Engineering Non-cytotoxic Delivery of Proteins by T cells via Fusion to NPC2

dc.contributor.committeeMemberVaradarajan, Navin
dc.contributor.committeeMemberWillson, Richard C.
dc.contributor.committeeMemberOrman, Mehmet A.
dc.contributor.committeeMemberPeng, Weiyi
dc.contributor.committeeMemberMerchant, Fatima Aziz
dc.creatorSaeedi, Arash 2022
dc.description.abstractEngineering cellular therapeutics by programming T cells has great potential in immunology. The primary mechanism employed by T cells for the specific transfer of proteins at the immunological synapse is via the lysosomal perforin pathway that facilitates the transfer of cytotoxic granzymes leading to apoptosis in target cells. Facilitating the delivery of non-cytotoxic proteins through perforin oligomers will dramatically expand the range of protein cargos that T cells can traffic to the target cells. Here, we have identified the intralysosomal protein, NPC2, as a chaperone that can facilitate the delivery of T-cell derived reporter proteins through perforin pores at the immunological synapse. Structural and biophysical considerations suggested that NPC2 could traverse through perforin pores and in vitro experiments confirmed the transport of purified NPC2 through perforin pores on cell membranes. To characterize the ability of NPC2 to facilitate the transfer of payloads in T cells, we constructed NPC2-mCherry fusion proteins in T cells. Using confocal microscopy and flow cytometry, we confirmed the colocalization of the NPC2 fused protein with lytic granules and the transfer of the fluorescent protein payload from T cells to target cells in co-culture experiments. The NPC2 fusion enabled the localization of mCherry to secretory lysosomes in mouse TCR CD8+ T cells and human CD4+ and CD8+ chimeric antigen receptor (CAR) T cells. Finally, we introduce a novel method for expression of NPC2 fused toxins within CAR T cells for treating immune resistant tumor cells. These results illustrate that by using NPC2 as a molecular chaperone, the NPC2-perforin pathway can be exploited as a programmable molecular delivery system for cell-based therapies.
dc.description.departmentChemical and Biomolecular Engineering, William A. Brookshire Department of
dc.format.digitalOriginborn digital
dc.rightsThe author of this work is the copyright owner. UH Libraries and the Texas Digital Library have their permission to store and provide access to this work. Further transmission, reproduction, or presentation of this work is prohibited except with permission of the author(s).
dc.subjectLive-cell delivery
dc.subjectProtein therapeutics
dc.subjectPerforin/granzyme pathway
dc.titleEngineering Non-cytotoxic Delivery of Proteins by T cells via Fusion to NPC2
dcterms.accessRightsThe full text of this item is not available at this time because the student has placed this item under an embargo for a period of time. The Libraries are not authorized to provide a copy of this work during the embargo period.
local.embargo.terms2024-12-01 College of Engineering and Biomolecular Engineering, William A. Brookshire Department of Engineering of Houston of Philosophy


License bundle

Now showing 1 - 2 of 2
No Thumbnail Available
4.43 KB
Plain Text
No Thumbnail Available
1.81 KB
Plain Text