Gene therapy of sympathetic nervous system hyperactivity-associated conditions

Abstract

The present invention relates to a gene therapy approach for the treatment of sympathetic nervous system hyperactivity-associated conditions. The invention provides an expression vector comprising a nucleotide sequence encoding an isolated botulinum toxin serotype A light chain operably linked to a promoter active in sympathetic neurons, for use in the treatment of sympathetic nervous system hyperactivity-associated conditions.

Description

[0001] I

[0002] GENE THERAPY OF SYMPATHETIC NERVOUS SYSTEM HYPERACTIVITY-ASSOCIATED CONDITIONS

[0003] FIELD OF THE INVENTION

[0004] The present invention relates to a gene therapy approach for the treatment of sympathetic nervous system hyperactivity-associated conditions such as erectile dysfunction and others.

[0005] BACKGROUND OF THE INVENTION

[0006] The sympathetic nervous system (SNS) is one of the divisions of the autonomic nervous system which controls many functions in the body such as heart rate, blood pressure, blood glucose levels, body temperature, digestion, urination, sweating and immune system function, among other functions. SNS is best known for its role in responding to danger and stress by increasing blood flow, and oxygen delivery and speeding up the heart rate.

[0007] There are two kinds of neurons involved in the transmission of any signal through the sympathetic system: pre-ganglionic and post-ganglionic. The shorter pre-ganglionic neurons originate in the thoracolumbar division of the spinal cord and travel to a ganglion, often one of the paravertebral ganglia, where they synapse with a postganglionic neuron. From there, the long post-ganglionic extend across most of the body. At the synapses within the ganglia, preganglionic neurons release acetylcholine a neurotransmitter that activates nicotinic acetylcholine receptors on postganglionic neurons. In response to this stimulus, the postganglionic neurons release norepinephrine, which activates adrenergic receptors that are present on the peripheral target tissues. The activation of target tissue receptors causes the effects associated with the sympathetic system.

[0008] An abnormal increase of sympathetic nervous system (SNS) activity (i.e. overactivity or hyperactivity) has been reported in numerous conditions cardiovascular diseases, including ischemic heart disease, chronic heart failure, obliterating arteriopathy of the lower limbs and hypertension; kidney disease; diabetes mellitus, in particular type II diabetes; obesity; metabolic syndrome; obstructive sleep apnea; depression; ulcerative colitis; erectile dysfunction; Raynaud’s syndrome and neurological disorders such as spinal cord injury (Fisher et al., Auton. Neurosci., 2009, 148, 5-15; WO 2018 / 220232). SNS hyperactivity may be of various origins: neurogenic, psychogenic, vasculogenic, cardio-metabolic, endocrine (or hormonal) or mixed origin. It is linked to aberrant norepinephrine levels.

[0009] Botulinum toxin has been used to inhibit the release of norepinephrine from sympathetic terminations as a therapeutic approach for erectile dysfunction treatment (WO 2018 / 220232).

[0010] Botulinum toxin is a highly potent neurotoxic protein produced by the anaerobic bacteria Clostridium botulinum. It inhibits the release of neurotransmitters by exocytosis such as acetylcholine, norepinephrine, dopamine or glycine from the presynaptic nerve terminal. When inhibiting the release of acetylcholine at the neuromuscular junctions, botulinum toxin causes flaccid paralysis.

[0011] The botulinum toxin comprises two chains: a light chain (LC) and a heavy chain (HC) both linked by a disulfide bond. The heavy chain comprises an N-terminal translocation membrane domain (HC-N) and a C-terminal receptor binding domain (HC-C). The light chain represents the active portion, while the heavy chain is necessary for light chain entry into neurons. The light chain is a zinc-dependant endoprotease that cleaves SNARE (soluble N-ethylmaleimide-sensitive attachment protein receptor) proteins responsible for the fusion of synaptic vesicles and release of neurotransmitters by exocytosis. There are more than seven serotypes: A, B, C, D, E, F, G, H and X which have different SNARE targets. Botulinum toxin serotypes A, and E cleave the SNAP -25 protein; serotypes B, D, F and G cleave the synaptobrevin (VAMP) protein; serotype C cleaves both SNAP -25 and syntaxin la. Botulinum toxin serotype A (Botox ®, Dy sport ®, Xeomin ®) is the most used in medicine as well as in cosmetics.

[0012] However, injecting an entire botulinum neurotoxin (BoNT) is not safe and serious side-effects from its use can occur, largely due to unwanted spread or diffusion of the toxin’s biological activity (paralysis) into adjacent and distal muscles. In addition, expression and purification of a functional native full length recombinant BoNT is problematic in terms of costs, biosafety requirements, yields, solubility and folding.

[0013] Consequently, there is a need to improve current therapeutic approaches for the treatment of sympathetic nervous system hyperactivity-associated conditions such as erectile dysfunction and others.

[0014] SUMMARY OF THE INVENTION The inventors have used expression vectors to deliver botulinum toxin light chain in sympathetic neurons.

[0015] The inventors have shown that the light chain of botulinum toxin serotype A (BoNT / A-LC) is expressed at higher levels specifically in sympathetic neurons. The BoNT / A-LC expression level in sympathetic neurons are significantly higher than the expression level of other botulinum light chain serotypes such as serotype F, whereas the expression level of BoNT / A-LC in other neurons such as sensory neurons is similar to that of other light chain serotypes such as BoNT / F-LC. Furthermore, the high expression level correlates with an enhanced inhibition of norepinephrine release from sympathetic neurons with BoNT / A-LC expression vector. Due to this advantageous effect, the use of ubiquitous promoters to drive the expression of BoNT / A-LC allows to achieve a strong expression and a strong inhibition of norepinephrine release, specifically in sympathetic neurons.

[0016] The use of botulinum toxin light-chain is safe because it does not cross the cellular membrane and is not capable of binding to neurons without the heavy chain, as compared to complete botulinum toxin. The use of replication-defective vectors to express botulinum toxin light-chain, in particular replication-defective viral vectors such as those derived from Herpes Simplex virus type 1 (HSV-1) provides a completely safe approach.

[0017] In view of these results, the use of an expression vector for BoNT / A-LC, in particular derived from a replication-defective viral vector such as Herpes Simplex virus type 1 (HSV-1) derived vector is a promising approach for the gene therapy of sympathetic nervous system hyperactivity-associated conditions.

[0018] One aspect of the invention relates to an expression vector comprising a nucleotide sequence encoding an isolated botulinum toxin serotype A light chain operably linked to a promoter active in sympathetic neurons, for use in the treatment of sympathetic nervous system hyperactivity-associated conditions.

[0019] In some embodiments, the vector is a viral vector, in particular a herpes simplex virus (HSV) vector or an adeno-associated virus (AAV) vector; preferably a defective HSV-1 derived vector such as a HSV-1 amplicon vector or a defective recombinant HSV vector.

[0020] In some embodiments, the promoter is a ubiquitous promoter, in particular human elongation factor 1 alpha (hEFl-a) promoter or human cytomegalovirus immediate early enhancer and promoter (hCMV); preferably wherein the hEFl-a promoter comprises the nucleotide sequence of SEQ ID NO: 3 and / or the hCMV promoter comprises the nucleotide sequence of SEQ ID NO: 4.

[0021] In some embodiments, the isolated botulinum toxin serotype A light chain comprises an amino acid sequence having at least 70% identity, preferably at least 75% identity, at least 80% identity, even more preferably at least 85% identity with the sequence of SEQ ID NO: 1.

[0022] In some embodiments, the sympathetic nervous system hyperactivity-associated conditions are selected from the group consisting of:: cardiovascular diseases, including ischemic heart disease, chronic heart failure, obliterating arteriopathy of the lower limbs and hypertension; kidney disease; diabetes mellitus, in particular type II diabetes; obesity; metabolic syndrome; obstructive sleep apnea; depression; ulcerative colitis; erectile dysfunction; Raynaud’s syndrome; and neurological disorders such as spinal cord injury.

[0023] In some embodiments, the vector is formulated into a pharmaceutical composition, comprising a therapeutically effective dose of the vector and at least one pharmaceutically acceptable excipient and / or carrier. In some particular embodiments, the pharmaceutical composition further comprises another therapeutic agent.

[0024] In some embodiments, the vector is administered by injection.

[0025] DETAILED DESCRIPTION OF THE INVENTION

[0026] The invention provides a gene therapy approach for the treatment of sympathetic nervous system hyperactivity-associated conditions using an expression vector for Botulinum toxin serotype A Light-Chain” (or BoNT / A-LC).

[0027] An expression vector for BoNT / A-LC has the effect of inhibiting the release of norepinephrine from sympathetic neurons more specifically and more efficiently than an expression vector for the Botulinum toxin light chain of other serotypes.

[0028] As used herein “Gene therapy” refers to a treatment of an individual which involves delivery of nucleic acid of interest into an individual's cells for the purpose of treating a disease. Delivery of the nucleic acid is generally achieved using a delivery vehicle, also known as a vector. Viral and non-viral vectors may be employed to deliver a gene to a patient's cells. The term "treating" or "treatment", as used herein, is defined as the application or administration of vector as per the present disclosure or of a composition comprising the vector to a patient in need thereof with the purpose to reverse, alleviate, inhibit the progress of, or prevent the disorder or condition to which such term applies, or reverse, alleviate, inhibit the progress of, or prevent one or more symptoms of the disorder or condition to which such term applies. As used herein, the terms “treatment” or “treat” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patients at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and include suppression of clinical relapse. The treatment may be administered to a patient having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a patient beyond that expected in the absence of such treatment.

[0029] As used herein, the term “patient” or “individual” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment. Preferably, a patient or individual according to the invention is a human.

[0030] The sympathetic nervous system also called “SNS”, “Sympathetic autonomic nervous system”, or “SANS” is one of the divisions of the autonomic nervous system. The autonomic nervous system is separated into parasympathetic, and sympathetic nervous system. Both divisions work alongside each other to regulate the body’s unconscious actions.

[0031] Activation of the SNS leads to a “fight or flight” response. In this process, blood flow increases, as well as oxygen delivery, vasoconstriction of most blood vessels, and heart rate. When a signal is sent, presynaptic neurons of SNS utilize acetylcholine (ACh) as their neurotransmitter to trigger a response. Postsynaptic sympathetic neurons generally produce norepinephrine (NE), also called noradrenaline, as their effector transmitter to act upon target tissues. NE acts on adrenergic receptors located in various organs and tissues such as the cortex, heart, blood vessels, bronchi, intestine, and much more.

