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BackgroundAlthough Immune checkpoint inhibitors (ICI)-targeting therapies have revolutionized the treatment of cancer, several tumors do not respond to those therapies. Preclinical and clinical evidences suggest that STING is a promising target to improve the immunogenicity of tumors, turning them responsive to ICI, and enhancing anti-tumor response. DMXAA failed to show efficacy in clinical trials, despite its encouraging anti-tumor response in preclinical phase, highlighting the need of accurate translational preclinical models. On top of the specificity barrier reported for STING-targeting agents, the heterogeneity of STING variants and their variability in the response to STING-targeting therapies, brings another level of complexity in preclinical evaluation of anti-STING therapies. Here, we report the generation of STING humanized (hSTING) mouse models enabling the in vivo assessment of STING-targeting agents.MethodsHuman STING variants show a high heterogenicity and population stratification. Different variants, and isoforms, respond differently to STING agonists. We developed mouse models expressing the main human STING variants and isoforms found in the population to recapitulate this complexity. Human STING was inserted by knock-in at the endogenous locus to enable a physiological expression pattern of STING while invalidating the mouse gene. Herein, we will focus on the human STING full length H323 model.ResultsT and B lymphocytes, NK, DCs and monocytes frequence in the spleen, bone marrow and blood were found to be similar in hSTING and WT mice, suggesting that the humanization of STING did not alter the immune cell distribution in these compartments. These cells express human STING, while no expression of mouse STING was observed. Splenocytes isolated from hSTING and WT mice produced IL-6 and IFN-γ upon activation with 2'3'-cGAMP, a cyclic dinucleotide with activity towards both mouse and human STING. Similarly, a mouse and human STING agonist induced the activation of DCs in both hSTING and WT mice, as observed by the increased expression of CD80/CD86 on DCs ex vivo and in vivo. Moreover, systemic production of IL-6, IFN-γ and TNF-α in response to this STING agonist was observed and suggest that human STING is functional in hSTING mice. As expected, hSTING mice did not respond to activation with DMXAA in vivo, whereas this agonist induced the systemic production of cytokines and activation of DCs in WT mice.ConclusionsThe novel hSTING model described here supports the assessment of human STING-targeting agents in immuno-oncology and inflammation. Intercrosses of this model with ICI humanized models could support the assessment of combination therapies

BackgroundThe breakthrough of immunotherapies has unleashed new hope and new success for cancer therapy. However, the choice of a preclinical model is one of the main challenges as they are important for evaluation of translatability to help support testing in clinical studies, including potential efficacy and tolerability of immunotherapies during preclinical development.The development of mouse models featuring a human immune system (HIS) provides new paths for the investigation of the efficacy of immunotherapies in preclinical models engrafted with human tumors. Although these models provided a breakthrough in the assessment of immune targeting agents, they also come with a few significant caveats. These include: a lack of a mature human myeloid compartment in the mouse, and a short life span of the model when this compartment is promoted at non-physiological levels via the over-expression of human cytokines. Here, we report a novel mouse model (BRGSF-HIS), featuring functional human lymphoid and myeloid compartments. The human immune response of this model was assessed through examination of the immune cells composition in the tumor microenvironment (TME), and its ability to respond to biologics known to trigger cytokine release syndrome (CRS).MethodsBRGSF (balb/C Rag2-/-, IL2Rg-/-, SIRPaNOD and Flt3-/-) is a highly immunodeficient mouse, with reduced murine myeloid cells, which allows long term CD34+ HSC-engraftment and development of human lymphoid and myeloid compartments (human CD141+ and CD1c+ DC subsets, CD123+ pDC, CD14+ monocytes), in blood, spleen and bone marrow. The engraftment is stable for over twelve months with no side effects. The effect of exogenous human Flt3 ligand (Flt3L) on the composition of TME in A549 model, and an anti-CD3 antibody (OKT3)-induced CRS, were assessed.ResultsExogenous human Flt3L significantly and transiently increased the proportion of human myeloid cells. This can be recalled by continuous dosing of Flt3L. Assessment of tumor immunobiology in A549 model, showed increased tumor-infiltrating T-cells (mainly CD8+ T-cells) and myeloid cells, while tumor-infiltrating NK cells were decreased. The presence of myeloid cells provides a new opportunity for assessment of myeloid targeted therapies, as proven using pDC-depleting antibodies. OKT3 administration resulted in CRS symptoms including a temperature drop, body weight loss and a change in serum cytokine levels. Symptoms were mitigated upon administration of tocilizumab, suggesting the contribution of the myeloid compartment in the response observed.ConclusionsThese data demonstrate that BRGSF-HIS mice support development of functional human myeloid cells and that this mouse model enables preclinical evaluation of cancer immunotherapy in vivo.

