Viral vectors for treating bladder diseases

Abstract

The present invention provides a method and pharmaceutical composition for the treatment of overactive bladder (OAB) and / or bladder pain syndrome (BPS), comprising a viral expression vector having a transcription cassette containing one or more transgenes that inhibit / silence neurotransmission or synaptic transmission of afferent neurons.

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 524,486, filed Jun. 30, 2023. The entire disclosure of the above application is incorporated herein by reference.

[0002] Sequence Listing The sequence listing filed via EFS in accordance with 37 CFR § 1.52(e)(5) of the United States Patent Laws is incorporated herein by reference. The XML file of the sequence listing filed via EFS contains the file "42873006 WO Seq List.xml" created on Jun. 27, 2024, with a size of 65,362 bytes.

Background Art

[0003] The present invention relates to viral expression vectors that selectively regulate or silence the afferent nerves of the bladder and pharmaceutical compositions thereof as gene therapy strategies for the treatment of overactive bladder (OAB) and / or interstitial cystitis (IC).

[0004] Abnormal bladder function is a common problem that greatly affects the quality of life of millions of men and women in the United States. Changes in bladder physiology have two main clinical manifestations: atonic bladder and hyperreflexic bladder. In atonic bladder, i.e., detrusor underactivity, the contractile force of the detrusor smooth muscle (the smooth muscle outside the bladder wall) is reduced, resulting in a reduced ability to empty its urine content. Conversely, a bladder showing hyperreflexia, uninhibited bladder, or detrusor overactivity contracts spontaneously during bladder filling, which can cause frequency of urination, urgency of urination, and urge incontinence (where the individual cannot control the passage of urine).

[0005] Overactive bladder is a very common condition affecting 15-33% of the general population, ranging from mild to severely debilitating forms. It is more prevalent in women and is characterized by an urgent need to urinate up to 40 times a day, with or without incontinence. The main impact on quality of life and self-esteem occurs concurrently with mental health effects such as depression and anxiety. The need to urinate frequently can occur during the day, at night, or both. Overactive bladder is characterized by four symptom groups: urgency, frequent urination, nocturia (frequent urination at night), and urge urinary incontinence.

[0006] Interstitial cystitis (IC), defined as chronic pain in the pelvic region in the absence of a urinary tract infection, is a type of painful bladder syndrome (PBS) characterized by chronic pain in the bladder and pelvic floor. Symptoms include frequent urges to urinate, a need to urinate often, and painful intercourse. IC / PBS is associated with depression and a poor quality of life. Many affected individuals also suffer from irritable bowel syndrome and fibromyalgia. IC / PBS is estimated to affect 0.9% of the US population, and its incidence has doubled in recent decades. However, these patients have very limited medical options, with only one approved medication showing limited efficacy.

[0007] The drugs used to treat hyperreflex bladder are usually only partially effective and have severe side effects, limiting patient use and enthusiasm. Currently accepted treatment options (e.g., oxybutynin and toltelladine) are largely nonspecific and most frequently involve blockade of the muscarinic receptor pathway and / or calcium channels in bladder muscle cells. Given the central importance of these two pathways in the cellular function of many organ systems in the body, such therapeutic strategies are not only crude ways of regulating bladder smooth muscle tone but are virtually guaranteed to have serious and undesirable systemic effects.

[0008] Over time, most OAB patients do not respond to standard pharmacological approaches to treatment, such as oral antimuscarinic agents and β-3 agonists, which have systemic off-target effects and are poorly tolerated by many patients. Local injections of botulinum toxin essentially paralyze the bladder muscle, requiring patients to empty their bladder 4-6 times a day via catheterization in 15% of cases, increasing the risk of associated urinary tract infections. For OAB, expensive implantable medical devices requiring surgical intervention are approved. Therefore, there is a great need for improved treatment options for bladder dysfunction.

[0009] The aforementioned and other objects, features and advantages of the present invention will become apparent from the following more detailed description of preferred embodiments of the invention, as shown in the accompanying drawings, where similar reference numerals in different figures refer to the same parts. The drawings are not necessarily to scale and instead focus on illustrating the principles of the invention.

[0010] The following examples are merely illustrative of the present invention. [Brief explanation of the drawing]

