Patent Description:
In particular, the present invention is related to the field of control of urine storage and bladder emptying or micturition, which is dependent upon the activity of two functional units in the lower urinary tract: (<NUM>) a reservoir (the urinary bladder) and (<NUM>) an outlet consisting of the bladder neck, urethra, and striated muscles of the external urethral sphincter (EUS) (Fowler et al. <NUM>; Morrison et al. These structures are controlled by three sets of efferent peripheral nerves: sacral parasympathetic (pelvic nerves), thoracolumbar sympathetic (hypogastric nerves and lumbo-sacral sympathetic chain), and somatic nerves (pudendal nerves) distributed bilaterally (de Groat <NUM>; Morrison et al. These nerves consist of efferent axons originating at thoracolumbar and sacral spinal levels. Parasympathetic efferent nerves contract the bladder and relax the urethra. Sympathetic efferent nerves relax the bladder and contract the urethra. Somatic efferent nerves contract the EUS. These nerves also contain afferent neurons that transmit information from the lower urinary tract to the lumbosacral spinal cord. The cellular bodies of the afferent neurons of the human lower urinary tract are located in the S2-S4 and T11-L2 dorsal root ganglia (DRG). Sensations of bladder fullness are conveyed to the spinal cord by the pelvic and hypogastric nerves, whereas sensory input from the bladder neck and the urethra is carried in the pudendal and hypogastric nerves.

A similar segmental organization occurs in nonhuman primates, cats and dogs. In rats, cellular bodies of the afferent neurons of pelvic, pudendal and hypogastric nerves are located in the L6-S1 and T11-L2 DRG respectively. The neural pathways that control lower urinary tract function are organized as simple on-off switching circuits that maintain a reciprocal relationship between the urinary bladder and the urethral outlet. Storage reflexes are activated during bladder filling and are organized primarily in the spinal cord, whereas voiding is mediated by reflex mechanisms that are organized in the brain (Fowler et al. Throughout bladder filling, the parasympathetic innervation of the detrusor is inhibited and the smooth and striated parts of the urethral sphincter are activated, preventing involuntary bladder emptying. This process is organized by urethral reflexes known collectively as the 'guarding reflex'. They are activated by bladder afferent activity that is conveyed through the pelvic nerves, and are organized by interneuronal circuitry in the spinal cord (Fowler et al.

NDO refers to a condition in which abnormal bladder function is observed in patients with neurological diseases, such as cerebrovascular disease or cerebral infarction, brain or spinal cord injury due to trauma, multiple sclerosis, Parkinson's disease, congenital malformation e.g. spina bifida, or disease e.g. hereditary spastic paraplegia of the central nervous system, peripheral neuropathy, and various spinal lesions, that is, spinal cord compression and injury due to vertebra(e) fracture, cervical and lumbar spondylosis, spondylosis deformans, spondylolisthesis, spinal stenosis, vertebral disk hernia and the like.

NDO is characterized by involuntary detrusor (bladder) contractions during the filling phase, which may be spontaneous or provoked due to a relevant neurological condition. It is often associated to bladder-sphincter dyssynergia.

NDO due to spinal cord injury (SCI) is the most severe form of NDO. Immediately after SCI there is a period of spinal shock lasting for <NUM>-<NUM> weeks during which the bladder is areflexic, accountable for complete urinary retention. Then, a spinal micturition reflex progressively develops that is responsible for NDO. For SCI patients, these impairments lead to urinary incontinence and increase in bladder pressure, which, if untreated, can damage upper urinary tract and precipitate renal failure. Urinary incontinence is associated with a significant burden and severely impairs quality of life. In SCI patients, recurrent urinary tract infections due to incomplete bladder emptying and renal failure remain the first cause of rehospitalization and second cause of mortality respectively. SCI disrupts voluntary control of voiding as well as the normal reflex pathways that coordinate bladder and sphincter functions. In suprasacral spinal lesion, NDO results of the unmasking of a segmental reflex at the level of the sacral cord, mediated by bladder afferent nociceptive C-fibers (de Groat and Yoshimura, <NUM>). These silent C-fibers become mechano-sensitive and initiate automatic micturition reflex after SCI. This reflex is facilitated after elimination of supraspinal control. Plasticity occurs in bladder afferents and is associated with changes in the properties of ion channels and electrical excitability of afferent neurons, and appears to be mediated in part by neurotrophic factors released in the spinal cord and the peripheral target organs. Overall, the neurobiological substrate for NDO comprises functional alterations in bladder urothelium and sub-urothelium as well as increased afferent sensory messages to the spinal cord, originating in the bladder. Exacerbated afferent bladder stimuli, resulting from hypertrophy and hyperactivity of non-myelinated type-C bladder afferent neurons, are the main mechanisms causing NDO in SCI subjects.

Standard of care for the treatment of NDO consists in inhibiting efferent neurotransmission at the detrusor level. Accordingly, NDO patients are currently treated with antimuscarinics, which block the activity of the muscarinic acetylcholine receptors thereby inhibiting detrusor contractions, and/or repeated intradetrusor injection of Clostridium botulinum neurotoxin A (BoNT-A), again to block detrusor contractions by acting on bladder efferents. Both treatments must be combined with intermittent bladder catheterization (<NUM>-<NUM> times/day).

BoNT-A injections suppress the formation of SNARE complex, blocking the fusion of neurotransmitter-filled vesicles with the plasma membrane of efferent neurons and their release during exocytosis. Accordingly, injection of BoNT-A is used as medication for treating patients with overactive bladder from neurogenic origin or not. For example, <CIT> and <CIT> disclose the use of BoNT-A injection to prevent a nerve from stimulating its target tissue, e.g., a muscle, a gland, or another nerve, for the treatment of various urinary disorders.

<CIT> discloses the use of a viral vector encoding a modified botulinum neurotoxin, thereby producing a protein that has improved binding properties to its human receptors. Following production in cell lines, once recovered and purified from the supernatants, this neurotoxin can be locally applied to treat a condition associated with unwanted neuronal activity such as NDO. However, these vectors are not conceived for a gene therapy approach.

Nevertheless, injection of botulinum neurotoxins presents the inconvenient of toxin diffusion, which is largely due to diffusion of toxins to other regions of the body. The adverse effects range from transient non-serious events such as ptosis and diplopia to life-threatening events even death. In addition, for NDO these injections must be repeated in average every <NUM> months because of decreased efficacy overtime.

Because NDO, with or without bladder-sphincter dyssynergia, caused by supra sacral spinal lesions is due to the emergence of an abnormal reflex mediated by bladder afferences (a8 and c fibers), an alternative approach for the treatment of NDO has been developed by Brindley (Brindley et al <NUM>). This approach combines posterior sacral rhizotomies and sacral anterior roots stimulation (SARS). This treatment appeared to be one of the most effective therapeutic methods for NDO caused by complete suprasacral spinal lesions: sacral rhizotomies permanently increases the compliance of the bladder and eliminates hyperactivity of the detrusor -and detrusor-sphincteric dyssynergia- which are the main causes of renal failure and urinary incontinence, while implantation of a stimulator of the anterior spinal roots enables the patient to elicit and to control micturition.

Deafferentation by posterior sacral rhizotomies, as proposed by Brindley (<NUM>), consists of the complete surgical transsection of all afferent neural fibers to the spinal S2-S4 segments, including those providing sensory input from the detrusor muscle. In this way, the sensory stimuli from the detrusor muscle cannot reach anymore the central nervous system, and consequently, reflex activities generated by the central nervous system causing uncontrolled bladder contractions can be inhibited. The procedure is necessary to prevent exacerbated reflex activities of detrusor and allows larger amount of urine to be stored at low bladder pressure. However, bladder deafferentation obtained from extensive, non-selective, irreversible pelvi-perineal deafferentation by posterior sacral rhizotomies (S2-S4) has many pitfalls and drawbacks, as it is responsible for loss of remaining pelvi-perineal sensation if present, impairing orgasm if present, reflex erection and ejaculation if present, and reflex micturition and defecation if present, and possibly facilitating bedsore because of loss of skin sensory innervation. In addition, the magnitude of neurosurgical procedure makes it expensive and can be responsible for cerebrospinal fluid fistulas and in the long-term for Charcot spinal arthropathy.

Consequently, there is a need for a new strategy to treat NDO in case of supraspinal lesion, targeting specifically its pathophysiology i.e., the abnormal spinal reflex mediated by bladder afferences, but without affecting other afferent neurons conveyed in the same nerves, while sparing the bladder efferent neurons. The strategy we propose is a gene therapy approach resulting in selective molecular bladder deafferentation, to restore continence and micturition in NDO patients when combined with sacral anterior roots stimulation. This has been achieved by a new strategy requiring a viral expression vector able to deliver therapeutic transgene(s) presenting:.

Any other aspects or embodiments set forth herein falling outside of the scope of the claims are provided for information purposes only. Any references in the description to methods of treatment refer to the compositions or cells of the present invention for use in a method for treatment of a subject.

