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The emergence of new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mutants and breakthrough infections despite available coronavirus disease 2019 (COVID-19) vaccines calls for antiviral therapeutics. The application of soluble angiotensin converting enzyme 2 (ACE2) as a SARS-CoV-2 decoy that reduces cell bound ACE2-mediated virus entry is limited by a short plasma half-life. This work presents a recombinant human albumin ACE2 genetic fusion (rHA-ACE2) to increase the plasma half-life by an FcRn-driven cellular recycling mechanism, investigated using a wild type (WT) albumin sequence and sequence engineered with null FcRn binding (NB). Binding of rHA-ACE2 fusions to SARS-CoV-2 spike protein subdomain 1 (S1) was demonstrated (WT-ACE2 KD = 32.8 nM and NB-ACE2 KD = 31.7 nM) using Bio-Layer Interferometry and dose-dependent in vitro inhibition of host cell infection of pseudotyped viruses displaying surface SARS-CoV-2 spike (S) protein. FcRn-mediated in vitro recycling was translated to a five times greater plasma half-life of WT-ACE2 (t½ β = 13.5 h) than soluble ACE2 (t½ β = 2.8 h) in humanised FcRn/albumin double transgenic mice. The rHA-ACE2-based SARS-CoV-2 decoy system exhibiting FcRn-driven circulatory half-life extension introduced in this work offers the potential to expand and improve the anti-COVID-19 anti-viral drug armoury. Statement of significance: The COVID-19 pandemic has highlighted the need for rapid development of efficient antiviral therapeutics to combat SARS-CoV-2 and new mutants to lower morbidity and mortality in severe cases, and for people that are unable to receive a vaccine. Here we report a therapeutic albumin ACE2 fusion protein (rHA-ACE2), that can bind SARS-CoV-2 S protein decorated virus-like particles to inhibit viral infection, and exhibits extended in vivo half-life compared to ACE2 alone. Employing ACE2 as a binding decoy for the virus is expected to efficiently inhibit all SARS-CoV-2 mutants as they all rely on binding with endogenous ACE2 for viral cell entry and, therefore, rHA-ACE2 constitutes a versatile addition to the therapeutic arsenal for combatting COVID-19.

Co-inhibitory immune receptors can contribute to T cell dysfunction in patients with cancer1,2. Blocking antibodies against cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and programmed cell death 1 (PD-1) partially reverse this effect and are becoming standard of care in an increasing number of malignancies3. However, many of the other axes by which tumours become inhospitable to T cells are not fully understood. Here we report that V-domain immunoglobulin suppressor of T cell activation (VISTA) engages and suppresses T cells selectively at acidic pH such as that found in tumour microenvironments. Multiple histidine residues along the rim of the VISTA extracellular domain mediate binding to the adhesion and co-inhibitory receptor P-selectin glycoprotein ligand-1 (PSGL-1). Antibodies engineered to selectively bind and block this interaction in acidic environments were sufficient to reverse VISTA-mediated immune suppression in vivo. These findings identify a mechanism by which VISTA may engender resistance to anti-tumour immune responses, as well as an unexpectedly determinative role for pH in immune co-receptor engagement. V-domain immunoglobulin suppressor of T cell activation (VISTA) selectively engages P-selectin glycoprotein ligand-1 (PSGL-1) and suppresses T cells at acidic pH similar to those in tumour microenvironments, thereby mediating resistance to anti-tumour immune responses.

