IMMU-37. DISRUPTION OF PD-L1 BY ENHANCED TWO-SGRNAS CRISPR/CAS9 IN TREATMENT OF GLIOBLASTOMA

2021 ◽  
Vol 23 (Supplement_6) ◽  
pp. vi100-vi100
Author(s):  
Javier Fierro ◽  
joshua Perez ◽  
Rocio Aguilar ◽  
Jake Dipasquale ◽  
An Tran ◽  
...  
Keyword(s):  
Base Pair ◽  
Gene Editing ◽  
Genomic Sequence ◽  
Macrophage Polarization ◽  
Homology Directed Repair ◽  
Immunotherapeutic Strategy ◽  
Reverse Strand ◽  
Repair Template ◽  
And Migration ◽  
U87 Cells

Abstract Anti-glioblastoma multiform (GBM) immunotherapy poses a great challenge due to immunosuppressive brain tumor environments and the blood brain barrier (BBB). Programmed death ligand 1 (PD-L1) plays a key role in GBM immunosuppression, vitality, proliferation, and migration. Targeting PD-L1 for immunotherapy is a promising new avenue for treating GBM. CRISPR/Cas9 gene editing can be used to knockout both membrane and cytoplasmic PD-L1, leading to an enhanced immunotherapeutic strategy. We identified two sgRNA sequences located on PD-L1 exon 3. The first sgRNA recognized the forward strand of human PD-L1 near the beginning of exon 3 and cuts at approximately base pair 82 (g82). The second sgRNA recognized the reverse strand of exon 3 and cuts at base pair 165 (g165). Two sgRNAs, g82 and g165, created an 83bp deletion in PD-L1 genomic sequence. Two sgRNAs combination with a homology-directed repair template (HDR) was designed to enhance PD-L1 knockout specificity and efficiency. Both g82 and g165 were cloned into one CRISPR/Cas9 plasmid, and was co-transfected with HDR. GFP tagged CRISPR/Cas9 plasmid containing of g82 and g165 (Cas9-g82/165) was loaded into Rhodamine labeled nanoparticles (Cas9-g82/165-NPs) and then treated to GBM U87 cells. The enhanced intracellular uptake and transfection of Cas9-g82/165-NPs were detected by a fluorescence microscopy. T7E1, qRT-PCR and western blot analysis determined that the dual sgRNA CRISPR/Ca9 system knocked out both endogenous (80%) and exogenous (64%) PD-L1 in U87 cells and PD-L1 overexpression U87 cells, respectively. Deletion of PD-L1 reduced U87 migration and proliferation, while PD-L1 overexpression promoted tumor growth and tumor-associated macrophage polarization. Together, deletion of both membrane and cytoplasmic PD-L1 altered the PD-L1-associated immunosuppressive environment and prevented tumor progression and migration. Thus, two-sgRNAs CRISPR/Cas9 gene-editing system is a promising avenue for anti-GBM immunotherapy.

scholarly journals NGMA-2. Dual sgRNA-directed PD-L1 knockout in human glioblastoma cells using the CRISPR/Cas9 system

2021 ◽  
Vol 3 (Supplement_2) ◽  
pp. ii4-ii5
Author(s):  
Javier Fierro ◽  
Jake Dipasquale ◽  
Rocio Aguilar ◽  
Joshua Perez ◽  
An Tran ◽  
...  
Keyword(s):  
Base Pair ◽  
Genomic Sequence ◽  
Stop Codon ◽  
Macrophage Polarization ◽  
Protein A ◽  
Immune Checkpoints ◽  
Reverse Strand ◽  
And Migration ◽  
Immunosuppressive Microenvironment ◽  
U87 Cells

