Journal article
Identification of host essential factors for recombinant AAV transduction of the polarized human airway epithelium
Journal of virology, Vol.97(12), e0133023
12/21/2023
DOI: 10.1128/jvi.01330-23
PMCID: PMC10734497
PMID: 37966249
Abstract
Recombinant (r)AAV2.5T was selected from the directed evolution of an adeno-associated virus (AAV) capsid library in the human airway epithelium (HAE). The capsid gene of rAAV2.5T is a chimera of the N-terminal unique coding sequence of AAV2 VP1 unique (VP1u) and the VP2- and VP3-coding sequences of AAV5 with a single amino acid mutation of A581T. We conducted two rounds of genome-wide CRISPR guide (g)RNA library screening for host factors limiting rAAV2.5T transduction in HeLa S3 cells. The screening identified several genes that are critical for rAAV2.5T, including the previously reported genes KIAA0319L, TM9SF2, VPS51, and VPS54, as well as a novel gene WDR63. We verified the role of KIAA0319L and WDR63 in rAAV2.5T transduction of polarized HAE by utilizing CRISPR gene knockouts. Although KIAA0319L, a proteinaceous receptor for multiple AAV serotypes, played an essential role in rAAV2.5T transduction of polarized HAE either from the apical or basolateral side, our findings demonstrated that the internalization of rAAV2.5T was independent of KIAA0319L. Importantly, we confirmed that WDR63 is an important player in rAAV2.5T transduction of HAE while not being involved in vector internalization and nuclear entry. Furthermore, we identified that the basal stem cells of HAE can be significantly transduced by rAAV2.5T.
IMPORTANCE The essential steps of successful gene delivery by recombinant adeno-associated viruses (rAAVs) include vector internalization, intracellular trafficking, nuclear import, uncoating, double-stranded (ds)DNA conversion, and transgene expression. rAAV2.5T has a chimeric capsid of AAV2 VP1u and AAV5 VP2 and VP3 with the mutation A581T. Our investigation revealed that KIAA0319L, the multiple AAV serotype receptor, is not essential for vector internalization but remains critical for efficient vector transduction to human airway epithelia. Additionally, we identified that a novel gene WDR63, whose cellular function is not well understood, plays an important role in vector transduction of human airway epithelia but not vector internalization and nuclear entry. Our study also discovered the substantial transduction potential of rAAV2.5T in basal stem cells of human airway epithelia, underscoring its utility in gene editing of human airways. Thus, the knowledge derived from this study holds promise for the advancement of gene therapy in the treatment of pulmonary genetic diseases.
Details
- Title: Subtitle
- Identification of host essential factors for recombinant AAV transduction of the polarized human airway epithelium
- Creators
- Siyuan HaoXiujuan Zhang - University of Kansas Medical CenterKang Ning - University of Kansas Medical CenterZehua FengSoo Yeun Park - University of IowaCagla Aksu KuzShane McFarlin - University of Kansas Medical CenterDonovan Richart - University of Kansas Medical CenterFang Cheng - University of Kansas Medical CenterElizabeth Yan ZhangAaron Zhang-ChenZiying Yan - University of IowaJianming Qiu - University of Kansas Medical Center
- Resource Type
- Journal article
- Publication Details
- Journal of virology, Vol.97(12), e0133023
- DOI
- 10.1128/jvi.01330-23
- PMID
- 37966249
- PMCID
- PMC10734497
- eISSN
- 1098-5514
- Grant note
- DOI: 10.13039/100000060, name: HHS | NIH | National Institute of Allergy and Infectious Diseases, award: AI150877; DOI: 10.13039/100000060, name: HHS | NIH | National Institute of Allergy and Infectious Diseases, award: AI56448; DOI: 10.13039/100000060, name: HHS | NIH | National Institute of Allergy and Infectious Diseases, award: AI56448; DOI: 10.13039/100000060, name: HHS | NIH | National Institute of Allergy and Infectious Diseases, award: AI166293
- Language
- English
- Electronic publication date
- 11/15/2023
- Date published
- 12/21/2023
- Academic Unit
- Anatomy and Cell Biology
- Record Identifier
- 9984512058802771
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