Jianhua Luo, MD, PhD, professor of pathology at the University of Pittsburgh Medical Center, Pennsylvania, told Cancer Therapy Advisor that he doubts preexisting immunity to Cas9 will affect the safety or efficacy of ex vivo CRISPR-based cancer therapies. John DiPersio, MD, PhD, deputy director of Siteman Cancer Center, Saint Louis, Missouri, also expressed a similar view to Cancer Therapy Advisor. Neither were involved in the current study.

“In ex vivo treatments,” Dr DiPersio explained, “the [Cas9] gene is expressed transiently. There’s no impact on the immune system since it’s done outside the body, and the expression of protein goes away in a very short period of time.”

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Although the general consensus in the field is that ex vivo CRISPR-based therapies are unaffected by preexisting immunity, Wagner said there is evidence that suggests otherwise. “There [are] some data hidden in papers and a few rumors suggesting Cas9 protein may stick on electroporated cells after ex vivo genome editing for days and sometimes even weeks,” Wagner explained, citing supplemental data from Annalisa Lattanzi, PhD, program manager at Stanford University School of Medicine, California, and colleagues.4 “If this proves to be true, the antibodies described by Charlesworth et al and Simhadri et al may be a bigger problem for ex vivo CRISPR therapies.”

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The real threat preexisting immunity poses is to in vivo CRISPR-based therapies because Cas9 doesn’t stay hidden within the target cell forever. Eventually the Cas9 protein breaks down, and fragments of the protein are displayed on the surface of the cell through major histocompatibility complex antigens. Roaming antigen-presenting cells then detect the fragments and activate a T cell, which returns to attack the genetically modified cell.

The impact of preexisting immunity to Cas9 depends on several factors, such as choice of vector and target tissue, Jeffrey Chamberlain, PhD, told Cancer Therapy Advisor. He is a professor at the University of Washington School of Medicine, Seattle, and was not involved in the current study.

For example, when the commonly used adeno-associated viral vector is used to deliver Cas9 to certain tissues that don’t turn over quickly, such as muscle and liver, Cas9 gene expression can persist for years. “If there is some preexisting immunity to Cas9, that’s plenty of time for that to get activated and potentially start attacking the cells that you have been trying to repair,” he said.

In the oncology space, it’s unclear how much preexisting immunity will affect in vivo CRISPR-based cancer therapies because the approach is rare, with one clinical trial slated to evaluate an in vivo CRISPR-based therapy for human papillomavirus-related cervical cancer (ClinicalTrials.gov Identifier: NCT03057912); the trial is not yet recruiting.

Furthermore, cancer is generally not driven by a single gene, making it more complicated to treat with in vivo gene editing than perhaps some other genetic diseases. “We know that in most cancers, there are many mutations and several driver mutations,” Dr DiPersio said. “It’s not really clear that if you targeted one gene of the many that are mutated, that would result in a complete resolution of the phenotype.” The exceptions to this, he said, may be chronic myelogenous leukemia and a few other rare cancers.

Even using in vivo gene editing to prevent inherited cancers, which on the surface, seems similar to treating a genetic disease, may be “very challenging” Dr DiPersio said, because doing so would require targeting multiple tissue types. For example, a germline TP53 mutation can result in tumors in many tissues, which means the mutation would have to be reversed in all those tissues.

Despite the mounting evidence in favor of preexisting immunity to Cas9, Wagner and colleagues may have also found a potential solution. They discovered that a substantial amount of the Cas9-reactive T cells were regulatory T cells, and these regulatory T cells could suppress cytotoxic Cas9-reactive T cells, suggesting a possible way to overcome preexisting immunity. Wagner said, “We hope that these [regulatory T] cells could be exploited to enable safe and successful CRISPR-Cas9 gene therapy.”


  1. Wagner DL, Amini L, Wendering DJ, et al. High prevalence of Streptococcus pyogenes Cas9-reactive T cells within the adult human population [published online October 29, 2018]. Nat Med. doi: 10.1038/s41591-018-0204-6
  2. Charlesworth CT, Deshpande PS, Dever DP, et al. Identification of pre-existing adaptive immunity to Cas9 proteins in humans [published online January 5, 2018]. bioRxiv. doi: 10.1101/243345
  3. Simhadri VL, McGill J, McMahon S, Wang J, Jiang H, and Sauna ZE. Prevalence of pre-existing antibodies to CRISPR-associated nuclease Cas9 in the USA population. Mol Ther Methods Clin Dev. 2018;10:105-112.
  4. Lattanzi A, Meneghini V, Pavani G, et al. Optimization of CRISPR/Cas9 delivery to human hematopoietic stem and progenitor cells for therapeutic genomic rearrangements [published online October 17, 2018]. Mol Ther. doi: 10.1016/j.ymthe.2018.10.008