[0032] The term “sympathetic nervous system hyperactivity” used herein refers to a significant increase in the activity of the sympathetic nervous system, the “fight or flight” response. Hyperactivity of the SNS leads to a significant release of norepinephrine by sympathetic nerve endings and aberrant norepinephrine levels. This can cause various pathological manifestations such as hypertension, tachyarrhythmias, hyperhidrosis, peripheral vasoconstriction, and hyperthermia or hypothermia and result in various diseases that are well-known in the art. A non-exhaustive list of sympathetic nervous system hyperactivity-associated conditions include cardiovascular diseases, including ischemic heart disease, chronic heart failure and hypertension; kidney disease; diabetes mellitus, in particular type II diabetes; obesity; metabolic syndrome; obstructive sleep apnea; depression; ulcerative colitis; erectile dysfunction; Raynaud’s syndrome; and neurological disorders such as spinal cord injury.

[0033] By “cardiovascular diseases” it means herein conditions affecting the heart or blood vessels. Norepinephrine increases heart rate, cardiac contractility, and vascular tone when released from postganglionic sympathetic neurons. Therefore, an increase in this release can cause a higher risk of cardiovascular diseases such as ischemic heart disease.

[0034] By “type II diabetes mellitus”, it means herein a chronic metabolic disorder characterized by high blood sugar levels (hyperglycemia) resulting from the body’s reduced ability to produce or use insulin effectively. In patients suffering from type II diabetes mellitus, a rise in norepinephrine release is seen when the diabetes is poorly controlled. There is an association between norepinephrine and insulin levels.

[0035] By “metabolic syndrome” it means herein a cluster of conditions that occur together, increasing the risk of heart disease, stroke, type II diabetes. These conditions include increased blood pressure, high blood sugar, excess body fat around the waist, and abnormal cholesterol or triglyceride levels.

[0036] By “obesity”, it means herein an abnormal or excessive accumulation of body fat. It is a chronic disease with several causes that lead to excessive body fat. It affects the metabolism, and can cause diabetes type II, cardiovascular diseases, kidney diseases due to high blood pressure, fatty liver diseases due to excess fat in the blood.

[0037] “Obstructive sleep apnea” means herein when the throat muscles relax and block the airway. One symptom of obstructive sleep apnea is high blood pressure, morning headaches, and decreased interest in sex.

[0038] By “Raynaud’s syndrome” it means herein a limited blood flow leading to color changes at extremities such as fingers and toes. Raynaud’s syndrome causes spasms in small blood vessels. The limitation of blood flow is triggered by cold weather and stress. The arterioles and capillaries tighten more than they should and prevent blood from circulating. When stressed or cold, the “fight or flight” response is activated, releasing a certain amount of norepinephrine. The vasoconstriction of arterioles and capillaries can be due to an increased release of norepinephrine, tightening and preventing the blood from passing.

[0039] By “spinal cord injury” it means herein a damage to the bundle of nerves and nerve fibers that sends and receives signals from the brain. By injuring the spinal cord, the sympathetic nervous system is disrupted and impaired, leading to possible hyperactivity.

[0040] “Erectile dysfunction” or “ED” refers to the inability to achieve or maintain a penile erection sufficient for satisfactory sexual performance and is a common sexual dysfunction in men. ED incidence increases with age and may be of psychogenic, organic (e.g. vasculogenic, neurogenic or else) or mixed origin. Organic etiologies associated with ED include cardiovascular diseases (CVD) and associated risk factors (e.g. smoking, obesity, dyslipidemia), diabetes mellitus, neurological diseases (e.g. spinal cord injury, multiple sclerosis, or else), depression, pelvic surgery or radiotherapy for cancer, lower urinary tract symptoms (LUTS), side effects of medications, and sleep apnea among others. Erection is a complex neuro-vasculo-tissular process in which, in response to sexual stimulation, brain signals transmitted via the spinal cord to the parasympathetic / sympathetic efferent neural pathways, induce neurotransmitter release by the parasympathetic terminations of the cavernous nerves while inhibiting neurotransmitter release by sympathetic terminations of the same nerves. This combination of neurotransmitter release and inhibition ultimately leads to erection via penile arterial dilation, which is responsible for increased blood flow to the erectile tissue, and cavernosal smooth muscle fibers relaxation, ultimately causing corporal veno-occlusion and penile rigidity. The sustained release of norepinephrine by sympathetic neural terminations within the erectile tissue is responsible for the permanent contraction of cavernosal smooth muscle fibers, maintaining the penis in a flaccid state. Thus, release of norepinephrine must be inhibited during penile erection.

[0041] “a”, “an”, and “the” include plural referents, unless the context clearly indicates otherwise. As such, the term “a” (or “an”), “one or more” or “at least one” can be used interchangeably herein; unless specified otherwise, “or” means “and / or”.

[0042] Expression vector The gene therapy approach according to the present disclosure uses an expression vector encoding an isolated botulinum toxin serotype A light chain (BoNT / A-LC) that is expressed in sympathetic neurons.

[0043] An isolated botulinum toxin serotype A light chain (BoNT / A-LC) refers to BoNT / A-LC devoid of other portions of a botulinum toxin. Isolated BoNT / A-LC lacks a botulinum toxin heavy chain (BoNT-HC) or any fragment thereof.

[0044] Representative examples of BoNT / A-LC include without limitation the amino acid sequence of SEQ ID NO: 1 and functional variants thereof. SEQ ID NO:1 corresponds to the amino acid sequence of BoNT / A-LC subtype Al. Other subtypes of serotype A exist, such as A2 to A8, and can also be used according to the present invention. For example, the amino acid sequences of BoNT / Al-LC (SEQ ID NO:1, which corresponds to residues 1 to 451 of SwissProt accession number P0DPI0.1) and BoNT / A2-LC (which corresponds to residues 1 to 451 of SwissProt accession number Q54894.3) share 95.3% identity.

[0045] In the following description, the amino acid residues are designated by the standard one letter amino acid code and the indicated positions are determined by alignment with SEQ ID NO:1. One skilled in the art can easily determine the positions in another BoNT / A-LC protein, by alignment with the reference sequence using appropriate software available in the art such as BLAST, CLUSTALW and others.

[0046] The term BoNT / A-LC protein according to the invention thus encompasses all variants of BoNT / A-LC comprising an amino acid sequence having at least 70% sequence identity with SEQ ID NO: 1 that have BoNT / A-LC activity (e.g. functional variants). BoNT / A-LC activity according to the present disclosure relates in particular to the capacity of BoNT / A-LC to inhibit norepinephrine (or noradrenalin) release from neurons, in particular sympathetic neurons. BoNT / A-LC activity can be assessed for example using the noradrenalin release assay as disclosed in the examples. Vectors expressing BoNT / A-LC have a higher inhibitory activity (i.e. enhanced inhibitory activity) on noradrenalin release from sympathetic neurons compared to vectors expressing the light chain of other botulinum toxin serotypes, in particular Bont / F-LC.

[0047] As used herein, the term “functional” with respect to a BoNT / A-LC protein, including variants and fragments thereof refers to a BoNT / A-LC protein having BoNT / A-LC activity as disclosed herein. As used herein, the term “variant” refers to a polypeptide comprising an amino acid sequence having at least 70% sequence identity with the native sequence; preferably having at least 75%, 80%, 85%, 90%, 95%, 98% or 99% sequence identity with the native sequence. The term “variant” refers to a functional variant having the activity of the native sequence. The activity of a variant may be assessed using methods well-known by the skilled person such as those disclosed in the examples (for example, SNARE cleavage activity).

[0048] The term “variant” includes fragments. As used herein, the term “fragment” with respect to a BoNT / A-LC protein, refers to a polypeptide having a sequence of at least 20 consecutive amino acids from a BoNT / A-LC protein sequence. A BoNT / A-LC fragment is also named truncated form of BoNT / A-LC.

[0049] In one embodiment, the BoNT / A-LC protein further comprises a Tag, such as a poly-Histidine tag.

[0050] The percent amino acid sequence or nucleotide sequence identity is defined as the percent of amino acid residues or nucleotides in a Compared Sequence that are identical to the Reference Sequence after aligning the sequences and introducing gaps if necessary, to achieve the maximum sequence identity and not considering any conservative substitutions for amino acid sequences as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways known to a person of skill in the art, for instance using publicly available computer software such as the GCG (Genetics Computer Group, Program Manual for the GCG Package, Version 7, Madison, Wisconsin) pileup program, or any of sequence comparison algorithms such as BLAST (Altschul et al., J. Mol. Biol., 1990, 215, 403-), FASTA or CLUSTALW. When using such software, the default parameters, are preferably used. The BLASTP program uses as default a word length (W) of 3 and an expectation (E) of 10.

[0051] In some embodiments, the term "variant" refers to a polypeptide having an amino acid sequence that differs from a native sequence by the substitution, insertion and / or deletion of less than 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10 or 5 amino acids. In a preferred embodiment, the variant differs from the native sequence by one or more conservative substitutions, preferably by less than 50, 40, 30, 25, 20, 15, 10 or 5 conservative substitutions. Conservative substitutions are substitutions of one amino acid with another having similar chemical or physical properties (size, charge or polarity), which substitution generally does not adversely affect the biochemical, biophysical and / or biological properties of the protein. Examples of conservative substitutions may be within the following groups: Group 1 -small aliphatic, non-polar or slightly polar residues (A, S, T, P, G); Group 2-polar, negatively charged residues and their amides (D, N, E, Q); Group 3 -polar, positively charged residues (H, R, K); Group 4-large aliphatic, nonpolar residues (M, L, I, V, C); and Group 5-large, aromatic residues (F, Y, W); or within the groups of basic amino acids (R, K, H), acidic amino acids (D, E), polar amino acids (Q, N), hydrophobic amino acids (M, L, I, V), aromatic amino acids (F, W, Y), and small amino acids (G, A, S, T).

[0052] In some particular embodiments, the isolated botulinum toxin serotype A light chain comprises an amino acid sequence having at least 70% identity, preferably at least 75% identity, at least 80% identity, even more preferably at least 85% identity, at least 90% identity, at least 94%, 95%, 96% 97%, 98%, 99%, 99,5% identity with the sequence of SEQ ID NO: 1.

[0053] The invention may use any vector suitable for the delivery and expression of nucleic acid into individual’s cells, wherein the vector is suitable for expression of nucleic acid into individual’s target cells (i.e. neurons). Such vectors that are well-known in the art include viral and non-viral vectors, wherein said vectors may be integrative or non-integrative; replicative or non- replicative. The vector is in particular suitable for gene therapy; more particularly gene therapy directed to neuron cells.

