Nucleic acid-based botulinum neurotoxine for therapeutic use

Description

[0001] The invention relates to a nucleic acid for therapeutic use that encodes botulinum neurotoxin. The invention also relates to the transfection of skeletal muscle cells, smooth muscle cells, and skin glands, as well as other skin cells, with nucleic acids (RNA or DNA) encoding botulinum neurotoxin (BoNT), with or without a secretory signal, for therapeutic and / or cosmetic purposes. State of the art

[0002] Botulinum neurotoxins (BoNT) are produced by spore-forming bacteria. Clostridium botulinumNeurotoxin is produced in at least seven serotypes and at least 30 subtypes. Expression occurs from a characteristic gene cluster along with a number of complex proteins, several hemagglutinins, and a non-toxin-non-hemagglutinin molecule of similar size to the neurotoxin itself. Neurotoxin serotype A (as an example) has a molecular weight of 150 kDa and consists of two domains: the heavy chain (100 kDa) and the light chain (50 kDa), which remain connected by a disulfide bond after activation. The heavy chain domain contains, at its N-terminus, the translocation domain through which the light chain is transported from the vesicle into the cytosol. At its C-terminus, it contains the binding domain, which binds to presynaptic gangliosides and to the vesicular SV2 protein. After translocation to the cytosol, the light chain develops catalytic activity and specifically cleaves the SNARE protein SNAP-25 downstream of glutamine 197< .As a result, the SNARE complex loses density and can no longer pull the vesicle containing the neurotransmitter firmly enough to the inner membrane of the presynaptic terminal, preventing fusion with the cell membrane and the corresponding release of the stored neurotransmitter into the synaptic cleft. The physiological consequence of this chemical denervation is postsynaptic muscle paralysis. The mechanism of action of botulinum neurotoxins comprises four steps: 1. Binding of the heavy chain and Gt1b and SV2 to the presynaptic terminal or the exposed release vesicle, 2. Uptake into the release vesicle during its retraction into the presynaptic terminal, 3. Translocation of the light chain from the vesicle into the presynaptic cytosol, and 4. Catalytic processing of SNAP-25 by the light chain in the cytosol.

[0003] The complex proteins expressed from the same gene cluster form various complexes with the neurotoxin. These complexes protect the neurotoxin against the low pH in the stomach and proteolytic enzymes until the complex dissociates in the duodenum at alkaline pH. The neurotoxin is then likely reabsorbed paracellularly through the mucosa into the bloodstream via hemagglutins. This mechanism is only relevant in the naturally occurring function of the neurotoxin as a stomach poison. In therapeutic applications, it is injected intramuscularly or intraglandularly, and the toxin's effects are also present without the complex proteins.

[0004] Botulinum neurotoxin has long been approved as a drug (e.g., BOTOX, Xeomin, Dysport, Myobloc).

[0005] Botulinum neurotoxin (BoNT) is particularly well known for its use in aesthetic medicine; less well known is its extraordinarily important role in the treatment of spasticity and dystonia, as well as the reduction of hyperhidrosis or drooling, which is usually caused by reduced swallowing ability (e.g., in Parkinson's disease, amyotrophic lateral sclerosis, infantile cerebral palsy).

[0006] The effect is based on the inhibition of excitation transmission at the presynaptic part of the motor endplate and the associated paralysis of the muscle cell or, in the case of the salivary glands, the autonomous innervation of the gland cells.

[0007] On the other hand, BoNT is one of the most potent toxins, and a dose of less than 1 mg can already lead to a person's death.

[0008] The duration of action varies depending on the dose and the BoNT serotype used. BoNT / A is generally used, but there are also BoNT / B preparations, such as Neurobloc, with a shorter duration of action. BoNT / E products are not yet on the market, but advanced development work is underway.

[0009] In chronic diseases, a particularly long duration of action is desired, as the treatment procedure is unpleasant and painful for the patient. Furthermore, the treatment costs are quite high, since it can only be performed by physicians with specialized experience. Additionally, the risk of the patient developing neutralizing antibodies against BoNT and thus becoming a secondary non-responder increases with the dose and number of treatments, rendering this valuable and often only therapy unavailable.

[0010] To achieve a long duration of action, it is necessary to administer the highest possible dose. However, with the current therapeutic method, the application of BoNT in solution, increasing the local dose also increases the systemic dose. Increasing the systemic dose can lead to serious side effects and is therefore risky. The duration of action of injected BoNT / A protein is generally limited to approximately 3-4 months.

[0011] The production of BoNT drugs takes place either in clostridia or recombinantly in a suitable expression system such as E. coli. Both methods require appropriately controlled environments and involve handling large quantities of toxin. This also poses a societal risk, as the presence of large quantities of toxin always carries the risk of theft and misuse. Furthermore, BoNT is classified as a Category A biological warfare agent by the Centers for Disease Control and Prevention (CDC) in the USA. Complicating matters further is the fact that even the highly regulated, industrial-scale pharmaceutical manufacturing process is subject to biological variations, necessitating activity testing for each batch and / or biological standardization of the preparations.One "unit" corresponds to the amount of botulinum toxin that, after intraperitoneal administration, is lethal to mice and leads to the death of 50% of the treated animals (LD50). This test is therefore used that has been largely phased out in other areas of toxicology. Although cell-based alternative methods have recently been developed, many animal experiments are still currently being conducted for these tests.

[0012] One disadvantage is that BoNT has a very narrow therapeutic range; this is naturally less significant in aesthetic applications such as frown lines on the forehead than in high-dose applications such as spasticity after a stroke or infantile cerebral palsy. Due to its high toxicity, there is always a risk of overdose with systemic or distant side effects. While the duration of action is long, it is still limited. Furthermore, because the drug is injected locally intramuscularly or intraglandularly, the treatment is unpleasant and associated with pain.

