Pre-existing adaptive immunity to the RNA-editing enzyme Cas13d in humans

Area of Science:

• Biotechnology
• Immunology
• Molecular Biology

Background:

• RNA-guided RNA-targeting nucleases, like CRISPR-Cas13, show promise for gene editing therapies.
• RfxCas13d, a Cas13d enzyme from Ruminococcus flavefaciens, is attractive for therapeutic use due to its small size and specificity.

Purpose of the Study:

• To investigate pre-existing antibody and T cell responses to RfxCas13d in healthy human donors.
• To assess the potential impact of pre-existing immunity on RfxCas13d-based therapeutic applications.

Main Methods:

• Enzyme-linked immunosorbent assays (ELISA) were used to detect RfxCas13d-reactive antibodies.
• T cell culture assays were employed to evaluate T cell responses, including CD4+ and CD8+ T cell activity.
• Cytokine production (IFN-γ, TNF-α, IL-17) by responding T cells was measured.

Main Results:

• A significant proportion of healthy donors exhibited pre-existing antibodies against RfxCas13d.
• CD4+ and CD8+ T cell responses to RfxCas13d were detected in most donors.
• These immune responses were comparable to those observed against commonly used Cas9 proteins (SaCas9 and SpCas9).
• RfxCas13d-reactive T cells produced inflammatory cytokines such as IFN-γ, TNF-α, and IL-17.

Conclusions:

• Pre-existing immunity to RfxCas13d is prevalent in the healthy population.
• The presence of anti-RfxCas13d antibodies and T cell responses may influence the efficacy and safety of RfxCas13d-based gene editing therapies.
• These immunological considerations are critical for the future development and clinical translation of RfxCas13d therapeutics.