[0054] Non-viral vector includes the various (non-viral) agents which are commonly used to either introduce or maintain nucleic acid into individual’s cells. Agents which are used to introduce nucleic acid into individual’s cells by various means include in particular polymer-based, particle-based, lipid-based, peptide-based delivery vehicles or combinations thereof, such as with no limitations cationic polymer, dendrimer, micelle, liposome, exosome, microparticle and nanoparticle including lipid nanoparticle (LNP); and cell penetrating peptides (CPP). CPP are in particular cationic peptides such as poly-L-Lysine (PLL), oligo-arginine, Tat peptides, Penetratin or Transportan peptides and derivatives thereof such as for example Pip. Agents which are used to maintain nucleic acid into individual’s cells (either integrated into chromosome(s) or else in extrachromosomal form) include in particular naked nucleic acid vectors such as plasmids, transposons and mini-circles, and gene-editing and RNA-editing systems. Transposon includes in particular the hyperactive Sleeping Beauty (SB100X) transposon system (Mates et al. 2009). Gene-editing and RNA-editing systems may use any site-specific endonuclease such as Cas nuclease, TALEN, meganuclease, zinc finger nuclease and the like. In addition, these approaches can advantageously be combined to introduce and maintain the nucleic acid of the invention into individual’s cells. Viral vectors are by nature capable of penetrating into cells and delivering nucleic acid(s) of interest into cells, according to a process named as viral transduction. As used herein, the term “viral vector” refers to a non-replicating, non-pathogenic virus engineered for the delivery of genetic material into cells. In viral vectors, viral genes essential for replication and virulence are replaced with an expression cassette for the transgene of interest. Thus, the viral vector genome comprises the transgene expression cassette flanked by the viral sequences required for viral vector production. As used herein, the term “recombinant virus” refers to a virus, in particular a viral vector, produced by standard recombinant DNA technology techniques that are known in the art. As used herein, the term “virus particle” or “viral particle” is intended to mean the extracellular form of a non-pathogenic virus, in particular a viral vector, composed of genetic material made from either DNA or RNA surrounded by a protein coat, called the capsid, and in some cases an envelope derived from portions of host cell membranes and including viral glycoproteins. As used herein, a viral vector refers to a viral vector particle, in particular recombinant viral vector particle.

[0055] A preferred vector for delivering the nucleic acid (nucleic acid construct) of the invention is a viral vector, in particular suitable for gene therapy, more particularly gene therapy directed to target cells (i.e., neurons) in the individual. In particular, the viral vector may be derived from a non-pathogenic parvovirus such as adeno-associated virus (AAV), a retrovirus such as a gammaretrovirus, spumavirus and lentivirus, an adenovirus, a poxvirus and an herpes virus. Lentivirus vector may be pseudotyped with an envelope glycoprotein from another virus for targeting the cells / tissues of interest. In some particular embodiments, the vector is an AAV vector or a herpes simplex virus (HSV) vector.

[0056] As used herein, the term AAV includes the different AAV serotypes including natural or synthetic AAV serotypes. Natural AAV serotypes may be isolated from both human and nonhuman primates. Synthetic AAV serotypes include variant, hybrid or chimeric AAV serotypes. Preferred AAV serotypes are human serotypes, more preferably human AAV of serotypes 2, 5, and 9, most preferably human AAV of serotype 5, which is the serotype displaying the highest level of neurotropism.

[0057] HSV vector is suitable for gene therapy, it is stable, easy to produce, and targets neurons including sympathetic neurons. HSV vector includes a HSV-1 vector or a HSV-2 vector. In some preferred embodiments, the vector is an HSV vector, preferably a defective HSV vector, even more preferably a defective non replicative HSV vector derived from HSV-1.

[0058] The term “defective HSV vector” refers both to defective recombinant HSV vectors and HSV amplicon vectors. The term “defective recombinant HSV”, as used herein describes a helperindependent vector, the genome of which comprises a complete deletion of the genes coding for two essential proteins, known as ICP4 and ICP27. The ICP4 gene is present in two copies, located in the inverted repeated sequences known as c and c' of the virus genome, and both copies of this gene are deleted. The gene encoding ICP27 is located in the unique long (UL) sequence of the virus genome. Preferentially, helper-independent vectors according to the invention carry the therapeutic expression cassette(s) embedded into the LAT (Latency Associated Transcripts) locus (Berthomme et al. 2000 and Berthomme et al. 2001), which is a repeated locus that is contained in the inverted repeated sequences known as b and b' of the virus genome. More preferentially, the expression cassette is placed between the LTE region and the DNA insulator (INS) sequence present downstream of the LTE. The b and b' sequences of the virus genome are also known as TRL (Terminal Repeat L) and IRL (Internal Repeat L) respectively, while the c' and c sequences are also known as IRS (Internal Repeat S) and TRS (Terminal Repeat S), where L and S refer respectively to the unique long (L) and unique short (S) sequences of preferably the HSV-1 genome. These HSV vectors are multiplied in cell lines simultaneously expressing the proteins ICP4 and ICP27 (Marconi et al, 2010).

[0059] By " Amplicon or amplicon vector" it means a helper-dependent vector, the genome of which lacks most or all HSV genes coding for virus proteins. The genome of amplicon vectors is a concatemeric DNA composed of multiple copies in tandem of a plasmid -known as the amplicon plasmid- that carries one origin of DNA replication and one packaging signal from HSV-1 genome, in addition to transgenic DNA (i.e. transcription cassettes) of interest.

[0060] In some more preferred embodiments, the defective HSV vector lacks at least the genes coding for the essential proteins ICP4 and ICP27, preferentially a vector lacking both ICP4 and ICP27. This vector can lack other genes coding for non-essential proteins, such as ICP34.5, UL55, UL56 and / or UL41.

[0061] In some more preferred embodiments, the expression cassette for the therapeutic transgene according to the present disclosure is introduced into the LAT locus. The expression vector according to the present disclosure comprises a transgene encoding an isolated botulinum toxin serotype A light chain (BoNT / A-LC) operably linked to a promoter active in sympathetic neurons.

[0062] The term "transgene" refers to a particular nucleic acid sequence encoding for an RNA and / or a polypeptide or a portion of a polypeptide to be expressed in a cell into which the nucleic acid sequence is introduced. The term "transgene" includes (1) a nucleic acid sequence that is not naturally found in the cell (i.e. a heterologous nucleic acid sequence); (2) a nucleic acid sequence that is a mutant form of a nucleic acid sequence naturally found in the cell into which it has been introduced; (3) a nucleic acid sequence that serves to add additional copies of the same (i.e., homologous) or a similar nucleic acid sequence naturally occurring in the cell into which it has been introduced; or (4) a silent naturally occurring or homologous nucleic acid sequence whose expression is induced in the cell into which it has been introduced. " Mutant form" means a nucleic acid sequence that contains one or more nucleotides that are different from the wild-type or naturally occurring sequence, i.e., the mutant nucleic acid sequence contains one or more nucleotide substitutions, deletions, and / or insertions. In some cases, the transgene may also include a sequence encoding a leader peptide or signal sequence such that the transgene product will be secreted from the cell, or the transgene may include both a leader peptide or signal sequence plus a membrane anchor peptide or even be a fusion protein between two naturally occurring proteins or part of them, such that the transgene will remain anchored to cell membranes.

[0063] The expression vector according to the present disclosure expresses the transgene in the individual’s target cells, i.e., in sympathetic neurons, in vivo.

[0064] The term "expressing a transgene" means when a transgene is transcribed to mRNA and the mRNA is translated to a polypeptide.

[0065] BoNT / A-LC transgene expression in the target cells (i.e., sympathetic neurons) may be measured as the RNA or protein level in appropriate cell system using standard assays that are well-known in the art, such as those disclosed in the examples. An example of suitable cell system is BE(2)-C cell line (SK-N-BE(2)-C; ATCC CRL-2268), a model of sympathetic neurons (Biedler & Spengler, Science (New York, N. Y.), 1976, 191(4223), 185-187; doi.org / 10.1126 / science.942798). For ease of detection, BoNT / A-LC can be co-expressed with a reporter protein such as GFP or luciferase. In some preferred embodiments, the transgene encoding BoNT / A-LC comprises the nucleotide sequence of SEQ ID NO: 2 or a sequence having at least 70%, preferably at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99,5% identity with the nucleotide sequence of SEQ ID NO:2.

[0066] The transgene encoding BoNT / A-LC is usually included in a nucleic acid construct comprising a nucleotide sequence encoding the molecule. The nucleic acid construct may comprise or consist of DNA, RNA or a synthetic or semi-synthetic nucleic acid which is expressible in the individual’s target cells (sympathetic neurons). The sequence encoding BoNT / A-LC may be codon-optimized for expression in the individual’s target cells, preferably a human individual. Appropriate softwares for codon optimization in the desired individual are well-known in the art and publicly available.

[0067] The nucleic acid construct comprises an expression cassette wherein the coding sequence is operably linked to appropriate regulatory sequences for expression of a transgene in the individual’s target cells. Such sequences which are well-known in the art include in particular a promoter, and further regulatory sequences capable of further controlling the expression of a transgene, such as without limitation, enhancer; terminator such as polyadenylation signal; intron; silencer, in particular tissue-specific silencer such as miRNAs; and post-translational regulatory element.

[0068] The term “promoter” refers herein to a DNA regulatory region capable of binding RNA polymerase in a mammalian cell and allowing transcription initiation of an operably linked downstream (3' direction) sequence. For purposes of the present invention, a promoter sequence includes at least the minimum number of bases or elements necessary to initiate transcription of a gene of interest at levels detectable above the background. Within the promoter sequence is a transcription initiation site, as well as RNA polymerase binding domains. Eukaryotic promoters will often, but not always, contain " TATA" boxes and other DNA motifs, such as " CAT" or " SP1" boxes.

[0069] By “active in sympathetic neurons” it is meant herein that the promoter is active in the sympathetic neurons and drives transcription (i.e. mRNA expression) of the BoNT / A-LC transgene to mRNA which is subsequently translated to a polypeptide (mRNA and protein expression, i.e., expression). The promoter may drive the transgene expression in other cells or tissues (i.e. non-specific, as long as it is active at least in sympathetic neurons) or may be specific for neurons or even specific for sympathetic neurons. The promoter may be a tissue-specific, ubiquitous, constitutive or inducible promoter that is functional in the individual’s target cells. Examples of tissue-specific promoter specific for sympathetic neurons include dopamine beta hydrolase (DBH) promoter, vesicular monoamine transporter 2 (VMAT2) promoter and norepinephrine transporter (NET) promoter.

