Nicolas Y Petit

Claude Baillou

Aude Burlion

Karim Dorgham

Béatrice Levacher

Corinne Amiel

Véronique Schneider

François M Lemoine

Guy Gorochov

Gilles Marodon
email: gilles.marodon@upmc.fr

Gene transfer of two entry inhibitors protects CD4 + T cell from HIV-1 infection in humanized mice -Running title: HIV-1 gene therapy in humanized mice

Targeting viral entry is the most likely gene therapy strategy to succeed in protecting the immune system from pathogenic HIV-1 infection. Here, we evaluated the efficacy of a gene transfer lentiviral vector expressing a combination of viral entry inhibitors, the C46 peptide (an inhibitor of viral fusion) and the P2-CCL5 intrakine (a modulator of CCR5 expression), to prevent CD4 + T cell depletion in vivo. For this, we used two different models of HIV-1-infected mice, one in which ex vivo genetically-modified human T cells were grafted into immunodeficient NOD.SCID.c -/-mice before infection and one in which genetically-modified T cells were derived from CD34 + hematopoietic progenitors grafted few days after birth. Expression of the transgenes conferred a major selective advantage to genetically-modified CD4 + T cells, the frequency of which could increase from 10 to 90% in the blood following HIV-1 infection.

Moreover, these cells resisted HIV-1-induced depletion, contrary to non-modified cells that were depleted in the same mice. Finally, we report lower normalized viral loads in mice having received genetically-modified progenitors. Altogether, our study documents that targeting viral entry in vivo is a promising avenue for the future of HIV-1 gene therapy in humans.

Introduction

Although there is no consensus on a definitive immune correlate of protection, there are multiple convincing examples linking human genetics and susceptibility to HIV-1 infection. The best example of a genetic predisposition protecting from HIV-1 remains the ∆32 mutation that prevents CCR5 expression at the cell surface, and thus completely protects 1% of Europeans from being infected. [START_REF] Martinson | Global distribution of the CCR5 gene 32-basepair deletion[END_REF] The so-called 'Berlin patient' was grafted with CCR5-deficient bone marrow to treat his leukemia and was subsequently cured of both diseases. [START_REF] Holt | Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo[END_REF] Simultaneously, genetic interventions targeting chemokine receptors using DNA -nucleases gave encouraging results in humanized mice (HuMice) [START_REF] Holt | Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo[END_REF][START_REF] Didigu C A, Wilen | Simultaneous zinc-finger nuclease editing of the HIV coreceptors ccr5 and cxcr4 protects CD4+ T cells from HIV-1 infection[END_REF][START_REF] Perez | Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases[END_REF] but the long term impact of this procedure, as well as the concerns with off-target cleavages, is still unknown. Clinical trials applying this strategy to lymphocytes or stem cells have shown that modified cells possessed a selective advantage compared with non-modified cells, [START_REF] Tebas | Gene Editing of CCR5 in Autologous CD4 T Cells of Persons Infected with HIV[END_REF] which is one criterion of success for the therapy. Thus, there is a strong rationale to use gene therapy as an adjunct to current and future treatments. [START_REF] Peterson | Combinatorial anti-HIV gene therapy: using a multipronged approach to reach beyond HAART[END_REF] Maraviroc, a CCR5 chemical antagonist, is a powerful medication in vitro but resistant variants rapidly emerge in treated patients for complex reasons, such as mutations in the gp120 coding sequence affecting CCR5 docking. [START_REF] Colin | HIV-1 exploits CCR5 conformational heterogeneity to escape inhibition by chemokines[END_REF] Similarly, the fusion inhibitor Enfuvirtide (a gp41 analog), which is delivered in solution to patients, rapidly becomes ineffective because gp41 mutates to escape Enfuvirtide binding. [START_REF] Greenberg | Resistance to enfuvirtide, the first HIV fusion inhibitor[END_REF] Thus, the therapeutic arsenal targeting viral entry is scarce and poorly efficient. However, strategies based on blocking entry are perhaps the most promising to rapidly restore a pool of functional T cells, the main goal to prevent AIDS. [START_REF] Laer | Gene therapy for HIV infection: what does it need to make it work?[END_REF] More recently, it was shown that HIV-1 infection needs not to be productive in CD4 + T cells to induce cell death by pyroptosis [START_REF] Doitsh | Cell death by pyroptosis drives CD4 T-cell depletion in HIV-1 infection[END_REF] . This mechanism of HIV-1-induced cell death highlights the interest of strategies aimed at preventing viral entry. We proposed developing a gene transfer vector in which two viral entry inhibitors in combination would have a better efficacy at preventing viral entry. In support of this hypothesis, a synergistic effect of Enfuvirtide was demonstrated in cells with low levels of CCR5 [START_REF] Heredia | CCR5 density levels on primary CD4 T cells impact the replication and Enfuvirtide susceptibility of R5 HIV-1[END_REF] . Importantly, viral variants able to escape gp41 analogs and CCR5 inhibitors at the same time have only been described in vitro with a drastic cost on viral fitness, [START_REF] Anastassopoulou | Effects of sequence changes in the HIV-1 gp41 fusion peptide on CCR5 inhibitor resistance[END_REF] illustrating the difficulty for the virus to escape both inhibitors at the same time. Using monocistronic lentiviral vectors, we previously showed a synergistic effect of the P2-CCL5 intrakine with the C46 peptide on HIV-1 infection in vitro [START_REF] Petit | Targeting Both Viral and Host Determinants of Human Immunodeficiency Virus Entry, Using a New Lentiviral Vector Coexpressing the T20 Fusion Inhibitor and a Selective CCL5 Intrakine[END_REF] . The P2-CCL5 intrakine, originally described as a high affinity CCL5 (RANTES) variant, [START_REF] Hartley | Human immunodeficiency virus type 1 entry inhibitors selected on living cells from a library of phage chemokines[END_REF] was later modified to incorporate an ER retention sequence, sequestering CCR5 away from the cell surface [START_REF] Schroers | Lentiviral transduction of human Tlymphocytes with a RANTES intrakine inhibits human immunodeficiency virus type 1 infection[END_REF] . The C46 peptide is the optimized membrane-bound form of Enfuvirtide and has been used in several gene therapy studies since it is effective on both CCR5-or CXCR4-tropic HIV-1, and can be accommodated in several gene transfer vectors, including lentiviral vectors [START_REF] Egelhofer | Inhibition of Human Immunodeficiency Virus Type 1 Entry in Cells Expressing gp41-Derived Peptides[END_REF][START_REF] Kimpel | Survival of the Fittest: Positive Selection of CD4+ T Cells Expressing a Membrane-Bound Fusion Inhibitor Following HIV-1 Infection[END_REF][START_REF] Burke | Engineering Cellular Resistance to HIV-1 Infection In Vivo Using a Dual Therapeutic Lentiviral Vector[END_REF][START_REF] Trobridge | Protection of stem cell-derived lymphocytes in a primate AIDS gene therapy model after in vivo selection[END_REF] . Here, we aimed to evaluate the in vivo efficacy of an optimized lentiviral vector co-expressing those two entry inhibitors. We used two pre-clinical models of HIV-1 gene therapy, either infusing genetically-modified T cells in adult immunocompromised NOD.SCID.gc -/-(NSG/PBL) mice or grafting genetically-modified hematopoietic progenitors in NSG neonates (NSG/CD34).

