DEVELOPING A CRISPR-CAS9 TOOL TO INACTIVATE THE GLUCOCORTICOID RECEPTOR AND BROADEN THE APPLICABILITY OF ANTIGEN-SPECIFIC T-CELL IMMUNOTHERAPY
Author(s): ,
Chrysoula Pantazi
Affiliations:
Gene and Cell Therapy Center, Heamatology Department - Stem Cell Transplantation Unit,George Papanikolaou Hospital,Thessaloniki,Greece;Department of Genetics, Development and Molecular Biology,Aristotle University of Thessaloniki,Thessaloniki,Greece
,
Maria Alvanou
Affiliations:
Gene and Cell Therapy Center, Heamatology Department - Stem Cell Transplantation Unit,George Papanikolaou Hospital,Thessaloniki,Greece;University General Hospital of Patras,Patras,Greece
,
Kiriakos Koukoulias
Affiliations:
Gene and Cell Therapy Center, Heamatology Department - Stem Cell Transplantation Unit,George Papanikolaou Hospital,Thessaloniki,Greece;Department of Genetics, Development and Molecular Biology,Aristotle University of Thessaloniki,Thessaloniki,Greece
,
Nikoletta Psatha
Affiliations:
Altius Institute for Biomedical Sciences,Seattle,United States
,
Georgios Tsiolas
Affiliations:
Institute of Applied Biosciences (INAB),Centre for Research and Technology Hellas (CERTH),Thessaloniki,Greece
,
Minas Yiangou
Affiliations:
Department of Genetics, Development and Molecular Biology,Aristotle University of Thessaloniki,Thessaloniki,Greece
,
Alexandros Spyridonidis
Affiliations:
University General Hospital of Patras,Patras,Greece
,
Kostas Stamatopoulos
Affiliations:
Institute of Applied Biosciences (INAB),Centre for Research and Technology Hellas (CERTH),Thessaloniki,Greece
,
Achilles Anagnostopoulos
Affiliations:
Gene and Cell Therapy Center, Heamatology Department - Stem Cell Transplantation Unit,George Papanikolaou Hospital,Thessaloniki,Greece
,
Antonios Makris
Affiliations:
Institute of Applied Biosciences (INAB),Centre for Research and Technology Hellas (CERTH),Thessaloniki,Greece
,
Evangelia Yannaki
Affiliations:
Gene and Cell Therapy Center, Heamatology Department - Stem Cell Transplantation Unit,George Papanikolaou Hospital,Thessaloniki,Greece
Anastasia Papadopoulou
Affiliations:
Gene and Cell Therapy Center, Heamatology Department - Stem Cell Transplantation Unit,George Papanikolaou Hospital,Thessaloniki,Greece
EHA Library. Pantazi C. 06/15/19; 266824; PS1207
Chrysoula Pantazi
Chrysoula Pantazi
Contributions
Abstract

Abstract: PS1207

Type: Poster Pitch

Presentation during EHA24: On Saturday, June 15, 2019 from 17:30 - 19:00

Location: Poster area

Background
Viral and fungal infections are among the most fatal complications in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). While pharmacotherapy often fails or is associated with toxicity, adoptive immunotherapy (AI) with pathogen-specific T cells (pSTs) represents an attractive alternative. However, T cells perform suboptimally under immunosuppression, mainly steroids, which represent the first-line treatment of transplant-associated immunological complications. The latter generates the obvious paradox of depriving the most susceptible to infections patients of the potential benefits of AI and the need to improve current T-cell AI.

Aims
We here aimed to evaluate the impact of dexamethasone (DEX) on primary T cells and develop a CRISPR/Cas9 system to genetically disrupt the glucocorticoid receptor (GR) in T-cells and confer resistance to steroids. The optimal CRISPR/Cas9 tool to be delivered by these studies will be subsequently used to generate steroid-resistant pSTs.

Methods
T cell proliferation, phenotype and apoptosis were evaluated upon culture with DEX. Human T cells were stimulated through CD3 ligation and co-stimulated directly by CD80. To inactivate the GR gene, 10 guide RNAs (gRNAs) were prepared to target different genomic sequences corresponding to various domains of the GR  (a transcription start site, and exons 2, 3, 4 and 5) and delivered either in pooled libraries or separately, into the T2 cell line using lentiviral vectors. Cells transduced with a viral vector expressing Cas9 but no gRNA (empty vector) was used as negative control.

