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Cited by 260Virus-based gene therapy researchRNA Interference and Gene DeliveryParvovirus B19 Infection Studies

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Capsid Modifications for Targeting and Improving the Efficacy of AAV Vectors

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Büning, Hildegard, Srivastava, Arun·2019·Molecular Therapy - Methods & Clinical Development
DOI 10.1016/j.omtm.2019.01.008PMC PMC6378346
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Abstract

In the past decade, recombinant vectors based on a non-pathogenic parvovirus, the adeno-associated virus (AAV), have taken center stage as a gene delivery vehicle for the potential gene therapy for a number of human diseases. To date, the safety of AAV vectors in 176 phase I, II, and III clinical trials and their efficacy in at least eight human diseases are now firmly documented. Despite these remarkable achievements, it has also become abundantly clear that the full potential of first generation AAV vectors composed of naturally occurring capsids is not likely to be realized, since the wild-type AAV did not evolve for the purpose of therapeutic gene delivery. In this article, we provide a brief historical account of the progress that has been made in the development of capsid-modified , next-generation AAV vectors to ensure both the safety and efficacy of these vectors in targeting a wide variety of human diseases.

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Figure 1 AAV Serotype 2 Vector Genome Conformation The AAV viral genome is flanked by inverted terminal repeats (ITRs), which serve as packaging signal and origin of replication. The rep gene encodes a family of multifunctional proteins (Rep proteins) responsible for controlling viral transcription, replication, packaging, and integration in AAVS1. For AAV2, four Rep proteins are described. Expression of Rep78 and Rep68 is controlled by the AAV2-specific p5 promoter, while p19 controls expression of the smaller Rep proteins (Rep52 and Rep40). Rep68 and Rep40 are splice variants of Rep78 and Rep52, respectively. Numbers indicate the molecular weight. Expression of AAP and the viral capsid proteins VP1 (90 kDa), VP2 (72 kDa), and VP3 (60 kDa), all encoded in the cap gene, is controlled by the p40 promoter. The X gene is located at the 3′ end of the genome within a region shared with the cap gene and possesses its own promoter (p81). While the X protein seems to enhance viral replication, AAP is essential for capsid assembly. The three different VPs contribute in a 1 (VP1):1 (VP2):10 (VP3) ratio to the icosahedral AAV2 capsid.
Figure 1 AAV Serotype 2 Vector Genome Conformation The AAV viral genome is flanked by inverted terminal repeats (ITRs), which serve as packaging signal and origin of replication. The rep gene encodes a family of multifunctional proteins (Rep proteins) responsible for controlling viral transcription, replication, packaging, and integration in AAVS1. For AAV2, four Rep proteins are described. Expression of Rep78 and Rep68 is controlled by the AAV2-specific p5 promoter, while p19 controls expression of the smaller Rep proteins (Rep52 and Rep40). Rep68 and Rep40 are splice variants of Rep78 and Rep52, respectively. Numbers indicate the molecular weight. Expression of AAP and the viral capsid proteins VP1 (90 kDa), VP2 (72 kDa), and VP3 (60 kDa), all encoded in the cap gene, is controlled by the p40 promoter. The X gene is located at the 3′ end of the genome within a region shared with the cap gene and possesses its own promoter (p81). While the X protein seems to enhance viral replication, AAP is essential for capsid assembly. The three different VPs contribute in a 1 (VP1):1 (VP2):10 (VP3) ratio to the icosahedral AAV2 capsid.CC BY-NC-ND · PubMed Central
Figure 2 Capsid Engineering for Re-targeting or Expansion of AAV2 Vector Tropism The N terminus of VP2 accepts large-peptide insertions as well as proteins. When combined with natural receptor-binding motif knock-out mutations, insertion of targeting ligands at VP2 re-targets vector tropism; otherwise, vector tropism is expanded. The N′ terminus of VP2 can also be used to label AAV vector particles for ex vivo and in vivo infection biology analyses. For that purpose, EGFP and luciferase were used as fusion partners, while the remaining capsid was left unmodified. Frequently used sites for insertions of targeting ligands within the common VP3 regions are I-453, I-587, and I-588 (not shown). In case of peptide insertions at I-587, R585 and R588 are separated, thereby destroying the natural HSPG binding ability of AAV2. Insertion at I-453 requires simultaneous mutations at R585 and R588 for re-targeting and efficient presentation of targeting ligands. ITR, inverted terminal repeat; REP, rep gene; CAP, cap gene; VP1, VP2, VP3, viral capsid proteins; R, arginine residue. Numbers represent amino acid position according to VP1 numbering.
Figure 2 Capsid Engineering for Re-targeting or Expansion of AAV2 Vector Tropism The N terminus of VP2 accepts large-peptide insertions as well as proteins. When combined with natural receptor-binding motif knock-out mutations, insertion of targeting ligands at VP2 re-targets vector tropism; otherwise, vector tropism is expanded. The N′ terminus of VP2 can also be used to label AAV vector particles for ex vivo and in vivo infection biology analyses. For that purpose, EGFP and luciferase were used as fusion partners, while the remaining capsid was left unmodified. Frequently used sites for insertions of targeting ligands within the common VP3 regions are I-453, I-587, and I-588 (not shown). In case of peptide insertions at I-587, R585 and R588 are separated, thereby destroying the natural HSPG binding ability of AAV2. Insertion at I-453 requires simultaneous mutations at R585 and R588 for re-targeting and efficient presentation of targeting ligands. ITR, inverted terminal repeat; REP, rep gene; CAP, cap gene; VP1, VP2, VP3, viral capsid proteins; R, arginine residue. Numbers represent amino acid position according to VP1 numbering.CC BY-NC-ND · PubMed Central