[0011] [Figure 1]This figure shows the genome of a recombinant deficient HSV-1 vector. (A): The top of the figure illustrates the skeleton of the HSV-1 genome used in the present invention. The HSV-1 genome contains two unique regions known as unique long (UL) and unique short (US), each bounded by repeating reverse sequences known as terminal repeat L / reverse repeat L (TRL / IRL) and reverse repeat S / terminal repeat S (IRS / TRS). TRL / IRL is also called ab / b'a' and IRS / TRS is also called a'c' / ca. Thus the genome begins and ends with a direct repeat sequence "a". The black square in UL indicates the deletion of the gene encoding the essential ICP27 protein in the vector used in the present invention. Similarly, the two black squares in the IRS / TRS repeat sequence indicate the deletion of two genes encoding the essential ICP4 protein. The white circles in UL, as well as the two white circles in the IRS and TRS regions, indicate the DNA replication origins of HSV-1 (OriL and two copies of OriS, respectively). Other genes encoding non-essential proteins such as UL41, UL55, and UL56 can also be deleted. Furthermore, copies of both IE4 / 5 promoters localized in IRS and TRS are modified so that these promoters are expressed at early kinetics (instead of immediate kinetics like the wild-type viral genome) (deletion of one TAATGARAT sequence). (B): The central portion of the figure shows details of the b'a'a'c' region of the viral genome, in particular showing the localization of the LAT locus in the IRL region containing the gene expressing the latent-associated transcript (LAT). (C and C'): The lower part of the figure shows detailed structure of the 5' portion of the LAT locus with a therapeutic DRG-specific transcription cassette (shown in the figure as a transgene labeled arrow). This locus contains an upstream DNA insulator (INS) sequence, a latent infection-associated promoter (LAP), a region conferring long-term expression (LTE), and a downstream DNA insulator (INS). The therapeutic DRG-specific transcription cassette is introduced either between the LAP and LTE (site 1 of C) or between the LTE and a second DNA insulator (site 2 of C').Other genes near the LAT are also shown by B (arrow). Various DRG-specific transcription cassettes introduced into the LAT region to construct recombinant vectors are shown in Figure 3. It should be emphasized that region b'a'a'c' is identical to the inverted caab region formed when the viral genome is circularized in the cell nucleus at the onset of infection. This means that both copies of ICP4 can be deleted and the transgenic transcription cassette can be introduced into both copies of the LAT locus. The cassette can also be introduced into the LAT locus of TRL. Such an embodiment is shown in Figure 1D. [Figure 2] This figure shows the genome of an amplicon vector (amplicon plasmid). An amplicon plasmid is a standard E. coli (E. coli) plasmid that generally has three modules: (1) a bacterial module containing the Col E1 sequence for plasmid replication in bacteria and a gene (black) that confers resistance to antibiotics, generally ampicillin; (2) an amplicon module (pink) containing the HSV-1 DNA replication origin, generally OriS(O), and a packaging signal (a) that enables amplification and packaging of the concatemer form of the amplicon plasmid. Furthermore, this module generally expresses a reporter protein (in our case, either a GFP protein or a fused GFP / sea urticaria luciferase (rLuc) protein) driven by the HSV-1 pre-initial promoter IE4 / 5. (3) The third module (white) is a transcription unit containing a DRG-specific transcription cassette (gray arrow labeled as transgene) positioned between the sequenced LTE and INS, designed to stably and selectively inhibit or silence neurotransmission in sensory neurons, as described in the present invention. Various transcription cassettes introduced into the amplicon plasmid to construct an amplicon vector are shown in Figure 3. [Figure 3A]This figure represents a region of the genome of the amplicon vector used in the present invention, which has two eukaryotic transcription cassettes. One of these expresses the reporter GFP (or fused GFP-rLuc) gene under the control of the HSV-1 IE4 / 5 pre-initial promoter. The second transcription cassette expresses either a therapeutic function that inhibits or silences neurotransmission, as described in the present invention. A DRG-specific promoter drives the expression of the transcription cassette, but the entire cassette is surrounded by a sequence (black square) that confers long-term expression. [Figure 3B] This figure shows some of the transcription cassettes used in this study to demonstrate the efficacy and selectivity of the gene constructs. These are as follows: Vector A: HCMV-TeNT light chain, (LC); Vector B: HCMV-BoNT-A(LC); Vector C: HCMV-BoNT-C(LC); Vector D: SNAP25 antisense RNA; Vector E: HCMV-luciferase; Vector F: TRPV1-luciferase. BoNT-A(LC) and BoNT-C(LC) are fusion proteins expressing a C-terminal HIS tag because effective anti-BoNT antibodies are not available. HCMV is a potent and ubiquitous viral promoter, while TRPV1 is a DRG-selective promoter. Other vectors expressing other botulinum toxins, or fusion SNARE / light chain toxins, or antisense RNA addressed to other SNARE proteins, or human GAD67 protein, or RIP proteins such as saporin S6, are not shown in this figure. [Figure 4]Figure 4 shows the expression of BoNT-A(LC), BoNT-C(LC), and TeNT(LC) in Gli36 (human glioblastoma-derived cell line) and BHK21 (hamster fibroblast cell) cell lines. Gli36 and BHK21 cells were infected with the amplicon vectors HCMV-Luc, HCMV-BoNT-A(LC), HCMV-BoNT-C(LC), or HCMV-TeNT(LC) (shown in Figure 2B). The infected cells were then fixed, and toxin expression was demonstrated by Western blotting using specific antibodies. TeNT(LC) was identified using anti-TeNT antibodies. Both BoNT-A(LC) and BoNT-C(LC) were identified using anti-HIS antibodies. [Figure 5] This figure shows that the toxin TeNT(LC), expressed in Gli36 cells and present in cell extracts, has proteolytic activity against VAMP2. Gli36 cells were infected with an amplicon vector expressing HCMV-TeNT(LC) at a multiple of infection (MOI) of 1. Infection was terminated after 2 days, and protein extracts were prepared. These extracts were incubated in a suitable buffer (50 mM Hepes, 400 mM NaCl, 5 mM dithiothreitol, and 2 μM ZnSO4) containing the target protein of TeNT, namely VAMP2. After incubation at 37°C for 24 hours with 2.5, 5, and 10 μL of cell extracts, Western blotting was performed using an anti-VAMP2 antibody to reveal the proteolytic activity of TeNT(LC) expression. [Figure 6A]This figure shows that SNAP25 protein levels were significantly reduced intracellularly 48 hours (hpi) after infection (see Figure 3B) of human neuroblastoma SH-S5Y5 cells with an amplicon vector expressing HCMV-BoNT-A(LC) compared to control cells (Mock) infected with a luciferase-expressing vector (HCMV-Luc) or uninfected cells. Protein levels were detected by a Western blot assay using an anti-SNAP25 antibody. Note that BoNT-A(LC) cleaves SNAP25 into two fragments. The antibodies used in these experiments recognize both the native SNAP25 protein (upper band) and the larger fragment of the cleaved protein (lower band). [Figure 6B] This figure shows that at 48 hours (hpi) after infection (hpi) of human neuroblastoma SH-S5Y5 cells with an amplicon vector expressing HCMV-BoNT-C(LC) (see Figure 3B), intracellular protein levels of both SNAP25 and syntaxin (STX) were significantly reduced compared to control cells infected with a luciferase-expressing vector (HCMV-Luc) or uninfected control cells (Mock). Protein levels were detected by Western blot assays using anti-SNAP25 and anti-STX antibodies. Note that BoNT-C(LC) cleaves SNAP25 into two fragments. The antibodies used in these experiments recognize both the native SNAP25 protein (upper band) and the larger fragment of the cleaved protein (lower band). [Figure 7A] This figure shows transcription cassettes delivered by recombinant and amplicon vector genomes. [Figure 7B] This figure shows transcription cassettes delivered by recombinant and ampliconvector genomes. [Figure 7C]This figure shows transcription cassettes carried by recombinant and amplicon vector genomes. This figure shows some of the transcription cassettes carried and expressed by recombinant and amplicon HSV-1 vectors. These transcription cassettes are classified into three families. Members of the A2 family are transcription cassettes driven by a potent, ubiquitous HCMV promoter and expressing a variety of therapeutic gene products (proteins or antisense RNA or miRNA). Vectors with A2 transcription cassettes are used to study the effects of these gene products on neurotransmission (cleavage of SNARE proteins and inhibition of neurotransmitter release), thus enabling the selection of the most efficient transgene in the context of the present invention. Members of the A5 family are transcription cassettes expressing the reporter gene firefly luciferase (fLuc) driven by different DRG-selective candidate promoters. These vectors are used to study the intensity, selectivity, and duration of expression in cultured neurons and explanted peripheral ganglia, thus enabling the identification of the most selective vector in the context of the present invention. Finally, members of the vector's A8 family express therapeutic transcription cassettes (therapeutic gene products driven by a DRG-selective promoter), thus allowing for the selection of vectors with high therapeutic potential in vivo, within the context of the present invention. It should be noted that, as shown in Figure 2, the amplicon vector also expresses GFP / rLuc transgenes driven by the HSV-1 IE4 / 5 promoter. Abbreviations: ·TeNT: tetanus neurotoxin light chain ·BoNT-X: botulinum neurotoxin light chain BoNT-A, -B, -C, -D, -E, or F ·BoNT-X-SNARE-Y: fusion protein in which the botulinum neurotoxin light chain is fused to the signal peptide and transmembrane peptide of the SNARE protein. More precisely, these transgenes express BoNT-A-syntaxin, BoNT-B-syntaxin, or BoNT-C-Vamp2.• GAD67: 67kD glutamate decarboxylase • NTR: Nitroreductase • Luc: Firefly luciferase (fLuc) • Antisense-SNARE: Antisense RNA against SNARE proteins SNAP25, VAMP2, or syntaxin • Human elongation factor 1 promoter (EF1A), rat transient receptor potential vanilloid 1 (rTRPV1), human and rat calcitonin gene-related peptides (hCGRP and rCGRP), rat acid-sensing ion channel 3 (rASIC3), and human and rat advilin (hADVL and rADVL) promoters. [Figure 8] This figure shows that BoNT-A expressed from an amplicon vector cleaves the SNARE protein SNAP25 in SH-SY5Y cells. Human neuroblastoma cells (SH-S5Y5) were infected with amplicon vectors expressing the transcription unit A2-CMV-BoNT-A(LC) or A2-CMV-Luc (both HCMV promoter-driven) at MOIs of 0.1, 1.0, and 10.0 pfu / cell. The following day, infection was stopped, and cellular proteins were analyzed by Western blotting using antibodies specific to BoNT-A LC and SNAP25. The upper part of the Western blot shows that the increase in the amount of BoNT-A LC corresponds to the increase in MOI, demonstrating that the vector used expresses this protein in infected cells. The lower part of the blot shows cleavage of SNAP25, a protein derived from the SNARE complex, which is the native target of BoNT-A, thus showing that two fragments are produced. At lower MOIs, mainly the native (uncleaved) form of SNAP25 is observed. At intermediate MOIs, both the native and cleaved forms (a slightly lower band) are observed, but at high MOIs, only the lower fragments of the doublet may be observed, indicating that the majority of the SNAP25 protein is cleaved. This demonstrates that BoNT-A LC synthesized in SH-S5Y5 cells can cleave SNAP25. In contrast, no cleavage of SNAP25 is observed in SH-S5Y5 cells infected with a vector expressing Luc. [Figure 9]This figure shows that the light chain of botulinum neurotoxin cleaves SNARE proteins in infected neurons. Primary cultures of rat embryonic dorsal root ganglion (DRG) neurons were infected 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 at a MOI of 10 pfu / cell. 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). A vector expressing A2-CMV-Luc was used as a negative control. In all cases, the HCMV promoter promoted the expression of the transcription cassette. The infection was stopped the following day, and cellular proteins were analyzed by Western blotting. As shown in the figure, each BoNT LC synthesized in the neuron cleaved the expected SNARE protein. Thus, as evidenced by the reduction in the size of this protein, the light chains of BoNT-A, -C, and -E cleaved SNP25, while the light chains of BoNT-B and -F cleaved VAMP2, which was no longer detectable in the blot. Furthermore, BoNT-C also cleaved syntaxin (STX), which was also not visible in the blot. BoNT-C is the only botulinum toxin described as being able to cleave two different SNARE proteins (SNAP25 and STX). The botulinum toxin light chain fused to the signal peptide and transmembrane peptide of the SNARE protein cleaved the corresponding SNARE protein in exactly the same way as the parent unfused toxin. Lane Luc shows the location of the native uncleaved SNARE protein (arrow). Thus, this figure demonstrates that the botulinum toxin light chain synthesized in sensory neurons upon vector infection (whether fused to a fragment of the SNARE protein or not) can cleave its corresponding target protein. [Figure 10]This figure shows that the light chain of botulinum toxin inhibits the release of neuropeptides in sensory neurons. Primary cultures of rat embryonic DRG neurons were infected 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 at increased MOI (0.5-3 pfu / cell). Neurons were also infected with amplicons expressing A2-CMV-BoNT-A-syntaxin, A2-CMV-BoNT-B-syntaxin, and A2-CMV-BoNT-C-VAMP2. A vector expressing A2-CMV-Luc was used as a negative control. The day after adding only the medium (mock) to the neurons, infected neurons were treated with 75 mM KCl to stimulate the release of CGRP, a neuropeptide normally synthesized in DRG neurons. 100 microliter aliquots were taken from the culture medium 30 minutes before and 30 minutes after KCl treatment, and the presence of CGRP was evaluated by ELISA (using the Spi Bio CGRP ELISA kit, reference number A05482). The results, expressed as a log-transformed linear regression profile, show that all toxins inhibited CGRP release, but to different intensities, with BoNT-F, BoNT-C, and BoNT-A being the most effective in this regard. No inhibition of CGRP release was observed in mock-infected neurons, as well as in neurons infected with a vector expressing Luc. These results clearly demonstrate that cleavage of SNARE proteins by BoNT LC leads to inhibition of neuropeptide release, and that BoNT-F is the most efficient in this regard. [Figure 11A]This figure shows that GAD67 expressed from an amplicon vector induces the synthesis and extracellular release of GABA (gamma-aminobutyric acid). Glioblastoma cells (Gli36) were infected with amplicon vectors expressing A2-CMV-GAD67 or A2-CMV-Luc at MOI 0.1, 1.0, and 10 pfu / cell. The following day, infection was stopped, and cellular proteins were analyzed by Western blotting using antibodies specific to GAD67 and GAPDH (a housekeeping gene used as an internal control). Endogenous GAD67 was identified using rat brain extracts as a positive control. Figure 11A shows that GAD67 expression increases with MOI, demonstrating that the vector A2-CMV-GAD67 expresses this protein. [Figure 11B] This figure shows that GAD67 expressed from an amplicon vector induces the synthesis and extracellular release of GABA (gamma-aminobutyric acid). Primary cultures of rat embryonic DRG neurons were infected with vectors expressing A2-CMV-GAD67 or A2-CMV Luc at MOI 0.1, 1.0, and 10 pfu / cell. The following day, infection was stopped, and both intracellular and extracellular GABA concentrations were evaluated using the Resazurine assay, a fluorescence-conjugated assay for GABA (the assay was performed as described in Ippolito et al., 2014). The upper panel shows that the amount of intracellular GABA increases with MOI, while the lower panel shows the increase in extracellular GABA. Channels labeled with GABA are positive controls for the Resazurine assay. These results clearly demonstrate that expression of GAD67 from the A2-CMV-GAD67 vector increases intracellular GABA synthesis and its release into the extracellular medium. [Figure 12A]This figure shows that nitroreductase (NTR) activates the nitro compound 7'-nitrocoumarin, inducing cell death in the presence of mitronidazole (MTZ). Human glioblastoma (Gli36) cells were infected with amplicon vectors expressing A2-CMV-NTR or A2-CMV-Luc at an MOI of 1.0 pfu / cell. After 2 days, infection was stopped, protein extracts were prepared, and used to evaluate 7'-nitrocoumarin activation using a fluorescence-conjugated assay (assay performed as in Muller et al., 2015). Figure 12A shows that only the protein extracted from cells infected with the vector A2-CMV-NTR induced significant 7'-nitrocoumarin activation, demonstrating that functional NTR was expressed in Gli36 cells infected with A2-CMV-NTR. [Figure 12B] This figure shows that nitroreductase (NTR) activates the nitro compound 7'-nitrocoumarin, inducing cell death in the presence of mitronidazole (MTZ). To evaluate whether NTR expression induces cell death in the presence of metronidazole (MTZ), Gli36 cells were infected with the amplicon vector A2-CMV-NTR or A2-CMV-Luc at an MOI of 1.0 pfu / cell. The following day, cells were incubated for 24 hours with and without MTZ (0.5 mM). Infection was then stopped, and cell viability was assessed using the MTT assay (as shown in Carmichael et al., 1987). This figure shows that MTZ significantly increased cell death in infected cells. Mock: Uninfected cells. [Figure 13]This figure shows the analysis of the selectivity of DRG-selective promoter candidates in the autonomic and sensory ganglia of adult rats. Adult rat sensory ganglia (DRG), autonomic sympathetic ganglia (superior cervical ganglia, SCG), and autonomic parasympathetic ganglia (paracervical ganglia, GPC) were explanted and maintained as organoid cultures. After 3 days, the ganglia were individually infected with vectors expressing A5-TRPV1-Luc, A5-rCGRP-Luc, A5-ASIC3-Luc, or A5-EF1A-Luc. All of these express firefly luciferase (fLuc), but are driven by the following promoters, respectively: rat TRPV1 (rTRPV1), rat CGRP (rCGRP), rat ASIC3 (rASIC3), and EF1a, a non-selective promoter serving as a general control. Each ganglion was infected with 10⁶ vector particles. The vector also expresses sea urchin luciferase (rLuc) driven by the viral promoter (HSV-1 IE4 / 5). The following day, infection was stopped, and cell extracts were prepared for luciferase testing using a Promega dual luciferase reporter assay system. Results were expressed as the fLuc / rLuc ratio and normalized as the proportion of EF1a promoter expression in DRG (left), SCG (center), and GPC (right), respectively. Figure 13 shows that several 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 show preferential activity in DRG. These results suggest that rTRPV1 and rCGRP promoters exhibit selective activity for DRG, while rASIC3 does not exhibit such selectivity when expressed from the viral genome. [Figure 14] This figure shows that an amplicon vector expressing the reporter protein GFP can infect primary cultures of embryonic rat DRG neurons and adult rat DRG explants, enabling the expression of the transgene GFP within these neurons. [Figure 15]Inoculation of the bladder wall with defective HSV-1 vectors reaches the dorsal root ganglia (DRGs) and expresses the transgene in sensory neurons innervating the bladder. Viral vectors expressing IE4 / 5-GFP and HCMV-luciferase (shown in Figure 3B) can penetrate into bladder afferent neurons and express both transgenic proteins after inoculation into the bladder wall of spinal cord injury (SCI) rats. DRG neurons expressing both GFP and luciferase (Luc) are shown in DRG ganglion L6, from which neurons innervating the bladder extend. However, in DRG ganglion T13, which does not innervate the bladder, the results are negative. One week after infection, the animals were sacrificed, and the transgene proteins were revealed by IHC using specific antibodies against GFP and luciferase. These results indicate that after inoculation into the bladder wall, the vector enters bladder afferent neurons, is retrogradely transported through the axons to the cell bodies of the neurons to the L6 ganglion in the dorsal root ganglia (DRGs), and from there the viral genome expresses both transgenic proteins. The vector cannot reach or be expressed in neurons that do not innervate the bladder (T13). [Figure 16] Figure showing high cell selectivity of viral vector expression in dorsal root ganglia (DRGs) when luciferase is driven by the DRG-selective TRPV1 promoter. Luciferase is significantly expressed only in afferent neurons and not in autonomic neurons (sympathetic or parasympathetic). The results were normalized as the ratio of luciferase expression to luciferase expression from a vector expressing luciferase under the control of the strong but non-specific HCMV promoter (both vectors are shown in Figure 3B). [Figure 17] Figure showing that BoNT / F-LC is expressed in DRGs after intradermal injection of the vector according to the present invention into the bladder wall. DRG L6 / S1 was collected and pooled 1 week and 5 weeks after injection. ddPCR was performed to quantify the absolute number of copies of BoNT / F-LC (mean + / − SD). [Figure 18A]Figure showing the maximum pressure (MP) and baseline pressure (BP) of urinary contractions in rats treated with buffer (black squares, n = 11) and 2.2 x 108 PFU of vector (dark blue triangles, n = 12) 5 weeks later, expressed as a percentage of the stabilization period. Data are mean ± SEM. The MP parameter was significantly decreased in rats treated with the vector according to the present invention. [Figure 18B] Figure showing the maximum pressure (MP) and baseline pressure (BP) of urinary contractions in rats treated with buffer (black squares, n = 11) and 2.2 x 108 PFU of vector (dark blue triangles, n = 12) 5 weeks later, expressed as a percentage of the stabilization period. Data are mean ± SEM. The BP parameter was significantly decreased in rats treated with the vector according to the present invention. [Figure 19A] Figure showing the area under the curve (AUC) and duration of urinary contractions in rats treated with buffer (black squares, n = 11) and 2.2 x 108 PFU of vector (dark blue triangles, n = 12) 5 weeks later, expressed as a percentage of the stabilization period. Data are mean ± SEM. The AUC was significantly decreased in vector-treated rats 5 weeks after treatment compared to the buffer group (two-way ANOVA: *p < 0.05). [Figure 19B] Figure showing the area under the curve (AUC) and duration of urinary contractions in rats treated with buffer (black squares, n = 11) and 2.2 x 108 PFU of vector (dark blue triangles, n = 12) 5 weeks later, expressed as a percentage of the stabilization period. Data are mean ± SEM. In contrast, the duration of urinary contractions was not significantly modified in vector-treated rats 5 weeks after treatment compared to the buffer group (two-way ANOVA: ns p > 0.05). [Figure 20]This figure shows the delta pressure threshold (ΔPT) of rats treated with buffer (black squares, n=11) and 2.2 × 10⁸ PFU of vector (dark blue triangles, n=12) after 5 weeks, expressed as a percentage of the stabilization period. Data are mean ± SEM. ΔPT was not altered in the vector-treated rats after 5 weeks compared to the buffer group (two-way ANOVA: ns p>0.05). [Figure 21] This figure shows the intercostal interval (ICI) of rats 5 weeks after treatment with buffer (black squares, n=11) and 2.2 × 10⁸ PFU of vector (dark blue triangles, n=12), expressed as a percentage of the stabilization period. Data are mean ± SEM. ICI was significantly increased with the EG110A vector 5 weeks after treatment compared to the buffer group (two-way ANOVA: **p<0.01). [Figure 22] This figure shows the infusion volume (IV) as an indicator of bladder capacity in rats 5 weeks after treatment with buffer (black squares, n=11) and 2.2 × 10⁸ PFU of vector (dark blue triangles, n=12), expressed as a percentage (%) of the stabilization period. Data are mean ± SEM. As reported for the ICI parameter, IV was significantly increased with the vector treatment 5 weeks after treatment compared to the buffer control group (two-way ANOVA: **p<0.01). [Figure 23A] This figure shows the urinary volume (VV) and compliance of rats 5 weeks after treatment with buffer (black squares, n=11) and 2.2 × 10⁸ PFU vector (dark blue triangles, n=12), expressed as a percentage (%) of the stabilization period. Data are mean ± SEM. As shown in Figure 23A, VV was significantly increased in the vector group 5 weeks after treatment compared with the buffer control group (two-way ANOVA: ***, p<0.001), and particularly significantly increased 30 minutes after the start of capsaicin infusion (Bonferroni multiple comparison test against the buffer group: $, p<0.05 at 30–45 minutes and 45–60 minutes). [Figure 23B]This figure shows the urination volume (VV) and compliance of rats treated with buffer (black squares, n=11) and 2.2 × 10⁸ PFU of vector (dark blue triangles, n=12) 5 weeks later, expressed as a percentage (%) of the stabilization period. Data are mean ± SEM. Similarly, compliance in the vector group 5 weeks later was significantly increased compared to the buffer control group (two-way ANOVA:*, p<0.05 (Figure 23B)). [Figure 24] This figure shows the urination efficiency (VE) of rats 5 weeks after treatment with buffer (black squares, n=11) and 2.2 × 10⁸ PFU of vector (dark blue triangles, n=12), expressed as a percentage of the stabilization period. Data are mean ± SEM. VE was not altered by the EG110A vector 5 weeks after treatment compared to the buffered control group (two-way ANOVA: ns, p>0.05). [Figure 25A] This figure shows (B) the post-void volume (PVR) expressed as raw data and (A) the percentage of total volume at 5 weeks in rats treated with buffer (black squares, n=11) and 2.2 × 10⁸ PFU of vector (dark blue triangles, n=12). Data are mean ± SEM. Considering the PVR at the last urination, this parameter was not significantly altered in vector-treated rats at 5 weeks compared to the buffer control group (60.1 ± 9.3 μl in buffer-injected rats, 59.6 ± 9.5 μl in vector-injected rats; unpaired t-test: ns p>0.05). [Figure 25B]This figure shows (B) the post-urination volume (PVR) expressed as raw data and (A) the percentage of total volume after 5 weeks in rats treated with buffer (black squares, n=11) and 2.2 × 10⁸ PFU of vector (dark blue triangles, n=12). Data are mean ± SEM. Note that if the PVR at the time of the last urination is taken into account, the same results will be obtained if this parameter is expressed as a percentage of the total volume injected before this last urination (28.0 ± 4.8% for rats injected with buffer and 21.7 ± 3.5% for rats injected with vector; unpaired t-test: ns p>0.05). [Modes for carrying out the invention]

