Vhh suitable to vectorize molecules intracellularly

Abstract

The invention relates to a particular modified camelid single chain antibody called VHHNPX and uses of VHHNPX in new methods for the efficient delivery of molecular cargos inside target cells (in particular neurons and eye retina cells). The invention further relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, in particular a Neurovita peptide short fragment encoding a PDZ-binding motif (PBM) inhibiting the PDZ domain of the human MAST2 & MAST1 enzymes. The invention also relates to polypeptides comprising this Neurovita short peptide, notably peptides further comprising a cell-penetrating molecule (CPM), and uses thereof.

Description

[0001] VHH SUITABLE TO VECTORIZE MOLECULES INTRACELLULARLY

[0002] FIELD OF THE INVENTION

[0003] The invention relates to a particular modified camelid single chain antibody called VHHNPX and uses of VHHNPX in new methods for the efficient delivery of molecular cargos inside target cells (in particular neurons and eye retina cells). The invention further relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, in particular a Neurovita peptide short fragment encoding a PDZ-binding motif (PBM) inhibiting the PDZ domain of the human MAST2 & MAST1 enzymes. The invention also relates to polypeptides comprising this Neurovita short peptide, notably peptides further comprising a cell-penetrating molecule (CPM), and uses thereof.

[0004] DESCRIPTION OF THE PRIOR ART

[0005] Most of the drugs do not enter cells spontaneously; they need to be vectorized by a Cell Penetrating Molecule (CPM). Use of Cell Penetrating Molecules (CPMs) to deliver drugs into a cell is a widely used method, whose yield is unfortunately low because most CPMs send the drug into the cellular degradation pathways. When drugs vectorized by CPM do manage to enter the cell, they are indeed often directed into the endosomal organelles, which are the cell’s cleaning system. Consequently, the drugs are often eliminated and never reach the cytoplasm of the target cells.

[0006] For example, it is known that the Phosphorodiamidate Morpholino Oligomer (PMO) eteplirsen only restores 1.93% of the dystrophin gene to normal levels in patients’ muscles after 180 weeks of treatment with a weekly 30 or 50mg / kg dose (Lim et al., 2017). Most ASOs are administered intravenously to maximize bioavailability, leading to rapid distribution in highly vascularized organs such as the liver, kidney, and spleen, but to slow distribution to other organs such as muscle.

[0007] One solution to improve the bioavailability and cell penetration of PMOs is to conjugate them to CPMs (Moulton & Moulton, 2010), in particular with cationic CPMs that can improve the efficiency of PMO delivery and uptake (Moulton et al., 2004).

[0008] However, the use of positively charged peptides [TAT, (RXR)4, R9 F2C (Moulton et al., 2004) (RXR)4-B, Hph-1 (Choi et al., 2006), Transportan, TP10 (Andaloussi et al., 2011; Mae et al., 2009), P007, or B peptide (Yin et al., 2008)] mainly directs the cargoes into the cell degradation pathway. This results in drug degradation and requires increased PMO concentrations (above lOpM) to achieve therapeutic antisense activity. Such doses approaching cytotoxic levels (Amantana et al., 2007), use of this type of CPMs became an obstacle for clinics (Abes et al., 2008). In addition, administration of these PMOs caused side effects characterized by lethargy, weight loss, and kidney damage. Their toxicity could result from membrane disruption influenced by the amino acid (aa) composition of CPMs when they contain arginine or 6- aminohexanoic acid residue (Wu et al., 2007, Wang et al, 2020 and Shadid et al 2021).

[0009] In this context, on the first hand, it is required to identify new means to vectorize drugs, in particular therapeutic peptides or PMOs, into target cells without using the endocytic / lysosomal pathway which results in drug degradation. More precisely, it is required to identify new CPMs that can bypass endosomal entrapment and have non-toxic amino acid compositions.

[0010] On the second hand, the protein-protein interactions (PPIs) play a crucial role in cell metabolism by promoting scaffold and contact between proteins such as enzymes and their substrate ligands. Among the different PPIs types, many are mediated by PDZ domains allowing the interaction of two distinct proteins through the recognition of the PDZ encoded by one protein by the PDZ Binding Motives (PBM) present on the second protein, often located at the C-terminus.

[0011] Although PDZ domains are frequent in nature, they have been described as difficult to target with high affinity / high specificity in pharmacological approaches. However, the structures of several PDZ / PBM interactions have been solved by Nuclear Magnetic Resonance (NMR) or crystallography, making the PDZ / PBM interaction now druggable. PDZ / PBM interactions can be modulated by blocking agents and therefore developed as drug targets (Dev KK. Making protein interactions druggable: targeting PDZ domains. Nat Rev Drug Discov. 2004 Dec;3(12): 1047-56). In this context, blocking peptides, such as interfering peptides have been developed to modulate the PDZ / PBM interactions of proteins that are often involved in neurological disorders (Christensen N, Calyseva J, Fernandes E, Liichow S, Clemmensen L, Haugaard-Kedstrbm L, et al. PDZ Domains as Drug Targets. Adv Then 2019 Apr 24;2: 1800143). Hence, modulation of PDZ interactions with interfering peptides is a powerful strategy for developing therapeutical approaches for neurological diseases.

[0012] One interfering peptide showing great interest is the Neurovita peptide. Its C-terminal PBM domain blocks the interaction of the PDZ domain of the Microtubule Associated Serine Threonine kinase 2, MAST2, and kinase 1, MAST1 with the PBM domain of its ligand PTEN. It is sufficient to launch a cascade of signalling pathway (PI3K / Akt / mT0R) allowing the activation of survival and protein translation mechanisms in neurons, and to function as a neuroprotective and neuroregenerative biomolecule, allowing to keep injured nerves alive (neurosurvival) and to induce axon regrowth after injury (neuroregeneration) (WO 2010 / 116258 and WO 2013 / 068430).

[0013] It has been demonstrated in application WO2010 / 116258 that the nature of the amino acid residues at positions 491 (H / L) and 521 (Q / E) of the G protein of rabies viruses is important for the effects on neuron survival and on neurite outgrowth. The G protein of a virulent rabies virus strain presenting a H residue at position 491 and a Q residue at position 521 is non-apoptotic and favors neurite outgrowth. In contrast, the G protein of an attenuated rabies virus strain presenting a L residue at position 491 and a E residue at position 521 is apoptotic and does not promote neurite outgrowth.

[0014] Furthermore, WO 2013 / 068430 proposed to use, as therapeutic peptide, the whole cytoplasmic domain of the G protein of rabies virus comprising a PBM formed by two anchors (a C-terminal QTRL in position -3, -2, -1, 0 and a sequence of 2 residues (serine, S and tryptophan, W) linked by a flexible part (Terrien et al, 2012) and a cytoplasmic part upstream of the PBM, said cytoplasmic part upstream of the PBM containing 20 to 40 amino acids, preferably 31 amino acids. For example the peptide as defined in SEQ ID NO:2 or a variant having at least 80% identity with SEQ ID NO:2 of WO 2013 / 068430. Thus, the Neurovita polypeptide described in WO 2013 / 068430 contains at least 31 amino acids (at least 20 amino acids for the upstream part of the PBM and 11 amino acids for the C-terminal PBM).

[0015] More generally, all biological assays involving the Neurovita peptides in the past have been carried out by using long polypeptide containing between 30-44 amino acids of the cytoplasmic domain of the pro-survival rabies virus G protein (including the PBM in the C-terminal part). Of note, the biological effect of several long polypeptides (Neurovita 1, 44 amino acid long, Neurovita 2 (42 amino acids long) and Neurovita 3 (42 amino acids long), were tested in W02013 / 068430, and the same long peptides were still preferred few years later as shown in Khan et al, Structure-based optimization of a PDZ-binding motif within a viral peptide stimulate neurite outgrowth, J. Biol. Chem. 2019, 294(3) 13755-13768. This is because the cytoplasmic domain of the G protein from which the Neurovita peptide is derived is a protein that is known to be intrinsically disordered. As a result, it was expected that, by reducing its size too much, the conformation of its C-terminal part would be modified, and the resulting peptide would thus lose the biological activity of the whole cytoplasmic domain of the rabies virus G protein. Examples of such observations were described in the art, see e.g., Flanagan et al., 1992 (Flanagan JM, Kataoka M, Shortle D, Engelman DM. Truncated staphylococcal nuclease is compact but disordered. Proc Natl Acad Set USA. 1992 89(2):748-752) and more recently An et al., 2020 (An SY, Ka D, Kim I, Kim EH, Kim NK, Bae E, Suh JY. Intrinsic disorder is essential for Cas9 inhibition of anti-CRISPR AcrIIA5. Nucleic Acids Res. 2020 48(13):7584- 7594).

[0016] Moreover, the yield of production of a fusion of the Neurovita peptide with a VHH, as a Cell Penetrating Molecule (CPM), was too low for drug development. Moreover, the fusion was found to form aggregates when contacted with physiological fluids such as the vitreous humor of the eye.

[0017] Thus, on the second hand, there is still a need to provide a therapeutically active molecule based on the Neurovita peptide.

[0018] SUMMARY

[0019] In a first aspect, the invention provides a VHH antibody (VHHNPX) of SEQ ID NO:5, or a variant thereof whose sequence displays at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity with SEQ ID NO:5, said variant comprising i) the CDR1-3 of sequence SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:2 respectively and ii) at least one disulfide bond formed by two cysteines.

[0020] Preferably, said VHH antibody has a basic isoelectric point. More preferably, the sequence of the VHHNPX chain is SEQ ID NO: 5.

[0021] In another aspect, the invention relates to a complex containing a molecular cargo conjugated or fused to the VHH antibody disclosed above.

[0022] In an embodiment, said molecular cargo is a peptide, a protein, a chemical molecule, a small molecule, an oligonucleotide of interest such as antisense oligonucleotides (ASO) (e.g. an uncharged phosphodiamidate morpholino oligo (PMO) or other DNA or RNA analogue with neutral electrostatic charge).

[0023] In an embodiment, said VHH antibody is used in combination with or coupled to a homing molecule. Preferably, said homing molecule is a Rabies virus Derived Peptide (RDP) derived from the rabies virus envelope G protein of the CVS-NIV sequence, preferably the peptide having the sequence SEQ ID NO: 14, or a variant thereof displaying at least 90%, more preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity with SEQ ID NO: 14, but not containing any cysteine residue.

[0024] In an embodiment, the molecular cargo is the Neurovita short peptide having the sequence SEQ ID N0:21.

[0025] In an embodiment, the molecular cargo is the Neurovita short peptide having a sequence selected in the group consisting of: SEQ ID NO:22-28.

[0026] In an embodiment, the molecular cargo is an uncharged phosphodiamidate morpholino oligo (PMO).

[0027] In an embodiment, the PMO has the sequence SEQ ID NO:39.

[0028] In another aspect, the invention relates to an in vitro use of the VHH antibody described herein to vectorize a molecular cargo in a non-endosomal / non-lysosomal pathway inside a target cell.

[0029] In another aspect, the invention relates to the VHH antibody described herein for use for delivering a molecular cargo in a non-endosomal / non-lysosomal pathway inside a target cell, for preventing and / or treating a disease or condition involving the Peripheral Nervous System (PNS) or the Central Nervous System (CNS) or the evolution of such disease or condition, such as optic neuropathies (glaucoma, NAION), motor neuron diseases (ALS, SMA) muscular disease (Duchenne Disease) or neurodegenerative diseases (Parkinson, Alzheimer).

[0030] In another aspect, the invention relates to a pharmaceutical composition comprising a therapeutically effective amount of the complex described herein, and a pharmaceutically acceptable carrier.

[0031] In another aspect, the invention relates to the complex or the pharmaceutical composition described herein, for use as a medicament.

[0032] In an embodiment, the complex or the pharmaceutical composition is used for preventing and / or treating a disease or condition involving the Peripheral Nervous System (PNS) or the Central Nervous System (CNS) or the evolution of such disease or condition, such as optic neuropathies (glaucoma, NAION), motor neuron diseases (ALS, SMA) muscular disease (Duchenne Disease) or neurodegenerative diseases (Parkinson, Alzheimer). Preferably, said disease or condition is a neurodegenerative disease, or a cell damage-associated condition such as stroke or injury including optic nerve disease, retina diseases, muscular, motor neuron disease.

[0033] In a further aspect, the present invention provides a Neurovita polypeptide comprising or consisting of the NVshort peptide of sequence:

[0034] XnSWXVXXQQTRL (SEQ ID NO:21), wherein X is any amino acid and n is an integer comprised between 0 and 4 (i.e., n is 0, 1,

[0035] 2, 3, or 4), and wherein the sequence of the Neurovita polypeptide is not SEQ ID NO: 17, 18, or 19

[0036] In an embodiment, the NVshort polypeptide has a sequence selected in the group consisting of SEQ ID NOs: 22-28.

[0037] In an embodiment, the polypeptide of the invention further comprises at least one cellpenetrating molecule (CPM) and optionally a homing molecule. Preferably, the NVshort polypeptide has a basic isoelectric point.

[0038] In an embodiment, the polypeptide of the invention consists of a fusion protein between the NVshort peptide of sequence RIISSWEVHGQQTRL (SEQ ID NO: 24 and a VHH CPM.

[0039] Preferably, said CPM is chosen in the group consisting of:

[0040] A heavy chain polypeptide of a VHH camelid antibody, whose sequence is mutated to contain at least one disulfide bond and a CDR3 containing three or more mutations as compared with SEQ ID NO: 1, so as to inactivate the antibody function of said VHH camelid antibody,

[0041] A Rabies virus Derived Peptide (RDP) whose sequence displays at least 90% similarity with SEQ ID NO: 13, and in which the only cysteine amino acid has been substituted by another amino acid, or

[0042] A protein transduction domain of the HIV trans-activator of transcription L-TAT having at least 90% similarity with SEQ ID NO: 16.

[0043] More preferably, said CPM and / or Neurovita peptides are separated by a linker.

[0044] In an embodiment, the polypeptide of the invention contains: a. the VHHNPX CPM having the SEQ ID NO : 5 or a variant thereof, whose sequence displays at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity with SEQ ID NO:5, said variant comprising i) the CDRs of SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:2 as CDR1-3 respectively, and ii) at least one disulfide-bridge formed by two cysteines, and b. the NVshort polypeptide of sequence RIISSWEVHGQQTRL (SEQ ID NO:24).

[0045] In an embodiment, the polypeptide of the invention has the sequence SEQ ID NO:30.

[0046] Preferably, the polypeptide of the invention is soluble in vitreous humor, non-aggregated, and able to bind and block with high affinity the PDZ domain of the human MAST1 and MAST2 proteins in neuronal cells.

[0047] In another aspect, the invention provides a vector, preferably a viral vector, DNA or mRNA, encoding the polypeptide described above.

[0048] In another aspect, the invention relates to a pharmaceutical composition comprising the polypeptide or the vector described above.

[0049] In another aspect, the invention relates to the pharmaceutical composition described above for its use as a medicament. Preferably, the pharmaceutical composition is used for preventing and / or treating a disease or condition involving the Peripheral Nervous System (PNS) or the Central Nervous System (CNS) or the evolution of such disease or condition, such as optic neuropathies (glaucoma, NAION), motor neuron diseases (ALS), muscular diseases (Duchenne Disease) or neurodegenerative diseases (Parkinson disease, Alzheimer’s Disease). More preferably, said disease or condition is a neurodegenerative disease, or a cell damage-associated condition such as stroke or injury including optic nerve disease, retinal diseases, muscular diseases, motor neuron disease.

[0050] FIGURE LEGENDS

[0051] Figure 1 A shows in non-fixed cells, by live-cell confocal microscopy imaging, that L-TAT- NV-conjugated to carboxyrhodamine (LTAT-NV-Rho) is directed to lysosomes as early as 10 minutes after Ih incubation with human retina cells (ARPE19, retinal pigment epithelial cells). In contrast, in the same experimental conditions, VHHNPX-NV conjugated to carboxyrhodamine (VHH-NV-Rho) is not directed to these degradation cellular organelles. The white dots (arrows) show colocalization between lysotracker, the marker of lysosomes and LTAT-NV-Rho. N indicates the cell nuclei. Size marker is 10pm.

[0052] Figure IB shows in fixed ARPE-19 cells, by confocal microscopy imaging, that NV (as detected by the NV specific antibody SYCO22) colocalizes with the lysosome marker, LAMP1 (Lysosomal Associated Membrane Proteinl) when NV is transported by L-TAT (image on the right) and not by VHHNPX (image on the left). N indicates the cell nuclei. Size marker is 200pm. Cells were treated with 3nmol of CPM-NV for 1 hour. The plot on the right illustrates that Manders coefficients of colocalization were higher in LTAT-NV treated cells than in VHHNPX- NV treated cells.

[0053] Figure 2 shows that NV can be detected longer in ARPE 19 cells when it has been transported by VHHNPX than by L-TAT. ARPE19 cells (450 000 cells) were treated with vehicle or 15nmol of CPM-NV for 1 hour before to be lysed in RIPA buffer, separated on gel, transferred to nitrocellulose membrane and immunoblotted using SYCO22 a NV specific antibody and an actin-specific antibody. Actin serves as a control for cell lysate loading. Membranes were revealed with the ECL western blot and chemiluminescence signals were detected at different exposure times with a G-Box.

[0054] Figure 3 Comparison of the PI3K / AKT signaling pathway activation by NV depending on whether NV is transported by VHHNPX or by L-TAT. Activation of the PI3K / AKT pathway by NV was evaluated by measuring the phosphorylated AKT / panAKT ratio in ARPE19 cells treated for 30min with different molar concentrations (3.6nM- 5 M) of VHHNPX-NV or L-TAT- NV or vehicle-treated (CT for control). CT is PBS for VHH-NV and MES Buffer for LTAT-NV. After washes, cells were lysed in RIPA buffer, separated on gel, transferred to nitrocellulose membrane and immunoblotted with pan AKT, phosphorylated AKT specific antibodies and actin-specific antibody. Actin serves as a control for cell lysate loading. Membranes were revealed with the ECL western blot and chemiluminescence signals were detected at different exposure times with a G-Box. Dose responses curves of phosphoAKT / AKT ratio standardized to CT (value 1) were presented on the left for VHHNPX-NV and on the right for L-TAT-NV. The black arrows indicate the estimated Efficacy Dose 50 (ED50) for each compound. These data show that less biomolecule (NV) is required to activate the PI3K / AKT pathway when NV is transported into cells by VHHNPX than when NV is transported by L-TAT. This observation could be linked to a more rapid degradation of NV (Figure 2) and a driving of NV into the degradation pathways of the cell (Figurel A&B) when it is transported by L-TAT.

[0055] Figure 4 compares the toxicity of the two constructs L-TAT NV or VHHNPX NV (NPX461) in ARPE19 cells (measured by Alamar blue viability analysis). The Toxicity Dose 50, ToxDso. is of 3.5 nmol for L-TAT-NV and of 22 nmol for VHHNPXNV, indicating that LTAT-NV is almost 7-fold more toxic for ARPE19 than VHHNPXNV.

[0056] Figure 5 shows that the insertion of a disulfide bridge in VHH increases solubility and avoid aggregates formation upon contact with vitreous humor. The in vitro test of detection of aggregate formation upon contact with rabbit vitreous was used to compare the solubility of the compound VHH-NV not containing any disulfide bridge (“VHH w / o DS”) and those of the compound (“VHH with DS”) where the disulfide bridge was allowed to form after insertion of two cysteines (C22 and C95). In this test, 2 pl of several molar concentrations of each compound (9pM, 18pM and 36pM) were added to 5 pl of rabbit vitreous. After Ih incubation at 37°C, the presence of aggregates was detected with the camera of the DLS (Dynamic Light scattering) station.

[0057] It was observed that the compound VHH-NV- whose VHH does not contain any disulfide bridge (“VHH w / o DS”)- triggers aggregate formation at each tested concentration (9pM - 36pM). In contrast, when the disulfide bridge was allowed to form after insertion of the two cysteines (C22 and C95) (“VHH with DS”), no aggregates could be observed.

[0058] Figure 6 shows the fluorescence intensity of NV in the retina 6 hours after an intravitreal injection of different doses (10, 50, 250 pmol) of VHHNPX-NV (NPX461) or L-TAT-NV, as detected by immunohistology of flatmount retina with double stained retinas using a marker of RGCs (RBPMS specific antibody) and a NV specific antibody (SYCO22 antibody). Dose response curves are drawn for each compound. The calculated half maximal intensity of fluorescence (IC 50) is 6pmol forNPX461 (VHHNPX-NV) and 28pmol for L-TAT-NV, indicating that NV is better detected in the retina when it has been transported by VHHNPX than by L-TAT.

[0059] Figure 7 shows that NPX452 (VHHNPx-NVlong) and NPX461 (VHHNPx-NVshort) are efficiently addressed to RGCs layer following intravitreal injection. (A) 25, 50, 125, or 250 pmol of NPX461 or NPX452 are injected and 6 hours later an immunoanalysis of flatmount retina was performed with the Neurovita specific antibody (SYCO22). The calculated half maximal intensity of fluorescence (IC 50), 6pmol for NPX461 (VHHNPx-NVshort) and lOpmol for NPX452 (VHHNPx-NVlong) are very close, indicating that NVlong and NVshort are similarly detected in the retina when they have been transported by VHHNPX.

[0060] (B) 10 or 50 pmol of NPX461 or NPX452 are injected in the vitrea and the presence of the NV polypeptide is assessed 3h after the injection with a specific antibody (SYCO22) and a RGC specific marker (RBPMS antibody). The graph shows that the proportion of RGCs labelled with SYCO22 is almost the same, independently of the size of the molecular cargo. These data indicate that VHHNPX can efficiently vectorize into the retina and RGCs both NVshort and NVlong, suggesting that transport capacity of VHHNPX is independent of the nature of the peptide cargo.

[0061] Figure 8 shows the in vivo preclinical properties of the VHHNPx-NVshort of the invention in two experiments: (A) on RGC protection and (B) on optic nerve regeneration of the mouse model of optic nerve crush.

[0062] Figure 9 shows that the replacement of the cysteine in the RDP sequence by a serine improves the solubility of RDP -NVshort. Two constructs of RDP -NVshort (ML49c and ML49s) were chemically synthesized. ML49c results of the fusion of RDPc with NVshort, while ML49s results of the fusion of a modified RDP, RDPs, with NVshort. RDPs contains a serine instead of the cysteine. An in vitro test performed in 384 well plates was set up to test the solubility of compounds upon contact with rabbit vitreous humor. Solubilization of ML49c and ML49s in 60nM MES buffer 300mM NaCl, was obtained in pH 6.5 condition and addition of 7.5% DMSO. 2pl of each compound or vehicle were added to 5pl of rabbit vitreous. After Ih incubation at 37°C, the presence of aggregates was detected with the camera of the DLS (Dynamic Light scattering) station.

[0063] Figure 10 shows the structure of the VHH-NV fusion protein of the prior art, as described in W02013 / 068430 and in WO2016 / 038122 and in 2 publications: Khan Z et al, 2019 and da Costa A et al, 2020. The term “RDP” designates a short polypeptide derived from the RAB V G protein extracellular domain, which interacts with the alpha 7 subunit of nAchR. In the fusion proteins of the prior art, the polypeptide RDP contained a cysteine (Synthetic peptides corresponding to sequences of snake venom neurotoxins and rabies virus glycoprotein bind to the nicotinic acetylcholine receptor. Proteins 2, 298-307, (1987); Lentz, T. L., Wilson, P. T, Hawrot, E. & Speicher, D. W. Amino acid sequence similarity between rabies virus glycoprotein and snake venom curare mimetic neurotoxins. Science 226, 847-848, (1984); Donnelly-Roberts, D. L. & Lentz, T. L. Synthetic peptides of neurotoxins and rabies virus glycoprotein behave as antagonists in a functional assay for the acetylcholine receptor. Pept Res 2, 221-226 (1989)). The term “VHH” designates an alpaca single chain antibody that can cross the blood-brain barrier. It is derived from the VHH A12 described by Li T et al, 2017. In the fusion proteins of the prior art, the CDR3 is native. The “strep tag” is a streptavidin tag used to monitor the presence and the expression of the molecule in cells. The term “Neurovita long” stands for the neuroprotective and neurodegenerative biomolecule called “Neurovita 3” (or “NV3-Cyto”) derived from the cytoplasmic domain of RABV G protein, as displayed on figure 11 of WO2013 / 068430.

[0064] Figure 11 shows how the removal of RDP improves the production of the fusion protein in recombinant bacteria E. coli. The HISRDP-VHH-NV and HIS-VHH-NV constructs leading to the expression of eHIS-RDP VHH-NV (C3.1b) and the expression of eHIS-VHH-NV lacking RDP (C3.3) constructs were produced in T7 shuffle Express bacteria. Cultures were harvested after 4h or after over-night. Bacteria were lysed and centrifuged to separate pellet (whole cell fraction) from supernatant (soluble fraction). The fractions were separated on SDS PAGE under denaturating conditions and stained with Coomassie Blue. Left lanes correspond to C3.1b bacteria production. Right lanes correspond to C3.3 (no RDP) production. Molecular weight was run on both sides of the gel. Product Of Interest (POI) (here the VHH-NV) could be detected at the expected molecular weight (18kDa), arrow, in whole cell and soluble fraction of the C3.3 bacteria cultures and not in the C3.1 bacteria cultures. Note that, in the two fusion proteins C3.1b and C3.3, the encoded VHH lacks disulfide bonds.

[0065] Figure 12 shows that RDPc forms dimers and aggregates, but not RDPs. Three constructs of RDP-NVshort (ML49c, MG49c, and ML49s) were chemically synthesized. ML49c results of the fusion of RDPc with NVshort ML49c-APBM (or MG49c) corresponds to a ML49c lacking the C-terminal PBM, while ML49s results of the fusion of a modified RDP, RDPs, with NVshort. RDPs contains a serine instead of the cysteine. Gels were run and immunoblotted. NVs was detected using a Neurovita-specific rabbit antibody. Under denaturing conditions (left gel), semi-denaturating conditions (middle gel) ML49c forms both monomers and dimers. This is still observed with ML49c-APBM, indicating that PBM is not involved in the dimer formation. In native conditions (Right gel) ML49c forms a smear of aggregates.