Results

A lentiviral vector expressing two inhibitors of HIV-1 entry

With the general aim to validate the combination of the C46 peptide and the P2-CCL5 intrakine for HIV-1 gene therapy in vivo, we used an optimized version of our previously described lentiviral vector, which efficiently inhibited HIV-1 infection in vitro. [START_REF] Petit | Targeting Both Viral and Host Determinants of Human Immunodeficiency Virus Entry, Using a New Lentiviral Vector Coexpressing the T20 Fusion Inhibitor and a Selective CCL5 Intrakine[END_REF] To facilitate detection of genetically-modified cells, we added the GFP reporter gene after the therapeutic cassette to generate the LvGFP-C46-P2 vector (Fig. 1a). A vector using the same strong promoter EF1 but in which the therapeutic cassette was omitted was used as a control (Fig. 1a). We transduced anti-CD3/CD28 activated PBMCs to monitor transgene expression and function in vitro and in vivo. Expression of the GFP reporter molecule was well correlated with the expression of the C46 peptide (detected with the 2F5 monoclonal antibody) (Fig. 1b) and was also associated with a lower median fluorescence intensity (MFI) of CCR5 in vitro (Fig. 1c).

Passive diffusion of the intrakine was ruled out by the observation that GFP -cells exhibited similar CCR5 MFI than non-transduced cells (Fig. 1c), suggesting that this reduction was due to ER retention of CCR5 through interaction with the P2-CCL5 intrakine.. The MFI of CCR5 was also reduced two-fold in genetically-modified PBMCs injected in vivo in NSG mice (Fig. 1d), reflecting the expected down-modulation of CCR5 surface expression. Thus, GFP expression was a faithful reporter of transgenes expression and function, and was thus used to follow genetically-modified cells in vivo.

Protection of genetically-modified human CD4 + T cells from HIV-1 infection in NSG/PBL mice

As a model for HIV-1 infection of human CD4 + T cells in vivo, we first used adoptive cell transfer (ACT) in immunocompromised NSG mice (NSG/PBL). A major problem with ACT of human T cells in NSG mice is the xenogeneic graft-versus-host disease (GVHD) that develops thereafter and that invariably leads to death. [START_REF] King | Human peripheral blood leucocyte non-obese diabetic-severe combined immunodeficiency interleukin-2 receptor gamma chain gene mouse model of xenogeneic graft-versus-host-like disease and the role of host major histocompatibility complex[END_REF] We tested various ACT protocols in NSG mice and found that injection of 6.10 6 activated T cells in 1 Gy-irradiated mice represented an optimal trade-off between survival and engraftment efficiency (protocol P3 in Figure S1). To normalize the number of genetically-modified cells across experiments and vectors, we diluted transduced cells into non-transduced cells ex vivo prior ACT, establishing a number of GFP + cells at 10% of the injected cells. Mice were infected i.v with a CCR5-tropic HIV-1 strain 12 days after ACT.

The frequencies of CD4 + GFP + cells steadily increased in the blood of LvGFP-C46-P2-treated animals during the course of the infection to reach a plateau where up to 95% of all CD4 + T cells expressed the transgene (Fig. 2a). In contrast, the frequencies of GFP + cells in control HIV-1infected LvGFP-treated mice remained close to the 10% input throughout the experiment (Fig. 2a). The increase in GFP + cells with the LvGFP-C46-P2 vector was dependent on HIV-1 infection because it was not observed in non-infected NSG/PBL mice (Fig. S2), showing that the therapeutic vector did not increase the proliferation of modified cells per se. The frequencies of GFP + cells were also superior in the spleen and in the bone marrow of LvGFP-C46-P2-treated mice compared with LvGFP-treated control mice (Fig. 2b). These increased frequencies translated into increased numbers of CD4 + GFP + cell in the spleen and the bone marrow of LvGFP-C46-P2-treated mice compared with LvGFP mice (Fig. S3). Altogether, the results demonstrate that LvGFP-C46-P2-transduced CD4 + T cells possess a selective advantage relative to LvGFP-modified T cells.

To test the hypothesis that genetically-modified cells resisted HIV-1-induced depletion, we analyzed longitudinally the frequencies of CD4 + cells in CD3 + GFP + and CD3 + GFP -T cells in the blood of LvGFP-C46-P2-and LvGFP-treated mice (Fig. 2c). The frequencies of CD4 + T cells in the GFP -subset rapidly dropped after HIV-1 infection, showing that non-protected CD4 + T cells underwent HIV-1-induced depletion as expected (Fig. 2d). In striking contrast, the frequency of CD4 + T cells in the GFP + fraction remained constant throughout the experiment, showing that these cells were protected from HIV-1-induced depletion. Resistance to depletion was also observed in the spleen and in the bone marrow of LvGFP-C46-P2-treated animals, with statistically significant differences in the frequencies of CD4 + T cells in GFP + vs GFP -T cells (Fig. 2e). In contrast, the frequencies of GFP + cells, like the GFP -subset, steadily decreased in the blood of control LvGFP-treated mice (Fig. S4a), showing that GFP expression per se did not protect from HIV-1-induced deletion. A similar depletion of GFP + cells were found in the spleen and in the bone marrow of control LvGFP-treated mice (Fig. S4b). Thus, CD4 + T cells expressing the combination of viral entry inhibitors were protected from HIV-1-induced depletion in NSG/PBL mice in the blood and in lymphoid tissues.

Resistance of genetically-modified human CD4 + T cells to HIV-1-induced depletion in NSG/CD34 HuMice

We next wanted to confirm the potency of the vector to prevent HIV-1-induced CD4 + T cell depletion in a more physiological setting. For this, we grafted LvGFP-C46-P2-transduced CD34-purified cells from cord blood into neonatal NSG mice and monitored human cell reconstitution and transgene expression overtime. At 17 weeks post-injection, 11.9 ± 11.0 % of total cells from the blood (excluding erythrocytes) were human CD45 + CD3 + T cells in the animals used for the experiment. The frequencies of CD4 + and CD8 + T cells among CD3 + cells were less variable representing 40.0 ± 5.8 % and 47.0 ± 5.3 % , respectively (Fig. S5). Among 14 NSG/CD34 HuMice generated with LvGFP-C46-P2-modified CD34 + cells, only 8 had detectable GFP + cells in CD4 + T cells 17 weeks after. Four of those mice were infected with a CCR5-tropic HIV-1 strain, whereas 4 were left uninfected. Because the frequency of GFP + cells was highly variable among NSG/CD34 HuMice, it was not possible to reliably measure a selective advantage in that setting. To directly assess resistance of genetically-modified CD4 + T cells to HIV-1-induced depletion, frequencies of CD4 + T cells were measured in GFP + and GFP -cells (Fig. 3). In non-infected mice, the frequencies of CD4 + T cells in the blood remained similar in GFP + vs GFP -T cells throughout the course of the experiment (Fig. 3a). As expected, frequencies of GFP -cells steadily decreased in HIV-1-infected animals whereas frequencies of GFP + remained stable, showing that CD4 + GFP + T cells resisted HIV-1-induced depletion in the blood of NSG/CD34 HuMice (Fig. 3b). As expected in non-infected mice, the frequencies of CD4 + T cells in lymphoid organs were similar in GFP + or GFP -subsets (Fig. 3c). In contrast, frequencies of CD4 + T cells among GFP + and GFP -cells significantly differed in the LN, spleen and bone marrow (Fig. 3d). Of note is the one mouse in which resistance to deletion was not evident in the blood did not show any sign of resistance in the lymphoid organs. Thus, gene transfer of two entry inhibitors in CD34 + cells conferred resistance to CD4 + T cells in 3 mice out of 4 analyzed.

Gene transfer of entry inhibitors impact viral replication in NSG/CD34 HuMice.

