Boosters for adeno-associated virus (AAV) vector (r)evolution

  • Joanna Szumska
    Department of Infectious Diseases/Virology, Section Viral Vector Technologies, Medical Faculty, University of Heidelberg, Heidelberg, Germany

    BioQuant Center and Center for Integrative Infectious Diseases Research (CIID), University of Heidelberg, Heidelberg, Germany
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  • Dirk Grimm
    Correspondence: Dirk Grimm, University of Heidelberg, BioQuant BQ0030, Im Neuenheimer Feld 267, 69120 Heidelberg, Germany. E-mail address: (D. Grimm).
    Department of Infectious Diseases/Virology, Section Viral Vector Technologies, Medical Faculty, University of Heidelberg, Heidelberg, Germany

    BioQuant Center and Center for Integrative Infectious Diseases Research (CIID), University of Heidelberg, Heidelberg, Germany

    German Center for Infection Research (Deutsches Zentrum für Infektionsforschung, DZIF) and German Center for Cardiovascular Research (Deutsches Zentrum für Herz-Kreislauf-Erkrankungen, DZHK), partner site Heidelberg, Heidelberg, Germany
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Published:August 20, 2022DOI:


      Adeno-associated virus (AAV) is one of the most exciting and most versatile templates for engineering of gene-delivery vectors for use in human gene therapy, owing to the existence of numerous naturally occurring capsid variants and their amenability to directed molecular evolution. As a result, the field has witnessed an explosion of novel “designer” AAV capsids and ensuing vectors over the last two decades, which have been isolated from comprehensive capsid libraries generated through technologies such as DNA shuffling or peptide display, and stratified under stringent positive and/or negative selection pressures. Here, we briefly highlight a panel of recent, innovative and transformative methodologies that we consider to have exceptional potential to advance directed AAV capsid evolution and to thereby accelerate AAV vector revolution. These avenues comprise original technologies for (i) barcoding and high-throughput screening of individual AAV variants or entire capsid libraries, (ii) selection of transduction-competent AAV vectors on the DNA level, (iii) enrichment of expression-competent AAV variants on the RNA level, as well as (iv) high-resolution stratification of focused AAV capsid libraries on the single-cell level. Together with other emerging AAV engineering stratagems, such as rational design or machine learning, these pioneering techniques promise to provide an urgently needed booster for AAV (r)evolution.