[0066] Figure 13 shows that NVshort is as efficient as NViongin promoting neurite outgrowth in vitro in NGF-pretreated neuroscreen cells. The property of NVshort and those of NViong to promote neurite outgrowth were compared in a 72h-assay on Neurotrophic factor (NGF) pre- treated neurosc reen cells (a subclone of rat PC12, see Khan Z et al, 2019). RKM is a Streptag- VHH-NV fusion protein encoding NViong(42 amino acids), RK2M is a Streptag-VHH-NV fusion protein encoding NVshort (15 amino acids) and AAA RK2M is a Streptag-VHH-NV fusion protein encoding NVshort (15 amino acids) and containing a linker formed by 3 Alanines inserted between the VHH sequence and the Neurovita sequence. Whatever the concentrations (0.22pM to 0.055pM), no difference in promoting neurite outgrowth could be observed between constructs encoding NV long or NV short. In addition, the insertion of a 3 A linker between VHH and Neurovita sequences does not alter the VHH-NVshort neurite outgrowth function.

[0067] Figure 14 shows the in vitro cell entry of the CPM-NVshort (7pmol) after 3h-contact with ARPE19 cells once NVshort has been vectorized with the CPM VHHNPX (NPX461) or with the CPM LTAT (LTAT-NV). After wash and PFA fixation, cells were incubated with a NVshort-specific guinea-pig antibody, followed by an Alexa 555- conjugated antibody directed against guinea-pig IgG. Slides were mounted with Fluoromount G containing DAPI (marker of nucleus) and analyzed by confocal microscopy imaging. The image on the left, illustrates that NVshort vectorized by LTAT enters the cytoplasm where it forms aggregates that accumulate in perinuclear regions (arrows). The image on the right, shows that NVshort vectorized with VHHNPX enters into the cytoplasm (stars) without forming aggregates. The non-treated condition (CT) shows no cytoplasm staining. N indicates the cell nucleus. Size marker is 50pm.

[0068] Figure 15 is a scheme showing the structure of the retina. An intravitreal injection allows delivery of therapeutics into the inner part of retina, in particular in the first layer of cells, consisting mainly of retinal ganglion cells (RGC) whose axons form the optic nerve. After intravitreal injection, compound has to cross the vitreous humor, (a gel at pH 7.5), escapes enzymatic degradation and engulfment by macrophages located in the vitreous, and finally crosses the inner limitans membrane at the vitreo-retinal interface before to reach the RGCs located in the RGC layer (RGL in the scheme). CPM-NVs is delivered by the intravitreal route to target the RGCs.

[0069] Figure 16 shows that both NPX461 (VHHNpx-NVshort) and NPX452 (VHHNpx-NVlong) are efficiently addressed to retina RGCs layer following intravitreal injection of the mouse eye. 250 pmol of NPX461 or NPX452 are injected in the vitreous and the presence of the NV polypeptide is assessed 3h, 8h, 24h or 30h after the injection with a specific NV antibody (SYCO22). The graph shows that the duration of the Neurovita detection in retina RGC layer is similar after NPX461 injection than after NPX452 injection, indicating that the shortening of NV has no impact on NVshort peptide detection in the retina.

[0070] Figure 17 shows the ex vivo effect of NPX461 on axon regeneration in retina explants as a model of axotomy. It shows that the number of axons able to regrowth on long distance (250- 550pm) is significantly enhanced after NPX461 treatment as compared with positive control (BDNF, CNTF, Forskoline) and negative control (DPBS vehicle) treatments.

[0071] Figure 18. Comparison of exon skipping activity in human neuroblastoma cells after treatment with naked PMO or VHHNPX conjugated PMO

[0072] A: RT-PCR analysis of SMN transcripts across exons 4 and 8 in SH-SY5Y following treatment with 2pM of VHH (non-conjugated VHHNPX), SSOAR2 (PMO-SSOAR2 conjugated VHHNPX 6HIS), SSOAR1 (PMO-SSOAR1 conjugated VHHNPX 6HIS), OAR2 (PMO-OAR2 conjugated VHHNPX 6HIS), PMO (naked PMO), OAR1 (PMO-OAR1 conjugated VHHNPX 6HIS) or without treatment (NT). A DNA ladder lOObp, Invitrogen, indicates the size of FL pathogenic transcript (404 bp) and of A7 exon skipped transcripts (350 bp). This experiment is representative of two distinct experiments.

[0073] B: Graphical representation by densitometry of impact of treatments on exon 7 skipped product (7) percentage compared to VHH treatment.

[0074] DETAILLED DESCRIPTION OF THE INVENTION

[0075] In a first aspect, the invention relates to a VHH suitable to vectorize molecules intracellularly.

[0076] The present inventors solved the issues above mentioned by providing a new efficient CPM having unique properties of cell internalization without entering the endosomal / lysosomal pathway. This new CPM is a mutated version of a single-chain camelid antibody called VHH A12 and is herein called VHHNPX.

[0077] The inventors herein show that, by mutating the VHH Al 2 chain as herein proposed, the VHHNPX is able to let cargo molecules bypass the cell degradation pathway (figure 1A, IB, figure 14), to let cargo longer detectable and active into cells (figures 2 and 3) and to confer a low toxic profile to the complex VHH-Cargo (figure 4), when compared to fusion of the cargo to a classical CPM such as TAT. In the examples below, the efficacy of the VHHNPX CPM of the invention has been studied in the context of the vectorization of the Neurovita peptide (NV).

[0078] This peptide of 42 amino acids is known to block the interaction of the PDZ domain of the Microtubule Associated Serine Threonine kinase 2, MAST2, and MAST1 with the PBM domain of its ligand PTEN and to launch a cascade of signalling pathway (PI3K / Akt / mTOR) in the cells where it is expressed. This peptide is advantageously introduced and expressed in neurons, where it functions as a neuroprotective and neuroregenerative biomolecule, allowing to keep injured nerves alive (neurosurvival) and to induce axon regrowth (neuroregeneration) (WO 2013 / 068430). The Neurovita peptide has been successfully fused to a cell penetrating molecule (CPM) so as to get them into the cell cytoplasm where the target MAST2 and MAST1 kinases are located. These fusion polypeptides (or proteins because of their size) typically contain a therapeutically active Neurovita peptide of 42 amino acids (“NVlong”) and a CPM consisting in an alpaca native VHH antibody (WO2016 / 038122). The VHH allows both the delivery of Neurovita inside the neuron cytoplasm by crossing the neuron plasma membrane and crossing of the Blood Brain Barrier (BBB) (WO2016 / 038122, Da Costa A et al, 2019). In addition, VHH-Neurovita was tagged with an RDP domain (i.e., a RABV Derived Peptide) allowing the interaction with the Alpha? sub-unit of the AchR present both on neurons membranes and on the endothelial cells of the BBB (Kumar P, Wu H, McBride JL, Jung KE, Kim MH, Davidson BL, et al. Transvascular delivery of small interfering RNAto the central nervous system. Nature. 2007 Jul 5 ;448(7149):39— 43). The role of the RDP was to facilitate the crossing of the BBB (endothelial cells of the BBB express AchR, see Kumar et al.2007) and to target cells such as neurons specifically (neurons express AchR). The last property was useful when tissues contained not only neurons but also other cell types, (astrocytes, microglia...), as in the nervous parenchyma (Khan Z, Terrien E, Delhommel F, Lefebvre-Omar C, Bohl D, Vitry S, et al. Structurebased optimization of a PDZ binding motif within a viral peptide stimulates neurite outgrowth. J Biol Chem. 2019 Jul 25;jbc.RAl 19.008238 ; da Costa A, Prehaud C, Bakoa F, Afonso P, Ceccaldi PE, Lafaye P, et al. A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain. J Vis Exp JoVE. 2019 09 (146)).

[0079] However, the yield of production of VHH-NV molecules produced in recombinant E. coli bacteria by expression of a single fused open reading frame (ORF) (WO2016 / 038122) (i.e. 5,6ng / pL in 250pL for a 800mL bacterial culture) was too low to consider further purification steps necessary for drug development. Moreover, the non-mutated VHH-NV precipitates forming aggregates in contact with physiological fluids such as the vitreous humor of the eye. The present invention also solves this problem, by providing a fusion protein containing a VHH CPM that does not precipitate during its production process and can therefore be produced with high yields.

[0080] In the examples below, the CPM efficiency of the VHHNPX of the invention has been compared to the efficacy of a widely used CPM, L-TAT (a sequence derived from the Human Immunodeficiency Virus, HIV, Trans Activator of Transcription), and to the efficacy of the VHH CPMs of the prior art as disclosed in WO2016 / 038122. The production and efficacy of the intracellular vectorization of the Neurovita peptide fused to these two CPMs have been tested.

[0081] The results obtained by the inventors demonstrate the significant superiority of VHHNPX over other CPMs in various read-outs:

[0082] 1) unlike L-TAT, VHHNPX is capable to let the vectorized NV bypass the endosomal degradation pathway and to accumulate in various cell types (figures 1 A&B, figure 14), thus leading to a longer detection of active cargo in the cells (figure 2) and a better biological activity (figure 3)

[0083] 2) the toxicity of the fusion protein VHHNPX - NV at therapeutic dosage is lower than the toxicity of the fusion protein L-TAT-NV (figure 4)

[0084] 3) the amount of NV in retina (figure 6) and in retinal neurons (Retina Ganglions Cells, RGCs (results section) after intravitreal injection in the mouse eye, is increased when vectorized in vivo by VHHNPX, as compared with L-TAT.

[0085] 4) the detection of NV by Mass spectrometry analysis in the retina lasts longer, when NV has been vectorized in vivo by VHHNPX, as compared with L-TAT (results section).

[0086] 5) the fusion protein of the invention, containing VHHNPX and a cargo molecule, has an enhanced solubility as compared with fusion proteins containing the VHH of the prior art (figure 5), so that i) its aggregation is prevented during its production process, what significantly enhances its production yield and ii) unlike prior art VHH-containing fusion proteins, it can be put in contact with physiological fluids without any risk of aggregation, therefore keeping intact its biological functions and physical properties even in vivo.

[0087] It is herein demonstrated that the capacity of VHHNPX to vectorize peptides into the Retina and RGCs is independent of the size of the peptide cargo, since VHHNPX can similarly vectorize short peptides (e.g., the NV short peptide containing 15 amino acids) or long peptides (e.g., the NV long polypeptide containing 42 amino acids) after an intravitreal injection into the mouse eye (figure 7).

[0088] Finally, it is herein demonstrated the capacity of VHHNPX to vectorize conjugated PMO into human neurons more efficiently than non-conjugated PMO (naked PMO).

[0089] The present invention therefore provides a solution for delivering peptides or other molecules (e.g., small molecules, ASO...) more efficiently into various cells (including neurons and muscle cells), wherein the vectorized peptides have a lower toxicity and a higher solubility compared to other CPMs in physiological fluids, what is advantageous for their in vitro production and their efficiency after their in vivo administration.

[0090] VHH of the invention

[0091] The prior art VHH CPM is the heavy chain polypeptide of the VHH Alpaca antibody (native VHH A12, cf. Li T. et al., 2017) which has been isolated from an immunized alpaca. It has a basic isoelectric point of 9.78, what is important to convey the peptides across the cell membrane and the Blood Brain Barrier (BBB). The overall sequence of the VHH of the prior art is displayed in SEQ ID NO:8; it contains the CDRs of SEQ ID NO:6, 7 and 1 as CDR1-3 respectively:

[0092] VHH CDR1 : IDVINNMA (SEQ ID NO: 6)

[0093] VHH CDR2: TITSGFSTNY (SEO ID NO: 7)

[0094] VHH CDR3: KVHLIRLGAARAYDY (SEQ ID NO: 1)

[0095] In addition to safety considerations, the recognition by the VHH A12 antibody of an unidentified antigen could decrease the efficacy of the molecular cargo and therefore lower its therapeutic activity. In a preferred embodiment, the inventors therefore propose to use a VHH chain in which the sequence of the CDR3 has been mutated so as to inactivate any potential antibody function of VHH A12 and thereby avoid all unwanted cross reaction or off-target side effects once administered in vivo.

[0096] In particular, the inventors propose to use a mutated VHH A12 in which the CDR3 sequence contains more than two mutated amino acids as compared with SEQ ID NO:1.

[0097] The inventors tested five different variants of VHH in which the antigen recognition CDR3 was mutated. Among these five CDR3 variants, the sequences chosen were those that gave the best yield of VHH-NVshort in recombinant E coli cultures. The preferred variant is herein called the “CDR3.4 variant” and it has the SEQ ID NO:2. The inventors discovered that, unlike the other tested variants, a VHH chain containing this particular CDR3 variant offers a good production yield and can be successfully fused to molecular cargos.

[0098] Thus, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the inventors propose to use specifically a VHHNPX chain in which the sequence of the CDR3 is mutated into KVHLGG'G'GAAG'AG'GY (SEQ ID NO:2), or any variant thereof displaying at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity with SEQ ID NO: 2 and containing more than two mismatches as compared with the VHH CDR3 native sequence SEQ ID NO: 1.

[0099] The present inventors propose to further mutate the sequence of the VHH Al 2 chain so as to promote its dimerization capacity by enabling intrachain disulfide bridge formation, in order to ensure proper folding of the polypeptide during its production and when in contact with physiological fluids such as vitreous humor. The inventors have indeed observed that, when disulfide-bond forming cysteines are added in the sequence of the VHH A12 antibody, the fusion proteins containing the VHHNPX sequence folds properly and, upon contact with the vitreous humor, does not precipitate any more (figure 5).

[0100] Thus, in another embodiment, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the VHHNPX chain of the invention contains at least two disulfide- bond forming cysteines. More particularly, these disulfide-bond forming cysteines can be located in position 22 and 95, as reflected in SEQ ID NO:4.

[0101] In a preferred embodiment, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the VHHNPX chain of the invention contains both two disulfide-bond forming cysteines and a mutated CDR3 sequence as described above. In this particular embodiment, said VHHNPX has for example the SEQ ID NO:5. It can also be a variant thereof, whose sequence displays at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity with SEQ ID NO:5, said variant comprising the CDR1-3 having the sequences SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:2 respectively.

[0102] Thus, in this embodiment, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the invention relates to a VHH antibody (the VHHNPX chain) of SEQ ID NO: 5 or of a variant thereof whose sequence displays at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity with SEQ ID NO:5, said variant comprising i) the CDR1-3 of sequence SEQ ID NO:6 or a sequence having up to two amino acid additions, deletions, and / or substitutions compared to SEQ ID NO:6, SEQ ID NO:7 or a sequence having up to two amino acid additions, deletions, and / or substitutions compared to SEQ ID NO:7 and SEQ ID NO:2 respectively, and ii) at least one disulfide bond formed by two cysteines.

[0103] In a particular embodiment, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the invention relates to a VHH antibody (the VHHNPX chain) of SEQ ID NO:5, or of a variant thereof whose sequence displays at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity with SEQ ID NO:5, said variant comprising i) the CDR1-3 of sequence SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:2 respectively and ii) at least one disulfide bond formed by two cysteines.

[0104] The VHHNPX of the invention is particularly useful to vectorize molecular cargo in a non- endosomal / non-lysosomal pathway inside a target cell.

[0105] Accordingly, the present invention also relates to an in vitro use of the VHHNPX of the invention to vectorize molecular cargo in a non-endosomal / non-lysosomal pathway inside a target cell.

[0106] In a particular embodiment, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the invention targets the VHHNPX chain of SEQ ID NO: 5 or of a variant thereof whose sequence displays at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity with SEQ ID NO:5, said variant comprising i) the CDR1-3 of sequence SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:2 respectively, and ii) one disulfide bond formed by two cysteines to vectorize a molecular cargo in a non- endosomal / non-lysosomal pathway inside a target cell.

[0107] Said VHHNPX chain or variant thereof has preferably a basic isoelectric point, said isoelectric point being more preferably comprised between 8.5-11, 8.5-10, 9-10 and 9-10.5.

[0108] The VHHNPX chain will be hereafter referred to as the “VHHNPX” of the invention or the “VHH CPM” of the invention, which should be fused to a molecular cargo in order to produce fusion complexes that are better therapeutic drug candidates, because i) they do not contain any antibody functional sequence that divert the fusion protein from the target cell, ii) they use a non-endosomal / non-lysosomal pathway and are therefore less prone to intracellular degradation, and iii) they are more soluble and therefore far more easier to produce and to administer than fusion proteins containing the natural VHH A12 antibody chain that was classically used in the art.

[0109] More preferably, said VHHNPX chain has the sequence displayed in SEQ ID NO:5, i.e.:

[0110] EVQLQASGGGLAQPGGSLRLSCTVSGSIDVINNMAWYRQAPGNARELVATITSGFST NYASSVKGRFTISRDNAKKAVYLQMNSLKPEDTADYYCKVHLGGGGAAGAGGYW GQGTQVTVS

[0111] The VHHNPX CPM of the invention enables to internalize the molecular cargo in any target cells, in particular, in retinal cells or in neuron cells.

[0112] In the examples below, the inventors have shown that the insertion of eHIS at the NH2 terminus of VHHNPX does not modify the capacity of the VHHNPX to let the molecular cargo targeting retina. This is not the case when the two amino acids Glycine (G) and Serine (S) are inserted at the NH2 terminus of VHHNPX. Therefore, the VHHNPX CPM of the invention and the complexes containing same may comprise a eHIS at their NH2 terminus. This tag can especially be used as a conjugation site when phosphodiamidate morpholino oligonucleotides (PMOs) are used as molecular cargos in the complex of the invention.

[0113] Molecular cargos to be vectorized.

[0114] As used herein, the term “complex of the invention” designates a complex containing the VHHNPX CPM of the invention as disclosed above, and a cargo molecule. Preferably, the cargo is fused or conjugated to the VHHNPX CPM in the complex of the invention.

[0115] In another aspect, the invention thus also relates to a complex containing the cargo fused or conjugated to the VHHNPX CPM of the invention as disclosed above.

[0116] The cargo molecule may be for example a peptide, a protein, a chemical molecule, a small molecule or an oligonucleotide of interest such as an antisense oligonucleotide (ASO) (e.g. an uncharged phosphodiamidate morpholino oligonucleotide (PMO) or other DNA or RNA analogue with neutral electrostatic charge).

[0117] The complexes of the invention may be produced by any technique known per se in the art, such as, without limitation, any chemical, biological, genetic or enzymatic technique, either alone or in combination. Knowing the amino acid sequence of the desired sequence, one skilled in the art can readily produce said complexes, polypeptides or fusion proteins, by standard techniques for production of amino acid or chemical sequences. For instance, they can be synthesized using well-known solid phase method, preferably using a commercially available peptide synthesis apparatus (such as that made by Applied Biosystems, Foster City, Calif.) and following the manufacturer's instructions.

[0118] In a preferred embodiment, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the complexes of the invention have a basic isoelectric point, in order to be able to cross the plasma membrane and the Blood Brain Barrier (BBB). Accordingly, the complexes of the invention have preferably a pl equal to or higher than 8, more preferably equal to or higher than 8.5 and even more preferably equal to or higher than 9. Preferably, the complexes of the invention have a pl equal to or lower than 11, more preferably equal to or lower than 10.5 and even more preferably equal to or lower than 10, in order to avoid nonspecific binding to the target cells. Preferred ranges for the pl of the complexes of the invention are therefore of 8.5-11, 8.5-10, 9-10 and 9-10.5.

[0119] Any kind of cargo can be vectorized by means of the VHHNPX CPM of the invention. It can be a long protein, a short peptide, a chemical drug, a short or long oligonucleotide, etc.

[0120] In case the cargo is a peptide or a polypeptide, it can be of any size, e.g., it can have a size comprised between 5 and 10 000 kDa, preferably between 500 and 7500 kDa, more preferably between 1000 and 5000kDa, even more preferably between 1500 and 4500kDa. In this case, the complex of the invention is a fusion protein that can be produced genetically by expressing in-frame protein fusion or chemically by conjugating the two peptides separately produced.

[0121] The VHHNPX of the invention can also be used to vectorize oligonucleotides. In particular, the VHHNPX of the invention can vectorize antisense oligonucleotides (ASOs). In a more particular embodiment, the VHHNPX of the invention can vectorize Phosphorodiamidate Morpholino Oligomers (PMOs), a subtype of non-charged ASOs, which encounter a poor cellular uptake while entry in the cytoplasm or the nucleus - where they can reach their targets. PMOs are the most advanced exon-skipping therapeutic treatments for genetic diseases such as Duchenne Muscular Dystrophy (DMD) or Spinal Muscular Atrophy (SMA). Thus, the molecular cargo in the complexes of the invention can be a Phosphorodiamidate Morpholino Oligomer (PMO) and can be used to treat any pathologies known to benefit from such a treatment (in particular, the DMD or SMA). In this case, the CPM polypeptide may be synthetized or produced in recombinant eukaryote or prokaryote cell cultures and chemically conjugated to the ASO.

[0122] In a particular embodiment, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the complexes of the invention contains such a molecular cargo conjugated or fused to the VHHNPX chain of SEQ ID NO:5, or of a variant thereof whose sequence displays at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity with SEQ ID NO:5, said variant comprising i) the CDR1-3 of sequence SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO:2 respectively and ii) at least one disulfide-bond formed by two cysteines.

[0123] In a preferred embodiment, the molecular cargo vectorized by the VHHNPX CPM of the invention is a Neurovita peptide.

[0124] As used herein, the term “Neurovita protein”, “Neurovita long peptide” or “NVlong” designates the cytoplasmic tail domain of the pro-survival rabies virus envelope G protein, said domain having a high affinity for the PDZ domain of the human MAST2 and MAST1 protein. This long peptide has been fully described in the previous applications of the same inventors, namely WO2010 / 116258 and WO 2013 / 068430, which are incorporated herein by reference.

[0125] It can be the “Neurovita 3” polypeptide described in the prior art document W02013 / 068430, which has the SEQ ID NO: 17.

[0126] It can also be the mutated polypeptide having the SEQ ID NO: 18, in which 5 amino acids have been mutated as compared with SEQ ID NO: 17, in order to modify its isoelectric point (pl).

[0127] It can also be the mutated polypeptide having the SEQ ID NO: 19, in which 5 amino acids have been mutated as compared with SEQ ID NO: 17 and in which one amino acid has been mutated as compared with SEQ ID NO: 18, so that its pl is even more adequate.

[0128] These particular Neurovita long peptides are the sequences provided below:

[0129] SEQ ID NO: 17: RRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWEVHGQQTRL (42 amino acids)

[0130] SEQ ID NO: 18: AAVNASEPTQHNLRGTGAEVSVTPQSGAIISSWEVHGQQTRL (the 5 underlined amino acids have been mutated as compared with SEQ ID NO: 17) SEQ ID NO: 19: AAVNASEPTQHNLRGTGAEVSVTPQSGRIISSWEVHGQQTRL (the 5 underlined amino acids have been mutated as compared with SEQ ID NO: 17) In this case, the fusion protein of the invention can have in particular the sequence displayed in SEQ ID NO:29 (also herein called NPX452).

[0131] In a preferred embodiment, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the complex of the invention contains the VHHNPX of the invention having the sequence SEQ ID NO: 5 and the Neurovita long peptide having the sequence SEQ ID NO: 19, or a variant thereof having at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity with the sequence SEQ ID NO: 19, and containing the first anchor peptide QTRL of the Neurovita peptide onto the MAST-1 / MAST- 2 PDZ and also a two aa sequence SW (serine & tryptophan) that forms the second anchor of PBM (Terrien et al, 2012, Khan et al, 2019).

[0132] All biological assays involving the Neurovita polypeptides in the past have been carried out by using the Neurovita long polypeptides as defined above, containing between 30-44 amino acids of the cytoplasmic domain of the pro-survival rabies virus G protein. This is because the cytoplasmic domain of the G protein from which the Neurovita polypeptide was derived was known to be intrinsically disordered and it was expected that, by reducing its size too much, the conformation of its C-terminal part would be so modified that the resulting peptide would lose its biological activity (see other examples in Flanagan, 1992 and more recently So Young An, 2020).

[0133] In this context, the present inventors nevertheless decided to reduce the size of the Neurovita polypeptide. Reducing Neurovita size avoid that non-essential parts of the peptide could be recognized by the immune system of the patient because they originate from a viral protein.

[0134] As used herein, in particular in the context of the VHH suitable to vectorize molecules intracellularly, the term “Neurovita short peptide” or “NVshort” thus designates a functional fragment of the cytoplasmic domain of the pro-survival rabies virus G protein, that comprises a first anchor (QTRL) onto MAST-2 PDZ, and two SW (Serine-Tryptophan) residues that form a second anchor onto the MAST-2 PDZ, these two anchoring subsites being separated by 5 amino acid, and that has been deprived of all non-essential residues. Preferably, the residues at positions -8 and -5 from the C-terminus of the peptide are V and Q, respectively. This functional fragment is efficient on its own in various biological assays (not shown), even when it is separated from 25-33 amino acids also present in the N-terminal part of the cytoplasmic domain of this G protein.

[0135] In a preferred embodiment, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the NVshort peptide of the invention corresponds to the Neurovita long peptide having the SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19 in which the 27 N-terminal amino acids upstream of the PBM have been removed, or a variant thereof in which the 15 amino acid in C-terminal position, preferably 7 amino acid of the 15 amino acid in C- terminal position, can be substituted by any amino acid, preferably similar aa, and / or deleted provided that: i) the first and second anchors described above are not mutated, ii) the two anchors are separated by 5 amino acids, and iii) the residues at positions -8 and -5 from the C-terminus of the peptide are V and Q, respectively.