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

T cell engagers show high efficacy in B cells malignances. High risk of immune-related adverse events, including cytokine release syndrome (CRS), is reported in patients due to on-target off-site effects of T cell engagers. Thus, reliable and translational mouse models are required to predict potential safety issues and investigate their rescue. Here we describe two preclinical models mimicking to some extent CRS induction upon activation with anti-CD3 antibodies.BRGSF (Balb/C Rag2-/-, IL2Rγ-/-, SIRPαNOD and Flt3-/-) is a highly immunodeficient mouse featuring reduced murine myeloid cells. Development of human lymphoid (B and T cells) and myeloid (NK, cDC, pDC and monocytes) compartments upon CD34+ HSC-engraftment are observed in blood, spleen and bone marrow and are stable for over a year, without side effects. Anti-hCD3 mAb, OKT3, administration in BRGSF-HIS mice induced a rapid release of human cytokines (i.e., IL-6, TNF-α, IFN-γ, IL-2) in serum. Pretreatment with Flt3L, enhancing the human myeloid compartment, boosted the production of cytokines. CRS clinical signs such as hypothermia and weight lost were reproduced in OKT3-injected BRGSF-HIS mice. Pretreatment with Tocilizumab (an anti-IL-6R) reduced cytokine production, suggesting a strong role of myeloid cells in the induced-CRS pathogenesis. The main limitation of this model is the lack of a fully functional crosstalk between human immune cells and mouse stroma, which can undermine endothelial cells contribution in CRS. Alternatively, the CD3ε N-terminal epitope knock-in mice expressing the humanized epitope of anti-CD3 clone SP34 (hCD3ε mice) enables the investigation of endothelial versus immune cells dialogue in CRS pathogenesis. hCD3ε mice display a functional CD3-TCR complex and a functional T and B cooperation, as shown by T cell proliferation upon SP34 activation and antigen-specific antibodies production, respectively. Cytotoxic responses were induced by various bispecific antibodies, ex vivo and in vivo. Splenocytes from hCD3ε mice activated with BsAb1 bispecific antibody targeting CD3ε and a tumor-associated antigen (TAA) induced mouse cytokines production (IFN-γ, TNF-α, IL-6, IL-1β, IL-10) in a concentration-dependent manner. A 2nd BSAb targeting CD3ε and an irrelevant TAA didn’t induce any cytokine release, confirming the specificity of the previous cytokine induction.These data suggest that both BRGSF-HIS and hCD3ε models could be used to assess T cell engager-induced CRS in a complementary setting: while BRGSF-HIS mice enable monitoring of any anti-hCD3 agent, the model might undermine the contribution of mouse endothelial cells due to specie specificity barrier of human cytokines and mouse cells. In contrast, hCD3ε mice enable monitoring of cytokine induction upon activation with SP34-derived anti-hCD3 taking into consideration a full crosstalk between immune and endothelial cells. Citation Format: Perrine Martin-Jeantet, Florence Renart-Depontieu, Ludovic Bourre, Gaëlle Martin, Angela Pappalardo, Dean O. Campbell, Astrid Doerner, Yacine Cherifi, Fabiane Sônego, Kader Thiam. BRGSF-HIS and CD3Ε humanized mice: Translatable preclinical mouse models for assessment of T-cell engagers-induced CRS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4036.

