Cas9 immunity creates challenges for CRISPR gene editing therapies

Crudele, Julie M.; Chamberlain, Jeffrey S.

doi:10.1038/s41467-018-05843-9

Cited by 198 publications

(137 citation statements)

References 15 publications

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“…126,127 The barriers to broad clinical translation of CRISPR-Cas systems include (1) imperfect DNA-targeting specificity leading to off-target effects, 128 (2) low efficiency of genome editing using HDR, 129 and (3) challenging delivery of CRISPR-Cas components. 130 More recently, concerns were raised about the immune response toward Cas9 and the presence of neutralizing antibodies; 131 the potential for large deletions and complex rearrangement induced by Cas9 cleavage; 132 and activation of the p53 pathway, which can antagonize the efficiency of Cas9-mediated gene editing. 133 The therapeutic protein itself can also be a source of immune response, in particular when treating genetic diseases.…”

Section: Biodegradability and Targeting Issues With Non-viral Vectorsmentioning

confidence: 99%

“…In addition, the transient nature of the mRNA expression limits the presence of the nuclease inside the cells, reducing the risks of off-target cutting and immune response toward Cas9 protein; however, the intracellular presence of Cas9 protein has been more persistent after mRNA expression as compared to the delivery of Cas9-RNPs. 131 Thus far several studies have demonstrated the therapeutic potential of local in vivo Cas9-RNP delivery, 136,137 while systemic RNP delivery still needs to be evaluated in contrast to more established mRNA-based approaches. 130 The use of mRNA could also mitigate obstacles associated with Cas9-RNPs, such as manufacturing and preserving the activity of Cas9 protein as well as challenging in vivo protein delivery.…”

Section: Biodegradability and Targeting Issues With Non-viral Vectorsmentioning

confidence: 99%

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Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery

et al. 2019

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mRNA has broad potential as a therapeutic. Current clinical efforts are focused on vaccination, protein replacement therapies, and treatment of genetic diseases. The clinical translation of mRNA therapeutics has been made possible through advances in the design of mRNA manufacturing and intracellular delivery methods. However, broad application of mRNA is still limited by the need for improved delivery systems. In this review, we discuss the challenges for clinical translation of mRNA-based therapeutics, with an emphasis on recent advances in biomaterials and delivery strategies, and we present an overview of the applications of mRNA-based delivery for protein therapy, gene editing, and vaccination. mRNA holds the potential to revolutionize vaccination, protein replacement therapies, and the treatment of genetic diseases. Since the first pre-clinical studies in the 1990s, 1 significant progress in the clinical translation of mRNA therapeutics has been made through advances in the design of mRNA manufacturing and intracellular delivery methods. 2 The translatability and stability of mRNA as well as its immunostimulatory activity are the key factors to be optimized for specific therapeutic application. 3 Increased translation and stability can be affected by many regions of the RNA. mRNA 5 0 and 3 0 UTRs are responsible for recruiting RNA-binding proteins and microRNAs, and they can profoundly affect translational activity. 2,4 The modification of rare codons in protein-coding sequences with synonymous frequently occurring codons, so-called codon optimization, can result in order-of-magnitude changes in expression levels. 5,6 Modification of the 5 0 mRNA cap can also enhance mRNA translation by inhibiting RNA decapping and improving resistance to enzymatic degradation. 7 Chemical modification of RNA bases can be used to modify mRNA immunostimulatory activity. 8,9 The importance of immunostimulation can depend on the application, 10 and, in some cases, it may actually improve performance, as in the case of vaccines. 11Finally, methods and vehicles for intracellular delivery remain the major barrier to the broad application of mRNA therapeutics. 12 With some exceptions, the intracellular delivery of mRNA is generally more challenging than that of small oligonucleotides, and it requires encapsulation into a delivery nanoparticle, in part due to the significantly larger size of mRNA molecules (300-5,000 kDa, 1-15 kb) as compared to other types of RNAs (small interfering RNAs [siRNAs], 14 kDa; antisense oligonucleotides [ASOs], 4-10 kDa). 10,13 In this review, we discuss the challenges for clinical translation of mRNAbased therapeutics, with an emphasis on recent advances in biomaterials and delivery strategies, and we present an overview of the applications of mRNA-based delivery for protein therapy, gene editing, and vaccination. Materials for mRNA Delivery Structural Aspects of Material DesignAmong the many barriers to function, mRNA must cross the cell membrane in order to reach the cytoplasm (Figure 1). The cell me...