To assess the impact that the therapy might have on viral loads, we measured viremia in LvGFP-C46-P2-treated mice in which GFP + cells were observed (n=4) or not (n=6) prior HIV-1 infection. To accommodate the various levels of human cells engrafment among the different mice (Fig. S5), viremia was corrected by the frequency of CD45 + CD3 + CD4 + T cells among total cells of the blood at the time of the analysis. Initially, normalized viremia was similar in both groups, showing that the therapy was not associated with an immediate effect on viral replication. However, we observed a tendency for lower normalized viral loads in mice bearing GFP + cells compared to mice in which no GFP + cells could be detected (Fig. 4a). To confirm that animals with GFP + cells carried less virus, we analyzed p24 expression in CD4 + T cells at the end of the experiment. We found that the frequencies of CD4 + cells expressing p24 in mice with GFP + cells were lower than in mice without GFP + cells and close to background staining obtained in noninfected HuMice (Fig. 4b). Altogether, we conclude that NSG/CD34 HuMice reconstituted with gene-modified CD34 + progenitors were protected from HIV-1-induced CD4 + T cell deletion and had a lower number of infected cells, corroborating with lower viral loads.

Discussion

Here, we show that a lentiviral vector encoding two viral entry inhibitors confers a selective advantage to genetically-modified cells in vivo, due to their resistance to HIV-1mediated depletion. We observed a strong and long-lasting selective advantage in the NSG/PBL model. A lower selective advantage was reported in a very similar model of NSG/PBL HuMice using a vector expressing only the C46 peptide. [START_REF] Kimpel | Survival of the Fittest: Positive Selection of CD4+ T Cells Expressing a Membrane-Bound Fusion Inhibitor Following HIV-1 Infection[END_REF] This observation suggest that two entry inhibitors might be better than one at protecting cells from HIV-1. However, a pre-clinical study in macaques reconstituted with progenitors expressing the C46 peptide alone showed lower viral loads correlated to a clear selective advantage [START_REF] Younan | Positive selection of mC46-expressing CD4+ T cells and maintenance of virus specific immunity in a primate AIDS model[END_REF] . Moreover, recent studies showed that inhibition of CCR5 expression by shRNA was sufficient to protect CD4 + T cells from infection and to confer a selective advantage in chimeric Bone marrow-Liver-Thymus (BLT) HuMice [START_REF] Shimizu | RNAi-Mediated CCR5 Knockdown Provides HIV-1 Resistance to Memory T Cells in Humanized BLT Mice[END_REF][START_REF] Myburgh | Lentivector knock-down of CCR5 in hematopoietic stem cells confers functional and persistent HIV-1 resistance in humanized mice[END_REF] . Thus, targeting gp41 and CCR5 have independently the potential to curb HIV-1 infection, highlighting the interest of using two inhibitors of this crucial step of HIV-1 infection in the same vector.

A strong selective advantage is not always associated with lower viral loads. In CD34reconstituted HuMice, Walker et al. reported that expression of a triple combination of anti-HIV-1 genes did not impact viral replication, although a significant selective advantage was observed. [START_REF] Walker | Generation of an HIV-1-Resistant Immune System with CD34+ Hematopoietic Stem Cells Transduced with a Triple-Combination Anti-HIV Lentiviral Vector[END_REF] A modest but significant effect on viral loads was reported following CCR5-specific ZFNmediated modification in NSG/PBL HuMice. [START_REF] Perez | Establishment of HIV-1 resistance in CD4+ T cells by genome editing using zinc-finger nucleases[END_REF] However, only one time point was analyzed in that study. A kinetics study showed that the reduction in viral loads using the same technology was much more discrete in NSG/CD34 HuMice despite a considerable selective advantage. [START_REF] Holt | Human hematopoietic stem/progenitor cells modified by zinc-finger nucleases targeted to CCR5 control HIV-1 in vivo[END_REF] Our PCR and p24 data concur to the hypothesis that selective advantage conferred by our vector had an impact on viral replication. Recently, a complete protection from HIV-1 was observed in BLT mice reconstituted with human cells modified with a vector very similar to our, encoding the C46 peptide and a shRNA targeting CCR5 [START_REF] Burke | Engineering Cellular Resistance to HIV-1 Infection In Vivo Using a Dual Therapeutic Lentiviral Vector[END_REF] . This is the first report showing that viral replication can be totally controlled in humanized mice by gene therapy without prior sorting of geneticallymodified cells, as recently shown for a CCR5 shRNA [START_REF] Myburgh | Lentivector knock-down of CCR5 in hematopoietic stem cells confers functional and persistent HIV-1 resistance in humanized mice[END_REF] . This surprising and unique result suggest that maximal efficacy of HIV-1 gene therapy might necessitate a functional immune response, that is present in monkeys and BLT HuMice but lacking or severely hampered in other HuMice models. One must keep in mind though that some HIV-1-specific PCR might amplify the vector as well [START_REF] De Ravin | False-Positive HIV PCR Test Following Ex Vivo Lentiviral Gene Transfer Treatment of X-linked Severe Combined Immunodeficiency Vector[END_REF] . The use of HIV-1-specific PCR discriminating HIV-1 from the vector such as the one employed in our study should become the gold standard.

Considering the recent developments of nucleases that target CCR5 in CD34 + progenitor cells , we believe that residual expression of the molecule such as the one observed with our intrakine, might allow for normal hematopoeisis and circulation of modified-cells while total ablation by genetic means may impact on these processes. Recent advances in lentiviral delivery of ZN finger nucleases might improve specific targeting of the nuclease to mature CD4 + T cells, a protocol that would limit bystander effects [START_REF] Abarrategui-Pontes | Codon swapping of zinc finger nucleases confers expression in primary cells and in vivo from a single lentiviral vector[END_REF] .

The selective advantage of genetically-modified cells would only be obtained in the context of high levels of viral replication. Although ART interruptions have been performed in the past to provoke selective growth of modified cells in small-scale clinical trials for gene therapy, [START_REF] Digiusto | RNA-based gene therapy for HIV with lentiviral vector-modified CD34(+) cells in patients undergoing transplantation for AIDS-related lymphoma[END_REF][START_REF] Podsakoff | Selective survival of peripheral blood lymphocytes in children with HIV-1 following delivery of an anti-HIV gene to bone marrow CD34+ cells[END_REF] an interruption in therapy is not foreseeable in patients in the long term. Gene therapy might thus be particularly suitable for patients experimenting treatment failure with high viral loads.

Materials and Methods

Lentiviral vector design and production

Second-generation self-inactivating (SIN) lentiviral vectors were used in this study. [START_REF] Zufferey | Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery[END_REF] The LvGFP-C46-P2 vector was constructed by adding an eGFP gene and 2A sequence upstream of the therapeutic cassette (construction encoding the C46 peptide and P2-CCL5 analog described in Petit et al., 2014) [START_REF] Petit | Targeting Both Viral and Host Determinants of Human Immunodeficiency Virus Entry, Using a New Lentiviral Vector Coexpressing the T20 Fusion Inhibitor and a Selective CCL5 Intrakine[END_REF] in the backbone of a lentiviral vector carrying the EF1a promoter. As a control, the LvGFP vector expressing GFP only was used. Details on the cloning procedures are available on request. Lentiviral vectors were produced in mycoplasma-free HEK-293T cells, as described previously. [START_REF] Marodon | Specific transgene expression in human and mouse CD4+ cells using lentiviral vectors with regulatory sequences from the CD4 gene[END_REF]

Mice and humanization

NOD Prkdc scid Il2rg tm1Wjl (NSG) mice (strain ≠05557; Jackson Laboratory, USA) were bred in animal facilities of Centre d'Expérimentation Fonctionnelle (CEF) according to the Jackson Laboratory handling practice specific to that strain. The regional ethical committee on animal experimentation Darwin approved all mouse protocols. Primary human cells were obtained from leukapheresis samples collected from healthy donors at the Etablissement Francais du Sang after informed consent. Cells were grown at a concentration of 1.10 6 cells/mL and activated in RPMI, 10% FCS, penicillin/streptomycin, interleukin-2 (Proleukin, 600 IU/mL; Novartis, Basel, Switzerland), and CD3/CD28 beads (Invitrogen, Carlsbad, CA) at 3 beads per cell. Two days after activation, cells were transduced by spinoculation for 2 hrs at 1000 g at 30°C, with the indicated lentiviral vectors at a multiplicity of infection (MOI) of 6 to 8 in the presence of protamine sulfate (2 µg/mL, Sigma). Three days after transduction, 1 Gy-irradiated female 8 to 12-weeks old NSG mice were injected with 6.10 6 cells. Twelve days post-adoptive cell transfer (ACT), mice were infected with 25 ng of p24 of NLAD8 HIV-1 strain in a final volume of 100 µL of 1X PBS. All mice used in this study were randomly assigned to experimental group and cages. Investigator was not blinded to the group allocation during the experiments.