In the context of the present invention, the inventors surprisingly found that, following injection of the viral expression vector in the bladder wall, it is possible to obtain selective and stable transgenes expression in the afferent neurons of the bladder, using a viral expression vector that stably expresses over time proteins and/or transcripts to treat NDO, by specifically inhibiting/silencing neurotransmission or synaptic transmission of bladder afferent neurons at the spinal cord level.

A method and a pharmaceutical composition for the treatment of the NDO comprising the viral expression vector carrying a transcription cassette that harbors transgene(s) inhibiting/silencing neurotransmission or synaptic transmission of afferent neurons are notably detailed herein. Preferably, the method and a pharmaceutical composition comprise a viral expression vector carrying a transcription cassette that harbors transgene(s) disrupting SNARE complex, and/or ribosomal complex, and/or activating GABA(A) receptors, and/or inducing conditionally targeted neuron ablation, when transcribed, that inhibit/silence neurotransmission or synaptic transmission of bladder afferent neurons.

The term "transcription cassette" as used herein refers to any nucleic acid sequence containing a promoter and a downstream coding sequence or transgene, which expression is driven by said promoter, which is followed by a polyadenylation signal. The term "transgene" refers to a particular nucleic acid sequence encoding for a 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 (<NUM>) a nucleic acid sequence that is not naturally found in the cell (i. , a heterologous nucleic acid sequence); (<NUM>) 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; (<NUM>) 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 (<NUM>) a silent naturally occurring or homologous nucleic acid sequence whose expression is induced in the cell into which it has been introduced. By "mutant form" is meant 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.

As used herein, the term "ribosomal complex" refers to a complex which is essentially composed of the subunits of ribosomes, such as <NUM> and <NUM> subunits that catalyzes the synthesis of proteins, referred as translation.

In a first aspect, not comprised within the scope of the claims, a viral expression vector is provided herein comprising at least:.

Specifically, the present invention provides a herpes simplex virus (HSV) viral expression vector comprising at least:.

In a preferred embodiment, the nucleotide sequence of the viral expression vector according to the invention silences or inhibits neurotransmission or synaptic transmission when transcribed or translated by disrupting the SNARE complex, and/or the ribosomes complex, and/or by activating GABA(A) receptors, and/or by inducing conditionally targeted neuron ablation.

In a preferred embodiment, the nucleotide sequence of viral expression vector according to the invention, when transcribed, disrupts at least one of the proteins selected from VAMP, SNAP-<NUM> or syntaxin 1a, which are part of the SNARE complex, or codes for the protein GAD67 or for an active fragment thereof, or codes for a protein disrupting the ribosome complex or for an active fragment thereof, or codes for a protein inducing conditionally targeted neuron ablation, or for an active fragment thereof.

In a particular embodiment, the protein disrupting the ribosome complex according to the invention is a wild-type or a modified ribosome inactivating protein (RIP) or an active fragment thereof, preferentially said RIP are selected from RIP of type <NUM> or type <NUM>, preferentially RIP of type <NUM> are selected from saporin, gelonin, dianthin, trichosanthin; and RIP of type <NUM> are selected from ricin, volkensin and abrin, more preferentially said RIP of type <NUM> is saporin S6 or an active fragment thereof.

The term "protein inducing conditionally targeted neuron ablation" relates to a protein which converts innocuous prodrug substrates, such as metronidazole (MTZ), into cytotoxic DNA crosslinking agents - providing cell-specific ablation of the targeted cell type i.e., afferent neuron of the bladder. Examples of such proteins inducing conditionally targeted neuron ablation are nitroreductases (NTR).

The protein inducing conditionally targeted neuron ablation according to the invention can therefore be selected from the group consisting of a wild-type or a modified NTR or an active fragment thereof. Preferentially, said NTR is selected from the group consisting of a wild-type or a modified oxygen-insensitive NAD(P)H nitroreductases or an active fragment thereof, more preferentially said NTR is selected from the group consisting of a wild-type or a modified F. coli nitroreductases, even more preferentially said NTR is a wild-type or a modified E. coli nfnB or an active fragment thereof.

The term "viral vector" or "viral expression vector" as used herein refers to a nucleic acid vector that includes at least one element of a virus genome and may be packaged into a viral particle. In the present context, the term "viral vector" has to be understood broadly as including nucleic acid vector (e.g., DNA viral vector) as well as viral particles generated thereof. The viral expression vector may be an adeno-associated virus (AAV) vector or a herpes simplex virus (HSV) vector. According to the present invention, the viral expression vector is a herpes simplex virus (HSV) vector, preferably a HSV-<NUM> vector or a HSV-<NUM> vector, even more preferably a defective viral vector derived from HSV-<NUM>. As used herein the term "defective viral vector" shall refer to viral vectors that are missing genes or parts of genes necessary to complete successfully the viral life cycle.

The term "AAV" refers to the Adeno-Associated Virus itself or to derivatives thereof including recombinant AAV vector particles. Furthermore, as used herein, the term "AAV" includes many different serotypes, which have been isolated from both human and non-human primate samples. Preferred AAV serotypes are the human serotypes, more preferably human AAV of serotypes <NUM>, <NUM> and <NUM>, most preferably human AAV of serotype <NUM>, which is the serotype displaying the highest level of neurotropism.

According to the present invention, the term "defective viral vector derived from HSV" refers both to defective recombinant HSV vectors and amplicon HSV vectors. The term "defective recombinant HSV", as used herein, describes a helper-independent vector, the genome of which comprises at least complete deletions 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 transcription cassette(s) embedded in the LAT (Latency Associated Transcripts) locus (Berthomme et al. , <NUM> and Berthomme et al. , <NUM>), which is a repeated locus that is contained in the inverted repeated sequences known as b and b' of the virus genome. The transcription cassette is placed between the LTE region and the DNA insulator (INS) sequence present downstream of the LTE (site <NUM>) (as shown in <FIG>). Defective recombinant HSV-<NUM> vectors according to the present invention carry transcription cassette(s) expressing the different transgenes described above in order to inhibit/silence neurotransmission, i.e., expressing wild type or modified light chain botulinum toxins, and/or antisense RNA (AS-RNA) targeting SNARE proteins, and/or GAD67, and/or RIPs, and/or NTRs, all of them driven by long-term DRG-specific promoters as described in the present invention. 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 the HSV-<NUM> genome. Moreover, helper-independent vectors according to the invention can comprise additional deletions in genes encoding non-essential proteins such as ICP34. <NUM>, UL55, UL56, and UL41 proteins. These defective HSV vectors are multiplied in cell lines expressing simultaneously the proteins ICP4 and ICP27 (Marconi et al, <NUM>).

<CIT> and <CIT> disclose the use of a defective HSV-<NUM> vector for gene therapy of pain.

However, the vectors according to the invention differ from the vectors described in <CIT> or <CIT> in several significant respects, which are important in regard to the usefulness and efficacy of the vectors according to the invention. Most important, transgenic transcription cassettes according to the invention are introduced into the LAT locus, as this region contains both the LTE and the DNA insulator sequences (INS) that confer long-term expression to the DRG-specific promoters driving transgene expression in transcription cassettes according to the invention, in contrast to those provided in <CIT> and <CIT>. Of note, the vector described in <CIT> was conceived and proved for short-term action and, therefore, their transcription cassettes are driven by ubiquitous promoters and were not introduced into the LAT regions.

By "amplicon or amplicon vector" it is meant 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-<NUM> genome, in addition to transgenic DNA (i.e., transcription cassettes) of interest. Amplicon plasmids according to the present invention carry transcription cassettes expressing the different transgenes described above in order to inhibit/silence neurotransmission, i.e., expressing wild type or modified light chain botulinum toxins, and/or interfering RNA (RNAi) targeting SNARE proteins, and/or GAD67, and/or RIPs, and/or NTRs, all of them driven by long-term promoters, preferentially a long-term DRG-specific promoter as described in the present invention (see <FIG>).

In a preferred embodiment, the vector according to the invention is a defective recombinant vector lacking 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. <NUM>, UL55, UL56 and/or UL41 gene proteins, and carries the DRG-specific transcription cassette(s), described in <FIG>, embedded in the LAT regions of the vector genome.

In another embodiment, the vector according to the invention is an amplicon vector carrying the above-described transcription cassettes driven by long-term DRG-specific promoters, as described in other parts of this document.

The transcription cassette according to the invention is introduced into the LAT locus.

The expression "recombinant DNA" as used herein describes a nucleic acid molecule, i.e., a polynucleotide of genomic, cDNA, viral, semisynthetic, and/or synthetic origin, which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term "recombinant" as used with respect to virus means a virus carrying a recombinant genome or a genome that has been manipulated to introduce mutations, deletions or one or more heterologous polynucleotides, including genes. The term "recombinant" as used with respect to a protein or polypeptide, means a polypeptide produced by expression of a recombinant nucleic acid. The term "recombinant" as used with respect to a host cell means a recombinant vector that carries recombinant DNA within the host cell or a cell that contains recombinant DNA inserted in its genome. The term "infection" refers to the ability of a viral vector to enter into a host cell or subject.