[0070] Ubiquitous promoters usually induce higher levels of expression of the transgene in the target cells compared to other promoters. The fact that BoNT / A-LC is preferentially expressed in sympathetic neurons advantageously allows the use of a ubiquitous promoter to provide both higher levels and higher specificity of expression of BoNT / A-LC in the target cells (sympathetic neurons).

[0071] Representative ubiquitous promoters which can be used in the present invention include without limitation: phosphoglycerate kinase promoter (PGK); elongation factor-1 alpha (EF-1 alpha) promoter; dihydrofolate reductase promoter; P-actin promoter; viral promoters such as cytomegalovirus (CMV) immediate early enhancer and promoter, cytomegalovirus enhancer / chicken beta actin (CAG) promoter, SV40 early promoter and retroviral 5’ and 3’ LTR promoters including hybrid LTR promoters. The promoters are advantageously human promoters, z.e., promoters from human cells or human viruses. Such promoters are well-known in the art and their sequences are available in public sequence data base.

[0072] In some particular embodiments, the functional promoter used to control the expression of the transgene is a ubiquitous promoter, preferably human elongation factor 1 alpha (hEFl-a) promoter or human cytomegalovirus immediate early enhancer and promoter (hCMV). In some preferred embodiments, the hEFl-a promoter comprises the nucleotide sequence of SEQ ID NO: 3 and / or the hCMV promoter comprises the nucleotide sequence of SEQ ID NO: 4.

[0073] Pharmaceutical compositions and treatment

[0074] The invention encompasses the use of a pharmaceutical composition comprising a vector according to the present disclosure, as an active component.

[0075] The vector and derived pharmaceutical composition of the present disclosure may be used for treating diseases by gene therapy, in particular targeted gene therapy directed to neurons.

[0076] The pharmaceutical composition comprises a therapeutically effective amount of the vector and a pharmaceutically acceptable carrier and / or vehicle. In the context of the invention, a therapeutically effective amount refers to a dose sufficient for reversing, alleviating or inhibiting the progress of the disorder or condition to which such term applies, or reversing, alleviating or inhibiting the progress of one or more symptoms of the disorder or condition to which such term applies. The term "effective dose" or "effective dosage" is defined as an amount sufficient to achieve, or at least partially achieve, the desired effect. The effective dose is determined and adjusted depending on factors such as the composition used, the route of administration, the physical characteristics of the individual under consideration such as sex, age and weight, concurrent medication, and other factors, that those skilled in the medical arts will recognize. The effective dose can be determined by standard clinical techniques. In addition, in vivo and / or in vitro assays may optionally be employed to help predict optimal dosage ranges.

[0077] A "pharmaceutically acceptable carrier” refers to a vehicle that does not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.

[0078] The pharmaceutical vehicles and carriers are those appropriate to the planned route of administration, which are well known in the art. The pharmaceutical composition is formulated for administration by a number of routes, including but not limited to parenteral and local. The pharmaceutical composition of the present invention is generally administered according to known procedures, at dosages and for periods of time effective to induce a therapeutic effect in the patient. The pharmaceutical composition may be administered by any convenient route, such as in a non-limiting manner by injection, perfusion or implantation. The administration can be systemic, local or systemic combined with local; systemic includes parenteral and oral, and local includes local and loco-regional. Systemic administration is preferably parenteral such as subcutaneous (SC), intramuscular (IM), intravascular such as intravenous (IV) or intraarterial; intraperitoneal (IP); intradermal (ID), epidural or else. Local is for example intracavemosally, i.e., in the corpora cavernosa of the penis.

[0079] In some particular embodiments, the pharmaceutical composition or vector is administered by injection, preferably by local administration, more preferably intracavemosally.

[0080] The pharmaceutical composition may also comprise an additional therapeutic agent, in particular an agent useful for the treatment of a disease according to the present disclosure. The additional therapeutic agent is preferably selected from the group consisting of PDE5 inhibitors or prostaglandin El. Typically, said PDE5 inhibitor or prostaglandin El is locally delivered i.e. intracavernosally or intra-urethrally. In an alternative embodiment, said PDE5 inhibitor is delivered by systemic administration i.e. intravenously, intranasally or orally. Preferably, the PDE5 inhibitor is administered orally.

[0081] The pharmaceutical composition or vector may be used for treating various conditions associated with sympathetic nervous system hyperactivity. In some particular embodiments, the sympathetic nervous system hyperactivity-associated conditions are selected from the group consisting of: of: cardiovascular diseases, including ischemic heart disease, chronic heart failure and hypertension; kidney disease; diabetes mellitus, in particular type II diabetes; obesity; metabolic syndrome; obstructive sleep apnea; depression; ulcerative colitis; erectile dysfunction; Raynaud’s syndrome; and neurological disorders such as spinal cord injury; preferably erectile dysfunction.

[0082] The present invention also relates to the use of a vector or composition according to the present disclosure in the manufacture of a medicament for treatment of a disease as disclosed herein in a subject in need thereof.

[0083] The present invention also relates to the use of a vector or composition according to the present disclosure for the treatment of a disease as disclosed herein, in a subject in need thereof.

[0084] The present invention also relates to a pharmaceutical composition for treatment of a disease as disclosed herein, comprising a vector according to the disclosure as active component.

[0085] The present invention also relates to a pharmaceutical composition comprising a vector according to the disclosure for treating a disease as disclosed herein.

[0086] The vector or pharmaceutical composition of the invention may be used in combination with another therapy, wherein the combined therapies may be simultaneous, separate or sequential.

[0087] In a preferred embodiment, the vector or pharmaceutical composition of the invention may be used in combination with another therapy, wherein the combined therapies may be simultaneous, separate or sequential, wherein the vector or pharmaceutical composition of the invention may be administered intracavernosally, and another therapy may be administered orally. In a preferred embodiment, the other therapy may be to increase blood flow such as PDE5-inihibitors (PDE5-Is) (such as sildenafil, vardenafil, tadalafil, avanafil, udenafil or mirodenafil), preferably sildenafil or tadalafil.

[0088] In an embodiment, the present invention also relates to a vector or a pharmaceutical composition of the invention used in combination with a PDE5 inhibitor such as sildenafil, wherein the combined therapies may be simultaneous, separate, or sequential, preferably wherein the vector or pharmaceutical composition of the invention is administered intracavernosally, and the PDE5 inhibitor such as sildenafil is administered orally.

[0089] The various embodiments of the present disclosure can be combined with each other, and the present disclosure encompasses the various combinations of embodiments of the present disclosure.

[0090] The practice of the present invention will employ, unless otherwise indicated, conventional techniques, which are within the skill of the art. Such techniques are explained fully in the literature.

[0091] The invention will now be exemplified with the following examples, which are not limitative, with reference to the attached drawings in which:

[0092] BRIEF DESCRIPTION OF THE FIGURES

[0093] Figure 1. Schematic map of Puc57 plasmid (A), HSV1 vector amplicon of PA2 (B) and PA7 (C) types. A. In Puc57 plasmid, the luciferase is under the control of EFl a promoter. B. In PA2 vector amplicons, the transgenes (BoNT / A-LC, BoNT / F-LC or BoNT / D-LC) are under the control of human CMV promoter (hCMV). In order to titrate and normalize the transgene expression, a GFP expression cassette is under the control of the viral promoter IE4 / 5. C. In PA7, the BoNT / A-LC or BoNT / F-LC transgene (or luciferase) are under the control of EFla promoter while the viral promoter IE4 / 5 controls the expression of a fusion protein with GFP and renilla luciferase, RLuc.

[0094] Figure 2. A. NE release inhibition in BE(2)-C post transduction with HSV-1 amplicon derived vectors expressing BoNT / A / F / D-LC under the control of hCMV promoter (PA2-CMV-BoNTA-LC, PA2-CMV-BoNT / F-LC, PA2-CMV-BoNT / D-LC) (n=5). The result is expressed in percentage of NE release inhibition considering PA2-CMV-BoNT / D-LC as negative control. B. qPCR quantifying the expression of the BoNTA / F-LC RNA post BE(2)-C transduction with HSV-1 amplicon derived vectors (PA2-CMV-BoNT / A-LC and PA2-CMV-BoNT / F-LC) (n=3).

[0095] Figure 3. A. NE release inhibition in BE(2)-C post transduction with HSV-1 amplicon derived vectors expressing BoNT / A-LC or BoNT / F-LC under the control of EFla promoter (PA7-EFla-BoNT / A-LC or PA7-EFla-BoNT / F-LC (n=6)). B. qPCR quantifying the expression of the BoNT / A-LC or / F-LC RNA post BE(2)-C transduction with HSV-1 amplicon derived vectors (PA7-EFla-BoNT / A-LC or PA7-EFla-BoNT / F-LC (n=3)). C. Western blot quantifying SNAP-25 (up) and VAMP -2 (down) cleavage with GAPDH as control at increasing MOI (0.3, 1, 3). Vectors expressing Luciferase are used as negative control (MOI 3).

[0096] Figure 4. A. qPCR quantifying the expression of the BoNT / A-LC or BoNT / F-LC RNA posttransduction of primary cultures from sympathetic ganglia (SCG) from adult rats with HSV-1 amplicon derived vectors (PA2-CMV-BoNT / A-LC and PA2-CMV-BoNT / F-LC), n=4. B. qPCR quantifying the expression of the BoNT / A-LC or BoNT / F-LC RNA post-transduction of primary cultures from sensory ganglia (DRG) from adult rats with HSV-1 amplicon derived vectors (PA2-CMV-BoNT / A-LC and PA2-CMV-BoNT / F-LC, n=4).

[0097] Figure 5: Erectile responses elicited by electrical stimulation of the cavernous nerve (ES CN) at increasing frequencies in anaesthetized spontaneously hypertensive rats (SHR) treated by either saline (n=ll), control vectors at 2.107PFU (n=13), or BoNT / A LC HSV1 Non-Replicating Recombinant (NR-Rec) vectors (GenED vectorat 2.106PFU (n=12) at 5 weeks post intracavernosal (ic) injection. A. Erectile responses are expressed as AICP / MAP. B. Erectile responses are expressed as AUC45 / MAP. C. Erectile responses are expressed as AUCtot / MAP. Data are mean ± SEM. **p<0.01, ***p<0.001, ns: non-significant, two-way ANOVA.