[0012] The present invention relates to a viral expression vector and its pharmaceutical composition for selectively modulating or silencing afferent nerves in the bladder as a gene therapy strategy for the treatment of overactive bladder (OAB) and / or bladder pain syndrome (BPS). In embodiments, BPS is interstitial cystitis (IC).

[0013] The proposed strategy is a gene therapy approach that results in selective molecular afferent bladder pathway blockade for treating OAB and / or IC / BPS patients. Targeting sensory nerves, the inventors also aim to alleviate the urgent symptoms of OAB and selectively eliminate the sensation of pain in IC / PBS, thus addressing the most important issues in both cases. This can be achieved by using a viral expression vector capable of delivering a therapeutic transgene presenting the following: • The ability to inhibit / silence neurotransmission or synaptic transmission of afferent neurons. • In particular, high selectivity for afferent neurons in the bladder, • High efficiency, • Stability of expression over time, and • Absence of off-target denervation.

[0014] In the context of the present invention, it is possible to obtain selective and stable transgene expression in afferent neurons of the bladder by specifically inhibiting / silencing neurotransmission or synaptic transmission of bladder afferent neurons at the spinal cord level, using a viral expression vector that stably expresses proteins and / or transcripts over time to treat OAB and / or IC / BPS after injection of the viral expression vector into the bladder wall.

[0015] The present invention provides a method and pharmaceutical composition for treating OAB and / or IC / BPS, comprising a viral expression vector having a transcription cassette containing a transgene that inhibits / silences neurotransmission or synaptic transmission of afferent neurons. Preferably, the method and pharmaceutical composition according to the present invention comprises a viral expression vector having a transcription cassette containing one or more transgenes that disrupt SNARE complexes and / or ribosome complexes, and / or, when transcribed, activate GABA(A) receptors, and / or induce ablation of conditionally targeted neurons that inhibits / silences neurotransmission or synaptic transmission of bladder afferent neurons.

[0016] As used herein, the term “transcription cassette” refers to any nucleic acid sequence comprising a promoter and a downstream coding sequence or transgene whose expression is driven by the promoter, followed by a polyadenylation signal. The term “transgene” refers to a specific nucleic acid sequence that codes for RNA and / or polypeptides or portions of polypeptides expressed in the cell into which the nucleic acid sequence is introduced. The term “transgene” includes (1) nucleic acid sequences not found naturally in the cell (i.e., heterologous nucleic acid sequences); (2) nucleic acid sequences that are variant forms of naturally occurring nucleic acid sequences in the cell into which they are introduced; (3) nucleic acid sequences that serve to add further copies of identical (i.e., homologous) or similar nucleic acid sequences naturally present in the cell into which they are introduced; or (4) silent, naturally occurring or homologous nucleic acid sequences whose expression is induced in the cell into which they are introduced. “Variant form” means a nucleic acid sequence having one or more nucleotides that differ from the wild-type or naturally occurring sequence; i.e., a variant nucleic acid sequence has one or more nucleotide substitutions, deletions and / or insertions. In some cases, a transgene may contain a sequence encoding a leader peptide or signal sequence so that the transgene product is secreted from the cell; or it may contain both a leader peptide or signal sequence and a membrane-anchored peptide; or it may even be a fusion protein consisting of two naturally occurring proteins or parts thereof so that the transgene remains fixed to the cell membrane.

[0017] As used herein, the term “ribosome complex” refers to a complex essentially composed of ribosome subunits, such as the 80S and 70S subunits, which catalyze the synthesis of proteins called translation.

[0018] Therefore, in the first aspect, the present invention provides at least, a) A single promoter that is selectively activated in afferent neurons of the bladder, b) A transcription cassette comprising a nucleotide sequence operably linked to the promoter, wherein the nucleotide sequence, when transcribed, silences or inhibits the transmission of neurotransmitter signals in a postsynaptic cell. c) A sequence operably linked to the transcription cassette that confers long-term expression, such as a sequence known as LTE (Lokensgard et al., 1997) and / or a sequence containing a DNA insulator derived from the HSV-1 genome (Amelio et al., 2006). The present invention provides a viral expression vector containing the following:

[0019] In preferred embodiments, the nucleotide sequence of the viral expression vector according to the present invention, upon transcription or translation, silences or inhibits neurotransmission or synaptic transmission by disrupting SNARE complexes and / or ribosome complexes, and / or activating GABA(A) receptors, and / or inducing ablation of conditionally targeted neurons.

[0020] In a preferred embodiment, the nucleotide sequence of the viral expression vector according to the present invention, when transcribed, disrupts at least one of the proteins selected from VAMP, SNAP-25, or syntaxin 1a, which are part of the SNARE complex, or encodes the protein GAD67 or its active fragment, or a protein or its active fragment that disrupts the ribosome complex, or a protein or its active fragment that induces ablation of conditionally targeted neurons.

[0021] In certain embodiments, the protein that disrupts the ribosome complex according to the present invention is a wild-type or modified ribosome inactivating protein (RIP) or an active fragment thereof, wherein the RIP is preferably selected from type 1 or type 2 RIPs, preferably type 1 RIPs are selected from saporin, geronin, dianthine, and tricosanthine, and type 2 RIPs are selected from lysine, volkensin, and abrin, and more preferably type 1 RIPs are saporin S6 or an active fragment thereof.

[0022] The term "protein-induced conditional targeting neuronal ablation" refers to proteins that convert harmless prodrug substrates, such as metronidazole (MTZ), into cytotoxic DNA crosslinkers to provide cell-specific ablation of target cell types, namely afferent neurons in the bladder. An example of such a protein that induces conditional targeting neuronal ablation is nitroreductase (NTR).

[0023] Therefore, the protein that induces ablation of conditionally targeted neurons according to the present invention is selected from the group consisting of wild-type or modified NTR or its active fragment. Preferably, the NTR is selected from the group consisting of wild-type or modified oxygen-insensitive NAD(P)H nitroreductase or its active fragment; more preferably, the NTR is selected from the group consisting of wild-type or modified Escherichia coli (E. coli) nitroreductase; and even more preferably, the NTR is wild-type or modified Escherichia coli (E. coli) nfnB or its active fragment.

[0024] As used herein, the terms “viral vector” or “viral expression vector” refer to a nucleic acid vector comprising at least one element of a viral genome and packaged within a viral particle. In the context of the present invention, the term “viral vector” should be broadly understood to include not only nucleic acid vectors (e.g., DNA viral vectors) but also viral particles produced therefrom. According to the present invention, a viral expression vector is an adeno-associated virus (AAV) vector or a herpes simplex virus (HSV) vector, preferably an HSV-1 vector or an HSV-2 vector, and more preferably an HSV-1-derived deficient viral vector. As used herein, the term “deficient viral vector” refers to a viral vector lacking a gene or part of a gene necessary for successfully completing the viral life cycle.

[0025] According to the present invention, the term "AAV" refers to the adeno-associated virus itself or its derivatives, including recombinant AAV vector particles. Furthermore, as used herein, the term "AAV" includes many different serotypes isolated from both human and non-human primate specimens. Preferred AAV serotypes are human serotypes, more preferably human AAV serotypes 2, 5, and 9, and most preferably human AAV serotype 5, which exhibits the highest level of neurotropism.

[0026] According to the present invention, the term “HSV-derived deficient viral vector” refers to both deficient recombinant HSV vectors and amplicon HSV vectors. As used herein, the term “deficient recombinant HSV” refers to a helper-independent vector whose genome contains at least an inactivating deletion of genes encoding two essential proteins known as ICP4 and ICP27. The ICP4 gene is present in two copies within the reverse repeat sequences known as c and c' of the viral genome, and both copies of this gene have been deleted. The gene encoding ICP27 is located within the unique long (UL) sequence of the viral genome. Preferably, the helper-independent vector according to the present invention has a therapeutic transcription cassette embedded in the LAT (latency-associated transcript) locus (Berthomme et al., 2000 and Berthomme et al., 2001), which is a repeating gene locus contained within the reverse repeat sequences known as b and b' of the viral genome. More preferably, the transcription cassette is located either between the latent infection-associated promoter (LAP) and the long-term expression (LTE) region (site 1), or between the LTE region and the DNA insulator (INS) sequence located downstream of the LTE (site 2) (as shown in Figure 1). As used herein, a “deletion” of a gene is a complete or partial deletion of a gene, or any substitution or addition of a gene that prevents the expression of a functional protein.

[0027] The deficient recombinant HSV-1 vector according to the present invention has one or more transcription cassettes expressing the various transgenes described above to inhibit / silence neurotransmission, namely wild-type or modified light chain botulinum toxin, and / or SNARE protein-targeting antisense RNA (AS-RNA), and / or GAD67, and / or RIP, and / or NTR, all of which are driven by the long-term DRG-specific promoter described in the present invention. The b and b' sequences of the viral genome are also known as TRL (terminal repeat L) and IRL (internal repeat L), respectively, and the c' and c sequences are also known as IRS (internal repeat S) and TRS (terminal repeat S), respectively. Here, L and S refer to the unique long (L) and unique short (S) sequences of the HSV-1 genome, respectively.

[0028] Furthermore, the helper-independent vectors according to the present invention may include additional deletions in genes encoding non-essential proteins such as ICP34.5, UL55, UL56, and UL41 proteins. These deletion HSV vectors are grown in cell lines that simultaneously express proteins ICP4 and ICP27 (Marconi et al., 2010).

[0029] International Publication No. 2006 / 050211 discloses the use of a deficient HSV-1 vector for gene therapy of pain. However, the vector according to the present invention differs from the vector described in the International Publication No. 2006 / 050211 brochure in several important respects concerning the usefulness and efficacy of the vector according to the present invention. Most importantly, the transgenic transcription cassette according to the present invention is introduced into the LAT locus. This is because this region contains both the LTE and DNA insulator sequence (INS), which confer long-term expression to the DRG-specific promoter that drives transgene expression within the transcription cassette according to the present invention. On the other hand, the vector described in International Publication No. 2006 / 050211 was designed and proven for short-term action, and therefore its transcription cassette is driven by a ubiquitous promoter and is not introduced into the LAT region.

[0030] An "amplicon or amplicon vector" refers to a helper-dependent vector whose genome lacks most or all HSV genes encoding viral proteins. The genome of an amplicon vector is a concatemer DNA consisting of multiple serial copies of a plasmid known as an amplicon plasmid, which, in addition to the target transgenic DNA (i.e., the transcription cassette), has one DNA replication origin and one packaging signal derived from the HSV-1 genome. The amplicon plasmid according to the present invention has a transcription cassette to express the various transgenes described above for the purpose of inhibiting / silencing neurotransmission, namely wild-type or modified light chain botulinum toxin, and / or interfering RNA (RNAi) targeting SNARE proteins, and / or GAD67, and / or RIP, and / or NTR, all of which are driven by a long-term promoter, preferably a long-term DRG-specific promoter as described in the present invention (see Figure 2).

[0031] In a preferred embodiment, the vector according to the present invention is a deletion recombinant vector comprising inactivation deletions of genes encoding at least the essential proteins ICP4 and ICP27. In an embodiment, the vector further comprises an inactivation deletion of at least one copy of the gene encoding the essential protein ICP0. In some embodiments, the deletion HSV-1 vector further lacks one copy of the ICP0 gene. In a preferred embodiment, one copy of the ICP0 gene in the LAT, ICP0, UL34.5 cluster of the IRL (Internal Repeat Long) region of the HSV vector is removed.

[0032] This vector may lack other genes encoding non-essential proteins such as ICP34.5, UL55, UL56, and / or UL41 gene proteins, and may have one or more DRG-specific transcription cassettes, as shown in Figure 7, integrated into the LAT region of the vector genome.

[0033] In another embodiment, the vector according to the present invention is an amplicon vector having the transcription cassette driven by a long-term DRG-specific promoter, as described in other parts of this specification.

[0034] In a preferred embodiment, the transcription cassette according to the present invention is introduced into the LAT gene locus.

