Mechanism Driven Early Stage Identification and Avoidance of Antisense Oligonucleotides Causing TRL9 Mediated Inflammatory Responses in Bjab cells

Nucleic acid-based PS-ASOs have the potential to activate cellular innate immune responses, and the level of activation can vary quite dramatically with sequence. Minimizing degree of proinflammatory effect is one of the main selection criteria for compounds intended to move into clinical trials. While a recently developed hPBMC-based assay showed excellent ability to detect innate immune active PS-ASOs, which can then be discarded from the developmental process, this assay is highly resource-intensive and easily affected by subject variability. This compelled us to develop of a more convenient high-throughput assay. Here, we describe a new in vitro assay, utilizing a cultured human Bjab cell line, which was developed and validated to identify PS-ASOs that may cause innate immune activation. The assay was calibrated to replicate results from the hPMBC assay. The Bjab assay was designed to be high throughput and more convenient by using RT-qPCR readout of mRNA of the chemokine Ccl22. The Bjab assay was also shown to be highly reproducible and to provide a large dynamic range in determining the immune potential of PS-ASOs via comparison to known benchmark PS-ASO controls that were previously shown to be either safe or inflammatory in clinical trials. In addition, we demonstrate that Bjab cells can be used to provide mechanistic information on PS-ASO-TLR9 dependent innate immune activation.

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Nucleic Acid Immunity and DNA Damage Response: New Friends and Old Foes

Frontiers in Immunology ◽

10.3389/fimmu.2021.660560 ◽

2021 ◽

Vol 12 ◽

Author(s):

Clara Taffoni ◽

Alizée Steer ◽

Johanna Marines ◽

Hanane Chamma ◽

Isabelle K. Vila ◽

...

Keyword(s):

Dna Damage ◽

Nucleic Acid ◽

Nucleic Acids ◽

Dna Damage Response ◽

Immune Activation ◽

Inflammatory Responses ◽

Innate Immune ◽

Fine Tuning ◽

Innate Immune Activation ◽

Damage Response

The maintenance of genomic stability in multicellular organisms relies on the DNA damage response (DDR). The DDR encompasses several interconnected pathways that cooperate to ensure the repair of genomic lesions. Besides their repair functions, several DDR proteins have emerged as involved in the onset of inflammatory responses. In particular, several actors of the DDR have been reported to elicit innate immune activation upon detection of cytosolic pathological nucleic acids. Conversely, pattern recognition receptors (PRRs), initially described as dedicated to the detection of cytosolic immune-stimulatory nucleic acids, have been found to regulate DDR. Thus, although initially described as operating in specific subcellular localizations, actors of the DDR and nucleic acid immune sensors may be involved in interconnected pathways, likely influencing the efficiency of one another. Within this mini review, we discuss evidences for the crosstalk between PRRs and actors of the DDR. For this purpose, we mainly focus on cyclic GMP-AMP (cGAMP) synthetase (cGAS) and Interferon Gamma Inducible Protein 16 (IFI16), as major PRRs involved in the detection of aberrant nucleic acid species, and components of the DNA-dependent protein kinase (DNA-PK) complex, involved in the repair of double strand breaks that were recently described to qualify as potential PRRs. Finally, we discuss how the crosstalk between DDR and nucleic acid-associated Interferon responses cooperate for the fine-tuning of innate immune activation, and therefore dictate pathological outcomes. Understanding the molecular determinants of such cooperation will be paramount to the design of future therapeutic approaches.

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