Figure 3 AAV Peptide Display Library Concept Schematic representation of AAV peptide display library production and selection. A pool of oligonucleotides with random sequence is inserted into the cap gene, for example for presentation of peptides with random sequence at I-587. Thereby, a plasmid pool is generated that is used to package the AAV peptide display library. Subsequently, the library is used for high-throughput screenings in cell culture or in vivo (not shown). Commonly, multiple rounds of selections are done. Sublibraries either originate from viral library progeny produced by helper virus co-infection (helper virus co-infection approach) or from viral library genomes isolated from target cells or target tissue. The genomes are amplified by PCR, cloned as a sublibrary plasmid pool, and then used for sublibrary production (PCR-based amplification approach). wt, wild-type.
Figure 3 AAV Peptide Display Library Concept Schematic representation of AAV peptide display library production and selection. A pool of oligonucleotides with random sequence is inserted into the cap gene, for example for presentation of peptides with random sequence at I-587. Thereby, a plasmid pool is generated that is used to package the AAV peptide display library. Subsequently, the library is used for high-throughput screenings in cell culture or in vivo (not shown). Commonly, multiple rounds of selections are done. Sublibraries either originate from viral library progeny produced by helper virus co-infection (helper virus co-infection approach) or from viral library genomes isolated from target cells or target tissue. The genomes are amplified by PCR, cloned as a sublibrary plasmid pool, and then used for sublibrary production (PCR-based amplification approach). wt, wild-type.CC BY-NC-ND · PubMed Central
Figure 4 The Life Cycle of AAV Vectors and the Basis of the Development of Tyrosine-, Serine/Threonine-, and Lysine-Modified Next Generation of Recombinant AAV Vectors (A) Following AAV2 binding to its primary cell surface receptor, heparan sulfate proteoglycan (HSPG), the vector interacts with human fibroblast growth factor receptor 1 (FGFR1) as one of the co-receptors to gain entry into cells via a Clathrin-coated pit, traffics through early and late endosomes and becomes phosphorylated by epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK). Phosphorylation of the capsid is a signal for ubiquitination, following which a large fraction of AAV vectors is degraded by the cellular proteasome machinery, leading to impaired intracellular trafficking and inefficient nuclear entry of AAV2 vectors. Inhibitors of EGFR-PTK prevent phosphorylation of the capsid, bypassing the ubiquitination- and proteasome-mediated degradation pathway and thereby improving intracellular trafficking and nuclear transport of AAV2 vectors. (B) In addition to surface-exposed Y residues, surface-exposed specific serine (S) and threonine (T) residues on AAV capsids can also be phosphorylated, which is a signal for ubiquitination. Surface-exposed specific lysine (K) residues on AAV capsids can be ubiquitinated and subsequently degraded by the host cell proteasome machinery. Site-directed mutagenesis of these residues led to the generation of AAV2 vectors, which were more efficient.
Figure 4 The Life Cycle of AAV Vectors and the Basis of the Development of Tyrosine-, Serine/Threonine-, and Lysine-Modified Next Generation of Recombinant AAV Vectors (A) Following AAV2 binding to its primary cell surface receptor, heparan sulfate proteoglycan (HSPG), the vector interacts with human fibroblast growth factor receptor 1 (FGFR1) as one of the co-receptors to gain entry into cells via a Clathrin-coated pit, traffics through early and late endosomes and becomes phosphorylated by epidermal growth factor receptor protein tyrosine kinase (EGFR-PTK). Phosphorylation of the capsid is a signal for ubiquitination, following which a large fraction of AAV vectors is degraded by the cellular proteasome machinery, leading to impaired intracellular trafficking and inefficient nuclear entry of AAV2 vectors. Inhibitors of EGFR-PTK prevent phosphorylation of the capsid, bypassing the ubiquitination- and proteasome-mediated degradation pathway and thereby improving intracellular trafficking and nuclear transport of AAV2 vectors. (B) In addition to surface-exposed Y residues, surface-exposed specific serine (S) and threonine (T) residues on AAV capsids can also be phosphorylated, which is a signal for ubiquitination. Surface-exposed specific lysine (K) residues on AAV capsids can be ubiquitinated and subsequently degraded by the host cell proteasome machinery. Site-directed mutagenesis of these residues led to the generation of AAV2 vectors, which were more efficient.CC BY-NC-ND · PubMed Central
Figure 5 Schematic Representation of the Most Efficient Capsid-Modified Next Generation of Recombinant AAV Serotype Vectors (A) Various surface-exposed specific Y, S, T, and K residues in AAV2, AAV3, and AAV6 capsids are denoted, the site-directed mutagenesis of which has led to the generation of Y, S, T, and K mutant AAV2, AAV3, and AAV6 vectors. (B) Specific examples of the most efficient AAV2,99 AAV3,104 and AAV6107 serotype vectors containing various permutations and combinations of the Y, S, and T mutations generated thus far, are depicted.
Figure 5 Schematic Representation of the Most Efficient Capsid-Modified Next Generation of Recombinant AAV Serotype Vectors (A) Various surface-exposed specific Y, S, T, and K residues in AAV2, AAV3, and AAV6 capsids are denoted, the site-directed mutagenesis of which has led to the generation of Y, S, T, and K mutant AAV2, AAV3, and AAV6 vectors. (B) Specific examples of the most efficient AAV2,99 AAV3,104 and AAV6107 serotype vectors containing various permutations and combinations of the Y, S, and T mutations generated thus far, are depicted.CC BY-NC-ND · PubMed Central

