Compositions and methods for delivery and expression of active peptides and use thereof

Abstract

The present invention provides a chimeric polypeptide comprising (i) a large dense core vesicle (LDV) targeting element and (ii) an ion channel targeting peptide.

Description

[0001] P1789PC00

[0002] - 1 - COMPOSITIONS AND METHODS FOR DELIVERY AND EXPRESSION OF ACTIVE PEPTIDES AND USE THEREOF FIELD OF THE INVENTION

[0003] The present invention provides a chimeric polypeptide comprising: (i) a first polypeptide portion which comprises a large dense core vesicle (LDV) targeting element; and (ii) a second polypeptide portion which comprises an ion channel targeting peptide. The present invention further provides a nucleic acid sequence that encodes a pre-propeptide, an active peptide and a cleavage site. Suitably, the present invention provides a nucleic acid sequence that encodes a prepropeptide and an active peptide. These elements work in concert: only the active peptide is secreted and, upon release, the same acts retroactively on the cell which released it. Moreover, delivery vectors for transferring said sequence to a cell in vitro, ex vivo or in vivo are provided.

[0004] BACKGROUND

[0005] Many neurological disorders such as chronic pain and epilepsy are characterized by sustained, high frequency neuronal firing. Current treatments such as antiepileptic drugs or opioids which reduce this firing often display poor efficacy and cause severe side effects.

[0006] Neuropeptides are a class of neurotransmitters whose release is calcium-dependent and is triggered by high-frequency neuronal activity. They are initially produced as immature peptides with signal sequences and pro-peptides, then processed for mature neuropeptide secretion only.

[0007] WO2019162942 describes signal and propeptides, fused to inhibitory peptides.

[0008] W02024069010 describes dynorphin fusion peptides.

[0009] Alternative drugs capable to be delivered in target cells, to be released as functional and active peptides, and to modulate their own release and action by inhibiting the cells that released them are needed.

[0010] DESCRIPTION

[0011] The present invention is based - at least in part - on the provision of a chimeric polypeptide comprising (i) a large dense core vesicle (LDV) targeting element and (ii) an ion channel targeting peptide. In particular, the ion channel targeting peptide may be capable of acting on a large proportion of neurons, such as Dorsal Root Ganglion (DRG) neurons. Following expression in neurons, the LDV targeting element directs the chimeric polypeptide into LDVs, where the chimeric polypeptide undergoes ma tu ration and removal of the LDV targeting element. The ion channel targeting peptide is subsequently released from the neuron only upon a series of action potentials that exceed a certain excitation threshold, which occur - for example - during epilepsy episodes or bouts of pain. Following release, the ion channel targeting peptide acts on the neuron that has released it, enabling the ion channel targeting peptide to modulate its own release andP1789PC00

[0012] - 2 -action. Without wishing to be bound be theory, such a self -modulating mechanism is not considered to be appropriately achieved by opioid peptides such as dynorphin, for example. The primary receptor for dynorphin, kappa opioid receptor (KOR, gene name oprkl) has a narrow expression pattern in the central and peripheral nervous system, with expression only in a small population of DRG neurons. As such, the proportion of neuron cells that can be targeted to express dynorphin in a controllable manner and also naturally express requisite receptors to be inhibited by the dynorphin once it is released is expected to be insufficient to provide an appropriate self-regulation mechanism.

[0013] Accordingly, in a first aspect the present invention provides a chimeric polypeptide comprising: (i) a first polypeptide portion which comprises a large dense core vesicle (LDV) targeting element; and (ii) a second polypeptide portion which comprises an ion channel targeting peptide.

[0014] Without wishing to be bound by theory, the present invention is considered to provide a number of advantages. For example, the self-regulation mechanism allows for a controllable release of the ion channel targeting peptide, which is subsequently able to precisely inhibit the neuron that it was released from. This effect may be particularly advantageous for providing a targeted inhibition of increased neuronal firing frequency which occurs in indications such as epilepsy and chronic pain. In addition, the self-inhibition means that the release of the ion channel targeting peptide may be time-restricted. Such a feature may be advantageous in allowing the use of an ion channel targeting peptide which could have potentially detrimental effects if its release were not controlled.

[0015] Ion channel targeting peptides typically have IC50s in the sub nanomolar range and thus provide an advantageous potency profile. Further, the ion channel targeting peptide may be selected to target a specific ion channel, allowing for selective action. Targeting ion channels may also facilitate precise modulation of target cells, as the effect will be directed to the specific function of the ion channel targeted, in contrast to broad downstream signalling induced by KOR, for example.

[0016] The invention further provides a polynucleotide encoding the chimeric polypeptide of the invention.

[0017] In another aspect, the invention provides a vector comprising said polynucleotide. Preferably, the vector is an AAV vector.

[0018] In a further aspect, the invention provides a pharmaceutical composition comprising the chimeric polypeptide, the polynucleotide, or the vector of the invention and a pharmaceutically acceptable carrier, diluent and / or excipient.

[0019] The invention further provides the chimeric polypeptide, the polynucleotide, the vector, or the pharmaceutical composition of the invention for use in treating a disease. The disease may be aP1789PC00

[0020] - 3 -neurological disorder associated with, or characterised by, high frequency neuronal firing. The disease may be chronic pain or epilepsy.

[0021] In further aspects, the present invention, in some embodiments thereof, relates to a chimeric polynucleotide encoding a protein precursor, a polynucleotide encoding a protease / con vertase cleavage site and a polynucleotide encoding protein or peptides of 2 - 500 amino acids, or 2 - 250 amino acids, preferably of 2 - 50 amino acids.

[0022] In an embodiment, the invention relates to compositions comprising said chimeric polynucleotide.

[0023] In a different embodiment, the invention relates to methods for selectively modifying specific cell activity comprising administering such compositions to target cells.

[0024] According to one aspect, there is provided a chimeric polynucleotide molecule comprising: a first polynucleotide encoding a pro-peptide domain of a first polypeptide; and a second polynucleotide encoding a second polypeptide or protein of 2 - 500 amino acids, or 2 - 250 amino acids, or 2 - 50 amino acids having a modulating activity, wherein the pro-peptide domain is a specific cell predominantly expressed protein precursor.

[0025] In some embodiments, the pro-peptide domain comprises a signal peptide sequence and a protease / con vertase motif.

[0026] In some embodiments, the modulating peptide is an endogenous peptide. In some embodiments, the modulating peptide is an exogenous peptide. In some embodiments, the modulating peptide is a synthetic peptide.

[0027] In some embodiments, a delivery vector for expression of the disclosed chimeric polynucleotide is provided.

[0028] In some embodiments, the delivery vector comprises a promoter polynucleotide for driving the expression of the disclosed chimeric polynucleotide predominantly in a specific cell.

[0029] In some embodiments, the specific cell is a neural cell.

[0030] In some embodiments, the first polynucleotide sequence encoding the pro-peptide domain of the first polypeptide is selected from the group consisting of neurotrophic factors.

[0031] DRAWINGS DESCRIPTION

[0032] Figure 1: Mechanism of PreProBDNF-MVIIA action, schematic representation. PreProBDNF-MVIIA sequence was engineered and packaged into an adeno-associated viral vector (AAV) for gene delivery. Transduced target cells, by reading such information, produce MVIIA and sort it into the regulated secretory pathway. MVIIA is only released upon sustained neuronal activityP1789PC00

[0033] -4 -in the cell and then acts retroactively against the same cells that produced it, reducing the information passed to the post-synaptic neuron while modulating its own release as well.

[0034] Figure 2: Quantification of release events in DCV s transfected either with a control or MVIIA, under the control of PreProBDNF. The dashed line represents the media; One-sample t-test, p<0.0001; n=6

[0035] Figure 3: Representative images showing cells response to 30 Hz stimulation in presence of a control (PreProBDNF-mEGFP) or MVIIA (PreProBDNF-MVIIA) (A) and response to a range of tested frequencies (B). A) On the left, the panel shows the release in the presence of a sustained stimulation (30 Hz). Scale bar is 10 pm. Notably, in presence of MVIIA (bottom panel) cells reacted less than the control, showing that at 30 Hz MVIIA is released and able to inhibit its own release. Cellular exocytosis as fluorescence signal is quantified in the graph on the right. B) Both in control (mEGFP) and MVIIA cells, release is detected only at sustained stimulation (15 and 30 Hz), but not at low-frequency stimuli (0.3, 1, 3 and 10 Hz). The arrow indicated the timepoint where the electric field was provided. Both sets of data were analysed through impaired two-tailed t-test on AUG values; p<0.0001; n=3.

[0036] Figure 4: Panel of nociception behavioural experiments comparing wild-type (WT) animals and PreProBDNF-MVIIA treated ones. The clip test (A) was performed to assess reaction to acute, mechanical, painful stimulation. The rotarod test (B) did not report any effect on balance and locomotion, excluding off-target effects. When mechanical sensitivity was assessed (C), treated animals reported a higher sensitivity threshold which was confirmed in the CFA pain model, with high statistical significance. Animals showed higher tolerance to rising temperatures (D), which was again confirmed in the CFA pain model. The Formalin test (E) revealed decreased pain behaviour especially in the late phase of the reaction, highlighting the relevance of PreProBDNF-MVIIA for chronic pain conditions.

[0037] Figure 5: Dissociated DRG neuron infected by rAAV:PreProBDNF-GpTx-l-71, quantification of the peak of fluorescence during KC1 stimulation. PreProBDNF sequence is driving GpTx-1-71 into DCVs. When excited with KC1, the cells show exocytotic events reported by an increase of almost 3-fold in fluorescence. Histograms show the level of fluorescence in each vesicle (A). Scale bar is 10 pm. Paired two-tailed t-test; p<0.0001. (B) Graph showing the occurrence of action potentials: in the presence of GpTx-1-71, neurons were markedly less responsive to gradually increasing electrical stimulation.

[0038] Figure 6: Clip test (A) and Rotarod (B) experiments comparing wild-type (WT) animals and PreProBDNF-GpTx-1-71 treated ones. The clip test was performed to assess reaction to acute, mechanical, painful stimulation. Treated animals displayed a prolonged tolerance to the nociceptive stimulation compared to the control group (student t-test; **p= 0.0059). The rotarod test did not report any effect on balance and locomotion, excluding off-target effects.P1789PC00

[0039] - 5 - Figure 7: Panel of behavioural tests performed to assess pain-associated behaviours in rAAV2:PreProBDNF-MVIIA injected animals in comparison to the control rAAV2:PreProBDNF-mEGFP cohort. (A) PreProBDNF-MVIIA injected animals reported an increased tolerance to punctate, mechanical stimulation in the Von Frey test (unpaired two-tailed t-test, p=0.0036) compared to the mock group. (B) PreProBDNF-MVIIA injected animals reported an increased tolerance to noxious heat (55°C, unpaired two-tailed t-test, p=0.0092) compared to the mock group. (C) No difference in locomotion and balance behaviour has been detected in the rotarod test between PreProBDNF-MVIIA injected animals and mock ones (Two-way Anova with Sidak's multiple comparison test, p>0.999). (D) Von Frey test reveals effect carried by MVIIA on mechanical sensitivity also in the CFA pain model compared to the mEGFP mock control (Mann-Whitney test, p=0.0023). n =6.

[0040] DEFINITIONS

[0041] As used herein, the term "polynucleotide" refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and / or a chimeric polynucleotide sequence (e.g., a combination of the above).

