Immunity to CRISPR Cas9 and Cas12a therapeutics

Chew, Wei Leong

doi:10.1002/wsbm.1408

Cited by 112 publications

(92 citation statements)

References 306 publications

(503 reference statements)

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“…However, this is the first study that experimentally validates predicted immunodominant epitopes. None of the epitopes we report overlap with the peptides previously predicted 42 . This is not unsurprising since we restricted our analysis to one HLA haplotype.…”

Section: Discussioncontrasting

confidence: 61%

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Multifunctional CRISPR/Cas9 with engineered immunosilenced human T cell epitopes

Ferdosi

Ewaisha

Moghadam

et al. 2018

Preprint

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The application of Cas9 for genetic and epigenetic therapies in humans raises concerns over immunogenicity of this foreign protein. We report pre-existing human CD8+ T cell immunity to Streptococcus pyogenes Cas9 in the majority of healthy individuals screened. In a proof-ofprinciple study, we demonstrate that Cas9 protein can be modified to eliminate immunodominant epitopes through targeted mutation while preserving its function and specificity.Of the Cas9 orthologs derived from bacterial species, the SpCas9 is the best characterized. S. pyogenes is a ubiquitous pathogen, with an annual incidence of 700 million worldwide 26 , but immunity to SpCas9 in humans has not been reported. Here, we sought to characterize the pre-existing immune response to SpCas9 in healthy individuals and to identify the immunodominant T cell epitopes with the aim of developing SpCas9 proteins that have diminished capacity to invoke human adaptive response.CRISPR application for human therapies will span its use both for gene editing (through DNA double-strand breaks) or epigenetic therapies (without DNA double-strand breaks). In fact, recent reports shed light on CRISPR's ability to activate or repress gene expression in mice [27][28][29] , which opens the door to a variety of new therapeutic applications such as activating silent genes, compensating for disrupted genes, cell fate reprogramming, or silencing disrupted genes, without the concern over permanent change in DNA sequence. However, unlike the use of Cas9 for gene editing, which may only require Cas9 presence in cells for a few hours, current techniques for CRISPR-based epigenetic therapies require longer term expression of Cas9 in vivo, possibly for weeks and months 28,29 , which poses the challenge of combating pre-existing immune response towards Cas9. This challenge will need to be addressed before CRISPR application for human therapies, especially for epigenetic therapies, can be fully implemented. Delivery of CRISPR in vivo by incorporating its expression cassette in adeno-associated virus (AAV), will most likely shape many of the initial clinical trials as AAV-based gene delivery is one of the safest and most prevalent forms of gene therapies in human. AAV will enable longer term expression of Cas9, desirable for epigenetic therapies. Therefore, unlike Cas9 delivery in the form of ribonucleoprotein complexes (which are short term), it is highly likely that CRISPR delivery through AAV and its expression within target cells will engage CD8+ T cell immunity.

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Section: Discussioncontrasting

confidence: 61%

“…The top binding T cell epitopes within Cas9 that are most promiscuous for common HLA class I and class II alleles have been recently predicted in silico using IEDB 42 . However, this is the first study that experimentally validates predicted immunodominant epitopes.…”

Section: Discussionmentioning

confidence: 99%

Multifunctional CRISPR/Cas9 with engineered immunosilenced human T cell epitopes

Ferdosi

Ewaisha

Moghadam

et al. 2018

Preprint

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“…The Cas9 protein is derived from the bacteria Streptococcus pyogenes (Zhang 2019) and can trigger an immune response in mice (Chew et al 2016;Chew 2018) . During the gene drive multiplication process, germline cells produce Cas9 proteins to cut the DNA and insert the gene drive cassette (Fig.…”

Section: Probability That the Immune System Does Not Eliminate Cas9-ementioning

confidence: 99%

“…In vertebrates, if Cas9 proteins accumulate in somatic tissues at a late stage during development due to leakage of the cis-regulatory regions controlling Cas9 gene expression, Cas9 fragments may be recognized as foreign molecules and trigger an adaptive immune T cell response, leading to the potential elimination of the Cas9-expressing cells and a probable decrease in fitness of the individual carrying the gene drive (Chew 2018) . The expression of Cas9 at an early stage of development may also activate an immune response if the gene drive carrier has herited anti-Cas9 antibodies from its mother, as maternal immunoglobulins G have been shown to cross the placental barrier and the intestine, and to be maintained for a long time in the fetus after birth (Madani and Heiner 1989;Roopenian and Akilesh 2007).…”

Section: Probability That the Immune System Does Not Eliminate Cas9-ementioning

confidence: 99%

Evaluating the Probability of CRISPR-based Gene Drive Contaminating Another Species