Human hematopoietic progenitor cells were obtained from cord blood samples collected from healthy donors after informed consent. Mononuclear cells from human cord blood were isolated by Ficoll density gradient and centrifuged at 200 g during 13 min to remove platelets.

Then, CD34 + progenitors were sorted with the human CD34 MicroBeads kit, according to the manufacturer's instructions (Miltenyi). CD34 + cells were incubated at a concentration of 1.10 6 cells/mL over night into StemSpan SFEMII medium (StemCell technologies) complemented with human recombinant cytokines (IL-6 and TPO at 20 ng/mL; SCF and FLT3-L at 100 ng/mL; Peprotech) and antibiotics. Cells were transduced with the LvGFP-C46-P2 lentiviral vector in StemSpan medium in the presence of cytokines, the proteasome inhibitor MG-132 (1 µM; Sigma), antibiotics and protamine sulfate (8 µg/mL; Sigma). CD34 + cells underwent two rounds of transduction separated by 3 hours incubation at 37°C and 5% CO 2 . For each transduction cycle, cells were centrifuged at 1000 g at 30°C for 2 hours with the lentiviral vector at a MOI of 15. Twenty-four to 48-hour-old NSG mice were irradiated at 0.9 Gy and grafted with 0.5.10 5 to 2.5.10 5 transduced CD34 + cells by the intra-hepatic route. Ten ng of the p24 NL-AD8 HIV-1 strain were injected into the retro-orbital sinus of 17 weeks-old mice in a final volume of 100 µL of 1X PBS.

HIV-1 production and quantification

HIV-1 molecular clone NL-AD8 was obtained through the AIDS Research and Reference Reagent Program. HIV-1 stocks were prepared with 30 µg of plasmid transfected into 15.10 6 mycoplasma-free HEK 293T cells in T-175 flasks by calcium phosphate precipitation. The supernatant was frozen at -80°C and viral titers were quantified by p24 ELISA according to the manufacturer's instructions (Zeptometrix, Buffalo, NY). Mice were bled on ACD (acid-Citrate-Dextrose) anticoagulant and plasma HIV-1 RNA viral loads were measured using the Abbott RealTime HIV-1 RT-PCR assay that do not amplify genomic regions present in lentiviral vectors contrary to the Roche Cobas PCR (our unpublished observations and De Ravin et al [START_REF] De Ravin | False-Positive HIV PCR Test Following Ex Vivo Lentiviral Gene Transfer Treatment of X-linked Severe Combined Immunodeficiency Vector[END_REF] ). Due to the small volumes of plasma from the mice, a dilution was necessary to reach the volume needed for the assay. Thus, this detection limit varied between 200 and 2000 copies/mL depending on the initial volume of mouse plasma.

Flow cytometry

Red blood cells from whole blood were lysed with 4.5 ml of water for 15 s before adding 0.5 ml of 10X PBS. Red blood cells from spleen or bone marrow were lysed with ACK buffer (NH 4 Cl 0.15 M, KHCO3 10 mM, EDTA 0.1 mM). Cell suspensions were stained with an optimal quantity of antibodies at a concentration of 10 7 cells/mL in a final volume of 100 µL of PBS/FCS 3%. Incubation was performed in the dark at 6°C for 20 min. The following anti-human mAbs were used for cell surface staining: CD45 PE-CF594 (clone HI30; catalog number (cat ≠) 562279, BD Biosciences) anti-CCR5 Alexa Fluor 647 (HEK/1/85a; cat ≠ 313712, Biolegend), anti-CD4 PerCP (RPA-T4, cat ≠ 300528, Biolegend), anti-CD8 Alexa Fluor 700 (HIT8a, cat ≠ 300920, Biolegend), CD3 PE-Cy7 (UCHT1, cat≠ 300420, Biolegend). The human IgG1 mAb 2F5 specific for a gp41 epitope (cat ≠ AB001, Polymun, Austria) was used to detect the C46 peptide. The KC57-RD1 (cat≠ 6604667, Beckman Coulter) antibody was used to detect intracellular p24 after cells were treated with permeabilization buffer (eBioscience, fixation and permeabilization kit). All cell preparations were acquired on an LSRII cytometer (BD) and analyzed with FlowJo software (Tree Star, Portland, OR). The frequencies of positive cells were determined according to the fluorescence minus one (FMO) staining negative control.

Statistical analysis

No statistical method was used to assess sample size needed to detect an effect. Except for the NSG/CD34 model, which is a single experiment, all the results shown in this study are compiled from 2 independent experiments. Two-tailed p values indicated on the graphs were calculated with Prism version 6.0 software (GraphPad Software, San Diego, CA), using the unpaired Mann-Whitney test with a confidence interval of 95%. The median value are indicated by horizontal bars on the graphs. Linear and non linear regression analysis were performed using Prism 6.0 to determine whether slopes significantly differed. Plateau with one phase decay association or dissociation equations were used to model the data.

  Briefly, 23.3 µg of the ∆8.9 packaging plasmid, 30 µg of the vector plasmid, and 10 µg of the vesicular stomatitis virus (VSV)-G envelope were transfected into 15.10 6 cells in T-175 flasks by calcium phosphate precipitation. Vector supernatants were collected 48 hours post-transfection and concentrated by ultrafiltration (Centricon Plus-70; Millipore, Molsheim, France) at 3500 g at 4°C. Viral stocks were kept frozen at -80° C. Viral titers were determined on HEK-293T cells with various concentrations of vector supernatants in the presence of Polybrene (8 µg/mL; Sigma-Aldrich, Saint-Quentin-Fallavier, France). Seventy-two hours after transduction, the percentage of cells expressing the transgenes was determined by flow cytometry and used to calculate a viral titer as the number of infectious particles per milliliter.

Figure 1 .

 1 Figure 1. Lentiviral vector design and co-expression of anti-HIV-1 genes and eGFP into a lentiviral vector. (a) A schematic representation of the structure of the lentiviral vectors used in the present study is shown. (LTR: long terminal repeat; cppT: central polypurine tract of HIV-1; EF1α: Elongation factor 1 promoter; C46: membrane-bound form of T20 (C46 peptide); 2A: 2A sequence of the foot-and-mouth disease virus; P2i: P2-CCL5 intrakine; WPRE: Woodchuck Hepatitis virus regulatory element; ΔLTR: U3 deleted LTR). Indicated is the reference of the vector used throughout the study. Not to scale. (b) Co-expression of the C46 peptide (detected with the 2F5 mAb) and of eGFP and (c) co-expression of CCR5 and eGFP in human CD4 + PBMC activated by CD3/CD28 beads and IL-2 21 to 29 days post transduction with the LvGFP-C46-P2 vector (NT: Non-transduced; FMO: fluorescence minus one; MFI: median fluorescence intensity) (d) In vivo CCR5 expression on CD45 + CD3 + CD4 + T cells in GFP + and GFP -cells from non-irradiated NSG mice grafted with 2.10 6 LvGFP-C46-P2 transduced T lymphocytes and analyzed in the blood and the spleen 34 to 53 days post-graft.

