Heat shock protein 70/peptide complexes: potent mediators for the generation of antiviral T cells particularly with regard to low precursor frequencies

<p>Abstract</p> <p>Background</p> <p>Heat shock protein 70 (HSP70) has gained major attention as an adjuvant capable of inducing antigen-specific CD8⁺and CD4⁺T-cell responses. The ability of HSP70/peptide complexes to elicit cytotoxic T-cell (CTL) responses by cross-presentation of exogenous antigens via HLA class I molecules is of central interest in immunotherapy. We examined the role of HSP70/CMVpp65_495-503-peptide complex (HSP70/CMV-PC) in HLA class I-restricted cross-presentation for <it>ex vivo </it>expansion of CMV-specific CTLs.</p> <p>Methods</p> <p>CMV-specific T cells generated from PBMCs of HLA-A*02:01/CMV-seropositive donors were stimulated for 21 days with HSP70/CMV-PC and analyzed in functional assays. As a control PBMCs were cultured in the presence of CMVpp65_495-503peptide or HSP70. Increase of CMV-specific CTLs was visualized by pentameric HLA-A*02:01/CMVpp65_495-503complex.</p> <p>Results</p> <p>About 90% of HSP70/CMV-PC generated T cells were CMV-specific and exhibited significantly higher IFN-γ secretion, cytotoxic activity, and an increased heme oxygenase 1 (HO-1) gene expression as compared to about 69% of those stimulated with CMVpp65_495-503peptide. We decided to classify the HLA-A*02:01/CMV-seropositive donors as weak, medium, and strong responder according to the frequency of generated A2/CMV-pentamer-positive CD8⁺T cells. HSP70/CMV-PC significantly induces strong antiviral T-cell responses especially in those donors with low memory precursor frequencies. Blockage of CD91 with α2-macroglobulin markedly reduced proliferation of antiviral T cells suggesting a major role of this receptor in the uptake of HSP70/CMV-PC.</p> <p>Conclusion</p> <p>This study clearly demonstrates that HSP70/CMV-PC is a potent mediator to induce stronger T-cell responses compared to antiviral peptides. This simple and efficient technique may help to generate significant quantities of antiviral CTLs by cross-presentation. Thus, we propose HSP70 for chaperoning peptides to reach an efficient level of cross-presentation. HSP70/peptide complexes may be particularly useful to generate stronger T-cell responses in cases of low precursor frequencies and may help to improve the efficiency of antigen-specific T-cell therapy for minor antigens.</p

Minimal 2'-O-methyl phosphorothioate linkage modification pattern of synthetic guide RNAs for increased stability and efficient CRISPR-Cas9 gene editing avoiding cellular toxicity

<div><p>Since its initial application in mammalian cells, CRISPR-Cas9 has rapidly become a preferred method for genome engineering experiments. The Cas9 nuclease is targeted to genomic DNA using guide RNAs (gRNA), either as the native dual RNA system consisting of a DNA-targeting CRISPR RNA (crRNA) and a <i>trans</i>-activating crRNA (tracrRNA), or as a chimeric single guide RNA (sgRNA). Entirely DNA-free CRISPR-Cas9 systems using either Cas9 protein or Cas9 mRNA and chemically synthesized gRNAs allow for transient expression of CRISPR-Cas9 components, thereby reducing the potential for off-targeting, which is a significant advantage in therapeutic applications. In addition, the use of synthetic gRNA allows for the incorporation of chemical modifications for enhanced properties including improved stability. Previous studies have demonstrated the utility of chemically modified gRNAs, but have focused on one pattern with multiple modifications in co-electroporation with Cas9 mRNA or multiple modifications and patterns with Cas9 plasmid lipid co-transfections. Here we present gene editing results using a series of chemically modified synthetic sgRNA molecules and chemically modified crRNA:tracrRNA molecules in both electroporation and lipid transfection assessing indel formation and/or phenotypic gene knockout. We show that while modifications are required for co-electroporation with Cas9 mRNA, some modification patterns of the gRNA are toxic to cells compared to the unmodified gRNA and most modification patterns do not significantly improve gene editing efficiency. We also present modification patterns of the gRNA that can modestly improve Cas9 gene editing efficiency when co-transfected with Cas9 mRNA or Cas9 protein (> 1.5-fold difference). These results indicate that for certain applications, including those relevant to primary cells, the incorporation of some, but not all chemical modification patterns on synthetic crRNA:tracrRNA or sgRNA can be beneficial to CRISPR-Cas9 gene editing.</p></div

Modest improvement of gene editing efficiency with some MS-modified gRNAs was observed using Cas9 mRNA and Cas9 protein in lipid co-transfections.