Defective vectors derived from HSV allow for the infection of neighbouring sensory neurons and establish latent infections in the nucleus of these neurons, located in the trigeminal or the dorsal root ganglia (DRG), depending on the site of infection. In particular, HSV-<NUM> naturally infects sensory neurons and establishes lifelong latent infections in the nucleus of these neurons. It could thus be hypothesized that following injection in the bladder wall, the vectors, such as vector derived from HSV-<NUM>, will reach the sensory DRG innervating the bladder from where they will stably express the therapeutic transgene, provided that adequate bladder afferent neuron-specific promoters drive their expression. However, HSV-<NUM> can also infect and establish latent infections in autonomic neurons (Furuta et al. , <NUM>; Warren et al. <NUM>), and preliminary results demonstrate that this is actually the case when the vector is inoculated into the bladder. Therefore, it is mandatory that expression from the vectors be utterly controlled by afferent-specific promoters, also called selective promoters or selective afferent neuron-specific promoters, in order to obtain significant transgene expression only in these neurons (i.e., afferent neuron), thus avoiding expression in autonomic, also called efferent, neurons. Selective molecular or biochemical (as opposed to surgical) deafferentation of bladder afferent neurons is the most critical aspect of the present invention as it is important to preserve remaining pelvi-perineal sensation if present, orgasm if present, reflex erection and ejaculation if present, and reflex micturition and defecation if present, all of which are conveyed by sensory nerves of the pelvis that do not originate in the bladder. Further, selective bladder deafferentation would also allow for the preservation of bladder efferent neurons, which could be later stimulated by electrical stimulation for example via electrodes. Some studies describe the use of HSV-<NUM> -based vectors in which the transcription cassettes comprise either transient (Miyazato et al. , <NUM>) or long-term (Puskovic et al. , <NUM>; Miyagawa et al. , <NUM>; <CIT>) promoters. However, the promoters used in the studies of Puscovic (LAP2), Miyazato (HCMV promoter) and Miyagawa (artificial CAG promoter) are non-selective, leading to expression of their transgenes in many cell types, including autonomic neurons, brain neurons, and non-neuronal cells. In contrast, by combining viral regulatory sequences and an afferent neuron-specific cellular promoter, the vectors according to the present invention enable a significantly higher afferent neuron-specific expression of the transgenes of interest (see <FIG>). <CIT> teaches the use of the LAT region for long-term expression of the transgene and suggests to use tissue-specific or inducible promoters, but does not cite the promoter of substance P.

The vectors used in the practice of the invention include at least one promoter selectively active in afferent neurons that is operationally linked to nucleotides (usually DNA) encoding an RNA molecule. By "operationally linked" it is meant herein that, in the vector, the promoter is associated with the nucleotides encoding the RNA in a manner that allows the promoter to drive transcription (i.e., expression) of the RNA from the nucleotides. Transcription of RNA from, e.g., a DNA template is well-understood.

A "promoter," as used herein, is a DNA regulatory region capable of binding RNA polymerase in a mammalian cell and initiating transcription of an operably linked downstream (<NUM>' 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 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. The promoter according to the invention comprises DNA sequence starting at least <NUM> kb, preferably <NUM> kb, more preferably <NUM> kb upstream of the initiation site of the messenger coding for specific, relevant gene products. These sequences preferably contain known promoter sequence elements, such as specific transcription binding sites, and distal sequences upstream of the gene, containing additional regulatory elements.

By "active selectively in afferent neurons" it is meant herein that the promoter is active mainly or only in the afferent neurons, preferably in afferent neurons of the bladder and drives transcription (i.e., expression) of the RNA.

Also, those of skill in the art will recognize that many such mammalian afferent neuron specific promoters are known, and additional afferent neuron specific promoters are continually being discovered. All such afferent neuron specific promoters are encompassed by the present disclosure. However, many cell-specific promoter candidates have been shown to display selectivity only when they express from their endogenous location in the cellular chromosomes (McCart et al. , <NUM>; Vassaux et al. There is no way to predict how these promoters will behave when introduced into the genome of a non-integrative expression vector, such as HSV vectors. Notably, it cannot be anticipated whether afferent neuron-specific promoters will retain the same afferent neuron-specific activity. This is both because (a) the nucleosomes bound to the promoter could differ in several respects (for example they can be in a repressive or a permissive configuration) according to the location of the promoter (in the chromosomes versus in the extra-chromosomal vector genome, or even between different positions in cellular chromosomes) and also (b) because the accessibility of positive or negative transcription factors could also differ. This means that every promoter candidate should be thoroughly studied in each specific setting (i.e., episomal vector vs. chromosomal location) to establish whether or not it retains its afferent neuron-specific activity when placed in the vector genome, as we have done experimentally (see results in Example <NUM> and <FIG>).

According to the invention, the promoter is the promoter of Substance P. This promoter was used by Sin et al. (<NUM>) to drive the expression of the GFP in an AAV vector injected into the rat striatum. In this study, the authors observed a broad expression of the GFP transgene in different cell types, contrary to the authors' prediction. In this article, the authors concluded that the determining factor in the pattern of transgene expression observed during transduction is the nature of the viral vector, while the influence of the transgene promoter appears to be secondary. While not included in the scope of the appended claims, the promoter may be selected from promoters of genes coding for sensory neuroreceptors, such as Transient Receptor Potential Vanilloid <NUM> (TRPV1) or Transient Receptor Potential cation channel subfamily M member <NUM> (TRPM8), or from promoters of genes coding for other sensory neuromodulators or sensory neurotransmitters, such as the promoters of PACAP, Calcitonin Gene Related Peptide (CGRP) of SEQ ID NO: <NUM> or SEQ ID NO: <NUM>. Promoters of the TRP gene family, such as the promoter TRPV1 of SEQ ID NO: <NUM> or TRPM8 of SEQ ID NO: <NUM> and promoters of genes coding for sensory neuromodulators or sensory neurotransmitters, such as the CGRP of SEQ ID NO: <NUM> or SEQ ID NO: <NUM>, are also disclosed but not included in the scope of the appended claims.

The viral expression vector of the invention is directed more particularly to vertebrates, preferably to mammals, more preferably primates and humans. Therefore, those skilled in the art will recognize that such promoters are specific to species and would be able to select homologous sequences of a particular species of interest.

By "long-term expression sequence" or "long-term expression element (LTE)" it is meant a nucleotide sequence operably linked to the transcription cassette included in the sequence of the viral expression vector, allowing the expression of a gene product to be sustained for more than <NUM> to <NUM> days or <NUM> to <NUM> days, preferably <NUM> to <NUM> days, more preferably <NUM> to <NUM> days, even more preferably <NUM> days to several years or even more preferably during the life of the patient.

Long-term expression (LTE) sequences were identified in HSV-<NUM> as a region of the latency-associated transcripts (LAT), which originate from the LAT-associated promoter (LAP). This LTE is located downstream of the LAT transcription start site. Indeed, viruses harboring a DNA fragment <NUM>' of the LAT promoter maintained detectable promoter expression throughout latency (Lokensgard et al, <NUM>, Berthomme et al. , <NUM>, <NUM>). Preferably, the LTE is comprised between about <NUM> kb to about <NUM> kb downstream of the LAT transcription start site (Perng et al. More recently, additional sequences, known as DNA insulators, have also been described both upstream and downstream the LTE region (Amelio et al. These sequences also contribute to providing long-term expression to a given transcription cassette, probably by inhibiting epigenetic silencing, and also will be incorporated in the present invention as part of the LTE elements, to confer long-term expression to the transcription cassette. Interestingly, sequences conferring long-term expression to the transcription cassette (both the LTE and the DNA insulator sequences) can be placed either upstream and/or downstream the transcription cassette.

Those of skill in the art will recognize that other LTE-like sequences, as well as other DNA insulator sequences, have been described and are continually being discovered. All such LTE-like sequences and DNA insulator sequences are encompassed.

In a preferred embodiment, the viral expression vector of the invention comprises at least one nucleotide sequence that is transcribed into a non-coding nucleotide sequence inhibiting the synthesis of at least one protein selected from VAMP, SNAP-<NUM> and syntaxin, which are part of the SNARE complex.

The SNARE complex (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) is one of the two key components of the membrane fusion machinery with the SM (Sec1 /Munc18) proteins. The SNARE complex comprises the vesicle-associated "v-SNAREs" (Vesicle Associated Membrane Proteins, VAMPs, particularly VAMP1, <NUM>, and <NUM>) and the target membrane-associated "t-SNAREs" Syntaxins (Syn-<NUM>, <NUM>, <NUM>, and <NUM>) and Synaptosome-Associated Protein of <NUM> kDa (SNAP-<NUM>) that assemble into complexes to mediate different fusion events.

Therefore, methods able to silence a specific gene and/or to disrupt the corresponding encoded protein (a "gene of interest" or "targeted gene" or "selected gene") are provided herein. By "silencing" a gene, we mean that expression of the gene product is reduced or eliminated, in comparison to a corresponding control gene that is not being silenced. Those of skill in the art are familiar with the concept of comparing results obtained with control vs. experimental results. Without being bound by theory, it is believed that silencing is characterized by specific mRNA degradation or mRNA block in translation after the expression of a non-coding complementary sequence such as antisense RNA (asRNA), a small hairpin RNA (shRNA), a microRNA (miRNA), or any other form of interfering RNA (iRNA) in cells.