[0136] The NVshort peptide can thus be defined by the sequence:

[0137] XnSWXVXXQQTRL (SEQ ID NO:21), wherein X is any amino acid and n is an integer comprised between 0 and 4 (i.e., n is 0, 1, 2, 3, or 4).

[0138] In particular, the Neurovita short peptide (NVshort) has the sequence:

[0139] - SEQ ID NO:22, or a variant thereof in which 1, 2, 3, 4, 5, 6, 7 amino acids can be substituted by any amino acid, preferably similar aa, and / or deleted provided that: i) the first and second anchors described above are not mutated, and ii) the two anchors are separated by 5 amino acids and iii) the residues at positions -8 and -5 from the C-terminus of the peptide are V and Q, respectively. - SEQ ID NO:23, or a variant thereof in which 1, 2, 3, 4, 5, 6, 7 amino acids can be replaced by any amino acid, preferably similar aa, and / or deleted provided that: i) the first and second anchors described above are not mutated, and ii) the two anchors are separated by 5 amino acids and iii) the residues at positions -8 and -5 from the C-terminus of the peptide are V and Q, respectively.

[0140] - SEQ ID NO:24, or a variant thereof in which 1, 2, 3, 4, 5, 6, 7 amino acids can be replaced by any amino acid, preferably by similar aa, and / or deleted provided that: i) the first and second anchors described above are not mutated, and ii) the two anchors are separated by 5 amino acids and iii) the residues at positions -8 and -5 from the C-terminus of the peptide are V and Q, respectively.

[0141] More preferably, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the NVshort peptide has the sequence SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28 corresponding respectively to the 14, 13, 12, 11 C-terminal amino acids of SEQ ID NO: 19, or variants thereof preferably displaying:

[0142] - the sequence of said NVshort peptide corresponding to a sequence of 14 amino acids of SEQ ID NO:25 which can be replaced by any amino acid, preferably by similar amino acids, provided that i) the first and second anchors described above are not mutated, ii) the two anchors are separated by 5 amino acids, and iii) the residues at positions -8 and -5 from the C-terminus of the peptide are V and Q, respectively;

[0143] - the sequence of said NVshort peptide corresponding to a sequence of 13 amino acids of SEQ ID NO:26: which can be replaced by any amino acid, preferably by similar amino acids, provided that i) the first and second anchors described above are not mutated, ii) the two anchors are separated by 5 amino acids, and iii) the residues at positions -8 and -5 from the C-terminus of the peptide are V and Q, respectively;

[0144] - the sequence of said NVshort peptide corresponding to a sequence of 12 amino acids of SEQ ID NO:27: which can be replaced by any amino acid, preferably by similar amino acids, provided that i) the first and second anchors described above are not mutated, ii) the two anchors are separated by 5 amino acids, and iii) the residues at positions -8 and -5 from the C-terminus of the peptide are V and Q, respectively; or

[0145] - the sequence of said NVshort peptide corresponding to a sequence of 11 amino acids of SEQ ID NO:28: which can be replaced by any amino acid, preferably by similar amino acids, provided that i) the first and second anchors described above are not mutated, ii) the two anchors are separated by 5 amino acids, and iii) the residues at positions -8 and -5 from the C-terminus of the peptide are V and Q, respectively.

[0146] In a preferred embodiment, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, -once the Neurovita short peptide has been fused to VHH, the fusion molecule has a basic isoelectric point (pl), in order to be able to cross the Blood Brain Barrier (BBB) and the cell membranes. A protein is said to have a basic pl when the latter is higher than 7, more preferably equal to or higher than 8 and even more preferably equal to or higher than 8.5 or equal to or higher than 9. Accordingly, the Neurovita short peptide once it has been fused to VHH used in the invention has preferably a pl equal to or higher than 8, more preferably equal to or higher than 8.5 and even more preferably equal to or higher than 9. Also, the Neurovita short peptide used in the invention once it has been fused to a VHH has a pl equal to or lower than 11, more preferably equal to or lower than 10.5 and even more preferably equal to or lower than 10, in order to avoid non-specific binding to the target cells. Preferred ranges for the pl of the Neurovita short peptide once it has been fused to a VHH of the invention are therefore of 8.5-11, 8.5-10, 9-10 and 9-10.5.

[0147] In a preferred embodiment, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the complex of the invention contains the Neurovita short peptide having the sequence SEQ ID NO:21.

[0148] In a further preferred embodiment, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the complex of the invention contains the Neurovita short peptide having a sequence selected in the group consisting of SEQ ID NO:22-28.

[0149] In this case, the complex of the invention can be a fusion protein having in particular the sequence displayed in SEQ ID NO:30 (also herein called NPX461), or a variant thereof whose sequence displays at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity with the sequence SEQ ID NO: 30, and containing the essential amino acids disclosed above. Association with homing molecule.

[0150] In the context of the invention, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, it is possible to associate the VHHNPX CPM of the invention with a homing molecule or homing sequence, or with any other drug delivery systems known in the art. Accordingly, the complexes of the invention may also contain, in addition to the VHHNPX CPM of the invention, one or more other peptide(s) addressing the molecular cargo to a particular organ, tissue, or cell.

[0151] In this particular embodiment, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the VHH antibody (VHHNPX chain) is used in combination with or coupled to a homing molecule in the complexes of the invention.

[0152] This means that the complexes of the invention may contain, in addition to the VHHNPX CPM of the invention and the molecular cargo as described above, any peptide or other delivery systems known in the art that may transport said complex across the Blood Brain Barrier and / or address said complex to a particular organ, tissue, or cell.

[0153] Therefore, in a particular embodiment, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the complexes of the invention contain the Cell Penetrating Molecule (CPM) of the invention, a molecular cargo and optionally, at least one homing molecule that will bypass the Blood Brain Barrier and / or target or address the molecular cargo toward cells or region of interest.

[0154] Preferably, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, this homing molecule is the Rabies virus Derived Peptide (RDP) derived from the G protein of the CVS-NIV sequence, which is known to bypass the Blood-Brain barrier and to target nAChR-expressing cells (Kumar P, Wu H, McBride JL, Jung KE, Kim MH, Davidson BL, et al. Transvascular delivery of small interfering RNAto the central nervous system. Nature. 2007 Jul 5;448(7149):39-43), The last property is useful when tissues contain not only neurons but also other cell types, (astrocytes, microglia...), as in the nervous parenchyma; da Costa A, Prehaud C, Bakoa F, Afonso P, Ceccaldi PE, Lafaye P, et al. A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain. J Vis Exp JoVE. 2019 09 (146)). 1

[0155] In this case, it is recommended to combine a molecular cargo with a RDP to target nAChR- expressing cells and with a VHH CPM according to the invention to enter in these cells in a non-endocytic / lysosomal pathway.

[0156] In a particular embodiment, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the VHHNPX CPM of the invention can be thus used in combination with or coupled to a RDP derived from the G protein of the CVS-NIV sequence (hereafter “RDP domain”) that may carry the molecular cargo into nAChR-expressing cells (e.g., neuronal cells) and increase the specificity of the peptide toward nAChR-expressing cells (e.g., neurons) (see WO2016 / 038122 when the cargo is the Neurovita peptide; see Kumar P. et al, Nature, 2007 when the cargo is a small interfering RNA).

[0157] Any RDP sequences can be used in this respect, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, in particular those recited page 43 of WO2016 / 038122 (see the SEQ ID NO: 1, SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34 of WO2016 / 038122). A particular sequence is for example SEQ ID NO: 13 in the enclosed sequence listing.

[0158] Preferably, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the RDP sequence comprises a mutation compared to SEQ ID NO: 13. More preferably, this mutation is a substitution, e.g., by a serine, of the cysteine residue at position 15 of the sequence of SEQ ID NO:13. Indeed, this substitution prevents the aggregation of a Neurovita peptide-containing fusion protein (figure 9). Therefore, it is particularly advantageous to associate the Neurovita peptide used in the invention with a mutated RDP, as described herein in order to avoid the protein aggregation induced by the domain and therefore to enhance the production yield and the biological activity of the Neurovita peptide.

[0159] This is surprising because it was thought so far that the cysteine in neuron cell-targeting peptides derived from a Rabies virus G protein was required to maintain its biological activity (Lentz et al., 1984, 1987 and 1989). Therefore, all the RDPs carrying Neurovita peptides in the past contained a cysteine residue (see in particular SEQ ID NO: 1, SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34 of WO2016 / 038122).

[0160] In a preferred embodiment, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the Neurovita peptide used in the invention is therefore fused to the VHHNPX CPM of the invention and to a RDP sequence that does not contain any cysteine residue, nor any amino acid that may create unwanted disulfide bridge(s). For example, the cysteine residue contained in the RDP sequence of said RDP can be replaced by a serine residue (SEQ ID NO: 14).

[0161] In a more preferred embodiment, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the mutated RDP used in the complexes of the invention has the sequence YTIWMPENPRLGMSSDIFTNSRGKRASKG (SEQ ID NO: 14) or a variant thereof displaying at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity with SEQ ID NO: 14, but not containing any cysteine residue. Of note, this similar sequence of RDP should be chosen so that the resulting peptide keeps the loop structure of SEQ ID NO: 14, in order to favor its anchoring to nAChR receptors at the surface of target cells (Lentz et al, 1987, Lentz et al, 1984, Donnelly-Roberts et al, 1989).

[0162] In a more preferred embodiment, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the complex of the invention comprises the VHHNPX CPM, the NVshort peptide, and the mutated RDP described above. A preferred example of the complex of the invention is represented by the complex of sequence:

[0163] YTIWMPENPRLGMSSDIFTNSRGKRASKGEVQLQASGGGLAQPGGSLRLSCTVSGSID VINNMAWYRQAPGNARELVATITSGF STNYAS S VKGRFTI SRDN AKK AV YLQMN SLK PEDTADYYCKVHLGGGGAAGAGGYWGQGTQVTVS RIISSWEVHGQQTRL (SEQ ID NO:31).

[0164] Linkers

[0165] Advantageously, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, linkers may be used between the molecular cargo (e.g. a protein) and the VHHNPX CPM associated thereto and / or between two homing molecules / CPM. Accordingly, the last N-terminal residue of the cargo can be linked by a peptide bond to the first C-terminal residue of a peptide linker, the last N-terminal residue of which being linked to the first C- terminal residue of the CPM / homing molecule. In this particular embodiment, a peptide linker consisting of one to ten amino acids, preferably of one to five amino acids, more preferably of one to three amino acids can be added between the CPM / homing molecule and the cargo, or between the two CPM / homing molecules. In a most preferred embodiment, said linker has the three amino acid sequence “AAA”. In a preferred embodiment, the linker has the sequence SEQ ID NO:36.

[0166] Accordingly, the complexes of the invention may contain linkers between the different peptide components of their sequence.

[0167] Polynucleotides

[0168] In another aspect, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the present invention also relates to the polynucleotides encoding the VHHNPX CPM or complexes, in particular fusion proteins, disclosed above. These polynucleotides are preferably integrated in vectors, in particular in expression vectors.

[0169] The term "vector", as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are in the form of plasmids. In the present specification, "plasmid" and "vector" may be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such forms of expression vectors, such as bacterial plasmids, YACs, cosmids, retrovirus, EBV-derived episomes, and all the other vectors that the skilled man will know to be convenient for ensuring the expression of the VHHNPX CPM or complexes of the invention.

[0170] Selection of an appropriate vector will depend mainly on the size of the nucleic acids to be inserted into the vector and the particular host cell to be transformed with the vector. Each vector contains various components, depending on its function (e.g., expression of heterologous polynucleotide) and its compatibility with the particular host cell in which it resides. The vector components generally include, but are not limited to: an origin of replication, a selection marker gene, a promoter, a ribosome binding site (RBS), a signal sequence, the heterologous nucleic acid insert and a transcription termination sequence. Said vectors or cassettes of the invention preferably contain regulatory sequences allowing the expression of the polypeptides of the invention in a host cell, preferably in a bacterial host cell, e.g., a Escherichia coli bacterium. These regulatory sequences are for example a promoter, which is inducible or constitutive. Promoters suitable for use with prokaryotic hosts include E. coli promoters such as lac, trp, tac, trc and ara, viral promoters recognized by E. coli such as lambda and T5 promoters, and the T7 and TTlac promoters derived from T7 bacteriophage.

[0171] In another aspect, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the invention provides vectors comprising polynucleotides of the invention encoding the VHHNPX CPM and complexes, in particular fusion proteins of the invention. In order to express them, these vectors are “expression vectors”, in which the sequences encoding the VHHNPX CPM and complexes, in particular fusion proteins of the invention are operatively linked to transcriptional and translational sequences. These vectors may remain free (i.e., nonintegrated into a host cell genome), or may become integrated ("stably” or “temporary” incorporated) into the host cell genome, either as a result of the original transformation of the host cells, or as the result of subsequent recombination and / or repair events. Still, they remain “heterologous” as compared to the host cell and its natural genome. As used therein, the term “operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0172] The invention and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, therefore relates to a vector, DNA or mRNA, encoding the VHHNPX CPM or complexes, in particular the fusion proteins, of the invention, as defined above.

[0173] Pharmaceutical compositions

[0174] In another aspect, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the invention relates to a pharmaceutical composition comprising the complexes of the invention which contain at least the VHHNPX and a molecular cargo as disclosed above, and a pharmaceutically acceptable carrier or excipient. These pharmaceutical compositions are hereafter called “pharmaceutical compositions of the invention”.

[0175] In these pharmaceutical compositions, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the complexes of the invention are preferably formulated in an effective amount. An "effective amount" refers to an amount effective, at dosages and for periods of time, necessary to achieve the desired result, such as prevention or treatment of the target disease. A “therapeutically effective amount” means an amount sufficient to influence the therapeutic course of said target disease. A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects.

[0176] As used herein, the term "pharmaceutically acceptable carrier" refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any conventional type. A "pharmaceutically acceptable carrier" is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation; suitable carriers include, but are not limited to, phosphate buffered saline solutions, distilled water, emulsions such as an oil / water emulsions, various types of wetting agents, sterile solutions and the like, dextrose, glycerol, saline, ethanol, and combinations thereof. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like. Carriers for parenteral administration include aqueous solutions of dextrose, mannitol, mannose, sorbitol, saline, pure water, ethanol, glycerol, propyleneglycol, peanut oil, sesame oil, polyoxyethylenepolyoxypropylene block polymers, and the like.

[0177] As used herein the term "pharmaceutically acceptable excipient” refers to a substance that is used as a carrier or for the manufacturing of the administrable form of the complexes of the invention. Suitable excipients include fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize, wheat, rice, or potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as crosslinked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. These pharmaceutical compositions contain preferably the fusion proteins of SEQ ID NO:29, SEQ ID NO:30, or SEQ ID NO:31, and therefore a Neurovita peptide that can be administered in the eye of a subject in need thereof.

[0178] In particular, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, when the composition of the invention is intended to be administered in the eye (see below), the preferred excipients are those authorized by the FDAfor eye injection, namely: Calcium chloride, carboxymethylcellulose sodium, hyaluronate sodium, hydrochloric acid, magnesium chloride, magnesium stearate, microcrystalline cellulose, poly(dl-lactic-co-glycolic acid, poly(dl-lactic-co-glycolic acid, polysorbate 80, polyvinyl alcohol, potassium chloride, sodium acetate, sodium bicarbonate, sodium chloride, sodium hydroxide, sodium phosphate, trisodium citrate dihydrate.

[0179] Therapeutic uses

[0180] In another aspect, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the present invention relates to the VHHNPX CPM of the invention, as defined above, for use for vectorizing a molecular cargo in a non-endosomal / non-lysosomal pathway inside a target cell.

[0181] The VHHNPX CPM of the invention can thus be used for delivering therapeutic molecules inside the target cell. The VHHNPX CPM of the invention can be used notably for delivering therapeutic molecules inside the target cell in a non-endosomal / non-lysosomal pathway. This is particularly useful for preventing and / or treating any type of disease or condition. Notably, this use can be contemplated for preventing and / or treating neurodegenerative diseases or cell damage-associated conditions, preferably affecting the brain or the muscle, for example a disease or condition involving the Peripheral Nervous System (PNS) or the Central Nervous System (CNS) or the evolution of such disease or condition, such as optic neuropathies (glaucoma, NAION), motor neuron diseases (ALS, SMA), muscular disease (Duchenne Disease) or neurodegenerative diseases (Parkinson, Alzheimer).

[0182] This embodiment relates to the VHHNPX of the invention, as defined above, for use for vectorizing a molecular cargo in a non-endosomal / non-lysosomal pathway inside a target cell, for preventing and / or treating a disease or condition involving the Peripheral Nervous System (PNS) or the Central Nervous System (CNS), or the evolution of such disease or condition, such as optic neuropathies (glaucoma, NAION), motor neuron diseases (ALS, SMA), muscular disease (Duchenne Disease) or neurodegenerative diseases (Parkinson, Alzheimer).

[0183] The complexes of the invention (as described above) are characterized by the fact that they are produced under a functional form in vitro and remain functional in vivo when administered, in particular injected, to a subject in need thereof.

[0184] A “subject” in the present invention can be any mammal (human or animal). Preferably, it is a human being.

[0185] By “functional”, it is herein meant that the complexes of the invention exhibit a significant biological activity.

[0186] As far as Neurovita is concerned, said “biological activity” corresponds to the inactivation of the MAST1 / MAST2 / PTEN pathway, by binding with high affinity the PDZ domain of the human MAST 1 / MAST2 proteins and sterically blocking the PDZ domain of the human MAST1 / MAST2 proteins, especially in neuronal cells. Importantly, the significant biological activity of fusion proteins containing a Neurovita peptide is due to the fact that these fusion proteins are soluble in physiological fluids, e.g., in vitreous humor. As a matter of fact, the present results show that, contrary to the VHH-Neurovita peptides of the prior art, the fusion proteins of the invention do not aggregate when put in contact with these fluids (figure 5). The fusion proteins of the invention are moreover “stably” soluble, as they remain non-aggregated for a long-term in solution.

[0187] In addition, the complexes of the invention are designed to target and enter various eukaryotic cells such as neuron cells, due to the VHHNPX CPM they contain. They preferably target and penetrate neuron cells, such as RGCs in the retina.

[0188] The fusion protein of the invention is preferably SEQ ID NO:30 or SEQ ID NO:31 and therefore contains a VHHNPX and a Neurovita short peptide. It was shown in WO2016 / 038122 that Neurovita polypeptides fused at the C-terminal extremity of the VHH A12 or VHH A12- comprising polypeptide are efficiently delivered across the Blood Brain Barrier (BBB), when they display a basic isoelectric point pl. As explained above, most of the fusion proteins of the invention containing a Neurovita peptide display a basic pl. These fusion proteins are therefore able to cross the Blood Brain Barrier (BBB). Accordingly, they should reach the brain after being injected intravenously.

[0189] As shown in the examples below, the complexes of the invention are not toxic on human cells, they target retina cells and persist in the mouse eye after injection, where they can promote neurosurvival and / or neuroprotection and allow the repair / recovery of neuron cells after a lesion.

[0190] In one aspect, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the invention therefore relates to the complexes of the invention, for their use as a medicament. Also, the invention relates to the pharmaceutical composition of the invention, containing said complexes, for its use as a medicament.

[0191] In one particular aspect, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the present invention targets the complexes as defined above, notably complexes comprising a NVshort peptide as a cargo, or pharmaceutical composition comprising same, in order to prevent and / or treat diseases involving the Peripheral Nervous System (PNS) or Central Nervous System (CNS), or the evolution of such disease or condition. Said disease or condition is preferably a neurodegenerative disease, or a cell damage-associated condition such as stroke or injury including optic nerve disease, retina diseases, motor neuron disease, or the evolution of such disease or condition. The invention also provides therapeutic methods using the complexes of the invention, notably complexes comprising a NVshort peptide as a cargo, or pharmaceutical compositions comprising same, for use for preventing and / or treating Peripheral Nervous System (PNS) or Central Nervous System (CNS), or the evolution of such disease or condition. Said disease or condition is preferably a neurodegenerative disease, or a cell damage-associated condition such as stroke or injury including optic nerve disease, retina diseases, motor neuron disease. The invention also provides the complexes of the invention, notably complexes comprising a NVshort peptide as a cargo, or pharmaceutical composition comprising same, for use in the manufacture of a medicament, especially a medicament for use in the therapy of diseases involving the Peripheral Nervous System (PNS) or Central Nervous System (CNS), or the evolution of such disease or condition. Said disease or condition is preferably a neurodegenerative disease, or a cell damage-associated condition such as stroke or injury including optic nerve disease, retina diseases, motor neuron disease. This therapy comprises advantageously the intravenous injection of the complexes of the invention, notably complexes comprising a NVshort peptide as a cargo, or pharmaceutical compositions comprising same, in patients in need thereof.

[0192] In another particular aspect, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, the present invention relates to the use of the complexes of the invention, notably complexes comprising a NVshort peptide as a cargo, or pharmaceutical compositions comprising same, in order to prevent and / or treat diseases involving the PNS or the CNS, such as neurodegenerative diseases, or brain cell damage-associated conditions such as brain stroke or injury, or the evolution of such disease or condition. The invention also provides therapeutic methods using the complexes of the invention, notably complexes comprising a NVshort peptide as a cargo, or pharmaceutical compositions comprising same, for use for preventing and / or treating neurodegenerative diseases or brain cell damage- associated conditions. The invention also provides the complexes of the invention, notably complexes comprising a NVshort peptide as a cargo, or pharmaceutical compositions comprising same, for use in the manufacture of a medicament, especially a medicament for use in the therapy of neurodegenerative diseases or brain cell damage- associated conditions. This therapy comprises advantageously the most appropriate route of injection (such as intravitreal injection, intrathecal injection, intra-meningeal or intravenous injection) of the complexes of the invention, notably complexes comprising a NVshort peptide as a cargo, or pharmaceutical compositions comprising same, in patients in need thereof.

[0193] The complexes of the invention, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, notably complexes comprising a NVshort peptide as a cargo, or pharmaceutical compositions comprising same are preferably for use as a neuroregenerative (reparation and regrowth of neuronal axons,) and / or neuroprotective agent (protection of neurons from apoptosis or degeneration).

[0194] The complexes of the invention, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, notably complexes comprising a NVshort peptide as a cargo, or pharmaceutical compositions comprising same are preferably for use in the treatment and / or palliation and / or prevention of a disease, disorder or condition, which alters the Central Nervous System (CNS) and / or the Peripheral Nervous System (PNS), for example as a neurorestorative therapy and / or prevention and / or palliation.

[0195] The expression "Central Nervous System" or "CNS" is herein intended as meaning the brain and (in case of a vertebrate animal) the spinal cord. The Peripheral Nervous System (PNS) is the vast network of spinal and cranial nerves linking the body to the brain and spinal cord. The PNS is subdivided into the autonomic nervous system (sympathetic NS and parasympathetic NS) and the somatic nervous system. The PNS consists of sensory neurons running from stimulus receptors to the CNS and motor neurons running from the CNS to the muscle and glands.

[0196] According to an embodiment of the invention, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, said disease or disorder is or involves a neurodegenerative disease or disorder (for example, a chronic neurodegenerative disease or disorder), such as non-viral encephalopathy, Alzheimer's disease, Parkinson's disease, ALS, SMA, Huntington disease, multiple sclerosis (MS) or rare genetic diseases. According to an embodiment of the invention, said condition is or involves a neurodegenerative condition, such as aging.

[0197] According to an embodiment of the invention, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, said disease, disorder or condition is or involves a physical or ischemic injury of the nervous system, such as seizure, stroke, trauma, epilepsy.

[0198] According to an embodiment of the invention, and in particular insofar as it relates to VHH suitable to vectorize molecules intracellularly, said disease, disorder or condition involves the presence of a chemical neurotoxic agent and / or of an oxidative stress.

[0199] Definitions

[0200] A VHH antibody (also called camelid single domain antibody, single domain VHH antibody, VHH, or nanobody®) is an antibody that comprises the variable domain of a camelid antibody that contains two heavy chains and lacks the two light chains usually found in antibodies. The VHH is an antibody with three complementarity determining regions (CDR). The VHH has the following structure from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and where CDR1 to CDR3 refer to complementarity determining region 1 to 3.

[0201] The isoelectric point (pl) of a peptide, polypeptide or protein is defined as the pH at which the protein carries no net charge (i.e. the negative charges compensate the positive charges). Methods to determine the pl of a peptide, polypeptide or protein, either through experimental determination or through theoretical calculation based on the sequence of the protein, are known to the skilled person. In particular, the pl can be measured experimentally using isoelectric focusing. Alternatively, or in addition, the pl may be calculated using a computer program such as the EMBOSS iep software, available from the European Bioinformatics Institute, Genome Campus, Hinxton, Cambridge CB10 1SD, UK and / or the Compute PI tool from the Expasy software, available from the Swiss Institute of Bioinformatics, Quartier Sorge - Batiment Genopode, 1015 Lausanne, Switzerland. A protein is said to have a basic pl when the latter is higher than 7, more preferably equal to or higher than 8 and even more preferably equal to or higher than 8.5 or equal to or higher than 9. Since the pl of a fusion protein or complexe (e.g. the VHH-Neurovita peptide of the invention, including other CAMs or CPMs, if any), as well as the pl of each separate peptide thereof, is relevant to the permeability of the entire construct across the BBB, the pl is to be assessed (measured and / or calculated) for the entire construct.

