Therapeutic potential of fetal liver cell transplantation in hemophilia A mice.

Hemophilia A (HA) cell therapy approaches in pediatric individuals require suitable factor (F)VIII-producing cells for stable engraftment. Liver sinusoidal endothelial cells (LSEC) and hematopoietic stem cells (HSC) have been demonstrated to be suitable for the treatment of adult HA mice. However, after transplantation in busulfan (BU)-conditioned newborn mice, adult LSEC/HSC cannot efficiently engraft, while murine fetal liver (FL) hemato/vascular cells from embryonic day 11-13 of gestation (E11-E13), strongly engraft the hematopoietic and endothelial compartments while also secreting FVIII. Our aim was to investigate the engraftment of FL cells in newborn HA mice to obtain a suitable "proof of concept" for the development of a new HA treatment in neonates. Hence, we transplanted FL E11 or E13 cells and adult bone marrow (BM) cells into newborn HA mice with or without BU preconditioning. Engraftment levels and FVIII activity were assessed starting from 6 weeks after transplantation. FL E11-E13+ BU transplanted newborns reached up to 95% engraftment with stable FVIII activity levels observed for 16 months. FL E13 cells showed engraftment ability even in the absence of BU preconditioning, while FL E11 cells did not. BM BU transplanted newborn HA mice showed high levels of engraftment; nevertheless, in contrast to FL cells, BM cells cannot engraft HA newborns in BU non-conditioning regimen. Finally, none of the transplanted mice developed anti-FVIII antibodies. Overall, this study sheds some light on the therapeutic potential of healthy FL cells in the cure of HA neonatal/pediatric patients.

Deciphering the Ets-1/2-mediated transcriptional regulation of F8 gene identifies a minimal F8 promoter for hemophilia A gene therapy.

A major challenge in the development of a gene therapy for hemophilia A (HA) is the selection of cell type- or tissue-specific promoters to ensure factor VIII (FVIII) expression without eliciting an immune response. As liver sinusoidal endothelial cells (LSECs) are the major FVIII source, understanding the transcriptional F8 regulation in these cells would help optimize the minimal F8 promoter (pF8) to efficiently drive FVIII expression. In silico analyses predicted several binding sites (BS) for the E26 transformation-specific (Ets) transcription factors Ets-1 and Ets-2 in the pF8. Reporter assays demonstrated a significant up-regulation of pF8 activity by Ets-1 or Ets-1/Est-2 combination, while Ets2 alone was ineffective. Moreover, Ets-1/Ets-2-DNA binding domain mutants (DBD) abolished promoter activation only when the Ets-1 DBD was removed, suggesting that pF8 up-regulation may occur through Ets-1/Ets-2 interaction with Ets-1 bound to DNA. pF8 carrying Ets-BS deletions unveiled two Ets-BS essential for pF8 activity and response to Ets overexpression. Lentivirus-mediated delivery of GFP or FVIII cassettes driven by the shortened promoters led to GFP expression mainly in endothelial cells in the liver and to long-term FVIII activity without inhibitor formation in HA mice. These data strongly support the potential application of these promoters in HA gene therapy.

A Novel Platform for Immune Tolerance Induction in Hemophilia A Mice.

Hemophilia A (HA) is an X-linked bleeding disease caused by factor VIII (FVIII) deficiency. We previously demonstrated that FVIII is produced specifically in liver sinusoid endothelial cells (LSECs) and to some degree in myeloid cells, and thus, in the present work, we seek to restrict the expression of FVIII transgene to these cells using cell-specific promoters. With this approach, we aim to limit immune response in a mouse model by lentiviral vector (LV)-mediated gene therapy encoding FVIII. To increase the target specificity of FVIII expression, we included miRNA target sequences (miRTs) (i.e., miRT-142.3p, miRT-126, and miRT-122) to silence expression in hematopoietic cells, endothelial cells, and hepatocytes, respectively. Notably, we report, for the first time, therapeutic levels of FVIII transgene expression at its natural site of production, which occurred without the formation of neutralizing antibodies (inhibitors). Moreover, inhibitors were eradicated in FVIII pre-immune mice through a regulatory T cell-dependent mechanism. In conclusion, targeting FVIII expression to LSECs and myeloid cells by using LVs with cell-specific promoter minimized off-target expression and immune responses. Therefore, at least for some transgenes, expression at the physiologic site of synthesis can enhance efficacy and safety, resulting in long-term correction of genetic diseases such as HA.