As herein used, the term "antisense" relates to unmodified or chemically modified single-stranded nucleic acid molecules which are relatively short in general and which are able to hybridize to a unique sequence in the total pool of targets present in cells, the sequence of said nucleic acid molecule being complementary, by virtue of Watson-Crick bp hybridization, to a specific mRNA and able to inhibit said mRNA expression and then induce a blockade in the transfer of genetic information from DNA to protein.

In the context of the invention, "RNA interference" (hereinafter referred to as RNAi) is interpreted as a process by which a double stranded RNA (dsRNA) with a given sense nucleic sequence leads to the breakdown of all messenger RNA (mRNA) comprising said nucleic sequence, in a manner specific to said nucleic sequence. Although the RNAi process was originally demonstrated in Caenorhabditis elegans, it is now clear that the RNAi process is a very general phenomenon, and inhibition of human genes by RNAi has been achieved.

The process of RNAi can be achieved using small interfering RNA (or siRNA). These siRNAs are dsRNA of less than <NUM> nucleotides long, comprising in their sense sequence a sequence that is highly complementary to a fragment of the target mRNA. When a siRNA crosses the plasma membrane, the reaction of the cell is to destroy the siRNA and all the sequences comprising a highly complementary sequence. Thus, an mRNA with a fragment that is highly complementary to the siRNA sequence will be destroyed, the expression of this gene being thus inhibited.

shRNA may be also used as inhibitor according to the present invention. As used herein, an "shRNA molecule" includes a conventional stem-loop shRNA, which forms a precursor miRNA (pre-miRNA). "shRNA" also includes micro-RNA embedded shRNAs (miRNA-based shRNAs), wherein the guide strand and the passenger strand of the miRNA duplex are incorporated into an existing (or natural) miRNA or into a modified or synthetic (designed) miRNA. When transcribed, a conventional shRNA forms a primary miRNA (pri-miRNA) or a structure very similar to a natural pri-miRNA. The pri-miRNA is subsequently processed by Drosha and its cofactors into pre-miRNA. Therefore, the term "shRNA" includes pri-miRNA (shRNA-mir) molecules and pre-miRNA molecules.

In general, "reduced or eliminated" refers to a reduction or elimination of detectable amounts of the gene product by an amount in the range of at least about <NUM>% to about <NUM>%, or preferably of at least about <NUM>% to <NUM>%, or more preferably about <NUM>% to about <NUM>%, and most preferably from about <NUM>% to about <NUM>%. If desired, a reduction or elimination may be determined by any of several methods that are well-known to those of skill in the art, and may vary from case to case, depending on the gene that is being silenced. For example, such a reduction or elimination of the expression of the gene may be determined by quantification of the gene product (e.g., by determining the quantity of a protein, polypeptide, or peptide that is made) or quantification of an activity of the gene product (e.g. an activity such as signaling or transport activity, activity as a structural component of the cell, activity such as enzymatic activity, etc.), or by observation and quantification of a phenotypic characteristic of the targeted cell in comparison to a control cell (e.g., the presence or absence of a protein using specific antibodies). Any suitable means to determine whether or not a targeted gene has been silenced may be used.

In one embodiment, the non-coding nucleotide sequence according to the invention is selected from antisense RNA (asRNA), a small hairpin RNA (shRNA), a micro RNA (miRNA), or any other interfering RNA (iRNA), which inhibits the synthesis of at least one protein selected from VAMP, SNAP-<NUM> and syntaxin.

In one embodiment, the viral expression vector comprises at least one nucleotide sequence that is transcribed into an asRNA inhibiting the synthesis of VAMP, SNAP-<NUM> and/or syntaxin. In particular, the sequences of the asRNA used in the context of the present invention are VAMP2 antisense of SEQ ID NO: <NUM>, SNAP25 antisense of SEQ ID NO: <NUM> and syntaxin antisense of SEQ ID NO: <NUM>.

In a particular embodiment, the viral expression vector comprises at least one nucleotide sequence that is transcribed into an shRNA inhibiting the synthesis of VAMP, SNAP-<NUM> and/or syntaxin.

In another embodiment, the viral expression vector comprises at least one nucleotide sequence that is transcribed into an miRNA inhibiting the synthesis of VAMP, SNAP-<NUM> and/or syntaxin.

The RNA molecule that is encoded by the construct of the present invention ultimately forms a double-strand RNA molecule within the cell in which it is transcribed. In general, one strand of the double-strand RNA structure will be in the range of from about <NUM> to about <NUM> ribonucleotides in length, and preferably from about <NUM> to about <NUM> ribonucleotides in length. In the case of asRNA, one of the double-strand RNA structure will be in the range of from about <NUM> to several hundreds of ribonucleotides in length. It could actually be as long as the target mRNA. Those of skill in the art will recognize that several viable strategies exist for forming such double-stranded RNA.

Moreover, provision of multiple viral vectors with the same afferent neuron-specific promoter but which encode different silencing RNAs may be used within the practice of the invention.

Further, it should be possible to express more than one silencing RNA in a single viral vector, driven by a single afferent neuron-specific promoter, or by more than one promoter arranged in tandem (e.g., two or more promoters). Thus, the invention contemplates using a single viral vector for silencing more than one gene.

In another embodiment, the viral expression vector according to the invention comprises at least one nucleotide sequence coding for a wild-type or a modified toxin disrupting the SNARE complex or the ribosome complex or for an active fragment thereof.

Advantageously, the active fragment of the toxin is a bacterial neurotoxin, preferentially said bacterial neurotoxin is the light chain of said bacterial neurotoxin. In particular, the sequences of the toxin light chains used in the context of the present invention are the protein sequence light chain of the botulinum neurotoxin A (BoNT-A) of SEQ ID NO: <NUM> (coding nucleotide sequence SEQ ID NO: <NUM>), the protein sequence light chain of the botulinum neurotoxin B (BoNT-B) of SEQ ID NO: <NUM> (coding nucleotide sequence SEQ ID NO: <NUM>), the protein sequence light chain of the botulinum neurotoxin C1 (BoNT-C1) of SEQ ID NO: <NUM> (coding nucleotide sequence SEQ ID: <NUM>), the protein sequence light chain of the botulinum neurotoxin E3 (BoNT-E3) of SEQ ID NO: <NUM> (coding nucleotide sequence SEQ ID NO: <NUM>), the protein sequence light chain of the botulinum neurotoxin F1 (BoNT-F1) of SEQ ID NO: <NUM> (coding nucleotide sequence SEQ ID NO: <NUM>), and the protein sequence light chain of the tetanic neurotoxin (TeNT) of SEQ ID NO: <NUM> (coding nucleotide sequence SEQ ID NO: <NUM>).

In a preferred embodiment, the viral expression vector according to the invention comprises at least one nucleotide sequence coding for a wild-type or a modified GAD67 protein or for an active fragment thereof, preferentially nucleotide sequence coding for a wild-type GAD67 protein of SEQ ID NO: <NUM> (coding nucleotide sequence SEQ ID NO: <NUM>) or an active fragment thereof.

In a preferred embodiment, the viral expression vector according to the invention comprises at least one nucleotide sequence coding for a wild-type or a modified RIP or for an active fragment thereof, preferentially said RIP is Saporin S6 protein of SEQ ID NO: <NUM> (coding nucleotide sequence SEQ ID NO: <NUM>) or an active fragment thereof.

In a preferred embodiment, the viral expression vector according to the invention comprises at least one nucleotide sequence coding for a wild-type or a modified NTR or an active fragment thereof, preferentially said NTR is nitroreductase NfnB protein of SEQ ID NO: <NUM> (coding nucleotide sequence SEQ ID NO: <NUM>) or an active fragment thereof.

As used herein, the term "coding sequence" refers to a ribonucleic acid (e.g., RNA) sequence that, when it is translated, produces the polypeptide of interest. The polypeptide can be encoded by a full-length coding sequence or by any portion of the coding sequence so long as the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signal transduction, etc.) of the full-length or fragment is retained.

In one embodiment, the invention relates to a viral expression vector that comprises at least one nucleotide sequence coding for a wild-type or a modified botulinum neurotoxin of Clostridium botulinum of any serotype or for an active fragment thereof, preferably the light chain of Clostridium botulinum neurotoxin of any serotype.

In another embodiment, the invention is directed to a viral expression vector that comprises at least one nucleotide sequence coding for a wild-type or a modified tetanus neurotoxin of Clostridium tetani or for an active fragment thereof, preferably the light chain of Clostridium tetani neurotoxin.