Abstract Glioblastoma multiforme (GBM) is an astrocyte derived brain tumor. It induces an immunosuppressive microenvironment by exploiting immune checkpoints such as the PD-1/PD-L1 pathway. Targeting the PD-1/PD-L1 pathway for immunotherapy is a promising new avenue for treating GBM, but more work is needed to develop a safe and effective method for clinical applications. We identified two sgRNA sequences located on PD-L1 exon 3. The first sgRNA recognized the forward strand of human PD-L1 near the beginning of exon 3 and cuts at approximately base pair 82 (g82). The second sgRNA recognized the reverse strand of exon 3 and cuts at base pair 165 (g165). Two sgRNAs, g82 and g165, created an 83bp deletion in the genomic sequence that can lead to the production of a non-functional PD-L1 protein. A homology-directed repair template (HDR) containing an in-frame stop codon was designed to enhance PD-L1 knockout specificity and efficiency. Both g82 and g165 were cloned into the CRISPR/Cas9 plasmid, and was co-transfected with the added HDR template. T7E1, qRT-PCR and western blot analysis determined that the dual sgRNA CRISPR/Ca9 system knocked out both endogenous (80%) and exogenous (64%) PD-L1 in U87 cells and PD-L1 overexpression U87 cells, respectively. Deletion of PD-L1 reduced U87 migration and proliferation, while PD-L1 overexpression promoted tumor growth and tumor-associated macrophage polarization. Together, deletion of both membrane and cytoplasmic PD-L1 altered the PD-L1-associated immunosuppressive environment and prevented tumor progression and migration. Thus, a dual sgRNA CRISPR/Cas9 gene-editing system is a promising avenue for anti-GBM immunotherapy.

scholarly journals OTME-18. Targeted CRISPR/Cas9 gene-editing regulates the brain tumor environment

2021 ◽  
Vol 3 (Supplement_2) ◽  
pp. ii17-ii17
Author(s):  
Joshua Perez ◽  
Javier Fierro ◽  
Rocio Aguilar ◽  
Huanyu Dou
Keyword(s):  
Brain Tumor ◽  
Gene Editing ◽  
Transfection Efficacy ◽  
Programmed Death ◽  
Tumor Environment ◽  
Human Gbm ◽  
And Migration ◽  
The Brain ◽  
U87 Cells

Abstract Glioblastoma multiform (GBM) is the most common malignant brain tumor. Recent immunotherapy has demonstrated potential to treat GBM. However, the immune suppressive tumor environment in the brain represents a significant barrier for the treatment of GBM. Overexpression of programmed death ligand-1 (PD-L1) in GBM tumor cells and macrophages plays a key role in GBM vitality, proliferation, and migration, while also suppressing the immune system. We developed a CRISPR/Cas9 gene-editing system to delete whole cell PD-L1. Human PD-L1 targeted sgRNA were cloned into CRISPR/Cas9 plasmids with or without an HDR templet. CRISPR/Cas9 were treated to human GBM U87 cells for 15, 30, 60, 120 and 240 minutes. The intracellular concentration of CRISPR/Cas9 exhibited a time-dependent increases. A GFP tagged CRISPR/Cas9 plasmid was developed to test the transfection efficacy. Higher levels of GFP+ U87 cells were observed at day 3. CRISPR/Cas9 showed a greater PD-L1 knockout at day 3. The PD-L1 reduction limited the proliferation of U87 cells. A scratch assay showed that PD-L1 deletion inhibited the migration of U87 cells. An in vitro GBM model was developed by co-cultivation of U87 cells and macrophages. CRISPR/Cas9 treated co-cultures changed the ratios of U87 cells and macrophages and polarized tumor associated macrophages (TAM) from M2 toward M1. CRISPR/Cas9 gene-editing effectively deleted PD-L1 in U87 cells. Successful deletion of PD-L1 prevented U87 cells growth and migration, and altered the TAMs plasticity and the tumor environment.

scholarly journals OMRT-12. Nanoparticle-based CRISPR-Cas9 delivery for anti-glioblastoma immunotherapy

