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Updated: Oct 2, 2025
Quantification of Immunostained Caspase-9 in Retinal Tissue
Published on: July 25, 2022
Investigation of Cas9 antibodies in the human eye
Marcus A Toral1,2, Carsten T Charlesworth3, Benjamin Ng1,4
1Molecular Surgery Program, Department of Ophthalmology, Byers Eye Institute, Stanford University, Palo Alto, CA, USA.
Abstract:
Preexisting immunity against Cas9 proteins in humans represents a safety risk for CRISPR-Cas9 technologies. However, it is unclear to what extent preexisting Cas9 immunity is relevant to the eye as it is targeted for early in vivo CRISPR-Cas9 clinical trials. While the eye lacks T-cells, it contains antibodies, cytokines, and resident immune cells. Although precise mechanisms are unclear, intraocular inflammation remains a major cause of vision loss. Here, we used immunoglobulin isotyping and ELISA platforms to profile antibodies in serum and vitreous fluid biopsies from human adult subjects and Cas9-immunized mice. We observed high prevalence of preexisting Cas9-reactive antibodies in serum but not in the eye. However, we detected intraocular antibodies reactive to S. pyogenes-derived Cas9 after S. pyogenes intraocular infection. Our data suggest that serum antibody concentration may determine whether specific intraocular antibodies develop, but preexisting immunity to Cas9 may represent a lower risk in human eyes than systemically.
Insights
Preexisting antibodies against Cas9 proteins are common systemically but rare in the human eye. This suggests a potentially lower risk for CRISPR-Cas9 gene editing therapies targeting the eye.
Area of Science:
- Immunology
- Ophthalmology
- Gene Therapy
Background:
- Preexisting immunity to Cas9 proteins poses a safety concern for CRISPR-Cas9 gene editing applications.
- The relevance of systemic Cas9 immunity to ocular tissues, targeted in early clinical trials, remains largely unknown.
- The eye possesses immune components like antibodies and cytokines, and intraocular inflammation can cause vision loss.
Purpose of the Study:
- To investigate the prevalence and characteristics of preexisting Cas9-reactive antibodies in human serum and ocular fluid.
- To assess the potential risk of Cas9 immunity in the context of in vivo CRISPR-Cas9 ocular therapies.
- To explore the relationship between systemic and intraocular antibody responses to Cas9.
Main Methods:
- Utilized immunoglobulin isotyping and ELISA platforms to analyze serum and vitreous fluid from human subjects and Cas9-immunized mice.
- Quantified Cas9-reactive antibodies in both systemic and ocular compartments.
- Correlated findings with intraocular infection models.
Main Results:
- High prevalence of preexisting Cas9-reactive antibodies was detected in human serum.
- Conversely, Cas9-reactive antibodies were largely absent in ocular fluid from non-infected subjects.
- Intraocular antibodies reactive to Streptococcus pyogenes Cas9 were detected following S. pyogenes intraocular infection.
- Serum antibody levels may influence the development of intraocular antibodies.
Conclusions:
- Preexisting systemic immunity to Cas9 may pose a lower risk to the human eye than previously assumed for CRISPR-Cas9 therapies.
- The development of intraocular antibodies appears dependent on systemic antibody concentrations and local inflammatory triggers.
- Further research is warranted to fully elucidate the implications of Cas9 immunity for ocular gene editing.