e14504 Background: Active cancer immunotherapeutics frequently give rise to immune-mediated adverse events (AEs), including cytokine release syndrome (CRS) or immune effector cell-associated neurotoxicity syndrome (ICANS). On-target, off-tumor activation of myeloid lineage cells (e.g. monocytes) has been implicated as a factor in the generation of these immune-mediated AEs. VISTA (V-domain Ig suppressor of T-cell activation) is an immune checkpoint, which is highly expressed on myeloid cells, and binds PSGL-1 on T-cells, but only when ‘activated’ by protonation at low pH (̃pH 6). Inhibition of the VISTA:PSGL-1 interaction in the acidic tumor microenvironment has been shown to be efficacious in multiple syngeneic murine tumor models. However, antibodies binding to VISTA at physiological pH 7.4 have significant potential to induce dose-limiting toxicities such as CRS and/or ICANS and be prematurely eliminated from circulation through targeted-mediated drug disposition (TMDD), reducing the likelihood of reaching efficacious drug occupancy levels. Among several non-pH-selective antibodies in clinical development, JNJ-61610588 (now CI-8993) induced dose-limiting on-target CRS at subtherapeutic dose levels and exhibited TMDD. To mitigate potential CRS and prevent TMDD, we developed SNS-101, a highly selective monoclonal IgG1 antibody for “active” VISTA, which inhibits the critical interaction with PSGL-1. Methods: Using flow cytometry at physiological pH, we compared SNS-101 with clinical stage non-pH-selective anti-VISTA antibodies and examined binding to VISTA-positive cell populations in human PBMCs as well as in human CD34+ cord blood cells reconstituted BRGSF-HIS mice (humanized BRGSF-HIS mice), which develop both human lymphoid and myeloid compartments. Furthermore, we performed in vitro and in vivo CRS studies in a HUVEC/PBMC co-culture system and humanized BRGSF-HIS mice, respectively. Results: Under physiological pH, non-pH-selective antibodies bound to monocytes, neutrophils and NK cells whereas SNS-101 did not exhibit any significant interactions. CRS assays indicate that the magnitude of induced cytokine levels was significantly higher with non-pH-selective antibodies compared to SNS-101. Conclusions: We assessed the binding profile of SNS-101 vs. non-pH-selective VISTA antibodies. Our results demonstrate that SNS-101 does not bind to VISTA-positive cells in circulating blood. In addition, in vitro and in vivo CRS studies suggest that SNS-101 has a lower risk of inducing CRS compared to non-pH selective anti-VISTA antibodies, alleviating liabilities previously associated with anti-VISTA antibodies. IND-enabling studies, including pharmacokinetic and toxicology studies, are ongoing.

Background T-cell engagers have proved to be a promising therapeutic strategy in immunotherapy, for redirecting T cells activity against tumor cells. To facilitate the preclinical assessment of novel T-cell engagers and their translatability, we have developed an immunocompetent CD3 epsilon N-terminal epitope humanized mouse model. Methods This model was developed to express the human epitope of the CD3 epsilon chain, which is recognized by approximately 70% of the T-cell engagers (clone SP34). The rest of the extracellular domain was kept from mouse origin to preserve the amino acids involved in the interaction with CD3 gamma and delta. Similarly, the transmembrane domains and the intracellular domains where kept murine to enable salt bridges interaction, interaction with the CD3 zeta and the signaling into mouse cells. Results T cells from CD3 epsilon epitope humanized mice are found in comparable frequency in spleen, blood and bone marrow from WT mice. B cells, monocytes, dendritic cells and NK frequencies are also similar to the frequencies of these cell types in WT mice, suggesting that the humanization of the epitope of CD3 epsilon did not alter the immune cells distribution in these mice. Activation of T cells with antibodies targeting human CD3 (clone SP34) induced CD4 and CD8 T cell proliferation, as well as production of IL-2 and IFN-gamma. The CD3 functionality was demonstrated in vitro by the ability of B cells to produce IgM upon activation of T cells, suggesting a proper cooperation between T and B cells. Additionally, a first class of T-cell engagers targeting both human CD3 and a tumoral antigen, induced tumor cell lysis of MC38-Ag in a concentration-dependent manner. A second class of T cell engagers, also targeting CD3 and a tumoral antigen, showed an anti-tumor effect in vivo, and this effect was also shown to be dose-dependent. Conclusions These data suggest that the CD3 epsilon N-terminal epitope humanized mouse model enables the assessment of efficacy and mechanism of action of T-cell engagers.This model is currently being intercrossed with immunostimulatory humanized mouse models to provide new opportunities for assessment of bi-specific antibodies targeting the CD3 and immunostimulatory molecules. This model is the first generation of a broader program aiming at developping a Pan CD3 humanized model, where the gamma, delta and epsilon chains of the CD3 complex will be humanized. The Pan CD3 humanized mice are currently being investigated for immune responses and would provide a broader tool for assessment of T-cell engagers.