2021 ◽  
Vol 3 (Supplement_2) ◽  
pp. ii9-ii9
Author(s):  
Rocio Aguilar ◽  
Javier Fierro ◽  
Joshua Perez ◽  
Huanyu Dou
Keyword(s):  
Gene Editing ◽  
Nuclear Accumulation ◽  
Dependent Manner ◽  
Western Blot Assay ◽  
Immune Resistance ◽  
Programmed Death ◽  
Immunotherapeutic Strategy ◽  
Human Gbm ◽  
A Charge ◽  
U87 Cells

Abstract Anti-glioblastoma GBM) immunotherapy poses a great challenge due to immunosuppressive brain tumor environments and the blood brain barrier (BBB). Programmed death ligand 1 (PD-L1) is an immune checkpoint that mediated the immune resistance. Inhibition of PD-L1 by antibodies was widely studied to treat many type of cancers. However, the inefficient therapeutic immune response became a significant barrier for treatment of GBM. CRISPR/Cas9 gene editing can be used to knockout both membrane and cytoplasmic PD-L1, leading to an enhanced immunotherapeutic strategy. It is extremely difficulty to deliver CRISPR/Cas9 containing plasmid for translational and clinic applications. We have been developed a core-shell nanoparticle (NP) to carry CRISPR/Cas9 plasmid for PD-L1 knockout. The different NP formulations were made and optimized to deliver CRISPR/Cas9 plasmid. NPs were prepared by modifying the water temperature, sonication power and time and formulation time. The obtained NPs had a size of 115-160nm and a charge of 40-50mV. The size and charge were significantly altered after CRISPR/Cas9 plasmids were loaded into NPs (Cas9-NPs). Agarose gel electrophoresis showed that CRISPR/Cas9 plasmids were fully encapsulated by NPs with 1 and 2 ug. The positive DNA bands occurred with 4ng, indicating the overloaded CRISPR/Cas9 plasmid. Fluorescence microscopy determined Cas9-NPs uptake by U87 cells under a time-dependent manner. GFP tagged Cas9-NPs were treated to U87 cells for transfection evaluation. The obtained different NPs delivery of CRISPR/Cas9 exhibited various transfection efficiencies in U87 cells. Visualization of intracellular Cas9-NPs showed increases in uptake by U87 cells from 0.5, 1, 2, and 4 hours. The greater nuclear accumulation of Cas9-NPs was seen at 24 hours. A western blot assay determined the success of PD-L1 deletion by Cas9-NPs in human GBM U87 cells. NPs-based CRISPR/Cas9 gene-editing system has great potential as an immunotherapeutic platform to treat GBM.

scholarly journals Fusobacterium nucleatum Facilitates M2 Macrophage Polarization and Colorectal Carcinoma Progression by Activating TLR4/NF-κB/S100A9 Cascade

2021 ◽  
Vol 12 ◽  
Author(s):  
Lijun Hu ◽  
Yan Liu ◽  
Xuehua Kong ◽  
Rui Wu ◽  
Qi Peng ◽  
...  
Keyword(s):  
Colorectal Carcinoma ◽  
Molecular Mechanisms ◽  
Macrophage Polarization ◽  
Fusobacterium Nucleatum ◽  
M2 Macrophage ◽  
Molecular Pattern ◽  
Immunotherapeutic Strategy ◽  
And Migration

Fusobacterium nucleatum (Fn) has been considered as a significant contributor in promoting colorectal carcinoma (CRC) development by suppressing host anti-tumor immunity. Recent studies demonstrated that the aggregation of M2 macrophage (Mφ) was involved in CRC progress driven by Fn infection. However, the underlying molecular mechanisms are poorly characterized. Here, we investigated the role of Fn in Mφ polarization as well as its effect on CRC malignancy. Fn infection facilitated differentiation of Mφ into the M2-like Mφ phenotype by in vitro study. Histological observation from Fn-positive CRC tissues confirmed the abundance of tumor-infiltrating M2-like Mφ. Fn-induced M2-like Mφ polarization was weakened once inhibiting a highly expressed damage-associated molecular pattern (DAMP) molecule S100A9 mainly derived from Fn-challenged Mφ and CRC cells. In addition, Fn-challenged M2-like Mφ conferred CRC cells a more malignant phenotype, showing stronger proliferation and migration characteristics in vitro and significantly enhanced tumor growth in vivo, all of which were partially inhibited when S100A9 was lost. Mechanistic studies further demonstrated that activation of TLR4/NF-κB signaling pathway mediated Fn-induced S100A9 expression and subsequent M2-like Mφ activation. Collectively, these findings indicate that elevated S100A9 in Fn-infected CRC microenvironment participates in M2-like Mφ polarization, thereby facilitating CRC malignancy. Furthermore, targeting TLR4/NF-κB/S100A9 cascade may serve as promising immunotherapeutic strategy for Fn-associated CRC.