Clostridial neurotoxins are produced by various species of the genus Clostridium, for example several strains of C. botulinum and C. When Clostridium toxin molecules enter into the neuron, the light chain disrupts the proteins that form the SNARE complex located at the presynaptic nerve terminal. This prevents the neurotransmitter filled synaptic vesicles from attaching to the presynaptic membrane, therefore inhibiting exocytosis of the neurotransmitter from the presynaptic nerve terminal. At present, there are eight different classes of the neurotoxins known: tetanus toxin and botulinum neurotoxin in its serotypes A, B, C, D, E, F, and G, all of which share homology and similar molecular structures. Within said serotypes, sub-types are also well documented, such as subtypes A<NUM>-A<NUM>, B<NUM>-B<NUM>, etc..

Botulinum neurotoxin serotypes A, C, and E cleave the SNAP-<NUM> protein located on the plasma membrane of the presynaptic nerve terminals. Because SNAP-<NUM> is necessary for the fusion of neurotransmitter-filled vesicles with the plasma membrane and their release during exocytosis, its cleavage causes a highly specific blockade of vesicular neurotransmitter release at somatic and autonomic presynaptic nerve terminals. Botulinum neurotoxin serotypes B, D, F, and G cleave the synaptobrevin (VAMP) protein, so that the vesicles cannot fuse to the cell membranes. Each botulinum neurotoxin or its light chain fragment cleaves one of the SNARE proteins except for botulinum neurotoxin C, or its light chain fragment, which cleaves both SNAP-<NUM> and syntaxin 1a. Preferably, according to the invention the serotypes of botulinum neurotoxin are A, B, C, E, and F.

The structure of Clostridial neurotoxins has been well-documented (Habermann et al, <NUM>; Sugiyama et al <NUM>). In this regard, Clostridial neurotoxins comprise two polypeptide chains, the heavy chain (H-chain), which has a molecular mass of approximately <NUM> kDa, and the light chain (L-chain), which has a molecular mass of approximately <NUM> kDa, joined together by a disulphide bond.

The different serotypes of botulinum toxin vary in the animal species that they affect and in the severity and duration of the paralysis they evoke. For example, it has been determined that botulinum toxin type A is <NUM> times more potent, as measured by the LD<NUM> in mice, than botulinum toxin type B. Additionally, botulinum toxin type B has been determined to be nontoxic in primates at a dose of <NUM> U/kg which is about <NUM> times the primate LD<NUM> for botulinum toxin type A. Naturally, botulinum toxin binds with high affinity to neurons, is translocated into the neuron and blocks the release of neurotransmitters.

In a particular embodiment, the invention is directed to a viral expression vector that comprises at least one nucleotide sequence coding for a wild-type or a modified tetanus neurotoxin of Clostridium tetani or for an active fragment thereof to cleave the protein VAMP-<NUM>.

In a particular embodiment, the invention is directed to a viral expression vector that comprises at least one nucleotide sequence coding for a wild-type or a modified botulinum neurotoxin of Clostridium botulinum of serotype B, D, F, and G or for an active fragment thereof to cleave the protein VAMP-<NUM>.

In a particular embodiment, the invention is directed to a viral expression vector that comprises at least one nucleotide sequence coding for a wild-type or a modified botulinum neurotoxin of Clostridium botulinum of serotype A and E or for an active fragment thereof to cleave the protein SNAP-<NUM>.

In a preferred embodiment, the invention is directed to a viral expression vector that comprises at least one nucleotide sequence coding for a wild-type or a modified botulinum neurotoxin of Clostridium botulinum of serotype C or for an active fragment thereof to cleave the proteins SNAP25 and syntaxin 1a.

Preferably, the nucleotide sequence of the transgene codes for a wild-type or a modified protein silencing or inhibiting the transduction of the neurotransmitter signal in postsynaptic cell which is fused to a signal peptide domain. The signal peptide is selected according to the intracellular compartment where transcript or protein targeted to silence or inhibit the transduction of the neurotransmitter signal in postsynaptic cell is located. Therefore, those skilled in the art will recognize that such signal peptides are specific to intracellular compartment and would be able to select the appropriate corresponding nucleotide sequences to be fused to the nucleotide sequence coding for the protein silencing or inhibiting neurotransmission or synaptic transmission. In particular, the signal peptides comprise at least the luminal, transmembrane, or cytoplasmic domains of proteins selected from VAMP2 or Syntaxin 1a.

Preferably, the fusion protein comprises a signal peptide domain selected from luminal, transmembrane, or cytoplasmic signal peptide domains, preferentially the luminal, transmembrane, or cytoplasmic signal peptide domains of the SNARE proteins, the substance P, or CGRP sequences. Such signal peptide domains notably include the signal peptide of syntaxin 1a (BoNTB-STX) of SEQ ID NO: <NUM> (coding nucleotide sequence SEQ ID NO: <NUM>) and the signal peptide of VAMP2 (BoNTC-VAMP) of SEQ ID NO: <NUM> (coding nucleotide sequence SEQ ID NO: <NUM>). Thus, the fusion protein preferably comprises a modified bacterial neurotoxin, such as e.g., a modified botulinum neurotoxin, and a signal peptide such as e.g., the signal peptide of syntaxin 1a (BoNTA-STX) of SEQ ID NO: <NUM> or (BoNTB-STX) of SEQ ID NO: <NUM> (coding nucleotide sequence SEQ ID NO: <NUM>) and the signal peptide of VAMP2 (BoNTC-VAMP) of SEQ ID NO: <NUM> (coding nucleotide sequence SEQ ID NO: <NUM>).

In one specific example, the fusion protein comprises the wild-type or modified Clostridium botulinum neurotoxin of serotype A, B, C, E, or F linked to the signal peptide of syntaxin 1a, preferentially the fusion protein comprises the wild-type or modified Clostridium botulinum neurotoxin of serotype A linked to the signal peptide of syntaxin 1a (BoNTA-STX) of SEQ ID NO: <NUM> (coding nucleotide sequence SEQ ID NO: <NUM>) or the fusion protein comprises the wild-type or modified Clostridium botulinum neurotoxin of serotype B linked to the signal peptide of syntaxin 1a (BoNTB-STX) of SEQ ID NO: <NUM> (coding nucleotide sequence SEQ ID NO: <NUM>).

In one example, the fusion protein comprises the wild-type or modified Clostridium botulinum neurotoxin of serotype A, C, and E linked to the signal peptide of VAMP2, preferentially the fusion protein comprises the wild-type or modified Clostridium botulinum neurotoxin of serotype C linked to the signal peptide of VAMP2 (BoNTC-VAMP) of SEQ ID NO: <NUM> (coding nucleotide sequence SEQ ID NO: <NUM>).

In one example, the fusion protein comprises the wild-type or modified Clostridium botulinum neurotoxin of any serotype linked to the signal peptide of Substance P.

In one example, the fusion protein comprises the wild-type or modified Clostridium botulinum neurotoxin of any serotype linked to the signal peptide of CGRP sequence.

The present invention is also directed to a viral expression vector according to the invention, comprising at least:.

In a preferred embodiment, the viral expression vector according to the invention comprises:.

In a particular embodiment, the invention relates to a viral expression vector, wherein.

In a preferred embodiment, the invention relates to a viral expression vector, wherein at least one of said transgenic transcription cassettes according to the invention harbors a promoter and a sequence coding for the wild-type protein GAD67 or for an active fragment thereof.

In a more preferred embodiment, the invention relates to a viral expression vector, wherein one of the said transgenic transcription cassettes according to the invention harbors a promoter and a sequence coding for the wild-type protein GAD67 or for an active fragment thereof; and one of the said transgenic transcription cassettes according to the invention harbors a promoter and a sequence coding for a wild type or modified neurotoxin of Clostridium tetani and/or botulinum or for an active fragment thereof.

In a preferred embodiment, the invention relates to a viral expression vector, wherein at least one of said transgenic transcription cassettes according to the invention harbors a promoter and a sequence coding for the wild-type RIP or for an active fragment thereof.

In a more preferred embodiment, the invention relates to a viral expression vector, wherein one of the said transgenic transcription cassettes according to the invention harbors a promoter and a sequence coding for the wild-type RIP or for an active fragment thereof; and one of the said transgenic transcription cassettes according to the invention harbors a promoter and a sequence coding for a wild type or modified neurotoxin of Clostridium tetani and/or botulinum or for an active fragment thereof; and/or a sequence coding for the wild-type protein GAD67 or for an active fragment thereof, and/or a sequence coding for the wild-type NTR or for an active fragment thereof.

In a preferred embodiment, the invention relates to a viral expression vector, wherein at least one of the transgenic transcription cassettes according to the invention comprises a promoter and a sequence coding for the wild-type NTR or for an active fragment thereof.

In a more preferred embodiment, the invention relates to a viral expression vector, wherein said viral expression vector comprises at least <NUM> transgenic transcription cassettes, wherein:.

In a second aspect, the invention relates to a composition comprising the viral expression vector of the present invention for use as a medicament.

In a third aspect, the invention is directed to a pharmaceutical composition comprising at least one viral expression vector according to the invention.

Advantageously, the pharmaceutical composition according to the invention is used for the treatment of NDO.

The invention also relates to a pharmaceutical composition comprising:.