Immune tolerance promotion by LSEC-specific lentiviral vector-mediated expression of the transgene regulated by the stabilin-2 promoter.

Liver sinusoidal endothelial cells (LSECs) are specialized endocytic cells that clear the body from blood-borne pathogens and waste macromolecules through scavenger receptors (SRs). Among the various SRs expressed by LSECs is stabilin-2 (STAB2), a class H SR that binds to several ligands, among which endogenous coagulation products. Given the well-established tolerogenic function of LSECs, we asked whether the STAB2 promoter (STAB2p) would enable us to achieve LSEC-specific lentiviral vector (LV)-mediated transgene expression, and whether the expression of this transgene would be maintained over the long term due to tolerance induction. Here, we show that STAB2p ensures LSEC-specific green fluorescent protein (GFP) expression by LV in the absence of a specific cytotoxic CD8+ T cell immune response, even in the presence of GFP-specific CD8+ T cells, confirming the robust tolerogenic function of LSECs. Finally, we show that our delivery system can partially and permanently restore FVIII activity in a mouse model of severe hemophilia A without the formation of anti-FVIII antibodies. Overall, our findings establish the suitability of STAB2p for long-term LSEC-restricted expression of therapeutic proteins, such as FVIII, or to achieve antigen-specific immune tolerance in auto-immune diseases.

GP64-pseudotyped lentiviral vectors target liver endothelial cells and correct hemophilia A mice.

Lentiviral vectors (LV) are efficient vehicles for in vivo gene delivery to the liver. LV integration into the chromatin of target cells ensures their transmission upon proliferation, thus allowing potentially life-long gene therapy following a single administration, even to young individuals. The glycoprotein of the vesicular stomatitis virus (VSV.G) is widely used to pseudotype LV, as it confers broad tropism and high stability. The baculovirus-derived GP64 envelope protein has been proposed as an alternative for in vivo liver-directed gene therapy. Here, we perform a detailed comparison of VSV.G- and GP64-pseudotyped LV in vitro and in vivo. We report that VSV.G-LV transduced hepatocytes better than GP64-LV, however the latter showed improved transduction of liver sinusoidal endothelial cells (LSEC). Combining GP64-pseudotyping with the high surface content of the phagocytosis inhibitor CD47 further enhanced LSEC transduction. Coagulation factor VIII (FVIII), the gene mutated in hemophilia A, is naturally expressed by LSEC, thus we exploited GP64-LV to deliver a FVIII transgene under the control of the endogenous FVIII promoter and achieved therapeutic amounts of FVIII and correction of hemophilia A mice.

Ischemia/Reperfusion Injury of Fatty Liver Is Protected by A2AR and Exacerbated by A1R Stimulation through Opposite Effects on ASK1 Activation.