[0042] According to some embodiments, the present invention is directed to the delivery and expression of a modulating peptide. In one embodiment, the modulating peptide is encoded by a polynucleotide. In one embodiment, the modulating peptide is encoded by a chimeric polynucleotide of the invention. In one embodiment, the modulating peptide is an endogenous peptide. In one embodiment, the modulating peptide is an exogenous peptide. In one embodiment, the modulating peptide is a synthetic peptide.

[0043] "Modulating activity" is an activity attributed to a modulating peptide. In one embodiment, a modulating peptide is an inhibitory peptide that reduces electrical excitability. In one embodiment, a modulating peptide is a stimulatory peptide. In some embodiments, a modulating peptide induces cell death. In one embodiment, a modulating peptide induces cell apoptosis. In one embodiment, a modulating peptide induces cell survival. In one embodiment, a modulating peptide enhances a signal transduction pathway in a target cell. In one embodiment, a modulating peptide blocks an intracellular cascade. In one embodiment, a modulating peptide blocks cell to cell communication. In one embodiment, a modulating peptide is a hormone. In one embodiment, a modulating peptide is a secreted peptide. In one embodiment, a modulating peptide is an antigen. In one embodiment a modulating peptide is an antibody or a fragment thereof. In one embodiment, a modulating peptide is a toxin. In one embodiment, a modulating peptide inhibits protein-protein interaction. In one embodiment, a modulating peptide inhibits protein translocation from the cytosol into the nucleus, inhibits translocation from the nucleus to the cytosol, or both.

[0044] The terms "precursor" and "pro-peptide" are used herein interchangeably.P1789PC00

[0045] - 6 - Sequence identity comparisons may be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percent identity between two or more sequences.

[0046] Percent identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0047] Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the amino acid or nucleotide sequence may cause the following residues or codons to be put out of alignment, thus potentially resulting in a large reduction in percent identity when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall identity score. This is achieved by inserting "gaps" in the sequence alignment to try to maximise local identity.

[0048] However, these more complex methods assign "gap penalties" to each gap that occurs in the alignment so that, for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. "Affine gap costs" are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will of course produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0049] Calculation of maximum percent identity therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al. (1984) Nucleic Acids Research 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al. (1999) ibid - Ch. 18), FASTA (Atschul et al. (1990) J. Mol. Biol. 403-410), EMBOSS Needle (Madeira, F., et al., 2019. Nucleic acids research, 47(W1), pp.W636-W641) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al. (1999) ibid, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. Another tool, BLAST 2 Sequences, is also available for comparingP1789PC00

[0050] -7-protein and nucleotide sequences (FEMS Microbiol. Lett. (1999) 174(2):247-50; FEMS Microbiol. Lett. (1999) 177(l):187-8).

[0051] Although the final percent identity can be measured, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix (the default matrix for the BLAST suite of programs). GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table if supplied (see the user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.

[0052] Once the software has produced an optimal alignment, it is possible to calculate percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. The percent sequence identity may be calculated as the number of identical residues as a percentage of the total residues in the SEQ ID NO referred to.

[0053] "Fragments" typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. "Fragment" thus refers to an amino acid or nucleic acid sequence that is a portion of a full-length polypeptide or polynucleotide.

[0054] DETAILED DESCRIPTION

[0055] The present invention provides a chimeric polypeptide comprising: (i) a first polypeptide portion which comprises a large dense core vesicle (LDV) targeting element; and (ii) a second polypeptide portion which comprises an ion channel targeting peptide.

[0056] A particular object of the present invention provides a nucleotide sequence of a precursor named PreProBDNF-active, wherein said sequence comprises at least two auxiliary sequences isolated from BDNF (Pre- and Pro-) and a sequence codifying for the mature form of an active peptide, or protein. Said active is a peptide, or a protein, of 2- 500 amino acids, or 2 - 250 amino acids, preferably of 2- 50 amino acids.

[0057] Chimeric polypeptide

[0058] As used herein, a "chimeric polypeptide" may refer to a protein created through the joining of two or more peptides or polypeptides which provide separate functional elements, wherein the two or more polypeptides are not found co-joined in a natural polypeptide.

[0059] The LDV targeting element and the ion channel blocker polypeptide portions of the present chimeric polypeptide thus do not co-exist in a naturally occurring polypeptide. For example, the LDV targeting element and the ion channel blocker polypeptide of the present chimeric polypeptide are not a naturally occurring prepropeptide.P1789PC00

[0060] -8 - Ion channel targeting peptide

[0061] An "ion channel targeting peptide" may be referred to as an "ion channel modulating peptide". Suitably, the ion channel targeting peptide interacts directly with (e.g. binds to) an ion channel to modulate, such as decrease or increase, the flux of ions through the ion channel.

[0062] The ion channel targeting peptide may be an antagonist or an agonist of the ion channel. An antagonist may be referred to as an "ion channel blocker". An ion channel blocker may inhibit the flux of ions through an ion channel by binding to and blocking the ion pathway or binding to a voltage sensor on the ion channel, inducing the ion channel into a closed conformation. As used herein "inhibit" is synonymous with "reduce". The ion channel agonist may bind to an ion channel, such as a ligand-gated ion channel, or a voltage sensor on an ion channel to induce the ion channel into an open conformation.

[0063] An ion channel blocker may reduce the flux of ions through the ion channel by, for example, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% compared to the ion flux achieved in corresponding, control conditions and the absence of the ion channel blocker.

[0064] An ion channel agonist may increase the flux of ions through the ion channel by, for example, at least 1.2-fold, at least 1.5-fold, at least 2-fold, at least 5-fold, at least 10-fold, at least 15-fold, at least 20-fold, at least 25-fold, at least 50-fold, or at least 100-fold compared to the ion flux achieved in corresponding, control conditions and the absence of the ion channel agonist.

[0065] Assays for determining the activity of an ion channel targeting peptide are known in the art. Such assays include, but are not limited to, the use membrane permeable markers to detect changes in membrane potential and patch clamp assays. Illustrative assays include, but are not limited to, FLIPR (Fluorescence imaging plate reader) assays and the QPatch system. Illustrative markers include, but are not limited to, FluxOR and DiBAC4.

[0066] Suitably, the ion channel targeting peptide may comprise 10-50, 10-45, 10-40, 10-35, 15-50, 15-45, 15-40, 15-35, 20-50, 20-45, 20-40, or 20-35 amino acids.

[0067] The ion channel targeting peptide may comprise a toxin peptide or a venom peptide. Suitably, the ion channel targeting peptide may comprise a conotoxin, a scorpion venom peptide, a pufferfish peptide (e.g. Tetrodotoxin), an anemones peptide, a snake venom peptide or a spider venom peptide.

[0068] Conotoxins include, but are not limited to, a-conotoxins, 5-conotoxins, k-conotoxins, and p-conotoxins.

[0069] Sea anemones peptides may be referred to as kunitz-type inhibitors and include, for example, dendrotoxin and Mambaquaretin-1.

[0070] Suitably, the ion channel may be a voltage gated calcium channel, a voltage gated sodiumP1789PC00

[0071] -9-channel, a TRP channel, a voltage activated potassium channel, an acid-sensing ion channel, a Piezo channel, and a ligand-gated ion channel.

[0072] Voltage gated calcium channels include, but are not limited to, L-type channels, such as Cavl.l, Cavl.2, Cavl.3, and Cavl.4; P-type channels, such as Cav2.1; N-type channels, such as Cav2.2; R-type channels such as Cav2.3; and T-type channels, such as Cav3.1, Cav3.2, Cav3.3. Suitably, the voltage gated calcium channel may be Cav2.2.

[0073] Voltage gated sodium channels include, but are not limited to, Navl.l, Navi.2, Navl.3, Navl.4, Navi.5, Navi.6, Navi.7, Navl.8, Navl.9, and Nax. Suitably, the voltage gated sodium channel may be Navi.7.

[0074] Voltage gated potassium channels include, but are not limited to, Delayed rectifiers (such as Kval.l - Kval.8, Kvl.5, Kva2.1, Kva3.1) and A-type potassium channels (such as Kval.4, Kva4.1, Kva4.2, Kva4.3).

[0075] ASIC channels include ASIC1, ASIC2, ASIC3, ASIC4, and ASIC5.

[0076] TRP channels include, but are not limited to, TRPA family (e.g. TRPA1); TRPC family (e.g. TRPC1, TRPC2, TRPC3, TRPC4, TRPC5, TRPC6, TRPC7); TRPML family (e.g. TRPML1, TRPML2, TRPML3); TRPP family (e.g. PKDl-like, PKD2-like); and TRPV family (e.g. TRPV1, TRPV2, TRPV3, TRPV4, TRPV5, TRPV6).

[0077] Piezo channels include PIEZO1 and PIEZO2.

[0078] Ligand gated ion channels include, but are not limited to, Gaba A, Glycine (GlyR), AMPA (GluA), Kainate (GluK), NMDA (GluN), Orphan (gluD), Serotonin (5-HT), Nicotinic acetylcholine (nAChR), and Zinc-activated ion channel.

[0079] Examples of ion channel targeting peptides and the ion channels they regulate are provided in Table 1.

[0080] Table 1

[0081]

[0082] P1789PC00

[0083] - 10 -

[0084]

[0085] Suitably, the ion channel targeting peptide may be an ion channel targeting peptide from Table 1.

[0086] Suitably, the ion channel targeting peptide may comprise or consist of a polypeptide shown as one of SEQ ID NO: 31, 32 or 39-47 or a variant or fragment thereof. As will be apparent, the variant or fragment should maintain the ability to modulate the target ion channel.

[0087] Suitably, the variant may comprise at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to one of SEQ ID NO: 31, 32 or 39-47. Suitably, the variant may comprise one, two or three amino acid alterations compared to one of SEQ ID NO: 31, 32 or 39-47. Suitably, the amino acid alterations may be conservative amino acid alterations.

[0088] Suitably, the ion channel targeting peptide may comprise or consist of a polypeptide shown as one of SEQ ID NO: 31 or a variant or fragment thereof as defined herein.

[0089] Suitably, the ion channel targeting peptide may comprise or consist of a polypeptide shown as one of SEQ ID NO: 32 or a variant or fragment thereof as defined herein.

[0090] Illustrative ion channels involved in pain and examples of ion channel targeting peptides against them are summarised in Table 2.

[0091] Table 2

[0092]

[0093] Illustrative ion channels involved in epilepsy and examples of ion channel targeting peptides against them are summarised in Table 3.P1789PC00

[0094] - 11 - Table 3

[0095]

[0096] An ion channel targeting peptide described herein does not bind to a G-protein coupled receptor (GPCR). As such, an ion channel targeting peptide is not an opioid peptide. Suitably, the ion channel targeting peptide is not a dynorphin peptide.

[0097] Large dense core vesicle (LDV) targeting element

[0098] The LDV targeting element directs the present chimeric polypeptide into LDVs, where the chimeric polypeptide undergoes maturation and removal of the LDV targeting element. The ion channel targeting peptide is subsequently released from the LDV, and therefore the neuron, only upon a series of action potentials that exceed a certain excitation threshold, which occur - for example - during epilepsy episodes or bouts of pain.

[0099] Large dense core vesicle (LDCVs) are lipid vesicles in neurons and secretory cells which may be filled with neurotransmitters, such as catecholamines or neuropeptides. LDVCs release their content through SNARE-mediated exocytosis similar to synaptic vesicles.

[0100] At a cellular level, delivery - for example through viral vectors - provides the target cells (e.g. neuronal cells) with the genetic information for a chimeric polypeptide of the invention (for example a PreProBDNF-active). The active, for example the ion channel targeting peptide, is then produced by the target cells and localized in dense core vesicles where the active is stored and ready for release upon sustained neuronal stimulation. In an embodiment, said active peptide, once secreted from the cells, triggers a retrograde signalling effect on the same cells that released it, thereby modulating their activity and influencing further signalling events.

