Dataset Information

Efficient shRNA-based knockdown of multiple target genes for cell therapy using a chimeric miRNA cluster platform.

ABSTRACT: Genome engineering technologies are powerful tools in cell-based immunotherapy to optimize or fine-tune cell functionalities. However, their use for multiple gene edits poses relevant biological and technical challenges. Short hairpin RNA (shRNA)-based cell engineering bypasses these criticalities and represents a valid alternative to CRISPR-based gene editing. Here, we describe a microRNA (miRNA)-based multiplex shRNA platform obtained by combining highly efficient miRNA scaffolds into a chimeric cluster, to deliver up to four shRNA-like sequences. Thanks to its limited size, our cassette could be deployed in a one-step process along with all the CAR components, streamlining the generation of engineered CAR T cells. The plug-and-play design of the shRNA platform allowed us to swap each shRNA-derived guide sequence without affecting the system performance. Appropriately choosing the target sequences, we were able to either achieve a functional KO, or fine-tune the expression levels of the target genes, all without the need for gene editing. Through our strategy we achieved easy, safe, efficient, and tunable modulation of multiple target genes simultaneously. This approach allows for the effective introduction of multiple functionally relevant tweaks in the transcriptome of the engineered cells, which may lead to increased performance in challenging environments, e.g., solid tumors.

SUBMITTER: Rossi M

PROVIDER: S-EPMC10548280 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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Efficient shRNA-based knockdown of multiple target genes for cell therapy using a chimeric miRNA cluster platform.

Rossi Matteo M Steklov Mikhail M Huberty Fanny F Nguyen Thuy T Marijsse Jérôme J Jacques-Hespel Céline C Najm Paul P Lonez Caroline C Breman Eytan E

Molecular therapy. Nucleic acids 20230920

Genome engineering technologies are powerful tools in cell-based immunotherapy to optimize or fine-tune cell functionalities. However, their use for multiple gene edits poses relevant biological and technical challenges. Short hairpin RNA (shRNA)-based cell engineering bypasses these criticalities and represents a valid alternative to CRISPR-based gene editing. Here, we describe a microRNA (miRNA)-based multiplex shRNA platform obtained by combining highly efficient miRNA scaffolds into a chimer ...[more]

PMID: 37799328

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Project description:BackgroundControlling and limiting the expression of short hairpin RNA (shRNA) by using constitutive or tissue-specific polymerase II (pol II) expression can be a promising strategy to avoid RNAi toxicity. However, to date detailed studies on requirements for effective pol II shRNA expression and processing are not available. We investigated the optimal structural configuration of shRNA molecules, namely: hairpin location, stem length and termination signal required for effective pol II expression and compared it with an alternative strategy of avoiding toxicity by using artificial microRNA (miRNA) scaffolds.ResultsHighly effective shRNAs targeting luciferase (shLuc) or Apolipoprotein B100 (shApoB1 and shApoB2) were placed under the control of the pol II CMV promoter and expressed at +5 or +6 nucleotides (nt) with reference to the transcription start site (TSS). Different transcription termination signals (TTS), namely minimal polyadenylation (pA), poly T (T5) and U1 were also used. All pol II- expressed shRNA variants induced mild inhibition of Luciferase reporters carrying specific targets and none of them showed comparable efficacy to their polymerase III-expressed H1-shRNA controls, regardless of hairpin position and termination signal used. Extending hairpin stem length from 20 basepairs (bp) to 21, 25 or 29 bp yielded only slight improvement in the overall efficacy. When shLuc, shApoB1 and shApoB2 were placed in an artificial miRNA scaffold, two out of three were as potent as the H1-shRNA controls. Quantification of small interfering RNA (siRNA) molecules showed that the artificial miRNA constructs expressed less molecules than H1-shRNAs and that CMV-shRNA expressed the lowest amount of siRNA molecules suggesting that RNAi processing in this case is least effective. Furthermore, CMV-miApoB1 and CMV-miApoB2 were as effective as the corresponding H1-shApoB1 and H1-shApoB2 in inhibiting endogenous ApoB mRNA.ConclusionOur results demonstrate that artificial miRNA have a better efficacy profile than shRNA expressed either from H1 or CMV promoter and will be used in the future for RNAi therapeutic development.

Dataset Information

Efficient shRNA-based knockdown of multiple target genes for cell therapy using a chimeric miRNA cluster platform.

Publications

Efficient shRNA-based knockdown of multiple target genes for cell therapy using a chimeric miRNA cluster platform.

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