Courtier‐Orgogozo

Danchin

Gouyon

et al. 2019

Preprint

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The probability D that a given CRISPR-based gene drive element contaminates another, non-target species can be estimated by the following Drive Risk Assessment Quantitative Estimate (DRAQUE) Equation: D = ( hyb+transf).express.cut.flank.immune.nonextinct with hyb = probability of hybridization between the target species and a non-target species transf = probability of horizontal transfer of a piece of DNA containing the gene drive cassette from the target species to a non-target species (with no hybridization) express = probability that the Cas9 and guide RNA genes are expressed cut = probability that the CRISPR-guide RNA recognizes and cuts at a DNA site in the new host flank = probability that the gene drive cassette inserts at the cut site immune = probability that the immune system does not reject Cas9 -expressing cells nonextinct = probability of invasion of the drive within the populationWe discuss and estimate each of the seven parameters of the equation, with particular emphasis on possible transfers within insects, and between rodents and humans. We conclude from current data that the probability of a gene drive cassette to contaminate another species is not insignificant. We propose strategies to reduce this risk and call for more work on estimating all the parameters of the formula.

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“…1,2 Immunity against therapeutic gene vectors or gene-modifying cargo nullifies the effect of a possible curative treatment and may pose significant safety issues. 3-5 Immunocompetent mice treated with CRISPR/Cas9-encoding vectors exhibit humoral and cellular immune responses against the Cas9 protein, that impact the efficacy of treatment and can cause tissue damage. 5,6 Most applications aim to temporarily express the Cas9 nuclease in or deliver the protein directly into the target cell.…”

mentioning

confidence: 99%

High prevalence of S. pyogenes Cas9-specific T cell sensitization within the adult human population – A balanced effector/regulatory T cell response

Wagner

Amani

Wendering

et al. 2018

Preprint

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Paragraph 32The field of gene therapy has been galvanized by the discovery of the highly efficient and 33 adaptable site-specific nuclease system CRISPR/Cas9 from bacteria. 1,2 Immunity against 34 therapeutic gene vectors or gene-modifying cargo nullifies the effect of a possible curative 35 treatment and may pose significant safety issues. [3][4][5] Immunocompetent mice treated with 36 CRISPR/Cas9-encoding vectors exhibit humoral and cellular immune responses against the 37 Cas9 protein, that impact the efficacy of treatment and can cause tissue damage. 5,6 Most 38 applications aim to temporarily express the Cas9 nuclease in or deliver the protein directly into 39 the target cell. Thus, a putative humoral antibody response may be negligible. 5 However, 40 intracellular protein degradation processes lead to peptide presentation of Cas9 fragments on 41 the cellular surface of gene-edited cells that may be recognized by T cells. While a primary T 42 cell response could be prevented or delayed, a pre-existing memory would have major impact. 43Here, we show the presence of a ubiquitous memory/effector T cell response directed towards 44 the most popular Cas9 homolog from Streptococcus pyogenes (SpCas9) within healthy human 45 subjects. We have characterized SpCas9-reactive memory/effector T cells (TEFF) within the 46 CD4/CD8 compartments for multi-effector potency and lineage determination. Intriguingly, 47SpCas9-specific regulatory T cells (TREG) profoundly contribute to the pre-existing SpCas9-48 directed T cell immunity. The frequency of SpCas9-reactive TREG cells inversely correlates with 49 the magnitude of the respective TEFF response. SpCas9-specific TREG may be harnessed to 50 ensure the success of SpCas9-mediated gene therapy by combating undesired TEFF response 51 in vivo. Furthermore, the equilibrium of Cas9-specific TEFF and TREG cells may have greater 52 importance in Streptococcus pyogenes-associated diseases. Our results shed light on the T 53 cell mediated immunity towards the much-praised gene scissor SpCas9 and offer a possible 54 solution to overcome the problem of pre-existing immunity. 55 Text 56SpCas9 was the first Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) 57 associated nuclease hijacked to introduce DNA double-strand breaks at specific DNA sequences. 1 58Through the ease of target adaption and the remarkable efficacy, it advanced to the most popular 59 tool for re-writing genes in research and potential clinical applications. The major concern for 60 clinical translation of CRISPR/Cas9 technology is the risk for off-target activity causing potentially 61 harmful mutations or chromosomal aberrations. 2,7 High-fidelity Cas9 enzymes were developed to 62 reduce the probability of these events. 8 Furthermore, novel Cas9-based fusion proteins allow base 63 editing or specific epigenetic reprogramming without inducing breaks in the DNA. 9,10 Most 64 approaches are based on the original SpCas9 enzyme that originates in the facultatively 65 2 pathogenic bacterium Streptococcus ...

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Immunity to CRISPR Cas9 and Cas12a therapeutics

Cited by 112 publications

References 306 publications

Multifunctional CRISPR/Cas9 with engineered immunosilenced human T cell epitopes

Multifunctional CRISPR/Cas9 with engineered immunosilenced human T cell epitopes

Evaluating the Probability of CRISPR-based Gene Drive Contaminating Another Species

High prevalence of S. pyogenes Cas9-specific T cell sensitization within the adult human population – A balanced effector/regulatory T cell response

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