[0101] As such, the ion channel targeting peptide is released from the target cell "on demand"; for example, after high frequency stimulation and / or upon a frequency of action potentials thatP1789PC00

[0102] - 12 -exceed a certain threshold. The threshold may be, for example, at least 6 Hz, at least 7 Hz, at least 8 Hz, at least 9 Hz, at least 10 Hz, at least 15 Hz, at least 20 Hz or at least 30 Hz. Suitably, the release-on-demand is triggered by increased neuronal firing frequency. Said increased neuronal firing frequency may be measured by EEG (electroencephalography) as spike trains. Suitably, "increased" may refer to a frequency of spikes in a train measured by EEG in said subject that is at least 6 Hz, at least 7 Hz, at least 8 Hz, at least 9 Hz, at least 10 Hz, at least 15 Hz, at least 20 Hz or at least 30 Hz.

[0103] Suitable assays to determine that a peptide is an element capable of directing a polypeptide in which it is comprised to an LDV are known in the art. Suitable assays include, for example, immunofluorescence and confocal microscopy or subcellular fractionation. In these assays, localisation to LDVs may be confirmed by determining co-localisation with proteins known to localise to LDVs, for example neuropeptide Y, chromogranin B, Brain-Derived Neurotrophic Factor (BDNF), enkephalin, tachykinin, somatostatin, vasoactive intestinal peptide (VIP), cholecystokinin, nociceptin or dynorphin. For assays such as immunofluorescence and confocal microscopy, the present chimeric polypeptide may further comprise a suitable marker peptide, for example a fluorescent polypeptide such as GFP. Illustrative methods to confirm LDV localisation are described in present Examples 1 and 3.

[0104] The LDV targeting element is preferably located at the N-terminus of the ion channel targeting peptide in order to mediate localisation to the LDV.

[0105] Suitably, the LDV targeting element may comprise a signal peptide which is capable of directing the chimeric polypeptide to the endoplasmic reticulum, in particular the luminal side of the endoplasmic reticulum. Localisation to the endoplasmic reticulum in target cells precedes localisation to the LDV. Suitable signal peptides are known in the art. The core of the signal peptide may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix. The signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation. At the end of the signal peptide there is typically a stretch of amino acids that is recognized and cleaved by signal peptidase. Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein. The free signal peptides are then digested by specific proteases. The signal peptide is preferably at the amino terminus of the chimeric polypeptide. The signal peptide may comprise SEQ ID NO: 6-19 or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions or additions) provided that the signal peptide still functions to cause endoplasmic reticulum localisation. Said sequences are listed in Table 4.

[0106] Table 4

[0107]

[0108] P1789PC00

[0109] - 13 -

[0110]

[0111] Suitably, the signal peptide comprises or consists of SEQ ID NO: 6 or a variant thereof having 5, 4, 3, 2 or 1 amino acid mutations (insertions, substitutions or additions) provided that the signal peptide still functions to cause endoplasmic reticulum localisation. SEQ ID NO: 6 is an illustrative human BDNF signal peptide.

[0112] Suitably, the LDV targeting element comprises a sorting motif which is capable of targeting the present chimeric polypeptide to a LDV.

[0113] The sorting motif may comprise the elements DL (SEQ ID NO: 20) and EXyL, suitably wherein the sorting motif comprises the amino acid sequence DLXxEXyL, where x is an integer from 1 to 20, y is an integer from 1 to 10, and each instance of X may independently be any amino acid.

[0114] Examples of polypeptides which comprise an LDV targeting element which may be utilised in the present invention are known in the art. For example, the LDV targeting element may comprise or consist of the "prepro" targeting elements from a preproneuropeptide. Examples of suitable preproneuropeptides include, but are not limited to, Brain-Derived Neurotrophic Factor (BDNF), Enkephalin, Tachykinin, Somatostatin, Vasoactive intestinal peptide (VIP), Cholecystokinin, Nociceptin, dynorphin and Neuropeptide Y.

[0115] The LDV targeting element may comprise or consist of an LDV targeting element of Brain-Derived Neurotrophic Factor (BDNF), Enkephalin, Tachykinin, Somatostatin, Vasoactive intestinal peptide (VIP), Cholecystokinin, Nociceptin, dynorphin or Neuropeptide Y; or variant or a fragment thereof. As will be apparent, the variant or fragment should be capable of targeting the chimeric polypeptide to LDVs when the chimeric polypeptide is expressed in a cell, for example a neuronal cell.

[0116] Illustrative LDV targeting elements are listed in Table 5.

[0117] Table 5

[0118]

[0119] P1789PC00

[0120] - 14 -

[0121]

[0122] Suitably, the LDV targeting element may comprise or consist of a polypeptide shown as one of SEQ ID NO: 21-30 or a variant or fragment thereof. As will be apparent, the variant or fragment should maintain the ability to target the chimeric polypeptide to LDVs.

[0123] Suitably, the variant may comprise at least 80%, at least 85%, at least 90%, at least 95%, or at least 98% sequence identity to one of SEQ ID NO: 21-30.

[0124] Suitably, the LDV targeting element may comprise or consist of a polypeptide shown as one of SEQ ID NO: 21 or a variant or fragment thereof as defined herein.

[0125] Suitably, the LDV targeting element may comprise or consist of a polypeptide shown as one of SEQ ID NO: 22 or a variant or fragment thereof as defined herein.

[0126] Suitably, the present chimeric polypeptide may comprise the LDV targeting element from human BDNF and the MVIIA ion channel targeting peptide. An illustrative amino acid sequence for such a chimeric polypeptide is shown as SEQ ID NO: 33.

[0127] SEQ ID NO: 33 MTILFLTMVISYFGCMKAAPMKEANIRGQGGLAYPGVRTHGTLESVNGPKAGSRGLTSLADT FEHVIEELLDEDQKVRPNEENNKDADLYTSRVMLSSQVPLEPPLLFLLEEYKNYLDAANMSM RVRRCKGKGAKCSRLMYDCCTGSCRSGKCP1789PC00

[0128] -15 - Suitably, the present chimeric polypeptide may comprise a polypeptide shown as SEQ ID NO: 33 or a variant which has at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 33. As detailed herein, the variant is capable of localising to LDVs and inhibiting Cav2.2 ion channels following release from the target cell.

[0129] Suitably, the present chimeric polypeptide may comprise the LDV targeting element from human BDNF and the GpTx-1-71 ion channel targeting peptide. An illustrative amino acid sequence for such a chimeric polypeptide is shown as SEQ ID NO: 34.

[0130] SEQ ID NO: 34 MTILFLTMVISYFGCMKAAPMKEANIRGQGGLAYPGVRTHGTLESVNGPKAGSRGLTSLADT FEHVIEELLDEDQKVRPNEENNKDADLYTSRVMLSSQVPLEPPLLFLLEEYKNYLDAANMSM RVRRDCLGFMRKCIPDNDKCCRPNLVCSRTHKWCKYVF

[0131] Suitably, the present chimeric polypeptide may comprise a polypeptide shown as SEQ ID NO: 34 or a variant which has at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% sequence identity to SEQ ID NO: 34. As detailed herein, the variant is capable of localising to LDVs and inhibiting Navi.7 ion channels following release from the target cell.

[0132] Polynucleotide

[0133] The present invention provides a polynucleotide encoding a chimeric polypeptide according to the invention.

[0134] As used herein, the terms "polynucleotide" and "nucleic acid" are intended to be synonymous with each other. The nucleic acid sequence(s) may be RNA or DNA sequences.

[0135] The nucleic acid sequence(s) may be single-stranded or may be double-stranded. The nucleic acid sequence(s) may be, for example, genomic, recombinant, mRNA or cDNA. The nucleic acid sequence(s) may comprise synthetic nucleotides and / or modified nucleotides. These synthetic nucleotides and / or modified nucleotides may enhance in vivo activity and / or stability.

[0136] Due to the redundancy of the genetic code, variations in nucleic acid sequences are possible that encode for the same polypeptide. These variations in nucleic acid sequences are encompassed by the present invention. Therefore, multiple nucleic acid sequence(s) are envisaged, each of which may be different, but which still encode a chimeric polypeptide according to the present invention. It is known in the art how to design and produce such nucleic acid sequences.

[0137] In some embodiments, the nucleic acid sequence(s) may be codon optimised for production in the host, or target, cell of choice. In some embodiments, the nucleic acid sequence(s) may be operably linked to further sequence(s) such as control sequence(s), e.g. promoter sequence(s), enhancer sequence(s), polyadenylation signal sequence(s) and / or other regulatory sequence(s), which control transcription and / or translation. The nucleic acid sequence(s) may be in the form of one or more expression cassettes. The nucleic acid sequences may be suitable for expression inP1789PC00

[0138] - 16 -prokaryotic cells or in eukaryotic cells, such as mammalian cells. Any promoter may be used, such as a strong promoter that is functional in prokaryotic cells or in eukaryotic cells. Suitable promoters will be known in the art. The promoter may be a constitutive promoter. The promoter may be a tissue specific promoter.

[0139] Suitably, the nucleic acid sequence encoding the LDV targeting element may comprise (i) SEQ ID NO: 1 or a variant with at least 70% identity thereto; and (ii) SEQ ID NO: 2 or a variant with at least 70% identity thereto. Suitably, the variant of SEQ ID NO: 1 may have at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity thereto. Suitably, the variant of SEQ ID NO: 2 may have at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity thereto. Suitably, the nucleic acid sequence encoding the LDV targeting element may comprise SEQ ID NO: 35 or a variant with at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity thereto.

[0140] SEQ ID NO: 35 atgaccatccttttccttactatggttatttcatacttcggttgcatgaaggcggcgcccatgaaagaagtaaacgtccacggacaaggcaact tggcctacccaggtgtgcggacccatgggactctggagagcgtgaatgggcccagggcaggttcgagaggtctgacgacgacatcactg gctgacacttttgagcacgtcatcgaagagctgctggatgaggaccagaaggttcggcccaacgaagaaaaccataaggacgcggactt gtacacttcccgggtgatgctcagcagtcaagtgcctttggagcctcctctactctttctgctggaggaatacaaaaattacctggatgccgca aacatgtctatgagggttcggcgc

[0141] Suitably, the nucleic acid sequence encoding the LDV targeting element may comprise SEQ ID NO: 36 or a variant with at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity thereto.