[0098] Figure 6: Erectile responses elicited by ES CN at increasing frequencies in anaesthetized SHR treated by either control vectors ic at 2.107PFU (5 weeks post ic injection, n=13), BoNT / A LC HSV1 NR-Rec vectors ic at 2.106PFU (5 weeks post ic injection, n=12), sildenafil iv at 0.3 mg / kg (on the day of erectile function (EF) evaluation, n=l 1) or combined BoNT / A LC HSV1 NR-Rec vectors ic at 2.106PFU and sildenafil iv at 0.3 mg / kg (n=l 1).

[0099] A. Erectile responses are expressed as AICP / MAP. B. Erectile responses are expressed as AUC45 / MAP. C. Erectile responses are expressed as AUCtot / MAP. Data are mean ± SEM. *p<0.05, **p<0.01, ***p<0.001, ns: non-significant, two-way ANOVA. EXAMPLES

[0100] Example 1

[0101] Material and methods

[0102] 1. Cell and bacteria models

[0103] 1.1 Bacteria

[0104] For plasmid amplification the NEB® 5-alpha competent E. coli bacteria were used. For Takara In-Fusion® Snap Assembly Cloning kit (Takara bio), chemically competent Stellar™ cells were used.

[0105] 1.2 Cell lines

[0106] Vero 7B is a Vero (African green monkey epithelial cell line) cell line expressing ICP27 and ICP4 (Krisky et al., Gene Therapy, 1998, 5, 1593-1603) used as a transcomplementing cell line for HSV-1 amplicon vector production. Vero 7B cells were cultured in DMEM, 10% FBS, penicillin, streptomycin.

[0107] BE(2)-C (SK-N-BE(2)-C) (ATCC CRL-2268) is a model of sympathetic neurons (Biedler & Spengler, Science (New York, N. Y.), 1976, 191(4223), 185-187; doi.org / 10.1126 / science.942798). BE(2)-C were cultured in DMEM F12, 10% FBS, penicillin, streptomycin.

[0108] Gli36 is a human glioblastoma cell line (Rothenberg et al., Cancer Research, 2010, 70(6), 2158— 2164; doi.org / 10.1158 / 0008-5472. CAN-09-3458). Gli36 was used to titrate the virus production and as a non-sympathetic cell line control. Gli36 cells are cultured in DMEM, 10% FBS, penicillin, streptomycin.

[0109] 1.3 Primary culture of adult Rat superior cervical Ganglia (SCG) neurons and rat dorsal root ganglia (DRG)

[0110] Adult Wistar rats (250 g) were sacrificed in accordance with the European Communities Council Directives 2010 / 63 / UE on the use of laboratory animal and care regulation in force in France (Ministry of Agriculture). SCG and DRG were obtained from the same rats. The SCG were used as models of sympathetic neurons whereas the DRG were used as a control for non-sympathetic neurons (sensory neurons). 1.4 Dissection of rat SCG

[0111] After death, the animals were decapitated, the head was placed on the dissecting pad and the trachea was exposed, the skin and fat were removed from the trachea under microscope, the muscle were then dissected and the carotid artery was exposed and makes a Y shape ribbon along the trachea, The SCG were then dissected at the bifurcation between the trachea and the carotid, the other ganglia was dissected on the other side of the trachea.

[0112] 1.5 Dissection of rat DRG

[0113] After death, a dorsal middle incision was performed to expose S3-L4 vertebrae. Supraspinous, interspinous, and yellow ligaments, were sectioned. A laminectomy was then performed to expose the spinal cord and T13 to S2 DRG. T13 to SI DRG were harvested and dissected in a Petri dish containing sterile ice-cold Hanks balanced salt solution containing 0.4% dextrose (HBSS-D)

[0114] 1.6 Culture of rat SCG and DRG

[0115] The SCG or DRG were transferred to F12 medium and dissociated with trypsin / collagenase enzymes, filtered with 50 pm strainers, and plated on poly-D-lysine coated plates in DMEM-F12 medium, lx penicillin / streptomycin, lOOmM sodium pyruvate, 10% fetal bovine serum and lOOng / ml NGF. After 2 days, the medium was renewed and 10 pM PAR. AC and lOOng / ml NGF was added. After 4 days, once neurite outgrowth allowed infection of internal neurons, a suspension of 2 * 106vector particles was added to the ganglia and left for 48 hours.

[0116] 2. Construction and Titration of Non-Replicative HSV-1 Amplicon Vectors

[0117] 2.1 Construction of HSV-1 amplicon plasmids

[0118] Two different HSV-1 amplicon plasmids, PA2, and PA7 (nomenclature from the laboratory) were used and only differ from the reporter gene controlled by the viral promoter IE4 / 5, GFP for PA2 and GFP fused with luciferase for PA7 (Figure IB and 1C). The HSV-1 amplicon plasmid PA2 containing the transgenes BoNT / A-LC, BoNT / F-LC and BoNT / D-LC, under the control of the CMV promoter were disclosed previously (Joussain et al., Toxins, 2019, 11(2), 123; doi.org / 10.3390 / toxinsl 1020123). The HSV-1 amplicon plasmids PA7 are identical to the HSV-1 amplicon plasmid PA5 previously described in Joussain et al., 2022 (International Journal of Molecular Sciences, 2022, 23(15), 8474; doi.org / 10.3390 / ijms23158474), except for the replacement of the ampicillin resistance gene by a kanamycin resistance gene. pUC57 plasmids containing the transgenes BoNT / A-LC, and BoNT / F-LC were ordered from GenScript. In order to test the transgenes under the control of EFla promoter, a pUC57 EFla-Luc was also ordered from GenScript. pUC57 EFla-Luc comprises the EFla-Luc construct flanked by Spel and Notl restriction sites, a chimeric intron from human P-globin and immunoglobulin heavy chain genes included in between EFla promoter and the Luc coding sequence and a Nhel restriction site included between the chimeric intron and the Luc coding sequence (Figure 1A).

[0119] To generate plasmid amplicon PA7-EFla-Luc, the EFla-Luc construct flanked by Spel and Notl restriction sites was PCR-amplified from PUC57-EFla-Luc and cloned into PA7 backbone flanked by Spel and Notl restriction sites enzymes using the In-Fusion® Snap Assembly Cloning kit (Takara bio). Since all the transgenes were flanked with Nhel and Notl sites, luciferase in PA7-EFla-Luc was replaced by BoNT / A-LC or BoNT / F-LC using the in-fusion cloning kit. Plasmid amplicons PA7-EFla-BoNT / A-LC and PA7-EFla-BoNT / F-LC were thus obtained (Figure 1C).

[0120] 2.2 HSV-1 amplicon vector production and titration

[0121] HSV-1 amplicon vectors derived from the various PA2 and PA7 amplicon plasmid constructs were produced as already described (Joussain et al., Toxins, 2019, 11(2), 123; doi.org / 10.3390 / toxins 11020123). Amplicons are defective, helper-dependent, herpes simplex virus type 1 (HSV-1 )-derived vectors that are packaged into viral particles in the presence of helper function. Non-replicative HSV-lLaLAJ virus was used as a helper. Its genome lacks the gene encoding the essential ICP27 and ICP4 proteins, which have been cloned into a bacterial artificial chromosome (BAC) and contain loxPl sites surrounding the packaging signals (Zaupa et al., Human Gene Therapy, 2003, 14(11), 1049-1063. https: / / doi.org / 10.1089 / 104303403322124774). To produce HSV-l-derived amplicon vector, trans-complementing eukaryotic VERO 7B cells expressing ICP27 and ICP4 are used. The 7B cells were independently transfected with 5 to 10 pg of each amplicon plasmid using lipofectamine. The day after, transfected 7B cells were infected with the HSV-lLaLAJ helper virus. When the cytopathic effect (CPE) was maximal, cells were collected by centrifugation, disrupted by three freeze / thaw cycles to release vector stocks, and re-centrifuged at lOOOxg for 10 min to pellet the cell debris. Helper and vector particles in the supernatants where then titrated as already described. Cells (Gli 36 cell line) expressing fluorescent GFP were scored at 24 h postinfection using an inverted fluorescence microscope (Olympus, Tokyo, Japan) yielding transducing units (TU). Titers of helper virus particles were provided as plaque-forming units (PFU) per milliliter. Serial passages of the vector populations were then carried out on 7B cells, thus increasing the TU / PFU ratio to 99 / 1 or higher.

[0122] 3. Cell infection with HSV-1 amplicon vectors

[0123] 3.1 Primary cultures

[0124] After 4 days of primary culture of rat superior cervical Ganglia (SCG) or dorsal root ganglia (DRG) neurons, or once explanted SCG or DRG displayed neurite outgrowth (usually after 3 days in culture), the culture medium was withdrawn and replaced with a minimal amount of medium containing the vector suspension. After two hours of contact, fresh medium was added to the wells, and the cultures were kept at 37°C in a 5% CO2 for 48 hours.

[0125] 3.2 Cell lines

[0126] After passage, the BE(2)-C cells were infected for 48 hours and were kept at 37°C in a 5% CO2 for 48 hours.

[0127] 4. Transgene expression analysis

[0128] The same primer set located in the 3’ UTR shared by BoNT / A-LC and BoNT / F-LC was used to analyze the expression of both transgenes by qPCR. The expression was normalized by GFP expression to avoid bias due to the variability of infection. RNA extraction was performed with RNeasy Mini kit (Qiagen GmbH,) according to the manufacturer protocol and cDNA was obtained using Luna Superscriptlll (Thermofisher) CA, USA), qRT-PCR was performed using iTAQ universal SYBR green supermix (Biorad) and using the oligonucleotides shown in Table 1 (Eurogentech).

[0129] 5. SNARE protein cleavage analysis

[0130] After 48 hours of infection, the culture medium was removed, and cells were Lysed into RIPA lysis buffer (Thermofisher). Then, homogenates were centrifuged at 10,000 rpm for 10 min at 4 °C. Cell lysates were resuspended into Laemmli buffer (Bio-Rad)). After protein quantification, 50 pg of protein was used to perform electrophoresis (Tris-HCl PAGE-Gel, Bio-Rad). Transfer of proteins was performed using an activated-methanol membrane (Immobilon FL Transfer Membrane, Merck Millipore at 4 °C during lh30 with 0.35A. Blocking and antibody incubation were performed using the IBindTM Flex Western System SFL2000 (Thermofisher). The intensity of each band was determined by pixel density integration using LI-COR® Odyssey Western Blotting Kits.

