Pre-arrayed Pan-AAV Peptide Display Libraries for Rapid Single-Round Screening

Börner, Kathleen; Kienle, Eike; Huang, Li-Hsin; Weinmann, Jonas; Sacher, Anna; Bayer, Philipp; Stüllein, Christian; Fakhiri, Julia; Zimmermann, Laura; Westhaus, Adrian; Beneke, Jürgen; Beil, Nina; Wiedtke, Ellen; Schmelas, Carolin; Miltner, Dominik; Rau, Alexander; Erfle, Holger; Kräusslich, Hans‐Georg; Müller, Martin; Agbandje‐McKenna, Mavis; Grimm, Dirk

doi:10.1016/j.ymthe.2020.02.009

Cited by 55 publications

(84 citation statements)

References 53 publications

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“…Subsequent quality control by NGS revealed up to 3600-fold differences in the abundance of a specific barcode for individual capsids versus the mean, most evident for peptide-modified AAV6 and AAV12, or capsids with the 9-mer insertion CDCRGDCFC (peptide P2 in Supplementary Fig. 1 and Supplementary Table 1, and as reported 15 ). Considering that the 91 variants in this first library were not titrated prior to pooling and that AAV capsids are widely known to produce with different efficiencies (in particular, AAV6 is hard to scale-up), we were not surprised to observe this variation.…”

Section: Resultsmentioning

confidence: 80%

“…During vector production, each barcode was assigned to a unique AAV capsid from the list of 183 variants in Supplementary Table 1, comprising 12 AAV wild types (AAV1 to AAV9, AAVrh.10, AAVpo.1, AAV12), as well as 94 peptide display mutants and 71 chimeric capsids created through DNA family shuffling 2 . The synthetic capsids have previously been isolated by others or us in specific tissues (e.g., AAV-PHP.B 3 , AAV2-ESGHGYF 4 , AAVM41 5 , AAV-LK03 6 , AAV-DJ 7 , AAV2-BR1 8 , AAV 587 MTP 9 , AAV-Anc80L65 10 , AAV2-7m8 11 , AAV2HBKO 12 , AAV2YF 13 , or AAV6.2 14 ) or have emerged in our recent screens of AAV libraries in cultured cells or in murine liver or muscle, respectively 15 . This includes a set of 12 AAV serotypes that we have previously modified by insertion of over 20 different peptides in exposed capsid loops, and that we have recently studied and characterized extensively in cultured cell lines or primary cells 15 .…”

Section: Resultsmentioning

confidence: 99%

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Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants

et al. 2020

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Adeno-associated virus (AAV) forms the basis for several commercial gene therapy products and for countless gene transfer vectors derived from natural or synthetic viral isolates that are under intense preclinical evaluation. Here, we report a versatile pipeline that enables the direct side-by-side comparison of pre-selected AAV capsids in high-throughput and in the same animal, by combining DNA/RNA barcoding with multiplexed next-generation sequencing. For validation, we create three independent libraries comprising 183 different AAV variants including widely used benchmarks and screened them in all major tissues in adult mice. Thereby, we discover a peptide-displaying AAV9 mutant called AAVMYO that exhibits superior efficiency and specificity in the musculature including skeletal muscle, heart and diaphragm following peripheral delivery, and that holds great potential for muscle gene therapy. Our comprehensive methodology is compatible with any capsids, targets and species, and will thus facilitate and accelerate the stratification of optimal AAV vectors for human gene therapy.

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Section: Resultsmentioning

confidence: 80%

Section: Resultsmentioning

confidence: 99%

Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants

et al. 2020

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“…Plasmid pNNHIV (pNLC4-3 IND64N/D116N tat∆33-64bp) for production of non-infectious, RT-competent HIV-1 particles, was constructed by successive introduction of integrase catalytic mutations D64N and D116N by Quickchange PCR into pUC19 NL4-35'CA-3'Vpr (generated by subcloning a 4296 bp SphI/EcoRI fragment from pNL4-3 into pUC19, from which the NdeI site has been removed by Klenow insertion and religation). Plasmids for AAV production, AAV helper plasmid encoding rep and 1P5 cap gene (Börner et al, 2020), the vector for AAV expression of triple short hairpin RNA (shRNA) targeting three CPSF6 sequences (Bejarano et al, 2019) and vector for expression of a single non-silencing…”

