@article{604f3c7b07814067a928e81e0ed067e8,
title = "AAV vectors displaying bispecific DARPins enable dual-control targeted gene delivery",
abstract = "Precise delivery of genes to therapy-relevant cells is crucial for in vivo gene therapy. Receptor-targeting as prime strategy for this purpose is limited to cell types defined by a single cell-surface marker. Many target cells are characterized by combinations of more than one marker, such as the HIV reservoir cells. Here, we explored the tropism of adeno-associated viral vectors (AAV2) displaying designed ankyrin repeat proteins (DARPins) mono- and bispecific for CD4 and CD32a. Cryo-electron tomography revealed an unaltered capsid structure in the presence of DARPins. Surprisingly, bispecific AAVs transduced CD4/CD32a double-positive cells at much higher efficiencies than single-positive cells, even if present in low amounts in cell mixtures or human blood. This preference was confirmed when vector particles were systemically administered into mice. Cell trafficking studies revealed an increased cell entry rate for bispecific over monospecific AAVs. When equipped with an HIV genome-targeting CRISPR/Cas cassette, the vectors prevented HIV replication in T cell cultures. The data provide proof-of-concept for high-precision gene delivery through tandem-binding regions on AAV. Reminiscent of biological products following Boolean logic AND gating, the data suggest a new option for receptor-targeted vectors to improve the specificity and safety of in vivo gene therapy.",
keywords = "CRISPR-Cas, DART-AAV, Designed ankyrin repeat protein, FcγRIIA, HIV reservoir, Receptor-targeting",
author = "Theuerkauf, {Samuel A} and Elena Herrera-Carrillo and Fabian John and Zinser, {Luca J} and Molina, {Mariano A} and Vanessa Riechert and Thalheimer, {Frederic B} and Kathleen B{\"o}rner and Dirk Grimm and Petr Chlanda and Ben Berkhout and Buchholz, {Christian J}",
note = "Funding Information: This work was supported by grant 1R01AI145045-01 from the National Institutes of Health (NIH) to B.B., E.H. and C.J.B. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health . F.B.T. and C.J.B. received funding by the State of Hessen within the LOEWE program. D.G. and C.J.B. are funded by the BMBF project COMMUTE ( 16GW0339 ). P.C. received funding from the Chica and Heinz Schaller Foundation and acknowledges support from the German Research Foundation ( DFG ) project number 437060729 . P.C. and D.G. acknowledge support by the DFG SFB1129 (project number 240245660 ). K.B. and D.G. are grateful for support from the German Center for Infection Research (DZIF, BMBF; TTU-HIV 04.819 ). Funding Information: The authors acknowledge Gundula Braun and Manuela Gallet (Paul-Ehrlich-Institut) for their excellent technical assistance in AAV vector generation and quantification of VCNs, as well as Regina Eberle (Paul-Ehrlich-Institut) for technical assistance at the electron microscope. The authors gratefully access to the infrastructure and support provided by the Cryo-EM Network at the Heidelberg University (HD-cryoNet) and the data storage service SDS@hd supported by the Ministry of Science, Research and the Arts Baden-W{\"u}rttemberg (MWK) and the German Research Foundation ( DFG ) through grant INST 35/1314-1 FUGG and INST 35/1503-1 FUGG . Figures in the manuscript were partially created with BioRender.com . Publisher Copyright: {\textcopyright} 2023 The Authors",
year = "2023",
month = dec,
doi = "https://doi.org/10.1016/j.biomaterials.2023.122399",
language = "English",
volume = "303",
pages = "122399",
journal = "Biomaterials",
issn = "0142-9612",
publisher = "Elsevier BV",
}