scholar

  7. Balint Szalontai
    View author publications

    Search author on:PubMed Google Scholar

  8. Emina Ucambarlic
    View author publications

    Search author on:PubMed Google Scholar

  9. Catharina Gandor
    View author publications

    Search author on:PubMed Google Scholar

  10. Thomas Heigl
    View author publications

    Search author on:PubMed Google Scholar

  11. Martin Biel
    View author publications

    Search author on:PubMed Google Scholar

  12. Elvir Becirovic
    View author publications

    Search author on:PubMed Google Scholar

Contributions

D.M.M. L.M.R. and Z.G. designed and performed the experiments. D.M.M, L.M.R., Z.G., V.J.W., V.M., B.S., E.U., D.Y.O., C.G., T.H., M.B. and E.B. wrote the manuscript.

Corresponding author

Correspondence to Elvir Becirovic.

Ethics declarations

Competing interests

E.B. and M.B. are authors on a patent application covering the splice site module and its applications (PCT/EP2019/086454, filed by ViGeneron GmbH, status: published). The other authors declare no competing interests.

Peer review

Peer review information

Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.

Additional information

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

Key reference

Riedmayr, L. M. et al. Nat. Commun. 14, 6578 (2023): https://doi.org/10.1038/s41467-023-42386-0

Supplementary information

Source data

Source Data Figs. 5 and 6 (download XLSX )

Statistical source data.

Source Data Fig. 5 (download PDF )

Unprocessed western blots and gels.

Source Data Fig. 6 (download PDF )

Unprocessed western blots.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Mittas, D.M., Riedmayr, L.M., Gavrilov, Z. et al. Dual AAV vectors for efficient delivery of large transgenes. Nat Protoc 21, 1466–1522 (2026). https://doi.org/10.1038/s41596-025-01243-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Version of record:

  • Issue date:

  • DOI: https://doi.org/10.1038/s41596-025-01243-8

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research