Hepatic ischemia/reperfusion injury (IRI) is aggravated by steatosis and is a main risk factor in fatty liver transplantation. Adenosine receptors (ARs) are emerging as therapeutic targets in liver diseases. By using cellular and in vivo systems of hepatic steatosis and IRI, here we evaluated the effects of pharmacological A2AR and A1R activation. The A2AR agonist CGS21680 protected the primary steatotic murine hepatocyte from IR damage and the activation of ASK1 and JNK. Such an effect was attributed to a phosphatidylinositol-3-kinase (PI3K)/Akt-dependent inhibition of ASK1. By contrast, the A1R agonist CCPA enhanced IR damage, intracellular steatosis and oxidative species (OS) production, thereby further increasing the lipid/OS-dependent ASK1-JNK stimulation. The CGS2680 and CCPA effects were nullified by a genetic ASK1 downregulation in steatotic hepatoma C1C7 cells. In steatotic mice livers, CGS21680 protected against hepatic IRI and ASK1/JNK activation whereas CCPA aggravated hepatic steatosis and IRI, and enhanced ASK1 and JNK stimulation. These results evidence a novel mechanism of CGS21680-mediated hepatoprotection, i.e., the PI3K/AKT-dependent inhibition of ASK1, and they show that CGS21680 and CCPA reduces and enhances the IRI of fatty liver, respectively, by preventing or increasing the activation of the cytotoxic ASK1/JNK axis. They also indicate the selective employment of A2AR agonists as an effective therapeutic strategy to prevent IRI in human fatty liver surgery.

Identification and functional characterization of a novel splicing variant in the F8 coagulation gene causing severe hemophilia A.

BACKGROUND: We have identified a synonymous F8 variation in a severe hemophilia A (HA) patient who developed inhibitors following factor VIII (FVIII) prophylaxis. The unreported c.6273 G > A variant targets the consensus splicing site of exon 21. OBJECTIVES: To determine the impact of c.6273 G > A nucleotide substitution on F8 splicing and its translated protein. METHODS: Patient peripheral blood mononuclear cells were isolated and differentiated into monocyte-derived macrophages (MDMs). FVIII distribution in cell compartments was evaluated by immunofluorescence. The splicing of mutated exon 21 was assessed by exon trapping. Identified FVIII splicing variants were generated by site-directed mutagenesis, inserted into a lentiviral vector (LV) to transduce Chinese hamster ovary (CHO) cells, and inject into B6/129 HA-mice. FVIII activity was assessed by activated partial thromboplastin time, whereas anti-FVIII antibodies and FVIII antigen, by ELISA. RESULTS: HA-MDMs demonstrated a predominant retention of FVIII around the endoplasmic reticulum. Exon trapping revealed the production of two isoforms: one retaining part of intron 21 and the other skipping exon 21. These variants, predicted to truncate FVIII in the C1 domain, were detected in the patient. CHO cells transduced with the two FVIII transcripts confirmed protein retention and absence of the C2 domain. HA mice injected with LV carrying FVIII mutants, partially recovered FVIII activity without the appearance of anti-FVIII antibodies. CONCLUSIONS: Herein, we demonstrate the aberrant impact of a FVIII synonymous mutation on its transcription, activity, and pathological outcomes. Our data underline the importance of increasing the knowledge regarding the functional consequences of F8 mutations and their link to inhibitor development and an effective replacement therapy.

FVIII expression by its native promoter sustains long-term correction avoiding immune response in hemophilic mice.

Here we describe a successful gene therapy approach for hemophilia A (HA), using the natural F8 promoter (pF8) to direct gene replacement to factor VIII (FVIII)-secreting cells. The promoter sequence and the regulatory elements involved in the modulation of F8 expression are still poorly characterized and biased by the historical assumption that FVIII expression is mainly in hepatocytes. Bioinformatic analyses have highlighted an underestimated complexity in gene expression at this locus, suggesting an activation of pF8 in more cell types than those previously expected. C57Bl/6 mice injected with a lentiviral vector expressing green fluorescent protein (GFP) under the pF8 (lentiviral vector [LV].pF8.GFP) confirm the predominant GFP expression in liver sinusoidal endothelial cells, with a few positive cells detectable also in hematopoietic organs. Therapeutic gene delivery (LV.pF8.FVIII) in hemophilic C57/Bl6 and 129-Bl6 mice successfully corrected the bleeding phenotype, rescuing up to 25% FVIII activity, using a codon-optimized FVIII, with sustained activity for the duration of the experiment (1 year) without inhibitor formation. Of note, LV.pF8.FVIII delivery in FVIII-immunized HA mice resulted in the complete reversion of the inhibitor titer with the recovery of therapeutic FVIII activity. Depletion of regulatory T cells (Tregs) in LV-treated mice allowed the formation of anti-FVIII antibodies, indicating a role for Tregs in immune tolerance induction. The significant blood loss reduction observed in all LV.pF8.FVIII-treated mice 1 year after injection confirmed the achievement of a long-term phenotypic correction. Altogether, our results highlight the potency of pF8-driven transgene expression to correct the bleeding phenotype in HA, as well as potentially in other diseases in which an endothelial-specific expression is required.