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        • Sonntag F
        • Schmidt K
        • Kleinschmidt JA.
        A viral assembly factor promotes AAV2 capsid formation in the nucleolus.
        Proc Natl Acad Sci U S A. 2010; 107: 10220-10225
        • Tse LV
        • Moller-Tank S
        • Meganck RM
        • Asokan A.
        Mapping and engineering functional domains of the assembly-activating protein of adeno-associated viruses.
        J Virol. 2018; 92
        • Ogden PJ
        • Kelsic ED
        • Sinai S
        • Church GM.
        Comprehensive AAV capsid fitness landscape reveals a viral gene and enables machine-guided design.
        Science. 2019; 366: 1139-1143
        • Grimm D
        • Lee JS
        • Wang L
        • Desai T
        • Akache B
        • Storm TA
        • et al.
        In vitro and in vivo gene therapy vector evolution via multispecies interbreeding and retargeting of adeno-associated viruses.
        J Virol. 2008; 82: 5887-5911
        • Li W
        • Asokan A
        • Wu Z
        • Van Dyke T
        • DiPrimio N
        • Johnson JS
        • et al.
        Engineering and selection of shuffled AAV genomes: A new strategy for producing targeted biological nanoparticles.
        Mol Ther. 2008; 16: 1252-1260
        • Koerber JT
        • Jang JH
        • Schaffer DV.
        DNA shuffling of adeno-associated virus yields functionally diverse viral progeny.
        Mol Ther. 2008; 16: 1703-1709
        • Muller OJ
        • Kaul F
        • Weitzman MD
        • Pasqualini R
        • Arap W
        • Kleinschmidt JA
        • et al.
        Random peptide libraries displayed on adeno-associated virus to select for targeted gene therapy vectors.
        Nat Biotechnol. 2003; 21: 1040-1046
        • Perabo L
        • Buning H
        • Kofler DM
        • Ried MU
        • Girod A
        • Wendtner CM
        • et al.
        In vitro selection of viral vectors with modified tropism: The adeno-associated virus display.
        Mol Ther. 2003; 8: 151-157
        • Zinn E
        • Pacouret S
        • Khaychuk V
        • Turunen HT
        • Carvalho LS
        • Andres-Mateos E
        • et al.
        In silico reconstruction of the viral evolutionary lineage yields a potent gene therapy vector.
        Cell Rep. 2015; 12: 1056-1068
        • Grimm D
        • Zolotukhin S
        E Pluribus Unum: 50 years of research, millions of viruses, and one goal-tailored acceleration of AAV evolution.
        Mol Ther. 2015; 23: 1819-1831
        • Li C
        • Samulski RJ.
        Engineering adeno-associated virus vectors for gene therapy.
        Nat Rev Genet. 2020; 21: 255-272
        • Wang D
        • Tai PWL
        • Gao G.
        Adeno-associated virus vector as a platform for gene therapy delivery.
        Nat Rev Drug Disc. 2019; 18: 358-378
        • Weinmann J
        • Grimm D.
        Next-generation AAV vectors for clinical use: An ever-accelerating race.
        Virus Genes. 2017; 53: 707-713
        • Buning H
        • Srivastava A.
        capsid modifications for targeting and improving the efficacy of AAV vectors.
        Mol Ther Methods Clin Dev. 2019; 12: 248-265
        • Zolotukhin S
        • Vandenberghe LH.
        AAV capsid design: A Goldilocks challenge.
        Trends Mol Med. 2022; 28: 183-193
        • Adachi K
        • Enoki T
        • Kawano Y
        • Veraz M
        • Nakai H.
        Drawing a high-resolution functional map of adeno-associated virus capsid by massively parallel sequencing.
        Nat Commun. 2014; 5: 1-14
        • Marsic D
        • Méndez-Gómez HR
        • Zolotukhin S.
        High-accuracy biodistribution analysis of adeno-associated virus variants by double barcode sequencing.
        Mol Ther Methods Clin Dev. 2015; 2: 15041
        • Weinmann J
        • Weis S
        • Sippel J
        • Tulalamba W
        • Remes A
        • El Andari J
        • et al.
        Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants.
        Nat Commun. 2020; 11: 1-12
        • Kremer LPM
        • Cerrizuela S
        • Dehler S
        • Stiehl T
        • Weinmann J
        • Abendroth H
        • et al.
        High throughput screening of novel AAV capsids identifies variants for transduction of adult NSCs within the subventricular zone.
        Mol Ther Methods Clin Dev. 2021; 23: 33-50
        • Kondratov O
        • Kondratova L
        • Mandel RJ
        • Coleman K
        • Savage MA
        • Gray-Edwards HL
        • et al.
        A comprehensive study of a 29-capsid AAV library in a non-human primate central nervous system.
        Mol Ther. 2021; 29: 2806-2820
        • Westhaus A
        • Cabanes-Creus M
        • Rybicki A
        • Baltazar G
        • Navarro RG
        • Zhu E
        • et al.
        High-throughput in vitro, ex vivo, and in vivo screen of adeno-associated virus vectors based on physical and functional transduction.
        Hum Gene Ther. 2020; 31: 575-589