scholarly journals A Consensus Model of Homology-Directed Repair Initiated by CRISPR/Cas Activity

2021 ◽  
Vol 22 (8) ◽  
pp. 3834
Author(s):  
Kevin Bloh ◽  
Natalia Rivera-Torres
Keyword(s):  
Dna Repair ◽  
Mechanism Of Action ◽  
Gene Editing ◽  
Strand Break ◽  
Consensus Model ◽  
Homology Directed Repair ◽  
Dna Repair Mechanisms ◽  
Repair Mechanisms ◽  
Repair Template ◽  
The Many

The mechanism of action of ssODN-directed gene editing has been a topic of discussion within the field of CRISPR gene editing since its inception. Multiple comparable, but distinct, pathways have been discovered for DNA repair both with and without a repair template oligonucleotide. We have previously described the ExACT pathway for oligo-driven DNA repair, which consisted of a two-step DNA synthesis-driven repair catalyzed by the simultaneous binding of the repair oligonucleotide (ssODN) upstream and downstream of the double-strand break. In order to better elucidate the mechanism of ExACT-based repair, we have challenged the assumptions of the pathway with those outlines in other similar non-ssODN-based DNA repair mechanisms. This more comprehensive iteration of the ExACT pathway better described the many different ways where DNA repair can occur in the presence of a repair oligonucleotide after CRISPR cleavage, as well as how these previously distinct pathways can overlap and lead to even more unique repair outcomes.

scholarly journals Hypoxic glioma-derived exosomes promote M2-like macrophage polarization by enhancing autophagy induction

2021 ◽  
Vol 12 (4) ◽  
Author(s):  
Jianye Xu ◽  
Jian Zhang ◽  
Zongpu Zhang ◽  
Zijie Gao ◽  
Yanhua Qi ◽  
...  
Keyword(s):  
Macrophage Polarization ◽  
Autophagy Induction ◽  
Antitumor Immunotherapy ◽  
Novel Biomarkers ◽  
And Migration ◽  
Immunosuppressive Microenvironment ◽  
Proliferation And Migration ◽  
Glioma Progression

AbstractExosomes participate in intercellular communication and glioma microenvironment modulation, but the exact mechanisms by which glioma-derived exosomes (GDEs) promote the generation of the immunosuppressive microenvironment are still unclear. Here, we investigated the effects of GDEs on autophagy, the polarization of tumor-associated macrophages (TAMs), and glioma progression. Compared with normoxic glioma-derived exosomes (N-GDEs), hypoxic glioma-derived exosomes (H-GDEs) markedly facilitated autophagy and M2-like macrophage polarization, which subsequently promoted glioma proliferation and migration in vitro and in vivo. Western blot and qRT-PCR analyses indicated that interleukin 6 (IL-6) and miR-155-3p were highly expressed in H-GDEs. Further experiments showed that IL-6 and miR-155-3p induced M2-like macrophage polarization via the IL-6-pSTAT3-miR-155-3p-autophagy-pSTAT3 positive feedback loop, which promotes glioma progression. Our study clarifies a mechanism by which hypoxia and glioma influence autophagy and M2-like macrophage polarization via exosomes, which could advance the formation of the immunosuppressive microenvironment. Our findings suggest that IL-6 and miR-155-3p may be novel biomarkers for diagnosing glioma and that treatments targeting autophagy and the STAT3 pathway may contribute to antitumor immunotherapy.

scholarly journals Genetic analysis of the human thymidine kinase gene promoter.