PPARs are mediators of anti-cancer properties of superparamagnetic iron oxide nanoparticles (SPIONs) functionalized with conjugated linoleic acid.

Breast cancer chemotherapy can cause side effects due to nonspecific drug delivery, low solubility and fast metabolism of drugs used in conventional therapy. Moreover, the therapeutic effect of the drugs is often reduced by the strengthening of chemoresistance, which occurs via a variety of mechanisms. Different strategies have been developed to reduce multidrug resistance (MDR)-associated gene expressions including the use of surfactants and polymers. In this study superparamagnetic iron oxide nanoparticles (SPIONs) functionalized with conjugated linoleic acid (CLA) reduced the number and viability of cells in comparison with both untreated cells or cells treated with SPIONs alone. This cytostatic effect correlated with the increase of peroxisome proliferator-activated receptors γ (PPARγ). The necrotic cell death induced, as a consequence, an inflammatory process, as evidenced by the decrease of the anti-inflammatory PPARα and increase of pro-inflammatory TNFα and IL-1ß. PPARs were examined because CLA is one of their natural ligands. The antitumor effect observed was accompanied by a down-regulation of p-glycoprotein (P-gp), which was the first important discovered efflux transporter belonging to MDR, and of ALDH3A1, an enzyme able to metabolize some drugs, reducing their effects. The down-regulation of P-gp correlated with the increase of cytokines. The ALDH3A1 decrease correlated with the increase of PPARγ. Based on these results, PPARs are molecular mediators of anti-cancer effect of SPIONs functionalized with CLA, being changes in these nuclear receptors correlated with induction of cytotoxicity and inflammation, and decreased ability of cancer cells in blocking anti-cancer drug effect.

Innovative superparamagnetic iron-oxide nanoparticles coated with silica and conjugated with linoleic acid: Effect on tumor cell growth and viability.

One of the goals for the development of more effective cancer therapies with reduced toxic side effects is the optimization of innovative treatments to selectively kill tumor cells. The use of nanovectors loaded with targeted therapeutic payloads is one of the most investigated strategies. In this paper superparamagnetic iron oxide nanoparticles (SPIONs) coated by a silica shell or uncoated, were functionalized with single-layer and bi-layer conjugated linoleic acid (CLA). Silica was used to protect the magnetic core from oxidation, improve the stability of SPIONs and tailor their surface reactivity. CLA was used as novel grafting biomolecule for its anti-tumor activity and to improve particle dispersibility. Mouse breast cancer 4T1 cells were treated with these different SPIONs. SPIONs functionalized with the highest quantity of CLA and coated with silica shell were the most dispersed. Cell viability was reduced by SPIONs functionalized with CLA in comparison with cells which were untreated or treated with SPIONs without CLA. As regards the types of SPIONs functionalized with CLA, the lowest viability was observed in cells treated with uncoated SPIONs with the highest quantity of CLA. In conclusion, the silica shell free SPIONs functionalized with the highest amount of CLA can be suggested as therapeutic carriers because they have the best dispersion and ability to decrease 4T1 cell viability.

Tumor targeting by lentiviral vectors combined with magnetic nanoparticles in mice.