[0142] SEQ ID NO: 36 (from human BDNF - Uniprot P23560-1) atgaccattctgtttctgaccatggtgattagctattttggctgcatgaaagcggcgccgatgaaagaagcgaacattcgcggccagggcg gcctggcgtatccgggcgtgcgcacccatggcaccctggaaagcgtgaacggcccgaaagcgggcagccgcggcctgaccagcctggc ggatacctttgaacatgtgattgaagaactgctggatgaagatcagaaagtgcgcccgaacgaagaaaacaacaaagatgcggatctgt ataccagccgcgtgatgctgagcagccaggtgccgctggaaccgccgctgctgtttctgctggaagaatataaaaactatctggatgcggc gaacatgagcatgcgcgtgcgccgc

[0143] In an embodiment, said sequence comprises:

[0144] Pre-BDNF, the signal sequence driving the first steps of the immature peptide processing, towards the endoplasmic reticulum, (SEQ ID NO: 1):

[0145] ATGACCATCCTTTTCCTTACTATGGTTATTTCATACTTCGGTTGCATGAAGGCG

[0146] Pro-BDNF, the auxiliary pro-peptide, driving peptide processing in the Golgi and driving dense core vesicles localization, (SEQ ID NO: 2):

[0147] GCGCCCATGAAAGAAGTAAACGTCCACGGACAAGGCAACTTGGCCTACCCAGGTG TGCGGACCCATGGGACTCTGGAGAGCGTGAATGGGCCCAGGGCAGGTTCGAGAGG TCTGACGACGACATCACTGGCTGACACTTTTGAGCACGTCATCGAAGAGCTGCTGG ATGAGGACCAGAAGGTTCGGCCCAACGAAGAAAACCATAAGGACGCGGACTTGTAP1789PC00

[0148] - 17- CACTTCCCGGGTGATGCTCAGCAGTCAAGTGCCTTTGGAGCCTCCTCTACTCTTTCTG CTGGAGGAATACAAAAATTACCTGGATGCCGCAAACATGTCTATGAGGGTTCGGCG C

[0149] The nucleotide sequence codifying for the active.

[0150] In one embodiment, the GKR, glycine-lysine-arginine cleavage site (SEQ ID NO: 4): GGCAAACGA is comprised in said precursor, too.

[0151] The auxiliary sequences (Pre- and Pro-) isolated from BDNF are substituting the endogenous Pre-Pro-sequences from the active. These drive the peptide processing and localization as a neuropeptide.

[0152] In one embodiment, said precursor is PreProBDNF-MVIIA, wherein said sequence codifying for the active peptide is MVIIA, the sequence coding the mature MVIIA peptide (SEQ ID NO: 3): TGCAAAGGGAAAGGTGCTAAATGTTCAAGGCTGATGTACGACTGTTGTACTGGTTCTTG TAGGAGTGGCAAATGC

[0153] Suitably, the sequence coding the MVIIA peptide may comprise SEQ ID NO: 3 or a variant with at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity thereto.

[0154] oo-conotoxin MVIIA, a venom peptide from fish-hunting snails, is a highly selective and reversible blocker of Cav2.2, the voltage-gated calcium channels that regulate release of neurotransmitters. From MVIIA's full sequence, only the segment encoding for the mature peptide was kept, allowing a functional and active MVIIA to be released upon proper stimulation and following the regulated secretory pathway.

[0155] The unique combination of these elements (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 and, optionally, SEQ ID NO: 4) constitutes PreProBDNF-MVIIA sequence. Each of it secures a key step in MVIIA processing as a neuropeptide.

[0156] Suitably, the polynucleotide encoding the chimeric polypeptide may comprise SEQ ID NO: 37 or a variant with at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity thereto. SEQ ID NO: 37 (Human BDNF LDV targeting element + MVIIA) Atgaccattctgtttctgaccatggtgattagctattttggctgcatgaaagcggcgccgatgaaagaagcgaacattcgcggccagggcg gcctggcgtatccgggcgtgcgcacccatggcaccctggaaagcgtgaacggcccgaaagcgggcagccgcggcctgaccagcctggc ggatacctttgaacatgtgattgaagaactgctggatgaagatcagaaagtgcgcccgaacgaagaaaacaacaaagatgcggatctgt ataccagccgcgtgatgctgagcagccaggtgccgctggaaccgccgctgctgtttctgctggaagaatataaaaactatctggatgcggc gaacatgagcatgcgcgtgcgccgctgcaaagggaaaggtgctaaatgttcaaggctgatgtacgactgttgtactggttcttgtaggagt ggcaaatgc

[0157] In one embodiment, the MVIIA DNA sequence, engineered to include the PreProBDNF sequence, has been packaged into an adeno-associated viral (AAV) vector.

[0158] At a cellular level, delivery through viral vectors provides the target cells with the geneticP1789PC00

[0159] - 18 -information for PreProBDNF-MVIIA. This is then produced by the target cells and localized in dense core vesicles where MVIIA is stored and ready for release upon sustained neuronal stimulation. When this occurs, a correct post-translational modification and the secretion of only a functional and mature MVIIA peptide is observed. Once released, MVIIA is retroactive and targets Cav2.2 expressed on the membranes of the cells that secreted it. This causes modulation of the pool of signals released by that neuron, including MVIIA itself. This mechanism of action describes PreProBDNF-MVIIA as a controlled, targeted and self-inhibiting biopharmaceutical delivered through a gene therapy approach directed to sustained neuronal activity modulation. This effect is schematically depicted in Figure 1.

[0160] In vitro experiments demonstrated that PreProBDNF sequence managed to drive MVIIA localization into dense-core vesicles (DCVs), the selected storage station for neuropeptides, as well as being released upon sustained stimulation and inhibit its own release.

[0161] This capability has been confirmed in in vivo experiments.

[0162] In an alternative embodiment, said precursor is PreProBDNF- GpTx-1-71, wherein said sequence codifying for the active is GpTx-1-71, the sequence coding the mature GpTx-1-71 peptide (SEQ ID NO: 5):

[0163] GACTGCCTGGGCGCCTTCAGGAAGTGCATCCCCGACAACGACAAGTGCTGCAGGCCCA ACCTGGTGTGCAGCAGGCTGCACAGGTGGTGCAAGTACGTGTTC

[0164] Suitably, the sequence coding the GpTx-1-71 peptide may comprise SEQ ID NO: 5 or a variant with at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity thereto. GpTx-1-71 originates from a 34-residue peptide isolated from the venom of the tarantula Grammostola Porteri and its potent activity against the voltage-gated sodium channel Navi.7 is known. Navi.7 is a channel that contributes to tuning neuronal excitability, and it is implicated in pain signalling. Suitably, the polynucleotide encoding the chimeric polypeptide may comprise SEQ ID NO: 38 or a variant with at least 70%, at least 80%, at least 90%, at least 95% or at least 99% identity thereto. SEQ ID NO: 38 (Human BDNF LDV targeting element + GpTx-1-71) atgaccattctgtttctgaccatggtgattagctattttggctgcatgaaagcggcgccgatgaaagaagcgaacattcgcggccagggcg gcctggcgtatccgggcgtgcgcacccatggcaccctggaaagcgtgaacggcccgaaagcgggcagccgcggcctgaccagcctggc ggatacctttgaacatgtgattgaagaactgctggatgaagatcagaaagtgcgcccgaacgaagaaaacaacaaagatgcggatctgt ataccagccgcgtgatgctgagcagccaggtgccgctggaaccgccgctgctgtttctgctggaagaatataaaaactatctggatgcggc gaacatgagcatgcgcgtgcgccgcgactgcctgggcgccttcaggaagtgcatccccgacaacgacaagtgctgcaggcccaacctgg tgtgcagcaggctgcacaggtggtgcaagtacgtgttc

[0165] In vitro experiments demonstrated that PreProBDNF sequence managed to drive GpTx-1-71 localization into DCVs: the peptide localization was confirmed in cells expressing both MVIIA and NPY, used as marker for DCVs, and showing colocalization of the two.

[0166] VectorP1789PC00

[0167] - 19 - The present invention further provides a vector comprising a polynucleotide of the invention. The vector may be used to introduce nucleic acid sequence(s) into a cell so that the cell expresses and / or produces the chimeric polypeptide according to the invention.

[0168] As used herein, the term "vector" may be considered interchangeable with the term "expression vector" and "expression construct". The vector may be any vector that is suitable for introducing and / or expressing a nucleic acid sequence in a cell. The vector may comprise regulatory sequences, enhancer sequences and / or promoter sequences that promote expression of a nucleic acid sequence in a cell.

[0169] In some embodiments, the vector may be an expression cassette or construct.

[0170] The vector according to the invention may be any agent capable of delivering polynucleotide according to the invention to a cell and / or expressing polynucleotide according to the invention in a cell. Examples of suitable vectors include but are not limited to plasmids, cosmids, phages, viruses or artificial chromosomes.

[0171] In some embodiments, the vector may be a plasmid or a viral vector. In some embodiments, the viral vector is any of a parvoviral vector, an adenoviral vector, a herpes simplex viral vector, an anelloviral vector, a retroviral vector, or a lentiviral vector. In some embodiments, the vector may be an adeno-associated virus (AAV), a retroviral vector or a lentiviral vector.

[0172] The vector may be capable of transfecting or transducing a cell. Suitably, the vector is capable of transducing a neuronal cell. Suitably, the neuronal cell is a DRG neuron.

[0173] Preferably, the vector is an adeno-associated virus (AAV) vector.

[0174] The vector of the present invention may be in the form of a viral vector particle. In some embodiments, the viral vector is any of a parvoviral vector particle, an adenoviral vector particle, a herpes simplex viral vector particle, an anelloviral vector particle, a retroviral vector particle, or a lentiviral vector particle. Preferably, the vector is an AAV vector particle.

[0175] Adeno-associated virus (AAV)

[0176] AAV genomes

[0177] The AAV vector or AAV vector particle may comprise an AAV genome or a fragment or derivative thereof. An AAV genome is a polynucleotide sequence, which may encode functions needed for production of an AAV particle. These functions include those operating in the replication and packaging cycle of AAV in a host cell, including encapsidation of the AAV genome into an AAV particle. Naturally occurring AAVs are replication-deficient and rely on the provision of helper functions in trans for completion of a replication and packaging cycle. Accordingly, the AAV genome of the AAV vector of the invention is typically replicationdeficient.P1789PC00

[0178] -20 - The AAV genome may be in single-stranded form, either positive or negative-sense, or alternatively in double-stranded form. The use of a double-stranded form allows bypass of the DNA replication step in the target cell and so can accelerate transgene expression.

[0179] AAVs occurring in nature may be classified according to various biological systems. The AAV genome may be from any naturally derived serotype, isolate or clade of AAV.

[0180] AAV may be referred to in terms of their serotype. A serotype corresponds to a variant subspecies of AAV which, owing to its profile of expression of capsid surface antigens, has a distinctive reactivity which can be used to distinguish it from other variant subspecies. Typically, an AAV vector particle having a particular AAV serotype does not efficiently cross-react with neutralising antibodies specific for any other AAV serotype. AAV serotypes include AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11. In some embodiments, the AAV vector of the present invention is an AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 serotype, or a variant thereof. In some embodiments, the AAV vector of the present invention is an AAV2 serotype, or a variant thereof.

[0181] The AAV genome may also comprise packaging genes, such as rep and / or cap genes which encode packaging functions for an AAV particle. The rep gene encodes one or more of the proteins Rep78, Rep68, Rep52 and Rep40 or variants thereof. The cap gene encodes one or more capsid proteins such as VP1, VP2 and VP3 or variants thereof. These proteins make up the capsid of an AAV particle, which determines the AAV serotype.

[0182] The AAV genome may be the full genome of a naturally occurring AAV. For example, a vector comprising a full AAV genome may be used to prepare an AAV vector or vector particle.

[0183] Preferably, the AAV genome is derivatised for the purpose of administration to patients. Such derivatisahon is standard in the art and the invention encompasses the use of any known derivative of an AAV genome, and derivatives which could be generated by applying techniques known in the art. The AAV genome may be a derivative of any naturally occurring AAV. Suitably, the AAV genome is a derivative of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11. Suitably, the AAV genome is a derivative of AAV2.

[0184] Derivatives of an AAV genome include any truncated or modified forms of an AAV genome which allow for expression of a transgene from an AAV vector of the invention in vivo. Typically, it is possible to truncate the AAV genome significantly to include minimal viral sequence yet retain the above function. This is preferred for safety reasons to reduce the risk of recombination of the vector with wild-type virus, and also to avoid triggering a cellular immune response by the presence of viral gene proteins in the target cell.