<p>Unmodified (unmod) and modified guide RNAs were co-transfected with Cas9 mRNA (<b>A</b> and <b>B</b>) or Cas9 protein (<b>C</b> and <b>D</b>). Gene editing efficiencies were estimated for <i>PPIB</i>-targeting guide RNAs in U2OS cells (light blue bars) and HeLa cells (dark blue bars; <b>A</b> and <b>C</b>). Functional gene knockout was quantified using a phenotypic analysis with <i>PSMD7-</i> (light blue bars) or <i>PSMD11</i>- (dark blue bars) targeting guide RNAs in a Ubi-EGFP U2OS cell line (<b>B</b> and <b>D</b>). Cell viability was assessed for each lipid transfection experiment: U2OS cells (dark green boxes) and HeLa cells (light green boxes; <b>A</b> and <b>C</b>) and <i>PSMD7</i> (dark green boxes) and <i>PSMD11</i> (light green boxes; <b>B</b> and <b>D</b>). NTC = Non-targeting control. Error bars representative of biological triplicates.</p

Minimal modification of guide RNA improves stability to increase gene editing efficiency with Cas9 mRNA in co-electroporation.

<p><b>A.</b> Modification patterns of synthetic sgRNA (100-mer), crRNA (42-mer) and tracrRNA (74-mer) with one to three 2’-O-methyl modifications with 3’ phosphorothioate linkages (MS, denoted with red *). 1x-3xMS modifications were added to both ends of crRNA, tracrRNA and sgRNA (left column) or the 5’ or 3’ end of crRNA and tracrRNA (right column) and tested in the different combinations shown. Unmodified and modified synthetic guide RNAs targeting genes <i>PPIB</i>, <i>PSMD7</i> and <i>PSMD11</i> were co-electroporated with Cas9 mRNA into K-562 cells (duplicate samples shown; <b>B-D</b>). <b>B.</b> Co-electroporation of Cas9 mRNA and 1x-3xMS-modified synthetic sgRNA all showed similar levels of gene editing as estimated from a DNA mismatch detection assay. <b>C.</b> Unmodified (unmod) crRNA:tracrRNA produced no detectable (n.d.) editing for any of the gene targets, while 1x-3xMS-modified crRNA and tracrRNA had detectable, but varying, levels of gene editing efficiencies. <b>D.</b> Single-end modifications on crRNA and tracrRNA indicated that modification of the 5’ end of crRNA is important for stability for efficient gene editing. The numbers under each gel image are percentage of gene editing. UT = Untreated; NTC = Non-targeting control, M = DNA ladder.</p

Summary of results from modifying gRNAs in different applications compared to unmodified gRNAs.

<p>A comparison of unmodified and modified gRNAs in different applications, from electroporations to lipid co-transfections, based on the results in this paper. N.d. = not determined, * some modification patterns can be toxic to cells.</p

Addition of MS modifications on guide RNAs may improve gene editing efficiency with Cas9 protein in co-electroporation of RNPs.

<p>Cas9 protein and unmodified or modified synthetic guide RNAs targeting <i>PPIB</i>, <i>PSMD7</i> and <i>PSMD11</i> were complexed and delivered as RNPs into K-562 cells using co-electroporation (duplicate samples shown). <b>A.</b> For most modification patterns, similar gene editing efficiencies were detected with both ends modified sgRNA when compared to unmodified (unmod) sgRNA; only one gene (<i>PSMD7</i>) showed increased (1.8-fold) gene editing with 1x-2xMS modifications. <b>B.</b> Co-electroporation of both ends and single-end modified crRNA:tracrRNA for genes <i>PSMD7</i> and <i>PSMD11</i> with Cas9 protein did not result in consistent increase or decrease in gene editing efficiencies compared to unmodified, but increased gene editing with some modification patterns targeting <i>PPIB</i>. The numbers under each gel image are percentage of gene editing. UT = Untreated, NTC = Non-targeting control, M = DNA ladder.</p

Unmodified crRNA:tracrRNA require a sequential electroporation method with Cas9 mRNA for gene editing.

<p>Sequential electroporation involves electroporation of Cas9 mRNA, followed 6 hours later by electroporation of synthetic guide RNA and harvested 2–3 days later for analysis. With a co-electroporation method, both Cas9 mRNA and synthetic guide RNA are delivered into cells at the same time, then harvested 2–3 days later. No detectable (n.d.) gene editing was observed with unmodified synthetic crRNA:tracrRNA in co-electroporation (Co) with Cas9 mRNA into K-562 cells for three gene targets, but resulted in a significant increase when with sequential electroporation (Seq, duplicate samples). UT = Untreated, NTC = Non-targeting control, M = DNA ladder.</p

Strongest PAMs including degenerate base designations.

<p>Strongest PAMs including degenerate base designations.</p

Representative agarose gel images from the <i>in vitro</i> PAM walk experiment conducted with the PSMD7 gene target.

<p>The percent cutting for active sequences is indicated below the gel. (PC) positive control. (NC) negative control. The target sequence and surrounding sequence is indicated for both isolated active sites as well as an example of consecutive active target sites. Target sites are underlined for all active sequences and for some, are paired with their lane on the gel for orientation. The percent cutting measurement can only be interpreted as a semi-quantitative estimate due to small differences in lysate batches in the <i>in vitro</i> assay measurement, as well as staining differences in gels. Comparisons of the efficacy of one target site/PAM versus another are not made except to conclude whether or not they have cutting activity.</p