[0035] As used herein, the term “recombinant DNA” refers to nucleic acid molecules, i.e., polynucleotides of genome origin, cDNA origin, viral origin, semi-synthetic origin, and / or synthetic origin, which, due to their origin or manipulation, do not have all or some of the polynucleotides that are bound together in nature. As used with respect to viruses, the term “recombinant” means a virus possessing a recombinant genome, i.e., a genome manipulated to introduce one or more heterologous polynucleotides, including mutations, deletions, or genes. As used with respect to proteins or polypeptides, the term “recombinant” means a polypeptide produced by the expression of recombinant nucleic acid. As used with respect to host cells, the term “recombinant” means a recombinant vector possessing recombinant DNA within a host cell, or a cell containing recombinant DNA inserted into its genome. The term “infection” refers to the ability of a viral vector to enter a host cell or subject.

[0036] Deficit vectors derived from HSV can infect adjacent sensory neurons, allowing them to establish latent infection in the nuclei of these neurons located in the trigeminal ganglion or dorsal root ganglion (DRG), depending on the site of infection. In particular, HSV-1 spontaneously infects sensory neurons and establishes lifelong latent infection in the nuclei of these neurons. Therefore, it can be assumed that after injection into the bladder wall, vectors such as those derived from HSV-1 can reach the sensory DRG innervating the bladder, and if appropriate bladder afferent neuron-specific promoters drive their expression, they can stably express therapeutic transgenes from there. However, HSV-1 can also infect autonomic neurons and establish latent infection (Furuta et al., 1993; Warren et al., 1978), and preliminary results have demonstrated that this is indeed the case when vectors are inoculated into the bladder. Therefore, in order to obtain significant transgene expression only in these neurons (i.e., afferent neurons), and thus to avoid expression in autonomic neurons (also called efferent neurons), it is essential that expression from the vector is completely controlled by an afferent-specific promoter, also called a selective promoter or selective afferent neuron-specific promoter. Selective molecular or biochemical (as opposed to surgical) afferent pathway blockade of bladder afferent neurons is the most important aspect of the present invention because it is important to preserve the remaining pelvic perineal sensation, if present, orgasm, if present, reflex erection and ejaculation, and if present, reflex urination and defecation (all transmitted by pelvic sensory neurons that do not originate from the bladder). Furthermore, selective bladder afferent pathway blockade also makes it possible to preserve bladder efferent neurons that can later be stimulated, for example, by electrical stimulation via electrodes. Several studies have described the use of HSV-1-based vectors in which the transcription cassette contains either a transient promoter (Miyazato et al., 2009) or a long-term promoter (Puskovic et al., 2004; Miyakawa et al., 2015; International Publication No. 2015 / 009952).However, the promoters used in studies such as Puscovic (LAP2), Miyazato (HCMV promoter), and Miyagawa (artificial CAG promoter) are non-selective and result in the expression of their transgenes in many cell types, including autonomic neurons, cerebral neurons, and non-neuronal cells. In contrast, by combining a viral regulatory sequence with an afferent neuron-specific cell promoter, some of the vectors according to the present invention enable remarkably high afferent neuron-specific expression of the target transgene (see Figure 13).

[0037] The vector used in the embodiment of the present invention comprises at least one promoter selectively active in afferent neurons, operably ligated to a nucleotide (typically DNA) encoding an RNA molecule. “Operatably ligated” means, as used herein, that in the vector, the promoter is associated with the nucleotide encoding the RNA, so that the promoter can drive the transcription (i.e., expression) of RNA from the nucleotide. For example, the transcription of RNA from a DNA template is well understood.

[0038] As used herein, “promoter” is a DNA regulatory region that can bind to RNA polymerase in a mammalian cell and initiate transcription of a operably linked downstream (3' direction) sequence. In the present invention, a promoter sequence contains at least the minimum number of bases or elements necessary to initiate transcription of a gene of interest at a level detectable beyond the background. Within the promoter sequence, in addition to the transcription start site, there is an RNA polymerase binding domain. Eukaryotic promoters often, though not always, contain other DNA motifs such as “TATA” boxes and “CAT” or “SP1” boxes. The promoter according to the present invention includes a DNA sequence that begins at least 2 kb, preferably 3 kb, more preferably 4 kb, upstream of the start site of a messenger encoding a particular gene product of interest. These sequences preferably include known promoter sequence elements, such as a specific transcription binding site, and distal sequences upstream of the gene that include further regulatory elements.

[0039] "Selectively active in afferent neurons" means, as used herein, that the promoter is active primarily in afferent neurons, or only in afferent neurons, preferably in afferent neurons of the bladder, and drives the transcription (i.e., expression) of RNA.

[0040] Furthermore, those skilled in the art will recognize that numerous such mammalian afferent neuron-specific promoters are known, and that new afferent neuron-specific promoters are constantly being discovered. All such afferent neuron-specific promoters are encompassed within the scope of this invention.

[0041] In preferred embodiments, the promoter according to the present invention is selected from promoters of genes encoding sensory neuron receptors, promoters of the TRP gene family such as transient receptor potential vanilloid 1 (TRPV1) or transient receptor potential cation channel subfamily M member 8 (TRPM8), or promoters of genes encoding sensory neuromodulators or sensory neurotransmitters, such as substance P, PACAP, or calcitonin gene-related peptide (CGRP), or promoters of genes involved in neurite outgrowth and stress response of sensory neurons, preferably promoters of genes encoding advilin (ADVL). In preferred embodiments, the promoter according to the present invention is selected from promoters of the TRP gene family or calcitonin gene-related peptide (CGRP) promoters. In preferred embodiments, the promoter according to the present invention is a calcitonin gene-related peptide (CGRP) promoter. In preferred embodiments, the promoter according to the present invention is a promoter of the TRP gene family.

[0042] In preferred embodiments, the promoter according to the present invention is selected from promoters of genes encoding sensory nerve receptors, such as transient receptor potential vanilloid 1 (TRPV1) or transient receptor potential cation channel subfamily M member 8 (TRPM8), or promoters of genes encoding sensory neuromodulators or sensory neurotransmitters, such as substance P, PACAP, or the promoter of calcitonin gene-related peptide (CGRP) of SEQ ID NO: 3 or SEQ ID NO: 4. Preferably, the promoter of a gene encoding a sensory nerve receptor according to the present invention is a promoter of the TRP gene family, more preferably the promoter of TRPV1 of SEQ ID NO: 1 or the promoter of TRPM8 of SEQ ID NO: 2. Preferably, the promoter of a gene encoding a sensory neuromodulator or sensory neurotransmitter according to the present invention is CGRP of SEQ ID NO: 3 or SEQ ID NO: 4. Preferably, the promoter of a gene involved in neurite outgrowth and stress response of sensory neurons, preferably the promoter of a gene encoding advilin (ADVL) of SEQ ID NO: 5 or SEQ ID NO: 6.

[0043] The viral expression vectors of the present invention are more specifically intended for vertebrates, preferably mammals, more preferably primates and humans. Those skilled in the art will therefore recognize that such promoters are species-specific and that homologous sequences of a particular species of interest can be selected. In particular, the promoters according to the present invention include, among others, the human homolog of rat TRPV1 of SEQ ID NO: 1 or human TRPM8 of SEQ ID NO: 2, or rat CGRP of SEQ ID NO: 3, or human CGRP of SEQ ID NO: 4, or rat advilin of SEQ ID NO: 5, or human advilin of SEQ ID NO: 6.

[0044] "Long-term expression sequence" or "Long-term expression element (LTE)" means a nucleotide sequence that is operably ligated to a transcription cassette contained in the sequence of a viral expression vector, enabling the expression of a gene product to be sustained for 15 to 45 days or 30 to 45 days, preferably 45 to 90 days, more preferably 90 to 365 days, even more preferably 365 days to several years, or even more preferably for the lifetime of the patient.

[0045] Long-term expression (LTE) sequences have been identified in HSV-1 as regions of the latent-associated transcript (LAT) originating from the LAT-associated promoter (LAP). This LTE is located downstream of the LAT transcription start site. Indeed, viruses with a DNA fragment at 3' of the LAT promoter maintained detectable promoter expression throughout the incubation period (Lokensgard et al., 1997; Berthomme et al., 2000, 2001). Preferably, the LTE is located between approximately 1.5 kb and 3 kb downstream of the LAT transcription start site (Perng et al., 1996). More recently, further sequences known as DNA insulators located both upstream and downstream of the LTE region have also been described (Amelia et al., 2006). These sequences also contribute to long-term expression of a given transcription cassette, possibly by inhibiting epigenetic silencing, and are further intended to be incorporated into the present invention as part of the LTE element to confer long-term expression to the transcription cassette. Interestingly, the sequences that confer long-term expression to the transcription cassette (both LTE sequences and DNA insulator sequences) can be located either upstream or / or downstream of the transcription cassette.

[0046] Those skilled in the art will recognize that other LTE-like sequences and other DNA insulator sequences have been described and are constantly being discovered. All such LTE-like sequences and DNA insulator sequences are encompassed within the scope of this invention.

[0047] In a preferred embodiment, the viral expression vector of the present invention comprises at least one nucleotide sequence that is transcribed to a non-coding nucleotide sequence that inhibits the synthesis of at least one protein selected from VAMP, SNAP-25, and syntaxin, which are part of the SNARE complex.

[0048] The SNARE complex (soluble N-ethylmaleimide-sensitive factor-attached protein receptor) is one of the two key components of the membrane fusion mechanism with the SM(Sec1 / Munc18) protein. The SNARE complex includes vesicle-associated "v-SNARE" (vesicle-associated membrane proteins, VAMPs, particularly VAMP1, 2, and 3), as well as target membrane-associated "t-SNARE" syntaxins (Syn-1, 2, 3, and 4) and a 25 kDa synaptosome-associated protein (SNAP-25) that form the complex and mediate different fusion events.

[0049] Therefore, one embodiment of the present invention relates to a method for silencing a particular gene and / or disrupting the corresponding encoded protein ("Desired Gene," "Target Gene," or "Selected Gene"). "Silencing" a gene means that the expression of the gene product is reduced or eliminated compared to the corresponding control gene that has not been silenced. Those skilled in the art are familiar with the concept of comparing experimental results with those obtained with a control. While not bound by theory, silencing is thought to be characterized by specific mRNA degradation or mRNA blocking at translation after expression of a non-coding complementary sequence such as antisense RNA (asRNA), small hairpin RNA (shRNA), microRNA (miRNA), or any other form of interfering RNA (iRNA) in cells.

[0050] As used herein, the term “antisense” generally refers to an unmodified or chemically modified single-stranded nucleic acid molecule that is relatively short and capable of hybridizing to a specific sequence in the total pool of targets present within a cell, wherein the sequence of the nucleic acid molecule is complementary to a particular mRNA by Watson-Crick bp hybridization and can inhibit the mRNA expression, thereby inducing a blockage of the transfer of genetic information from DNA to protein.

[0051] In the context of this invention, "RNA interference" (hereinafter referred to as RNAi) is interpreted as a process in which a double-stranded RNA (dsRNA) having a given sense nucleic acid sequence degrades all messenger RNA (mRNA) containing the nucleic acid sequence in a manner specific to the nucleic acid sequence. The RNAi process was first demonstrated in the nematode Caenorhabditis elegans, but it is now clear that the RNAi process is a very common phenomenon, and RNAi-mediated inhibition of human genes has been achieved.

[0052] RNAi processes can be achieved using small interfering RNAs (or siRNAs). These siRNAs are dsRNAs less than 30 nucleotides in length, and their sense sequence contains a sequence highly complementary to the target mRNA fragment. When the siRNA crosses the cell membrane, the cell's response is to disrupt the siRNA and all sequences containing the highly complementary sequence. Therefore, mRNA with a fragment highly complementary to the siRNA sequence is disrupted, and thus the expression of this gene is inhibited.

[0053] According to the present invention, shRNA can also be used as an inhibitor. As used herein, “shRNA molecule” includes conventional stem-loop shRNA that forms precursor miRNA (pre-miRNA). “shRNA” also includes shRNA embedded in microRNA (miRNA-based shRNA), in which the guide and passenger strands of the miRNA double helix are incorporated into existing (or native) miRNA or modified or synthetic (designed) miRNA. Upon transcription, conventional shRNA forms a structure very similar to primary miRNA (pri-miRNA) or native pri-miRNA. The pri-miRNA is then processed into pre-miRNA by drotherapeutic compounds and their cofactors. Thus, the term “shRNA” includes both pri-miRNA (shRNA-mir) molecules and pre-miRNA molecules.

[0054] Generally, “reduced or eliminated” means that a detectable amount of the gene product is reduced or eliminated by at least about 10% to about 100%, preferably at least about 25% to 100%, more preferably about 50% to about 100%, and most preferably about 75% to about 100%. Where necessary, reduction or elimination may be determined by any of several methods well known to those skilled in the art, and may vary depending on the gene being silenced. For example, such reduction or elimination of gene expression may be determined by quantification of the gene product (e.g., by determining the amount of protein, polypeptide, or peptide produced) or by quantification of the gene product’s activity (e.g., activity such as signaling or transport activity, activity as a structural component of cells, activity such as enzymatic activity), or by observation and quantification of the phenotypic characteristics of the target cell compared to a control cell (e.g., the presence or absence of the protein using a specific antibody). Any suitable means may be used to determine whether the target gene has been silenced.

[0055] In one embodiment, the non-coding nucleotide sequence according to the present invention is selected from antisense RNA (asRNA), small hairpin RNA (shRNA), microRNA (miRNA), or any other interfering RNA (iRNA) and inhibits the synthesis of at least one protein selected from VAMP, SNAP-25, and syntaxin.

[0056] In one embodiment, the viral expression vector includes at least one nucleotide sequence that is transcribed into an asRNA that inhibits the synthesis of VAMP, SNAP-25, and / or syntaxin. In particular, the asRNA sequences used in the context of the present invention are the VAMP2 antisense of SEQ ID NO: 7, the SNAP25 antisense of SEQ ID NO: 8, and the syntaxin antisense of SEQ ID NO: 9.

[0057] In certain embodiments, the viral expression vector comprises at least one nucleotide sequence that is transcribed into shRNA that inhibits the synthesis of VAMP, SNAP-25, and / or syntaxin.

[0058] In another embodiment, the viral expression vector includes at least one nucleotide sequence which is transcribed into a miRNA that inhibits the synthesis of VAMP, SNAP-25, and / or syntaxin.