[0185] Typically, a derivative will include at least one inverted terminal repeat sequence (ITR), preferably more than one ITR, such as two ITRs or more. One or more of the ITRs may be derived from AAV genomes having different serotypes or may be a chimeric or mutant ITR. A preferred mutant ITR is one having a deletion of a trs (terminal resolution site). This deletion allows forP1789PC00

[0186] -21 -continued replication of the genome to generate a single-stranded genome which contains both coding and complementary sequences, i.e. a self-complementary AAV genome. This allows for bypass of DNA replication in the target cell and so enables accelerated transgene expression. The AAV genome may comprise one or more ITR sequences from any naturally derived serotype, isolate or clade of AAV or a variant thereof. The AAV genome may comprise at least one, such as two, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, or AAV11 ITRs, or variants thereof.

[0187] In some embodiments, the AAV genome comprises a 5TTR having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% identity to SEQ ID NO: 48. In some embodiments, the AAV genome comprises a 5TTR comprising or consisting of SEQ ID NO: 48 or 55. ctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtgagcgagcgagc gcgcagagagggagtggccaactccatcactaggggttcct

[0188] Example AAV 5TTR (SEQ ID NO: 48) cctgcaggcagctgcgcgctcgctcgctcactgaggccgcccgggcaaagcccgggcgtcgggcgacctttggtcgcccggcctcagtg agcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct

[0189] Example AAV 5TTR (SEQ ID NO: 55)

[0190] In some embodiments, the AAV genome comprises a 3TTR having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity, or 100% identity to SEQ ID NO: 49. In some embodiments, the AAV genome comprises a 3TTR comprising or consisting of SEQ ID NO: 49 or 56. attaactacaaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgccc gacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcag

[0191] Example AAV 3TTR (SEQ ID NO: 49) aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccgggcgaccaaaggtcgcccgacgcccgg gctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcagg

[0192] Example AAV 3TTR (SEQ ID NO: 56)

[0193] The inclusion of one or more ITRs is preferred to aid concatamer formation of the AAV vector in the nucleus of a host cell, for example following the conversion of single-stranded vector DNA into double-stranded DNA by the action of host cell DNA polymerases. The formation of such episomal concatamers protects the AAV vector during the life of the host cell, thereby allowing for prolonged expression of the transgene in vivo.

[0194] Suitably, the AAV genome may comprise one or more ITR sequences flanking the nucleotide sequence encoding the chimeric polypeptide of the invention.

[0195] Suitably, ITR elements will be the only sequences retained from the native AAV genome in theP1789PC00

[0196] - 22 -derivative. A derivative will preferably not include the rep and / or cap genes of the native genome and any other sequences of the native genome. This is preferred for the reasons described above, and also to reduce the possibility of integration of the vector into the host cell genome. Additionally, reducing the size of the AAV genome allows for increased flexibility in incorporating other sequence elements (such as regulatory elements) within the vector in addition to the transgene.

[0197] The following portions could therefore be removed in a derivative of the invention: one inverted terminal repeat (ITR) sequence, the replication (rep) and capsid (cap) genes. However, derivatives may additionally include one or more rep and / or cap genes or other viral sequences of an AAV genome. Naturally occurring AAV integrates with a high frequency at a specific site on human chromosome 19, and shows a negligible frequency of random integration, such that retention of an integrative capacity in the AAV vector may be tolerated in a therapeutic setting.

[0198] The invention additionally encompasses the provision of sequences of an AAV genome in a different order and configuration to that of a native AAV genome. The invention also encompasses the replacement of one or more AAV sequences or genes with sequences from another virus or with chimeric genes composed of sequences from more than one virus. Such chimeric genes may be composed of sequences from two or more related viral proteins of different viral species.

[0199] AAV capsid proteins

[0200] The AAV vector particle may be encapsidated by capsid proteins. The serotype may facilitate the transduction of neuronal cells (e.g. DRG neuronal cells), for example specific transduction of neuronal cells, such as DRG neuronal cells. The AAV vector particle may be a neuron-specific vector particle. The AAV vector particle may be encapsidated by a neuron-specific capsid. The AAV vector particle may comprise a neuron -specific capsid protein.

[0201] Suitably, the AAV vector particles may be transcapsidated forms wherein an AAV genome or derivative having an ITR of one serotype is packaged in the capsid of a different serotype. The AAV vector particle may also include mosaic forms wherein a mixture of unmodified capsid proteins from two or more different serotypes makes up the viral capsid. The AAV vector particle may also include chemically modified forms bearing ligands adsorbed to the capsid surface. For example, such ligands may include antibodies or ligands for targeting a particular cell surface receptor. Suitably, the receptor may be a receptor expressed on neuron cells. Suitably, the ligand may be nerve growth factor (NGF).

[0202] Where a derivative comprises capsid proteins i.e. VP1, VP2 and / or VP3, the derivative may be a chimeric, shuffled or capsid-modified derivative of one or more naturally occurring AAVs. In particular, the invention encompasses the provision of capsid protein sequences from different serotypes, clades, clones, or isolates of AAV within the same vector (i.e. a pseudotyped vector). The AAV vector may be in the form of a pseudotyped AAV vector particle.P1789PC00

[0203] -23 - Chimeric, shuffled or capsid-modified derivatives will be typically selected to provide one or more desired functionalities for the AAV vector. Thus, these derivatives may display increased efficiency of gene delivery, decreased immunogenicity (humoral or cellular), an altered tropism range and / or improved targeting of neuronal cells compared to an AAV vector comprising a naturally occurring AAV genome. Increased efficiency of gene delivery may be effected by improved receptor or co-receptor binding at the cell surface, improved internalisation, improved trafficking within the cell and into the nucleus, improved uncoating of the viral particle and improved conversion of a single-stranded genome to double-stranded form. Increased efficiency may also relate to an altered tropism range or targeting of neuronal cells, such that the vector dose is not diluted by administration to tissues where it is not needed.

[0204] The capsid protein may be an artificial capsid protein. The term "artificial capsid" as used herein means that the capsid particle comprises an amino acid sequence which does not occur in nature or which comprises an amino acid sequence which has been engineered (e.g. modified) from a naturally occurring capsid amino acid sequence. In other words, the artificial capsid protein comprises a mutation or a variation in the amino acid sequence compared to the sequence of the parent capsid from which it is derived where the artificial capsid amino acid sequence and the parent capsid amino acid sequences are aligned.

[0205] Suitably, the AAV vector particle of the present invention is an AAV1, AAV2, AAV5, AAV8, AAV9 or AAVrhlO vector particle, or a variant thereof. Suitably, the AAV vector particle may be an engineered AAV, such as AAV-PHP.EB, AAV-PHP.S, AAV.CAP-B10, AAV.CAP-B22, or AAV2-Retro. AAV vector particles with these serotypes can transduce neuron cells.

[0206] The AAV vector particle of the present invention may comprise AAV1, AAV2, AAV5, AAV8, AAV9 or AAVrhlO capsid proteins, or variants thereof. Suitably, the AAV vector particle may comprise AAV1, AAV2, AAV5, AAV8, AAV9 or AAVrhlO capsid proteins VP1, VP2 and VP3, or variants thereof.

[0207] Modified viral particles for gene therapy, which may be used in the present invention, are described in WO 2020 / 225363 and WO 2022 / 101363.

[0208] Regulatory elements

[0209] The vector of the present invention may comprise one or more regulatory elements which may act pre- or post-transcriptionally. Suitably, the nucleotide sequence encoding the chimeric polypeptide is operably linked to one or more regulatory elements which may act pre- or post-transcriptionally.

[0210] As used herein, a "regulatory element" may refer to any nucleotide sequence that facilitates expression of a polypeptide, e.g. acts to increase expression of a transcript or to enhance mRNA stability. Suitable regulatory elements include for example promoters, enhancer elements, post-transcriptional regulatory elements, introns, polyadenylation sites, and Kozak sequences.P1789PC00

[0211] -24 - Promoters

[0212] The vector of the present invention may comprise a promoter. Suitably, the promoter may be operably linked to the nucleotide sequence encoding the chimeric polypeptide. The term "operably linked" may mean that the components described are in a relationship permitting them to function in their intended manner.

[0213] A "promoter" may refer to a region of DNA that leads to initiation of transcription of a gene. Promoters are typically located near the transcription start sites of genes, upstream on the DNA (towards the 5' region of the sense strand). Any suitable promoter may be used, the selection of which may be readily made by the skilled person.

[0214] A promoter typically comprises a "core" and a "proximal" region. The "core promoter region" may comprise promoter elements such as a transcription start site, RNA polymerase binding sites and general transcription factor binding sites (e.g. TATA box, B recognition element). The "proximal promoter region" may comprise primary regulatory elements and specific transcription factor binding sites which are required, for example, to facilitate effective and controllable transcription. The size and components of both the core and proximal promoter regions typically vary in a gene specific manner.

[0215] In some embodiments, the promoter is a constitutive promoter. As used herein, a "constitutive promoter" is a promoter which is always active. Exemplary constitutive promoters include a chicken beta-actin (CBA) promoter, or a variant or fragment thereof. The promoter may comprise or consist of a CAG promoter, or a variant or fragment thereof.

[0216] An illustrative polynucleotide sequence of a CBA promoter is provided as SEQ ID NO: 50.

[0217] SEQ ID NO: 50 tcgaggtgagccccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgat gggggcggggggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtg cggcggcagccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgc gcggcgggcg

[0218] In some embodiments, the promoter is a neuron-specific promoter. As used herein, an "neuronspecific promoter" is a promoter which preferentially facilitates expression of a gene in neuron cells (e.g. DRG neurons). DRG neuron specific promoters include, but are not limited to, Navi.7, Navi.9, TRPV1, Mrgprd, ScnlOa (Navl.8), and Advillin promoters.

[0219] Enhancers

[0220] The vector of the present invention may comprise an enhancer. Suitably, the nucleotide sequence encoding the chimeric polypeptide is operably linked to an enhancer. The enhancer may facilitate expression of the TNF inhibitor neuron cells (e.g. DRG neurons).

[0221] An "enhancer" or "enhancer element" may refer to a region of DNA that can be bound byP1789PC00

[0222] -25 -proteins (activators) to increase the likelihood that transcription of a particular gene will occur. Enhancers are cis-acting. They can be located up to 1 Mbp (1,000,000 bp) away from the gene, upstream or downstream from the start site.

[0223] The vector of the present invention may comprise a neuron-specific enhancer.

[0224] Polyadenylation sequences

[0225] The vector of the present invention may comprise a polyadenylation sequence. Suitably, the nucleotide sequence encoding the chimeric polypeptide is operably linked to a polyadenylation sequence. A polyadenylation sequence may be inserted after the nucleotide sequence to improve transgene expression.

[0226] A polyadenylation sequence typically comprises a polyadenylation signal, a polyadenylation site and a downstream element: the polyadenylation signal comprises the sequence motif recognised by the RNA cleavage complex; the polyadenylation site is the site of cleavage at which a poly-A tails is added to the mRNA; the downstream element is a GT-rich region which usually lies just downstream of the polyadenylation site, which is important for efficient processing.

[0227] Suitable polyadenylation sequences will be known to those of skill in the art (see e.g. Schambach, A., et al., 2007. Molecular Therapy, 15(6), pp.1167-1173; and Choi, J.H. et al., 2014. Molecular brain, 7(1), pp.1-10). Example polyadenylation sequences include the bovine growth hormone (bGH) polyadenylation sequence, the SV40 polyadenylation sequence, and the rabbit beta-globin polyadenylation sequence.

