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| Status |
Public on Dec 05, 2024 |
| Title |
Multiplexed inhibition of immunosuppressive genes with Cas13d for on-demand combinatorial cancer immunotherapy |
| Organisms |
Mus; Mus musculus; synthetic construct |
| Experiment type |
Expression profiling by high throughput sequencing
Other
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| Summary |
We devise Multiplex Universal Combinatorial Immunotherapy via Gene-silencing (MUCIG), as a versatile approach for combinatorial cancer immunotherapy. We harness CRISPR-Cas13d to efficiently target multiple endogenous immunosuppressive genes on demand, allowing us to silence various combinations of multiple immunosuppressive factors in the TME. Intra-tumoral AAV-mediated administration of MUCIG elicits significant anti-tumor activity with several Cas13d gRNA compositions (AAV-Cas13d-MUCIG). Optimization of TME targets by expression analysis led to a simplified off-the-shelf MUCIG targeting a four gene combination (PGGC: Pdl1/Cd274, Galectin9/Lgals9, Galectin3/Lgals3 and Cd47). AAV-Cas13d-PGGC showed significant in vivo anti-tumor efficacy in syngeneic tumor models. Single cell and flow profiling revealed that AAV-Cas13d-PGGC remodeled the TME by increasing CD8+ T cell infiltration and reducing neutrophils. MUCIG thus serves as a universal method to silence multiple immune genes in vivo, and can be delivered via AAV as a therapeutic approach. We devised an approach MUCIG (Multiplex Universal Combinatorial Immunotherapy via Gene-silencing) and designed MUCIG for targeting immunosuppressive genes at different scales of library pools. The first MUCIG pool included 313 genes selected based on these criteria. Subsequently, with a tiered approach, we designed three additional MUCIG pools (pool2: 152 genes; pool3: 55 genes; pool4: 19 genes). The distribution and coverage of gRNAs across all four pools were verified, confirming the robustness and specificity of this approach in the context of cancer immunosuppressive gene targeting therapy.
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| Overall design |
29 days post tumor injection , CD45+ cells were isolated from the colon26 or E0771 tumor following treatment with PBS, AAV-Cas13d-Vector or AAV-Cas13d-PGGC. The gRNA library readout PCR was performed for the MUCIG pool plasmids. And MUCIG gRNA library pool representation was analyzed.
*** Please note that the records have been updated with scRNAseq raw data on Mar 6,2025 ****
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| Contributor(s) |
Zhang F, Chow R, He E, Dong C, Xin S, Mirza D, Feng Y, Tian X, Verma N, Majety M, Zhang Y, Wang G, Chen S |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
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| Submission date |
Jun 10, 2024 |
| Last update date |
Mar 06, 2025 |
| Contact name |
Chuanpeng Dong |
| E-mail(s) |
chuanpeng.dong@yale.edu
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| Organization name |
Yale University School of Medicine
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| Department |
Genetics
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| Street address |
850 West Campus Dr
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| City |
New Haven |
| State/province |
CT |
| ZIP/Postal code |
06516 |
| Country |
USA |
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| Platforms (3) |
| GPL23787 |
Illumina HiSeq 4000 (Mus) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
| GPL26526 |
Illumina NovaSeq 6000 (synthetic construct) |
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| Samples (46)
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| Relations |
| BioProject |
PRJNA1122176 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE269516_MUCIG_pools_readCounts.txt.gz |
12.0 Kb |
(ftp)(http) |
TXT |
| GSE269516_RAW.tar |
309.5 Mb |
(http)(custom) |
TAR (of TAR, TXT) |
| GSE269516_RNAseq_STAR_count_matrix.tsv.gz |
1.4 Mb |
(ftp)(http) |
TSV |
SRA Run Selector |
| Raw data are available in SRA |
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