Short hairpin RNA (shRNA) technology offers a differentiated strategy to future development of CAR T
Manipulating protein expression to generate cells with a specific desired feature is one of the central goals of engineered cell therapy. Short hairpin RNA (shRNA) is a well-established approach to reduce protein expression by silencing genes in a process called RNA interference.
Celyad Oncology’s shRNA technology allows for the modulation of gene expression in our CAR T constructs without the need for gene-editing, an advantage of being able to modulate the target gene expression without completely removing genetic sequences. We are currently engineering T-cells with specific desired features, including the inhibition of alloreactivity, increased persistence and enhanced anti-tumor activity or potentially improved tolerability. We have validated the utility, versatility and safety of our shRNA platform through our clinical pipeline candidates. Our next-generation NKG2D receptor CAR T clinical candidate, CYAD-02, incorporates a single shRNA hairpin targeting the NKG2D ligands MICA/MICB within the construct, and our BCMA-targeting CAR T, CYAD-211, incorporates a single shRNA hairpin targeting the CD3ζ component of the TCR complex
Our shRNA platform provides a versatile approach to the design and development of next-generation CAR T candidates with the potential to work with a broad array of CAR T constructs with customized features
Innovative multiplexing technology goes beyond allogeneic CAR T therapy
While we first validated our shRNA technology by creating off-the-shelf allogeneic CAR T cells by modulating the expression of CD3ζ (see section below), this technology also allows us to regulate protein expression of multiple targets at the same time, whether cell source is of autologous or allogeneic origin. Thereby multiplexing allows us to optimize CAR T cell features (phenotype), persistence, efficacy, or ability to evade complex or immunosuppressive tumor microenvironments.
This is of particular interest in the context of solid tumors, like ovarian cancer or colorectal cancer, which are still a huge challenge for current cell immunotherapies.
This technology is complementary to our All-in-One Vector approach, which allows for the expression of multiple shRNAs in a single construct within a single transduction step. Data from preclinical studies demonstrate simultaneous knockdown of multiple genes in a single multiplexed vector with our second-generation scaffold, without loss of function of the CAR itself. In contrast, targeting multiple genes using gene editing would require multiple double-strand breaks in the DNA, inducing stress which might impact their viability or function.
Finally, combining multiplexed shRNAs with CARs and additional genes of choice also provides potential for broad therapeutic functionality.
shRNA leads the future of allogeneic CAR T
Our CYAD-200 series of allogeneic CAR T candidates use shRNA to interfere with the expression of the TCR by knockdown of the RNA encoding the CD3ζ component of the TCR complex. Data from preclinical studies have shown shRNA knockdown targeting CD3ζ is as effective as gene-editing methods such as CRISPR/Cas9. In addition, preclinical data demonstrated expression of a single shRNA hairpin provides prolonged TCR knockdown.
Celyad Oncology’s lead shRNA-based allogeneic CAR T candidate, CYAD-211, which uses a single shRNA to knockdown the CD3ζ component of the TCR complex, is currently in the Phase 1 IMMUNICY-1 trial.