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.

Targeting CTLA-4 has shown remarkable long-term benefits and thus remains a valuable approach for combating cancers of many types. A number of preclinical models have been developed over the years to evaluate the efficacy of immune checkpoint blockade in promoting antitumor immunity. In particular, knock-in (KI) humanized mouse models offer the possibility to study clinical grade immune checkpoint inhibitors (ICI) in the context of a fully functional immune system. Here we show the response to ipilimumab in a newly developed hCTLA-4 KI humanized mouse model. Our results demonstrate significant tumor growth inhibition as well as complete tumor regressions in the MC38 colorectal cancer model following treatment with ipilimumab. We have extended these studies by re-challenging the tumor-free surviving animals with tumors cells implanted opposite to the original tumor site. We established that all re-challenged hCTLA-4 KI mice remained tumor free suggesting potent T cell memory was maintained. To identify the immune cells and cytokines that could be responsible for the durable responses, we used multiparameter flow cytometry to characterize and compare blood, spleen and tumors tissues from the control, ipilimumab and mCTLA-4 treated mice. Comprehensive multichromatic phenotyping and functional intracellular cytokine staining (ICS) using validated 18-color flow cytometry panels showed a significant increase in CD8+ T cell frequencies when mice were treated with anti-hCTLA-4 but not with the mouse counterpart or the isotype control. Importantly, hCTLA-4 blockade reduced the regulatory T cell (FoxP3+ Treg) frequency and increased leukocyte infiltration into the tumor in the hCTLA-4 treated mice but not in the other two treated groups. Although equivalent numbers of CD4+ and CD8+ total T lymphocytes where found in the blood of all groups after each treatment, the quality of the T cell responses was found to be significantly different. CD45+ lymphocytes from the blood analyzed from the hCTLA-4 treated mice showed significant increases in both central memory (CM) CD8+ and CD4+ T cells consistent with increased anti-tumor efficacy seen in these mice. Furthermore, treatment of MC38 tumor-bearing mice with ipilimumab enhanced significantly the secretion of IFNγ from CD8+ and CD4+ T cells and the proliferation of both T cell populations as shown by the upregulation of Ki67 staining. These results indicate that immune cells were actively proliferating and had the appropriate effector functions to impact tumor growth. Altogether, the data presented here demonstrates that hCTLA-4 KI humanized mice are a robust model for evaluating the immune-modulatory effects and the activity of clinical grade ICI against tumors. Citation Format: Elizabeth Reap, Fabiane SÔNEGO, Emily O'Koren, Martin Gaëlle, Anya Avrutskaya, Jacob Hauser, Robin Ball, Thi Bui, Ian Belle, Chassidy Hall, Paula L. Miliani De Marval, Kader Thiam. Enhanced anti-tumor lymphocyte function and frequencies measured by multichromatic flow cytometry in human CTLA-4 knock-in mice in a colorectal carcinoma model after treatment with ipilimumab [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5635.

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.