Results
We first tested the effect of DEX in primary T-cells. A 3-day exposure to DEX impaired the proliferation of CD80-pulsed primary T cells and induced early apoptosis over the “no DEX” condition (p=0.0001, p=0.02 respectively). These effects seemed to correlate with a DEX-induced transition of T cells from a central memory to a more differentiated effector memory phenotype. DEX also upregulated PD-1 and CTLA-4, the major coinhibitory molecules in antigen-specific immune responses, over the “no DEX” condition (p<0.05). Then, in order to inactivate the GR and render cells steroid-resistant, T2 cells were transduced with viral vectors encoding Cas9 and 2 pooled libraries of 10 different gRNAs targeting GR and subsequently incubated in the presence or absence of DEX. T2 cells edited with either gRNA pool, presented normal proliferation and reduced apoptosis on DEX treatment as contrasted to their untreated counterparts and the empty-transduced cells, suggesting that both pools contained gRNAs which functionally disrupted GR. To clarify whether DEX-resistance was a single-gRNA effect or an additive/synergistic action of the pooled gRNAs, T2 cells were transduced by viral vectors expressing each single gRNA. In the presence of DEX, 8/10 tested gRNAs in CRISPR/Cas9-edited T2 cells presented similar apoptosis and proliferation rates to the untreated T2 cells, suggesting functional resistance to DEX. Among these functional gRNAs, an optimal gRNA will be selected on the basis of higher GR disruption efficiency and lower off-target effects for further studies. Data will be presented.

Conclusion
Overall, we provide a series of gRNAs to CRISPR/Cas9-disrupt the GR and confer resistance of T-cells to steroids. By this approach, we endeavor to ultimately offer the benefits of AI to the most vulnerable to infections patients, as those who receive high-dose steroids post allo-HSCT, by generating steroid-resistant, pSTs.

Session topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Res

Keyword(s): Infection

Abstract: PS1207

Type: Poster Pitch

Presentation during EHA24: On Saturday, June 15, 2019 from 17:30 - 19:00

Location: Poster area

Background
Viral and fungal infections are among the most fatal complications in patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HSCT). While pharmacotherapy often fails or is associated with toxicity, adoptive immunotherapy (AI) with pathogen-specific T cells (pSTs) represents an attractive alternative. However, T cells perform suboptimally under immunosuppression, mainly steroids, which represent the first-line treatment of transplant-associated immunological complications. The latter generates the obvious paradox of depriving the most susceptible to infections patients of the potential benefits of AI and the need to improve current T-cell AI.

Aims
We here aimed to evaluate the impact of dexamethasone (DEX) on primary T cells and develop a CRISPR/Cas9 system to genetically disrupt the glucocorticoid receptor (GR) in T-cells and confer resistance to steroids. The optimal CRISPR/Cas9 tool to be delivered by these studies will be subsequently used to generate steroid-resistant pSTs.

Methods
T cell proliferation, phenotype and apoptosis were evaluated upon culture with DEX. Human T cells were stimulated through CD3 ligation and co-stimulated directly by CD80. To inactivate the GR gene, 10 guide RNAs (gRNAs) were prepared to target different genomic sequences corresponding to various domains of the GR  (a transcription start site, and exons 2, 3, 4 and 5) and delivered either in pooled libraries or separately, into the T2 cell line using lentiviral vectors. Cells transduced with a viral vector expressing Cas9 but no gRNA (empty vector) was used as negative control.

Results
We first tested the effect of DEX in primary T-cells. A 3-day exposure to DEX impaired the proliferation of CD80-pulsed primary T cells and induced early apoptosis over the “no DEX” condition (p=0.0001, p=0.02 respectively). These effects seemed to correlate with a DEX-induced transition of T cells from a central memory to a more differentiated effector memory phenotype. DEX also upregulated PD-1 and CTLA-4, the major coinhibitory molecules in antigen-specific immune responses, over the “no DEX” condition (p<0.05). Then, in order to inactivate the GR and render cells steroid-resistant, T2 cells were transduced with viral vectors encoding Cas9 and 2 pooled libraries of 10 different gRNAs targeting GR and subsequently incubated in the presence or absence of DEX. T2 cells edited with either gRNA pool, presented normal proliferation and reduced apoptosis on DEX treatment as contrasted to their untreated counterparts and the empty-transduced cells, suggesting that both pools contained gRNAs which functionally disrupted GR. To clarify whether DEX-resistance was a single-gRNA effect or an additive/synergistic action of the pooled gRNAs, T2 cells were transduced by viral vectors expressing each single gRNA. In the presence of DEX, 8/10 tested gRNAs in CRISPR/Cas9-edited T2 cells presented similar apoptosis and proliferation rates to the untreated T2 cells, suggesting functional resistance to DEX. Among these functional gRNAs, an optimal gRNA will be selected on the basis of higher GR disruption efficiency and lower off-target effects for further studies. Data will be presented.

Conclusion
Overall, we provide a series of gRNAs to CRISPR/Cas9-disrupt the GR and confer resistance of T-cells to steroids. By this approach, we endeavor to ultimately offer the benefits of AI to the most vulnerable to infections patients, as those who receive high-dose steroids post allo-HSCT, by generating steroid-resistant, pSTs.

Session topic: 24. Gene therapy, cellular immunotherapy and vaccination - Biology & Translational Res

Keyword(s): Infection

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