RESUMO
BACKGROUND: Animal models are essential to understand the physiopathology of human diseases but also to evaluate new therapies. However, for several diseases there is no appropriate animal model, which complicates the development of effective therapies. HPV infections, responsible for carcinoma cancers, are among these. So far, the lack of relevant animal models has hampered the development of therapeutic vaccines. In this study, we used a candidate therapeutic vaccine named C216, similar to the ProCervix candidate therapeutic vaccine, to validate new mouse and dog HPV preclinical models. ProCervix has shown promising results with classical subcutaneous murine TC-1 cell tumor isografts but has failed in a phase II study. RESULTS: We first generated E7/HPV16 syngeneic transgenic mice in which the expression of the E7 antigen could be switched on through the use of Cre-lox recombination. Non-integrative LentiFlash® viral particles were used to locally deliver Cre mRNA, resulting in E7/HPV16 expression and GFP reporter fluorescence. The expression of E7/HPV16 was monitored by in vivo fluorescence using Cellvizio imaging and by local mRNA expression quantification. In the experimental conditions used, we observed no differences in E7 expression between C216 vaccinated and control groups. To mimic the MHC diversity of humans, E7/HPV16 transgenes were locally delivered by injection of lentiviral particles in the muscle of dogs. Vaccination with C216, tested with two different adjuvants, induced a strong immune response in dogs. However, we detected no relationship between the level of cellular response against E7/HPV16 and the elimination of E7-expressing cells, either by fluorescence or by RT-ddPCR analysis. CONCLUSIONS: In this study, we have developed two animal models, with a genetic design that is easily transposable to different antigens, to validate the efficacy of candidate vaccines. Our results indicate that, despite being immunogenic, the C216 candidate vaccine did not induce a sufficiently strong immune response to eliminate infected cells. Our results are in line with the failure of the ProCervix vaccine that was observed at the end of the phase II clinical trial, reinforcing the relevance of appropriate animal models.
RESUMO
BACKGROUND: The application of CRISPR/Cas9 technology in human induced pluripotent stem cells (hiPSC) holds tremendous potential for basic research and cell-based gene therapy. However, the fulfillment of these promises relies on the capacity to efficiently deliver exogenous nucleic acids and harness the repair mechanisms induced by the nuclease activity in order to knock-out or repair targeted genes. Moreover, transient delivery should be preferred to avoid persistent nuclease activity and to decrease the risk of off-target events. We recently developed bacteriophage-chimeric retrovirus-like particles that exploit the properties of bacteriophage coat proteins to package exogenous RNA, and the benefits of lentiviral transduction to achieve highly efficient, non-integrative RNA delivery in human cells. Here, we investigated the potential of bacteriophage-chimeric retrovirus-like particles for the non-integrative delivery of RNA molecules in hiPSC for CRISPR/Cas9 applications. RESULTS: We found that these particles efficiently convey RNA molecules for transient expression in hiPSC, with minimal toxicity and without affecting the cell pluripotency and subsequent differentiation. We then used this system to transiently deliver in a single step the CRISPR-Cas9 components (Cas9 mRNA and sgRNA) to generate gene knockout with high indel rate (up to 85%) at multiple loci. Strikingly, when using an allele-specific sgRNA at a locus harboring compound heterozygous mutations, the targeted allele was not altered by NHEJ/MMEJ, but was repaired at high frequency using the homologous wild type allele, i.e., by interallelic gene conversion. CONCLUSIONS: Our results highlight the potential of bacteriophage-chimeric retrovirus-like particles to efficiently and safely deliver RNA molecules in hiPSC, and describe for the first time genome engineering by gene conversion in hiPSC. Harnessing this DNA repair mechanism could facilitate the therapeutic correction of human genetic disorders in hiPSC.