[0059] The RNA molecule encoded by the construct of the present invention ultimately forms a double-stranded RNA molecule in the cell where it is transcribed. Generally, the length of one strand of the double-stranded RNA structure is in the range of about 10 to about 30 ribonucleotides, preferably about 19 to about 25 ribonucleotides.

[0060] In the case of asRNA, the length of one strand of the double-stranded RNA structure ranges from approximately 100 to several hundred ribonucleotides. It can actually be as long as the target mRNA. Those skilled in the art will recognize that several viable strategies exist for forming such double-stranded RNA.

[0061] Furthermore, providing multiple viral vectors that have the same afferent neuron-specific promoter but encode different silencing RNAs may also be used within the scope of the present invention.

[0062] Furthermore, it should be possible to express two or more silencing RNAs within a single viral vector, either driven by a single afferent neuron-specific promoter or by multiple promoters arranged in series (e.g., two or more promoters). Therefore, the present invention intends to use a single viral vector to silence two or more genes.

[0063] In another embodiment, the viral expression vector according to the present invention comprises at least one nucleotide sequence encoding a wild-type or modified toxin or an active fragment thereof that disrupts the SNARE complex or ribosome complex.

[0064] Advantageously, the active fragment of the toxin is a bacterial neurotoxin, and preferably the bacterial neurotoxin is the light chain of the bacterial neurotoxin. In particular, the toxin light chain sequences used in the context of the present invention are the protein sequence light chain of botulinum neurotoxin A (BoNT-A), the protein sequence light chain of botulinum neurotoxin B (BoNT-B), the protein sequence light chain of botulinum neurotoxin C1 (BoNT-C1), the protein sequence light chain of botulinum neurotoxin E3 (BoNT-E3), the protein sequence light chain of botulinum neurotoxin F1 (BoNT-F1), and the protein sequence light chain of tetanus neurotoxin (TeNT). In embodiments, the toxin light chain sequence used in the context of the present invention is the protein sequence light chain of botulinum neurotoxin A (BoNT-A). In embodiments, the toxin light chain sequence used in the context of the present invention is the protein sequence light chain of botulinum neurotoxin B (BoNT-B). In some embodiments, the toxin light chain sequence used in the context of the present invention is the protein sequence light chain of botulinum neurotoxin C1 (BoNT-C1). In some embodiments, the toxin light chain sequence used in the context of the present invention is the protein sequence light chain of botulinum neurotoxin E3 (BoNT-E3). In some embodiments, the toxin light chain sequence used in the context of the present invention is the protein sequence light chain of botulinum neurotoxin F1 (BoNT-F1). In some embodiments, the toxin light chain sequence used in the context of the present invention is the protein sequence light chain of tetanus neurotoxin (TeNT).

[0065] In embodiments, the toxin light chain sequences used in the context of the present invention are the protein sequence light chain of botulinum neurotoxin A (BoNT-A) of SEQ ID NO: 10 (coding nucleotide sequence is SEQ ID NO: 11), the protein sequence light chain of botulinum neurotoxin B (BoNT-B) of SEQ ID NO: 12 (coding nucleotide sequence is SEQ ID NO: 13), the protein sequence light chain of botulinum neurotoxin C1 (BoNT-C1) of SEQ ID NO: 14 (coding nucleotide sequence is SEQ ID NO: 15), the protein sequence light chain of botulinum neurotoxin E3 (BoNT-E3) of SEQ ID NO: 16 (coding nucleotide sequence is SEQ ID NO: 17), the protein sequence light chain of botulinum neurotoxin F1 (BoNT-F1) of SEQ ID NO: 18 (coding nucleotide sequence is SEQ ID NO: 19), and the protein sequence light chain of tetanus neurotoxin (TeNT) of SEQ ID NO: 20 (coding nucleotide sequence is SEQ ID NO: 21).

[0066] In a preferred embodiment, the viral expression vector according to the present invention comprises at least one nucleotide sequence encoding a wild-type or modified GAD67 protein or an active fragment thereof, preferably the wild-type GAD67 protein of SEQ ID NO: 22 (coding nucleotide sequence is SEQ ID NO: 23) or an active fragment thereof.

[0067] In a preferred embodiment, the viral expression vector according to the present invention comprises at least one nucleotide sequence encoding wild-type or modified RIP or an active fragment thereof, preferably RIP being the saporin S6 protein of SEQ ID NO: 24 (coding nucleotide sequence of SEQ ID NO: 25) or an active fragment thereof.

[0068] In a preferred embodiment, the viral expression vector according to the present invention comprises at least one nucleotide sequence encoding a wild-type or modified NTR or an active fragment thereof, wherein the NTR is preferably the nitroreductase nfnB protein of SEQ ID NO: 26 (coding nucleotide sequence of SEQ ID NO: 27) or an active fragment thereof.

[0069] As used herein, the term “coding sequence” refers to a ribonucleic acid (e.g., RNA) sequence that, when translated, produces the polypeptide of interest. Polypeptides may be encoded by a full-length coding sequence or by any portion of a coding sequence, provided that the desired activity or functional properties (e.g., enzymatic activity, ligand binding, signaling, etc.) of the full-length or fragment are preserved.

[0070] In one embodiment, the present invention relates to a viral expression vector comprising a wild-type or modified botulinum neurotoxin or an active fragment thereof of any serotype of Clostridium botulinum, preferably at least one nucleotide sequence encoding the light chain of any serotype of Clostridium botulinum neurotoxin.

[0071] In another embodiment, the present invention relates to a viral expression vector comprising a wild-type or modified tetanus neurotoxin of Clostridium tetani or an active fragment thereof, preferably at least one nucleotide sequence encoding the light chain of the Clostridium tetani neurotoxin.

[0072] Clostridium neurotoxins are produced by various species of the genus Clostridium, such as C. botulinum and several strains of C. tetani. When Clostridium toxin molecules enter a neuron, their light chains cleave proteins that form the SNARE complex located at the presynaptic nerve terminal. This prevents synaptic vesicles filled with neurotransmitters from attaching to the presynaptic membrane, thus inhibiting the exocytosis of neurotransmitters from the presynaptic nerve terminal. Currently, there are eight distinct classes of known neurotoxins. These are serotypes A, B, C, D, E, F, and G of tetanus and botulinum neurotoxins, all of which share homology and similar molecular structures. Within these serotypes, subtypes such as A1-A3 and B1-B3 are also well documented.

[0073] Serotypes A, C, and E of botulinum neurotoxin cleave the SNAP-25 protein located in the cell membrane of presynaptic nerve terminals. Since SNAP-25 is necessary for the fusion of neurotransmitter-filled vesicles with the cell membrane and their release during exocytosis, its cleavage causes a highly specific blockage of vesicular neurotransmitter release in presynaptic nerve terminals of somatic and autonomic nerves. Serotypes B, D, F, and G of botulinum neurotoxin cleave the synaptobrevin (VAMP) protein, resulting in the inability of vesicles to fuse with the cell membrane. Each botulinum neurotoxin or its light chain fragment cleaves both SNAP25 and syntaxin 1a, while others cleave one of the SNARE proteins. Preferably, according to the present invention, the serotypes of botulinum neurotoxin are A, B, C, E, and F.

[0074] The structure of Clostridium neurotoxin has been well demonstrated (Habermann et al., 1986; Sugiyama et al., 1980). Each of these references is incorporated herein by reference in its entirety. In this regard, Clostridium neurotoxin consists of two polypeptide chains linked to each other by disulfide bonds: a heavy chain (H chain) with a molecular weight of approximately 100 kDa and a light chain (L chain) with a molecular weight of approximately 50 kDa.

[0075] Different serotypes of botulinum toxin differ in the animal species they affect, as well as in the severity and duration of paralysis they induce. For example, in mice, LD 50 Measurements have shown that botulinum toxin type A is 500 times more potent than botulinum toxin type B. Furthermore, botulinum toxin type B has been determined to be non-toxic to primates at a dose of 480 U / kg, which is the LD50 of botulinum toxin type A in primates. 50 This is approximately 12 times stronger. Normally, botulinum toxin binds to neurons with high affinity, moves to neurons, and blocks the release of neurotransmitters.

[0076] In certain embodiments, the present invention relates to a viral expression vector comprising at least one nucleotide sequence encoding wild-type or modified tetanus neurotoxin or an active fragment of Clostridium tetani for cleaving the protein VAMP-2.

[0077] In certain embodiments, the present invention relates to a viral expression vector comprising at least one nucleotide sequence encoding wild-type or modified botulinum neurotoxin of serotypes B, D, F, and G of Clostridium botulinum, or an active fragment thereof, for cleaving the protein VAMP-2.

[0078] In certain embodiments, the present invention relates to a viral expression vector comprising at least one nucleotide sequence encoding wild-type or modified botulinum neurotoxin of serotypes A and E of Clostridium botulinum, or an active fragment thereof, for cleaving the protein SNAP-25.

[0079] In preferred embodiments, the present invention relates to a viral expression vector comprising at least one nucleotide sequence encoding wild-type or modified botulinum neurotoxin of serotype C Clostridium botulinum, or an active fragment thereof, for cleaving the protein SNAP25 and syntaxin 1a.

[0080] In preferred embodiments, the nucleotide sequence of the transgene according to the present invention encodes a wild-type or modified protein that silences or inhibits the transmission of neurotransmitter signals in postsynaptic cells, which is fused to a signal peptide domain. The signal peptide according to the present invention is selected according to the intracellular compartment in which the transcript or protein intended to silence or inhibit the transmission of neurotransmitter signals in postsynaptic cells is located. Thus, those skilled in the art will recognize that such a signal peptide is specific to an intracellular compartment and that an appropriate corresponding nucleotide sequence can be selected to be fused to the nucleotide sequence encoding the protein that silences or inhibits neurotransmission or synaptic transmission according to the present invention. In particular, the signal peptide according to the present invention comprises at least the luminal domain, transmembrane domain, or cytoplasmic domain of a protein selected from VAMP2 or syntaxin 1a.

[0081] In certain embodiments, the fusion protein according to the present invention comprises a luminal, transmembrane, or cytoplasmic signal peptide domain of a SNARE protein, preferably a signal peptide domain selected from the luminal, transmembrane, or cytoplasmic signal peptide domains of a SNARE protein, substance P, or CGRP sequence. Examples of such signal peptide domains include the syntaxin 1a (BoNTB-STX) signal peptide of SEQ ID NO: 30 (coding nucleotide sequence: SEQ ID NO: 31) and the VAMP2 (BoNTC-VAMP) signal peptide of SEQ ID NO: 32 (coding nucleotide sequence: SEQ ID NO: 33). Thus, according to certain embodiments of the present invention, the fusion protein comprises a modified bacterial neurotoxin, for example, a modified botulinum neurotoxin, and a signal peptide, for example, the syntaxin 1a (BoNTB-STX) signal peptide of SEQ ID NO: 28 or the syntaxin 1a (BoNTB-STX) signal peptide of SEQ ID NO: 30 (coding nucleotide sequence: SEQ ID NO: 31) and the VAMP2 (BoNTC-VAMP) signal peptide of SEQ ID NO: 32 (coding nucleotide sequence: SEQ ID NO: 33).

[0082] In one specific embodiment, the fusion protein according to the present invention comprises a wild-type or modified Clostridium botulinum neurotoxin of serotype A, B, C, E, or F linked to a syntaxin 1a signal peptide, preferably comprising a wild-type or modified Clostridium botulinum neurotoxin of serotype A linked to a syntaxin 1a (BoNTA-STX) signal peptide (coded nucleotide sequence is SEQ ID NO: 29), or comprising a wild-type or modified Clostridium botulinum neurotoxin of serotype B linked to a syntaxin 1a (BoNTB-STX) signal peptide (coded nucleotide sequence is SEQ ID NO: 31), according to SEQ ID NO: 30.

[0083] In one specific embodiment, the fusion protein according to the present invention comprises wild-type or modified Clostridium botulinum neurotoxin of serotypes A, C, and E linked to a signal peptide of VAMP2, and preferably the fusion protein comprises wild-type or modified Clostridium botulinum neurotoxin of serotype C linked to a signal peptide of VAMP2 (BoNTC-VAMP) of SEQ ID NO: 32 (coding nucleotide sequence of SEQ ID NO: 33).

[0084] In one specific embodiment, the fusion protein according to the present invention comprises a wild-type or modified Clostridium botulinum neurotoxin of any serotype linked to a signal peptide of substance P.

[0085] In one specific embodiment, the fusion protein according to the present invention comprises a signal peptide of any serotype of wild-type or modified Clostridium botulinum neurotoxin linked to a CGRP sequence.

[0086] The present invention also includes at least, a) A single nucleotide sequence encoding the wild-type or modified neurotoxin or an active fragment of Clostridium tetani or Clostridium botulinum, and / or b) A single nucleotide sequence in the transcript that inhibits the synthesis of the proteins VAMP, SNAP-25 and / or syntaxin, and / or c) A single nucleotide sequence encoding the wild-type or modified GAD67 protein or an active fragment thereof, and / or d) A single nucleotide sequence encoding wild-type or modified RIP or an active fragment thereof, and / or e) A single nucleotide sequence encoding the wild-type or modified NTR or its active fragment. The present invention also relates to a viral expression vector, including the present invention.

[0087] In a preferred embodiment, the viral expression vector according to the present invention is i. One of the long-term expression sequences operably linked to two transcription cassettes according to the present invention, or ii. The present invention comprises two long-term expression sequences, both operably linked to one of the transcription cassettes, wherein, a) One transcription cassette according to the present invention has a coding sequence according to the present invention, and a second transcription cassette according to the present invention has a sequence that is transcribed to a non-coding nucleotide according to the present invention, or b) Both transcription cassettes according to the present invention have a nucleotide sequence that encodes a non-coding nucleotide sequence according to the present invention, or c) Both transcription cassettes according to the present invention have nucleotide sequences encoding wild-type or modified neurotoxins of Clostridium tetani and / or Clostridium botulinum, or their active fragments; or wild-type or modified GAD67 protein or its active fragments; or wild-type or modified RIP or its active fragments; or wild-type or modified NTR or its active fragments.

