The repurposing of the CRISPR/Cas microbial adaptive immune system for gene editing has resulted in an exponential rise in new technologies and promising approaches for treating numerous human diseases. While some of the approaches being currently developed involve ex vivo editing by CRISPR/Cas9, many more potential applications will require in vivo editing. The in vivo use of this technology comes with challenges, one of which is the immune response to Cas9, a protein of microbial origin. Thus, the prevalence of pre-existing antibodies to Cas9 could also be a relevant parameter. There are many avenues for how CRISPR/Cas9 technologies will be applied in vivo, including the mode of delivery. These may be expected to invoke different immunological pathways. Nonetheless, as with all protein therapeutics, it may be desirable to monitor for anti-Cas9 antibodies during clinical development. This will require the development of robust and reliable assays. Here, we describe ELISA-based assays that are capable of detecting antibodies to Cas9 from Staphylococcus aureus (SaCas9) and Streptococcs pyogenes (SpCas9) in human sera. Furthermore, using these assays to screen for pre-existing antibodies in 200 human serum samples, we found the prevalence of anti-SaCas9 and anti-SpCas9 antibodies to be 10% and 2.5%, respectively.
The immunogenicity of protein therapeutics is an important safety and efficacy concern during drug development and regulation. Strategies to identify individuals and subpopulations at risk for an undesirable immune response represent an important unmet need. The major histocompatibility complex (MHC)–associated peptide proteomics (MAPPs) assay directly identifies the presence of peptides derived from a specific protein therapeutic on a donor’s MHC class II (MHC-II) proteins. We applied this technique to address several questions related to the use of factor VIII (FVIII) replacement therapy in the treatment of hemophilia A (HA). Although >12 FVIII therapeutics are marketed, most fall into 3 categories: (i) human plasma-derived FVIII (pdFVIII), (ii) full-length (FL)–recombinant FVIII (rFVIII; FL-rFVIII), and (iii) B-domain–deleted rFVIII. Here, we investigated whether there are differences between the FVIII peptides found on the MHC-II proteins of the same individual when incubated with these 3 classes. Based on several observational studies and a prospective, randomized, clinical trial showing that the originally approved rFVIII products may be more immunogenic than the pdFVIII products containing von Willebrand factor (VWF) in molar excess, it has been hypothesized that the pdFVIII molecules yield/present fewer peptides (ie, potential T-cell epitopes). We have experimentally tested this hypothesis and found that dendritic cells from HA patients and healthy donors present fewer FVIII peptides when administered pdFVIII vs FL-rFVIII, despite both containing the same molar VWF excess. Our results support the hypothesis that synthesis of pdFVIII under physiological conditions could result in reduced heterogeneity and/or subtle differences in structure/conformation which, in turn, may result in reduced FVIII proteolytic processing relative to FL-rFVIII.
BACKGROUND: Hemophilia A (HA) is associated with mutations in the F8 gene that expresses factor VIII (FVIII). Unexpectedly, HA also manifests in a small subset of individuals with no mutations (exonic or intronic) in their F8 gene. MicroRNAs (miRNAs) cause translational interference, affecting protein quality and stoichiometry. Here, by analyzing miRNAs of two patients from this subset, we evaluated miRNA-based FVIII suppression as a testable hypothesis to explain FVIII deficiency in patients with HA with no F8 gene mutations. STUDY DESIGN AND METHODS:To test the hypothesis, miRNA sequencing from two patients with mild and moderate HA with no mutations in their F8 gene, followed by experimental verification, was used to identify a group of upregulated miRNAs in patients with HA compared to normal controls; with binding sites in the 3 0 untranslated region (UTR) of F8 messenger RNA (mRNA), a prerequisite for miRNA-based gene regulation. From this pool, miR-374b-5p and miR-30c-5p, known to be expressed in human liver, where FVIII is expressed, were subjected to extensive characterization. RESULTS:In two cell lines that constitutively express FVIII, we demonstrated that overexpression of miR-374b or miR-30c decreased FVIII expression, while an miR-30c inhibitor partially restored FVIII expression.CONCLUSION: These data support a role for microRNAs in fine-tuning F8 gene regulation. Based on our findings, our current model suggests that in HA cases where the F8 gene is normal and is predicted to express normal levels of FVIII, F8 mRNA 3 0 UTR targeting miRNAs may be responsible for a FVIIIdeficiency phenotype clinically manifesting as HA. ABBREVIATIONS: AT = antithrombin; cDNA = complementary DNA; DMEM = Dulbecco's Modified Essential Medium; eGFP = enhanced green fluorescent protein; FBS = fetal bovine serum; FV = factor V; FVII = factor VII; FVIII = factor VIII; GAPDH = glyceraldehyde 3-phosphate dehydrogenase; HA = hemophilia A; HUVEC = human umbilical vein endothelial cells; LCL = lymphoblastoid cell line; MEM = Minimum Essential Medium; miRNAs = MicroRNAs; MS1 = Mile Seven 1; PCR = polymerase chain reaction; UTR = untranslated region; VWF = von Willebrand factor.
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