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. 2018 Jun 15;10:105-112.
doi: 10.1016/j.omtm.2018.06.006. eCollection 2018 Sep 21.

Prevalence of Pre-existing Antibodies to CRISPR-Associated Nuclease Cas9 in the USA Population

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Free PMC article

Prevalence of Pre-existing Antibodies to CRISPR-Associated Nuclease Cas9 in the USA Population

Vijaya L Simhadri et al. Mol Ther Methods Clin Dev. .
Free PMC article

Abstract

The repurposing of the CRISPR/Cas microbial adaptive immune system for gene editing has resulted in an exponential rise in new technologies and promising approaches for treating numerous human diseases. While some of the approaches being currently developed involve ex vivo editing by CRISPR/Cas9, many more potential applications will require in vivo editing. The in vivo use of this technology comes with challenges, one of which is the immune response to Cas9, a protein of microbial origin. Thus, the prevalence of pre-existing antibodies to Cas9 could also be a relevant parameter. There are many avenues for how CRISPR/Cas9 technologies will be applied in vivo, including the mode of delivery. These may be expected to invoke different immunological pathways. Nonetheless, as with all protein therapeutics, it may be desirable to monitor for anti-Cas9 antibodies during clinical development. This will require the development of robust and reliable assays. Here, we describe ELISA-based assays that are capable of detecting antibodies to Cas9 from Staphylococcus aureus (SaCas9) and Streptococcs pyogenes (SpCas9) in human sera. Furthermore, using these assays to screen for pre-existing antibodies in 200 human serum samples, we found the prevalence of anti-SaCas9 and anti-SpCas9 antibodies to be 10% and 2.5%, respectively.

Keywords: CRISPR/Cas9; anti-drug antibodies; drug development; gene editing; immunogenicity; pre-existing antibodies.

Figures

Figure 1
Figure 1
Standard Curve of Anti-SaCas9 and Anti-SpCas9 Antibody and Cross-reactivity (A and B) The plates were coated with either SaCas9 (A) or SpCas9 (B) and incubated with increasing concentrations of rabbit polyclonal anti-SaCa9 (red) or mouse monoclonal anti-SpCas9 (blue) antibodies. Data are presented as mean ± SD. The limits of quantitation for the antibodies (0.73 ng/nL for rabbit anti-SaCa9 and 0.24 ng/mL for mouse anti-SpCas9) are indicated by the green arrows.
Figure 2
Figure 2
The Distribution of OD450 Values for SaCas9 in Serum Samples from 48 Donors in the Training Set for the Evaluation of Assay Precision The values are binned by machine (green), analyst (orange), run (blue), and plate position (pink). The box and error bar indicate the distribution of OD450 values for the subset of data identified. The central line shows the median OD450 value, the boxes indicate the range from the 25th to 75th percentiles, and the whiskers extend to 1.5 times the interquartile range of the data. The screening cut point for this data is shown by the red dotted line.
Figure 3
Figure 3
Minimum Required Dilution of Serum for the Measurement of Anti-Cas9 Antibody Levels (A–D) Five individual serum samples were spiked with increasing concentrations of anti-SaCas9 (A and B) and anti-SpCas9 (C and D) antibodies. Eight dilutions of each serum sample were used, as indicated by the colored lines. The 7-point standard curve of antibody concentration was generated by spiking the antibodies into each of 5 serum samples tested at each antibody concentration. For the detection of both anti-SaCas9 and anti-SpCas9 antibodies, a minimum serum dilution of 1:20 exhibited 80% of the dynamic range of the serum-free sample (black dashed line) (B and D). At each antibody concentration, individual OD450 values from serum samples are presented. Our target range for acceptable serum dilutions was the lowest 20% of the dynamic range.
Figure 4
Figure 4
Inhibition of Anti-Cas9 Antibody Binding with Excess Antigen (A and B) The anti-SaCas9 (188 ng/mL) and anti-SpCas9 (250 ng/mL) antibodies were pre-incubated with increasing concentrations of SaCas9 (A) and SpCas9 (B) antigen. The highest concentration of antigen used (200 μg/mL) inhibited binding of anti-SaCas9 and anti-SpCas9 antibody with immobilized Cas9 by 74.7% and 87.8%, respectively.
Figure 5
Figure 5
Anti-Cas9 Antibody-Positive Samples Identified in the Screening Assay The cut points defined with (orange) or without (green) inhibition by excess Cas9 competition are shown as dotted lines. (A and B) The ELISA was carried out using plates coated with SaCas9 (A) or SpCas9 (B). The bars on the extreme left indicate the values obtained using the training set as a reference point. Screening results from the 200 donors are shown for samples whose mean value for the triplicate OD450 reading was above the appropriate cut point. Samples identified as positive based on the cut point calculated without inhibition are shown in green. Additional positive samples identified based on the lower cut point following inhibition with excess Cas9 are shown in orange. Note that samples shown in green are also identified as positive when the lower cut point is used.

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