[0088] In certain embodiments, the present invention is i. One of the long-term expression (LTE) sequences is operably linked to two transgenic transcription cassettes according to the present invention, or ii. Relating to a viral expression vector in which two distinct long-term expression (LTE) sequences are each operably linked to one of the transcription cassettes according to the present invention, At this time, a) One transcription cassette according to the present invention comprises a sequence encoding a bacterial neurotoxin, GAD67, RIP, or NTR according to the present invention, and a second transgenic transcription cassette according to the present invention comprises a sequence that is transcribed to a non-coding nucleotide sequence according to the present invention that inhibits the synthesis of proteins VAMP, SNAP-25, and / or syntaxin; or b) Both transcription cassettes according to the present invention have a nucleotide sequence encoding a non-coding nucleotide sequence according to the present invention that inhibits the synthesis of at least one protein selected from VAMP, SNAP-25 and / or syntaxin; or c) Both transgenic transcription cassettes according to the present invention comprise a promoter and a nucleotide sequence encoding a wild-type or modified neurotoxin or active fragment of Clostridium tetani and / or botulinum; or a wild-type or modified GAD67 protein or its active fragment; or a wild-type or modified RIP or its active fragment; or a wild-type or modified NTR or its active fragment.

[0089] In a preferred embodiment, the present invention relates to a viral expression vector in which at least one of the transgenic transcription cassettes according to the present invention comprises a sequence encoding a promoter and the wild-type protein GAD67 or an active fragment thereof.

[0090] In a more preferred embodiment, the present invention relates to a viral expression vector wherein one of the transgenic transcription cassettes according to the present invention comprises a promoter and a sequence encoding the wild-type protein GAD67 or an active fragment thereof; and one of the transgenic transcription cassettes according to the present invention comprises a promoter and a sequence encoding the wild-type or modified neurotoxin of Clostridium tetani and / or Clostridium botulinum or an active fragment thereof.

[0091] In a preferred embodiment, the present invention relates to a viral expression vector in which at least one of the transgenic transcription cassettes according to the present invention comprises a sequence encoding a promoter and a wild-type RIP or an active fragment thereof.

[0092] In a more preferred embodiment, the present invention relates to a viral expression vector in which one of the transgenic transcription cassettes according to the present invention comprises a promoter and a sequence encoding wild-type RIP or an active fragment thereof; and one of the transgenic transcription cassettes according to the present invention comprises a promoter and a sequence encoding wild-type or modified neurotoxin of Clostridium tetani and / or Clostridium botulinum or an active fragment thereof; and / or a sequence encoding wild-type protein GAD67 or an active fragment thereof; and / or a sequence encoding wild-type NTR or an active fragment thereof.

[0093] In a preferred embodiment, the present invention relates to a viral expression vector in which at least one of the transgenic transcription cassettes according to the present invention comprises a promoter and a sequence encoding a wild-type NTR or an active fragment thereof.

[0094] In a more preferred embodiment, the present invention relates to a viral expression vector, wherein the viral expression vector comprises at least two transgenic transcription cassettes, At least one of the transgenic transcription cassettes according to the present invention comprises a promoter and a sequence encoding a wild-type NTR or an active fragment thereof; and At least one of the transgenic transcription cassettes according to the present invention comprises a promoter and a sequence encoding a wild-type or modified neurotoxin or an active fragment thereof of Clostridium tetani and / or Clostridium botulinum; and / or a sequence encoding the wild-type protein GAD67 or an active fragment thereof; and / or a sequence encoding wild-type RIP or an active fragment thereof.

[0095] In a second aspect, the present invention relates to a composition comprising the viral expression vector of the present invention for use as a pharmaceutical.

[0096] In a third aspect, the present invention relates to a pharmaceutical composition comprising at least one viral expression vector according to the present invention.

[0097] Advantageously, the pharmaceutical compositions according to the present invention are used for the treatment of Alzheimer's disease (AD).

[0098] The present invention also relates to a pharmaceutical composition comprising: a) At least one viral expression vector, preferably selected from antisense RNA (asRNA), small hairpin RNA (shRNA), or microRNA (miRNA), more preferably selected from antisense RNA (asRNA), and comprising at least one nucleotide sequence transcribed into a non-coding nucleotide sequence that inhibits the synthesis of at least one protein selected from VAMP, SNAP-25, and syntaxin, and / or b) At least one viral expression vector comprising a wild-type or modified bacterial neurotoxin or an active fragment thereof that disrupts the SNARE complex, preferably at least one nucleotide sequence encoding the light chain of the bacterial neurotoxin, wherein the bacterial neurotoxin is advantageously any serotype, preferably serotypes A, B, C, E, and F of Clostridium tetani and / or Clostridium botulinum, and / or c) At least one viral expression vector comprising at least the following: • A single nucleotide sequence encoding the wild-type or modified neurotoxin of Clostridium tetani or Clostridium botulinum, or its active fragment, and • The transcript contains a single nucleotide sequence that inhibits the synthesis of the proteins VAMP, SNAP-25, and / or syntaxin, and / or d) At least one viral expression vector according to the present invention, i. One of the long-term expression (LTE) sequences is operably linked to two transgenic transcription cassettes according to the present invention; or ii. Two long-term expression (LTE) sequences are operably linked to one of the transgenic transcription cassettes according to the present invention; in this case, One transgenic transcription cassette according to the present invention comprises a promoter and a sequence encoding the neurotoxin, and a second transgenic transcription cassette according to the present invention comprises a promoter and a sequence nucleotide that inhibits the synthesis of the proteins VAMP, SNAP-25 and / or syntaxin, or Both transgenic transcription cassettes according to the present invention have a promoter and a nucleotide sequence encoding a non-coding nucleotide sequence that inhibits the synthesis of at least one protein selected from VAMP, SNAP-25 and / or syntaxin, or • For use simultaneously, separately, or with a time delay to treat AD, both transgenic transcription cassettes according to the present invention have nucleotide sequences encoding wild-type or modified neurotoxins or active fragments of Clostridium tetani and / or Clostridium botulinum, respectively, comprising at least one viral expression vector according to the present invention.

[0099] In certain embodiments, the pharmaceutical composition according to the present invention further comprises at least one viral expression vector comprising at least one nucleotide sequence encoding the wild-type protein GAD67 and / or RIP and / or NTR, or an active fragment thereof.

[0100] In certain embodiments, the pharmaceutical composition according to the present invention comprises at least one viral expression vector comprising at least one nucleotide sequence encoding wild-type protein GAD67 or an active fragment thereof; and / or at least one nucleotide sequence encoding wild-type or modified neurotoxin of Clostridium tetani and / or Clostridium botulinum, or an active fragment thereof; and / or at least one nucleotide sequence encoding wild-type RIP or an active fragment thereof; and / or at least one nucleotide sequence encoding wild-type NTR or an active fragment thereof.

[0101] example Example 1: Construction of a deletion recombinant HSV-1 vector and an amplicon HSV-1 vector. material and method The present invention provides a set of deficient recombinant HSV-1 vectors containing inactivation deletions of ICP27 and ICP4 (both copies) and ICP0 (1 copy), the set of deficient recombinant HSV-1 vectors further having a therapeutic transcription cassette embedded in either the LAT locus between the LAP sequence and the LTE sequence (site 1) or between the LTE sequence and the INS sequence (site 2), as shown in Figures 1 and 2, to provide long-term expression to the cassette. Some of the transcription cassettes used to construct these vectors are shown in Figure 3.

[0102] The transcription cassette expresses Clostridium toxin light chain (LC), TeNT (LC), BoNT-A (LC), BoNT-B (LC), BoNT-C (LC), BoNT-E (LC), BoNT-F (LC), or antisense RNA (asRNA) against SNARE proteins, VAMP2, SNAP25, and syntaxin, or fused SNARE / light chain toxins, or RIP proteins such as human GAD67 protein or saporin S6, or Escherichia coli (E. coli) NTR nfnB, and when controlled by an afferent neuron-specific promoter, it specifically inhibits / silences neurotransmission in afferent neurons.

[0103] To construct the vectors, the inventors used the full-length HSV-1 genome of strain F cloned into a bacterial artificial chromosome (BAC), such as that described by Tanaka et al., 2003. Gene deletions and insertions were introduced by homologous recombination within the bacteria, and the vectors were then reconstituted by transfection with a tolerant cell line as previously described (Tanaka et al., 2003). The general structures of these vectors are shown in Figure 1A.

[0104] Genome of the HSV-1 ampliconvector. Figure 2 The present invention also provides a set of deficient amplicon vectors that express the same transgenic therapeutic transcription cassette as the recombinant vector, as enumerated in Figure 7. Sequences conferring long-term expression (LTE and INS) surround the transcription cassette (Figure 2). Figure 2 also shows that, in addition to the therapeutic transcription cassette, the amplicon vector has a second transcription cassette and in all cases expresses a reporter protein (either GFP or the fusion protein GFP / sea lizard luciferase) driven by the HSV-1 IE4 / 5 promoter.

[0105] The amplicon vector is generated using a deficient LaLdeltaJ virus and a complementary cell line, which have already been described by Epstein and his collaborators (Zaupa, Revol-Guyot, and Epstein, 2003), as helpers, and the complementary cell line expresses the set of proteins necessary for the amplification and packaging of the vector genome.

[0106] Transcription cassettes delivered by recombinant vector genomes and amplicon vector genomes. Figure 7. The recombinant vectors and amplicon vectors described in the present invention, as shown in Figures 1 and 2, both types of vectors have and express a transgenic transcription cassette embedded in an HSV-1 sequence (LAP, LTE, INS) that confers long-term expression. Some examples of transcription cassettes used in the present invention are listed in Figure 7.

[0107] Example 2: HSV-1 Ampliconvector The present invention also provides a set of knockout amplicon vectors, some of which express either a reporter protein (luciferase) or the light chain (LC) of Clostridium toxin (TeNT(LC), BoNT-A(LC), BoNT-B(LC), BoNT-C(LC), BoNT-E(LC), BoNT-F(LC)), or antisense RNA (asRNA) against SNARE proteins, VAMP2, SNAP25, and syntaxin, or a chimeric SNARE / light chain toxin, or a RIP protein such as human GAD67 protein, or Saporin S6, or a nitroreductase (NTR) protein such as nfnB. The promoter (prom) driving the expression of these transgenes is either a non-specific promoter (HCMV, EF1A) or an afferent neuron-specific promoter (TRPV1, TRPM8, ASIC3, GCRP, ADVl). Further sequences conferring long-term expression (LTE and DNA insulator sequences) are added to some of these promoters (Figure 3A). The promoters that control the expression of the reporter GFP or GFP-rLuc fusion protein present in the amplicon vector are previral early promoters known as the HSV-1 IE4 / 5 promoter. Some general structures of the amplicon vectors used herein are shown in Figure 3B.

[0108] Example 3: Expression of BoNT-A, BoNT-C, and TeNT. Figure 4 BoNT-A(LC), BoNT-C(LC), and TeNT(LC) were expressed in Gli36 (a cell line derived from human glioblastoma) and BHK21 (hamster fibroblast) cell lines. Gli36 and BHK21 cells were infected with amplicon vectors expressing HCMV-Luc, HCMV-BoNT-A(LC), HCMV-BoNT-C(LC), or HCMV-TeNT(LC). The cells were then fixed, and toxin expression was demonstrated by Western blotting using an anti-TeNT antibody to identify TeNT(LC) and an anti-HIS antibody to identify BoNT-A(LC) and BoNT-C(LC). In fact, no effective anti-BoNT antibodies are available; therefore, BoNT-A(LC) and BoNT-C(LC) are expressed as fusion proteins with a C-terminal HIS tag. Figure 4 shows that viral vectors containing genes encoding HCMV-BoNT-A(LC), HCMV-BoNT-C(LC), and HCMV-TeNT(LC) express BoNT-A(LC), BoNT-C(LC), and TeNT(LC), respectively, in both Gli36 and BHK21 cells.

[0109] Example 4: In vitro proteolytic activity of recombinant toxin TeNT. Figure 5 The proteolytic activity of the toxin TeNT(LC) against VAMP2 was evaluated by Western blotting using an anti-VAMP2 antibody. The toxin TeNT(LC) was expressed in Gli36 cells after infection with a viral expression vector expressing HCMV-TeNT(LC). Infection was terminated after 2 days, and protein extracts were prepared. These extracts were incubated in a suitable buffer containing the target protein of TeNT, namely VAMP2 (containing 50 mM Hepes, 400 mM NaCl, 5 mM dithiothreitol, and 2 μM ZnSO4). Western blotting (Figure 5) was performed using 2.5 μL, 5 μL, and 10 μL of cell extracts. Untreated samples, samples of cells infected with a vector that did not express the transgene (pA-1), and samples of cells infected with a vector expressing HCMV-Luc (10 μL) were used as negative controls. Various amounts of recombinant TeNT (recTeNT) were used as positive controls. The results show that an increase in the protein extract expressing TeNT(LC) leads to a decrease in the amount of VAMP2, demonstrating that the toxin present in the protein extract exhibits proteolytic activity against VAMP2.

[0110] Example 5: Intracellular proteolytic activity of recombinant toxins BoNT-A(LC) and BoNT-C(LC). Figure 6 SH-S5Y5 human neuroblastoma cell lines were used to track intracellular SNAP25 and syntaxin 1a (STX) cleavage after infection with amplicon vectors expressing BoNT-A(LC) or BoNT-C(LC), due to their characteristic of spontaneously expressing SNARE proteins. SNAP25 and STX levels were detected by Western blot assays using anti-SNAP25 or anti-STX antibodies, respectively. Negative controls included uninfected cells (mock) or cells infected with a vector expressing HCMV-Luc. The results (Figures 6a and 6b) show that 48 hours (hpi) after infection (BPI) of SH-S5Y5 cells with vectors expressing BoNT-A or BoNT-C light chains, intracellular SNAP25 (Figure 6a) or SNAP25 and STX (Figure 6b) protein levels were significantly reduced, respectively, compared to cells infected with a control vector expressing luciferase.