Random errors in protein synthesis are prevalent and ubiquitous, yet their effect on organismal health has remained enigmatic for over five decades. Here, we studied whether mice carrying the ribosomal ambiguity (ram) mutation Rps2-A226Y, recently shown to increase the inborn error rate of mammalian translation, if at all viable, present any specific, possibly aging-related, phenotype. We introduced Rps2-A226Y using a Cre/loxP strategy. Resulting transgenic mice were mosaic and showed a muscle-related phenotype with reduced grip strength. Analysis of gene expression in skeletal muscle using RNA-Seq revealed transcriptomic changes occurring in an age-dependent manner, involving an interplay of PGC1α, FOXO3, mTOR, and glucocorticoids as key signaling pathways, and finally resulting in activation of a muscle atrophy program. Our results highlight the relevance of translation accuracy, and show how disturbances thereof may contribute to age-related pathologies., Communications Biology, 4 (1), ISSN:2399-3642

Background Immuno-intervention through targeting of activating and inhibitory immune checkpoints (ICP), has shown promising results in the clinic over the last years. To facilitate these researches, mouse models expressing humanized ICP instead of their mouse counterparts were developed. Herein, we describe a novel CD28 humanized mouse model (hCD28 model), designed to test compounds targeting human CD28 (hCD28). Methods Human and mouse CD28 (mCD28) have different signalling responses, with hCD28 being known for inducing higher levels of pro-inflammatory cytokines upon stimulation with ligands/superagonists. This can be explained by the expression of CD28i, a hCD28 amplifier isoform which is not found in mouse. Additionally, evidences suggested that the different signalling between human and mCD28 relies on one amino acid change in the intracellular domain (ICD).1 Because the hCD28 model was developed to assess hCD28-targeting therapeutics, we decided to keep the expression of both canonical and CD28i isoforms to avoid undermining the biological effects of the testing antibodies. Although keeping the human ICD could favour the evaluation of cytokine production and therefore the safety of the test therapeutics, we decided to keep the mouse ICD to enable a proper interaction of CD28 with its signalling partners, allowing a physiological stimulation of CD28 in efficacy studies. Results hCD28 mice express hCD28 on T cells and the frequency of CD3 T cells is comparable in both WT and hCD28 mice. Stimulation of hCD28 mice-isolated T cells with hCD28 ligands and agonist antibodies resulted in T cell proliferation and cytokine production, suggesting that hCD28 is functional in mouse cells. MC38 uptake rate and kinetic of growth were comparable in WT and hCD28 mice, suggesting no major defect in the immune response in the hCD28 mice. Importantly, splenocytes and tumor draining lymph nodes cells isolated from tumor-bearing hCD28 mice showed higher production of IL-2 and IFN-gamma upon in vitro re-challenged with MC38 when compared to WT cells. Since the frequency of CD3 cells (Treg, CD4+ and CD8+) is comparable to WT mice, this could be explained by the expression of the amplifier CD28i isoform, which is absent in WT mice. Conclusions The hCD28 model described here supports the efficacy assessment of hCD28-targeting biologics, enabling PK/PD studies as hCD28 expression levels and pattern are physiological. However, after careful consideration of the CD28 biology, we decided to keep the mouse ICD, although it triggers lower pro-inflammatory cytokine production than CD28 human ICD. As such, this model is not suitable for toxicology/safety studies. Reference Porciello N, Grazioli P, Campese AF, et al. A non-conserved amino acid variant regulates differential signalling between human and mouse CD28. Nat Commun 2018; 9:1–16.