Over the past decade there has been an increasing demand in the use of syngeneic models for evaluating the efficacy of checkpoint inhibition-based cancer immunotherapies. During tumor development, immune cells can became unresponsive to the presence of tumor cells due to chronic activation and expression of the programmed cell death protein-1 (PD-1) or the T-lymphocyte-associated antigen 4 (CTLA4) in T-cells or the presence of FoxP3+ Treg cells resulting in tumor immune-tolerance. Our previous studies have demonstrated that murine anti-PD-1 and CTLA-4 therapy can effectively re-activate the antitumor response against multiple syngeneic tumor models. While these models proved instrumental for evaluating murine immune-checkpoint inhibitors (ICI), there is a clear need for additional mouse models to evaluate the efficacy of ICI specific for human targets. To address this need, we describe the development of a humanized PD-1 knock-in (KI) mouse model. This mouse model has the advantage of expressing the human PD-1 protein in the context of a fully functional immune system. We also show the response to clinically relevant immune checkpoint inhibitors in two preclinical tumor models. We evaluated the anti-tumor activity of pembrolizumab in the MC38 colorectal carcinoma and the GL261 glioblastoma models. We observed significant tumor growth inhibition and growth delay in the MC38 tumor model when treated with pembrolizumab monotherapy, but not when treated with the murine counterpart (anti-PD-1 clone RPM1-14). To extend our validation studies to other tumor models, we implanted GL261 glioblastoma orthotopically in the brain of PD-1 KI mice and achieved a significant increased life span in the group treated with pembrolizumab compared to both the control group and the group treated with murine anti-PD-1 antibody. The immune profile from control and treated these animals were also characterized in these studies by flow cytometry. In summary, the results shown here underscore the value of the humanized PD-1 knock-in (KI) mouse model as a tool to evaluate human specific immune-checkpoint based therapeutics alone and in combination with other agents. Citation Format: Anya Avrutskaya, Fabiane Sonego, Jacob Hauser, Emily O’Koren, Gaëlle Martin, Julie Chaix, Robin Ball, Thi Bui, Ian Belle, Chassidy Hall, Kader Thiam, Paula L. Miliani De Marval. Validation of humanized PD-1 knock-in mice as an emerging model to evaluate human specific PD-1 therapeutics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-063.

Because of its relevance to human physiology, the rat may provide highly predictable models for the pharmaceutical industry. Until recently, the lack of efficient tools to manipulate the rat genome has drastically limited the use of this research model. Recent advances in gene expression and transgenic systems have provided new possibilities for the generation of informative rat models. This review presents a state-of-the-art transgenic technologies in the rat and their application to biomedical research. Novel technologies enabling the faithful expression of human genes in rats are focussed on specifically.

The delineation of the minimal requirements for efficient delivery of functional cytotoxic epitopes into APC could be a step toward the definition of “minimal length” lipopeptides for the modulation of CTL activity. Several analogues of the HLA-A*0201-restricted HIV-1 polymerase (pol476–484) minimal cytotoxic epitope were obtained by modifying P0, P1, or P10 positions by a single Nε-palmitoyl-lysine residue. The use of fluorescent derivatives confirmed the cell-permeating activities and suggested that a P0- and a P1-modified lipopeptide possessing ionizable extremities fulfills the structural requirements for MHC loading. The expressions of HLA-peptide complexes at the surface of TAP-deficient cells incubated with the parent epitope or lipopeptide derivatives were compared, in terms of intensity and stability. Both lipopeptides induced a considerably prolonged expression of conformationally correct complexes, which were dependent on the integrity of the exocytosis pathway, suggesting a dynamic mechanism of formation or reloading of the complexes from an intracellular pool. The agonistic activities of the different HLA-peptide complexes were evaluated using two independent T cell lines from HIV-infected donors. We report that a lipodecapeptide obtained by N-terminal addition of a Nε-palmitoyl-lysine to the pol476–484 epitope was able to increase the life span of functional presentation to cytotoxic T cells specific for the parent peptide.

We describe the isolation of a synthetic agonist of IFN-gamma (L-mIFN-gamma-CT) active on mouse and human cells. Its biological activity is the result of the ability of the C-terminal extremity of murine IFN-gamma to interact with the intracellular part of IFNgamma-R and the observation that the modification of peptides by a palmitic acid enables their cytoplasmic delivery. L-mIFN-gamma-CT stimulated murine cells exhibited an increase in MHC class II molecules and FcgammaRII/III expression and conferred protection against viral lysis. Unresponsiveness to L-mIFN-gamma-CT of cells recovered from IFNgamma-R alpha-chain knockout mice indicated the involvement of IFNgamma-R in the biological activities observed. Induction of VCAM-1, ICAM-1, and HLA-DR expression on human cells stimulated with L-mIFN-gamma-CT demonstrated an abrogation of species specificity. These results describe the development of a new synthetic agonist of IFN-gamma, which substitutes for the native cytokine in any IFN-gamma responsive cells, by acting intracellularly on IFN-gammaR.