[0111] Example 6, Figure 8 BoNT-A, expressed from an amplicon vector, cleaves the SNARE protein SNAP25 in SH-SYS5 cells. This experiment was designed to evaluate whether a vector expressing the light chain of BoNT-A expresses this protein and to test whether this toxin has the same biological activity as a complete neurotoxin (light chain + heavy chain), i.e., the ability to cleave its target SNARE protein (SNAP25). As shown in Figure 8, cells infected with an amplicon expressing A2-CMV-BoNT-A express an increasing amount of toxin as the multiplicity increases. Furthermore, when cells are infected with a high MOI, virtually all SNAP25 is cleaved, clearly demonstrating the functional activity of the BoNT-A light chain.

[0112] Example 7, Figure 9 The light chain of botulinum neurotoxin cleaves SNARE proteins in infected neurons.

[0113] This experiment was designed to confirm that all BoNT light chains synthesized in vector-infected neurons could cleave their native SNARE target proteins in sensory neurons. For this purpose, primary cultures of rat embryonic DRG neurons were infected with amplicon vectors expressing A2-CMV-BoNT-A, -B, -C, -D, -E, and -F, or A2-CMV-Luc as a negative control, at a MOI of 10. Infection was stopped the following day, and cell extracts were analyzed by Western blotting. As shown in Figure 9, each botulinum neurotoxin expressed by the vector cleaved its native target SNARE protein. Thus, BoNT-A and -E cleaved SNAP25, BoNT-B, -D, and -F cleaved VAMP2, and BoNT-C cleaved both SNAP25 and syntaxin. This clearly demonstrates that the light chains of all neurotoxins exhibit the same biological activity as the complete neurotoxin (light chain + heavy chain).

[0114] Example 8, Figure 10 The light chain of botulinum toxin inhibits the release of neuropeptides in sensory neurons.

[0115] This experiment was designed to evaluate whether the light chain of botulinum neurotoxin induces inhibition of neurotransmitter release and to assess its comparative effect in this regard. Primary cultures of rat embryonic DRG neurons were infected with a vector with increased MOI, as shown in Figure 10. The following day, infected neurons were treated with KCl to stimulate the release of the neuropeptide CGRP, and the extracellular concentration of CGRP was assessed by ELISA. As shown in Figure 10, all neurotoxins induced inhibition of CGRP release. Furthermore, Figure 6 shows that BoNT-F was the most effective in this regard, followed by BoNT-A and BoNT-C.

[0116] Example 9, Figure 11 GAD67 expressed from an amplicon vector induces GABA synthesis and extracellular release.

[0117] The objective of this experiment was to evaluate whether a vector expressing GAD67 induces the synthesis and release of the inhibitory neurotransmitter GABA. To this end, glioblastoma cells (Gli36) were infected with an amplicon vector at increasing MOIs, as described in Figure 11. The following day, infected cell extracts were analyzed by Western blotting using antibodies specific to GAD67 and GAPDH. Figure 11 shows that GAD67 expression increases with MOI, demonstrating that the vector A2-CMV-GAD67 expresses this protein. Furthermore, primary cultures of rat embryonic DRG neurons were infected with the same vector at various MOIs. The following day, infection was stopped, and both intracellular and extracellular GABA concentrations were evaluated using a Resazurine assay (as shown in the legend of Figure 11). The upper panel of this figure shows that the amount of intracellular GABA increases with MOI, while the lower panel shows the increase in extracellular GABA. This clearly demonstrates that the expression of GAD67 from the A2-CMV-GAD67 vector increases intracellular GABA synthesis and its release into the extracellular medium.

[0118] Example 10 Figure 12 Nitroreductase (NTR) activates the nitro compound 7'-nitrocoumarin, inducing cell death in the presence of mitronidazole (MTZ).

[0119] This experiment was designed to evaluate whether nitroreductase expressed from an amplicon vector induces cell death in the presence of metronidazole but not in its absence. There are no available antibodies specific to nitroreductase (NTR). Therefore, to assess whether this protein is expressed in A2-CMV-NTR-infected cells, we used a functional in vitro test based on the evaluation of 7'-nitrocoumarin reduction (Muller et al., 2015). Figure 12 shows that an amplicon vector expressing A2-CMV-NTR certainly activates nitro compounds. Furthermore, Figure 12 shows that NTR expression induced significant cell death in the presence of metronidazole (MTZ). This is explained by the fact that NTR can activate MTZ and thus convert this molecule into a cytotoxic drug.

[0120] Example 11 Figure 13 Analysis of the selectivity of DRG-selective promoter candidates in the autonomic and sensory ganglia of adult rats.

[0121] This study was designed to investigate whether afferent neuron-specific promoter candidates, which are normally active only in afferent neurons or primarily in afferent neurons, retain their afferent neuron-specific activity when expressed from a non-replicating HSV-1 vector genome. Adult rat afferent ganglia (DRG), autonomic sympathetic ganglia (SCG), and autonomic parasympathetic ganglia (GPC) were explanted and maintained as organoid cultures. After 3 days, the time required for neurite extension, 3 × 10⁶ cells were placed in the ganglia, as described in the legend in Figure 13. 6The vector particles were individually infected. These vectors express firefly luciferase (fLuc) driven by the following promoters: rat TRPV1 (rTRPV1), rat CGRP (rCGRP), rat ASIC3 (rASIC3) (all of which are thought to be afferent neuron-specific promoters), and EF1a, a non-selective promoter that serves as a general control. In addition to fLuc, these vectors also express sea urchin luciferase (rLuc) driven by the viral promoter (HSV-1 IE4 / 5). The following day, infection was stopped and cell extracts were prepared for luciferase testing. The results are expressed as the fLuc / rLuc ratio and the percentage of luciferase activity driven by EF1a. Figure 13 shows that rTRPV1 and rCGRP preferentially express firefly luciferase activity in the DRG and can therefore be considered DRG-specific even when expressed from the vector genome. In contrast, rASIC3 does not exhibit such preferential expression in DRGs, demonstrating that this promoter does not preserve its selectivity when expressed from a vector genome. Therefore, this example shows that while some DRG-specific promoter candidates, such as rTRPV1 and rCGRP promoters, preserve their selectivity for DRGs, other promoter candidates, such as rASIC3, appear to be DRG-specific promoters when expressed from cell chromosomes but do not preserve this specificity when expressed from a vector genome. Consequently, the behavior of any particular DRG-specific promoter candidate cannot be predicted and needs to be evaluated experimentally.

[0122] Example 12: Infection and expression of recombinant proteins in cell cultures Primary rat neuron cultures derived from embryonic DRGs and organ-type cultures of adult rat DRG explants were infected with an amplicon vector expressing GFP driven by the HSV-1 pre-early IE4 / 5 promoter. The results showed that the viral expression vector infected both primary rat sensory neuron cultures and adult rat ganglion (DRG) explants, and expressed the transgene (GFP) (Figure 14).

[0123] Example 13: In vivo expression of recombinant proteins in neurons Spinal cord injury (SCI) rats were infected with the amplicon vector HCMV-Luc, which simultaneously expresses GFP and Luc reporter proteins. One week after infection, the animals were sacrificed, and transgenic protein expression was revealed by intracellular heat therapy (IHC). As shown by IHC, upon inoculation into the bladder, the amplicon vector enters afferent neurons innervating the bladder and is subsequently transported retrogradely through the axons to the cell bodies of neurons in the dorsal ganglion (DRG), where the viral genome expresses both transgenic proteins. The results demonstrate that the amplicon vector HCMV-Luc can thus induce and specifically express transgenic proteins within bladder afferent neurons after inoculation into the bladder wall (Figure 15). Furthermore, neurons expressing GFP and Luc were observed only in the ganglion (L6 ganglion) from which neurons innervating the bladder extend. In contrast, no transgene expression was observed in ganglion T13, which does not innervate the bladder (data not shown).

[0124] Example 14: Cell-specific expression of viral expression vectors We infected sensory ganglia or autonomic ganglia (both sympathetic and parasympathetic) using amplicon vectors TRPV1-Luc, which express luciferase under the control of the TRPV1 promoter (a promoter selectively active in afferent neurons), and HCMV-Luc, which expresses luciferase under the control of a non-selective HCMV promoter. The results showed that luciferase expression under the TRPV1 promoter was specifically expressed in afferent neurons of sensory ganglia (dorsal root ganglia, DRGs) and not in autonomic neurons (sympathetic or parasympathetic) (Figure 16). The results were expressed as the percentage of expression driven by the non-selective HCMV promoter, which was equally high in all ganglia types.

[0125] Example 15 - Transgene expression by ddPCR in DRGs of capsaicin research In a capsaicin study, dorsal root ganglia (DRG) L6 and S1 (including the cell bodies of sensory neurons innervating the bladder) were analyzed by digital droplet PCR (ddPCR) to measure the expression (transcriptional level) of the botulinum toxin F light chain (BONT / F-LC) after intravesical administration of the vector according to the present invention.

[0126] In short, total RNA was extracted and purified from pooled L6 and S1 DRGs. Following the reverse transcription step, ddPCR was performed on cDNA using primers and probes for the 3'-UTR region of the BONT / F-LC transcript. The absolute copy number of BONT / F-LC was measured. DNA contamination was accounted for by subtracting the copy number measured by ddPCR without the reverse transcription step. The data are reported as the total number of BONT / F-LC copies per pooled L6 and S1 DRG.

[0127] ddPCR in the DRG showed a significant number of BoNT / F-LC transcript copies one week after intravesical injection in all rats (Figure 17). There was no significant difference in BoNT / F-LC expression levels between one week and five weeks after injection. No expression was observed in the medium-injected rats. Therefore, after local administration of the vector to the detrusor muscle, the vector can transduce afferent fibers to achieve stable BoNT / F-LC expression in L6 / S1 DRG sensory neurons for at least five weeks.

[0128] Example 16 The effect of the vector according to the present invention on urinary flow dynamics parameters 5 weeks after administration to a bladder stimulation model (30 μM capsaicin) was investigated compared to a control administered with a buffer. The vector contains a deficient recombinant HSV-1 vector having a cassette containing the light chain of botulinum neurotoxin F1 (BoNT-F1) operably linked to the CGRP promoter, with the transcription cassette located at the latent-associated transcript (LAT) locus of the HSV-1 genome. At the end of the bladder manometry experiment, dorsal root ganglia (DRG) L6-S1 and L1-L2, as well as bladders from all groups, were collected for histological and molecular biological experiments.

[0129] On day 1, all rats except for the negative control rats that did not undergo surgery were anesthetized with isoflurane (1.5-2.0%), and the vector or positive control was injected into the following 10 locations within the detrusor muscle using a 30-gauge needle: - Two injection sites each within the bladder trigone on both the anterior and posterior sides of the bladder (four injection sites in total), and - Three injection sites on each side of the bladder, towards the bladder trigone (six injection sites in total).

[0130] A 35-minute postoperative recovery period was observed before initiating bladder irrigation. Subsequently, the bladder was continuously irrigated with saline solution (50 μl / min) for a 90-minute stabilization period to obtain a stable urination cycle. The bladder irrigation was then switched to 30 μM capsaicin to induce continuous bladder stimulation. A 60-minute bladder stimulation period was then recorded. Throughout the experiment, intravesical and arterial pressures were recorded.

[0131] The following parameters of reflex-induced bladder contraction were measured: • Maximum amplitude of urination contraction (mmHg), MP • Area under the curve (mmHg × s), AUC during urination contraction, • Duration of urination contraction (s), • Baseline bladder pressure (mmHg), BP (measured immediately after bladder emptying) • Dysfunction delta pressure threshold (mmHg), ΔPT (calculated as the difference between bladder pressure and BP at the start of urination) • Contraction interval, ICI (s, closely correlated with the frequency of urination contractions), • Urine volume, VV (volume of urine released from the urethral opening during urination contractions) • Infusion volume (μl), IV, (infusion rate × ICI), (indicator of bladder capacity during each voiding contraction) • Urinary efficiency (%), VE, (VV / IV ratio (×100)) • Bladder compliance (%), BC (ratio of volume threshold to ΔPT), • Post-void residual volume (μl), PVR (the amount of urine remaining in the bladder at the end of urination).

[0132] These parameters were measured and expressed for each urination cycle. Raw data were then calculated every 15 minutes during all bladder pressure measurement experiments to show the effect of saline or capsaicin over perfusion time. Furthermore, for all groups, each urodynamic parameter was normalized to the stabilization period, and the results were expressed as mean ± SEM percentage. During the stabilization period, these parameters were measured from the last stable control urination cycle and averaged over a stabilization period of approximately 30 minutes. During the capsaicin bladder stimulation setting period, these parameters were measured every 15 minutes for 60 minutes and averaged. Comparisons were performed using two-way ANOVA, one-way ANOVA, and Student's t-test. Statistical analysis was performed using GraphPad Prism® 6.07 software. A p-value < 0.05 was considered statistically significant.

[0133] At the end of the experimental period, the DRG (left and right) and bladder were carefully dissected from all rats. All samples were then immediately placed in saline solution at +4°C to remove excess blood, and carefully dissected, ensuring that no surrounding tissue was included. Next, the entire bladder was weighed and then cut longitudinally.

[0134] For histological evaluation, the left DRG T13-S2, L6-S1, and L1-L2, as well as the bladder portion, were fixed in a 4% neutral buffered formalin solution for 24 hours, and then stored in a 70% ethanol solution at +4°C in Pelvipharm.

[0135] For molecular evaluation, the right DRG T13-S2, L6-S1, and L1-L2, as well as the bladder portion, were weighed (wet weight) and placed in RNA Later® (Invitrogen®) at +4°C according to the manufacturer's instructions. After one week, the tissue was removed from RNA Later and frozen at -80°C.

[0136] As shown in Table 1 and Figures 18-25, five weeks after IVI injection at 10 sites (10 × 4 μl) with 2.2 × 10⁸ PFU in 40 μl, the vector did not induce significant changes in urodynamic parameters compared to the buffer group during the stabilization period, indicating that the vector maintained bladder voiding function. Conversely, this treatment induced significant effects on MP, AUC, ICI, IV, VV, BP, and compliance compared to rats injected with the buffer, particularly during the last 30 minutes of capsaicin perfusion.