Figure 2 .

 2 Figure 2. Protection of genetically-modified human CD4 + T cells from HIV-1 infection in NSG/PBL mice. (a-b) Frequencies of GFP + cells in human CD45 + CD3 + CD4 + T cells in the blood at various days after HIV-1 infection (a) and in the spleen or bone marrow (BM) (b) 35 to 45 days after injection of LvGFP-or LvGFP-C46-P2-modified T cells in NSG mice. (c) Representative histograms and dot plots showing the gating strategy to determine the frequencies of CD4 + T cells in GFP + and GFP -human CD3 + T cells. (d-e) Frequencies of CD4 + cells in the CD3 + GFP + and CD3 + GFP -populations were determined in LvGFP-C46-P2-injected mice in the blood at various days after infection (d) and in the spleen or bone marrow (BM) (e) at the end of the experiment. The results are compiled from 2 independent experiments using the P3 ACT protocol (Fig. S1). Non linear regression analysis curve fit are shown. The p value indicate the significant difference between the two slopes.

Figure 3 .

 3 Figure 3. Resistance of genetically-modified CD4 + T cells to HIV-1-induced depletion in vivo in NSG/CD34 HuMice. (a) Blood frequencies of CD4 + cells in CD3 + GFP + or CD3 + GFP -

Figure 4 .

 4 Figure 4. Gene transfer of entry inhibitors impacts viral replication in NSG/CD34 HuMice (a) Viral load was measured by qPCR after HIV-1 infection in LvGFP-C46-P2-treated mice with undetectable (-GFP) or detectable GFP + cells (+GFP) in CD4 + T cells prior to infection. Shown is the viral load value normalized by the frequency of human CD45 + CD3 + CD4 + T cells present in total cells of the blood sample for each time point. (b) Frequencies of p24 + cells in CD4 + T cells from the lymph node of NSG HuMice with (+GFP) or without GFP + cells (-GFP) 77 days after infection with NL-AD8 HIV-1 (HIV+) or non infected (HIV-). A representative CD4 vs p24 staining is shown above each group. One mouse from the (+GFP) group was excluded from the graph since it did not show any protection against HIV-1-induced depletion in the periphery.

Acknowledgments

This work was supported by grants from Agence Nationale de la Recherche to Guy Gorochov and Gilles Marodon. Nicolas Petit was supported by a doctoral fellowship from Fond Pierre Bergé /SIDAction and by the ANRS. The authors would like to thank David Klatzmann (INSERM U959, Paris, France) for his initial support on this project, Dorothée van Laer (Innsbruck University, Austria) for kind gift of the C46 peptide, Arnaud Moris (CIMI-PARIS, France) for the kind gift of NL-AD8 HIV-1 strain, Dr Hans Yssel (CIMI-PARIS) for performing Hoechst-based detection test of mycoplasma, Aude Burlion (CIMI-PARIS, France) for technical help. Nicolas Petit, Claude Baillou and Gilles Marodon performed experiments; Béatrice Levacher constructed the vectors; Corinne Amiel and Véronique Schneider performed HIV-1 qPCR; Karim Dorgham, Francois Lemoine, Guy Gorochov conceived experiments, contributed to essential reagents and corrected the manuscript; Nicolas Petit and Gilles Marodon conceived experiments, analyzed the data and wrote the manuscript.

This work was supported by grants from Agence Nationale de la Recherche to Guy Gorochov and Gilles Marodon. Nicolas Petit was supported by a doctoral fellowship from Fond Pierre Bergé /SIDAction and by the ANRS.

Conflict of Interest

The authors declare no conflict of interest Supplementary information is available at Gene Therapy's website


Petr Červenka

Karel Brinda
email: karel.brinda@univ-mlv.fr

Radek Seifert

Petr Hofman

Karel Břinda

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Sophie Nicole

Prof Bertrand Fontaine
email: bertrand.fontaine@upmc.fr.

Skeletal Muscle Sodium Channelopathies

Keywords:

Purpose of review

This is an update on skeletal muscle sodium channelopathies since knowledge in the field have dramatically increased the last years.

Recent finding

The relationship between two phenotypes and SCN4A have been confirmed with additional cases that remain extremely rare: 1) severe neonatal episodic laryngospasm mimicking encephalopathy, which should be actively searched for since patients respond well to sodium channel blockers; 2) congenital myasthenic syndromes, which have the particularity to be the first recessive Nav1.4 channelopathy. Deep DNA sequencing suggests the contribution of other ion channels in the clinical expressivity of sodium channelopathies, which may be one of the factors modulating the latter. The increased knowledge of channel molecular structure, the quantity of sodium channel blockers, and the availability of preclinical models would permit a most personalized choice of medication for patients suffering from these debilitating neuromuscular diseases.

Summary

Advances in the understanding of the molecular structure of voltage-gated sodium channels as well as availability of preclinical models would lead to improve medical care of patients suffering from skeletal muscle as well as other sodium channelopathies.

Introduction

Excitable cells are the hallmark of animals, found both in Protozoa and Metazoa, which give them the capacity of sensing and of reacting to an environmental modification. In Metazoa, excitable cells communicate with other cells by a unique mechanism common to perception, brain information processing, and motor action [START_REF] Cook | From membrane excitability to metazoan psychology[END_REF]. This latter relies on the functioning of transmembrane voltage-gated channels that regulate rapid changes in ion fluxes, resulting in transient and drastic changes in cell membrane potential, the so-called action potential (AP).

APs lead to liberation of a chemical signal that binds to specific receptors in neighboring cells, or of calcium within the cell to promote cell response such as secretion (neuron) or contraction (muscle). Due to their unique role in the depolarizing phase of AP, voltage-gated sodium channels (VGSC) are the direct targets for natural toxins and synthetic drugs that modulate cell excitability, or are the site for mutations causing diseases. In humans, distinct diseases are associated with mutations of nearly all isoforms of VGSC depending upon their cellular expression: epilepsies or hemiplegic migraines for neuronal isoforms (Nav1.1, Nav1.2, Nav1.6), cardiac arrhythmias for the heart isoform (Nav1.5), and myotonia or paralyses for the skeletal muscle isoform (Nav1.4) (2). However, the picture is far more complex than initially expected with distinct disease expressivity for each isoform. In this review, we will focus on recent advances in the comprehension of skeletal muscle sodium channelopathies. They were the first examples of defective VGSC as the direct cause of human diseases despite the late general belief that defective function of such an important channel would be lethal [START_REF] Fontaine | Hyperkalemic periodic paralysis and the adult muscle sodium channel alpha-subunit gene[END_REF][START_REF] Ptacek | Identification of a mutation in the gene causing hyperkalemic periodic paralysis[END_REF][START_REF] Rojas | A Met-to-Val mutation in the skeletal muscle Na+ channel alpha-subunit in hyperkalaemic periodic paralysis[END_REF], and are still good drivers for a better understanding of VGSCs.