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Section: Biodegradability and Targeting Issues With Non-viral Vectorsmentioning

confidence: 99%

Section: Biodegradability and Targeting Issues With Non-viral Vectorsmentioning

confidence: 99%

Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery

et al. 2019

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“…However, unexpected nuances of the CRISPR-Cas9 editing system continue to emerge. Varied biological responses to CRISPR-Cas9, such as DNA damage repair (Haapaniemi et al, 2018), enzymatic immunity (Crudele and Chamberlain, 2018), and alternative templating (Ma et al, 2017) exemplify our still nascent understanding of DNA and RNA editing. Furthermore, natural human genetic variation has been shown to influence both the efficaciousness of on-target editing and the frequency of off-target editing (Lessard et al, 2017).…”

Section: Discussionmentioning

confidence: 99%

Analysis of single nucleotide variants in CRISPR-Cas9 edited zebrafish embryos shows no evidence of off-target inflation

Mooney

Davis

Katsanis

2019

Preprint

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1Therapeutic applications of CRISPR-Cas9 gene editing have spurred innovation in Cas9 2 enzyme engineering and single guide RNA (sgRNA) design algorithms to minimize potential off-3 target events. While recent work in rodents outlines favorable conditions for specific editing and 4 uses a trio design to control for the contribution of natural genome variation, the potential for 5 CRISPR-Cas9 to induce de novo mutations in vivo remains a topic of interest. In zebrafish, we 6 performed whole exome sequencing (WES) on two generations of offspring derived from the 7 same founding pair: 54 exomes from control and CRISPR-Cas9 edited embryos in the first 8 generation (F0), and 16 exomes from the progeny of inbred F0 pairs in the second generation 9 (F1). We did not observe an increase in the number of transmissible variants in edited 10 individuals in F1, nor in F0 edited mosaic individuals, arguing that in vivo editing does not 11 precipitate an inflation of deleterious point mutations. 12 Results 34 Generating and sequencing CRISPR-Cas9-edited F0 and F1 individuals 35We focused on three different genes (anln, kmt2d, and smchd1) for which (a) we have 36 substantial experience in this model organism and (b) which give reproducible, quantitative 37 defects in kidney morphogenesis (Hall et al., 2018), mandibular and neuronal development 38 (Tsai et al., 2018), and craniofacial morphogenesis (Shaw et al., 2017). For each locus, we used 39 sgRNAs that had the following three characteristics. First, for each of the three genes, we 40 selected an sgRNA with demonstrated high efficiency (100%) and an sgRNA with low efficiency 41 (~30%), as determined by heteroduplex analysis and Sanger sequencing of cloned PCR 42 products (Hall et al., 2018; Shaw et al., 2017; Tsai et al., 2018) (Suppl. Figure 1). Second, we 43 mandated that all sgRNAs have a high specificity score (MIT specificity score 79-99 for each 44 sgRNA; Suppl. Table S1). Finally, we required that each sgRNA was predicted to generate off-45 target effects with low cutting frequency determination (CFD) scores (mean = 0.17, range = 0-46 0.73; Suppl. Figure 2). 47 289

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“…Recent reports have highlighted pre-existing immunity towards both SpCas9 and SaCas9 in the human population, with a high prevalence of both Cas9-reactive T cells and antibodies [71]. Although it is still unclear whether adeno-associated virus delivery of Cas9 leads to the immune rejection of transduced cells in vivo , strategies to control the anti-Cas9 T cell responses, such as transient immunosuppression or engineering Cas9 proteins with mutated T cell epitopes, are being considered [72]. Another limitation of Cas9 for its use in gene therapy resides in its rather large size, which is incompatible for efficient packaging into adeno-associated virus vectors, the most commonly used delivery systems in gene therapy.…”

Section: Future Perspectivementioning

confidence: 99%

Guidelines for Optimized Gene Knockout Using CRISPR/Cas9

et al. 2019

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CRISPR/Cas9 technology has evolved as the most powerful approach to generate genetic models both for fundamental and preclinical research. Despite its apparent simplicity, the outcome of a genome-editing experiment can be substantially impacted by technical parameters and biological considerations. Here, we present guidelines and tools to optimize CRISPR/Cas9 genome-targeting efficiency and specificity. The nature of the target locus, the design of the single guide RNA and the choice of the delivery method should all be carefully considered prior to a genome-editing experiment. Different methods can also be used to detect off-target cleavages and decrease the risk of unwanted mutations. Together, these optimized tools and proper controls are essential to the assessment of CRISPR/Cas9 genome-editing experiments.

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Cas9 immunity creates challenges for CRISPR gene editing therapies

Cited by 198 publications

References 15 publications

Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery

Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery

Analysis of single nucleotide variants in CRISPR-Cas9 edited zebrafish embryos shows no evidence of off-target inflation

Guidelines for Optimized Gene Knockout Using CRISPR/Cas9

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