[0137] Importantly, the vector was able to desensitize the bladder and bring the intersystolic interval closer to baseline without affecting urination; that is, there was no increase in post-void residual volume. This demonstrates the selectivity of the vector in the bladder. [Table 1]

[0138] While the present invention has been specifically illustrated and described with reference to its preferred embodiments, it will be understood by those skilled in the art that various modifications in form and detail can be made without departing from the scope of the invention as encompassed in the appended claims. References

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Claims

1. A viral expression vector for use in the treatment of overactive bladder (OAB) and / or bladder pain syndrome (BPS), wherein the viral vector comprises at least: a) A single promoter that is selectively activated in afferent neurons of the bladder, b) At least one transcription cassette comprising a nucleotide sequence operably linked to the promoter, wherein the nucleotide sequence, when transcribed, silences or inhibits the transmission of neurotransmitter signals in a postsynaptic cell, c) A sequence that operatively connects to the transcription cassette to confer long-term expression and A viral expression vector containing a virus.

2. The viral expression vector according to claim 1, wherein the vector is an adeno-associated virus (AAV) vector or a herpes simplex virus (HSV) vector.

3. The viral expression vector according to claim 1 or 2, wherein the vector is a herpes simplex virus (HSV) vector.

4. The viral expression vector according to claim 2 or 3, wherein the herpes simplex virus (HSV) vector is an HSV-1 vector.

5. A viral expression vector according to any one of claims 1 to 4, for use in the treatment of OAB.

6. A viral expression vector according to any one of claims 1 to 4, for use in the treatment of BPS.

7. The viral expression vector according to claim 6, wherein the BPS is interstitial cystitis (IC).

8. The viral expression vector according to any one of claims 1 to 7, wherein the nucleotide sequence, when transcribed by disrupting the SNARE complex and / or the ribosome complex, and / or by activating the GABA(A) receptor, and / or by inducing ablation of conditionally targeted neurons, silences or inhibits neurotransmission or synaptic transmission of afferent neurons.

9. The viral expression vector according to any one of claims 1 to 8, wherein the promoter is a promoter selected from promoters of genes encoding sensory nerve receptors.

10. The viral expression vector according to any one of claims 1 to 9, wherein the at least one nucleotide sequence is transcribed to a non-coding nucleotide sequence that inhibits the synthesis of at least one protein selected from the group consisting of VAMP, SNAP-25, and syntaxin.

11. The viral expression vector according to claim 10, wherein the non-coding nucleotide sequence is selected from the group consisting of antisense RNA (asRNA), small hairpin RNA (shRNA), and microRNA (miRNA).

12. The viral expression vector according to any one of claims 1 to 9, wherein the at least one nucleotide sequence encodes a wild-type or modified bacterial neurotoxin or an active fragment thereof that disrupts the SNARE complex, a wild-type or modified GAD67 protein or an active fragment thereof, a wild-type or modified ribosome inactivating protein (RIP) or an active fragment thereof, or a wild-type or modified nitroreductase (NTR) or an active fragment thereof.

13. The viral expression vector according to claim 12, wherein the active fragment of the wild-type or modified bacterial neurotoxin is the light chain of the bacterial neurotoxin.

14. The viral expression vector according to claim 12, wherein the bacterial neurotoxin is a neurotoxin of any serotype of Clostridium botulinum or a tetanus neurotoxin of Clostridium tetanis.

15. The viral expression vector according to claim 12, wherein the bacterial neurotoxin is the light chain of botulinum neurotoxin A (BoNT-A), the light chain of botulinum neurotoxin B (BoNT-B), the light chain of botulinum neurotoxin C1 (BoNT-C1), the light chain of botulinum neurotoxin E3 (BoNT-E3), the light chain of botulinum neurotoxin F1 (BoNT-F1), and the light chain of tetanus neurotoxin (TeNT).

16. at least, a) A single nucleotide sequence encoding the wild-type or modified neurotoxin or an active fragment of Clostridium tetanus or Clostridium botulinum, and / or b) A single nucleotide sequence in which the transcript inhibits the synthesis of the proteins VAMP, SNAP-25 and / or syntaxin, and / or c) A single nucleotide sequence encoding the wild-type or modified GAD67 protein or an active fragment thereof, and / or d) A single nucleotide sequence encoding wild-type or modified RIP or an active fragment thereof, and / or e) A single nucleotide sequence encoding the wild-type or modified NTR or its active fragment. A viral expression vector according to claim 1, comprising:

17. A viral expression vector according to claim 1, i. The one sequence conferring long-term expression is operably linked to two of the at least one transcription cassette, or ii. Both sequences conferring long-term expression are operably linked to one of the at least one transcription cassettes, in this case, a) One of the at least one transcription cassettes has a coding nucleotide sequence for a wild-type or modified bacterial neurotoxin or its active fragment, a wild-type or modified GAD67 protein or its active fragment, a wild-type or modified ribosome inactivating protein (RIP) or its active fragment, or a wild-type or modified nitroreductase (NTR) or its active fragment that disrupts the SNARE complex, and the second of the at least one transcription cassette has a nucleotide sequence that is transcribed to a non-coding nucleotide sequence that inhibits the synthesis of at least one protein selected from the group consisting of VAMP, SNAP-25, and syntaxin, or b) Both transcription cassettes contain nucleotide sequences encoding non-coding nucleotide sequences of wild-type or modified bacterial neurotoxins or their active fragments that disrupt the SNARE complex, wild-type or modified GAD67 protein or its active fragment, wild-type or modified ribosome inactivating protein (RIP) or its active fragment, or wild-type or modified nitroreductase (NTR) or its active fragment, or c) Both transcription cassettes contain nucleotide sequences encoding wild-type or modified neurotoxins or active fragments of Clostridium tetanus and / or Clostridium botulinum, or wild-type or modified GAD67 protein or active fragments, or wild-type or modified RIP or active fragments, or wild-type or modified NTR or active fragments. Virus expression vector.

18. The viral expression vector according to any one of claims 1 to 17, wherein the at least one sequence conferring long-term expression is an LTE and / or DNA insulator derived from the HSV-1 genome.

19. The viral expression vector according to claim 9, wherein the promoter is a promoter of the TRP gene family, or a promoter of a gene encoding a sensory neuromodulator or sensory neurotransmitter.

20. The viral expression vector according to claim 19, wherein the promoter is the promoter of TRPV1 or TRPM8, or the promoter of Substance P, PACAP, CGRP, or ADVL.

21. The viral expression vector according to claim 19, wherein the promoter is a promoter for CGRP or ADVL.

22. A viral expression vector according to claim 1, wherein the viral expression vector is a) A promoter that is selectively activated in the afferent neurons of the bladder; and b) at least one transcription cassette comprising a nucleotide sequence operably linked to the promoter, wherein the at least one transcription cassette is introduced into a latent-associated transcript (LAT) locus of the HSV-1 genome, The LAT locus is a transcription cassette comprising at least one long-term expression sequence, preferably a latent-associated promoter (LAP), a long-term expression (LTE) region, and / or a DNA insulator (INS) sequence. A viral expression vector that contains herpes simplex virus (HSV).

23. The viral expression vector according to claim 22, wherein the transcription cassette is inserted between the LAP and the LTE, or between the LTE and the DNA insulator sequence.

24. A pharmaceutical composition comprising a viral expression vector according to any one of claims 1 to 23, for use in the treatment of overactive bladder (OAB) and / or bladder pain syndrome (BPS).

25. A method for treating patients requiring treatment for overactive bladder (OAB) and / or bladder pain syndrome (BPS), comprising at least: a) A single promoter that is selectively activated in afferent neurons of the bladder, b) at least one transcription cassette comprising a nucleotide sequence operably linked to the promoter, wherein the nucleotide sequence, when transcribed, silences or inhibits the transmission of neurotransmitter signals in a postsynaptic cell, and c) A sequence that confers long-term expression, operably linked to the transcription cassette. A method comprising administering a viral expression vector containing [a specific substance].

26. The method according to claim 25, wherein the vector is an adeno-associated virus (AAV) vector or a herpes simplex virus (HSV) vector.

27. The method according to claim 25, wherein the vector is a herpes simplex virus (HSV) vector.

28. The method according to claim 25, wherein the herpes simplex virus (HSV) vector is an HSV-1 vector.

29. The method according to any one of claims 25 to 28, wherein the patient is suffering from OAB.

30. The method according to any one of claims 25 to 28, wherein the patient is suffering from BPS.

31. The method according to claim 30, wherein the BPS is interstitial cystitis (IC).

32. The method according to any one of claims 25 to 31, wherein the nucleotide sequence is transcribed by disrupting the SNARE complex and / or the ribosome complex, and / or by activating the GABA(A) receptor, and / or by inducing ablation of a conditionally targeted neuron, thereby silencing or inhibiting neurotransmission or synaptic transmission of afferent neurons.

33. The method according to any one of claims 25 to 32, wherein the promoter is a promoter selected from promoters of genes encoding sensory nerve receptors.

34. The method according to any one of claims 25 to 33, wherein the at least one nucleotide sequence is transcribed to a non-coding nucleotide sequence that inhibits the synthesis of at least one protein selected from the group consisting of VAMP, SNAP-25, and syntaxin.

35. The method according to claim 34, wherein the non-coding nucleotide sequence is selected from the group consisting of antisense RNA (asRNA), small hairpin RNA (shRNA), and microRNA (miRNA).

36. The method according to any one of claims 25 to 33, wherein the at least one nucleotide sequence encodes a wild-type or modified bacterial neurotoxin or an active fragment thereof that disrupts the SNARE complex, a wild-type or modified GAD67 protein or an active fragment thereof, a wild-type or modified ribosome inactivating protein (RIP) or an active fragment thereof, or a wild-type or modified nitroreductase (NTR) or an active fragment thereof.

37. The method according to claim 36, wherein the active fragment of the wild-type or modified bacterial neurotoxin is the light chain of the bacterial neurotoxin.

38. The method according to claim 36, wherein the bacterial neurotoxin is a neurotoxin of any serotype of Clostridium botulinum or a tetanus neurotoxin of Clostridium tetanis.

39. The method according to claim 36, wherein the bacterial neurotoxin is the light chain of botulinum neurotoxin A (BoNT-A), the light chain of botulinum neurotoxin B (BoNT-B), the light chain of botulinum neurotoxin C1 (BoNT-C1), the light chain of botulinum neurotoxin E3 (BoNT-E3), the light chain of botulinum neurotoxin F1 (BoNT-F1), and the light chain of tetanus neurotoxin (TeNT).

40. The aforementioned virus expression vector comprises at least, a) A single nucleotide sequence encoding the wild-type or modified neurotoxin or an active fragment of Clostridium tetanus or Clostridium botulinum, and / or b) A single nucleotide sequence in which the transcript inhibits the synthesis of the proteins VAMP, SNAP-25 and / or syntaxin, and / or c) A single nucleotide sequence encoding the wild-type or modified GAD67 protein or an active fragment thereof, and / or d) A single nucleotide sequence encoding wild-type or modified RIP or an active fragment thereof, and / or e) A single nucleotide sequence encoding the wild-type or modified NTR or its active fragment. The method according to claim 25, including the method described in claim 25.

41. The method according to claim 25, i. The one sequence conferring long-term expression is operably linked to two of the at least one transcription cassette, or ii. Both sequences conferring long-term expression are operably linked to one of the at least one transcription cassettes, in this case, a) One of the at least one transcription cassettes has a coding nucleotide sequence for a wild-type or modified bacterial neurotoxin or its active fragment, a wild-type or modified GAD67 protein or its active fragment, a wild-type or modified ribosome inactivating protein (RIP) or its active fragment, or a wild-type or modified nitroreductase (NTR) or its active fragment that disrupts the SNARE complex, and the second of the at least one transcription cassette has a nucleotide sequence that is transcribed to a non-coding nucleotide sequence that inhibits the synthesis of at least one protein selected from the group consisting of VAMP, SNAP-25, and syntaxin, or b) Both transcription cassettes contain nucleotide sequences encoding non-coding nucleotide sequences of wild-type or modified bacterial neurotoxins or their active fragments that disrupt the SNARE complex, wild-type or modified GAD67 protein or its active fragment, wild-type or modified ribosome inactivating protein (RIP) or its active fragment, or wild-type or modified nitroreductase (NTR) or its active fragment, or c) A method wherein both transcription cassettes contain nucleotide sequences encoding wild-type or modified neurotoxins or active fragments of Clostridium tetanus and / or Clostridium botulinum, or wild-type or modified GAD67 protein or active fragments, or wild-type or modified RIP or active fragments, or wild-type or modified NTR or active fragments.

42. The method according to any one of claims 25 to 41, wherein the at least one sequence conferring long-term expression is an LTE and / or DNA insulator derived from the HSV-1 genome.

43. The method according to claim 33, wherein the promoter is a promoter of the TRP gene family or a promoter of a gene encoding a sensory neuromodulator or sensory neurotransmitter.

44. The method according to claim 43, wherein the promoter is the promoter of TRPV1 or TRPM8, or the promoter of substance P, PACAP, CGRP, or ADVL.

45. The method according to claim 43, wherein the promoter is a promoter for CGRP or ADVL.

46. The method according to claim 25, wherein the virus expression vector is a) A promoter that is selectively activated in the afferent neurons of the bladder; and b) at least one transcription cassette comprising a nucleotide sequence operably linked to the promoter, wherein the at least one transcription cassette is introduced into a latent-associated transcript (LAT) locus of the HSV-1 genome, The LAT locus is a transcription cassette comprising at least one long-term expression sequence, preferably a latent-associated promoter (LAP), a long-term expression (LTE) region, and / or a DNA insulator (INS) sequence. A method comprising a herpes simplex virus (HSV) virus expression vector containing [a specific substance].

47. The method according to claim 46, wherein the transfer cassette is inserted between the LAP and the LTE, or between the LTE and the DNA insulator sequence.

48. The method according to any one of claims 25 to 47, wherein the virus expression vector is part of the pharmaceutical composition.

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