Advances in basic understanding of Nav1.4 functioning

Na V 1.4 is the main VGSC isoform of mature and innervated skeletal muscle fibers. This heterodimer is composed of two not covalently bound transmembrane proteins, the  poreforming subunit encoded by SCN4A and a single auxiliary  subunit encoded by SCN1B. To summarize a complex picture, the  subunit forms the pore, and is the site for the mutations causing skeletal muscle sodium channelopathies. As all  subunits of VGSCs, it is made of four domains (DI to DIV), each constituted of six transmembrane segments (S1 to S6) with 3 possible conformations: closed, activated and inactivated (Figure 1). The channel opening (activation) in response to cell membrane depolarization is initiated by a motion into the extracellular space of positively charged amino acid residues (mainly arginine) at each third position on the transmembrane -helix within each S4 segment. Inactivation designs the mechanism by which the VGSC is closed after its opening despite the maintenance of cell membrane depolarization. There are two types of inactivation: rapid (few milliseconds range) and slow (hundreds of milliseconds to seconds range). Grossly, rapid and slow inactivation terminates action potential and regulates the number of channels that may open during repetitive firing, respectively. Recent reviews focusing on the molecular architecture and structure-function relationship of VGSC are available, and the readers are referred to them for a more complete overview [START_REF] Catterall | Voltage-gated sodium channels at 60: structure, function and pathophysiology[END_REF][START_REF] Catterall | Structure and function of voltage-gated sodium channels at atomic resolution[END_REF]. The  auxiliary subunit modulates the membrane expression level by interacting with extracellular matrix and cytoskeleton, influences the voltage-dependence and kinetics of channel gating, and might exert independent function in cell signaling [START_REF] Calhoun | The role of non-pore-forming beta subunits in physiology and pathophysiology of voltage-gated sodium channels[END_REF]. We will not further discuss on  despite its importance, since its dysfunction has surprisingly no effect on skeletal muscle in contrast to brain, peripheral nerve, and heart [START_REF] Wallace | Neuronal sodium-channel alpha1-subunit mutations in generalized epilepsy with febrile seizures plus[END_REF][START_REF] Chen | Mice lacking sodium channel beta1 subunits display defects in neuronal excitability, sodium channel expression, and nodal architecture[END_REF][START_REF] Watanabe | Sodium channel beta1 subunit mutations associated with Brugada syndrome and cardiac conduction disease in humans[END_REF][START_REF] Patino | A functional null mutation of SCN1B in a patient with Dravet syndrome[END_REF].

During the last few years, crystallization studies have dramatically increased knowledge of VGSCs. Crystals were obtained from the bacterial sodium channels NaChBac considered as the ancestors of VGSCs [START_REF] Payandeh | Bacterial voltage-gated sodium channels (BacNa(V)s) from the soil, sea, and salt lakes enlighten molecular mechanisms of electrical signaling and pharmacology in the brain and heart[END_REF]. In contrast to VGSCs, NaChBac are homotetramers, and only activation and fast inactivation can be studied, slow inactivation requiring physical linkage between the four domains. The first crystal structure of NaChBac was captured in the activated state [START_REF] Payandeh | The crystal structure of a voltagegated sodium channel[END_REF]. The rotation of the S4-S5 linkers responsible for the channel opening could be visualized as well as the mechanism of ion selectivity. The latter results from an anionic high field with ion dehydration by direct interaction with glutamate side chains.

Crystallization of NaChBac in inactivated state was most recently obtained [START_REF] Payandeh | Crystal structure of a voltage-gated sodium channel in two potentially inactivated states[END_REF][START_REF] Zhang | Crystal structure of an orthologue of the NaChBac voltage-gated sodium channel[END_REF]. A profound conformation change reshaping the ion selectivity filter was observed confirming the theorized mechanical coupling of activation and inactivation. Negatively charged amino acids were shown to obtrude the outer mouth of the channel as expected from biophysical models of VGSC inactivation. Crystallization studies of NaChBac have not only increased our basic understanding of VGSCs but also opened new avenues to design specific drugs modulating transmembrane Na + fluxes.

New SCN4A-associated diseases and modulators

Skeletal muscle sodium channelopathies are known since several decades to manifest as autosomal dominant syndromes with different degrees of recurrent attacks of muscle weakness (periodic paralyses) or muscle stiffness (myotonia) depending on the mutation. All described mutations are missense and affect the gating behavior or ion current passing through Nav1.4 [START_REF] Suetterlin | Muscle channelopathies: recent advances in genetics, pathophysiology and therapy[END_REF][START_REF] Cannon | Channelopathies of skeletal muscle excitability[END_REF]. Their prevalence has been recently estimated to be equal to 0.43/100.000 [START_REF] Horga | Prevalence study of genetically defined skeletal muscle channelopathies in England[END_REF] and 1.19/100.000 (unpublished data), in the UK and France, respectively. They display differential electroneuromyographic (ENMG) patterns at needle and compound muscle action potentials (CMAP) recording after short and long exercise [START_REF] Fournier | Electromyography guides toward subgroups of mutations in muscle channelopathies[END_REF][START_REF] Fournier | Cold extends electromyography distinction between ion channel mutations causing myotonia[END_REF]. According to variations of blood K + levels during attacks of muscle weakness, patients are classified into hypo-(HOKPP, OMIM#170400), normo-(NormoPP, OMIM#170600) or hyperkalemic (HYPP, OMIM#170500) periodic paralysis (PP). Several forms of sodium channel myotonia (SCM, OMIM#608390) are distinguished according to their intensity, fluctuating evolution, or response to acetazolamide, and may be difficult to clinically distinguish each other without ENMG and molecular characterization. In Paramyotonia Congenita (PMC, OMIM#168300), symptoms and signs are aggravated by exercise and cold, and include some degree of muscle weakness reminiscent of HYPP.

A new and severe form of SCM have emerged these last years. Severe Neonatal Episodic Laryngospasm (SNEL, OMIM#608390) is caused by de novo SCN4A mutations [START_REF] Mignot | Severe neonatal episodic laryngospasm due to de novo SCN4A mutations: a new treatable disorder[END_REF][START_REF] Simkin | Mechanisms underlying a life-threatening skeletal muscle Na+ channel disorder[END_REF]. In the neonatal phase, the disease is characterized by attacks of muscle stiffness affecting the upper airways. This causes cyanosis and hypoxemia and may be mistaken for an epileptic encephalopathy. Five additional cases have been described, leading to a total of 8 described patients with 2 distinct mutations, the p.Gly1306Glu accounting for all but one cases (Figure 1) [START_REF] Caietta | Diagnosis and outcome of SCN4A-related severe neonatal episodic laryngospasm (SNEL): 2 new cases[END_REF][START_REF] Singh | Mutations in SCN4A: a rare but treatable cause of recurrent life-threatening laryngospasm[END_REF]. The authors always emphasized the diagnostic difficulty because ENMG is not informative in babies, delaying efficient therapeutic intervention resulting to permanent deficiencies caused by hypoxia or even death of the patient. This rare condition should be systematically discussed as a cause of neonatal epileptic encephalopathy since sodium channel blockers (mexiletine and carbamazepine) dramatically ameliorate its course. When patients survive, they later develop a severe SCM affecting the limb muscles and easily diagnosed by ENMG. Why the upper airways are the first muscle groups to be affected in SNEL, distinguishing it from the severe myotonia permanens condition -caused by de novo SCN4A mutations including the p.Gly1306Glu one -is currently unknown.