[0131] BoNT / A-LC and BoNT / F-LC cleave 2 different SNARE proteins, to determine the activity of BoNT / F-LC Vamp2 cleavage was assessed using a rabbit anti-V Amp-2 antibody (ABCAM, AB3347) while assessing SNAP25 cleavage for BoNT / A-LC with a rabbit anti SNAP25 antibody (ABCAM, AB66066). The GAPDH was used to normalize the quantifications (Millipore CB1001). The quantification of the cleavage of VAMP2 by BoNT / F-LC was calculated as the percentage of the Vamp2 protein quantity observed in the control cells with no infection. In order to compare the efficacy of BoNT / A-LC with the cleavage of SNAP25, the same quantification was done with the percentage of the native form of SNAP25 observed with no infection. As SNAP25 protein is cleaved in 2 forms, the decrease of the native form was correlated with the increase of the cleaved form.

[0132] 6. Noradrenalin release assay

[0133] For noradrenaline release, BE(2)-C was differentiated with dibutyryl cAMP 3mM as it is done for other neuroblastoma cell lines (Kume et al., 2008). This differentiation allowed to optimize NA release to show NA release inhibition.

[0134] 48 hours after infection, the cells were stimulated with 75mM KC1, noradrenaline concentration was measured for BE(2)-C cells infected with PA2 CMV-BoNTA-LC, F or D amplicons or with the PA7-EFla BoNT / A-LCor BoNT / F-LC. The noradrenaline ELISA kit from Labor Diagnostika Nord, Nordhorn, Germany (LDN) (BA E-5200R) was used according to the manufacturer's protocol.

[0135] 7. Biosafety and biosecurity

[0136] All Genetically modified organisms (GMOs) have been declared to the French regulation agency ANSM, and the use of microorganisms and toxins highly pathogen (MOT) was authorized (ADE-182852024-6, ADE-182892024-1). To limit the risk of the use of Clostridium neurotoxins, only the light-chain (BoNT-LC) was used. BoNT-LC is not a threat because the proteolytic fragments do not cross the cellular membrane and are not capable of neuronal binding without the heavy chain (Teng et al., Gene Therapy, 2005, 12(2), 108-119; doi.org / 10.1038 / sj.gt.3302400). In addition, the HSV-1 vectors used in the study are defective and non-replicative in non- complementing cells (Oehmig et al., Molecular Therapy, 2004, 10(4), 630-643; doi.org / 10.1016 / j.ymthe.2004.06.641).

[0137] Results

[0138] The inhibition of norepinephrine (NE) (or Noradrenaline (NA)) from sympathetic neurons was studied using HSV-1 amplicon vectors expressing three different types of transgenes: botulinum toxin A light-chain (BoNT / A-LC), botulinum toxin D light chain (BoNT / D-LC) and botulinum toxin F light-chain (BoNT / F-LC).

[0139] The use of botulinum toxin light-chain is safe because it does not cross the cellular membrane and is not capable of binding to neurons without the heavy chain, as compared to complete botulinum toxin which is one of the most potent neurotoxin. In addition, the HSV-1 vectors used in the study are defective and non-replicative in vivo.

[0140] As human sympathetic primary cultures are not possible to culture for multiple experiments, the two closest models were chosen, primary culture of rat sympathetic neurons, and human cell lines from neuroblastoma tumors of the sympathetic neurons (BE(2)-C cell line).

[0141] In a first series of experiments, the ubiquitous human cytomegalovirus (hCMV) promoter was used to control the expression of the transgenes. In a second series of experiments, the EFla promoter was used to control the expression of the transgenes (BoNTA-Lc and BoNTF-Lc). Finaly, hCMV promoter was used in primary cultures of adult rats (sympathetic and sensory neurons).

[0142] Following transduction of BE(2)-C cells with HSV amplicon vector expressing BoNT / A-LC (PA2-CMV-BoNT / A-LC), a high inhibition of NE release (60 %) was observed. The inhibition of NE release observed with HSV amplicon vector expressing BoNT / A-LC (PA2-CMV-BoNT / A-LC) is at least twice higher than the inhibition (27 %) observed with the same HSV amplicon vector expressing BoNT / F-LC (PA2-CMV-BoNT / F-LC) (Figure 2A). A concordant pattern was observed at the RNA level with a significantly lower expression of BoNT / F-LC mRNA compared to BoNT / A-LC mRNA when normalized by internal control (GFP mRNA) (Figure 2B).

[0143] Analogous experiments were conducted with HSV-1 amplicon derived vectors expressing BoNT / A / F-LC driven by EFla promoter. Following the transduction of BE(2)-C cells with PA7-EFla-BoNT / A-LC, and PA7- EFla-BoNT / F-LC, a significant difference in terms of NE release was observed with a lower NE release (i.e. a higher inhibition of NE release) in cells transduced with vectors expressing BoNT / A-LC at low multiplicity of infection (MOI) (0.3). When cells were infected with higher MOI (1 or 3), no differences were observed (Figure 3A). A concordant pattern was observed at the RNA level with a significantly lower expression of BoNT / F-LC mRNA compared to BoNT / A-LC mRNA, when normalized by internal control (GFP mRNA) at MOI 0.3, and no differences or more expression of BoNT / F-LC mRNA at MOI 1 and 3 respectively (Figure 3B).

[0144] To verify if the mRNA levels of BoNT-LC were correlated with their activity, the cleavage of their SNARE protein targets, was assessed. As BoNT / A-LC and BoNT / F-LC cleave 2 different SNARE proteins, Vamp2 cleavage was assessed to determine BoNT / F-LC activity, while SNAP25 cleavage was assessed to determine BoNT / A-LC activity. To assess the role of the number of viral particles per cell on the light chains efficacy in cleaving SNARE proteins, different MOIs were used in this experiment. The quantification of the cleavage of VAMP2 by BoNT / F-LC was calculated as the percentage of the Vamp2 protein quantity observed in the control cells with no infection. In order to compare the efficacy of BoNT / A-LC with the cleavage of SNAP25, the same quantification was done with the percentage of the native form of SNAP25 observed with no infection. As SNAP25 protein is cleaved in 2 forms, the decrease of the native form was correlated with the increase of the cleaved form.

[0145] The biological effect of the botulinum toxin light chain is clearly demonstrated in Figure 3C, with a cleavage of SNAP -25 by BoNT / A-LC (see cleaved and uncleaved forms) and VAMP2 by BoNT / F-LC (see disappearance of uncleaved form) increasing with MOI. At a relatively low MOI (MOI=0.3), similar to the MOI aimed in vivo for a therapeutic approach, BoNT / A-LC displayed a better cleavage than BoNT / F-LC after infection of BE(2)-C with the transgenes under the control of EFla promoter.

[0146] Based on these results suggesting a higher expression of BoNT / A-LC than BoNT / F-LC in BE(2)-C, a comparison of this level of expression in primary cultures of sympathetic neurons versus sensory neurons was made.

[0147] Following the transduction of primary cultures of sympathetic cells (neurons from supra-cervical ganglia of adult rats) with PA2-CMV-BoNT / A-LC and PA2-CMV-BoNT / F-LC, a significant difference in terms of mRNA level was observed with a significantly lower expression of BoNT / F-LC mRNA compared to BoNT / A-LC mRNA when normalized by internal control (GFP mRNA) (Figure 4A). By contrast, no differences in terms of mRNA expression were observed in primary cultures of sensory neurons (neurons from dorsal root ganglia of adult rats) (Figure 4B)

[0148] To conclude, with both CMV and EFla promoters, the BoNT / A-LC vector performs better than the BoNT / F-LC vector in models of sympathetic neurons as shown by a higher expression level (mRNA expression level), as well as a higher SNARE cleavage activity that led to a higher noradrenaline release inhibition. These results are surprising since previous observations suggested a stronger inhibition of neuropeptide release with HSV-1 amplicon vectors expressing light chain of botulinum toxin serotype F as compared to HSV-1 amplicon vector expressing light chain of botulinum toxin of other serotypes (A, B, C, D and E; Joussain et al., 2019, precited; WO 2017 / 220800).

[0149] Example 2

[0150] Material and Method

[0151] 1. Research Design and Experimental Endpoints

[0152] Adult male spontaneously hypertensive rats (SHR) were housed at least 7 days prior to the beginning of the experiments with free access to regular diet and water, and maintained on an inversed 12h dark / light cycle (7:00 / 19:00).

[0153] Following acclimation period, a first set of SHR at 12 ± 2 weeks of age received a single intracavernosal (ic) injection with penile tourniquet application prior to the injection and left 30 mins post-injection of either saline, control vectors at 2.107PFU, or BoNT / A LC HSV1 NonReplicating Recombinant (NR-Rec) vectors at 2.106PFU.

[0154] On the day of erectile function (EF) evaluation, 4 minutes prior to the electrical stimulation of the cavernous nerve (ES CN), animals received an intravenous (zv) injection of either saline or sildenafil iv at 0.3 mg / kg.

[0155] The EF evaluation of SHR was performed 5 weeks post ic injection for the first set of rats, using the well-standard model of electrical stimulation of the cavernous nerve (ES CN)with the recording of intracavernosal pressure (ICP).

[0156] 2. Drugs and vectors Aliquots of 90pL of BoNT / A LC HSV1 NR-Rec vectors containing 2.8.106PFU were used. BoNT / A LC HSV1 NR-Rec vector is a vector wherein the BoNT / A-LC transgene is under the control of EFla promoter.

[0157] Likewise, eighteen aliquots of 90 pL of control vectors (Rec-EFla-Luc) were used, containing 3.107PFU each.

[0158] All aliquots were stored at -80°C until the day of use. On the day of administration, aliquots were thawed quickly in a dry bath at +37°C and injected into the corpus cavernosum (CC) of rats. Each aliquot was designated for a single rat, with 70 pL administered per injection, leading to 2.18.106PFU.

[0159] Sildenafil was purchased from Biosynth (ref. FS27829, Bratislava, Slovakia). Doses were chosen according to a previous study (Giuliano F, et al. J Sex Med. 2022 June; 19).