In a particular embodiment, the pharmaceutical composition according to the invention, further comprises at least one viral expression vector comprising at least one nucleotide sequence coding for the wild-type protein GAD67 and/or RIP and/or NTR, or for an active fragment thereof.

In a particular embodiment, the pharmaceutical composition according to the invention, comprises at least one viral expression vector comprising at least one nucleotide sequence coding for the wild-type protein GAD67 or for an active fragment thereof; and/or at least one nucleotide sequence coding for a wild type or modified neurotoxin of Clostridium tetani and/or botulinum or for an active fragment thereof; and/or for the wild-type RIP or for an active fragment thereof; and/or for the wild-type NTR or for an active fragment thereof.

In a fourth aspect, the present invention relates to a kit comprising at least one viral expression vector or the pharmaceutical composition according to the invention, or the pharmaceutical composition according to the invention, and an electrical stimulation system comprising electrodes to be implanted on the sacral anterior roots, such as S2-S3-S4, to apply intermittent stimulation pulse trains in order to achieve a sustained detrusor muscle contraction with intervals of urethral sphincter relaxation allowing urine to flow.

By "electrical stimulation" it is meant herein that an electrical stimulation is applied, via electrodes, in bursts of a few seconds, separated by longer gaps, to sustain pressure in the bladder, while allowing the external urethral sphincter to relax rapidly between bursts, causing urine to flow during these gaps. The preferred electrical stimulation system is the Finetech-Brindley stimulator (ref <NUM> to <NUM> in Ren et al, <NUM>).

While not comprised in the scope of the invention, a method for the treatment of patient suffering from NDO comprising the steps of:.

The following examples merely intend to illustrate the present invention.

Human elongation factor <NUM> promoter (EF1A), rat Transient Receptor Potential Vanilloide <NUM> (rTRPV1), human and rat Calcitonin Gene-Related Peptide (hCGRP and rCGRP), rat Acid-Sensing Ion Channel <NUM> (rASIC3), and human and rat Advillin (hADVL and rADVL) promoters.

BoNT-A expressed from amplicon vectors cleave the SNARE protein SNAP25 in SH-SY5Y cells.

Human neuroblastoma cells (SH-S5Y5) are infected at an MOI of <NUM>, <NUM>, and <NUM> pfu/cell with amplicon vectors expressing transcription units A2-CMV-BoNT-A (LC) or A2-CMV-Luc, driven in both cases by HCMV promoter. The following day, infections were stopped, and cell proteins were analysed by Western blots using antibodies specific for BoNT-A LC and SNAP25. The higher part of the Western blot shows that increasing amounts of BoNT-A LC correspond to increasing MOI, demonstrating that the vectors used do express this protein in the infected cells. The lower part of the blots shows cleavage of SNAP25, the protein from the SNARE complex that is the natural target of BoNT-A, thus producing two fragments. At the lower MOI, mainly the native (not cleaved) form of SNAP25 is observed. At intermediate MOI, both the native and the cleaved form (the slightly lower band) can be seen, while at the higher MOI most of the SNAP25 protein is cleaved, since only the lower fragment of the doublet can be observed. This demonstrates that BoNT-A LC synthesized in SH-S5Y5 cells is able to cleave SNAP25. In contrast, in SH-S5Y5 cells infected with the vector expressing Luc, no cleavage of SNAP25 is observed.

Light chains of botulin neurotoxins cleave SNARE proteins in infected neurons. Primary cultures of rat embryonic dorsal root ganglia (DRG) neurons are infected at an MOI of <NUM> pfu/cell with amplicon vectors expressing transcription units A2-CMV-BoNT-A, A2-CMV-BoNT-B, A2-CMV-BoNT-C, A2-CMV-BoNT-E, and A2-CMV-BoNT-F. Neurons were also infected with amplicon vectors expressing A2-CMV-BoNT-A-syntaxin (STX), A2-CMV-BoNT-B-syntaxin (STX), and A2-CMV-BoNT-C-VAMP2 (V2). Vector expressing A2-CMV-Luc was used as negative control. In all cases, HCMV promoter drove expression of the transcription cassettes. The following day, infections were stopped and cell proteins were analyzed by Westerns blots. As shown in the figure, each BoNT LC synthesized in the neurons cleaved the expected SNARE protein: thus, the light chains of BoNT-A, -C and -E, cleaved SNP25, as evidenced by the decrease in size of this protein, whereas the light chains of BoNT-B, and -F, cleaved VAMP2, which is no longer detectable in the blots. In addition, BoNT-C also cleaved Syntaxin (STX), also no longer visible in the blots. BoNT-C is the only botulin toxin described to cleave two different SNARE proteins (SNAP25 and STX). The light chains of botulinum toxins fused to the signal and transmembrane peptides of SNARE proteins cleaved the corresponding SNARE proteins exactly as the parental non-fused toxins did. The lane Luc shows the positions of native, non-cleaved, SNARE proteins (arrows). This figure therefore demonstrates that the light chains of botulin toxins (fused or not with fragments of the SNARE proteins) synthesized in sensory neurons upon vector infection, are able to cleave their corresponding target proteins.

Light chains of botulin toxins inhibit release of neuropeptides in sensory neurons.

Primary cultures of rat embryonic DRG neurons are infected at increasing MOI (from <NUM> to <NUM> pfu/cell) with amplicon vectors expressing A2-CMV-BoNT-A, A2-CMV-BoNT-B, A2-CMV-BoNT-C, A2-CMV-BoNT-D, A2-CMV-BoNT-E, and A2-CMV-BoNT-F. Neurons were also infected with amplicons expressing A2-CMV-BoNT-A-syntaxin, A2-CMV-BoNT-B-syntaxin, and A2-CMV-BoNT-C-VAMP2. Vector expressing A2-CMV-Luc was used as negative control. Neurons were also infected with vehicle only (mock). The following day, the infected neurons were treated with <NUM> KCl to stimulate release of CGRP, a neuropeptide normally synthesized in DRG neurons. Thirty minutes before and thirty minutes after KCl treatment, <NUM> microliter aliquots were taken from the culture media and assessed for the presence of CGRP by ELISA (using the CGRP ELISA kit from Spi Bio, ref N° A05482). Results, expressed as linear regression profiles after logarithm conversion, show that all toxins inhibited CGRP release but that they do it with different intensities, with BoNT-F, BoNT-C and BoNT-A being the most effective in this respect. In mock-infected neurons, as well as in neurons infected with the vector expressing Luc, no inhibition of CGRP release was observed. These results clearly indicate that cleavage of SNARE proteins by BoNT LC results in inhibition of neuropeptide release, and that BoNT-F is the most efficient in this respect.

GAD67 expressed from amplicon vectors induces synthesis and extracellular release of GABA (gamma amino-butyric acid).

7A) Glioblastoma cells (Gli36) were infected at MOI <NUM>, <NUM> and <NUM> pfu/cell with amplicon vectors expressing A2-CMV-GAD67 or A2-CMV-Luc. The following day, infections were stopped and cell proteins were analyzed by Western blots, using antibodies specific for GAD67 and GAPDH (a housekeeping gene used as internal control). Extracts from rat brain were used as positive controls to identify endogenous GAD67. <FIG> shows that expression of GAD67 increases with the MOI, demonstrating that vector A2-CMV-GAD67 does express this protein. 7B) Primary cultures of rat embryonic DRG neurons were infected at MOI <NUM>, <NUM> and <NUM> pfu/cell with vectors expressing A2-CMV-GAD67 or A2-CMV Luc. The following day infections were stopped and both intracellular and extracellular concentrations of GABA were evaluated using Resazurine assay, which is a fluorescence-coupled assay for GABA (the assay is performed as indicated in Ippolito et al. The upper panel shows that the amount of intracellular GABA increases with the MOI, while the lower panel shows the increase of extracellular GABA. The channel labeled GABA is a positive control for the Resazurine assay. This result clearly shows that expression of GAD67 from the A2-CMV-GAD67 vector increases synthesis of intracellular GABA and its release to the extracellular medium.

Nitroreductase (NTR) activates the nitro compound <NUM>'nitrocoumarin and induces cell death in the presence of mitronidazole (MTZ).

12A) Human glioblastoma (Gli36) cells were infected with amplicon vectors expressing A2-CMV-NTR or A2-CMV-Luc at an MOI of <NUM> pfu/cell. Two days later, infections were stopped and protein extracts were prepared and used to assess the activation of <NUM>'nitrocoumarin, using a fluorescence-coupled assay (assay performed as in Muller et al. <FIG> shows that only the proteins extracted from cells infected with vector A2-CMV-NTR induced significant activation of <NUM>'nitrocoumarin, demonstrating that functional NTR was expressed in Gli36 cells infected with A2-CMV-NTR.

12B) To assess whether expression of NTR induced cell death in the presence of metronidazole (MTZ), Gli36 cells were infected with amplicon vectors A2-CMV-NTR or A2-CMV-Luc at an MOI of <NUM> pfu/cell. The following day cells were incubated with or without MTZ (<NUM>. mM) for <NUM> hours. Infections were then stopped and cell viability was assessed using the MTT assay (as indicated by Carmichael et al. The figure shows that MTZ significantly increased cell death of infected cells. Mock: non-infected cells.

