We developed a reversible safe switch for CAR T therapy based on the tet-inducible CMV promoter and U24 protein of
Human Herpesvirus 6. To our knowledge, tet-inducible transcription system is a widely-used inducible expression system in mammalian
cell lines, yet few available part is in the registry, left alone limited information for those part. Aiming to benefit the iGEM
community, we standardized both the tet-ON promoter and the coding region of reverse tet-responsive transactivator in our project by
cloning the sequences into the standard plasmid backbone pSB1C3, and submitted them to the iGEM registry. To ensure the parts’
quality, we sequenced the part using VF2 as primer before submitting the plasmids. Hopefully, teams in the future that are ambitious
to conduct projects requiring inducible expression will find our parts useful.
We developed a reversible safe switch for CAR T therapy based on the tet-inducible CMV promoter and U24 protein of Human Herpesvirus 6. Since it is the major part of our project, we conducted extensive research on U24 and obtained valuable results. Yet we believed we did not discover the full potential of this protein. By submitting to the registry, we aim to add a new tool to the toolbox of T cells functions regulation. We are confident that it will benefit teams in the future ambitious to conduct T cell- related project.
U24 is a small (87aa) tail-anchored protein (Sullivan and Coscoy, 2010) that can downregulate
TCR/CD3 complex from the cell surface by exclusion of CD3 from Rab11-containing recycling endosomes and thus inhibiting TCR complex
recycling back to the surface (Sullivan and Coscoy, 2008) . While it was demonstrated later
that U24 also downregulate transferrin (Sullivan and Coscoy, 2010) , its action is relatively
specific, without affecting the surface level of ICAM-1, MHC class I, ULBP1, ULBP2, CD4 and CD8
(Sullivan and Coscoy, 2008) . Since U24 does not colocalized with CD3, it is believed that the downregulation does not rely on
the interaction of U24 with CD3 but instead results from interaction of U24 and the endosomal recycling machinery
(Sullivan and Coscoy, 2010) . In addition, unlike proteins from other herpesviruses that
downregulate TCRs or B cell receptors (BCRs), U24 does not activate lymphocyte signaling pathways
(Sullivan and Coscoy, 2008) . Furthermore, it was demonstrated that U24 can impair T cell activation by antigen presenting
cells (Sullivan and Coscoy, 2008) .
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This year, SYSU-CHINA developed a reversible safe switch for CAR T therapy based on the tet-inducible CMV promoter and U24 protein of
Human Herpesvirus 6. To our knowledge, tet-inducible transcription system is a widely-used inducible expression system in mammalian
cell lines, yet few available part is in the registry, left alone limited information for those part. Aiming to benefit the iGEM
community, we standardized both the tet-ON promoter and the coding region of reverse tet-responsive transactivator in our project by
cloning the sequences into the standard plasmid backbone pSB1C3, and submitted them to the iGEM registry. To ensure the parts'
quality, we sequenced the part using VF2 as primer before submitting the plasmids. In addition, we provided first-hand data on the
tet-ON promoter and rtTA. Hopefully, teams in the future that are ambitious to conduct projects requiring inducible expression will
find our parts useful.
Tet-ON promoter is the promoter sequence in the Tet-inducible transcription system (tet-ON system).
Tet-ON system(Gossen et al., 1995) is a inducible transcription system widely used in mammalian
cells. The tet-ON system utilizes the sequence-specific DNA binding property of tet repressor protein (tetR) from Escherichia coli in
the presence of tet or dox, and consists of two parts: The Tet-inducible CMV promoter (tet-ON promoter) and reverse tetracycline-
controlled transactivator (rtTA). The tet-ON promoter consists of tandem tetracycline-responsive elements (TRE) followed by a minimal
CMV promoter. The rtTA protein comprises of reverse-tetR (rtetR, mutant of tetR) and activation domains from herpes simplex virus
VP16. When tet or dox is added, the rtTA binds to TRE, and VP16 domain will recruit factors of RNA polymerase II to initiate
transcription. In the absence of tet or dox, the rtTA detaches from tetON promoter, and thus no transcription.
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rtTA is the transactivator in the Tet-inducible transcription system (tet-ON system) that selectively binds to tet-ON promoter in the
presence of tetracycline or doxycycline and activate transcription. By submitting this to the registry, we aims to benefit the
community that teams in the future can establish their own stable cell lines for for tet-inducible expression, or integrate it into
vectors containing tet-ON promoter (like we did!) for tet-inducible expression of gene of interest.
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