[0160] 3. Treatment administration

[0161] 3.1. Intracavernous injection with tourniquet application

[0162] All SHR received a single ic injection of one of the following compounds:

[0163] Saline

[0164] Control vectors at 2.107PFU

[0165] - BoNT / A LC HS VI NR-Rec vectors at 2.106PFU

[0166] Briefly, rats were anesthetized with isoflurane (Isovet® 2-2.5%, Piramal Critical Care, Holland) and their temperature maintained at 37°C using a homoeothermic blanket. A rubber band was wrapped around the base of penis, and a knot was tied by applying maximum tension. The tourniquet was applied in part on the penile sheath for 30 minutes.

[0167] To perform the ic injection, a tip of 30-gauge needle connected to a Hamilton syringe via a catheter was inserted into one CC (single administration site). 70 pL of one of the compounds was ic injected. An ic injection was validated when the filling of the CC was visible (erectionlike to the eye). Rats treated by control or BoNT / A LC HSV1 NR-Rec vectors received a single validated ic injection, which could be obtained following one or two ic injection attempts. As determined previously, the second ic injection, if needed, was performed at least 4 days after the first one.

[0168] Following treatment administration, SHR rats were monitored daily for any clinically observable abnormalities.

[0169] 3.2. Intravenous injection

[0170] On the day of erectile function (EF) evaluation, animals received an intravenous (iv) injection of either saline or sildenafil iv at 0.3 mg / kg. It is understood that, while the saline or sildenafil is administered by intravenous injection for rats, in human patients the administration is done orally.

[0171] Briefly, rats were anesthetized and their temperature maintained at 37°C using a homoeothermic blanket. One jugular vein was catheterized to allow the intravenous injection. Following dissection to prepare the EF evaluation, and 4 minutes before the first electrical stimulation, saline or sildenafil was intravenously injected.

[0172] 4. Erectile function evaluation: electrical stimulation of the cavernous nerve (ES CN) in anaesthetized animals

[0173] Five weeks post ic injection for groups saline, control vectors and BoNT / A LC HSV1 NR-Rec vectors (at D36), rats underwent an evaluation of their erectile function.

[0174] 4.1. Methodology

[0175] Erectile responses were elicited by ES CN and measured by monitoring intracavemous pressure (ICP) in anesthetized rats according to an experimental procedure previously described and well-standardized (Giuliano F, et al. J Urol. 1993 Aug;15; Giuliano F, et al.. J Auton Nerv Syst. 1995 Oct 5).

[0176] Rats were anesthetized and their temperature maintained at 37°C using a homoeothermic blanket. The carotid artery was catheterized with polyethylene tubings filled with heparinized saline (50 Ul / ml) to record blood pressure (BP) via a pressure transducer (Elcomatic 750, Glasgow, UK). One jugular vein was also catheterized to allow intravenous injections. Simultaneous computerized measure of BP and ICP were performed. CN was exposed at the lateral aspect of the prostate, with the aid of a dissecting microscope and mounted on a bipolar platinum electrode connected to an electrical stimulator (AMS 2100, Phymep, France).

[0177] At T= 0, the continuous recording of BP and ICP started.

[0178] At T= 5 min, 2 stimulations of the CN at 6V, 12.5 Hz, 1 ms for 45 s are performed to elicit an increase of ICP to certify the correct implantation of the catheter.

[0179] At T+ 16 min, saline or sildenafil (0.3 mg / kg) was intravenously injected.

[0180] At T+ 20 min, and at 3-minute intervals thereafter, the CN is stimulated at 6V, 1 ms for 45 s by different stimulation frequencies (0, 2, 3, 4, 5, 7.5, 10 and 12.5 Hz) in a randomized manner in order to assess the erectile responses. This set of electrical stimulations was repeated twice in view of establishing a frequency-response curve for each rat.

[0181] At T+ 60 min, the recording ends.

[0182] At the end of the ES CN experiments, the rats were euthanized by an intracardiac injection of pentobarbital (Eutasol®, Centravet, France).

[0183] 4.2. Experimental endpoints

[0184] Mean arterial pressure (MAP) and the amplitude of the erectile responses elicited by each ES CN were quantified for each rat and averaged for each experimental group by calculating:

[0185] • ICP (mmHg) / MAP (mmHg) x 100 with AICP being the difference between ICP in the flaccid state, i.e. before stimulation and ICP during the plateau phase of the erectile response, and MAP, the mean arterial pressure during the plateau phase.

[0186] • AUCtot / MAP with AUCtot, the area under the curve during the entire erectile response, measured from the beginning of the electrical stimulation until the end of the erectile response and determined using the ICP level in the flaccid state before the onset of the stimulation.

[0187] AUC45 / MAP with AUC45, the area under the curve during the first 45 seconds after the beginning of the electrical stimulation of the cavernous nerve. ICP increase and AUC were normalized to MAP during the erectile response to account for the close influence of the systemic blood pressure on the amplitude of ICP increase during the plateau phase of the erectile response.

[0188] 5. Statistical analysis

[0189] 5.1. Statistical analysis of in vivo evaluation of the erectile function

[0190] All results are presented as mean ± SEM. Grubbs’ test was used for the detection and exclusion of outliers. This statistical method allows determining when a value was unlikely to have come from the same Gaussian population as the other values in the group.

[0191] For erectile function evaluation, comparisons of frequency-response curves were performed with a two-way ANOVA statistical analysis test.

[0192] Statistical analysis was performed with GraphPad Prism® 6.05 software. P values < 0.05 was considered significant.

[0193] Results

[0194] 1. Pro-erectile effect of BoNT / A LC HSV1 NR-Rec vectors ic at 2.106PFU in SHR at 5 weeks post ic injection

[0195] Five weeks post ic injection, a single BoNT / A LC HSV1 NR-Rec vectors ic at 2.106PFU significantly increased erectile responses elicited by ES CN compared to the control vectors in terms of AICP / MAP ( / ?<0.01, 2-way ANOVA, figure 5A), AUC45 / MAP ( / ?<0.01, 2-way ANOVA, figure 5B) and AUCtot / MAP (p< Q. QQ 1, 2-way ANOVA, figure 5C).

[0196] 2. Additional pro-erectile effect of BoNT / A LC HSV1 NR-Rec vectors when combined with sildenafil First, as seen in Figure 6, BoNT / A LC HSV1 NR-Rec vectors treatment at 2.106PFU and sildenafil iv at 0.3 mg / kg exerted a similar pro-erectile effect, when considering all parameters of erectile responses elicited by ES CN (non-significant two-way ANOVA).

[0197] Next, the effect of a combined BoNT / A LC HSV 1 NR-Rec vectors ic and sildenafil iv was also investigated, and it was found that BoNT / A LC HSV1 NR-Rec vectors ic at 2.106PFU and sildenafil iv had an additional pro-erectile effect. In fact, as seen in figure 6, BoNT / A LC HSV1 NR-Rec vectors ic at 2.106PFU combined with sildenafil iv at 0.3 mg / kg significantly increased erectile responses elicited by ES CN in terms of AICP / MAP (p<0.0001, 2-way ANOVA, figure 6A) and AUCtot / MAP (p<0.0001, 2-way ANOVA, figure 6C) compared to BoNT / A LC HSV1 NR-Rec vectors ic at 2.106PFU alone.

[0198] 3. Conclusions

[0199] Following the ic injection with control vectors or BoNT / A LC HSV1 NR-Rec vectors, no abnormal clinical signs were observed throughout the study period. SHR rats remained in good general condition, with normal behavior and appearance, and no signs of distress or discomfort were detected during the 5 week-period.

[0200] 3.1. Pro-erectile effect of BoNT / A LC HSV1 NR-Rec vectors at 2.106PFU at 5 weeks post ic injection

[0201] This study aimed to investigate the effects of BoNT / A LC HSV1 NR-Rec vectors ic on ED in SHR, a well validated model of vasculogenic ED. First of all, BoNT / A LC HSV 1 NR-Rec vectors ic at 2.106PFU did not induce any clinical signs of distress in SHR throughout the 5-week experimental period. Furthermore, this observation was supported by the fact that BoNT / A LC HSV1 NR-Rec treatment at these doses had no effect on the body weight evolution of SHR.

[0202] As a main result, this study showed that BoNT / A LC HSV1 NR-Rec vectors ic at 2.106PFU had a pro-erectile effect in SHR, to the same extent as that of sildenafil iv at 0.3 mg / kg. Of note, sildenafil is the standard of care to treat ED, and this dose of sildenafil has previously been demonstrated to ameliorate erectile function in anesthetized rats. 3.2. BoNT / A LC HSV1 NR-Rec vectors mechanism of action

[0203] BoNT / A LC HSV1 NR-Rec vectors were designed to induce a local cell production of the light chain of onabotulinumtoxin A, which can interact with the SNARE complex and disable the exocytotic machinery, thereby inhibiting neurotransmitter (NT) release. Accordingly, in the penile tissue, BoNT / A LC HSV1 NR-Rec vectors was proposed to inhibit the release of NE, an anti-erectile NT, but not the release of NO, the pro-erectile messenger which does not require release from synaptic vesicles. Thus, BoNT / A LC HS V 1 NR-Rec vectors treatment was expected to exert its pro-erectile effect by inducing a transient cavernosal sympathectomy.

[0204] Interestingly, combined BoNT / A LC HSV1 NR-Rec vectors and sildenafil treatment improved ED in SHR compared to BoNT / A LC HSV1 NR-Rec vectors alone. These results confirm a BoNT / A LC HSV1 NR-Rec vectors mechanism of action different and additional from that of sildenafil.

[0205] In view of the above detailed study, the above examples show that BoNT / A LC HSV1 NR-Rec vector, alone or combined with sildenafil treatment, may efficiently inhibit NE release. This inhibition leads to a pro-erectile effect in SHR rats. Accordingly, an expression vector comprising a nucleotide sequence encoding an isolated botulinum toxin serotype A light chain alone or combined with a PDE5 inhibitor treatment is able to treat diseases associated with sympathetic nervous system hyperactivity-associated conditions such as Erectile Dysfunction.