According to a key role of Nav1.4 for the genesis of the muscle AP at the neuromuscular junction (NMJ) [START_REF] Mahmud | Na+ channels at postsynaptic muscle membrane affects synaptic transmission at neuromuscular junction: a simulation study[END_REF], Na v 1.4 dysfunction was recently confirmed in congenital myasthenic syndrome (CMS, OMIM#614198) -a heterogeneous group of inherited rare diseases resulting from functional or structural changes at the NMJ [START_REF] Hantai | Congenital myasthenic syndromes: an update[END_REF][START_REF] Engel | Congenital myasthenic syndromes: pathogenesis, diagnosis, and treatment[END_REF] -twelve years after the first report [START_REF] Tsujino | Myasthenic syndrome caused by mutation of the SCN4A sodium channel[END_REF][START_REF] Arnold | Defective fast inactivation recovery of Na 1.4 in congenital myasthenic syndrome[END_REF]. In the latter, only one (p. Val1442Glu) of the two compound SCN4A missense substitutions severely impaired Na v 1.4 gating, suggesting that the fatigable muscle weakness characteristic of CMS was dominantly inherited in the family [START_REF] Tsujino | Myasthenic syndrome caused by mutation of the SCN4A sodium channel[END_REF]. In the second patient, the phenotype resulting from one SCN4A missense mutation was for the first time recessively inherited [START_REF] Arnold | Defective fast inactivation recovery of Na 1.4 in congenital myasthenic syndrome[END_REF]. The inherited mutation was homozygous in the patient and substituted a charged amino acid residue of DIVS4 (p.Arg1457His). The two CMS-causing mutations dramatically favored the inactivation state, resulting in reduced availability of the mutant channels for opening and so a loss-of-function (Figure 2). This suggests that p.Val1442Glu would exert its dominant effect through haploinsufficiency, the amount of wild-type channels being not able to compensate for the inactivation of mutant channels.

Decremental muscle responses to repetitive nerve stimulation were recorded at ENMG investigations as expected for CMS but at nerve stimulation frequencies higher than usual in both patients (>10 Hz instead of 3 Hz). Why these two mutations cause a phenotype almost similar to CMS (i.e. blockade of neuromuscular transmission) rather than periodic paralysis (i.e. failure to generate/propagate muscle AP along sarcolemma), remains to be determined.

Patients suffering from PP may develop permanent muscle weakness related to late-onset permanent myopathy. In a recent survey, the percent of patients over 41 years with permanent muscle weakness was estimated to be equal to 68%, and with muscle fatigue to 89% [START_REF] Cavel-Greant | myopathies and cardiomyopathies : official journal of the Mediterranean Society of Myology / edited by the Gaetano Conte Academy for the study of striated muscle diseases[END_REF]. In addition, 89% of the surveyed patients reported muscle pain. The mechanisms at the origin of these symptoms are still unknown. Muscle weakness is probably the result of muscle changes including vacuolar myopathy and T-tubular aggregates. These myopathic features are direct consequences of the causing mutation since similar histopathological changes have been observed in all knock-in mouse models of PP developed so far [START_REF] Hayward | Targeted mutation of mouse skeletal muscle sodium channel produces myotonia and potassium-sensitive weakness[END_REF][START_REF] Wu | A sodium channel knockin mutant (NaV1.4-R669H) mouse model of hypokalemic periodic paralysis[END_REF][START_REF] Wu | A calcium channel mutant mouse model of hypokalemic periodic paralysis[END_REF][START_REF] Corrochano | Novel mutations in human and mouse SCN4A implicate AMPK in myotonia and periodic paralysis[END_REF].

The phenotype of each skeletal muscle sodium channelopathy is modulated by factors that may be genetics, epigenetics, and environmental, independently from their individual fluctuation with time. As physiologically expected, one genetic factor is the second SCN4A allele, the homozygosity of a dominant SCN4A mutation leading to a more severe phenotype [START_REF] Arzel-Hezode | Homozygosity for dominant mutations increases severity of muscle channelopathies[END_REF]. Another expected and recently documented genetics factor is the ClCN1 gene, which encodes the skeletal muscle chloride channel ClC-1 and is mutated in Myotonia Congenita, the most frequent form of non-dystrophic myotonia (NDM) [START_REF] Furby | Heterozygous CLCN1 mutations can modulate phenotype in sodium channel myotonia[END_REF]. SCN4A mutations may also modulate other neuromuscular phenotypes. Myotonic Dystrophy type 2 (DM2, OMIM#602668) is an adult onset muscle disease caused by a tetranucleotidic (CCTG) repeat expansion in the CNBP gene, combining muscle dystrophy and a mild myotonia often noticeable only by ENMG. DM2 are not classified as a primary channelopathy since the multisystemic phenotype results from defective posttranslational processing of many transcripts including ClCN1 one [START_REF] Mankodi | Expanded CUG repeats trigger aberrant splicing of ClC-1 chloride channel pre-mRNA and hyperexcitability of skeletal muscle in myotonic dystrophy[END_REF]. Nevertheless, a combination of DM2 repeat expansion and a SCN4A missense mutation (p.Pro72Leu) located within the N-terminus, results in earlier and most severe myotonia in DM2 [START_REF] Bugiardini | SCN4A mutation as modifying factor of Myotonic Dystrophy Type 2 phenotype[END_REF]. This missense variation modestly shifted the voltagedependence of activation toward hyperpolarization (-5 mV), which may favor membrane hyperexcitability.

Advances into pathophysiology understanding of skeletal muscle sodium channelopathies

The major advances made the last years in the field of skeletal muscle channelopathies concern HOKPP. HOKPP are typically associated with mutations of charged amino acid residues of the voltage sensor domain (VSD) -formed by the S4 segments -in the calcium Cav1.1 (60% of the cases) and Nav1.4 (20%) channels (Figure 1). The remaining 20% of cases, mostly isolated patients, remain not explained at the molecular level. The pathophysiology of HOKPP, especially the paradoxical sarcolemma depolarization in low extracellular K + concentration, had been remained mysterious until the demonstration of a potentiated gating pore current, also known as omega pore, at hyperpolarized potentials in all Nav1.4, then in Cav1.1, HOKPP mutant channels [START_REF] Wu | A calcium channel mutant mouse model of hypokalemic periodic paralysis[END_REF][START_REF] Sokolov | Gating pore current in an inherited ion channelopathy[END_REF][START_REF] Struyk | Gating pore currents in DIIS4 mutations of NaV1.4 associated with periodic paralysis: saturation of ion flux and implications for disease pathogenesis[END_REF][START_REF] Francis | Leaky sodium channels from voltage sensor mutations in periodic paralysis, but not paramyotonia[END_REF][START_REF] Jurkat-Rott | Pathophysiological role of omega pore current in channelopathies[END_REF][START_REF] Groome | Domain III S4 in closed-state fast inactivation: insights from a periodic paralysis mutation[END_REF]. To note that aberrant gating pore currents resulting from VSD mutations in Nav1.5 (DIS4) have recently been demonstrated in patients with mixed arrhythmia and dilated cardiomyopathy, indicating that this defective molecular mechanism is not restricted to skeletal muscle sodium channelopathies [START_REF] Moreau | Gating pore currents are defects in common with two Nav1.5 mutations in patients with mixed arrhythmias and dilated cardiomyopathy[END_REF]. Substitution of individual VSD gating charge allows inward cation current to flow through the gating pore at membrane potentials depending upon the position of the substituted amino acid.

In NaChBac, mutating the first or second charged residue resulted in gating pore current at hyperpolarized membrane potentials, where the homotetrameric channels are in resting states, but not at depolarized potentials [START_REF] El-Din | Tracking S4 movement by gating pore currents in the bacterial sodium channel NaChBac[END_REF]. Conversely, mutating the third charged residue resulted in closed gating pore at hyperpolarized membrane potentials and opened gating pore with the  activation. All S4 segments are also not equal regarding their potential to modulate gating pore current. Whereas substituting the charged residues of DI-DIIIS4 improved the gating pore currents at hyperpolarization, this had no effect when DIVS4 residues were replaced [START_REF] Gosselin-Badaroudine | Gating pore currents and the resting state of Nav1.4 voltage sensor domains[END_REF]. A critical component of the proposed pathophysiological mechanism to account for paradoxical depolarization is the other ion conductances active at the muscle resting potential, especially K + and Cl - [START_REF] Cannon | Channelopathies of skeletal muscle excitability[END_REF][START_REF] Jurkat-Rott | Pathophysiological role of omega pore current in channelopathies[END_REF]. Accordingly, inhibition of the Na-K-2Cl (NKCC) cotransporter was shown to prevent muscle weakness in the two available mouse models of HOKPP [START_REF] Wu | Beneficial effects of bumetanide in a CaV1.1-R528H mouse model of hypokalaemic periodic paralysis[END_REF][START_REF] Wu | Bumetanide prevents transient decreases in muscle force in murine hypokalemic periodic paralysis[END_REF].