[0202] The invention provides peptides, polypeptides, proteins or complexes whose amino acid sequences are or contains sequences that are “similar” or “substantially similar” to the reference sequences described herein. “Similarity” of two targeted amino acid sequences can be determined by calculating a similarity score for the two amino acid sequences. As used herein, the “similarity score” refers to the score generated for the two sequences using the BLOSUM62 amino acid substitution matrix, a gap existence penalty of 11, and a gap extension penalty of 1, when the two sequences are optimally aligned. Two amino acid sequences are substantially similar if their similarity score exceeds a certain threshold value. The threshold value can be any integer ranging from at least 1190 to the highest possible score for a particular reference sequence. For example, the threshold similarity score can be 1190, 1200, 1210, 1220, 1230, 1240, 1250, 1260, 1270, 1280, 1290, 1300, 1310, 1320, 1330, 1340, 1350, 1360, 1370, 1380, 1390, 1400, 1410, 1420, 1430, 1440, 1450, 1460, 1470, 1480, 1490, 1500, or higher. If in a particular embodiment of the invention, the threshold score is set at, for example, 1300, and the reference sequence is any of those described herein, then any amino acid sequence that can be optimally aligned with any of those described herein to generate a similarity score of greater than 1300 is be held as “similar” to those described herein. Amino acid substitution matrices and their use in quantifying the similarity between two sequences are well-known in the art and described, e.g., in Dayhoff et al. (1978), and in Henikoff et al. (1992). To generate accurate similarity scores using NCBI BLAST, it is important to turn off any filtering, e.g., low complexity filtering, and to disable the use of composition based statistics. One should also confirm that the correct substitution matrix and gap penalties are used. A “similar” sequence may comprise one or more conservative amino acid mutations. It is known in the art that one or more conservative amino acid mutations to a reference sequence may yield a modified polypeptide with no substantial change in physiological, chemical, or functional properties compared to the reference sequence; in such a case, the reference and modified sequences would be considered to be "similar" polypeptides. Conservative amino acid mutation may include addition, deletion, or substitution of an amino acid; in one nonlimiting example, the conservative amino acid mutation is a conservative amino acid substitution. A conservative amino acid substitution is defined herein as the substitution of an amino acid residue for another amino acid residue with similar chemical properties (e.g. size, charge, or polarity). A conservative amino acid substitution may substitute a basic, neutral, hydrophobic, or acidic amino acid for another of the same group. By the term "basic amino acid" it is meant hydrophilic amino acids having a side chain pK value of greater than 7, which are typically positively charged at physiological pH. Basic amino acids include histidine (His or H), arginine (Arg or R), and lysine (Lys or K). By the term "neutral amino acid" (also "polar amino acid"), it is meant hydrophilic amino acids having a side chain that is uncharged at physiological pH, but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Polar amino acids include serine (Ser or S), threonine (Thr or T), cysteine (Cys or C), tyrosine (Tyr or Y), asparagine (Asn or N), and glutamine (Gin or Q). The term "hydrophobic amino acid" (also "non-polar amino acid") is meant to include amino acids exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg (1984). Hydrophobic amino acids include proline (Pro or P), isoleucine (He or I), phenylalanine (Phe or F), valine (Vai or V), leucine (Leu or L), tryptophan (Trp or W), methionine (Met or M), alanine (Ala or A), and glycine (Gly or G). "Acidic amino acid" refers to hydrophilic amino acids having a side chain pK value of less than 7, which are typically negatively charged at physiological pH. Acidic amino acids include glutamate (Glu or E), and aspartate (Asp or D).

[0203] In a further aspect, the invention relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof.

[0204] A purpose of the invention described herein is to reduce the size of NV and to propose solutions to solve the issues of solubility, yield, formation of aggregates of NV-fused to CPM in order to transform the VHH-NV molecule with high therapeutic potential into an investigational drug candidate. In spite of the teaching of the art, the present inventors have surprisingly shown that a very short fragment of the cytoplasmic domain retains the PBM biological activity of the G protein. In particular, the PBM on its own is sufficient for the regeneration and protection of neurons. Accordingly, shorter peptides can be used for the regeneration of axons and protection of cells, in particular neurons, as long as these peptides contain the C-terminal PBM of type NV3 (SEQ ID NO:20) or a functional variant thereof (herein called “Neurovita short peptide”).

[0205] The Neurovita peptide (NV)

[0206] Prehaud et al. (Attenuation of rabies virulence: takeover by the cytoplasmic domain of its envelope protein, Sci Signal. 2010 Jan 19;3(105):ra5) reported that the C-terminal region of the cytoplasmic domain of the G protein of rabies viruses is involved in the binding of the G protein to the PDZ domain of the human MAST2 and MAST1 protein. This C-terminal region comprises a motif called PDZ-BM or PBM (PDZ binding motif) which has the sequence SEQ ID NO:20.

[0207] Subsequent analysis showed that the NV PBM is a non-classical PBM formed by two subsites (anchors), with a first anchor (QTRL residues in -4, -3, -2, and -1 from the C-terminus) binding the canyon on the surface of MAST-2 PDZ, and 2 residues SW (serine-tryptophan) in C- terminal position -10 and -9 forming a second anchor. For efficient binding to the MAST-2 PDZ, the two anchoring sub-sites should be separated by 5 amino acids (Terrien et al, 2012, Khan et al, 2019). In the present invention, the PBM of Neurovita has been optimized (it is called Neurovita 3) as described in Khan et al, 2019.

[0208] The NVlong, in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, can be the “Neurovita 3” long polypeptide described in the prior art document W02013 / 068430, which has the SEQ ID NO:17.

[0209] It can also be the mutated polypeptide having the SEQ ID NO: 18, in which 5 amino acids have been mutated as compared with SEQ ID NO: 17, in order to keep intact the value of the isoelectric point (pl), once it has been fused to a VHH to allow delivery into the cell cytoplasm.

[0210] It can also be the mutated polypeptide having the SEQ ID NO: 19, in which 5 amino acids have been mutated as compared with SEQ ID NO: 17, and in which one amino acid has been mutated as compared with SEQ ID NO: 18, so that the pl value is even more adequate. These particular long Neurovita polypeptides have the following sequences:

[0211] SEQ ID NO: 17: RRVNRSEPTQHNLRGTGREVSVTPQSGKIISSWEVHGQQTRL (42 amino acids)

[0212] SEQ ID NO: 18: AAVNASEPTQHNLRGTGAEVSVTPQSGAIISSWEVHGQQTRL (the 5 underlined amino acids have been mutated as compared with SEQ ID NO: 17)

[0213] SEQ ID NO: 19: AAVNASEPTQHNLRGTGAEVSVTPQSGRIISSWEVHGQQTRL (the 5 underlined amino acids have been mutated as compared with SEQ ID NO: 17)

[0214] NV short

[0215] All biological assays involving the Neurovita polypeptides in the past have been carried out by using the long Neurovita polypeptides as defined above, containing between 30-44 amino acids of the cytoplasmic domain of the pro-survival rabies virus G protein. This is because the cytoplasmic domain of the G protein from which the Neurovita polypeptide was derived was known to be intrinsically disordered and it was expected that, by reducing its size too much, the conformation of its C-terminal part would be so modified that the resulting peptide would lose its biological activity (see other examples in Flanagan, 1992 and more recently So Young An, 2020).

[0216] In this context, the present inventors nevertheless decided to reduce the size of the Neurovita polypeptide to avoid that non-essential parts thereof could be recognized by the immune system of the patient because they originate from a viral protein.

[0217] As used herein, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the term “Neurovita short peptide” or “NVshort” thus designates a functional fragment of the cytoplasmic domain of the pro-survival rabies virus G protein, that has been deprived of all non-essential residues and in which the C terminal PBM (original sequence called Neurovita 1) has been optimized into PBM of type Neurovita 3 as described in Kahn et al, 2019. The present inventors reveal in the examples below, for the first time, that the 11-15 amino acids long NV short sequences encoding a PDZ-binding motif (PBM) of type Neurovita 3, is efficient on its own in various biological assays (figures 13, and 7a, 16 and 8), even when it is separated from the 25-33 amino acids also present in the N-terminal part of the cytoplasmic domain of this G protein. In particular, NVshort is able to induce neurite elongation as well as the NVlong peptide (Figure 13). Surprisingly, NVshort fused to a VHH as a CPM displays a longer remanence in the retina after in vivo administration than NVlong fused to the same VHH (Figure 7a, 16).

[0218] In a first aspect, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the present invention relates to the “NVshort peptide” which consists in the C-terminal fragment of 11-15 amino acids of the “NVlong” polypeptide encoding a PBM of Neurovita 3 type (SEQ ID NO:20) disclosed above, or a variant thereof. In other words, the “NVshort” peptide of the invention corresponds to the “NVlong” polypeptide disclosed above, in which the 25, 26, 27, 28, 29, 30, 31, 32 or 33 consecutive N-terminal amino acids have been deleted. In other words, the NVshort peptide of the invention is a 11-15 amino acid-long peptide comprising a PBM of Neurovita 3 type (SEQ ID NO:20) and does not contain the 25 to 33 amino acids long N-terminal part of the cytoplasmic domain of the rabies virus G protein, or a variant of this peptide.

[0219] The NVshort peptide of the invention necessarily comprises the first anchor (QTRL) of the Neurovita peptide onto MAST-2 PDZ, and two SW (Serine-Tryptophan) residues that form the second anchor of the Neurovita peptide onto the MAST-2 PDZ. In the peptide of the invention, these two anchoring subsites are separated by 5 amino acids (Terrien et al, 2012, Khan et al, 2019). In a preferred embodiment, the residues at positions -8 and -5 from the C-terminus of the peptide are V and Q, respectively.

[0220] Preferably, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the value of the pl of the molecule- once the variant peptide has been fused to a Cell Penetrating Molecule, in particular a VHH, is preserved.

[0221] In a preferred embodiment, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the NVshort peptide of the invention corresponds to the long Neurovita peptide having the SEQ ID NO: 17, SEQ ID NO: 18, or SEQ ID NO: 19 in which the 27 N-terminal amino acids upstream of the PBM have been removed, or a variant thereof in which the 15 amino acid in C-terminal position, preferably 7 amino acid of the 15 amino acid in C-terminal position, can be substituted by any amino acid, preferably similar aa, and / or deleted provided that: i) the first and second anchors described above are not mutated, ii) the two anchors are separated by 5 amino acids, and iii) the residues at positions -8 and -5 from the C-terminus of the peptide are V and Q, respectively.

[0222] According to this preferred embodiment, the NVshort peptide has the sequence:

[0223] XnSWXVXXQQTRL (SEQ ID NO:21), wherein X is any amino acid and n is an integer comprised between 0 and 4 (i.e., n is 0, 1, 2, 3, or 4).

[0224] Preferably, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the value of the pl of the molecule- once the NVshort peptide has been fused to a Cell Penetrating Molecule, in particular a VHH, is preserved.

[0225] In a preferred embodiment, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the present invention targets a Neurovita short peptide (NVshort) having the sequence:

[0226] - SEQ ID NO:22, or a variant thereof in which the 15 amino acids in C-terminal position, preferably 7 amino acid of the 15 amino acid in C-terminal position, can be substituted by any amino acid, preferably similar aa, and / or deleted provided that: i) the first and second anchors described above are not mutated, ii) the two anchors are separated by 5 amino acids, and iii) the residues at positions -8 and -5 from the C-terminus of the peptide are V and Q, respectively.

[0227] Preferably, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the value of the pl of the molecule- once the NVshort peptide has been fused to a Cell Penetrating Molecule, in particular a VHH, is preserved.

[0228] - SEQ ID NO:23, or a variant thereof in which the 15 amino acids in C-terminal position, preferably 7 amino acid of the 15 amino acid in C-terminal position, can be replaced by any amino acid, preferably similar aa, and / or deleted provided that: i) the first and second anchors described above are not mutated, ii) the two anchors are separated by 5 amino acids, and iii) the residues at positions -8 and -5 from the C-terminus of the peptide are V and Q, respectively.

[0229] Preferably, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the value of the pl of the molecule- once the NVshort peptide has been fused to a Cell Penetrating Molecule, in particular a VHH, is preserved.

[0230] - SEQ ID NO:24, or a variant thereof in which the 15 amino acids in C-terminal position, preferably 7 amino acid of the 15 amino acid in C-terminal position, can be replaced by any amino acid, preferably by similar aas, and / or deleted provided that: i) the first and second anchors described above are not mutated, ii) the two anchors are separated by 5 amino acids, and iii) the residues at positions -8 and -5 from the C-terminus of the peptide are V and Q, respectively.

[0231] Preferably, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the value of the pl of the molecule- once the NVshort peptide has been fused to a Cell Penetrating Molecule, in particular a VHH, is preserved.

[0232] In a particular embodiment, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, said NVshort peptide variant may display the SEQ ID NO:22, 23, or 24 in which the SW, the QTRL amino acids (i.e., the first and second anchors), and the length of the linker (5aa) are maintained, and in which 1, 2, 3, 4, 5, 6, 7 of the other amino acids may be substituted, preferably by similar amino acids, provided that the residues at positions -8 and -5 from the C-terminus of the peptide are V and Q, respectively. More particularly, in said variants, 1, 2, 3, or 4 N-terminal amino acid(s) can be deleted from SEQ ID NO:22, 23, or 24 (cf. SEQ ID NO:25-27). In a further aspect, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the present invention also relates to a polypeptide containing the NVshort peptide as described above. This polypeptide will be designated as the “Neurovita polypeptide of the invention”.

[0233] In some embodiments, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the Neurovita polypeptide of the invention only contains the NVshort peptide of the invention, so that the Neurovita polypeptide of the invention consists in the NVshort peptide of the invention as described above. In a preferred embodiment, the Neurovita peptide of the invention does not correspond to a peptide of SEQ ID NO: 17, 18, or 19.

[0234] In other embodiments, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the Neurovita polypeptide of the invention contains the NVshort peptide of the invention, and also at least one other peptide(s), for example a Cell Penetrating Molecule (CPM). In this case, the Neurovita polypeptide of the invention can be also referred to as “fusion protein of the invention”.

[0235] In a particular embodiment, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the present invention thus provides a Neurovita polypeptide comprising or consisting of the NVshort peptide of sequence:

[0236] XnSWXVXXQQTRL (SEQ ID NO:21), wherein X is any amino acid and n is an integer comprised between 0 and 4 (i.e., n is 0, 1, 2, 3, or 4), and wherein the sequence of the Neurovita polypeptide is not SEQ ID NO: 17, 18, or 19.

[0237] As explained above, the NVshort variant contained in the Neurovita polypeptide of the invention should contain the four C-terminal amino acids residues of the cytoplasmic domain of the non-apoptotic rabies virus G protein, namely the PBM (having the sequence QTRL) and the SW (serine, tryptophan) residues that form the two anchors of the PBM to the MAST-2 PDZ, and the two anchors should be separated by 5 amino acids (Terrien et al, 2012, Khan et al, 2019). However, it is possible to remove 1, 2, 3 or 4 amino acids on their N-terminal side, as compared with SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:24, so that the NVshort variant contained in the Neurovita polypeptide of the invention eventually contains between 11-15 amino acids.

[0238] Thus, in a preferred embodiment, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the Neurovita polypeptide of the invention contains or consists in a NVshort peptide having the sequence SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28 corresponding respectively to the 14, 13, 12, 11 C-terminal amino acids of SEQ ID NO: 19, or variants thereof preferably displaying:

[0239] The sequence of said NVshort peptide corresponding to a sequence of 14 amino acids of SEQ ID NO:25 which can be replaced by any amino acid, preferably by similar amino acids, provided that i) the first and second anchors described above are not mutated, ii) the two anchors are separated by 5 amino acids, and iii) the residues at positions -8 and -5 from the C-terminus of the peptide are V and Q, respectively;

[0240] - The sequence of said NVshort peptide corresponding to a sequence of 13 amino acid of SEQ ID NO:26: which can be replaced by any amino acid, preferably by similar amino acids, provided that i) the first and second anchors described above are not mutated, ii) the two anchors are separated by 5 amino acids, and iii) the residues at positions -8 and -5 from the C-terminus of the peptide are V and Q, respectively;

[0241] The sequence of said NVshort peptide corresponding to a sequence of 12 amino acid of SEQ ID NO:27: which can be replaced by any amino acid, preferably by similar amino acids, provided that i) the first and second anchors described above are not mutated, ii) the two anchors are separated by 5 amino acids, and iii) the residues at positions -8 and -5 from the C-terminus of the peptide are V and Q, respectively;

[0242] The sequence of said NVshort peptide corresponding to a sequence of the 11 amino acid in C-terminal position SEQ ID NO:28: which can be replaced by any amino acid, preferably by similar amino acids, provided that i) the first and second anchors described above are not mutated, ii) the two anchors are separated by 5 amino acids, and iii) the residues at positions -8 and -5 from the C-terminus of the peptide are V and Q, respectively.

[0243] Preferably, the value of the pl of the molecule-once the NVshort peptide described above has been fused to a Cell Penetrating Molecule, in particular a VHH, is preserved. Thus, in a particular embodiment, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the Neurovita polypeptide of the invention contains or consists in a NVshort peptide variant whose sequence is the SEQ ID NO:22, 23, or 24, in which the SW and QTRL amino acids are maintained, and in which 1, 2, 3, 4, 5, 6, 7 of the other amino acids may be substituted, preferably by similar amino acids, the two anchors are separated by 5 amino acids, and the residues at positions -8 and -5 from the C-terminus of the peptide are V and Q, respectively. More particularly, in said variants, 1, 2, 3, or 4 N-terminal amino acid(s) can be deleted from SEQ ID NO:22, 23, or 24 (cf. SEQ ID NO:25-27).

[0244] In a particular embodiment, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the Neurovita polypeptide of the invention contains or consists in a NVshort peptide that has a sequence selected in the group consisting of SEQ ID NOs:22-28. Preferably, the Neurovita polypeptide of the invention contains or consists in a NVshort peptide that has SEQ ID NO:24

[0245] In a preferred embodiment, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the Neurovita polypeptide of the invention comprises a NVshort peptide and a CPM, preferably a VHH, more preferably VHHNPX, and has a basic isoelectric point (pl), allowing the molecule to cross the Blood Brain Barrier (BBB) and the cell membranes. A protein is said to have a basic pl when the latter is higher than 7, more preferably equal to or higher than 8 and even more preferably equal to or higher than 8.5 or equal to or higher than 9. Accordingly, the Neurovita polypeptide of the invention has preferably a pl equal to or higher than 8, more preferably equal to or higher than 8.5 and even more preferably equal to or higher than 9. Also, the Neurovita polypeptide of the invention has a pl equal to or lower than 11, more preferably equal to or lower than 10.5 and even more preferably equal to or lower than 10, in order to avoid non-specific binding to the target cells non-specifically. Preferred ranges for the pl of the Neurovita polypeptide of the invention are therefore of 8.5-11, 8.5-10, 9-10 and 9-10.5.

[0246] Fusion proteins [CPM- NVshort] In order to be conveyed into the cytoplasm of the cells where it is active, the Neurovita short peptide of the invention is preferably fused to a Cell Penetrating Molecule or to any other delivery system that has been described in the art, e.g. to bypass the Blood Brain Barrier (BBB) and convey cargo molecules into target cells.

[0247] This means that the polypeptides of the invention may contain, apart from the NVshort peptide as described above, any peptide or other delivery systems known in the art that may address said peptide toward and inside target cells such as neurons.

[0248] Therefore, in a particular embodiment, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the polypeptide of the invention contains the NVshort peptide as described above and a Cell Penetrating Molecule (CPM) that will mediate the vectorization of the NVshort peptide in the cytoplasm of target cells, where MAST1 / MAST2, the target proteins of the interfering peptide NV, are located. The polypeptide may further comprise a homing molecule.

[0249] In a particular embodiment, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the polypeptide of the invention comprising the NVshort peptide described herein and a CPM, and optionally a homing molecule, has a basic isoelectric point.

[0250] CPM=VHH

[0251] In a more particular embodiment, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the polypeptide of the invention contains the NVshort peptide as described above and a cam elid VHH antibody heavy chain as CPM.

[0252] The full sequence of the VHH is provided in WO2016 / 038122 and is put forth herein as SEQ ID NO:8. Its native CDRs (Complementary Determining Regions) are SEQ ID NO:6, 7 and 1 for CDR1-3 respectively:

[0253] VHH CDR1 : IDVINNMA (SEQ ID NO: 6)

[0254] VHH CDR2: TITSGFSTNY (SEQ ID NO: 7)

[0255] VHH CDR3: = KVHLIRLGAARAYDY (SEQ ID NO: 1) The polypeptide of the invention is preferably a fusion protein comprising or consisting of the polypeptide of sequence RIISSWEVHGQQTRL (SEQ ID NO:24) and a VHH CPM.

[0256] An optimized VHH, VHHNPX, is especially advantageous for the purposes of the present invention. In particular, VHHNPX when fused to the NV short polypeptide allows the fusion protein i) to be correctly folded, ii) to be produced in eukaryotic and prokaryotic cells, iii) to remain soluble in contact with physiological fluids, iv) to cross the cell membrane, and v) to deliver NVshort into the cytoplasm where it can reactivate intrinsic neuron survival and axon regeneration pathways by disrupting critical complexes formed between the PDZ of MAST2 / 1 kinases and their ligands.

[0257] In addition to safety considerations, the recognition by the VHH antibody of an additional (unidentified) antigen could decrease the efficacy of the molecular cargo and therefore lower its therapeutic activity. Because the nature of protein / epitope recognized by the VHH chain having not been identified (see Li et al, 2017), the inventors decided to inactivate antibody function of the VHH by changing several amino acids in the CDR3 domain. In a preferred embodiment, the inventors therefore propose to use VHH chain in which the sequence of the CDR3 has been mutated to inactivate any remaining antibody function of VHH and thereby avoid all unwanted cross reaction or off-target side effects once administered in vivo.

[0258] For example, it is possible to use a VHH in which the CDR3 sequence contains more than two mutated amino acids as compared with SEQ ID NO: 1.

[0259] The inventors tested five different variants of CDR3 for which the antigen recognition was abolished. Among these five variants, the best production of VHHNPx-NVshort was obtained when the VHHNPX comprises CDR3.4 (SEQ ID NO:2).

[0260] Thus, the inventors propose to bind the NVshort peptide of the invention with a VHH chain in which the sequence of the CDR3 is mutated into K VHL GGGGA AGAGGY (SEQ ID NO:2), or any variant thereof displaying at least 90% similarity with SEQ ID NO: 2 and containing more than two mismatches as compared with SEQ ID NO:1.

[0261] In a particular embodiment, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the VHH chain used in the Neurovita polypeptide of the invention is the mutated VHH displaying the sequence SEQ ID NO:3: EVQLQASGGGLAQPGGSLRLSVTVSGSIDVINNMAWYRQAPGNARELVATITSGFST NYASSVKGRFTISRDNAKKAVYLQMNSLKPEDTADYYSKVHLGGGGAAGAGGYW GQGTQVTVS

[0262] The present inventors also propose to mutate the sequence of the VHH chain so as to promote intrachain disulfide bridge formation, in order to ensure proper folding of the polypeptide during its production and no precipitation when it is in contact with physiological fluids such as vitreous humor. The insertion of an intramolecular disulfide bridge in the VHH sequence solves the solubility problems. The inventors observed that, when disulfide-bond forming cysteines are added in the sequence of the VHH antibody, the fusion protein containing the modified VHH sequence folds properly and, upon contact with the vitreous humor, does not precipitate any more (figure 5). Insertion of an intramolecular disulfide bridge in the VHH leads to a fusion protein that does not precipitate during its production process and can therefore be produced with high yields, and that does not form aggregates in vitreous humor (figure 5).

[0263] Thus, in another embodiment, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the VHH chain used in the polypeptides of the invention contains at least two disulfide-bond forming cysteines. More particularly, these disulfide-bond forming cysteines can be located in position 22 and 96, as reflected in SEQ ID NO:4.

[0264] In other terms, the VHH chain used in the polypeptides of the invention is preferably mutated to contain at least one disulfide bond to ensure proper folding and a CDR3 containing three or more mutations as compared with SEQ ID NO: 1, to inactivate the antibody function of said VHH camelid antibody.

[0265] In a preferred embodiment, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the VHH chain used in the polypeptides of the invention contains at least two disulfide-bond forming cysteines and a mutated CDR3 sequence as described above. In this particular embodiment, VHHNPX has for example the SEQ ID NO:5. The present disclosure also provides a variant of VHH-NPX, wherein the variant has a sequence displaying at least 90%, similarity with SEQ ID NO:5, the variant has the same pl as the VHH-NPX of SEQ ID NO:8, or of SEQ ID NO:5, preferably of sequence SEQ ID NO:5, and the variant comprises i) the CDRs of SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO: 1 as CDR1-3 respectively, and ii) at least one disulfide-bridge formed by two cysteines.

[0266] Said VHHNPX variant has preferably a basic pl comprised between 8.5-11, 8.5-10, 9-10 and 9- 10.5.

[0267] In this case, the Neurovita polypeptide of the invention can have the sequence displayed in SEQ ID NO:30 (also herein called NPX461).

[0268] CPM = L-TAT

[0269] In the context of the invention, it is also possible to associate the NVshort peptide as described above to the CPM called “L-TAT”, which is known to be the levogyre form (L) of the protein transduction domain of the HIV-1 derived trans-activator of transcription. This CPM has the sequence GRKKRRQRRR (SEQ ID NO: 16) and is different to the typical YGRKKRRQRRR (SEQ ID NO: 15) of the prior art in that it lacks the N-terminal Y amino acid residue (see EP1811033). Unlike the native L-TAT CPM of SEQ ID NO: 15, the L-TAT of SEQ ID NO: 16 does not address the molecular cargo to the cell nucleus.

[0270] The NVshort peptide of the invention can also be associated to a variant of SEQ ID NO: 16, whose sequence displays at least 90% similarity with SEQ ID NO: 16.

[0271] The L-TAT CPM used in the polypeptide of the invention has therefore preferably a sequence displaying at least 90% homology or similarity with SEQ ID NO: 16. It is preferably the L-TAT of SEQ ID NO: 16.

[0272] The sequence of the polypeptide of the invention can be for example as displayed on SEQ ID NO:32.

[0273] It is also possible to combine NVshort with a RDP (e.g., mutated as described below) acting as a homing molecule, and with a L-TAT CPM (e.g., mutated as described above) to target nAChR- expressing cells and to enter in these cells in the endocytic / lysosomal pathway by means of L- TAT.