[0206] Sequences disclosed in the application

[0207] SEQ ID NO: 1: Botulinum toxin serotype A Light Chain (BoNT / A-LC) protein MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDST YLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLV IIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHA GHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIV GTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVP KVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALN SEQ ID NO: 2: Tagged Botulinum toxin serotype A Light Chain (BoNT / A- LC) polynucleotide coding sequence atgcaccaccaccaccaccaccagttcgtgaacaagcagttcaactacaaggaccccgtgaacggcgtg gacatcgcctacatcaagatccccaacgtgggccagatgcagcccgtgaaggccttcaagatccacaac aagatctgggtgatccccgagagagacaccttcaccaaccccgaggagggcgacctgaacccccccccc gaggccaagcaggtgcccgtgagctactacgacagcacctacctgagcaccgacaacgagaaggacaac tacctgaagggcgtgaccaagctgttcgagagaatctacagcaccgacctgggcagaatgctgctgacc agcatcgtgagaggcatccccttctggggcggcagcaccatcgacaccgagctgaaggtgatcgacacc aactgcatcaacgtgatccagcccgacggcagctacagaagcgaggagctgaacctggtgatcatcggc cccagcgccgacatcatccagttcgagtgcaagagcttcggccacgaggtgctgaacctgaccagaaac ggctacggcagcacccagtacatcagattcagccccgacttcaccttcggcttcgaggagagcctggag gtggacaccaaccccctgctgggcgccggcaagttcgccaccgaccccgccgtgaccctggcccacgag ctgatccacgccggccacagactgtacggcatcgccatcaaccccaacagagtgttcaaggtgaacacc aacgc ct act a cgagat gagegg cctggaggt gage ttcgaggagctgagaacctt egg cggccacgac gccaagttcatcgacagcctgcaggagaacgagttcagactgtactactacaacaagttcaaggacatc gccagcaccctgaacaaggccaagagcatcgtgggcaccaccgccagcctgcagtacatgaagaacgtg ttcaaggagaagt acct get gag cgaggacaccagcggcaagttcagcgtggacaagctgaagttcgac aagctgtacaagatgctgaccgagatctacaccgaggacaacttcgtgaagttcttcaaggtgctgaac agaaagacctacctgaacttcgacaaggccgtgttcaagatcaacatcgtgcccaaggtgaactacacc atctacgacggcttcaacctgagaaacaccaacctggccgccaacttcaacggccagaacaccgagatc aacaacatgaacttcaccaagctgaagaacttcaccggcctgttcgagttctacaagctgctgtgcgtg agaggcatcatcaccagcaagaccaagagcctggacaagggctacaacaaggccctgaactga

[0208] SEQ ID NO: 3: EFla promoter (EPFla-3; Zheng et al., International Journal of Medical Sciences, 2014, 11 ( 5 ), 404-408; doi. org / 10. 7150 / ijms. 8033 ) GGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAAGTTGGGGGGAGGGGTCGGCAATT GAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAAAGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGA GGGTGGGGGAGAACCGTATATAAGTGCAGTAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACA CAGGTAAGTGCCGTGTGTGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTT CCACCTGGCTCCAGTACGTGATTCTTGATCCCGAGCTGGAGCCAGGGGCGGGCCTTGCGCTTTAGGAGCCCCTTCG CCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCGCCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTC TCGCTGCTTTCGATAAGTCTCTAGCCATTTAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAG TCTTGTAAATGCGGGCCAGGATCTGCACACTGGTATTTCGGTTTTTGGGCCCGCGGCCGGCGACGGGGCCCGTGCG TCCCAGCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCTCAAGCTG GCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGGCAAGGCTGGCCCGGTCGGC ACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTCCAGGGGGCTCAAAATGGAGGACGCGGCGCTCG GGAGAGCGGGCGGGTGAGTCACCCACACAAAGGAAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCA CGGAGTACCGGGCGCCGTCCAGGCACCTCGATTAGTTCTGGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGA GGGGTTTTATGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATGTAA TTCTCCTTGGAATTTGGCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAGTGGTTCAAAGTTTTT TTCTTCCATTTCAGGTGTCGTGA SEQ ID NO: 4: human cytomegalovirus (CMV) immediate early enhancer and promoter ( Schaack et al., Journal of Virology, 1995, 69 ( 6), 3920-3923 ) TAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCC CGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGG GACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATG CCAAGTCCGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTACGGG ACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACACCAA TGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGG CACCAAAATCAACGGGACTTTCCAAAATGTCGTAATAACCCCGCCCCGTTGACGCAAATGGGCGGTAGGCGTGTAC GGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATC SEQ ID NO: 5: Botulinum toxin serotype F Light Chain (BoNT / F-LC) protein MPWINSFNYNDPVNDDTILYMQIPYEEKSKKYYKAFEIMRNVWIIPERNTIGTDPSDFDPPASLENGSSAYYDPN YLTTDAEKDRYLKTTIKLFKRINSNPAGEVLLQEISYAKPYLGNEHTPINEFHPVTRTTSVNIKSSTNVKSSIILN LLVLGAGPDIFENSSYPVRKLMDSGGVYDPSNDGFGSINIVTFSPEYEYTFNDISGGYNSSTESFIADPAISLAHE LIHALHGLYGARGVTYKETIKVKQAPLMIAEKPIRLEEFLTFGGQDLNIITSAMKEKIYNNLLANYEKIATRLSRV NSAPPEYDINEYKDYFQWKYGLDKNADGSYTVNENKFNEIYKKLYSFTEIDLANKFKVKCRNTYFIKYGFLKVPNL LDDDIYTVSEGFNIGNLAVNNRGQNIKLNPKIIDSIPDKGLVEKIVKFCKSVIPRK SEQ ID NO: 6: Tagged Botulinum toxin serotype F Light Chain (BoNT / F-LC) polynucleotide coding sequence AT G C AC CAT CAT CAT C AC CAT CCCGTGGTTAT C AAT AG C T T T AAT T AT AAC GAT C C C GT GAAT GAT GAT AC AAT T C TCTACATGCAGATTCCATACGAGGAAAAGAGCAAGAAGTATTATAAAGCATTCGAAATAATGCGGAACGTTTGGAT TATTCCCGAGAGAAACACAATCGGAACCGACCCGTCCGATTTTGATCCACCCGCCTCATTGGAAAACGGCAGTAGC GCCTACTACGATCCCAATTATCTCACCACAGATGCTGAGAAGGACCGCTATCTGAAAACCACAATCAAGCTCTTTA AGAGAATCAACTCTAATCCAGCTGGCGAAGTCCTGCTGCAGGAAATTAGCTACGCAAAGCCATATCTCGGCAACGA GCATACACCTATTAATGAGTTCCATCCCGTCACTCGGACGACCTCTGTGAACATAAAAAGCTCTACAAACGTGAAG AGCTCTATAATACTGAACCTGCTCGTGCTGGGTGCTGGCCCAGACATTTTCGAGAATAGTTCCTATCCAGTTCGAA AGTTGATGGATTCTGGGGGCGTGTACGATCCTAGCAATGACGGATTTGGGAGTATTAATATAGTCACATTCAGTCC CGAGTATGAATACACCTTCAACGACATCAGCGGTGGCTACAATTCATCAACTGAGAGCTTCATTGCCGACCCAGCC ATCAGTCTGGCCCATGAGTTGATCCATGCCCTGCACGGCCTCTATGGGGCTAGAGGGGTTACCTACAAGGAAACAA TTAAAGTCAAGCAGGCTCCACTCATGATCGCTGAAAAGCCCATTCGGCTCGAGGAGTTTCTGACATTCGGCGGCCA GGATCTCAACATAATCACCAGTGCTATGAAAGAGAAGATCTACAATAACCTTCTTGCAAATTACGAAAAAATCGCA ACACGGCTGTCACGGGTGAATAGCGCTCCCCCTGAGTACGACATTAACGAGTATAAAGATTACTTCCAGTGGAAAT ACGGGCTGGACAAAAATGCCGACGGGAGCTACACAGTGAACGAGAACAAGTTTAACGAGATCTACAAGAAACTGTA CTCATTTACCGAGATTGACCTGGCTAATAAGTTTAAGGTCAAGTGCAGAAATACTTATTTCATCAAGTACGGATTT TTGAAAGTCCCTAATCTGCTGGACGACGATATTTATACTGTGTCTGAAGGTTTTAATATCGGAAACCTTGCCGTGA ATAACCGCGGGCAGAATATAAAGCTTAATCCTAAGATCATCGATTCAATTCCTGACAAAGGCCTCGTCGAGAAAAT AGT GAAAT T CT GCAAAAGT GT GAT T C CT AGAAAGT GA

Claims

CLAIMS1. An expression vector comprising a nucleotide sequence encoding an isolated botulinum toxin serotype A light chain operably linked to a promoter active in sympathetic neurons, for use in the treatment of sympathetic nervous system hyperactivity-associated conditions.

2. The vector for use according to claim 1, which is a viral vector.

3. The vector for use according to claim 2, wherein the viral vector is a herpes simplex virus (HSV) vector or an adeno-associated virus (AAV) vector.

4. The vector for use according to claim 3, wherein the HSV vector is a defective HSV vector.

5. The vector for use according to claim 4, wherein the defective HSV vector is a HSV amplicon vector or a defective recombinant HSV vector.

6. The vector for use according to any one of claims 1 to 5, wherein the promoter is a ubiquitous promoter.

7. The vector for use according to claim 6, wherein the ubiquitous promoter is human elongation factor 1 alpha (hEFl-a) promoter or human cytomegalovirus immediate early enhancer and promoter (hCMV).

8. The vector for use according to claim 7, wherein the hEFl-a promoter comprises the nucleotide sequence of SEQ ID NO: 3 and / or the hCMV promoter comprises the nucleotide sequence of SEQ ID NO: 4.

9. The vector for use according to any one of claims 1 to 8, wherein the isolated botulinum toxin serotype A light chain comprises an amino acid sequence having at least 85% identity with the sequence of SEQ ID NO: 1.

10. The vector for use according to any one of claims 1 to 9, wherein the sympathetic nervous system hyperactivity-associated conditions are selected from the group consisting of:: cardiovascular diseases, including ischemic heart disease, chronic heart failure, obliterating arteriopathy of the lower limbs and hypertension; kidney disease; diabetes mellitus, in particular type II diabetes; obesity; metabolic syndrome; obstructive sleep apnea; depression; ulcerative colitis; erectile dysfunction; Raynaud’s syndrome and neurological disorders such as spinal cord injury.

11. The vector for use according to claim 10, which is for the treatment of Erectile dysfunction.

12. The vector for use according to any one of claims 1 to 11, which is formulated in a pharmaceutical composition, comprising a therapeutically effective dose of the vector and at least one pharmaceutically acceptable excipient and / or carrier.

13. The vector for use according to claim 11, wherein the pharmaceutical composition further comprises another therapeutic agent.

14. The vector for use according to any one of claims 1 to 13, which is administered by injection.

15. The vector for use according to claims 1 to 14, wherein the vector is used in combination with another therapy, wherein the combined therapies may be simultaneous, separate, or sequential.

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