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Uncovering Immunogenic Peptides of Clinical Significance in CRISPR/Cas9

The risk of adverse immune responses following exposure to foreign-bacterial proteins is a potential hurdle to the successful broad implementation of CRISPR/Cas9 based gene and cell therapies. Anti-Cas9 adaptive responses, such as those mediated by neutralizing antibodies or cytotoxic T cell responses, can also impact the efficacy of CRISPR/Cas9 based therapies and potentially negate its benefits to some patients.

These risks have been highlighted by studies analyzing in vitro B and T cell responses against Cas9 in human samples. For example, work in the Porteus lab confirmed that, as expected, previous encounters with common pathogens such as Staphylococcus aureus and Staphylococcus pyogenes trigger anti-Cas9 adaptive immune responses against SaCas9 and SpCas9, respectively. In fact, the Porteus team found a high prevalence of antibody and T cell-mediated responses in serum and peripheral blood mononuclear cell (PBMC) samples from donors following in vitro testing (Charlesworth et al. 2019). These findings agree with previous work, which similarly supported the high prevalence of CD4+ and CD8+ T cell responses against SpCas9 amongst adult donors' samples (Wagner et al. 2018). 

Recognizing that adverse immune responses may limit the reach of CRISPR/Cas9 based therapies, more recently, Simhadri and colleagues took a closer look at Cas9 protein regions associated with preexisting adaptive immune reactivity (Simhadri et al. 2021). By leveraging two approaches: (1) T cell proliferation assays with full-length Cas9 protein and overlapping peptide pools, and (2) mass spectrometry (MS)-based MHC associated peptide proteomics (MAPPs) assay, Simhadri et al. elucidated which Cas9 derived peptides are more frequently and robustly expected to induce CD4+ T cell responses within a population.

T cell activation assays

T cell activation assays rely on the use of donor PBMCs, which are exposed to specific proteins or peptides (e.g., Cas9 protein or peptide libraries) for uptake by antigen-presenting cells (APCs) and eventual MHC dependent presentation of processed peptides to T cells.  Several methods may be leveraged to analyze preexistent T cell adaptive immune responses against bacterial proteins such as Cas9. Some of these assays include ELISPOT for detection of secreted cytokines (e.g., interferon-gamma) and immunostaining for intracellular cytokine and cell membrane activation makers (e.g., CD137 and CD154) (Charlesworth et al. 2019). Created with BioRender.com.

For this work, Simhadri and colleagues used a library of overlapping peptides synthesized by GenScript. A total of 21 pools of synthetic peptides spanning the SaCas9 sequence were used for T cell activation assays. Each pool consisted of 15mer peptides overlapping by 5 amino acids. Following incubation of donor-derived PBMCs with each peptide pool, T cell activation was determined based on positive immunostaining for cytokines, such as interferon-gamma, tumor necrosis factor-alpha, and interleukin 2.

Simhadri and colleagues found that peptide pool-induced T cell responses varied significantly amongst donors, with some donor samples mainly being insensitive to most peptide pools. Nevertheless, the majority of donors were responsive to several peptide pools. The team characterized combinations of MHC-II alleles and peptide pools underscoring CD4+ T cell reactivity in donor samples. Because the frequency of specific MHC-II alleles can be elucidated for any given population, this type of analysis promises to be a valuable approach to estimating the likelihood of T cell responses within a group.

Mass spectrometry (MS)-based MHC associated peptide proteomics (MAPPs) assay

Mass spectrometry (MS)-based MHC associated peptide proteomics (MAPPs) assay. Monocyte-derived dendritic cells cultured with full-length SaCas9 enabled peptide processing and presentation by MHC-II receptors. Recovery of MHC-II bound peptides and elution allowed the identification by LC-MS/MS of relevant Cas9 derived peptides. Modified from “MHC Immunoaffinity Assay,” by BioRender.com (2021). Retrieved from https://app.biorender.com/biorender-templates.

Next, MAPPs assays allowed the team to identify relevant SaCas9 derived peptides presented by MHC-II proteins. Lastly, findings from T cell activation and MAPPs assays supported the identification of 22 peptides from SaCas9, which may be relevant in the context of CRISPR/Cas9-based clinical applications, as they are likely to be presented by MHC-II receptors and induce CD4+ T cell activation.

Current CRISPR/Cas9 based clinical strategies include ex vivo cell editing (e.g., CAR T cell for immunotherapy) and in vivo gene editing applications (e.g., hereditary transthyretin amyloidosis). For instance, strategies relying on CRISPR/Cas9-based non-viral approaches are increasingly favored for CAR T cell therapeutics development. In this approach, the ribonucleoprotein (Cas9 and guide RNA) complex is delivered directly into T cells, which limits the persistence of the Cas9 protein within cells, and is thought to reduce the risk of potential adverse immune responses once cells are re-infused into patients (Charlesworth et al. 2019).

However, the risk of adverse anti-Cas9 immune reactions becomes a more significant concern for in vivo approaches. The very first Phase I trials for a non-viral CRISPR/Cas9 based therapy administered systemically were started in 2020. The study aimed to evaluate dosing, safety, tolerability, pharmacokinetics, and pharmacodynamics of NTLA-2001, consisting of a guide RNA targeting the transthyretin gene (TTR) and mRNA encoding the Cas9 protein delivered in lipid nanoparticles. A recent report by Gillmore et al. on findings from the first 6 patients dosed supports the efficacy and safety of the approach, albeit some mild adverse events were documented (Gillmore et al. 2021).

Overall, Simhadri and colleagues have developed methods and strategies to better understand and perhaps predict potential adverse immune responses in human patients upon exposure to Cas9 protein. Because CRISPR/Cas9 approaches continue to advance rapidly in the clinic, promising curative solutions for a broad range of rare diseases, understanding the potential for adverse immunological reactions is essential to ensure the safety of these gene and cell therapies.


Charlesworth, C. T. et al. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat. Med. (2019) doi:10.1038/s41591-018-0326-x.

Gillmore, J. D. et al. CRISPR-Cas9 In Vivo Gene Editing for Transthyretin Amyloidosis. N. Engl. J. Med. (2021) doi:10.1056/nejmoa2107454.

Simhadri, V. L. et al. Cas9-derived peptides presented by MHC Class II that elicit proliferation of CD4+ T-cells. Nat. Commun. (2021) doi:10.1038/s41467-021-25414-9.

Wagner, D. L. et al. High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population. Nat. Med. (2019) doi:10.1038/s41591-018-0204-6.

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