Analysis of the selectivity of expression of DRG-selective promoter candidates in autonomic and sensory ganglia from adult rats.

Rat adult sensory ganglia (DRG), autonomic sympathetic ganglia (superior cervical ganglia, SCG), and autonomic parasympathetic ganglia (paracervical ganglia, GPC) were explanted and kept as organotypic cultures. After <NUM> days, the ganglia were individually infected with vectors expressing A5-TRPV1-Luc, A5-rCGRP-Luc, A5-ASIC3-Luc, or A5-EF1A-Luc, all of them expressing firefly luciferase (fLuc), but driven respectively by the following promoters: rat TRPV1 (rTRPV1), rat CGRP (rCGRP), rat ASIC3 (rASIC3), and EF1a, a non-selective promoter serving as general control. Each ganglion was infected with <NUM><NUM> vector particles. The vectors also express renilla luciferase (rLuc) driven by a viral promoter (HSV-<NUM> IE4/<NUM>). The following day infections were stopped and cell extracts were prepared for luciferase tests using Dual-luciferase reporter assay system from Promega. Results are expressed as ratio of fLuc/rLuc and were normalized as percentage of expression of the EF1a promoter in each of DRG (left), SCG (center) and GPC (right). <FIG> shows that some candidate promoters, such as rTRPV1 and rCGRP promoters, express significantly higher levels of fLuc in DRG than in autonomic ganglia, while other promoters, such as rASIC3, do not display preferential activity in DRG. According to these results the rTRPV1 and the rCGRP promoters appear to display selective activity for DRG while rASIC3 does not display such selectivity when expressed from the virus genome.

<FIG> shows that an amplicon vector expressing the GFP reporter protein can infect primary cultures of embryonic rat DRG neurons and adult rat DRG explants, and express the GFP transgene within these neurons.

Intradetrusor inoculation of defective HSV-<NUM> vectors reach dorsal root ganglia (DRG) and express transgenes in sensory neurons innervating the bladder.

Viral vector expressing IE4/<NUM>-GFP and HCMV-Luciferase (shown in <FIG>) is capable of penetrating and expressing both transgenic proteins in the bladder afferent neurons following their inoculation into the bladder wall of spinal cord-injured (SCI) rats. DRG neurons expressing both GFP and Luciferase (Luc) are shown in DRG ganglion L6, from which neurons that innervate the bladder extend. However, in the DRG ganglion T13, which does not innervate the bladder, the results are negative. One week post-infection, the animals were sacrificed and transgenic proteins were revealed by IHC using specific antibodies for GFP and Luciferase. These results indicate that following inoculation into the bladder wall, the vectors enter the afferent neurons innervating the bladder and are retrogradely transported through the axons to the cell bodies of the neurons to the L6 ganglia, which lie in the dorsal root ganglia (DRG), from where the viral genome express both transgenic proteins. Vectors are not able to reach or to express in neurons not innervating the bladder (T13).

<FIG> shows the high cell selectivity of expression of the viral vector in the dorsal root ganglia (DRG) when Luciferase is driven by the DRG-selective TRPV1 promoter. Luciferase is significantly expressed only in the afferent neurons, and not in the autonomic neurons (sympathetic or parasympathetic). Results were normalized as percentage of luciferase expression relative to that from the vector expressing Luciferase under the control of the strong but not specific HCMV promoter (both vectors are shown in <FIG>).

The invention provides set of defective recombinant HSV-<NUM> vectors comprising complete deletions of ICP27 and ICP4 (both copies), and which carries, in addition, the therapeutic transcription cassettes embedded into the LAT locus, either between the LAP and LTE sequences (site <NUM>) or between the LTE and INS sequences (site <NUM>), as shown in <FIG>, to provide long-term expression to said cassette. Some of the transcription cassettes used to generate these vectors are shown in <FIG>.

Said transcription cassettes express the light chains (LC) of the Clostridium toxins TeNT (LC), BoNT-A (LC), BoNT B (LC), BoNT-C (LC), BoNT E (LC), BoNT-F (LC), or an antisense RNA (asRNA) directed to the SNARE proteins, VAMP2, SNAP25, and Syntaxin, or fusion SNARE/light-chain toxins, or the human GAD67 protein or a RIP protein such as Saporin S6, or the E. coli NTR nfnB, to inhibit/silence neurotransmission specifically in afferent neurons when placed under the control of an afferent neuron-specific promoter.

To generate the vectors, we used a full-length HSV-<NUM> genome of strain F cloned into a bacterial artificial chromosome (BAC) such as that described by Tanaka et al, <NUM>. Gene deletions and gene insertions were introduced by homologous recombination in bacteria and the vectors were then reconstituted by transfection of permissive cell lines as already described (Tanaka et al. The general structure of these vectors is illustrated in <FIG>.

Genome of HSV-<NUM> amplicon vectors.

The invention also provides a set of defective amplicon vectors, which express the same transgenic therapeutic transcription cassettes as the recombinant vectors, and listed in <FIG>. Sequences conferring long-term expression (LTE and INS) surround the transcription cassettes (<FIG> also shows that in addition to the therapeutic transcription cassettes, amplicon vectors carry a second transcription cassette, expressing a reporter protein (either GFP or the fusion protein GFP/renilla luciferase) driven in all cases by the HSV-<NUM> IE4/<NUM> promoter.

Amplicon vectors are produced using as helper the defective LaLdeltaJ virus and the complementing cell lines already described by Epstein and collaborators (Zaupa, Revol-Guyot and Epstein, <NUM>), which expresses the set of proteins required for amplification and packaging of the vector genome.

Transcription cassettes carried by recombinant and amplicon vector genomes. The recombinant and amplicon vectors described herein carry and express transgenic transcription cassettes embedded into HSV-<NUM> sequences that confer long-term expression (LAP, LTE, INS), in both types of vectors, as shown in <FIG>. Some examples of the transcription cassettes used herein are listed in <FIG>.

A set of defective amplicon vectors is also provided herein; some of these vectors express either reporter proteins (luciferase) or the light chains (LC) of the Clostridium toxins (TeNT (LC), BoNT-A (LC), BoNT B (LC), BoNT-C (LC), BoNT E (LC), BoNT-F (LC)), or an antisense RNA (asRNA) directed to the SNARE proteins, VAMP2, SNAP25, and Syntaxin, or chimeric SNARE/light-chain toxins, or the human GAD67 protein or a RIP protein such as Saporin S6 or a nitroreductase (NTR) protein such as NfnB. The promoters (prom) that drive the expression of these transgenes are either non-specific promoters (HCMV, EF1A), or afferent neuron-specific of promoters (TRPV1, TRPM8, ASIC3, GCRP, ADVl). Additional sequences conferring long-term expression (LTE and DNA insulator sequences) are added to some of these promoters (<FIG>). The promoter that governs the expression of the reporter GFP, or the GFP-rLuc fusion protein, present in amplicon vectors, is the viral immediate-early promoter known as HSV-<NUM> IE4/<NUM> promoter. The general structure of some of the amplicon vectors used herein is shown in <FIG>.

The expression of BoNT-A (LC), BoNT-C (LC) and TeNT (LC) is performed in Gli36 (a cell line derived from a human glioblastoma) and BHK21 (hamster fibroblast cells) cell lines. Gli36 and BHK21 cells are infected with the amplicon vectors expressing HCMV-Luc, HCMV-BoNT-A (LC), HCMV-BoNT-C (LC), or HCMV-TeNT (LC). The cells were then fixed and the expression of the toxin was demonstrated by Western blot using anti-TeNT antibodies to reveal TeNT (LC) and anti-HIS antibodies to reveal BoNT-A (LC) and BoNT-C (LC). Indeed, there are no efficient anti-BoNT antibodies available, therefore BoNT-A (LC) and BoNT-C (LC) are expressed as a fusion protein with a C-terminal HIS-tag. <FIG> shows that the viral vector carrying the genes coding for HCMV-BoNT-A (LC), HCMV-BoNT-C (LC), and HCMV-TeNT (LC) express respectively BoNT-A (LC), BoNT-C (LC) and TeNT (LC) in both Gli36 and BHK21 cells.

Proteolytic activity of the toxin TeNT (LC) with respect to VAMP2 was evaluated by Westerns blots using anti-VAMP2 antibody. The toxin TeNT (LC) was expressed in Gli36 cells after infection with the viral expression vector expressing HCMV-TeNT (LC). The infection was terminated <NUM> days later and protein extracts were prepared. These extracts were incubated in a suitable buffer (containing <NUM> Hepes, <NUM> NaCl, <NUM> dithiothreitol and <NUM> ZnSO<NUM>) containing the target protein of TeNT, i.e., VAMP2. Westerns blots (<FIG>) were performed using <NUM>, <NUM>, and <NUM>µL of cell extracts. Untreated sample, a sample from cells infected with a vector expressing no transgene (pA-<NUM>), and a sample from cells infected with a vector expressing HCMV-Luc (<NUM>µL) were used as a negative control. Varying amounts of recombinant TeNT (recTeNT) were used as a positive control. Results show that the quantity of VAMP2 decreases when the protein extract expressing TeNT (LC) is increased, which demonstrate that the toxin present in the protein extract exhibits a proteolytic activity toward VAMP2.