        • Xu M
        • Li J
        • Xie J
        • He R
        • Su Q
        • Gao G
        • et al.
        High-throughput quantification of in vivo adeno-associated virus transduction with barcoded non-coding RNAs.
        Hum Gene Ther. 2019; 30: 946-956
        • Pekrun K
        • De Alencastro G
        • Luo QJ
        • Liu J
        • Kim Y
        • Nygaard S
        • et al.
        Using a barcoded AAV capsid library to select for clinically relevant gene therapy vectors.
        JCI Insight. 2019; 4
        • Herrmann AK
        • Bender C
        • Kienle E
        • Grosse S
        • El Andari J
        • Botta J
        • et al.
        A robust and all-inclusive pipeline for shuffling of adeno-associated viruses.
        ACS Synth Bio. 2019; 8: 194-206
        • de Alencastro G
        • Pekrun K
        • Valdmanis P
        • Tiffany M
        • Xu J
        • Kay MA.
        Tracking Adeno-Associated Virus Capsid Evolution by High-Throughput Sequencing.
        Hum Gene Ther. 2020; 31: 553-564
        • Davidsson M
        • Wang G
        • Aldrin-Kirk P
        • Cardoso T
        • Nolbrant S
        • Hartnor M
        • et al.
        A systematic capsid evolution approach performed in vivo for the design of AAV vectors with tailored properties and tropism.
        Proc Natl Acad Sci U S A. 2019; 116: 27053-27062
        • Deverman BE
        • Pravdo PL
        • Simpson BP
        • Kumar SR
        • Chan KY
        • Banerjee A
        • et al.
        Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain.
        Nat Biotechnol. 2016; 34: 204-209
        • Ravindra Kumar S
        • Miles TF
        • Chen X
        • Brown D
        • Dobreva T
        • Huang Q
        • et al.
        Multiplexed Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types.
        Nat Methods. 2020; 17: 541-550
        • Ojala DS
        • Sun S
        • Santiago-Ortiz JL
        • Shapiro MG
        • Romero PA
        • Schaffer DV.
        In vivo selection of a computationally designed schema aav library yields a novel variant for infection of adult neural stem cells in the SVZ.
        Mol Ther. 2018; 26: 304-319
        • Hanlon KS
        • Meltzer JC
        • Buzhdygan T
        • Cheng MJ
        • Sena-Esteves M
        • Bennett RE
        • et al.
        Selection of an efficient AAV vector for robust CNS transgene expression.
        Mol Ther Methods Clin Dev. 2019; 15: 320-332
        • Beharry A
        • Gong Y
        • Kim JC
        • Hanlon KS
        • Nammour J
        • Hieber K
        • et al.
        The AAV9 variant capsid AAV-F mediates widespread transgene expression in nonhuman primate spinal cord after intrathecal administration.
        Hum Gene Ther. 2022; 33: 61-75
        • Nonnenmacher M
        • Wang W
        • Child MA
        • Ren XQ
        • Huang C
        • Ren AZ
        • et al.
        Rapid evolution of blood-brain-barrier-penetrating AAV capsids by RNA-driven biopanning.
        Mol Ther Methods Clin Dev. 2021; 20: 366-378
        • Tabebordbar M
        • Lagerborg KA
        • Stanton A
        • King EM
        • Ye S
        • Tellez L
        • et al.
        Directed evolution of a family of AAV capsid variants enabling potent muscle-directed gene delivery across species.
        Cell. 2021; 184 (e22): 4919-4938
        • Bauer A
        • Puglisi M
        • Nagl D
        • Schick JA
        • Werner T
        • Klingl A
        • et al.
        Molecular signature of astrocytes for gene delivery by the synthetic adeno-associated viral vector rAAV9P1.
        Adv Sci (Weinh). 2022; 9e2104979
        • Westhaus A
        • Cabanes Creus M
        • Jonker T
        • Sallard E
        • Navarro RG
        • Zhu E
        • et al.
        AAV-p40 bioengineering platform for variant selection based on transgene expression.
        Hum Gene Ther. 2022;
        • Brown D
        • Altermatt M
        • Dobreva T
        • Chen S
        • Wang A
        • Thomson M
        • et al.
        Deep parallel characterization of AAV tropism and AAV-mediated transcriptional changes via single-cell RNA sequencing.
        Front Immunol. 2021; 12730825
        • Öztürk BE
        • Johnson ME
        • Kleyman M
        • Turunc S
        • He J
        • Jabalameli S
        • et al.
        scAAVengr, a transcriptome-based pipeline for quantitative ranking of engineered AAVs with single-cell resolution.
        Elife. 2021; 10
        • Bryant DH
        • Bashir A
        • Sinai S
        • Jain NK
        • Ogden PJ
        • Riley PF
        • et al.
        Deep diversification of an AAV capsid protein by machine learning.
        Nat Biotechnol. 2021; 39: 691-696
        • Marques AD
        • Kummer M
        • Kondratov O
        • Banerjee A
        • Moskalenko O
        • Zolotukhin S.
        Applying machine learning to predict viral assembly for adeno-associated virus capsid libraries.
        Mol Ther Methods Clin Dev. 2021; 20: 276-286