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Metadata

Authors
Büning, H., & Srivastava, A.
Affiliations
  • Medizinische Hochschule Hannover (DE)
  • University of Florida Health (US)
Corresponding author
  • Büning, Hildegard
  • Srivastava, Arun
ORCID
Venue
Molecular Therapy - Methods & Clinical Development
Publisher
Elsevier BV
Published
2019-03
Volume
12
Pages
248-265
ISSN
2329-0501 (ISSN-L)
DOI
10.1016/j.omtm.2019.01.008
PMC
PMC6378346
OpenAlex
W2912684492
Cited by
260
References
147
Citations by year
2026: 28 · 2025: 34 · 2024: 38 · 2023: 40 · 2022: 31 · 2021: 32 · 2020: 48 · 2019: 9
FWCI
3.98
Type
journal article
Language
en
Open access
Licence
Locations
  • Molecular Therapy — Methods & Clinical Development (publishedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-nd)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Kitzman Foundation
  • Children's Miracle Network Hospitals
  • Deutsche Forschungsgemeinschaft
  • Bundesministerium für Bildung und Forschung
  • Ministerium für Wissenschaft, Forschung und Kunst Baden-Württemberg
  • National Institutes of Health (R01 HL-097088, R21 EB-015684, R41 AI-122735)
  • Niedersächsisches Ministerium für Wissenschaft und Kultur
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Virus-based gene therapy research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
  • Health Sciences › Medicine › Infectious Diseases › Parvovirus B19 Infection Studies
Keywords
adeno-associated virus vectors, capsid modification, gene therapy vectors, gene delivery, vector targeting, vector safety, parvovirus

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