Additional mechanisms have been proposed as contributing to the reduced fiber excitability in HOKPP. A shift of voltage-dependence of inactivation toward hyperpolarization and prolonged recovery were previously reported [START_REF] Jurkat-Rott | Voltagesensor sodium channel mutations cause hypokalemic periodic paralysis type 2 by enhanced inactivation and reduced current[END_REF][START_REF] Struyk | The human skeletal muscle Na channel mutation R669H associated with hypokalemic periodic paralysis enhances slow inactivation[END_REF]. The slower rate of recovery was recently confirmed [START_REF] Groome | NaV1.4 mutations cause hypokalaemic periodic paralysis by disrupting IIIS4 movement during recovery[END_REF], whereas decoupling between gating charge displacement and peak sodium current resulting in reduced Na + current density was reported [START_REF] Mi | Disrupted coupling of gating charge displacement to Na+ current activation for DIIS4 mutations in hypokalemic periodic paralysis[END_REF]. These mechanisms exert a loss-of-function effect contrasting with the gain-of-function effect of the gating pore current, and may contribute to the muscle hypoexcitability in HOKPP (Figure 2).

Finally, muscle hypoexcitability in PP may lead to secondary alterations that may themselves participate to the phenotype: careful investigation of a new mouse model with a single missense SCN4A mutation (p.Ile588Val) leading to myotonia and HYPP in humans and mice revealed general metabolic alterations that could result from abnormal AMPK activation [START_REF] Corrochano | Novel mutations in human and mouse SCN4A implicate AMPK in myotonia and periodic paralysis[END_REF].

Pharmacology and treatment of skeletal muscle sodium channelopathies

Binding of toxins or blockade of VGSC parts have historically played an important role in deciphering structure-function relationships, until more direct observation became possible through crystallization. It is noteworthy that most chemical compounds or toxins interact directly with the  pore. Mexiletine, the only treatment validated by a phase II randomized controlled study for NDM [START_REF] Statland | Mexiletine for symptoms and signs of myotonia in nondystrophic myotonia: a randomized controlled trial[END_REF], globally acts by blocking Nav1.4 in its open state. Tarentula and sea anemone toxins modify the gating pore current by complex interactions with the VSD [START_REF] Xiao | Gating-pore currents demonstrate selective and specific modulation of individual sodium channel voltage-sensors by biological toxins[END_REF]. Such fine knowledge of molecular structure is of importance since allosteric modifiers of Nav1.4 might be of safer use for therapy. The likelihood of repositioning known VGSC antagonists, the number of patented VGSC blockers and the possibility to chemically optimize them are promising approaches to improve drug-based treatments for NDM [START_REF] Bellis | Combined modifications of mexiletine pharmacophores for new lead blockers of Na(v)1.4 channels[END_REF][START_REF] Matthews | Repurposing of sodium channel antagonists as potential new anti-myotonic drugs[END_REF][START_REF] Zuliani | Sodium channel blockers: a patent review (2010 -2014)[END_REF][START_REF] Desaphy | Dramatic improvement of myotonia permanens with flecainide: a two-case report of a possible bench-to-bedside pharmacogenetics strategy[END_REF].

Developing new panels of drugs toward personalized medicine will be facilitated by the existence of preclinical models of NDM that could be used to test any new candidate drug [START_REF] Novak | Sodium channel slow inactivation as a therapeutic target for myotonia congenita[END_REF][START_REF] Desaphy | Preclinical evaluation of marketed sodium channel blockers in a rat model of myotonia discloses promising antimyotonic drugs[END_REF]. The sole limitation is the limited number of patients, which explains why funding of therapeutic trials have mainly relied on academia or charities even if promising molecules made their way from proofs of concept to preclinical models.

Clinical reasoning with some parts of chance led to the prophylactic use of carbonic anhydrase inhibitors (acetazolamide, dichlorphenamide) in PP since forty years without any pathophysiological evidence. This treatment has been shown only recently to be efficient in reducing the number of muscle weakness attacks in HOKPP with a much higher efficiency for Cav1.1 (≈56% of responders) than for Nav1.4 (<16%) related cases [START_REF] Sansone | Treatment for periodic paralysis. The Cochrane database of systematic reviews[END_REF][START_REF] Matthews | Acetazolamide efficacy in hypokalemic periodic paralysis and the predictive role of genotype[END_REF]. There were remaining questions on the safety of carbonic anhydrase inhibitors use in children, acidosis potentially interfering with bone development. A recent long-term follow-up of 3 children treated by acetazolamide during their first decade was recently reported with no side effect [START_REF] Markhorst | Clinical experience with long-term acetazolamide treatment in children with nondystrophic myotonias: a three-case report[END_REF]. Nevertheless, the reduced number of patients precludes from using this treatment without any precaution in children. Two drug-based therapeutic strategies for HOKPP now emerge with pathophysiological knowledge: i) the use of NKCC transporter inhibitor bumetanide -a drug approved for treatment of several conditions in humans found to be efficient on muscle weakness in the two mouse models of HOKPP -to prevent the paradoxical depolarization resulting from the aberrant gating pore current, and ii) to directly block this aberrant current using guanidinium analogues [START_REF] Wu | Beneficial effects of bumetanide in a CaV1.1-R528H mouse model of hypokalaemic periodic paralysis[END_REF][START_REF] Wu | Bumetanide prevents transient decreases in muscle force in murine hypokalemic periodic paralysis[END_REF][START_REF] Sokolov | Ion permeation and block of the gating pore in the voltage sensor of NaV1.4 channels with hypokalemic periodic paralysis mutations[END_REF]. For all, it remains to determine whether they prevent the permanent muscle deficit, a therapeutic need also expressed for HYPP with no solution currently proposed.

Conclusion

Skeletal muscle sodium channelopathies are model diseases that enabled to understand how mutations of the key-player in cell excitability modulate its functioning. Their essential features are the symptom fluctuation and their sensitivity to triggers such as exercise and hormones. These characteristics are still not well understood and deserve further studies. The more skeletal muscle sodium channelopathies -which affect a function easily investigated in animals and humans -are carefully studied, the more it may benefit to other, often more frequent, sodium channelopathies. Finally, skeletal muscle channelopathies should move to personalized medicine now that molecular characterization and preclinical models of individual mutation are available.

Key points

-Crystallography of bacterial voltage-gated sodium channel has increased the knowledge of mammalian sodium channel functioning.

-New phenotypes related to muscle sodium channelopathies have done recognized such as Severe Neonatal Episodic Laryngospasm (SNEL) and recessive forms of congenital myasthenia.

-New therapeutic targets have emerged from the studies of mouse models of periodic paralysis.

-Personalized treatments for muscle sodium channelopathies could emerge from screening of pharmacological compound libraries.

Acknowledgements

We would like to thank all the members of the "centre de référence maladies rarescanalopathies neuro-musculaires", especially Dr Savine Vicart, Prof Emmanuel Fournier, and Dr Damien Sternberg, and members of the Resocanaux network for fruitful discussions and collaborations.

Financial support and sponsorship

The authors received research grants from AFM-Telethon, Fondation Maladies Rares and "Investissement d'Avenir" ANR-10-IAIHU-06 program.

Conflicts of Interest

None.

Legends of the Figures