[0274] Association with homing molecules

[0275] Optionally, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the Neurovita polypeptide of the invention can also contain a homing molecule that will make the polypeptide of the invention (containing NVshort and optionally the CPM) cross the Blood Brain Barrier and / or target or address this polypeptide toward cells of interest.

[0276] As used herein, a “homing molecule” is a molecule which is capable of addressing a cargo to a specific organ, tissue or cell. Homing molecules are known in the art (see e.g., Yu H. et al, Drug Delivery 2020, Vol.27, N°l, 1425-1437). A number of other delivery systems have been described in the art (Milletti F. Cell-penetrating peptides: classes, origin, and current landscape. Drug Discov Today. 2012 Aug;17(15-16):850-60; Zhao et al, Cell 181, 2020).

[0277] In another particular embodiment, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the homing molecule is the Rabies virus Derived Peptide (RDP). According to this specific embodiment, the polypeptide of the invention contains the NVshort peptide as described above and RDP.

[0278] RDP is the Rabies virus Derived Peptide derived from the G protein of the CVS-NIV sequence. It is able to carry molecular cargo into neuronal cells and increase the specificity of peptides toward nAChR expressing cells only (see WO2016 / 038122 when the cargo is the long Neurovita peptide).

[0279] Any RDP sequences can be used in this respect, in particular those recited page 43 of WO2016 / 038122 (see the SEQ ID NO: 1, SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34 of WO2016 / 038122). A particular sequence is for example given as SEQ ID NO: 13 in the enclosed sequence listing.

[0280] Preferably, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the RDP sequence comprises a mutation compared to SEQ ID NO: 13. More preferably, this mutation is a substitution, e.g., by a serine, of the cysteine residue at position 15 of the sequence of SEQ ID NO: 13. Indeed, this substitution prevents the aggregation of a Neurovita peptide-containing fusion protein (figure 9). Therefore, it is particularly advantageous to associate the Neurovita peptide of the invention with a mutated RDP, as described herein in order to avoid the protein aggregation induced by the domain and therefore to enhance the production yield and the biological activity of the Neurovita peptide. This is surprising because it was thought so far that the cysteine in neuron cell-targeting peptides derived from a Rabies virus G protein was required to maintain its biological activity (Lentz et al., 1984,1987 and 1989). Therefore, all the RDPs carrying Neurovita polypeptides in the past contained a cysteine residue (see in particular SEQ ID NO: 1, SEQ ID NO:32, SEQ ID NO:33 and SEQ ID NO:34 of WO2016 / 038122).

[0281] In a preferred embodiment, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the Neurovita short peptide of the invention is therefore fused to a RDP sequence that does not contain any cysteine residue, nor any amino acid that may create unwanted disulfide bridge(s). For example, the cysteine residue contained in the sequence of said RDP can be replaced by a serine residue.

[0282] The RDP used in the polypeptide of the invention has thus preferably a sequence displaying at least 90% similarity with SEQ ID NO: 13, and in which the only cysteine amino acid has been substituted by another amino acid. Of note, this homologous or similar sequence of RDP should be chosen so that the resulting peptide retains the loop structure of SEQ ID NO: 13, in order to favor anchoring to nAChR receptors at the surface of target cells.

[0283] In a more preferred embodiment, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the mutated RDP used in the polypeptides of the invention has the sequence YTIWMPENPRLGMSSDIFTNSRGKRASKG (SEQ ID NO: 14) or a variant thereof displaying at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity with SEQ ID NO:22, but not containing any cysteine residue. Of note, this variant sequence should be chosen so that the resulting peptide retains the loop structure of SEQ ID NO: 14, in order to favor anchoring to nAChR receptors at the surface of target cells.

[0284] Said RDP variant has preferably a basic pl comprised between 8.5-11, 8.5-10, 9-10, 9-10.5, and 10-11.

[0285] The sequence of the polypeptide of the invention can be for example as displayed on SEQ ID NO:33 (RDP-AAA- NVshort).

[0286] In another aspect, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the present invention also relates to the mutated RDP of SEQ ID NO: 14 or a variant thereof displaying at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity with SEQ ID NO: 14, but not containing any cysteine residue, on its own. It is nevertheless possible to combine NVshort with a RDP (e.g., mutated as described above) acting as a homing molecule and with a VHH CPM (e.g., mutated as described above) to target nAChR-expressing cells and to enter in these cells in non-endocytic / lysosomal pathway by means of the VHHNPX.

[0287] It is also possible to combine NVshort with a RDP (e.g., mutated as described above) acting as a homing molecule with a L-TAT CPM (e.g., mutated as described above) to target nAChR- expressing cells and to enter in these cells in the endocytic / lysosomal pathway by means of L- TAT.

[0288] To conclude, the polypeptide of the invention can contain the NVshort peptide as described above and a CPM such as: VHHNPX, SEQ ID NO:5 or L-TAT SEQ ID NO: 16, (as exemplified in figure 14) or any variant thereof disclosed above. The polypeptide of the invention can also contain more than one CPM / homing molecule, for example a combination of RDP and VHH or of RDP and L-TAT, as disclosed above.

[0289] Linkers

[0290] In case a CPM is included in the polypeptide of the invention, or in case two or more CPM / homing molecule are used, said CPM(s) and the NVshort peptide are preferably separated with a linker.

[0291] Advantageously, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, linkers are used between the NVshort peptide of the invention and the CPM / homing molecule associated thereto. Accordingly, the last N-terminal residue of the NVshort peptide can be linked by a peptide bond to the first C-terminal residue of a peptide linker, the last N-terminal residue of which being linked to the first C-terminal residue of the CPM / homing molecule. In this particular embodiment, a peptide linker consisting of one to ten amino acids, preferably of one to five amino acids, more preferably of one to three amino acids can be added between the CPM / homing molecule and the NVshort peptide, or between the two CPM / homing molecule. In a most preferred embodiment, the linker has the three amino acid sequence “AAA”. Polynucleotides of the invention

[0292] In another aspect, the present invention also targets the polynucleotides encoding the peptides, polypeptides including fusion proteins disclosed above. These polynucleotides are preferably integrated in vectors, in particular in expression vectors.

[0293] In another aspect, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the invention provides vectors comprising polynucleotides of the invention encoding the peptides, polypeptides including fusion proteins of the invention. In order to express them, these vectors are “expression vectors”, in which the sequences encoding the peptides, polypeptides and fusion proteins of the invention are operatively linked to transcriptional and translational sequences. These vectors may remain free (i.e., non-integrated into a host cell genome), or may become integrated ("stably” or “temporary” incorporated) into the host cell genome, either as a result of the original transformation of the host cells, or as the result of subsequent recombination and / or repair events. Still, they remain “heterologous” as compared to the host cell and its natural genome. As used therein, the term “operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

[0294] The invention therefore relates to a vector, preferably a viral vector, DNA, or mRNA, encoding the Neurovita polypeptide or the Neurovita short peptide of the invention, as defined above.

[0295] Pharmaceutical compositions

[0296] As shown in the examples below, the polypeptide of the invention NVshort, once it has been fused to VHHNPX, is soluble in vitreous humor, non-aggregated, able to enter cell, be localized in the cytoplasm where it can bind and block with high affinity the PDZ domain of the human MAST1 / MAST2 enzymes in cells.

[0297] In another aspect, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the invention relates to a pharmaceutical composition comprising the Neurovita polypeptides or the fusion proteins of the invention, or the polynucleotides encoding same, which are as disclosed above, and a pharmaceutically acceptable carrier. These pharmaceutical compositions comprising the Neurovita polypeptides of the invention, including the fusion proteins of the invention, or the polynucleotides encoding same, are hereafter called “pharmaceutical compositions of the invention”.

[0298] In these pharmaceutical compositions, the Neurovita polypeptides including fusion proteins of the invention or polynucleotides are preferably formulated in an effective amount.

[0299] In particular, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, when the composition of the invention is intended to be administered in the eye (see below), the preferred excipients are those authorized by the FDA for eye injection, namely: Calcium chloride, carboxymethylcellulose sodium, hyaluronate sodium, hydrochloric acid, magnesium chloride, magnesium stearate, microcrystalline cellulose, poly(dl-lactic-co-glycolic acid, poly(dl-lactic- co-glycolic acid, polysorbate 80, polyvinyl alcohol, potassium chloride, sodium acetate, sodium bicarbonate, sodium chloride, sodium hydroxide, sodium phosphate, trisodium citrate dihydrate.

[0300] Therapeutic uses

[0301] The polypeptides of the invention including the fusion proteins of the invention (as described above) are characterized by the fact that they are under a functional form, not only in vitro but also in vivo when injected to a subject in need thereof.

[0302] The polypeptides including fusion proteins of the invention should reach the brain after being injected intravenously, intrathecaly or intranasaly.

[0303] The polypeptides including fusion proteins of the invention as well as the Neurovita peptides of the invention are moreover preferably less immunogenic so as not to be toxic to the treated subject when administered in vivo. As a matter of fact, the short form of the Neurovita peptide and some of the mutations introduced in the CPMs lower the size of the resulting protein at its minimum, and impair the aggregation of the polypeptides, avoiding to trigger any unwanted side effects. Also, their stable solubility in physiological fluids ensure that they will not aggregate in vivo after injection. As shown in the examples below, the polypeptides including fusion proteins of the invention they target retina cells and persist in the mouse eye after injection, where they can promote neurosurvival and / or neuroprotection and allow the repair of neuron cells after an axon injury.

[0304] In one aspect, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the invention therefore relates to the NVshort peptide or to the polypeptides and fusion proteins of the invention including the polynucleotides encoding same, for their use as a medicament. Also, the invention relates to the pharmaceutical composition of the invention, containing said NVshort peptides or said polypeptides including fusion proteins, for its use as a medicament.

[0305] In one particular aspect, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the present invention targets polypeptides including fusion proteins as defined above, or the polynucleotides encoding same, or pharmaceutical compositions comprising same, in order to prevent and / or treat diseases involving the nervous system (central nervous system and / or peripheral nervous system), such as neurodegenerative diseases, muscular dystrophies, or cell damage-associated conditions such as stroke or injury including optic nerve diseases, retina diseases, muscular diseases, motor neuron diseases, or the evolution of such disease or condition. The invention also provides therapeutic methods using the NVshort peptides of the invention, the polypeptides including fusion proteins containing same, the polynucleotides encoding same, or pharmaceutical compositions comprising same, for use for preventing and / or treating neurodegenerative diseases or cell damage-associated conditions, preferably affecting the brain. The invention also provides the NVshort peptides of the invention, the polypeptides including fusion proteins containing same, the polynucleotides encoding same, or pharmaceutical composition comprising same, for use in the manufacture of a medicament, especially a medicament for use in the therapy of neurodegenerative diseases or cell damage- associated conditions, preferably affecting the brain or the muscle. This therapy comprises advantageously the intravenous, intravitreal, intranasal injections or any other routes of injection, appropriate for the target disease, of the NVshort peptides of the invention, the polypeptides including fusion proteins containing same, the polynucleotides encoding same, or pharmaceutical compositions comprising same, in patients in need thereof.

[0306] In another particular aspect, and in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, the present invention targets the use of the NVshort peptides, polypeptides including fusion proteins of the invention, the polynucleotides encoding same, or pharmaceutical compositions comprising same, in order to prevent and / or treat diseases involving the nervous system (central or peripheral), such as neurodegenerative diseases, or cell damage-associated conditions such as stroke or injury including optic nerve disease, retina diseases, motor neuron disease, or muscular dystrophies, such as Duchenne disease or the evolution of such disease or condition. The invention also provides therapeutic methods using the NVshort peptides, polypeptides including fusion proteins of the invention, the polynucleotides encoding same, or pharmaceutical compositions comprising same, for use for preventing and / or treating neurodegenerative diseases or cell damage-associated conditions, preferably affecting the nervous system or the muscles. The invention also provides the NVshort peptides, polypeptides including fusion protein of the invention, the polynucleotides encoding same, or pharmaceutical compositions comprising same, for use in the manufacture of a medicament, especially a medicament for use in the therapy of neurodegenerative diseases or brain cell damage- associated conditions. This therapy comprises advantageously the most appropriate route of injection (such as intravitreal injection, intrathecal injection, intra-meningeal, intranasal or intravenous injection) of the polypeptides of the invention, the polynucleotides encoding same, or pharmaceutical compositions comprising same, in patients in need thereof.

[0307] The Neurovita peptides of the invention, the polypeptides including fusion proteins of the invention, the polynucleotides encoding same, or pharmaceutical compositions comprising same are preferably, in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, for use as a neuro- regenerative (regrowth of injured axons, restoration of synapses) and / or neuro-protective agent (protection of neurons and other cells expressing the target molecules MAST2 / MAST1 from apoptosis or decrease of viability).

[0308] The Neurovita peptides of the invention, the polypeptides including fusion proteins of the invention, the polynucleotides encoding same, or pharmaceutical compositions comprising same are preferably for use in the treatment and / or palliation and / or prevention of a disease, disorder or condition, which alters the Central Nervous System (CNS) and / or the Peripheral Nervous System (PNS), for example as a preventive therapy and / or palliation.

[0309] The Nervous System" as used herein, is composed of the Central Nervous System "CNS" and the Peripheral Nervous System (PNS). According to an embodiment of the invention, in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, said disease or disorder is or involves a neurodegenerative disease or disorder (for example, a chronic neurodegenerative disease or disorder), such as Alzheimer's disease, Parkinson's disease, ALS, Huntington disease, multiple sclerosis (MS) or rare genetic disease. According to an embodiment of the invention, said condition is or involves a neurodegenerative condition, such as aging. According to an embodiment of the invention, said disease or condition involving the CNS or the CNS is an optic neuropathy (e.g. glaucoma, NAION).

[0310] According to an embodiment of the invention, in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, said disease, disorder or condition is or involves a physical or ischemic injury of the nervous system, such as seizure, stroke, trauma, epilepsy.

[0311] According to an embodiment of the invention, said disease, disorder or condition involves the presence of a chemical neurotoxic agent and / or of an oxidative stress.

[0312] Host cells of the invention

[0313] For the VHH-NV proteins which large size exclude their production by chemical synthesis, the polynucleotides of the invention and vectors comprising same are used in vitro for the transformation of suitable host cells which are also encompassed by the present invention. The term "host cell", as used herein, is intended to refer to a cell into which a recombinant expression vector has been introduced to express the peptides, or polypeptides including fusion proteins of the invention.

[0314] The invention, in particular insofar as it relates to a Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof, therefore also relates to host cells, in particular eukaryotic and prokaryotic cells, containing the vector of the invention or the polynucleotides disclosed above. These cells are used to produce the peptides, or polypeptides including fusion proteins of the invention. Among the host cells which can be used in this respect, mention may of course be made of microbial cells, but also yeast cells, as well as animal cells, in particular mammalian cells, and insect cells. Preferably, said host cell is a prokaryote, for example, E. co / i, or a eukaryote, for example, a single-celled eukaryote (e.g., a yeast or other fungus), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell). Examples of host cells include the COS-7 line of monkey kidney cells (ATCC CRL 1651), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines which grow in serum- free media or CHO strain DX-B11, which is deficient in DHFR, HeLa cells, BHK (ATCC CRL 10) cell lines, the CV1ZEBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70), human embryonic kidney cells such as 293, 293 EBNA or MSR 293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK or Jurkat cells. Typically, a host cell is a cultured cell that can be transfected with a polypeptide-encoding nucleic acid, which can then be expressed in the host cell.

[0315] These host cells have been transfected or transformed with the polynucleotides encoding the sequences of the peptides, or polypeptides including fusion proteins of the invention, so as to eventually express them at high yield.

[0316] Transformation can be performed by any known method for introducing polynucleotides into a host cell. Such methods are well known of the man skilled in the art and include dextran- mediated transformation, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide into liposomes, biolistic injection and direct microinjection of DNAinto nuclei.

[0317] For long-term, high-yield production of recombinant peptides or polypeptides, including fusion proteins, stable expression is preferred. In one embodiment of the invention, cell lines which stably express the peptides or polypeptides, including fusion proteins, of the invention may be engineered. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with DNA under the control of the appropriate expression regulatory elements, including promoters, enhancers, transcription terminators, polyadenylation sites, and other appropriate sequences known to the person skilled in art, and a selectable marker. Following the introduction of the foreign DNA, engineered cells may be allowed to grow for one to two days in an enriched media, and then are moved to a selective media. The selectable marker on the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into a chromosome and be expanded into a cell line. Other methods for constructing stable cell lines are known in the art. In particular, methods for site-specific integration have been developed. According to these methods, the transformed DNA under the control of the appropriate expression regulatory elements, including promoters, enhancers, transcription terminators, polyadenylation sites, and other appropriate sequences is integrated in the host cell genome at a specific target site which has previously been cleaved (US 5,792,632; US 5,830,729; US 6,238,924; WO 2009 / 054985; WO 03 / 025183; WO 2004 / 067753). A number of selection systems may be used, including but not limited to the Herpes simplex virus thymidine kinase, hypoxanthine-guanine phosphoribosyltransferase, glutamate synthase selection in the presence of methionine sulfoximide and adenine phosphoribosyltransferase genes in tk, hgprt or aprt cells, respectively. Also, antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate; gpt, which confers resistance to mycophenolic acid; neo, which confers resistance to the aminoglycoside, G-418; and hygro, which confers resistance to hygromycin. Methods known in the art of recombinant DNA technology may be routinely applied to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons (2003). The expression levels of the peptides or polypeptides, including fusion proteins, of the invention can be increased by vector amplification. When a marker in the vector system is amplifiable, an increase in the level of inhibitor present in the culture will increase the number of copies of the marker gene. Since the amplified region is associated with the sequence encoding the peptide or polypeptide, including fusion protein, of the invention, production of said peptide or polypeptide, including fusion protein, will also increase. Alternative methods of expressing the polynucleotide of the invention exist and are known to the person of skills in the art. For example, a modified zinc finger protein can be engineered that is capable of binding the expression regulatory elements upstream of the nucleic sequence of the invention; expression of the said engineered zinc finger protein (ZFN) in the host cell of the invention leads to increases in peptide or polypeptide, including fusion protein, production. Moreover, ZFN can stimulate the integration of a DNA into a predetermined genomic location, resulting in high- efficiency site-specific gene addition.

[0318] Methods for introducing vectors into host cells are well known to the ordinarily skilled artisan. The host cells are first transformed or transfected with expression vectors or cassettes as those described above, by conventional means (e.g., electroporation). Then peptide or polypeptide, including fusion protein, production is induced by culturing the cells in conventional nutrient media that has been chosen or modified so as to induce promoters, select and / or maintain transformants, and / or express the nucleic sequences encoding the desired peptide or polypeptide, including fusion protein, sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: A Practical Approach, M. Butler, ed. (IRL Press, 19 1) and Molecular Cloning: A Laboratory Manual (Michael R. Green and Joseph Sambrook, 2012, Cold Spring Harbor Laboratory Press).

[0319] Particular transfer methods, such as phages, plasmids, and transposons, can be used to deliver and circulate engineered DNA sequences to microbial communities, via processes such as transduction, transformation, and conjugation. An engineered phage could be one possible delivery system for a polypeptide of the invention, by incorporating the nucleic acid encoding said polypeptide into the phage and utilizing the phage to deliver the nucleic acid to a host microbe that would then produce the polypeptide after having the phage deliver the nucleic acid into its genome.

[0320] One could also utilize a transposon delivery system to incorporate nucleic acids encoding a therapeutic polypeptide into a host microbe that is resident in a patient's microbiome (Sheth, et al., Trends in Genetics, 2016, Vol. 32, Issue 4, pgs, 189-200). In this case, the host cell of the invention is therefore a bacterium that is part of the microbiome.

[0321] Production method of the invention

[0322] The peptides or polypeptides, including fusion proteins, of the invention may be prepared by growing a culture of the transformed host cells under culture conditions necessary to express the desired peptides or polypeptides, including fusion proteins. The resulting expressed peptides or polypeptides, including fusion proteins, may then be purified from the culture medium or cell extracts. Soluble forms of the peptides or polypeptides, including fusion proteins, of the invention can be recovered from cell lysates or from the culture supernatant. In a further aspect, the present invention also targets a method for producing the Neurovita peptide or the polypeptides, including fusion proteins, of the invention.

[0323] The method of the invention is thus characterized in that it comprises the following steps: a) growing a host cell of the invention containing a vector encoding the polypeptides, including fusion proteins, or the Neurovita peptide of the invention in an appropriate culture medium and b) recovering or purifying said polypeptide, including fusion protein, or peptide.

[0324] Processes for recovering or purifying the polypeptide, including fusion protein, or peptide of the invention are known to those skilled in the art. For example, said recombinant polypeptide or peptide can be purified from cell lysates, cell extracts and / or from the culture medium supernatant, with methods such as fractionation, chromatography methods, with immunoaffinity techniques using specific monoclonal or polyclonal antibodies, centrifugation, differential solubility or by any other standard technique for the purification of proteins or peptides. Suitable methods of purification will be apparent to a person of ordinary skills in the art.

[0325] Some purification technologies are not recommended by Regulatory Safety agencies (e.g., those relying on nickel chromatography columns), so that it is herein preferred to use alternative techniques that do not use any toxic material. Also, it is preferred to avoid purification methods implying to add His Tag to the produced peptide or polypeptide, as this tag may increase the immunogenicity of the peptide or polypeptide. For these reasons, it is preferred to use for example a Ion Exchange column (IEX) purification technique (Boysen RI et al, Current protocols in Molecular Biology, 2001), which avoids using His-Tag and is authorized by the Regulatory Safety Agencies.

[0326] The present inventors have shown that using IEX purification technique enables to obtain adequate yields of the polypeptides of the invention (the downstream yield was in the range of 25mg of the proteins of interest per Liter of bacterial culture). Implementation of IEX facilitates the downstream purification steps and improve the acceptability of the CMC protocol by the Regulatory Safety Agencies.

[0327] Of note, the polypeptides, including fusion proteins, in particular when the CPM is TAT, and Neurovita peptides of the invention can also be obtained by chemical synthesis, by using any one of the many known synthesis processes, for example those involving partial solid phases, condensation of fragments or conventional synthesis in solution.

[0328] EXAMPLES

[0329] Example 1:VHH suitable to vectorize molecules intracellularly

[0330] 1. Material and Methods

[0331] Production of VHHNPX-NV, L-TAT-NV, RDPc-NVshort (ML49C) and RDPs-NV (ML49S).

[0332] The construct HIS-Sumo- VHHNPX-NV short, allowing the production of NPX461 (soluble VHH-NV short in which the disulfide bridge was restored and the native CDR3 was replaced by CDR3.4 of SEQ ID NO:2), was produced in T7 Shuffle Express E. coli. strain. After bacteria lysis and centrifugation, the soluble fraction was purified on affinity chromatography on nickel resin. Cleavage of HIS-Sumo tag was obtained with ULP1 protease at 1 : 10 protease / POI (W / v) ratio. Separation of Sumo tag was obtained by loading the digestion mixture on HIS Trap HP columns in the presence of imidazole in PBS buffer, pH 7.4. Then, Size Exclusion Chromatography (SEC) was performed, and the collected fractions were pooled and concentrated by Vivaspin concentrators and dialyzed in PBS buffer pH 7.5 (137mM NaCl, 2.7mM KCL, 8mM Na2HPO4, 2mM KH2PO4).

[0333] Alternatively, VHHNPx-NVshort was produced in E. coli. BL21XT7 with a signal peptide PelB to facilitate production in the periplasm. Purification of the production was performed on Amsphere A3 Protein A resin (JSR Light science). Endotoxins were captured on Ion exchange resin (Capto S, Cytiva) and Toxin Eraser resin (Genscript). The product was concentrated using a Vivaspin device and dialyzed in PBS buffer pH 7.5 (137mM NaCl, 2.7mM KCL, 8mM Na2HPO4, 2mM KH2PO4).

[0334] Alternatively, VHHNPx-NVshort was produced in Chinese Hamster Ovarian (CHO) cells by QMCF Technology. Cell culture supernatant was purified by Protein A affinity chromatography with Amsphere A3 column, and HPLC-SEC analysis. L-TAT-NVshort, RDPc-NVshort and RDPs-NV short were chemically synthesized (96-98% pure) in Acetonitrile (ACN / H20=l / 3). Quality control was performed with MS and HPLC. Lyophilized peptides were suspended either in PBS pH 7.5 for LTAT-NV or MES Buffer pH 6 (20mM, pH6 300mM NaCl), 7.5% DMSO for RDPc-NVshort and RDPs-NVshort production.

[0335] Live-cell imaging of Rhodamine-conjugated CPM association with lysosomes (lysotracker) in human retina ARPE19 cells. VHHNPX was conjugated to carboxyrhodamine using the NHS-Rhodamine Antibody labelling Kit, Pierce Biotechnology, USA. Carboxyrhodamine-NH2-conjugated L-TAT-NV was produced by Phoenix-France. 24h cultures of human retina pigmentated epithelial cell line ARPE19cells, ATCC CRL-2302, (20 000 cells per 8 chambers-Labteck well) were treated for Ihour with 75 nM of Lysotracker (marker of lysosomes) mixed with lOpM Rhodamine-conjugated VHHNPX-NV (VHH-NV- Rho) or with lOpM Rhodamine-conjugated-TAT-NV (LTAT-NV-Rho). After washes and addition of Hoechst to stain nuclei, imaging was performed using a confocal microscope (BC43 Oxford Instruments spinning disk confocal microscope) equipped with a thermo- and CO2- static chamber.

