TY  - JOUR
AB  - Genome engineering is a powerful tool for in vitro research and the creation of novel model organisms and has growing clinical applications. Randomly integrating vectors, such as lentivirus- or transposase-based methods, are simple and easy to use but carry risks arising from insertional mutagenesis. Here we present enhanced-specificity tagmentation-assisted PCR (esTag-PCR), a rapid and accurate method for mapping transgene integration and copy number. Using stably transfected HepG2 cells, we demonstrate that esTag-PCR has higher integration site detection accuracy and efficiency than alternative tagmentation-based methods. Next, we performed esTag-PCR on rhesus macaque embryos derived from zygotes injected with piggyBac transposase and transposon/transgene plasmid. Using low-input trophectoderm biopsies, we demonstrate that esTag-PCR accurately maps integration events while preserving blastocyst viability. We used these high-resolution data to evaluate the performance of piggyBac-mediated editing of rhesus macaque embryos, demonstrating that increased concentration of transposon/transgene plasmid can increase the fraction of embryos with stable integration; however, the number of integrations per embryo also increases, which may be problematic for some applications. Collectively, esTag-PCR represents an important improvement to the detection of transgene integration, provides a method to validate and screen edited embryos before implantation, and represents an important advance in the creation of transgenic animal models.
AU  - Ryu, J.*
AU  - Chan, W.*
AU  - Wettengel, J.M.
AU  - Hanna, C.B.*
AU  - Burwitz, B.J.*
AU  - Hennebold, J.D.*
AU  - Bimber, B.N.*
C1  - 64279
C2  - 52169
SP  - 241-254
TI  - Rapid, accurate mapping of transgene integration in viable rhesus macaque embryos using enhanced-specificity tagmentation-assisted PCR.
JO  - Mol.Ther.-Methods Clin. Dev.
VL  - 24
PY  - 2022
SN  - 2329-0501
ER  - 

TY  - JOUR
AB  - CD4+ T cells play an important role in the immune response against cancer and infectious diseases. However, mechanistic details of their helper function in hepatitis B virus (HBV) infection in particular, or their advantage for adoptive T cell therapy remain poorly understood as experimental and therapeutic tools are missing. Therefore, we identified, cloned, and characterized a comprehensive library of 20 MHC class II-restricted HBV-specific T cell receptors (TCRs) from donors with acute or resolved HBV infection. The TCRs were restricted by nine different MHC II molecules and specific for eight different epitopes derived from intracellularly processed HBV envelope, core, and polymerase proteins. Retroviral transduction resulted in a robust expression of all TCRs on primary T cells. A high functional avidity was measured for all TCRs specific for epitopes S17, S21, S36, and P774 (half-maximal effective concentration [EC50] <10 nM), or C61 and preS9 (EC50 <100 nM). Eight TCRs recognized peptide variants of HBV genotypes A to D. Both CD4+ and CD8+ T cells transduced with the MHC II-restricted TCRs were polyfunctional, producing interferon (IFN)-γ, tumor necrosis factor (TNF)-α, interleukin (IL)-2, and granzyme B (GrzB), and killed peptide-loaded target cells. Our set of MHC class II-restricted TCRs represents an important tool for elucidating CD4+ T cell help in viral infection with potential benefit for T cell therapy.
AU  - Schreiber, S.
AU  - Honz, M.*
AU  - Mamozai, W.*
AU  - Kurktschiev, P.*
AU  - Schiemann, M.*
AU  - Witter, K.*
AU  - Moore, E.*
AU  - Zielinski, C.*
AU  - Sette, A.*
AU  - Protzer, U.
AU  - Wisskirchen, K.
C1  - 63528
C2  - 51579
CY  - 50 Hampshire St, Floor 5, Cambridge, Ma 02139 Usa
SP  - 476-489
TI  - Characterization of a library of 20 HBV-specific MHC class II-restricted T cell receptors.
JO  - Mol.Ther.-Methods Clin. Dev.
VL  - 23
PB  - Cell Press
PY  - 2021
SN  - 2329-0501
ER  -