[0228] In some embodiments, the polyadenylation sequence comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 51 or a fragment thereof. Suitably, the polyadenylation sequence comprises or consists of a nucleotide sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 51 or a fragment thereof.

[0229] In some embodiments, the polyadenylation sequence comprises or consists of the nucleotide sequence SEQ ID NO: 51 or a fragment thereof. taagatacattgatgagtttggacaaaccacaactagaatgcagtgaaaaaaatgctttatttgtgaaatttgtgatgctattgctttatttgta accattataagctgcaataaacaagtt

[0230] Example SV40 polyadenylation sequence (SEQ ID NO: 51)

[0231] Post-transcriptional regulatory elements

[0232] The vector of the present invention may comprise a post-transcriptional regulatory element. Suitably, the nucleotide sequence encoding the chimeric polypeptide is operably linked to a post-transcriptional regulatory elements.

[0233] The vector of the present invention may comprise a woodchuck hepatitis post-transcriptional regulatory element (WPRE).P1789PC00

[0234] -26 - Suitable WPRE sequences will be known to those of skill in the art (see e.g. Zufferey, R., et al., 1999. Journal of virology, 73(4), pp.2886-2892; and Zanta-Boussif, M.A. et al., 2009. Gene therapy, 16(5), pp.605-619). Suitably, the WPRE is a wild-type WPRE or is a mutant WPRE. For example, the WPRE may be mutated to abrogate translation of the woodchuck hepatitis virus X protein (WHX) e.g. by mutating the WHX ORF translation start codon.

[0235] In some embodiments, the WPRE comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 52 or a fragment thereof. Suitably, the WPRE comprises or consists of a nucleotide sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 52 or a fragment thereof.

[0236] In some embodiments, the WPRE comprises or consists of the nucleotide sequence SEQ ID NO: 52 or a fragment thereof. gcttatcgataatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggatacgctgct ttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttgtggcccg ttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcctttccggga ctttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttgggcactgaca attccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtccttctgctacgtcc cttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgccctcagacgagtcg gatctccctttgggccgcctccccgc

[0237] Example WPRE sequence (SEQ ID NO: 52)

[0238] Introns

[0239] The vector of the present invention may comprise an intron. An intron may be inserted between the promoter and nucleotide sequence encoding the chimeric polypeptide to increase expression. Suitable introns will be known to those of skill in the art (see e.g. Powell, S.K., et al., 2015. Discovery medicine, 19(102), p.49) and may include an MVM intron, a F.IX truncated intron 1, a chimeric p-globin / immunoglobulin heavy chain intron, a chimeric adenovirus / immunoglobulin intron, and a SV40 intron.

[0240] In some embodiments, the intron is an SV40 intron.

[0241] In some embodiments, the intron comprises or consists of a nucleotide sequence which is at least 70% identical to SEQ ID NO: 80 or a fragment thereof. Suitably, the intron comprises or consists of a nucleotide sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to SEQ ID NO: 53 or a fragment thereof.

[0242] In some embodiments, the intron comprises or consists of the nucleotide sequence SEQ ID NO: 53 or a fragment thereof.P1789PC00

[0243] -27-gtaagtatcaaggttacaagacaggtttaaggagaccaatagaaactgggcttgtcgagacagagaagactcttgcgtttctgataggcac ctattggtcttactgacatccactttgccttctctccacag

[0244] Example SV40 intron (SEQ ID NO: 53)

[0245] Kozak sequences

[0246] The vector of the present invention may comprise a Kozak sequence. Suitably, the nucleotide sequence encoding the chimeric polypeptide is operably linked to a Kozak sequence. A Kozak sequence may be inserted before the start codon to improve the initiation of translation.

[0247] Suitable Kozak sequences will be known to those of skill in the art (see e.g. Kozak, M., 1987. Nucleic acids research, 15(20), pp.8125-8148). A consensus Kozak sequence in vertebrates may have the sequence of SEQ ID NO: 54.

[0248] Suitably, the Kozak sequence may comprise or consist of the nucleotide sequence of SEQ ID NO: 54, or variants thereof which have five or fewer deletions, substitutions or insertions. Suitably, the variants may have four or fewer, three or fewer, two or fewer, or one deletion(s), subshtution(s) or insertion(s). Suitably, the variants may have three or fewer, two or fewer, or one deletion(s) and / or three or fewer, two or fewer, or one substitution(s). Suitably, the variants may have three or fewer, two or fewer, or one deletion(s) and / or three or fewer, two or fewer, or one substitution(s). Suitably, the variants may have one deletion and / or one substitution. Suitably, the variants may have one deletion and one substitution.

[0249] gccaccatg

[0250] Example consensus Kozak sequence (SEQ ID NO: 54)

[0251] Example vectors

[0252] The vector of the present invention may comprise from 5' to 3': a promoter and a nucleotide sequence encoding the chimeric polypeptide of the invention.

[0253] The vector of the present invention may further comprise any other suitable elements, such as any other elements described herein or one or more spacer sequence. The spacer sequence(s) may comprise, for example, at least one (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10), at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten nucleotide bases. A spacer sequence may comprise a restriction site to enable the insertion of one or more further elements.

[0254] In preferred embodiments, the vector of the present invention is an AAV vector. In some embodiments, the AAV genome comprises from 5' to 3': a 5TTR; a promoter; a nucleotide sequence encoding a chimeric polypeptide of the invention; and a 3TTR. In some embodiments, the AAV genome comprises from 5' to 3': a 5TTR; a promoter; a nucleotide sequence encoding a chimeric polypeptide of the invention; a polyadenylation sequence; and a 3TTR. In some embodiments, the AAV genome comprises from 5' to 3': a 5TTR; a promoter; a nucleotideP1789PC00

[0255] - 28 -sequence encoding a chimeric polypeptide of the invention; a WPRE; a polyadenylation sequence and a 3'ITR.

[0256] Cells

[0257] In one aspect, the present invention provides a cell comprising the vector (e.g. viral vector) of the present invention. The cell may be an isolated cell. Suitably, the cell is a mammalian cell, for example a human cell. The cell may be an isolated human cell.

[0258] Suitably, the cell may be a producer cell. The term "producer cell" includes a cell that produces viral particles, after transient transfection, stable transfection or vector transduction of all the elements necessary to produce the viral particles or any cell engineered to stably comprise the elements necessary to produce the viral particles. In some embodiments, the producer cell is an AAV producer cell. Suitable producer cells will be known to those of skill in the art (see e.g. Martin, J., et al. 2013. Human gene therapy methods, 24(4), pp.253-269) and may include HEK293, COS-1, COS- 7, CV-1, HeLa, CHO, and A549 cell lines. In some embodiments, the producer cell is a HEK293 cell, or a derivative thereof (e.g. a HEK293T cell).

[0259] Suitably, the cell may be a packaging cell. The term "packaging cell" includes a cell which contains some or all of the elements necessary for packaging a recombinant virus genome. Typically, such packaging cells contain one or more vectors which are capable of expressing viral structural proteins (e.g. AAV rep and cap genes) and / or one or more genes encoding the viral structural proteins have been integrated into the genome of the packaging cell. Cells comprising only some of the elements required for the production of enveloped viral particles are useful as intermediate reagents in the generation of viral particle producer cell lines, through subsequent steps of transient transfection, transduction or stable integration of each additional required element. These intermediate reagents are encompassed by the term "packaging cell". In some embodiments, the packaging cell is an AAV packaging cell. Suitable packaging cells will be known to those of skill in the art (see e.g. Martin, J., et al. 2013. Human gene therapy methods, 24(4), pp.253-269).

[0260] As described herein, a vector (e.g. viral vector) is preferentially used to deliver a polynucleotide encoding the chimeric polypeptide of the invention to a target cell. Suitably, the chimeric polypeptide is then expressed in the target cell.

[0261] Suitably, the target cell is a neuronal cell. A neuron is an excitable cell that fires electric signals (action potentials) across a neural network in the nervous system. Neurons communicate with other cells via synapses, which are specialized connections that commonly use minute amounts of chemical neurotransmitters to pass the electric signal from the presynaptic neuron to the target cell through the synaptic gap.

[0262] Suitably, the neuronal cell is a dorsal root ganglion (DRG) neuron. DRG neurons may be identified by expression of one or more receptors such as: nociceptors such as Ntrkl (TrkA),P1789PC00

[0263] -29 - Trpm8, Trpvl; Mechanoreceptors, such as Ntrk2 (TrkB) and Ntrk3 (TrkC); proprioceptors such as Ntrk3 (TrkC) and vGlutl (Slcl7a7); Pruciceptors; Sst (Somatostatin), IL-31RA and Mrgpra3, non-peptiderfic nociceptors, Mrgprd and P2X3R, C-LTMRS, vGlut3 and Tyrosine hydroxylase. Further DRG neuron markers may include the ion channels described herein, for example. These receptors may be identified in combination with neuronal markers such as NeuN, TUJ1, neurofilament and / or Thy-1 (CD90).

[0264] Pharmaceutical composition

[0265] In one aspect, the present invention provides a pharmaceutical composition comprising the polynucleotide, polypeptide, or vector of the present invention. In preferred embodiments, the pharmaceutical composition comprises the vector of the present invention in the form of a viral vector particle.

[0266] A pharmaceutical composition is a composition that comprises or consists of a therapeutically effective amount of a pharmaceutically active agent e.g. the vector. A pharmaceutical composition preferably includes a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).

[0267] By "pharmaceutically acceptable" it is included that the formulation is sterile and pyrogen free. The carrier, diluent, and / or excipient must be "acceptable" in the sense of being compatible with the vector and not deleterious to the recipients thereof. Typically, the carriers, diluents, and excipients will be saline or infusion media which will be sterile and pyrogen free, however, other acceptable carriers, diluents, and excipients may be used.

[0268] Acceptable carriers, diluents, and excipients for therapeutic use are well known in the pharmaceutical art. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as (or in addition to) the carrier, excipient or diluent any suitable binder (s), lubricant(s), suspending agent(s), coating agent(s) or solubilising agent(s). Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatine, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.

[0269] The polynucleotide, polypeptide, vector or pharmaceutical composition according to the present invention may be administered in a manner appropriate for treating and / or preventing the diseases described herein. Suitable administration routes will be known to the skilled person. The quantity and frequency of administration may be determined by the skilled person, for example depending by such factors as the condition of the subject, and the type and severity of the subject's disease. The pharmaceutical composition may be formulated accordingly.P1789PC00

[0270] -30 - Suitably, the pharmaceutical composition may be administered by subcutaneous, intradermal, intravenous, intrathecal, intra-nerve, or intraarticular administration.

[0271] The pharmaceutical compositions may comprise polynucleotide, polypeptide, or vector of the invention in infusion media, for example sterile isotonic solution. The pharmaceutical composition may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0272] The polynucleotide, polypeptide, vector or pharmaceutical composition may be administered in a single or in multiple doses. Suitably, the vector or pharmaceutical composition may be administered in a single, one-off dose. The pharmaceutical composition may be formulated accordingly.