[0336] Imaging of CPM-NV association with lysosomes (LAMP1) in fixed human retina ARPE19 cells. 24h cultures of ARPE19 cells, ATCC CRL-2302 (10 000 cells per well of 16 chambers- Labteck) were treated for Ihour with 3 nmol of VHHNPX-NV or with 3 nmol of L-TAT-NV. After washes and fixation in 4%PFA, cells were stained with NV-specific antibody (SYCO22, guinea- pig antibody, 1 / 10 000) and Lysosomal-Associated-Membrane-Protein 1 (LAMP1) specific mouse mAb, (Cell Signalling #15665, l / 100) for Ihour at 37C. After washes, slides were treated with secondary antibodies [goat anti-guinea pig IgG- Alexa fluor 555 (#A21435, Invitrogen, 1 / 1000), and donkey anti-mouse IgG-Alexa fluor 488, (#A21202, Invitrogen 1 / 1000)]. Slides were mounted with Fluoromount G containing Dapi. Images were made on a LSM Zeiss confocal microscopy and analyzed with Image J. Manders split coefficients were calculated using LSM software.

[0337] Detection of NV by Western Blotting (WB) after delivery into human retinal cells ARPE 19 by L-TAT or VHHNPX. 24h cultures of ARPE19 cells, ATCC CRL-2302 (450 000 cells per well of 12 well-culture plate) were treated for Ihour with 15nmol of VHHNPX-NV, with 15nmol of L-TAT-NV or with vehicles (PBS for VHHNPX-NV, or MES buffer for L-TAT-NV). After washes, cells were lysed with RIPA buffer. Protein concentrations were measured using Mi croBC A technique. Gels (Nupage Bis-Tris 4-12%) were loaded with 20pg of cell lysates. The protein bands were blotted onto PVDF Immobilon-FL membranes (Millipore France #IPFL00010). Membranes were blocked with 4% non-fat dry milk in PBST buffer (PBS with 0.1% Tween 20) and incubated overnight at 4°C with primary antibodies in blocking buffer containing 0.1% Tween 20 (SIGMAUSA#P1379). The primary antibodies were an anti-RABV G-cyto domain guinea pig antibody (SYC022, 1 / 5 000 a custom antibody produced by Eurogentec), and an actin-specific mouse antibody (Sigma AC 40, 1 / 10 000). The membranes were washed with 0.1% Tween 20 in PBS and the antigen / antibody complex was detected with a horseradish peroxidase (HRP)-conjugated secondary antibodies, an HRP-conjugated antiguinea pig IgG antibody (Novex, USA, #A18775, 1 / 10 000) and an HRP-anti mouse IgG (Amersham #NA 9310, 1 / 10 000). Membranes were washed with PBST and visualized with the ECL western blot kit (Thermo Fisher Scientific France #35055). Chemiluminescence signals were snap-shot at different exposure times with a G-Box (SYNGENE USA).

[0338] Comparison of PI3K / AKT signaling pathway activation by NV when delivered by VHHNPX or by L-TAT into human retinal cells ARPE19.

[0339] 72h cultures of ARPE19cells (ATCC, CRL-2302) (500 000 cells per well of 6-wells-culture plates) were treated for 30min with different concentrations (3.6nM to 5pMol) of VHHNPX-NV or of L-TAT-NV or treated with vehicle (PBS for VHHNPX-NV and Mes buffer for L-TAT-NV). After washes, cells were lysed in RIPA buffer. Protein contents were measured using Micro BC A technique. Gels (Nupage Bis-Tris 4-12%) were loaded with 20pg of lysates and run by electrophoresis. The protein bands were blotted onto PVDF Immobilon-FL membranes (Millipore France #IPFL00010). Membranes were blocked with 4% non-fat dry milk in PBST buffer (PBS with 0.1% Tween 20) and incubated overnight at 4°C with primary antibodies in blocking buffer containing 0.1% Tween 20 (SIGMA USA #P1379). The primary antibodies were actin-specific mouse antibody (Sigma #AC 40, 1 / 10 000), pan AKT-specific IgGl mouse mAb (Cell Signalling #2920, 1 / 1000) or Serine 473 Phosphorylated AKT (PhosphoAKT) specific rabbit antibody (Cell Signalling #9271, 1 / 10 000) followed by secondary antibodies (HRP-conjugated antibodies directed against rabbit IgG (Amersham NA 934, 1 / 10 000) or mouse IgG (Amersham #NA 9310, 1 / 10 000). Actin was used as a gel loading control. Membranes were revealed with Thermo Scientific™ SuperSignal West ECL Femto. Chemiluminescence signals were detected at different exposure times with a G-Box (SYNGENE, USA). Images were analyzed with Imaged. Proportion of AKT which was phosphorylated (ratio PhosphoAKT / AKT) was standardized on signal obtained in vehicle- treated cultures (to which a value of “1” was attributed). Results are expressed as doseresponses curves.

[0340] In vitro toxicity assay in human ARPE19 retinal cells.

[0341] Toxicity of CPM-NV for ARPE19 (ATCC, CRL-2302) was tested in a 72h (3days) in vitro assay. ARPE19 cells (10 000 per well) were seeded on black 96-well plate, into 150 pl of DMEM / F12 + Pen-strep dlOOe + 1% fetal calf serum, at 37°C with 5% CO2. An internal control (non-treated cells) and a positive control triggering toxicity (H2O2 treatment) were included. After 24h, cells were treated with serial dilutions of NPX461 (1, 10, 20 and 30nmol), L-TAT- NV (1, 2.5 and 5nmol) and corresponding volume of DPBS 0.25x (vehicle for VHHNPX-NV) or MES buffer (vehicle for L-TAT-NV) for 3 days. Cell viability was measured with Alamar blue reagent (Invitrogen #DAL1100). Cells were washed 3 times with 200 pl of IxDPBS and 100 pl of 1 / 10thvolume of Alamar Blue into culture medium was added to measure the viability of ARPE19 cells. After a 3h incubation in the dark, fluorescence was recorded using an excitation wavelength of 570 nm and an emission reading at 600 nm. The average fluorescence values of the cell culture medium alone (background) was subtracted from the fluorescence values of experimental wells. The values of treated cells were then compared to the values of vehicle- treated cells (viability in % compared to control vehicle). Statistical analysis was conducted with Prism GraphPad.

[0342] Incidence of disulfide bridge insertion in VHH sequence of VHH-NV on the in vitro formation of aggregates upon contact with rabbit vitreous humor.

[0343] An in vitro test performed on 384 well plates was set up to test the solubility of VHHNPX-NV with disulfide bridge (WHH with DS) and VHH without disulfide bridge (VHH w / o DS) upon contact with rabbit vitreous humor. Three concentrations were tested (9, 18 and 36pM) Each compound (2 pl) was added to 5 pl of rabbit vitreous. After Ih incubation at 37°C, presence of aggregates was detected and imaged with the camera of the DLS (Dynamic Light scattering) station.

[0344] In vivo long-lasting detection of CPM-NV in mouse retina after an intravitreal injection of different concentrations of CPM-NV. CPM-NVs [1, 10, 50 and 250 pmol of L-TAT- NV or VHHNPX-NV (NPX461) in 1 pl] were injected intravitreally (IVT) in the eyes of C57B6 JR mice. Each CPM-NV concentration was tested in 3 eyes. The 4theye was injected with vehicle (PBS). Six hours after injection, mice were sacrificed. Mice were anesthetized with 250 pl of xylazine / ketamine injected in peritoneum. An intra-cardiac perfusion of PBS and then 4% PFA(PBS, 35ml at 12ml / min - 4% PFA, 50ml at 12ml / min) was performed to fix tissues. Mice heads were incubated over-night in 4% PFA at 4°C. Retina were dissected. Flat mount retinae were co-stained with SYCO22, 1 / 500, a NV-specific guinea pig antibody directed against the C-terminal of RAB V envelope protein G Cyto domain, and with a rabbit antibody (1 / 250) directed against RBPMS (RNA binding protein with multiple splicing) a specific marker of RGC (#1830, Phosphosolutions). The secondary species IgG specific antibodies were the goat anti-Rabbit - Alexa fluor 488 (#A11070, Thermo Fisher), 1 / 500, and the goat anti-Guinea pig - Alexa fluor 555 (#A21435, Thermo Fisher), 1 / 500. Retinae were mounted with Fluoromount G containing Dapi. Imaged on a LSM Zeiss confocal microscopy and analyzed with Image J. Intensity of fluorescence in retina was scored manually (scores 0.5-2). Results were presented as Fluorescence NV-intensity in retina 6 hours post IVT. The calculated half-maximal Fluorescence intensity (IC50) was calculated with Graph Pad.

[0345] Remanence of CPM-NV in mouse retina. Measure by mass spec analysis

[0346] For each CPM-NV protein, a group of twelve 8-week-old C57BL6 JR mice received an IVT injection of 4pg in a volume of 2pL. Groups of 3 mice were sacrificed 3, 6, 24 or 48h after IVT injection. Retina were lysed individually in RIPA and stored at -80°C before Mass Spec processing. Non-treated mouse retina and peptides in solution were provided as controls. A) Protein Digestion Protein samples were diluted in 1 M urea, 100 mM Tris HC1 pH 8.5. Samples were reduced using lOmM TCEP for 30 min at room temperature. Alkylation of the reduced disulfide bridges was performed using 45mM iodoacetamide for 30 min at RT in the dark. Proteins were then digested in two steps, first with r-LysC (Promega) at a protein: enzyme ratio of 40:1 for 4 h and then with modified sequencing grade trypsin (Promeg -V5111) at a protein: enzyme ratio of 40:1 overnight. Proteolysis was stopped by adding formic acid (FA, Fluka - 94318) at a final concentration of 5%. The resulting peptides were cleaned using Assay MAP Cl 8 cartridges on the Assay MAP Bravo platform (Agilent) according to the manufacturer's instructions. Peptides were concentrated to dryness and resuspended in 2% acetonitrile (ACN) / 0.1% FA just prior to LC-MS injection. B) LC-MS / MS acquisition. LC- MS / MS analysis was performed on a Q Exactive™ Plus Mass Spectrometer (Thermo Fisher Scientific) coupled with a Proxeon EASY-nLC 1200 (Thermo Fisher Scientific). One pg of peptides was injected onto a home-made 45 cm C18 column (1.9 pm particles, 100 A pore size, ReproSil-Pur Basic Cl 8, Dr. Maisch GmbH, Ammerbuch-Entringen, Germany). Column equilibration and peptide loading were done at 900 bars in buffer A (0.1% FA). Peptides were separated with a multi-step gradient from 3 to 6 % buffer B (80% ACN, 0.1% FA) in 5 min, 6 to 31 % buffer B in 130 min, 31 to 62 % buffer B in 30 min at a flow rate of 250 nL / min. Column temperature was set to 60°C. MS data were acquired using Xcalibur software using a data-dependent method. MS scans were acquired at a resolution of 70 000 and MS / MS scans (fixed first mass 100 m / z) at a resolution of 17,500. The AGC target and maximum injection time for the survey scans and the MS / MS scans were set to 3E6, 20ms and IE6, 60ms respectively. An automatic selection of the 10 most intense precursor ions was activated (Top 10) with a 30 s dynamic exclusion. The isolation window was set to 1.6 m / z and normalized collision energy fixed to 27 for HCD fragmentation. An underfill ratio of 1.0 % was used, corresponding to an intensity threshold of 1.7E5. Unassigned precursor ion charge states as well as 1, 7, 8 and >8 charged states were rejected and peptide match was disabled. C) Analysis. Acquired Raw data were analyzed using MaxQuant software version 1.6.6.0 using the Andromeda search engine. The MS / MS spectra were searched against the mouse database (Mus musculus Uniprot reference proteome 55493 entries) concatenated with the 2 Polypeptides of interest. All searches were performed with oxidation of methionine and protein N-terminal acetylation as variable modifications and cysteine carbamidomethylation as fixed modification. Trypsin was selected as protease allowing for up to two missed cleavages. The minimum peptide length was set to 5 amino acids and the peptide mass was limited to a maximum of 8000 Da. The false discovery rate (FDR) for peptide and protein identification was set to 0.01. The main search peptide tolerance was set to 4.5 ppm and to 20 ppm for the MS / MS match tolerance. Second peptides were enabled to identify co-fragmentation events and match between runs option selected with a match time window of 0.7 min over an alignment time window of 20 min. LFQ was enabled with default settings. Matching between runs of MSI features was enabled to improve the identification of Polypeptide 1 and 3 across all raw files.

[0347] In vivo retina and RGC targeting by CPM-NV after an intravitreal injection.

[0348] CPM-NV (10, 50, 125 or 250 pmol of NPX461 or NPX452 in Ipl) were injected intravitreally in the eye of C57B6 JR mice. Each dilution was tested in 3 eyes. The 4theye was injected with vehicle (PBS). After 6h, mice were anesthetized with 250 pl of xylazine / ketamine injected in peritoneum. An intra-cardiac perfusion of PBS and then 4% PFA(PBS, 35ml at 12ml / min - 4% PFA, 50ml at 12ml / min) was performed to fix tissues. Mice heads were incubated overnight in 4% PFA at 4°C. Retina were dissected. Flat mount retinae were co-stained with SYCO22, 1 / 500, a NV-specific guinea pig antibody directed against the C-terminal of RABV envelope G protein Cyto domain, and with a rabbit antibody (1 / 250) directed against RBPMS) a specific marker of RGC (#1830, RBPMS Phosphosolutions). The secondary species IgG specific antibodies were the goat anti-Rabbit - Alexa fluor 488 (Al 1070, Thermo Fisher), 1 / 500, and the goat antiGuinea pig - Alexa fluor 555 (A21435, Thermo Fisher), 1 / 500. Retina were mounted with Fluoromount G containing Dapi. Images were made on a LSM Zeiss confocal microscopy and analyzed with Image J. Intensity of fluorescence in retina was scored manually (scores 0.5-2). Results were presented as Fluorescence Neurovita Intensity in retina 6 hours post injection. Half-maximal Intensity of fluorescence (IC50) was calculated with Graph Pad.

[0349] Number of RGCs (RBPMS positive cells in green) having incorporated CPM-Neurovita (SYC022 positive cells in red) were counted. Results were expressed as proportion of RGCs labelled by SYC022 antibody (%).

[0350] In vivo neuroprotection and regeneration assay in the experimental mouse model of optic nerve compression.

[0351] The neuroprotective and neuroregenerative properties of NPX461 were studied in a mouse model of optic neuropathy, the optic nerve compression (ONC) model. The right optic nerve was crushed less than Ih before injection of NPX461 (5.6 pg) or BDNF (positive control) by the intravitreal (IVT) route. Negative control corresponds to mock injection or vehicle (PBS) injection. Mice were kept 14 days under regular supervision. At day 14, 2pg of CTB-555 (Cholera Toxin subunit B, Alexa Fluor™ 555 Conjugate, Fisher Scientific 11554267, resuspended at 2pg / pL in DPBS1X Ca2+- / Mg2+-) was injected in the right eye. CTB can penetrate RGCs and be transported in their axons forming the optic nerve, allowing axon regrowth visualization by confocal microscopy. Two days later, (at day 16) mice were terminally anesthetized. An intra-cardiac perfusion of PBS and then 4% PFA (PBS, 35ml at 12ml / min - 4% PFA, 50ml at 12ml / min) was performed to fix tissues. Eyes were harvested and dissected to isolate the retina and the optic nerves.

[0352] Flat mounted retinae were prepared and immunostained with a rabbit antibody (1 / 250) directed against RBPMS (#1830, Phosphosolutions), followed by a goat anti-Rabbit - Alexa fluor 488 (#A11070, Thermo Fisher, 1 / 500). Pictures of immunostained retina were taken in the periphery (8 pictures) and the mid-periphery (4 pictures) of upper and lower retina using a confocal Cell Voyager 1000 microscope (Yokagawa) and a lOx air objective. The RGC counting was performed using the Trackmate plugin on Fiji software. The number of RGCs was expressed in RGC / mm2and in percentage as compared to control (non-crushed optic nerve =left retina) respectively in each area (periphery / mid-periphery and upper / lower retina). Three retinae of the left eye served as control. Statistical analyses were performed using Graph Pad Prism version 9. Data were expressed as mean ± SEM. Data were analyzed by a Two-way ANOVA with a Tukey’s multiple comparison post hoc test.

[0353] Optic nerves were mounted on slides for confocal microscopy. The optic nerves highlighted by the CTB-555 were acquired using a confocal Nikon Ti2E microscope and a 20x air objective by tile scanning (from the crush site to the longest axon) and Z-stacking (3pm intervals). A Maximum Intensity Projection was done with the Z-stack images. The optic nerve regeneration was evaluated by the number of fibers measured on Icy software (de Chaumont, F. et al., 2012). On Icy, 10 rectangles of 100pm were drawn from the crush site. One rectangle is placed before the crush to measure the fluorescence before the crush and another one was placed at the end of the picture to measure the background. The CTB mean intensity was automatically measured by Icy in each rectangle, whereas the number of fibers was manually counted. All statistical analysis was performed using Graph Pad Prism version 9. Data were expressed as mean ± SEM. The number of fibers was analyzed by a Two-way ANOVA with a Tukey’s multiple comparison post hoc test.

[0354] Ex vivo axon regeneration assay on mouse retina explants (Figure 17).

[0355] Retinae were dissected and cut into square pieces (1 mm2) to form retina explants. The retina explants were set up on coverslip previously coated with poly-lysine and laminin, with the inner RGCs layer facing down. CPM-NV (here 1 500 ng of VHHNPx-NVshort, NPX461) or a cocktail of ciliary Neurotrophic factor, CNTF, Brain derived neurotrophic factor, BDNF and Forskolin, (positive control) were added to explants at day 1. A sham condition consists in addition of vehicle alone (here PBS). Treatment was performed a single treatment at day 1. Numbers of axons that regenerate and grow extensions at distances ranging from 50 pm up to 600 pm from center of the explant were numbered after 7 days of culture. Data from independent experiments were pooled. Normalization was made against the CNTF / BDNF / Forskolin condition. Statistical significance was obtained in Two ways Anova Dunnett assay (Graph pad): *p<0.05, ** p<0.01, ***p<0.001 and ****p<0.0001

[0356] In vitro incidence of the replacement of a cysteine by a serine on ML49 solubility on the upon contact with rabbit vitreous humor.

[0357] An in vitro test performed on 384 well plates was set up to test the solubility of compounds upon contact with rabbit vitreous humor. ML49c and ML49s solubility were tested for the formation of aggregates in contact with rabbit vitreous. Each compound (2 pl) was added to 5 pl of rabbit vitreous. After Ih incubation at 37°C, presence of aggregates was detected with the camera of the DLS (Dynamic Light scattering) station.

[0358] 2. Results

[0359] 2.1. Design of a CPM-VHH fusion protein whose folding and solubility are greatly improved by the insertion of an intra-disulfide bridge in the VHH sequence.

[0360] The previous VHH-NV fusion molecule is prone to form aggregates, and precipitates upon contact with the vitreous humor (lower line in figure 5).

[0361] The inventors hypothesized that the insolubility of the molecule and the formation of precipitates in vitreous humor might result from the structure of the VHH itself.

[0362] To overcome this folding issue, the sequence of VHH was modified by replacing two aa (V22 replaced by C22 and S95 replaced by C95) in order to form an intra-molecular disulfide bridge in canonical position (C22 and C95).

[0363] The sequence of mutated VHHNPX (with the cysteine residues located in C22 and C95 underlined) is (SEQ ID NO:4):

[0364] EVQLQASGGGLAQPGGSLRLSCTVSGSIDVINNMAWYRQAPGNARELVATITSGFSTN YASSVKGRFTISRDNAKKAVYLQMNSLKPEDTADYYCKVHLIRLGAARAYDYWGQG TQVTVS

[0365] Once the disulfide bridge has been inserted, the VHH-NV molecule folds properly, and, upon contact with the vitreous humor, does not aggregate and does not precipitate anymore (see Figure 5, upper line). Thus, it is possible to increase the solubility of the VHH-NV molecule drastically by inserting an intra-disulfide bridge in the VHH sequence, so as to keep the molecule soluble in physiological fluid such as the vitreous humor.

[0366] 2.2. Destruction of the VHH putative antibody function.

[0367] Although investigations to date have not identified any epitope recognized by the VHH A12 (Li et al, 2017), it cannot be totally excluded that administration of the antibody might lead to binding in vivo and potential side effects. Inactivating the antibody function of VHH would therefore improve therapeutic properties of the compound by further limiting any potential off- target side effects. In addition to safety considerations, the recognition by VHH of an additional (unidentified) antigen could also decrease the efficacy of NV targeting on MAST2 / MAST1 PDZ. Therefore, the putative antibody function carried by the CDR3 domain of VHH was inactivated, by replacing this CDR with a mutated variant devoid of any binding activity. Five variants were created and it was checked in protein sequence databanks (package EBI (UNIPROT Knowledgebase Swissprot, EMBL, Reference Proteome plus), USPTO, EPO, JPO, KIPO, NR 1 and 2) that these variants do not correspond to any existing CDR, strongly suggesting that they are not functional.

[0368] The inventors have therefore mutated the CDR3 domain of VHH A.12 to generate the five following mutants.

[0369] 1) CDR3.3 having the sequence KVHLIGLGAAGAGDY = SEQ ID NOV

[0370] No hits found.

[0371] 2) CDR3.4 KVHLGGGGAAGAGGY = SEQ ID NO:2

[0372] No hits found.

[0373] 3) CDR3.5 KVHGGGGYDY = SEQ ID NO: 10

[0374] No hits found.

[0375] 4) CDR3.6 KGPELR = SEQ ID NO : 11

[0376] No hits found.

[0377] 5) CDR3.7 RLYAEAIY = SEQ ID NO: 12. No hits found in 10 databases.

[0378] Among these five variants of CDR3 (SEQ ID NO:9, SEQ ID NO:2, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12), only one of them, the CDR3.4 variant of SEQ ID NO: 2, offered a good yield production of the VHH-NVshort.

[0379] 2.3. In vitro superiority of VHHNPX over the CPMs of the prior art (such as L-TAT)

[0380] Most of the drugs do not enter cells spontaneously, they need to be vectorized by a Cell Penetrating Molecule (CPM). Specifically, fusion to a CPM is mandatory for the entry of the Neurovita (NV) peptide inside cells, as shown previously (see e.g., WO2016 / 038122, W02013 / 068430).

[0381] Once drugs vectorized by CPM do manage to enter the cell, they are often directed into the cellular endosomal / lysosomal pathway, which is the cell’s cleaning system. As a result, this system eliminates a large part of the product that has entered the cytoplasm.

[0382] In contrast to L-TAT, VHHNPX does not drive NV into the lysosomal degradation pathway, as shown in Figure 1 (both in live cells, Figure 1 A, or in fixed cells, Figure IB)

[0383] Therefore, VHHNPX allows bypassing the cellular degradation pathway.

[0384] As a consequence, L-TAT NV is quickly degraded in ARPE19 cells whereas VHHNPX NV remains persistent (Figure 2). Therefore, the biological activity of NV (as measured by activation of the PI3K / AKT cell signaling pathway involved in survival) requires less NV molecules when it is vectorized by VHHNPX than when it is vectorized by L-TAT (Figure 3).

[0385] Finally, it was shown that the in vitro toxicity of the L-TAT NV for ARPE19 cells is much higher than the toxicity of VHHNPX NV in these cells (figure 4).

[0386] As shown in this table, the toxicity of the fusion protein of the invention is almost 7 times less than the toxicity of L-TAT NV.

[0387] 2.4. In vivo superiority of VHHNPX over the CPMs of the prior art such as (L-TAT) After intravitreal (IVT) injection, before reaching the retina, the CPM-NVs of the invention have to overcome:

[0388] - Precipitation upon contact with vitreous humor (a physiological gel at pH7.5);

[0389] - Degradation by the vitreous humor proteases;

[0390] Outflow (anterior and posterior) to the blood circulation;

[0391] Trapping in the inner limiting membrane (ILM);

[0392] Once retina is reached, misrouting to retinal cells other than Retinal Ganglion Cells (RGCs) can occur (astrocytes, displaced amacrine cells, Muller cells ...).

[0393] The properties of the polypeptides disclosed above (L-TAT NV and VHHNPX NV) were studied using a series of tests carried out in vivo which allowed to verify the:

[0394] Targeting of retina and Retinal Ganglion Cells (RGCs) by VHH-NV or L-TAT NV after injection into the mouse eye via the IVT route

[0395] - Neuroprotective and neuroregenerative properties in vivo in the mouse optic nerve compression model.

[0396] As shown in Figure 6, L-TAT is less efficient to let NV reaching the retina than VHHNPX. Also, the NV peptide enters RGCs more efficiently when it is vectorized by VHHNPX than with L- TAT (see table below).

[0397] Molecule RGC targeting

[0398] L-TAT-NV 38%

[0399] VHHWX-NV 70%

[0400] As shown in this table, RGC targeting was higher (by 2 folds) when NV is vectorized in the eye with VHHNPX rather than with L-TAT.

[0401] These targeting discrepancies are observed despite similar solubility in vitreous humor (not shown).

[0402] The inventors observed that the insertion of eHIS at the NH2 terminus of VHHNPX does not modify the capacity of the VHH to let NV targeting retina. This is not the case when the extension GS is inserted at the NH2 terminus of VHHNPX (data not shown). It is therefore possible to use a eHIS tag if necessary, as this could be the case for PMA conjugation. The inventors have then compared by Mass Spectrometry analysis the remanence of the eHIS - VHHNPX-NV fusion protein (4pg) and L-TAT-NV (4 pg) IVT injected. Mice were sacrificed 1 hour, 2 hours, 4 hours, 8 hours or 24 hours afterwards. Retinae were harvested and lysed with a RIPA buffer. The Mass spectrometry analysis was done after trypsin digestion with Orbitrap Q exactive Plus. The remanence of VHHNPX-NV was of at least 24h, while the detection of LTAT-NV was of 4hours maximum.

[0403] Thus, the fusion with VHHNPX enables to enhance very significantly the duration of the presence of the cargo protein in the target cells, even in vivo.