1986 ◽  
Vol 6 (8) ◽  
pp. 2903-2909 ◽  
Author(s):  
J A Kreidberg ◽  
T J Kelly
Keyword(s):  
Thymidine Kinase ◽  
Base Pair ◽  
Promoter Region ◽  
Genomic Sequence ◽  
Gene Promoter ◽  
Thymidine Kinase Gene ◽  
Rich Region ◽  
Kinase Gene ◽  
Inverted Orientation ◽  
Pair Sequence

The promoter of the human thymidine kinase gene was defined by DNA sequence and genetic analyses. Mutant plasmids with deletions extending into the promoter region from both the 5' and 3' directions were constructed. The mutants were tested in a gene transfer system for the ability to transform TK- cells to the TK+ phenotype. This analysis delimited the functional promoter to within an 83-base-pair region upstream of the mRNA cap site. This region contains sequences common to other eucaryotic promoters including G X C-rich hexanucleotides, a CAAT box, and an A X T-rich region. The CAAT box is in an inverted orientation and is part of a 9-base-pair sequence repeated twice in the promoter region. Comparison of the genomic sequence with the cDNA sequence defined the first exon of the thymidine kinase gene.

Genetic analysis of the human thymidine kinase gene promoter

1986 ◽  
Vol 6 (8) ◽  
pp. 2903-2909
Author(s):  
J A Kreidberg ◽  
T J Kelly
Keyword(s):  
Thymidine Kinase ◽  
Base Pair ◽  
Promoter Region ◽  
Genomic Sequence ◽  
Gene Promoter ◽  
Thymidine Kinase Gene ◽  
Rich Region ◽  
Kinase Gene ◽  
Inverted Orientation ◽  
Pair Sequence

The promoter of the human thymidine kinase gene was defined by DNA sequence and genetic analyses. Mutant plasmids with deletions extending into the promoter region from both the 5' and 3' directions were constructed. The mutants were tested in a gene transfer system for the ability to transform TK- cells to the TK+ phenotype. This analysis delimited the functional promoter to within an 83-base-pair region upstream of the mRNA cap site. This region contains sequences common to other eucaryotic promoters including G X C-rich hexanucleotides, a CAAT box, and an A X T-rich region. The CAAT box is in an inverted orientation and is part of a 9-base-pair sequence repeated twice in the promoter region. Comparison of the genomic sequence with the cDNA sequence defined the first exon of the thymidine kinase gene.

scholarly journals The Effect of Ginger on the Invasion and Migration of Glioma Cells

2020 ◽  
pp. 38-43
Author(s):  
Manar Zraikat ◽  
Munir Gharaibeh ◽  
Tasneem Alshelleh
Keyword(s):  
Cell Line ◽  
Tumour Progression ◽  
Glioma Cells ◽  
Three Dimension ◽  
Scratch Assay ◽  
Invasion And Migration ◽  
Invasion Model ◽  
Dimension 2 ◽  
And Migration ◽  
U87 Cells

Background: This work studies the effect of different concentrations of soaked ginger on the ability of the U87 glioma cells to invade collagen in a three dimension (3 D) invasion model and compare it with its effect on the ability of the same cell line to migrate in two-dimension (2 D) scratch assay. Methods: The hanging drop spheroids in 3D invasion assay were used to investigate the in invasion of the U87 cells. The 2D scratch assay was used to investigate the migration of the same cell line. Results: Gradual effect of the soaked ginger was noticed on the inhibition of the invasion of U87 in collagen and on the inhibition of the migration of the same cell line in scratch assay. Conclusion: The results in this article are promising and encourage further studies to investigate the effect of ginger active ingredients on tumour progression.

scholarly journals Adenine Base Editing of the Sickle Allele in CD34+ Hematopoietic Stem and Progenitor Cells Eliminates Hemoglobin S