[0273] The polynucleotide, polypeptide, vector or pharmaceutical composition may be administered at varying doses (e.g. measured in viral genomes (vg) per ml). The physician in any event may determine the actual dosage which will be most suitable for any individual subject, and the dosage may, for example, vary with the age, weight and response of the particular subject. Suitably, the vector of the present invention is administered at a dose of at least about 1010vg / mL, at least about 1011vg / mL, at least about 1012vg / mL, or at least about 5xl012vg / mL. Suitably, the vector of the present invention is administered at a dose of about 1013vg / mL or less, about 1012vg / mL or less, or about 1011vg / mL or less. Suitably, the vector of the present invention is administered in a dose of from about 1010to about 1013vg / mL, or from about 1011to about 1013vg / mL. Suitably, the vector of the present invention is administered in a dose of from about 1010to about 1012vg / mL. Suitably, the vector of the present invention is administered in a dose of from about 1011to about 1013vg / mL. Suitably, the vector of the present invention is administered in a dose of from about 1012to about 1013vg / mL. Suitably, the vector of the present invention is administered in a dose of from about 1012to about 5xl012vg / mL. The pharmaceutical composition may be formulated accordingly.

[0274] Suitably, the polynucleotide, polypeptide, or vector of the present invention may be administered in combination with one or more other therapeutic agents. The one or more other therapeutic agent may be administered separately, simultaneously or sequentially. The pharmaceutical composition may further comprise one or more other therapeutic agents.

[0275] The invention further includes kits comprising the vector, cell and / or pharmaceutical composition of the present invention. Preferably said kits are for use in the methods and used as described herein, e.g., the therapeutic methods as described herein. Preferably said kits comprise instructions for use of the kit components.

[0276] Methods for treating and / or preventing disease

[0277] In one aspect, the present invention provides the polynucleotide, polypeptide, vector and / or pharmaceutical composition according to the present invention for use as a medicament.P1789PC00

[0278] -31 - In one aspect, the present invention provides use of the polynucleotide, polypeptide, vector or pharmaceutical composition according to the present invention in the manufacture of a medicament.

[0279] In one aspect, the present invention provides a method of administering a therapeutically effective amount of the polynucleotide, polypeptide, vector or pharmaceutical composition according to the present invention to a subject in need thereof.

[0280] The polynucleotide, polypeptide, vector or pharmaceutical composition may be administered to any subject in need thereof. The subject may be a mammal (e.g. a human). The vector, cell or pharmaceutical composition according to the present invention may be administered to a subject with or at risk of a condition as described herein.

[0281] The disease may be a neurological disorder associated with, or characterised by, high frequency neuronal firing. Suitably, high frequency neuronal firing may refer to a frequency of spikes in a train measured by EEG in a subject that is at least 6 Hz, at least 7 Hz, at least 8 Hz, at least 9 Hz, at least 10 Hz, at least 15 Hz, at least 20 Hz or at least 30 Hz .

[0282] The disease may be selected from epilepsy; chronic pain, such as an example neuropathic pain or fibromyalgia; neurodegenerative diseases; neuroinflammation, such as multiple sclerosis; mood and psychiatric disorders; bipolar disorder; schizophrenia; stroke; cerebral palsy; Chronic Traumatic Encephalopathy (GTE), and Autism Spectrum Disorders (ASD).

[0283] The disease may be epilepsy.

[0284] The disease may be chronic pain, such as an example neuropathic pain or fibromyalgia.

[0285] In embodiments where the disease is chronic pain, the chimeric polypeptide may comprise an ion channel targeting peptide which modulates an ion channel as described in Table 2. Suitably, the ion channel targeting peptide may be MVIIA. Suitable MVIIA polypeptides are described herein. Suitably, the ion channel targeting peptide may be GPTX1. Suitable GPTX1 polypeptides are described herein.

[0286] In embodiments where the disease is epilepsy, the chimeric polypeptide may comprise an ion channel targeting peptide which modulates an ion channel as described in Table 3.

[0287] In an embodiment, the precursor according to the present invention is for use in the treatment of epilepsy, chronic pain, as an example neuropathic pain or fibromyalgia, neurodegenerative diseases, neuroinflammation, as an example multiple sclerosis, mood and psychiatric disorders, as an example, bipolar disorder, schizophrenia, stroke, cerebral palsy, Chronic Traumatic Encephalopathy GTE, Autism Spectrum Disorders ASD.

[0288] ASPECTS OF THE INVENTION

[0289] Aspects of the invention are described in the following numbered paragraphs (paras):P1789PC00

[0290] - 32 - 1. A nucleotide sequence of a precursor named PreProBDN F-acti ve, wherein said sequence comprises:

[0291] - Pre-BDNF, SEQ ID NO: 1;

[0292] - Pro-BDNF, SEQ ID NO: 2;

[0293] A sequence coding for the mature form of an active peptide or protein.

[0294] 2. The nucleotide sequence according to para 1, wherein said active peptide, once released by the cells, acts retroactively on the cells that releases it.

[0295] 3. The nucleotide sequence according to para 1 or 2, further comprising a Glycine-lysine- arginine cleavage site, SEQ ID NO: 4.

[0296] 4. The nucleotide sequence according to one of the paras 1-3, wherein said sequence encoding for the active encodes for an active peptide or protein of 2 - 500 amino acids, or 2 - 250 amino acids, preferably of 2 - 50 amino acids.

[0297] 5. The nucleotide sequence according to any one of the paras 1-4, wherein said sequence coding for the mature form of an active peptide is the sequence coding the mature MVIIA peptide (SEQ ID NO: 3).

[0298] 6. The nucleotide sequence according to any one of the paras 1-4, wherein said sequence coding for the mature form of an active peptide is the sequence coding the mature GpTx- 1-71 peptide (SEQ ID NO: 5).

[0299] 7. A delivery vector comprising the nucleotide sequence according to any one of the paras 1-6.

[0300] 8. A delivery vector according to para 7, wherein said delivery vector comprises in addition a recombinant adeno-associated virus (AAV) vector genome.

[0301] 9. A recombinant virus particle or a liposome or nanoparticle, comprising a delivery vector according to para 7 or 8.

[0302] 10. A delivery vector or recombinant virus particle or liposome or nanoparticle according to any of paras 7-9, for use in the treatment of epilepsy, chronic pain, as an example neuropathic pain or fibromyalgia, neurodegenerative diseases, neuroinflammation, as an example multiple sclerosis, mood and psychiatric disorders, as an example, bipolar disorder, schizophrenia, stroke, cerebral palsy, Chronic Traumatic Encephalopathy GTE, Autism Spectrum Disorders ASD.P1789PC00

[0303] - 33 - EXPERIMENTAL SECTION

[0304] Example 1: PreProBDNF-MVIIA, ex vivo test

[0305] Data in Figure 2 shows cells expressing PreProBDNF-MVIIA and their reaction to a stimulus which forces exocytosis, causing cells to release their neurotransmitters (including M VI I A, when present). This resulted in a reduced effect when MVIIA was present, due to the inhibitory feedback loop established by MVIIA as soon as it is released.

[0306] The peptide localization was confirmed in cells expressing both MVIIA and NPY, a marker for DCVs, and showing colocalization of the two peptides.

[0307] In Figure 3A, neurons experiencing a sustained stimulation (30 Hz delivered through an electric field) showed a similar result: cells expressing MVIIA are less reactive than the control ones. This, combined with the results obtained testing cells' reaction to lower frequency stimuli (Figure 3B) is indicative that the mechanism of action of the peptide according to the present invention is triggered only when sustained stimulation occurs.

[0308] Example 2: PreProBDNF-MVIIA, in vivo test

[0309] Gene delivery was achieved through AAV viral vectors (rAAV) packaging PreProBDNF-MVIIA to assess its mechanism of action in vivo, investigating any effect on nociception in mouse models.

[0310] Figure 4 reports a panel of behavioral tests performed in control (wild-type, WT) and PreProBDNF-MVIIA injected mice (n=6) which received a single subcutaneous injection for a total of 5xl010VG / mouse.

[0311] When exposed to painful stimuli of different origin, MVIIA injected mice reacted significantly less to acute mechanical stimulation in the clip test (Figure 4A), punctate mechanical stimulation (Von Frey test, Figure 4C) and noxious heat (hot plate test, Figure 4D). The increased tolerance during the Von Frey and the hot plate test was confirmed also in the CFA (Complete Freund's Adjuvant) inflammatory pain model.

[0312] The rotarod test (Figure 4B) did not report any effect on locomotion or balance, excluding off-target effects. Figure 4E shows the reaction of control and PreProBDNF-MVIIA injected animals to formalin injection, a widely used model for evaluating the effects of analgesics compounds in laboratory animals. The injection of formalin is known for triggering a biphasic response, which can be referred to as acute (early) and chronic (late) phases. The early phase of the formalin responses is caused by direct stimulation of sensory neurons, whereas the late phase involves spinal cord sensitization and inflammation.

[0313] Notably, while WT and injected animals seem to react with no relevant differences in the early phase, the two groups diverged more and more in the late phase, reaching a peak at 35 minutes after the injection, with MVIIA animals poorly reacting and WT ones showing an increase in pain-related behavior.P1789PC00

[0314] - 34 - The relevant difference observed at a late stage after exposure to formalin are indicative of the fact that MVIIA is modulating the signal reaching the spinal cord, therefore playing a role in central sensitization.

[0315] Notably, PreProBDNF-MVIIA exerts an effect on nociception mediated by direct activation of sensory neurons, to some extent, but a more relevant role is played during nociception associated with inflammation and spinal involvements, i.e., in the late phase of formalin response. This evidence indicates that the toxin is released upon noxious stimulation of different origin; the fact that the strongest inhibition was detected when testing mechanical and thermal noxious stimuli is consistent with literature reporting that the Cav2.2 channel contributes to heat and mechanical sensitivity.

[0316] At the end of the behavioral experiments, mEGFP, used as reporter for MVIIA, was observed in the spinal cord areas where neuros transmitting pain project, thus confirming the correct targeting (data not shown).

[0317] Example 3: PreProBDNF-GpTx-1-71, ex vivo test

[0318] Gene delivery was achieved through AAV viral vectors (rAAV) packaging PreProBDNF- driving GpTx-1-71 to assess its mechanism of action ex vivo on neuronal primary cultures.

[0319] Transduced cells revealed that the PreProBDNF sequence was driving GpTx-1-71 into vesicles as well (data not shown). Moreover, when transduced cells received a chemical depolarizing stimulus (potassium chloride, KC1), exocytosis was detected through the mEGFP reporter, demonstrating the functionality of the vectors and of the here proposed engineering strategy (Figure 5A). GpTx-1-71 secretion and ability to alter neuronal excitability was assessed via patchclamp experiments, where neurons experienced gradually increasing electrical stimuli before and after exposure to GpTx-1-71. These experiments demonstrated that, in the presence of GpTx-1-71, neurons were markedly less responsive to gradually increasing electrical stimulation, as action potentials were occurring at significantly lower frequency (Figure 5B). These results are indicative of the fact that PreProBDNF-GpTx-1 is being secreted and functionally active on the same cells that released it, altering neurons excitability due to its action on Navi.7. Altogether, this evidence suggests that the engineering of GpTx-1-71 sequence was successful and this peptide impacts neuronal activity upon secretion.

[0320] Example 4: PreProBDNF-GpTx-1-71, in vivo test

[0321] Six adult mice were injected intrathecally, in the sciatic nerve and subcutaneously in the hindpaw with rAAVs:PreProBDNF-GpTx-l-71 for a total of 8.75 x 1010VG / mouse. First behavioural experiments reported that GpTx-l-71-injected mice reacted significantly less to acute mechanical stimulation in the clip test) andre 6A), and did not show any alteration in balance and locomotion, thereby excluding off-target effects (Figure 6B).

[0322] The data demonstrates GpTx-1-71 is useful for disorders caused by sustained neuronal activityP1789PC00

[0323] -35 -due to its selective inhibitory action.