[0404] 2.5. Vectorization by VHHNPX of cargo peptides with different size

[0405] To ensure that VHHNPX can vectorize peptides independently of their size difference, it has been studied whether NPX452 (VHHNPx-NVlong) and NPX461 (VHHNPx-NVshort) could i) reach the retina (Figure 7A) and ii) be observed in RGCs (Figure 7B). As shown in Figure 7, whatever the dose tested (10, 50, 250 pmol), the ability of NPX461 and those of NPX452 to deliver NV in the retina 6 hours after IVT injection were not different (figure 7A). In addition, the proportion of RGCs in which NV was present was the same (Figure 7B).

[0406] Therefore, VHHNPX can vectorize peptides of short size (10-20 amino acids) and of longer size (20-500 amino acids).

[0407] 2.6. Functional properties ofNPX461

[0408] Regeneration of axons ex vivo in retina explants was greatly enhanced and sustained in presence of 105pmol (1.5pg) of NPX461 (Figure 17). Finally, in vivo preclinical properties of VHHNPX NVshort (NPX461) and L-TAT-NV were assessed in a mouse model of optic neuropathies, the optic nerve compression model.

[0409] In contrast to L-TAT-NV (data not shown), it has also been observed that the vectorization of NVshort with VHHNPX preserves the in vivo biological functions of NV, in so far as it is capable of RGC protection (Figure 8 A) and of optic nerve regeneration (Figure 8B).

[0410] 2.7. Alteration ofRDP sequence to use as an homing sequence

[0411] It can thus be advantageous to use the RDP domain, as a homing molecule to specifically target cells that express the nAChR receptor on their surfaces. However, the sequence ofRDP needs to be mutated in order to avoid the protein aggregation induced by the domain. Two fusion proteins RDPc-NVshort (alias ML49c, containing a Cysteine, Cl 5) and mutated RDPs- NVshort (alias ML49s) in which the C15 was replaced by a serine, SI 5 were constructed. As shown on Figure 9, when added to rabbit vitreous humor, ML49c precipitates while ML49s does not, indicating that the replacement of C15 by S15 solves the aggregate formation issue of the RDPc domain. This suggests that C15 and not S15 might favor the formation of sulfide bridges between molecules.

[0412] Example 2: Use of VHHNPX to improve PMO efficacy

[0413] 1-Context / state of the art

[0414] Anti-sense oligonucleotides (ASOs) are RNA-based therapeutics that must penetrate the cytoplasm or nucleus to reach their targets. ASOs are the most advanced exon-skipping therapeutic treatments for genetic diseases such as Duchenne Muscular Dystrophy (DMD) or Spinal Muscular Atrophy (SMA) (Wang et al., 2020). A sub-class of ASOs, the Phosphorodiamidate Morpholino Oligomers (PMOs), are uncharged molecules with high metabolic stability, sequence specificity, and no off-target effects (Shadid et al., 2021). They can distribute to the nucleus, bind pre-mRNA, and correct transcription through exon skipping by steric hindrance, leading to post-transcriptional modification of spliceosome RNAs. EteplirsenZExondys, developed by Sarepta Therapeutics, was the first FDA-approved PMO in 2016, indicated for the treatment of DMD by intravenous injection (zv) as a naked PMO. Since then, FDA has approved three other exon-skipping PMO therapies: golodirsen / Vyondys, viltolarsen / Viltepso, and casimersen / Amondys.

[0415] 1.1 Description of the experiment

[0416] The experiment consisted in demonstrating in vitro that a PMO can be more efficiently vectorized into human neurons when it has been conjugated with VHHNPX (PMO-VHH) than when it has not been conjugated (naked PMO).

[0417] The cellular in vitro model was the SH-SY5Y human neuroblastoma cells that express both full lenght transcripts (FL) of Survival Motor Neuron, SMN gene containing both Exon 7 and shortened transcripts of SMN gene missing exon7, A7). We chose the PMO inducing skipping of exon 7 from the SMN transcript as described by Flynn L et al., 2021. Cells were treated either with conjugated PMO (VHH-PMO) or with non-conjugated PMO (naked PMO). The read out of the experiment was to compare the amount of A7 transcripts in the two conditions. It was expected that after treatment with a PMO the amount of A7 transcripts will increase if the PMO has entered the cells.

[0418] PMO functionalization was performed with DBCO non-cleavable or reducible linkers. PMO conjugation on WHHNPX was obtained via HiPeg conjugation leading to the production of two types of conjugates: non cleavable (VHH-Hipeg non cleavable-PMO) and reducible (VHH- Hipeg reducible PMO). Conjugation protocol leads to the conjugation of 1 or 2 PMO per VHH molecule: conjugation of 1 PMO (OAR1) or conjugation of 2 PMOs (OAR2). (OAR means oligo antibody ratio).

[0419] Cells were treated individually with these different forms of VHH-PMOS:SSOAR2 (reducible PMO-SSOAR2 conjugated VHHNPX6HIS), SSOAR1 (reducible PMO-SSOAR1 conjugated VHHNPX 6HIS), OAR2 (non-cleavable PM0-0AR2 conjugated VHHNPX 6HIS), OAR1 (non- cleavable PM0-0AR1 conjugated VHHNPX 6HIS)

[0420] 1.2 Results

[0421] Result 1 . Comparison of the percentages of A7 exon skipping obtained in SH-SY5Y treated with naked PMO and VHHNPX conjugated PMO indicates that the use of VHHNPX improves PMO efficacy.

[0422] Result 2 . Comparison of the percentages of A7 exon skipping obtained in SH-SY5Y treated with OAR 2 species or with OAR1, indicates that the more molecules conjugated to the VHH, better the exon skipping efficiency.

[0423] Result 3. Comparison of the percentages of A7 exon skipping obtained in SH-SY5Y treated with SS OAR species (reducible form) or with OAR (non-cleavable) indicates that activity of the reducible molecule is better that the non-cleavable molecule. This may be related to the better access of PMO into the nucleus.

[0424] 1.3 Material and Methods

[0425] Choice of PMO. A PMO inducing skipping of exon 7 from the SMN transcript as described by Flynn L et al., 2021 was used in this study. The 20 base long 3’-NH2- hSMN H7A (+13+32) PMO (sequence SEQ ID NO: 39: CACCTTCCTTCTTTTTGATT) of 6.718 kD as molecular weight, was synthetized by GeneTools Oregon USA.

[0426] Conjugation of PMO to VHH-6His. The Abzena Cambridge LTD CMO operated functionalization of PMO using DBCO to obtain a non-cleavable and a reducible PMO (DBCO-PEG2-NHS and DBCO-CONH-S-S-NHS respectively), followed by conjugation of these 2 types of functionalized PMOs to VHH molecules expressing a 6HIS extension at the VHH COOH terminus. Purification was performed by preparative SEC (Supradex 16 / 75pg) eluted with DPBS. Conjugation of both types of PMO functionalization to VHH-6HIS generates PMO-VHHs conjugated with one PMO molecule (OAR1), VHH conjugated with 2PM0 molecules (OAR2) and negligible a of VHH conjugated with 3 PMO molecules (OAR3) that can be separated by SDS-PAGE before to be harvested and concentrated on Vivaspin 10K.

[0427] Choice of human cells to compare the in vitro activity of PMO unconjugated (naked PMO) and PMO conjugated to VHHNPX. The SH-SY5Y human neuroblastoma cell line (CRL2266 ATCC) expresses both pathogenic full-length SMN transcripts containing Exon 7 (FL SMN transcripts) and truncated SMN transcript missing Exon 7 (exon skipping A7 SMN transcripts). Cells were cultivated in Opti-MEM (medium without serum).

[0428] Comparison of in vitro A7 exon skipping activity after treatment of SH-SY5Y cells with naked PMO or VHHNPX conjugated PMOs. The comparison of naked PMO and VHHNPX conjugated PMO activities was evaluated in SH-SY5Y (1 xlO6cells per well of 6 well culture plate) treated with 2pM of PMOs. Non-treated cells (NT) and cells treated with non-conjugated VHHNPX were included as controls. Cultures in triplicate for each condition were incubated for three days at 37°C / 5% CO2, prior to cell harvesting. RNA was extracted using a total RNA isolation kit including a Dnase treatment. RT-PCR was performed on 1 pg total RNA with One-step Superscript with Platinium Taq Polymera using primers for Exon 4 Fwd (SEQ ID NO: 40: AGGTCCTGGAAATCAG) and Exon 8 Rev (SEQ ID NO: 41 : TGGTGTCATTTAGTGCTGCTCT). PCR products were separated on a 2% agarose gel stained with ReDSafe and visualized for densitometry analysis using Imaged software.lusion The experiment demonstrated that VHHNPX efficiently vectorizes PMO into human neurons when conjugated with VHHNPX (PMO-VHH) compared to unconjugated PMO (naked PMO).

[0429] 3. Applications

[0430] VHH is an efficient CPM for the intracellular transport of drugs (proteins and oligonucleotides). As such, it can be broadly used in a wide range of drugs and diseases.

[0431] Example 3: A Neurovita short interfering peptide suitable for neuroprotection and neuroregeneration and uses thereof

[0432] 1. Material and methods

[0433] Neurovita short interfering peptides were produced similarly as in Example 1.

[0434] Production of VHHNPx-NVshort, VHHNPx-NVlon , L-TAT-NVshort, RDPc-NVshort (ML49C), RDPs-NV short (ML49S) and RDPc-NVshort APBM (MG49c).

[0435] The construct HIS-Sumo-VHHNPx-NV short, allowing the production of NPX461 (soluble VHH-NV short, in which the disulfide bridge was restored and the native CDR3 was replaced by CDR3.4 sequence), was produced in T7 Shuffle Express E. coli. strain. After bacteria lysis and centrifugation, the soluble fraction was purified on affinity chromatography on nickel resin. Cleavage of HIS-Sumo tag was obtained with ULP1 protease at 1 : 10 protease / POI (W / v) ratio. Separation of Sumo tag was obtained by loading the digestion mixture on HIS Trap HP columns in the presence of imidazole in PBS buffer, pH 7.4. Then, Size Exclusion Chromatography (SEC) was performed and the collected fractions were pooled and concentrated by Vivaspin concentrators and dialysed in PBS buffer pH 7.5 (137mM NaCl, 2.7mM KCL, 8mM Na2HPO4, 2mM KH2PO4). Identical protocol was followed for the production and purification of construct HIS-Sumo-VHHNPx-NV long (NPX452).

[0436] Alternatively, VHHNPx-NVshort was produced in E. coli. BL21XT7 with a signal peptide PelB to facilitate production in the periplasm. Purification of the production was performed on Amsphere A3 Protein A resin (JSR Light science). Endotoxins were captured on Ion exchange resin (Capto S, Cytiva) and Toxin Eraser resin (Genscript). The product was concentrated using a Vivaspin device and dialysed in PBS buffer pH 7.5 (137mM NaCl, 2.7mM KCL, 8mM Na2HPO4, 2mM KH2PO4).

[0437] Alternatively, VHHNPx-NVshort was produced in Chinese Hamster Ovarian (CHO) cells by QMCF Technology. Cell culture supernatant was purified by Protein A affinity chromatography with Amsphere A3 column, and HPLC-SEC analysis.

[0438] L-TAT-NVshort, RDPc-NVshort, RDPs-NVshort and RDPc-NVshort APBM were chemically synthesized (96-98% pure) in Acetonitrile (ACN / H20=l / 3). Quality control was performed with MS and HPLC. Lyophilized peptides were suspended either in PBS pH 7.5 for LTAT-NV or MES Buffer pH 6 (20mM, pH6 300mM NaCl), 7.5% DMSO for the RDP-NVshort peptides.

[0439] Western blot analysis after gel electrophoresis detection in semi-native or native conditions (Figure 12).

[0440] For the semi-native condition, peptides were diluted in NuPAGE LDS buffer (Thermo Fisher Scientific France #NP0007) without denaturation. They were not heated. Protein samples (15 pg / pl) were loaded onto NuPAGE Novex 4-12% Bis-Tris gels (Thermo Fisher Scientific France #NP0341) and subjected to electrophoresis in MOPS SDS running buffer (Thermo Fisher Scientific France #NP0001). For the native condition, the NativePAGE™ Bis-Tris gel system (Thermo Fisher Scientific France #BN1002BOX) was used. This system contains Coomassie G-250 which confers a net negative charge while maintaining the peptide molecules in their native state without protein denaturation.

[0441] The protein bands were blotted onto PVDF Immobilon-FL membranes (Millipore France #IPFL00010). Membranes were blocked with 4% non-fat dry milk in PBST buffer (PBS with 0.1% Tween 20) and incubated overnight at 4°C with the primary antibody in blocking buffer containing 0.1% Tween 20 (SIGMA USA #P1379). The primary antibody was an anti-RABV G-cyto domain guinea pig antibody (SYC022, 1 / 10 000 a custom antibody produced by Eurogentec). The membrane was washed with 0.1% Tween 20 in PBS and the antigen / antibody complex was detected with a horseradish peroxidase (HRP)-conjugated secondary antibody, an HRP-conjugated anti-Guinea Pig IgG antibody (Novex, USA, #A18775, 1 / 10000). Membranes were washed with PBST and visualized with the ECL western blot kit (Thermo Fisher Scientific France #35055). Gels were imaged at different exposure times with a G-Box (SYNGENE USA). In vitro detection of aggregates upon contact with rabbit vitreous humor (Figure 9 and Figure 5)

[0442] An in vitro test performed on 384 well plates was set up to test the solubility of compounds upon contact with rabbit vitreous humor. Solubilization of CPM-NV diluted in vehicle. The vehicle was either 60nM MES buffer 300mM NaCl pH6, 7.5% DMSO for RDPc-NV, RDPs- NV or LTAT-NV assay, or DPBS, pH 7.5 for assay with VVH-NV with or without disulfide bridge. Each compound (2pl) or vehicle (2pl) were added to 5 pl of rabbit vitreous. After Ih incubation at 37°c, presence of aggregates was detected with the camera of the DLS (Dynamic Light scattering) station.

[0443] In vitro assay of neurite elongation in neuroscreen cells (Figure 13)

[0444] Neuroscreen cells (NS), a subclone of rat PC12 neuronal cells, were obtained from Cellomics (USA) and cultured according to the supplier’s instructions. NS cells were grown in nondifferentiating (NDF) medium (RPMI 1640, Thermo Fisher Scientific France #11875093; 5% fetal bovine serum, FBS; 10% horse serum; 1% 200 mM glutamine, Thermo Fisher Scientific France #25030149 and 1% 100 x Pen-Strep, Thermo Fisher Scientific France #15140148). The differentiating (DF) medium had the same composition with the addition of 200 ng / ml of nerve growth factor (NGF; SIGMA USA #N2513). Neurite outgrowth assay was performed as described in Khan et al, JBC 2019. Briefly, NS cells were seed on 24-well plates (CellBind plasticware, Corning USA# 83-3337) containing NDF medium at a density of 40 000 cells per well. They were cultured overnight at 37°C. The medium was then replaced with DF medium, and the cells were incubated for a further 6 h. They were then treated with CPM-Neurovita (three constructs: RKM containing NV long, RK2M containing NV short and AAA-RK2M containing NV short and a linker were tested) at three dilutions (0.055, 0.137 and 0.22 pM) in DF medium for 1 h. Each condition was assayed in triplicates. The cells were washed once with DF medium and incubated in DF medium for 24 h at 37°C. Seventy-two hours post treatment, the NS cells were fixed by incubation with 3% paraformaldehyde (PFA) in Dulbecco’s phosphate-buffered saline (DPBS, Thermo Fisher Scientific France # 14040141) for 20 min at room temperature and treated for 5 min with 0.1% Triton-X-100 and 50% normal goat serum in DPBS. Neuron-specific anti-pill-tubulin antibody (Promega France # G7121), followed with a Donkey anti-Mouse - Alexa fluor 647 (A31571, Thermo Fisher), 1 / 500 and Hoechst 33342 (Thermo Fisher Scientific France #62249) were used to stain the neurite processes and the nuclei, respectively. NS cells (36 fields, mean of 324 cells per field) were imaged with a Leica DM 5000B UV microscope equipped with a DC 300FX camera (x40 or x20 objective). The images were analyzed using ImageJ 1.38X Software (Wayne Rasband, NIH, USA, http: / / rsb.info.nih.gov / ij / ) and its plug-in NeuronJ

[0445] (http: / / www.imagescience.org / meijering / software / neuronj / ). The effects of bias were minimized by performing the experiment blind, with relabeling of the samples. The “neurite bearing cells” were numbered. A "neurite bearing cell" was defined as a cell that has at least one neurite whose length was three and a half times longer than the diameter of its cell body. Results were expressed as % of “neurite bearing cells” among total cells.

[0446] Information on RKM, R2KM and AAARK2M. (constructs used in Figure 13).

[0447] The sequences RKM, R2KM and AAAR2KM described here were used to establish that NViongand NVshort have similar biological function. Gen Bank (Banklt2739873) numbers: RKM OR506748 (SEQ ID NO:34); RK2M OR506749 (SEQ ID NO:35); AAARK2M OR506750 (SEQ ID NO:36).

[0448] The corresponding genes of the three constructs were synthesized chemically (MWG Operon, Germany) and inserted into the pASK-IBA2C plasmid (IB A, BioTAGnology, Germany) under the control of the tetracycline promoter. The ompA signal sequence added by this plasmid allowed the expression of the protein into the periplasmic space of the bacteria. The signal sequence was cleaved during translocation. The recombinant plasmids were used to transform DH5-a E. coli bacteria (Thermo Fisher Scientific, France). Recombinant bacteria were identified by PCR and glycerol stocks were generated. CPM-Neurovita peptides were expressed and purified from the bacterial periplasm as described by Lafaye et al.2009. The periplasmic extract was further purified by chromatography on Strep-Tactin®XT columns (IBA, BioTAGnology, Germany). The molecules were stored in 0.25x PBS at -80°C. CPM expression was monitored by western blotting using a mouse high-affinity anti-StrepTag IgGl mAb, C23.21 followed by a HRP- conjugated anti mouse IgG antibody (GE Health Care # NA 9310, 1 / 10 000. Gels were revealed with ECL (Thermo Fisher Scientific, France) and pictures were taken on a G-Box (SYNGENE USA). Quantification was obtained with running a standard VHH-StrepTag preparation on each gel.

[0449] Imaging of CPM-NV entry in fixed human retina ARPE19 cells (Figure 146) 24h cultures of ARPE19 cells, ATCC CRL-2302 (10 000 cells per well of 16 chambers-Labteck) were treated for 3hours with 7pmol of VHHNPX-NV or with 7pmol of L-TAT-NV or not treated (Control condition, CT). After washes and fixation in 4%PFA, cells were stained with NV- specific antibody (SYCO22, guinea-pig antibody, 1 / 10 000). After washes, slides were treated with secondary antibodies [goat anti-guinea pig IgG-Alexa fluor 555 (#A21435, Invitrogen, 1 / 1000), and donkey anti-mouse IgG-Alexa fluor 488, (#A21202, Invitrogen 1 / 1000)]. Slides were mounted with Fluoromount G containing Dapi. Images were made on a LSM Zeiss confocal microscopy and analyzed with Image J.

[0450] In vivo retina and RGC targeting by VHHNPx-NVshort (NPX461) or VHHNPx-NVlong (NPX452) after an intravitreal injection (Figure 7 A).

[0451] CPM-NV (10, 50, 125 or 250 pmol of NPX461 or NPX452 in 1 pl) were injected intravitreally in the eye of C57B6 JR mice. Each dilution was tested in 3 eyes. The 4theye was injected with vehicle (PBS). After 6h, mice were anesthetized with 250 pl of xylazine / ketamine injected in peritoneum. An intra-cardiac perfusion of PBS and then 4% PFA(PBS, 35ml at 12ml / min - 4% PFA, 50ml at 12ml / min) was performed to fix tissues. Mice heads were incubated overnight in 4% PFA at 4°C. Retina were dissected. Flat mount retinae were co-stained with SYCO22, 1 / 500, a NV-specific guinea pig antibody directed against the C-terminal of RABV envelope G protein Cyto domain, and with a rabbit antibody (1 / 250) directed against RBPMS) a specific marker of RGC (#1830, RBPMS Phosphosolutions). The secondary species IgG specific antibodies were the goat anti-Rabbit - Alexa fluor 488 (Al 1070, Thermo Fisher), 1 / 500, and the goat antiGuinea pig - Alexa fluor 555 (A21435, Thermo Fisher), 1 / 500 respectively. Retinae were mounted with Fluoromount G containing Dapi. Images were made on a LSM Zeiss confocal microscopy and analyzed with Image J. Intensity of fluorescence in retina was scored manually (scores 0.5-2). Results were presented as Fluorescence Neurovita Intensity in retina 6 hours post injection. Half-maximal Intensity of fluorescence (IC50) was calculated with Graph Pad.

[0452] In vivo kinetic of detection of VHHNPx-NVshort(NPX461) or VHHNPx-NVlong in mouse retina after an intravitreal injection of CPM-NV (Figure 16)

[0453] CPM-NVs (250 pmol of NPX452 or NPX461 in 1 pl) were injected intravitreally in the eye of C57B6JR mice. After 3, 8, 24 or 30h, mice were sacrificed. Each time point was tested in 3 eyes. The 4theye was injected with vehicle (PBS) injection. Mice were anesthetized with 250 pl of xylazine / ketamine injected in peritoneum. An intra-cardiac perfusion of PBS and then 4% PFA (PBS, 35ml at 12ml / min - 4% PF A, 50ml at 12ml / min) was performed to fix tissues. Mice heads were incubated over-night in 4% PFA at 4°C. Retina were dissected. Flat mount retinae were co-stained with SYCO22, 1 / 500, a NV-specific guinea pig antibody directed against the C-terminal of RABVG Cyto domain, and with a rabbit antibody (1 / 250) directed against RBPMS) a specific marker of RGC (1830, RBPMS Phosphosolutions #NB521y), 1 / 250. The secondary species IgG specific antibodies were the goat anti -Rabbit - Alexa fluor 488 (Al 1070, Thermo Fisher), 1 / 500, and the goat anti-Guinea pig - Alexa fluor 555 (A21435, Thermo Fisher), 1 / 500 respectively. Retinae were mounted with Fluoromount G containing Dapi. Images were made on a LSM Zeiss confocal microscopy and analyzed with Image J. Intensity of fluorescence in retina RGCs was scored manually (scores 1-3).

[0454] In vivo neuroprotection and regeneration assay in the experimental mouse model of optic nerve compression (Figure 8)

[0455] The neuroprotective and neuroregenerative properties of NPX461 were studied in a mouse model of optic neuropathy, the optic nerve compression (ONC) model. The right optic nerve was crushed less than Ih before injection of NPX461 (5.6 pg) or BDNF (positive control) by the intravitreal (IVT) route. Negative control corresponds to mock injection or vehicle (PBS) injection. Mice were kept 14 days under regular supervision. At day 14, 2pg of CTB-555 (Cholera Toxin subunit B, Alexa Fluor™ 555 Conjugate, Fisher Scientific 11554267, resuspended at 2pg / pL in DPBS1X Ca2+- / Mg2+-) was injected in the right eye. CTB can penetrate RGCs and be transported in their axons forming the optic nerve, allowing axon regrowth visualization by confocal microscopy. Two days later, (at day 16) mice were terminally anesthetized and intracardiacally perfused with 4% Paraformaldehyde. Eyes were harvested and dissected to isolate the retina and the optic nerves.

[0456] Flat mounted retinae were prepared and immunostained with a rabbit antibody (1 / 250) directed against RBPMS) a specific marker of RGC (1830, RBPMS Phosphosolutions #NB521y), 1 / 250, followed by a goat anti-Rabbit - Alexa fluor 488 (Al 1070, Thermo Fisher), 1 / 500). Pictures of immunostained retinae were taken in the periphery (8 pictures) and the midperiphery (4 pictures) of upper and lower retina using a confocal Cell Voyager 1000 microscope (Yokagawa) and a lOx air objective. The RGC counting was performed using the Trackmate plugin on Fiji. The number of RGC was expressed in RGC / mm2and in percentage as compared to control (non-crushed optic nerve =left retina) respectively in each area (periphery / mid- periphery and upper / lower retina). Three retinae of the left eye served as control. Statistical analysis was performed using Graph Pad Prism version 9. Data were expressed as mean ± SEM. Data were analyzed by a Two-way ANOVA with a Tukey’s multiple comparison post hoc test.

[0457] Optic nerves were mounted on slides for confocal microscopy. The optic nerves highlighted by the CTB-555 were acquired using a confocal Nikon Ti2E microscope and a 20x air objective by tile scanning (from the crush site to the longest axon) and Z-stacking (3pm intervals). A Maximum Intensity Projection was done with the Z-stack images. The optic nerve regeneration was evaluated by three parameters: (1) the length of the longest fiber measured by Fiji; (2) the CTB mean intensity and (3) the number of fibers measured on Icy software (de Chaumont F, Dallongeville S, Chenouard N, Herve N, Pop S, Provoost T, Meas-Yedid V, Pankajakshan P, Lecomte T, Le Montagner Y, Lagache T, Dufour A, Olivo-Marin JC. Icy: an open bioimage informatics platform for extended reproducible research. Nat Methods. 2012 9(7):690-6.). On Icy, 10 rectangles of 100pm were drawn from the crush site. One rectangle is placed before the crush to measure the fluorescence before the crush and another one was placed at the end of the picture to measure the background. The CTB mean intensity was automatically measured by Icy in each rectangle, whereas the number of fibers was manually counted. All statistical analysis was performed using Graph Pad Prism version 9. Data were expressed as mean ± SEM. The length of the longest fiber was expressed in pm, data were analyzed by an ordinary Oneway ANOVA with a Tukey’s multiple comparison post hoc test. The CTB mean intensity in each ROI (100pm) was normalized to the background and the fluorescence intensity before the crush and was expressed in percentage of maximum CTB intensity. Data were analyzed by a Two-way ANOVA with a Tukey’s multiple comparison post hoc test. The number of fibers was analyzed by a Two-way ANOVA with a Tukey’s multiple comparison post hoc test.