The SH-S5Y5 human neuroblastoma cell line was used for its property to spontaneously express SNARE proteins, in order to follow in cellulo SNAP25 and Syntaxin 1a (STX) cleavage following infection by amplicon vectors expressing BoNT-A (LC) or BoNT-C (LC). SNAP25 and STX levels were detected by Western blot assay using anti-SNAP25 or anti-STX antibodies, respectively. As negative controls, cells were not infected (Mock) or were infected with the vector expressing HCMV-Luc. Results (<FIG> and <FIG>) show that at <NUM> hours post-infection (hpi) of SH-S5Y5 cells with vectors expressing the light chains of BoNT-A or BoNT-C, there is respectively cleavage and significant decrease of in cellulo SNAP25 (<FIG>) or SNAP25 and STX (<FIG>) protein levels relative to cells infected with the control vector expressing Luciferase.

This experiment was designed to assess whether vectors expressing the light chain of BoNT-A do express this protein, and to study whether this toxin has the same biological activity as the complete neurotoxin (light chain + heavy chain), i.e., the ability to cleave its target SNARE protein (SNAP25). As shown in <FIG>, cells infected at increasing multiplicities with amplicon expressing A2-CMV-BoNT-A do express increasing amounts of the toxin. Moreover, when cells are infected at high MOI virtually all SNAP25 is cleaved, clearly demonstrating the functional activity of the light chain of BoNT-A.

Light chains of botulin neurotoxins cleave SNARE proteins in infected neurons.

This experiment was designed to confirm that all BoNT light chains synthesized in vector-infected neurons are able to cleave their natural SNARE target protein in sensory neurons. To this end, primary cultures of rat embryonic DRG neurons were infected at an MOI of <NUM> with amplicon vectors expressing A2-CMV-BoNT-A, -B, -C, -D, -E and -F, or A2-CMV-Luc as negative control. Infections were stopped the following day and cell extracts were analyzed by Western blots. As shown in <FIG>, each of the botulinum neurotoxin expressed by the vectors cleaved its natural target SNARE protein. Thus, BoNT-A and -E cleaved SNAP25, BoNT-B, -D, and -F cleaved VAMP2, while BoNT-C cleaved both SNAP25 and Syntaxin. This clearly demonstrates that the light chains of all neurotoxins display the same biological activity as the complete neurotoxins (light chain + heavy chain).

Light chains of botulin toxins inhibit release of neuropeptides in sensory neurons.

This experiment was designed to assess whether the light chains of botulinum neurotoxins induced inhibition of release of neurotransmitters and to evaluate their comparative efficacy in this respect. Primary cultures of rat embryonic DRG neurons were infected at increasing MOI with the vectors as described in <FIG>. The following day, infected neurons were treated with KCl to stimulated release of neuropeptide CGRP and the extracellular concentrations of CGRP were evaluated by ELISA. As shown in <FIG>, all neurotoxins induced inhibition of release of CGRP. Moreover, <FIG> shows that BoNT-F was the most effective in this respect, followed by BoNT-A and -C.

GAD67 expressed from amplicon vectors induces synthesis and extracellular release of GABA.

The goal of this experiment is to assess whether vectors expressing GAD67 induce synthesis and release of the inhibitory neurotransmitter GABA. To this end, glioblastoma cells (Gli36) were infected at increasing MOI with amplicon vectors as described in <FIG> and the following day infected cell extracts were analyzed by Western blots, using antibodies specific for GAD67 and GAPDH. <FIG> shows that expression of GAD67 increases with the MOI, demonstrating that vector A2-CMV-GAD67 does express this protein. In addition, primary cultures of rat embryonic DRG neurons were infected at different MOIs with the same vectors. The following day infections were stopped and both intracellular and extracellular concentrations of GABA were evaluated using Resazurine assay (as indicated in the legend to <FIG>). The upper panel of this figure shows that the amount of intracellular GABA increases with the MOI, while the lower panel shows the increase of extracellular GABA, clearly demonstrating that expression of GAD67 from the A2-CMV-GAD67 vector increases synthesis of intracellular GABA and its release to the extracellular medium.

Nitroreductase (NTR) activates the nitro compound <NUM>'nitrocoumarin and induces cell death in the presence of mitronidazole (MTZ).

This experiment was designed to assess whether nitroreductase expressed from amplicon vectors induced cell death in the presence, but not in the absence of metronidazole. There are no available antibodies specific for nitroreductase (NTR). Therefore, to assess that this protein is expressed in A2-CMV-NTR infected cells, we used a functional in vitro test based on the evaluation of reduction of <NUM>'nitrocoumarin (Muller et al. <FIG> shows that amplicon vectors expressing A2-CMV-NTR do activate the nitro compound. Furthermore, <FIG> shows that expression of NTR induced significant cell death in the presence of metronidazole (MTZ). This is explained by the fact that NTR can activate MTZ thus transforming this molecule into a cytotoxic drug.

Analysis of the selectivity of expression of DRG-selective promoter candidates in autonomic and sensory ganglia from adult rats.

This test was designed to investigate whether afferent neuron-specific promoter candidates, which normally are active only or mainly in afferent neurons, preserve their afferent neurons-specific activity also when they are expressed from the non-replicative HSV-<NUM> vector genome. Rat adult afferent ganglia (DRG), autonomic sympathetic ganglia (SCG), and autonomic parasympathetic ganglia (GPC) were explanted and kept as organotypic cultures. After <NUM> days, a time required for neurite outgrowth, the ganglia were individually infected with <NUM> × <NUM><NUM> vector particles as described in the legend to <FIG>. These vectors express firefly luciferase (fLuc) driven by the following promoters: rat TRPV1 (rTRPV1), rat CGRP (rCGRP), rat ASIC3 (rASIC3), all of which are considered as afferent-neuron specific promoters, and EF1a, a non-selective promoter serving as general control. In addition to fLuc, these vectors also express renilla luciferase (rLuc) driven by a viral promoter (HSV-<NUM> IE4/<NUM>). The following day infections were stopped and cells extracts were prepared for luciferase tests. Results are expressed as the ratio of fLuc/rLuc and as percentage of luciferase activity driven by EF1a. <FIG> shows that rTRPV1 and rCGRP express firefly luciferase activity preferentially in DRG and can thus be considered as DRG-specific even when they express from the vector genome. In contrast, rASIC3 does not display such preferential expression in the DRG demonstrating that this promoter does not preserve its selectivity when expressed from the vector genome. Therefore, this example shows that some DRG-specific promoter candidates, such as the rTRPV1 and rCGRP promoters, do preserve their selectivity for DRG while other promoter candidates, such as rASIC3, although considered a DRG-specific promoter when it expresses from the cellular chromosomes, does not preserve this specificity when expressed from the vector genome. Therefore, the behavior of any particular DRG-specific promoter candidate cannot be predicted and should be experimentally assessed.

Primary rat neuronal cultures from embryonic DRG and organotypic cultures of adult rat DRG explants were infected with an amplicon vector expressing GFP driven by the HSV-<NUM> immediate early IE4/<NUM> promoter. Results show that the viral expression vector infected and expressed the transgene (GFP) both in primary rat sensory neuronal cultures and in adult rat ganglion (DRG) explants (<FIG>).

Spinal cord injured (SCI) rats were infected by the amplicon vector HCMV-Luc, which simultaneously expresses GFP and Luc reporter proteins. One week post-infection, the animals were sacrificed and transgenic proteins expressions were revealed by IHC. As indicated by the IHC, when inoculated into the bladder the amplicon vector enters the afferent neurons innervating the bladder, and is then retrogradely transported through the axons to the cell bodies of the neurons, which lie in the dorsal ganglia (DRG), and where the viral genome expresses both transgenic proteins. Results indicate that amplicon vectors HCMV-Luc are thus capable of penetrating and specifically expressing transgenic proteins in the bladder afferent neurons following their inoculation into the bladder wall (<FIG>). Moreover, neurons expressing GFP and Luc are observed only in the ganglion from which neurons that innervate the bladder extend (the L6 ganglion). In contrast, in the ganglion T13, which does not innervate the bladder, no transgene expression could be observed (data not shown).

Claim 1:
A herpes simplex virus (HSV) viral expression vector comprising at least:
a) one promoter active selectively in afferent neurons of the bladder, wherein said promoter is the promoter of Substance P,
b) one transcription cassette comprising a nucleotide sequence operably linked to said promoter, wherein said nucleotide sequence silences or inhibits the transduction of the neurotransmitter signal in postsynaptic cell when transcribed, and
c) one long-term expression sequence, wherein said long-term expression sequence is an LTE and a DNA insulator from the HSV-<NUM> genome, and wherein said transcription cassette is placed between the LTE and the DNA insulator.