Nanomaterials conjugated or complexed with biological moieties such as antibodies, polymers or peptides appear to be suitable not only for drug delivery but also for specific cancer treatment. Here, biocompatible iron oxide magnetic nanoparticles (MNPs) with or without a silica shell coupled with lentiviral vectors (LVs) are proposed as a combined therapeutic approach to specifically target gene expression in a cancer mouse model. Initially, four different MNPs were synthesized and their physical properties were characterized to establish and discriminate their behaviors. MNPs and LVs strictly interacted and transduced cells in vitro as well as in vivo, with no toxicity or inflammatory responses. By injecting LV-MNPs complexes intravenously, green fluorescent protein (GFP) resulted in a sustained long-term expression. Furthermore, by applying a magnetic field on the abdomen of intravenous injected mice, GFP positive cells increased in livers and spleens. In liver, LV-MNPs were able to target both hepatocytes and non-parenchymal cells, while in a mouse model with a grafted tumor, intra-tumor LV-MNPs injection and magnetic plaque application next to the tumor demonstrated the efficient uptake of LV-MNPs complexes with high number of transduced cells and iron accumulation in the tumor site. More important, LV-MNPs with the application of the magnetic plaque spread in all the tumor parenchyma and dissemination through the body was prevented confirming the efficient uptake of LV-MNPs complexes in the tumor. Thus, these LV-MNPs complexes could be used as multifunctional and efficient tools to selectively induce transgene expression in solid tumor for therapeutic purposes. STATEMENT OF SIGNIFICANCE: Our study describes a novel approach of combining magnetic properties of nanomaterials with gene therapy. Magnetic nanoparticles (MNPs) coated with or without a silica shell coupled with lentiviral vectors (LVs) were used as vehicle to target biological active molecules in a mouse cancer model. After in situ injection, the presence of MNP under the magnetic field improve the vector distribution in the tumor mass and after systemic administration, the application of the magnetic field favor targeting of specific organs for LV transduction and specifically can direct LV in specific cells (or avoiding them). Thus, our findings suggest that LV-MNPs complexes could be used as multifunctional and efficient tools to selectively induce transgene expression in solid tumor for therapeutic purposes.

Symbolic dynamics of heart rate variability: a probe to investigate cardiac autonomic modulation.

BACKGROUND: Sympathetic and parasympathetic systems are considered the principal rapidly reacting systems that control heart rate. METHODS AND RESULTS: We propose a symbolic analysis series to quantify the prevalence of sympathetic or parasympathetic cardiac modulation. This analysis decomposes the heart rate variability series in patterns lasting 3 beats and classifies them into 3 categories: nonvariable, variable, and very variable patterns referred to as 0V, 1V, and 2V patterns. First, we applied this method to experimental and pharmacological conditions characterized by sympathetic activation (tilt test, handgrip, nitroprusside, and high-dose atropine administration) or parasympathetic activation (phenylephrine and low-dose atropine administration) in 60 healthy subjects. An increase in sympathetic modulation and a vagal withdrawal elicited a significant increase in 0V patterns and a decrease in 2V patterns, whereas parasympathetic dominance induced the opposite, reflecting a reciprocal sympathovagal balance. The second part of the study considered a series of 300 beats before the onset of major arrhythmic events in patients with an implantable cardioverter-defibrillator. Symbolic analysis detected an increase in the percentage of 0V patterns before the onset of major arrhythmias compared with baseline (41.6+/-3.9% and 24.4+/-2.9%, respectively; P<0.01), indicating a sympathetic prevalence. On the other hand, the 2V patterns did not decrease before major arrhythmias, suggesting the presence of nonreciprocal autonomic modulations. CONCLUSIONS: Symbolic analysis of 3 beat sequences takes into account the different time course of sympathetic and parasympathetic cardiac modulations and seems appropriate for elucidating the neural pathophysiological mechanisms occurring during the short periods that precede acute cardiac events.

Role of the autonomic nervous system in generating non-linear dynamics in short-term heart period variability.