Intracellular enzymes or receptors are interesting targets for thepharmacomodulation of cellular metabolism. We have previously shown thatmodification of relatively long peptides by a palmitoyl-lysine residue couldfacilitate their delivery into the cytoplasm of living cells. Severalpeptides containing pseudosubstrate sequences of protein kinase C (PKC) havebeen evaluated for their ability to modulate phosphorylation of modelsubstrate, neuronal morphology or tumor necrosis factor secretion. In thiswork we have evaluated the effect of palmitoyl-modified PKC-pseudosubstratepeptides on induction of apoptosis. We have established that these peptidesare able to induce apoptosis in different human cell types (primaryfibroblasts, T- and B-lymphocyte cell lines) as assessed by (terminal deoxynucleotidyl transferase dUTP nick-end labelling) and DNAfragmentation. In contrast, control peptides (non-lipidicPKC-pseudosubstrate peptides and irrelevant lipopeptides) had no or littleeffect on programmed cell death. This work highlights the pharmacologicalinterest of lipopeptides and argues in favor of the potential role of PKC(s)in the cell death machinery.

With the lack of tools available to manipulate the rat genome, alternative technologies have been investigated to generate loss-of-function rat models by gene invalidation. The recent demonstration that RNA interference (RNAi)-mediated gene silencing occurs in rodents has opened new opportunities for rat functional genetics. In this chapter, we provide some practical guidelines for RNAi working in rat, based on the recent design and development of mice and rat Knock down models.

With the lack of tools available to manipulate the rat genome, alternative technologies have been investigated to generate loss-of-function rat models by gene invalidation. The recent demonstration that RNA interference (RNAi)-mediated gene silencing occurs in rodents has opened new opportunities for rat functional genetics. In this chapter, we provide some practical guidelines for RNAi working in rat, based on the recent design and development of mice and rat Knock down models.

Autotaxin is a member of the phosphodiesterase family of enzymes, (NPP2). It is an important secreted protein found in conditioned medium from adipocytes. It also has a putative role in the metastatic process. Based on these observation, further validation of this potential target was necessary, apart from the classical biochemical ones. The construction of a knock out mouse strain for ATX was started. In this paper, we report the generation of a mouse line displaying an inactivated ATX gene product. The KO line was designed in order to generate a functional inactivation of the protein. In this respect, the threonine residue T210 was replaced by an alanine (T210A) leading to a catalytically inactive enzyme. If the experimental work was straight forward, we disappointedly discovered at the final stage that the breeding of heterozygous animals, ATX −/+, led to the generation of a Mendelian repartition of wild-type and heterozygous, but no homozygous were found, strongly suggesting that the ATX deletion is lethal at an early stage of the development. This was confirmed by statistical analysis. Although other reported the same lethality for attempted ATX−/− mice generation [van Meeteren, L.A., Ruurs, P., Stortelers, C., Bouwman, P., van Rooijen, M.A., Pradère, J.P., Pettit, T.R., Wakelam, M.J.O., Saulnier-Blache, J.S., Mummery, C.L., Moolenar, W.H. and Jonkers, J. (2006) Autotaxin, a secreted lysophospholipase D, is essential for blood vessel formation during development, Mol. Cell. Biol. 26, 5015–5022; Tanaka, M., Okudaira, S., Kishi, Y., Ohkawa, R., Isei, S., Ota, M., Noji, S., Yatomi, Y., Aoki, J., and Arai, H. (2006) Autotaxin stabilizes blood vessels and is required for embryonic vasculature by producing lysophosphatidic acid, J. Biol. Chem. 281, 25822–25830], they used more drastic multiple exon deletions in the ATX gene, while we chose a single point mutation. To our knowledge, the present work is the first showing such a lethality in any gene after a point mutation in an enzyme catalytic site.