Immunotherapies through targeting of activating and inhibitory immune checkpoints (ICP) has revolutionized the treatment of cancers. To facilitate the translation of basic research into clinical application of these immunotherapies, we developed a pipeline of immunocompetent mouse models expressing humanized ICP instead of their mouse counterparts, thus compounds can be tested in the absence of endogenous cross-reacting mouse targets. The humanization strategy is target-dependent but ensures that the biology of the target, its physiological regulation and interacting partners are preserved. Herein, we describe a novel OX40 humanized mouse model (hOX40 model), designed to assess the efficacy and mechanism of action of compounds targeting human OX40 (hOX40). The expression of the fully human OX40 protein (human extracellular, transmembrane and intracellular domains) did not alter the distribution of immune cells in spleen, blood and bone marrow. hOX40 was expressed on CD4 and CD8 T cells upon activation and the kinetic of expression mirrored the expression of mouse OX40 in WT cells activated under the same conditions. Treg cells isolated from hOX40 mice expressed hOX40 and this expression was upregulated upon activation, also recapitulating the expression of mouse OX40 in WT cells, and suggesting that the regulation of OX40 expression was kept in the hOX40 mice. hOX40 was also found to be functional on CD4 T cells, as evidenced by the increased proliferation and cytokine production upon stimulation with human OX40 ligand. Importantly, hOX40 mice bearing MC38 tumors showed a tumor growth inhibition upon treatment with an anti-human OX40 therapy with agonist activity. This model should also enable the investigation of mechanism of action as well as tumor immunological memory, as a similar design of the hOX40 model was used for the assessment of a bispecific therapy targeting OX40 and CTLA-4 (Kvarnhammar et.al, 2019). The hOX40 mouse model is currently being crossed with other models expressing humanized immune checkpoint to enable the assessment of bispecifics and combo therapies. Citation Format: Fabiane Sônego, Gaëlle Martin, Audrey Beringer, Chloé Beuraud, Yacine Cherifi, Alexandre Fraichard, Patricia Isnard Petit, Kader Thiam. A novel OX40 humanized mouse model for efficacy assessment of OX40-targeting therapies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1811.

The CRISPR/Cas9 gene editing tool enables accessible and efficient modifications which (re)ignited molecular research in certain species. However, targeted integration of large DNA fragments using CRISPR/Cas9 can still be challenging in numerous models. To systematically compare CRISPR/Cas9’s efficiency to classical homologous recombination (cHR) for insertion of large DNA fragments, we thoroughly performed and analyzed 221 experiments targeting 128 loci in mouse ES cells. Although both technologies proved efficient, CRISPR/Cas9 yielded significantly more positive clones as detected by overlapping PCRs. It also induced unexpected rearrangements around the targeted site, ultimately rendering CRISPR/Cas9 less efficient than cHR for the production of fully validated clones. These data show that CRISPR/Cas9-mediated recombination can induce complex long-range modifications at targeted loci, thus emphasizing the need for thorough characterization of any genetically modified material obtained through CRISPR-mediated gene editing before further functional studies or therapeutic use.

The 1555 A to G substitution in mitochondrial 12S A‐site rRNA is associated with maternally transmitted deafness of variable penetrance in the absence of otherwise overt disease. Here, we recapitulate the suggested A1555G‐mediated pathomechanism in an experimental model of mitoribosomal mistranslation by directed mutagenesis of mitoribosomal protein MRPS5. We first establish that the ratio of cysteine/methionine incorporation and read‐through of mtDNA‐encoded MT‐CO1 protein constitute reliable measures of mitoribosomal misreading. Next, we demonstrate that human HEK293 cells expressing mutant V336Y MRPS5 show increased mitoribosomal mistranslation. As for immortalized lymphocytes of individuals with the pathogenic A1555G mutation, we find little changes in the transcriptome of mutant V336Y MRPS5 HEK cells, except for a coordinated upregulation of transcripts for cytoplasmic ribosomal proteins. Homozygous knock‐in mutant Mrps5 V338Y mice show impaired mitochondrial function and a phenotype composed of enhanced susceptibility to noise‐induced hearing damage and anxiety‐related behavioral alterations. The experimental data in V338Y mutant mice point to a key role of mitochondrial translation and function in stress‐related behavioral and physiological adaptations.