We evaluated the role played by the autonomic nervous system in producing non-linear dynamics in short heart period variability (HPV) series recorded in healthy young humans. Non-linear dynamics are detected using an index of predictability based on a local non-linear predictor and a surrogate data approach. Different types of surrogates are utilized: (i) phase-randomized Fourier-transform based (FT) data; (ii) amplitude-adjusted FT (AAFT) data; and (iii) iteratively refined AAFT (IAAFT) data of two types (IAAFT-1 and IAAFT-2). The approach was applied to experimental protocols activating or blocking the sympathetic or parasympathetic branches of the autonomic nervous system or periodically perturbing cardiovascular control via paced respiration at different breathing rates. We found that short-term HPV was mostly linear at rest. Experimental protocols activating the sympathetic or parasympathetic nervous system did not produce non-linear dynamics. In contrast, paced respiration, especially at slow breathing rates, elicited significantly non-linear dynamics. Therefore, in short-term HPV ( approximately 300 beats) the use of non-linear models is not supported by the data, except under conditions whereby the subject is constrained to a slow respiratory rate.

Lentiviral vector interactions with the host cell.

Lentiviral vectors (LVs)-mediated gene transfer is an efficient method for ex vivo and in vivo gene therapy. Actually, LVs have been used in several clinical trials and therapeutic correction was reached in affected patients. However, in order to be effective gene therapy needs to be efficient without detrimental effects for target cells. Successful cell transduction by LVs can be hampered by several factors such as the activation of innate immune sensors during cell transduction and different restriction factors (RFs) inhibiting viral replication inside the cells. Therefore, a better knowledge of host-vector interactions is important for the development of more efficient gene therapy strategies improving the LVs platform by limiting harmful responses.

SIRT1 at the crossroads of AKT1 and ERß in malignant pleural mesothelioma cells.

In this report, we show that malignant pleural mesothelioma (MPM) patients whose tumors express high levels of AKT1 exhibit a significantly worse prognosis, whereas no significant correlation with AKT3 expression is observed. We provide data that establish a phosphorylation independent role of AKT1 in affecting MPM cell shape and anchorage independent cell growth in vitro and highlight the AKT1 isoform-specific nature of these effects.We describe that AKT1 activity is inhibited by the loss of SIRT1-mediated deacetylation and identify, by mass spectrometry, 11 unique proteins that interact with acetylated AKT1.Our data demonstrate a role of the AKT1/SIRT1/FOXM1 axis in the expression of the tumor suppressor ERß. We further demonstrate an inhibitory feedback loop by ERß, activated by the selective agonist KB9520, on this axis both in vitro and in vivo.Our data broaden the current knowledge of ERß and AKT isoform-specific functions that could be valuable in the design of novel and effective therapeutic strategies for MPM.

Different spectral components of 24 h heart rate variability are related to different modes of death in chronic heart failure.

AIMS: To assess whether analysis of heart rate variability (HRV) from 24 h Holter recordings provides information about the mode of death (pump failure vs. sudden death) in chronic heart failure (CHF). METHODS AND RESULTS: We analysed 24 h HRV in 330 consecutive CHF patients in sinus rhythm. Indices derived from time domain, spectral domain, and fractal analyses of 24 h automatic HRV were evaluated. Data from clinical assessment, echocardiography, right heart catheterization, exercise test, blood biochemical examination, and arrhythmia pattern were analysed. Patients were followed up for 3 years. Two simple multivariable models, both including 24 h spectral indices, were able to identify patients at higher risk of progressive pump failure and sudden death, respectively. Depressed power of night-time HRV (< or = 509 ms(2)) below 0.04 Hz [very low frequency (VLF)], high pulmonary wedge pressure (PWP > or = 18 mm Hg) and low left ventricular ejection fraction (LVEF < or = 24%) were independently related to death for progressive pump failure, while the reduction of power between 0.04 and 0.15 Hz at night (LF < or = 20 ms(2)) and increased left ventricular end-systolic diameter (LVESD > or = 61 mm) were linked to sudden mortality. CONCLUSION: Automatic spectral analysis of 24 h HRV provides independent risk indices related to mode of death in sinus rhythm CHF patients.