[0458] 2. Results

[0459] 2.1 Alteration of RDP sequence to improve yield of CPM-NV production and pharmacological properties.

[0460] A fusion protein comprising NVshort, VHH, and RDP was constructed. However, production of this polypeptide in bacterial cells was very inefficient, as the polypeptide was observed to aggregate. A better production can be obtained when the VHH-NV constructs lack RDP (see Figure 11, comparison on SDS PAGE stained with 2,2,2-Trichloroethanol (TCE) of RDP-VHH-NV and VHH-NV productions). It is therefore advantageous to remove the RDP domain from the construction for improving the production of NV.

[0461] It cannot be excluded that poor production of RDP-VHH-NV is related to the capacity of RDP to form dimers and aggregate triggered by the cysteine encoded in RDP.

[0462] The RDP sequence (YTIWMPENPRLGMSC15DIFTNSRGKRASKG, SEQ ID NO: 13) contains one single Cysteine residue (C15, underlined in the RDP sequence; it is herein also called RDPc). The inventors hypothesized that this Cysteine could alter production of the NV fusion protein by triggering the formation of inter-chain sulfide bridges. In order to test this hypothesis, two short polypeptides of 49aa long, ML49c and ML49s were chemically synthesized. The ML49c peptide is formed with a RDP encoding one cysteine (RDPc) and a shortened sequence of NV (NVshort)] while ML49c is formed with a mutated RDP domain called “RDPs” where the Cysteine was replaced by a Serine (serine does not form disulfide bridge) and NVshort. When added to rabbit vitreous humor, ML49c precipitates after being added to eye humor vitreous (pH 7.5) (black dots in the plate-well, Figure 9), while ML49s does not, indicating that the replacement of C15 by S15 solves the aggregate formation issue of the RDPc domain. This suggests that C15 might favor the formation of sulfide bridges between molecules.

[0463] When the ML49c was run on gels in non-denaturation conditions (left part of the Figure 12), or in native conditions (right part of the Figure 12), presence of dimers and aggregates was observed (Figure 12). In addition, the formation of dimers and aggregates was still observed with ML49cAPBM, an ML49c peptide which PBM, the active part of NV, was deleted, indicating that NV PBM is not involved in the aggregates and dimers formation.

[0464] 2.2. Shorten the pharmacophore size.

[0465] It cannot be excluded that the NV peptide, the pharmacophore, could be recognized by the immune system of the patient because it originates from a viral protein. To minimize this risk, the inventors reduced the size of the pharmacophore. The NVlong sequence encodes not only the PBM, critical for the biological function of NV, but also 27 residual amino acids of the cytoplasmic domain of the rabies virus G protein located upstream the 11 or 15 amino acid PBM domain. Deletion of these 27 aa upstream the PBM of NV leads to a shorten NV sequence called NVshort, (an example thereof has the sequence RIISSWEVHGQQTRL = SEQ ID NO:24).

[0466] To ensure that the shortening of NVlong to NVshort did not alter the biological properties of NV, the capacity of NVshort and NVlong to stimulate neurite outgrowth in a Neuroscreen cells assay was compared. As shown in Figure 13, whatever the dose tested (0.22 pM- 0.055 pM), the ability of VHH-NVshort and of VHH-NVlong to promote neurite elongation were identical.

[0467] In addition, the shortening of NV does not alter the retina RGCs targeting by NV (Figure 7A) nor the duration of detection of NV in retina RGC (Figure 16).

[0468] Moreover, VHHNPx-NVshort promotes axon regeneration in an ex vivo model of axotomy in retina explants (Figure 17) and in an in vivo preclinical model of mouse optic neuropathies (compression of the optic nerve) (figure 8B). In the same model, VHHNPx-NVshort confers protection of RGCs (Figure 8 A).

[0469] Therefore, the length of the pharmacophore can be reduced without losing the biological properties of the NV peptide.

[0470] 2.3. Design of an optimized VHH

[0471] 2.3-1 Insertion of a disulfide bridge in VHH

[0472] The yield of CPM-NV production was improved after the RDP was removed. However, the VHH-NV molecule was still prone to form aggregates, and precipitates upon contact with the vitreous humor, even in absence of the RDP domain.

[0473] The inventors hypothesized that the insolubility of the molecule and the formation of precipitates in vitreous humor must result from the structure of the VHH itself.

[0474] To overcome this folding issue, the sequence of VHH was modified by replacing two aa (V22 replaced by C22 and S95 replaced by C95) to form an intra-molecular disulfide bridge in canonical position (C22 and C95). The sequence of mutated VHH (with the cysteine residues located in C22 and C95 underlined) is (SEQ ID NO:5):

[0475] EVQLQASGGGLAQPGGSLRLSCTVSGSIDVINNMAWYRQAPGNARELVATITSGFSTN YASSVKGRFTISRDNAKKAVYLQMNSLKPEDTADYYCKVHLIRLGAARAYDYWGQG TQVTVS

[0476] Once the disulfide bridge has been inserted, the VHH-NVshort molecule folds properly, and, upon contact with the vitreous humor, does not aggregate and does not precipitate anymore (see Figure 5, upper line).

[0477] Protein was concentrated on Pierce Protein Concentrator PES (MWCO 3 kDa) in PBS buffer pH 7.4 and its concentration was continuously monitored using UV measurements with extinction coefficient specific for the amino acid sequence. Concentration cycles frequency was increasing correspondingly to POIs concentration (10 min to 1 min / cycle, 5 k ref, fixed angle rotor, 4°C, one PES filter / one test). A concentration of 130 mg / ml can be obtained with a VHH- NV encoding a DS.

[0478] Thus, it is also possible to increase the solubility of the VHH-NVshort molecule drastically by inserting an intra-disulfide bridge in the VHH sequence, to keep the molecule soluble in physiological fluid such as the vitreous humor.

[0479] 2.3.2 Destruction of the antibody function of VHH

[0480] Although investigations to date have not identified any epitope recognized by the VHH A12, (Li et al., 2017), it cannot be totally excluded that administration of the antibody might lead to binding in vivo and potential side effects. Inactivating the antibody function of VHH would therefore improve therapeutic properties of the compound by further limiting any potential off- target side effects. In addition to safety considerations, the recognition by VHH of an additional (unidentified) antigen could also decrease the efficacy of NV targeting on MAST2 / MAST1 PDZ. Therefore, the putative antibody function carried by the CDR3 domain of VHH was inactivated, by replacing this CDR with a mutated variant devoid of any binding activity. Five variants were created and it was checked in protein data banks (package EBI (UNIPROT Knowledgebase Swissprot, EMBL, Reference Proteome plus), USPTO, EPO, JPO, KIPO, NR 1 and 2) that these variants do not correspond to any existing CDR, strongly suggesting that they are not functional. The inventors have therefore mutated the CDR3 domain of VHH A.12 to generate five CDR3 variants.

[0481] 1) CDR3.3 having the sequence KVHLIGLGAAGAGDY = SEQ ID NO:9

[0482] No hits found.

[0483] 2) CDR3.4 KVHLGGGGAAGAGGY = SEQ ID NO :2

[0484] No hits found.

[0485] 3) CDR3.5 KVHGGGGYDY = SEQ ID NO: 10

[0486] No hits found.

[0487] 4) CDR3.6 KGPELR = SEQ ID NO: 11

[0488] No hits found.

[0489] 5) CDR3.7 RLYAEAIY = SEQ ID NO: 12

[0490] No hits found in 10 databases

[0491] Among these five variants of CDR3, (SEQ ID NO:9, SEQ ID NO:2, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12) only one of them, the CDR3.4 variant of SEQ ID NO: 2, offered a good yield production of the VHH-NV.

[0492] The VHH molecule (VHH mutated to form a single disulfide bridge and encoding the mutated CDR3.4), called VHHNPX corresponds to SEQ ID NO: 5.

[0493] Fusion of VHHNPX with NVshort, leads to the formation of VHHNPx-NVshort, named NPX461 (SEQ ID NO:30) that solves the issues of solubility, yield, formation of aggregates of VHH- NV and removal of offside effect by killing the antibody function of VHH. This series of improvements strengthens the high therapeutic potential of NPX461 as an investigational drug candidate.

[0494] 2.4. Analysis of the biological activity of the peptides of the invention in retinal cells

[0495] The following polypeptides were generated:

[0496] After Intravitreal (IVT) injection, before reaching the retina, the CPM-NVs of the invention have to overcome:

[0497] - Precipitation upon contact with vitreous humor (a physiological gel at pH7.5); - Degradation by the vitreous humor proteases;

[0498] Outflow (anterior and posterior) to the blood circulation;

[0499] Trapping in the inner limiting membrane (ILM) (see scheme of retina structure in figure 15); Once retina is reached, misrouting to retina cells other than Retinal Ganglion Cells (RGCs) can occur (astrocytes, displaced amacrine cells, Muller cells ...)

[0500] The properties of these polypeptides were studied using a series of tests carried out in vitro, ex vivo and in vivo which allowed to verify that they display the following properties:

[0501] Absence of toxicity of the products using in vitro tests (human ARPE-19 cells, Targeting of Retinal Ganglion Cells by VHH-NVs and their persistence after injection into the mouse eye via the intravitreal route (figure 16)

[0502] - Neuroprotective and neuroregenerative properties in appropriate models (ex vivo on mouse retina explants and in vivo in the mouse optic nerve compression model, cf. figure 17).

[0503] It has been observed that NPX461 has no toxicity at 3900pmol (55pg) in ARPE19 cells in vitro (not shown), and that the NPX461 molecule is able to target and penetrate retina cells in vivo (figure 16).

[0504] Regeneration of axons ex vivo in retina explants was greatly enhanced and sustained in presence of 105pmol (1.5pg) of NPX461 (Figure 17).

[0505] It has also been observed that the vectorization of NV with mutated VHH (NPX461) preserves the in vivo biological functions of NV, in so far as it is capable of RGC protection (Figure 8 A) and of optic nerve regeneration (Figure 8B).

[0506] The conservation of biological properties of NPX461 confirms that NVshort is sufficient to produce the desired therapeutic effect.

[0507] BIBLIOGRAPHY

[0508] Abes, R. et al. Delivery of steric block morpholino oligomers by (R-X-R)4 peptides: structureactivity studies. Nucleic Acids Res 36, 6343-6354, doi: 10.1093 / nar / gkn541 (2008). Amantana, A. et al. Pharmacokinetics, biodistribution, stability and toxicity of a cellpenetrating peptide-morpholino oligomer conjugate. Bioconjug Chem 18, 1325-1331, doi: 10.1021 / bc070060v (2007).

[0509] Andaloussi, S. E. et al. Design of a peptide-based vector, PepFect6, for efficient delivery of siRNA in cell culture and systemically in vivo. Nucleic Acids Res 39, 3972-3987, doi: 10.1093 / nar / gkql299 (2011).

[0510] An, S. Y. et al. Intrinsic disorder is essential for Cas9 inhibition of anti-CRISPR AcrIIA5. Nucleic Acids Res 48, 7584-7594, doi: 10.1093 / nar / gkaa512 (2020).

[0511] Ausubel, F. M. e. a. Current protocols in molecular biology. Current protocols December 4 (2003).

[0512] Boysen, R. I. e. a. HPLC of peptides and proteins: a standard operating conditions. Current protocols in molecular biology, 10.13.11 (2001).

[0513] Butler, M. Mammalian Cell biotechnology: a practical approach. Practical approach 76 (1991).

[0514] Christensen, N. R. et al. PDZ Domains as Drug Targets. Adv Ther (Weinh) 2, 1800143, doi: 10.1002 / adtp.201800143 (2019).

[0515] Choi, J. M. et al. Intranasal delivery of the cytoplasmic domain of CTLA-4 using a novel protein transduction domain prevents allergic inflammation. Nat Med 12, 574-579, doi: 10.1038 / nml385 (2006). da Costa, A. et al. A Human Blood-Brain Interface Model to Study Barrier Crossings by Pathogens or Medicines and Their Interactions with the Brain. J Vis Exp, doi: 10.3791 / 59220 (2019). de Chaumont, F. et al. Icy: an open bioimage informatics platform for extended reproducible research. Nat Methods 9, 690-696, doi: 10.1038 / nmeth.2075 (2012).

[0516] Dayhoff, M. O. e. a. A model of evolutionary change in proteins. National Biomed. Res. Found. Atlas of protein sequence and structure, 345-352 (1978).

[0517] Dev, K. K. Making protein interactions druggable: targeting PDZ domains. Nat Rev Drug Discov 3, 1047-1056, doi: 10.1038 / nrd!578 (2004). Donnelly-Roberts, D. L. & Lentz, T. L. Synthetic peptides of neurotoxins and rabies virus glycoprotein behave as antagonists in a functional assay for the acetylcholine receptor. Pept Res 2, 221-226 (1989).

[0518] Eisenberg, D. Three-dimensional structure of membrane and surface proteins. Annu Rev Biochem 53, 595-623, doi: 10.1146 / annurev.bi.53.070184.003115 (1984).

[0519] Eisenberg, D., Weiss, R. M. & Terwilliger, T. C. The hydrophobic moment detects periodicity in protein hydrophobicity. Proc Natl Acad Sci U S A 81, 140-144, doi: 10.1073 / pnas.81.1.140 (1984).

[0520] Flanagan, J. M., Kataoka, M., Shortle, D. & Engelman, D. M. Truncated staphylococcal nuclease is compact but disordered. Proc Natl Acad Sci U S A 89, 748-752, doi: 10.1073 / pnas.89.2.748 (1992).

[0521] Green, M. R. e. a. Molecular cloning: a laboratory manual. Laboratory manuals 4th edition (2012).

[0522] Henikoff, S. & Henikoff, J. G. Amino acid substitution matrices. Adv Protein Chem 54, 73-97, doi : 10.1016 / s0065-3233(00)54003-0 (2000).

[0523] Khan, Z. et al. Structure-based optimization of a PDZ-binding motif within a viral peptide stimulates neurite outgrowth. J Biol Chem 294, 13755-13768, doi: 10.1074 / jbc.RAl 19.008238 (2019).

[0524] Lafaye, P, Achour, I., England, P, Duyckaerts, C. & Rougeon, F. Single-domain antibodies recognize selectively small oligomeric forms of amyloid beta, prevent Abeta-induced neurotoxicity and inhibit fibril formation. Mol Immunol 46, 695-704, doi: 10.1016 / j.molimm.2008.09.008 (2009).

[0525] Lentz, T. L., Hawrot, E. & Wilson, P. T. Synthetic peptides corresponding to sequences of snake venom neurotoxins and rabies virus glycoprotein bind to the nicotinic acetylcholine receptor. Proteins 2, 298-307, doi: 10.1002 / prot.340020406 (1987).

[0526] Lentz, T. L., Wilson, P. T., Hawrot, E. & Speicher, D. W. Amino acid sequence similarity between rabies virus glycoprotein and snake venom curaremimetic neurotoxins. Science 226, 847-848, doi: 10.1126 / science.6494916 (1984). Li, T. et al. Selection of similar single domain antibodies from two immune VHH libraries obtained from two alpacas by using different selection methods. Immunol Lett 188, 89-95, doi:10.1016 / j.imlet.2017.07.001 (2017).

[0527] Meijering, E. et al. Design and validation of a tool for neurite tracing and analysis in fluorescence microscopy images. Cytometry A 58, 167-176, doi: 10.1002 / cyto.a.20022 (2004).

[0528] Prehaud, C. et al. Attenuation of rabies virulence: takeover by the cytoplasmic domain of its envelope protein. Sci Signal 3, ra5, doi:10.1126 / scisignal.2000510 (2010).

[0529] Sheth, R. U., Cabral, V, Chen, S. P. & Wang, H. H. Manipulating Bacterial Communities by in situ Microbiome Engineering. Trends Genet 32, 189-200, doi : 10.1016 / j .tig.2016.01.005 (2016).

[0530] Terrien, E. et al. Interference with the PTEN-MAST2 interaction by a viral protein leads to cellular relocalization ofPTEN. Sci Signal 5, ra58, doi: 10.1126 / sci signal.2002941 (2012).

[0531] Flynn L et al., Targeted SMN Exon Skipping : A Useful Control to Assess In Vitro and In Vivo Splice -Switching Studies. Biomedicines 2021.

[0532] Ghorai, S. M., Deep, A., Magoo, D., Gupta, C. & Gupta, N. Cell-Penetrating and Targeted Peptides Delivery Systems as Potential Pharmaceutical Carriers for Enhanced Delivery across the Blood-Brain Barrier (BBB). Pharmaceutics 15, doi: 10.3390 / pharmaceuticsl5071999 (2023).

[0533] Kumar, P. et al. Transvascular delivery of small interfering RNAto the central nervous system. Nature 448, 39-43, doi: 10.1038 / nature05901 (2007).

[0534] Lim, K. R., Maruyama, R. & Yokota, T. Eteplirsen in the treatment of Duchenne muscular dystrophy. Drug Des Devel Ther 11, 533-545, doi: 10.2147 / DDDT.S97635 (2017).

[0535] Mae, M. et al. A stearylated CPP for delivery of splice correcting oligonucleotides using a non- covalent co-incubation strategy. J Control Release 134, 221-227, doi : 10.1016 / j .j cornel .2008.11.025 (2009).

[0536] Milletti, F. Cell-penetrating peptides: classes, origin, and current landscape. Drug Discov Today 17, 850-860, doi: 10.1016 / j.drudis.2012.03.002 (2012). Moulton, H. M. & Moulton, J. D. Morpholinos and their peptide conjugates: therapeutic promise and challenge for Duchenne muscular dystrophy. Biochim Biophys Acta 1798, 2296- 2303, doi: 10.1016 / j.bbamem.2010.02.012 (2010).

[0537] Moulton, H. M., Nelson, M. H., Hatlevig, S. A., Reddy, M. T. & Iversen, P. L. Cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides. Bioconjug Chem 15, 290-299, doi: 10.1021 / bc034221g (2004).

[0538] Pothin, E., Lesuisse, D. & Lafaye, P. Brain Delivery of Single-Domain Antibodies: A Focus on VHH and VNAR. Pharmaceutics 12, doi: 10.3390 / pharmaceuticsl2100937 (2020).

[0539] Shadid, M., Badawi, M., & Abulrob, A. (2021). Antisense oligonucleotides: Absorption, distribution, metabolism, and excretion. Expert Opinion on Drug Metabolism & Toxicology, 77(11), 1281-1292. https: / / doi.org / 10.1080 / 17425255.2021.1992382

[0540] Vives, E., Schmidt, J. & Pelegrin, A. Cell-penetrating and cell-targeting peptides in drug delivery. Biochim Biophys Acta 1786, 126-138, doi: 10.1016 / j.bbcan.2008.03.001 (2008).

[0541] Wang, E, Zuroske, T., & Watts, J. K. (2020). RNA therapeutics on the rise. Nature Reviews. Drug Discovery, 19(7), 441-442. https: / / doi.org / 10.1038 / d41573-020-00078-0

[0542] Wu, R. P. et al. Cell-penetrating peptides as transporters for morpholino oligomers: effects of amino acid composition on intracellular delivery and cytotoxicity. Nucleic Acids Res 35, 5182- 5191, doi: 10.1093 / nar / gkm478 (2007).

[0543] Yin, H. et al. Cell-penetrating peptide-conjugated antisense oligonucleotides restore systemic muscle and cardiac dystrophin expression and function. Hum Mol Genet 17, 3909-3918, doi: 10.1093 / hmg / ddn293 (2008).

Claims

CLAIMS1. A VHH antibody (VHHNPX) of SEQ ID NO:5, or a variant thereof whose sequence displays at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity with SEQ ID NO:5, said variant comprising i) the CDR1-3 of sequence SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:2 respectively and ii) at least one disulfide bond formed by two cysteines.

2. The VHH antibody of claim 1, wherein said VHH antibody has a basic isoelectric point.

3. The VHH antibody of claim 1 or 2, wherein the sequence of the VHHNPX chain is SEQ ID NO:5.

4. A complex containing a molecular cargo conjugated or fused to the VHH antibody of any one of claims 1 to 3.

5. The complex of claim 4, wherein said molecular cargo is a peptide, a protein, a chemical molecule, a small molecule, an oligonucleotide of interest such as an uncharged phosphodiamidate morpholino oligo (PMO) or other DNA or RNA analogue with neutral electrostatic charge.

6. The complex of any one of claims 4 or 5, wherein said VHH antibody is used in combination with or coupled to a homing molecule.

7. The complex of claim 6, wherein said homing molecule is a Rabies virus Derived Peptide (RDP) derived from the rabies virus envelope G protein of the CVS-NIV sequence, preferably the peptide having the sequence SEQ ID NO: 14, or a variant thereof displaying at least 90%, more preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% similarity with SEQ ID NO: 14, but not containing any cysteine residue.

8. The complex of any one of claims 4 to 7, wherein said molecular cargo is the Neurovita short peptide having the sequence SEQ ID NO:21.

9. The complex of claim 8, wherein said molecular cargo is the Neurovita short peptide having a sequence selected in the group consisting of: SEQ ID NO:22-28.

10. An in vitro use of the VHH antibody of anyone of claims 1 to 3 to vectorize a molecular cargo in a non-endosomal / non-lysosomal pathway inside a target cell.

11. The VHH antibody of any one of claims 1 to 3, for use for delivering a molecular cargo in a non-endosomal / non-lysosomal pathway inside a target cell, for preventing and / or treating a disease or condition involving the Peripheral Nervous System (PNS) or the Central Nervous System (CNS), or the evolution of such disease or condition, such as optic neuropathies (glaucoma, NAION), motor neuron diseases (ALS, SMA), muscular disease (Duchenne Disease), or neurodegenerative diseases, (Parkinson, Alzheimer).

12. A pharmaceutical composition comprising a therapeutically effective amount of the complex of any one of claims 4-9, and a pharmaceutically acceptable carrier.

13. The complex of any one of claims 4-9, or the pharmaceutical composition of claim 12, for use as a medicament.

14. The complex of claims 4-9, or the pharmaceutical composition of claim 12, for use for preventing and / or treating a disease or condition involving the Peripheral Nervous System (PNS) or the Central Nervous System (CNS), or the evolution of such disease or condition, such as optic neuropathies (glaucoma, NAION), motor neuron diseases (ALS, SMA) muscular disease (Duchenne Disease) or neurodegenerative diseases (Parkinson, Alzheimer).

15. The VHH antibody according to claims 1 to 3, the complex according to claims 4 to 9 or the pharmaceutical composition according to claim 12, for use according to claims 11, 13 or 14, wherein said disease or condition is a neurodegenerative disease, or a cell damage- associated condition such as stroke or injury including optic nerve disease, retina diseases, muscular, motor neuron disease.

16. ANeurovita polypeptide comprising or consisting of the NVshort peptide of sequence:XnSWXVXXQQTRL (SEQ ID NO:21), wherein X is any amino acid and n is an integer comprised between 0 and 4 (i.e., n is 0, 1, 2, 3, or 4), and wherein the sequence of the Neurovita polypeptide is not SEQ ID NO: 17, 18, or 19.

17. The polypeptide of claim 16, wherein said NVshort polypeptide has a sequence selected in the group consisting of SEQ ID NOs:22-28.

18. The polypeptide of any one of claims 16 or 17, further comprising at least one cellpenetrating molecule (CPM) and optionally a homing molecule.

19. The polypeptide of claim 18, wherein the polypeptide has a basic isoelectric point.

20. The polypeptide of any of claims 16-19, consisting of a fusion protein between the NVshort peptide of sequence RIISSWEVHGQQTRL (SEQ ID NO:24) and a VHH CPM.

21. The polypeptide of claim 19 or 20, wherein said CPM is chosen in the group consisting of:A heavy chain polypeptide of a VHH camelid antibody, whose sequence is mutated to contain at least one disulfide bond and a CDR3 containing three or more mutations as compared with SEQ ID NO: 1, so as to inactivate the antibody function of said VHH camelid antibody,A Rabies virus Derived Peptide (RDP) whose sequence displays at least 90% homology or similarity with SEQ ID NO: 13, and in which the only cysteine amino acid has been substituted by another amino acid, orA protein transduction domain of the HIV trans-activator of transcription L-TAT having at least 90% homology or similarity with SEQ ID NO: 16.

22. The polypeptide of any of claims 16-21, wherein said CPM and / or Neurovita peptides are separated by a linker.

23. The polypeptide of any of claims 16-22, wherein it contains:- the VHHNPX CPM having the SEQ ID NO: 5 or a variant thereof, whose sequence displays at least 90%, preferably at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology or similarity with SEQ ID NO:5, said variant comprising i) the CDRs of SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:2 as CDR11-3 respectively, and ii) at least one disulfide-bridge formed by two cysteines, and- the NVshort polypeptide of sequence RIISSWEVHGQQTRL (SEQ ID NO:24).

24. The polypeptide of any of claims 16-21, wherein it has the sequence SEQ ID NO:30.

25. The polypeptide of any of claims 16-24, wherein it is soluble in vitreous humor, nonaggregated, and able to bind and block with high affinity the PDZ domain of the human MAST1 and MAST2 proteins in neuronal cells.

26. A vector, preferably a viral vector, DNA or mRNA, encoding the polypeptide as defined in any of claims 16-25.

27. A pharmaceutical composition comprising the polypeptide as defined in any of claims 16-25 or the vector of claim 26, for its expression in cells and its use as a medicament.

28. The pharmaceutical composition according to claim 27, for preventing and / or treating a disease or condition involving the Peripheral Nervous System (PNS) or the Central Nervous System (CNS) or the evolution of such disease or condition, such as optic neuropathies (glaucoma, NAION), motor neuron diseases (ALS), muscular diseases (Duchenne Disease) or neurodegenerative diseases (Parkinson disease, Alzheimer’s Disease).

29. The pharmaceutical composition for use according to claim 27 or 28, wherein said disease or condition is a neurodegenerative disease, or a cell damage-associated condition such as stroke or injury including optic nerve disease, retinal diseases, muscular diseases, motor neuron disease.