[0324] Example 5: PreProBDNF-MVIIA, in vivo test

[0325] The in vivo experiment with PreProBDNF-MVIIA of Example 2 has been repeated using, in parallel, PreProBDNF-MVIIA and a mock control, PreProBDNF-mEGFP (i.e. without the active MVIIA peptide).

[0326] A single rAAV subcutaneous administration into the plantar surface of the paw has been done. Nociception was then assessed using the Example 2 behavioural test panel in physiological conditions and in the inflammatory pain model induced by CFA injection.

[0327] In physiological condition, the MVIIA cohort showed delayed nociception to punctate mechanical stimuli in the Von Frey test (Figure 7A) and to noxious heat (Figure 7B, 55°C). No effects on motor coordination and balance were detected in the rotarod test (Figure 7C) suggesting that the observed effects were not due to motor alterations. Delayed responses to mechanical stimulation upon PreProBDNF-MVIIIA gene delivery were observed also in the CFA pain model.

[0328] No effects were observed in the PreProBDNF-mEGFP treated animals, confirming that the observed effects are due to the expression and release of MVIIA, and not merely by AAV administration or the presence of an inert construct. Overall, these results support the functional activity of MVIIA when delivered through the PreProBDNF- precursor and further demonstrate that a single subcutaneous administration of the recombinant AAV vector is sufficient to reduce nociceptive behaviour.

[0329] Moreover, the here presented experiment supports the versatility of the proposed technology, wherein the construct PreProBDNF-mEGFP can be relevant for studies in DCVs dynamics and related processes.

Claims

1. P1789PC00-36 - CLAIMS1. A chimeric polypeptide comprising:(i) a first polypeptide portion which comprises a large dense core vesicle (LDV) targeting element; and(ii) a second polypeptide portion which comprises an ion channel targeting peptide.

2. The chimeric polypeptide according to claim 1 wherein the ion channel targeting peptide is an ion channel blocker or an ion channel agonist.

3. The chimeric polypeptide according to claim 1 or 2 wherein the ion channel is selected from a voltage gated calcium channel, a voltage gated sodium channel, a TRP channel, a voltage activated potassium channel, an acid-sensing ion channel and a Piezo channel.

4. The chimeric polypeptide according to any preceding claim wherein the ion channel targeting peptide is an ion channel blocker which is capable of inhibiting a voltage gated calcium or sodium ion channel.

5. The chimeric polypeptide according to any preceding claim wherein the ion channel targeting peptide is a toxin peptide.

6. The chimeric polypeptide according to any of claims 2 to 5 wherein the ion channel blocker is selected from MVIIA, GpTx-1-71, GPTX1, protoxin-1, MrVIB, MfVIA, APHC1 and Psalmotoxin-1.

7. The chimeric polypeptide according to claim 6 wherein the ion channel blocker is MVIIA.

8. The chimeric polypeptide according to claim 7 wherein the MVIIA comprises SEQ ID NO: 31 or a variant comprising at least 80% identity thereto.

9. The chimeric polypeptide according to claim 6 wherein the ion channel blocker is GpTx-1-71.

10. The chimeric polypeptide according to claim 7 wherein the GpTx-1-71 comprises SEQ ID NO: 32 or a variant comprising at least 80% identity thereto.

11. The chimeric polypeptide according to any preceding claim wherein the LDV targeting element is a neuropeptide LDV targeting element.

12. The chimeric polypeptide according to any preceding claim wherein the LDV targeting element comprises (i) a signal peptide and (ii) a sorting motif which is capable of targeting the chimeric polypeptide to an LDV.P1789PC00-37 - 13. The chimeric polypeptide according to claim 12 wherein the signal peptide comprises an amino acid sequence selected from SEQ ID NO: 6 to 19 or a variant thereof comprising one, two or three amino acid alterations.

14. The chimeric polypeptide according to claim 12 or 13 wherein the sorting motif comprises the elements DL and EXyL, suitably wherein the sorting motif comprises the amino acid sequence DLXxEXyL (SEQ ID NO: 20), where x is an integer from 1 to 20, y is an integer from 1 to 10, and each instance of X may independently be any amino acid.

15. The chimeric polypeptide according to any preceding claim wherein the LDV targeting element comprises or consists of an LDV targeting element of Brain-Derived Neurotrophic Factor (BDNF), Enkephalin, Tachykinin, Somatostatin, Vasoactive intestinal peptide (VIP), Cholecystokinin, Nociceptin, dynorphin or Neuropeptide Y; or a fragment thereof.

16. The chimeric polypeptide according to any preceding claim wherein the LDV targeting element comprises or consists of an amino acid sequence selected from SEQ ID NO: 21 to 30 or a variant comprising at least 80% sequence identity thereto; or a fragment thereof; preferably wherein the LDV targeting element comprises or consists of an amino acid sequence selected from SEQ ID NO: 21 or 22 or a variant comprising at least 80% sequence identity thereto; or a fragment thereof17. The chimeric polypeptide according to any preceding claim wherein the chimeric polypeptide comprises SEQ ID NO: 33 or a variant comprising at least 70% identity thereto.

18. The chimeric polypeptide according to any of claims 1 to 16 wherein the chimeric polypeptide comprises SEQ ID NO: 34 or a variant comprising at least 70% identity thereto.

19. A polynucleotide encoding the chimeric polypeptide according to any preceding claim.

20. A polynucleotide comprising: a 5' ITR; a nucleotide sequence encoding a chimeric polypeptide according to any preceding claim; and a 3' ITR.

21. The polynucleotide according to claim 19 or 20, wherein the nucleotide sequence encoding the LDV targeting element comprises or consists of a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 35 or 36; or a fragment thereof.

22. The polynucleotide according to any of claims 19 to 21, wherein the nucleotide sequence encoding the ion channel targeting peptide comprises or consists of a nucleotide sequence having at least 70% sequence identity to SEQ ID NO: 37 or 38; or a fragment thereof.

23. The polynucleotide according to any of claims 19 to 22, wherein the nucleotide sequence encoding the chimeric polypeptide is operably linked to one or more regulatory elements.P1789PC00-38 - 24. The polynucleotide according to claim 23, wherein the one or more regulatory elements comprise one or more of a promoter, polyadenylation sequence, and optionally a WPRE.

25. The polynucleotide according to any of claims 19-24, wherein said nucleotide sequence comprises:- Pre-BDNF, SEQ ID NO: 1;- Pro-BDNF, SEQ ID NO: 2;A sequence coding for the mature form of an active peptide or protein.

26. The polynucleotide according to claim 25, wherein said sequence coding for the mature form of an active peptide is the sequence coding the mature MVIIA peptide (SEQ ID NO: 3).

27. The polynucleotide according to claim 25, wherein said sequence coding for the mature form of an active peptide is the sequence coding the mature GpTx-1-71 peptide (SEQ ID NO: 5).

28. A vector comprising the polynucleotide according to any of claims 19 to 27.

29. A recombinant adeno-associated virus (rAAV) vector comprising the polynucleotide according to any of claims 19 to 27.

30. A recombinant adeno-associated virus (rAAV) vector particle comprising the polynucleotide according to any of claims 19 to 27.

31. The rAAV vector particle according to claim 30, wherein the AAV vector particle is encapsidated by AAV9, AAV2 or AAV8 capsid proteins.

32. A pharmaceutical composition comprising the chimeric polypeptide according to any of claims 1 to 18, the polynucleotide according to any of claims 19 to 27, the vector according to claim 28, the rAAV vector according to claim 29, the rAAV vector particle according to claim 30 or 31, and a pharmaceutically acceptable carrier, diluent and / or excipient.

33. A chimeric polypeptide according to any of claims 1 to 18, the polynucleotide according to any of claims 19 to 27, the vector according to claim 28, the rAAV vector according to claim 29, the rAAV vector particle according to claim 30 or 31, or a pharmaceutical composition according to claim 32, for use as a medicament.

34. Use of a chimeric polypeptide according to any of claims 1 to 18, a polynucleotide according to any of claims 19 to 27, a vector according to claim 28, a rAAV vector according to claim 29, a rAAV vector particle according to claim 30 or 31, or a pharmaceutical composition according to claim 32, for the manufacture of a medicament.

35. A method of treating or preventing a disease in a subject, the method comprising administering chimeric polypeptide according to any of claims 1 to 18, a polynucleotideP1789PC00-39 -according to any of claims 19 to 27, a vector according to claim 28, a rAAV vector according to claim 29, a rAAV vector particle according to claim 30 or 31, or a pharmaceutical composition according to claim 32, to a subject in need thereof.

36. A chimeric polypeptide according to any of claims 1 to 18, a polynucleotide according to any of claims 19 to 27, a vector according to claim 28, a rAAV vector according to claim 29, a rAAV vector particle according to claim 30 or 31, or a pharmaceutical composition according to claim 32, for use in treating or preventing a disease selected from epilepsy; chronic pain, such as an example neuropathic pain or fibromyalgia; neurodegenerahve diseases; neuroinflammation, such as multiple sclerosis; mood and psychiatric disorders; bipolar disorder; schizophrenia; stroke; cerebral palsy; Chronic Traumatic Encephalopathy (CTE), and Autism Spectrum Disorders (ASD).

37. Use of chimeric polypeptide according to any of claims 1 to 18, a polynucleotide according to any of claims 19 to 27, a vector according to claim 28, a rAAV vector according to claim 29, a rAAV vector particle according to claim 30 or 31, or a pharmaceutical composition according to claim 32, for the manufacture of a medicament for treating or preventing a disease selected from epilepsy; chronic pain, such as an example neuropathic pain or fibromyalgia; neurodegenerahve diseases; neuroinflammation, such as multiple sclerosis; mood and psychiatric disorders; bipolar disorder; schizophrenia; stroke; cerebral palsy; Chronic Traumatic Encephalopathy (CTE), and Autism Spectrum Disorders (ASD).

38. A method of treating or preventing epilepsy; chronic pain, such as an example neuropathic pain or fibromyalgia; neurodegenerahve diseases; neuroinflammation, such as multiple sclerosis; mood and psychiatric disorders; bipolar disorder; schizophrenia; stroke; cerebral palsy; Chronic Traumatic Encephalopathy (CTE), and Autism Spectrum Disorders (ASD), the method comprising administering chimeric polypeptide according to any of claims 1 to 18, a polynucleotide according to any of claims 19 to 27, a vector according to claim 28, a rAAV vector according to claim 29, a rAAV vector particle according to claim 30 or 31, or a pharmaceutical composition according to claim 32, to a subject in need thereof.

39. The chimeric polypeptide, polynucleotide, vector, rAAV vector, rAAV vector particle or pharmaceutical composition for use, the use or method according to any of claims 33 to 38 wherein the disease is epilepsy.

40. The chimeric polypeptide, polynucleotide, vector, rAAV vector, rAAV vector particle or pharmaceutical composition for use, the use or method according to any of claims 33 to 38 wherein the disease is chronic pain, such as an example neuropathic pain or fibromyalgia.

41. A plasmid comprising the polynucleotide according to any of claims 19 to 27.

42. A kit for production of rAAV vector particles comprising, a plasmid according to claimP1789PC00-40 - 41, and one or more helper plasmids encoding AAV replication and capsid proteins.

43. An isolated cell comprising the chimeric polypeptide according to any of claims 1 to 18, the polynucleotide according to any of claims 19 to 27, the vector according to claim 28, the rAAV vector according to claim 29, the rAAV vector particle according to claim 30 or 31, or the plasmid according to claim 41.

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