In classical forward genetics, genes were defined by the resulting phenotype. Later, analysis of individual genes at the molecular level became possible. The develop-ment of high-throughput methods for the sequencing and annotation of cDNA and whole genomes has led to the expansion of reverse genetics, in which access to gene sequences makes it possible to elucidate their biological function. Although this approach has greatly extended our knowledge of simple organisms (Hannon 2004), it is of limited use for investigating the more complex mammalian biological systems. The generation of hypomorphic mutants using mouse knockout technology by homologous recombination provides a powerful means of elucidating gene function in vivo. To date, published knockouts exist for about 10% of mouse genes. These knockouts often display a complicated phenotype as the gene is inactivated at the single-cell stage. Better spatiotemporal control of knockouts has overcome this major limitation of in vivo gene inactivation. The recent discovery of RNA interference (RNAi) in cultured mammalian cells (Hammond 2001) and the demonstration that RNAi-mediated gene silencing can occur in rodents have led to new opportunities in mouse functional genetics (Lewis 2002). This chapter reviews the recent findings of in vivo RNAi and focuses on the advantages and limitations of the different approaches. New methods fulfilling major requirements for successful use of RNAi in vivo are also discussed. These methods combine the lessons learned from in vitro and in vivo studies that used RNAimediated gene knockdown and knowledge of the advantages and limitations of transgenic models. RNAi is a sequence-specific post-transcriptional gene silencing (PTGS) mechanism. It is triggered by double-stranded RNA (dsRNA), an important signaling molecule in eukaryotic cells in that it breaks down mRNAs that are homologous in sequence to itself. This elegant defense system evolved to eliminate non-self RNA molecules, such as cytoplasmic replicating RNA viruses, transcripts originating from transgenes, transposable elements, and viruses randomly integrated into the host genome (Hannon 2004; Mello 2004).

The ability of lipopeptides to passively cross the cell membrane opens new opportunities for the intracellular delivery of bioactive peptides. However, the production of large series of cell-permeable lipopeptides is not trivial due to their generally low solubility. We have evaluated the possibility of associating the fatty acid to the functional cargo using generally applicable ligation chemistries. To this end, we have designed an amphiphilic shuttle in which arginine residues served to solubilize the lipid part in aqueous media, during both the assembly of the lipopeptide and the cellular assays. Our model peptide, the pseudosubstrate sequence of protein kinase C-zeta (PKC-zeta), was associated to the pentapeptide Gly-Arg-Gly-Arg-Lys(Pam)-NH2 through thiazolidine, thioether, disulfide, or hydrazone linkages. The cytoplasm import of the resulting constructs was monitored through the quantification of the apoptosis specifically induced by PKC-zeta inhibition. Our observations suggested the interest of this noninvasive cellular import method to modulate the activity of an intracytoplasmic pharmacological target and showed the influence of a non-amide link created between the functional peptide and the lipidic vector: optimal results, in terms of both specific activity and low basal cytotoxicity, were obtained with the thiazolidine ligation product.

Two truncated analogues of a previously identified lipopeptide agonist toward the IFN-gamma receptor were synthesized in an attempt to determine the minimal compound able to induce expression of MHC class II molecules on murine and human cells and to study the role of the lipid tail. Circular dichroism studies were used to probe the induced conformationnal changes. Our results indicate at least a double role for the lipid modification that contributes to the stabilization of helical organization of the associated peptide and to its passive delivery into the cytoplasm. The persistence of biological activity in a truncated peptide of half of the residues present in the lead compound suggests that the lipid tail could also contribute to the stabilization of the peptide-receptor binding through additional hydrophobic interactions. This study allowed to readjust the minimal requirements for intracellular IFN-gamma receptor stimulation. More generally, we suggest that lipidated analogues of functional peptides could be utilized for intracellular target validation in the drug discovery process.