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 47-47
Author(s):  
S. Haihua Chu ◽  
Daisy Lam ◽  
Michael S. Packer ◽  
Jennifer Olins ◽  
Alexander Liquori ◽  
...  
Keyword(s):  
High Performance Liquid Chromatography ◽  
Liquid Chromatography ◽  
Base Pair ◽  
High Performance ◽  
Gene Editing ◽  
Publicly Traded ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Adenine Base

While there are several small molecule, gene therapy, and gene editing approaches for treating sickle cell disease (SCD), these strategies do not result in the direct elimination of the causative sickle β-globin (HbS) variant itself. The reduction or complete removal of this pathologic globin variant and expression of normal β-hemoglobin (HbB) or other non-polymerizing β-globin variant may increase the likelihood of beneficial outcomes for SCD patients. Adenine base editors (ABEs) can precisely convert the mutant A-T base pair responsible for SCD to a G-C base pair, thus generating the hemoglobin variant, Hb G-Makassar, a naturally occurring β-globin variant that is not associated with human disease. Our studies have identified ABEs that can achieve highly efficient Makassar editing (>70%) of the sickle mutation in both sickle trait (HbAS) and homozygous sickle (HbSS) patient CD34+ cells with high cell viability and recovery and without perturbation of immunophenotypic hematopoietic stem and progenitor cell (HSPC) frequencies after ex vivo delivery of guide RNA and mRNA encoding the ABE. Furthermore, Makassar editing was retained throughout erythropoiesis in bulk in vitro erythroid differentiated cells (IVED) derived from edited CD34+ cells. To gain an understanding of allelic editing at a single clone resolution, we assessed editing frequencies of clones from both single cell expansion in erythroid differentiation media, as well as from single BFU-E colonies. We found that we could achieve >70% of colonies with bi-allelic Makassar editing and approximately 20% of colonies with mono-allelic Makassar editing, while <3% of colonies remained completely unedited. Previously, conventional hemoglobin capillary electrophoresis and high-performance liquid chromatography (HPLC) were unable to distinguish between HbS and HbG-Makassar. Here, we developed an ultra-high-performance liquid chromatography (UPLC) method that resolves sickle globin (HbS) from Hb G-Makassar globin in IVED cells. The Makassar globin variant was further confirmed by liquid chromatography mass spectrometry (LC-MS). By developing this new method to resolve these two β-globin variants in edited HbSS cells, we were able to detect, in bulk IVED cultures, >80% abundance Hb G-Makassar of total β-globins, which corresponded to a concomitant reduction of HbS levels to <20%. Furthermore, we were also able to determine globin abundance as well as allelic editing at the level of single clones and found that HbS was completely eliminated in >70% of cells that had bi-allelic Makassar editing. Moreover, in the approximately 20% of colonies that were found to be mono-allelically edited for the Makassar variant, there was a 60:40 ratio of Hb G-Makassar:HbS globin abundance in individual clones, at levels remarkably similar to the HbA(wildtype HbB):HbS levels found in HbAS individuals, with minimal observable in vitro sickling when exposed to hypoxia. Thus, with our ABEs, we were able to reduce HbS to <40% on a per cell basis in >90% of IVED cells and found that in vitro sickling under hypoxia inversely correlated with the level of Hb G-Makassar globin variant installation and corresponding reduction in HbS levels. By converting HbS to Hb G-Makassar, our direct and precise editing strategy replaces a pathogenic β-globin with one that has been shown to have normal hematologic parameters. Coupled with autologous stem cell transplant, this next generation gene editing strategy presents a promising new modality for treating patients with SCD. Disclosures Chu: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lam:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Packer:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Olins:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Liquori:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Marshall:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lee:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Yan:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Decker:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gantzer:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Haskett:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bohnuud:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Born:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Barrera:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Slaymaker:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gaudelli:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hartigan:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ciaramella:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Sign in / Sign up

Export Citation Format

Share Document