Section: Plasmidsmentioning

confidence: 99%

Cone-shaped HIV-1 capsids are transported through intact nuclear pores

Žíla

Margiotta²,

Turoňová³

et al. 2020

Preprint

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Human immunodeficiency virus (HIV-1) remains a major health threat. Viral capsid uncoating and nuclear import of the viral genome are critical for productive infection. The size of the HIV-1 capsid is generally believed to exceed the diameter of the nuclear pore complex (NPC), indicating that capsid uncoating has to occur prior to nuclear import. Here, we combined correlative light and electron microscopy with subtomogram averaging to capture the structural status of reverse transcription-competent HIV-1 complexes in infected T cells. We demonstrate that the diameter of the NPC in cellulo is sufficient for the import of apparently intact, coneshaped capsids. Subsequent to nuclear import, we detected disrupted and empty capsid fragments, indicating that uncoating of the replication complex occurs by breaking the capsid open, and not by disassembly into individual subunits. Our data directly visualize a key step in HIV-1 replication and enhance our mechanistic understanding of the viral life cycle.

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“…Although targeted delivery can be achieved via viral vectors, they may be more suited for application in cells for ex vivo therapy due to safety concerns [ 80 , 84 ]. An example of AAV-mediated targeted delivery, which are considered the safest of viral vectors, involves differential peptide display on the surface of viral particles which can influence the interaction between the viruses and different cell-types [ 85 ]. By screening a matrix of 12 AAV serotypes and 6 peptides, the team has developed an online superior peptide insertions for improved targeting (SPIRIT) database which contains data required to target different cell-types [ 85 ].…”

Section: Non-viral Targeted Delivery Strategies For the Crispr-casmentioning

confidence: 99%

“…An example of AAV-mediated targeted delivery, which are considered the safest of viral vectors, involves differential peptide display on the surface of viral particles which can influence the interaction between the viruses and different cell-types [ 85 ]. By screening a matrix of 12 AAV serotypes and 6 peptides, the team has developed an online superior peptide insertions for improved targeting (SPIRIT) database which contains data required to target different cell-types [ 85 ]. However, AAVs have a limited packaging size, and may lead to prolong expression of the delivered CRISPR-encoding plasmids thereby increasing off-target effects [ 80 ].…”

Section: Non-viral Targeted Delivery Strategies For the Crispr-casmentioning

confidence: 99%

Tissue-Specific Delivery of CRISPR Therapeutics: Strategies and Mechanisms of Non-Viral Vectors

Shalaby

Aouida

El‐Agnaf

2020

IJMS

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The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) genome editing system has been the focus of intense research in the last decade due to its superior ability to desirably target and edit DNA sequences. The applicability of the CRISPR-Cas system to in vivo genome editing has acquired substantial credit for a future in vivo gene-based therapeutic. Challenges such as targeting the wrong tissue, undesirable genetic mutations, or immunogenic responses, need to be tackled before CRISPR-Cas systems can be translated for clinical use. Hence, there is an evident gap in the field for a strategy to enhance the specificity of delivery of CRISPR-Cas gene editing systems for in vivo applications. Current approaches using viral vectors do not address these main challenges and, therefore, strategies to develop non-viral delivery systems are being explored. Peptide-based systems represent an attractive approach to developing gene-based therapeutics due to their specificity of targeting, scale-up potential, lack of an immunogenic response and resistance to proteolysis. In this review, we discuss the most recent efforts towards novel non-viral delivery systems, focusing on strategies and mechanisms of peptide-based delivery systems, that can specifically deliver CRISPR components to different cell types for therapeutic and research purposes.

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Pre-arrayed Pan-AAV Peptide Display Libraries for Rapid Single-Round Screening

Cited by 55 publications

References 53 publications

Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants

Identification of a myotropic AAV by massively parallel in vivo evaluation of barcoded capsid variants

Cone-shaped HIV-1 capsids are transported through intact nuclear pores

Tissue-Specific Delivery of CRISPR Therapeutics: Strategies and Mechanisms of Non-Viral Vectors

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