[0013] Due to the numerous and varied disadvantages, it would therefore be beneficial to eliminate the need for toxin production and instead have a standardized, manufactured drug that poses no risk to humans or the environment. Furthermore, it would be advantageous to avoid the risk of overdose and significantly reduce systemic side effects. It would also be highly beneficial to have more options for adjusting the duration and strength of the drug's effects.

[0014] US 2002 / 107199 A1 describes DNA that encodes botulinum neurotoxin and is used for the direct transformation of cells and the treatment of various organs and diseases: pain, skeletal muscle, smooth muscle, glands, spasticity, dystonia, various movement disorders and impaired muscle activity (limb spasticity, eyelid tics, tremor, bruxism), hyperhidrosis, frown lines, skin wrinkles, etc. The botulinum toxin can be in any form A, B, C1, D, E, F, or G. For example, skin and muscle cells are transformed with BonNT / A, and inflammatory pain as well as gustatory hyperhidrosis (Frey's syndrome) are treated in human patients.

[0015] WO 2004 / 071538 A2 describes baculovirus constructs containing DNA encoding a botulinum toxin. These constructs are equipped with a promoter and secretion signal, enabling the toxin to be expressed and secreted in the keratinocytes (target cells) to relax the surrounding skeletal muscle tissue. Typically, this construct is applied to the skin via lotion or carrier, where it then exerts its anti-wrinkle effect.

[0016] The object underlying the invention was therefore to provide an alternative drug to the existing BoNT active ingredient that overcomes the disadvantages of the prior art. Description of the invention

[0017] The problem is solved by the independent claims. Preferred embodiments are found in the dependent claims.

[0018] In a first preferred embodiment, the invention relates to a nucleic acid for use as a drug and / or in therapeutic procedures, comprising or consisting of a botulinum neurotoxin encoding nucleic acid, wherein the BoNT encoding nucleic acid sequence is modified such that the encoded protein includes a recognition motif for a protease, preferably LVPRGS or LVPRGS, between the light and heavy chains in the linker region.

[0019] The invention therefore also relates to the use of BoNT nucleic acid for the transfection of cells so that these cells can produce the BoNT protein themselves and transport it to the site of action.

[0020] The dosage of a recombinant product cannot be controlled as precisely as the direct injection of a dissolved protein. This is likely one of the reasons why recombinant therapy using nucleic acids applied to the muscle or skin has not yet been described in the prior art. However, the fact that dosage accuracy is less critical in this gene therapy method because systemic availability is significantly reduced is an important insight and thus a central component of the invention.

[0021] The invention thus also relates to the first-time use in gene therapy of a nucleic acid described here which encodes BoNT.

[0022] One advantage of the invention is that the application of the nucleic acid allows for a longer duration of action. Since the prior art treatment procedure (administration of the toxin itself) is unpleasant and painful for the patient, this makes the treatment significantly more comfortable.

[0023] Treatment costs may also be reduced in the long term through this new approach.

[0024] The nucleic acid according to the invention can then be transfected into cells, e.g., muscle cells. The recombinant BoNT protein is then produced directly in the muscle cell and from there travels to its site of action, the synaptic cleft.

[0025] According to the invention, the nucleic acids or nucleic acid molecules can also be present in a vector.

[0026] Muscle cells are particularly easy to transfect. After intramuscular or intradermal application of naked plasmid DNA or RNA with a suitable transfection vector, expression of the recombinant protein in the target organ can be achieved relatively easily (see, e.g., Davis, Whalen & Demeneix "Direct gene transfer into skeletal muscle in vivo: factors affecting efficiency of transfer and stability of expression" Hum Gene Ther. 1993 Apr;4(2):151-9; or Danko & Wolff, "Direct gene transfer into muscle." Vaccine. 1994 Dec; 12(16):1499-502).

[0027] In a preferred embodiment, the nucleic acid of the invention is prepared for direct application into the organ and / or muscle to be treated.

[0028] In a particularly preferred embodiment, the nucleic acid is prepared as DNA or RNA. The use of RNA is especially preferred, as the best results have been achieved with this method to date.

[0029] The nucleic acid of the invention can also contain a promoter. However, experiments have also been conducted with mRNA that did not require a promoter. In experiments with plasmid DNA, a CMV promoter was used, which works well. Other promoters can also be used, so the invention is not limited in this respect.

[0030] Nucleic acid-based BoNT therapy eliminates the need to produce BoNT as a pharmaceutical in the laboratory. Only the nucleic acids are produced; the protein itself is synthesized in the body, for example, in the patient's muscle cells.

[0031] By treating with nucleic acids and thus administering a recombinant BoNT to the cells at the target site, not only can a higher local dosage be achieved, but the dose can also be maintained over a longer period of time.

[0032] In a further preferred embodiment, the invention relates to a nucleic acid comprising a BoNT-encoding nucleic acid and an S / MAR element. S / MAR stands for scaffold / matrix attachment region. These DNA sequences, which occur naturally in all organisms, ensure the anchoring of the DNA / chromatin to the nuclear matrix. Furthermore, these elements influence gene expression by interacting with or recruiting enhancers and by destabilizing the double helix structure.

[0033] It has been shown that S / MAR elements can prolong the expression of transfected DNA and result in a more homogeneous transfection of multiple cells. It is hypothesized that the S / MAR element, when transfected, prevents the transfected DNA from being integrated into the host genome, instead binding it directly to the nuclear matrix. Thus, the DNA is neither lost during cell division nor integrated at an unfavorable location within the host genome. The latter typically leads to the silencing of the transfected gene or to adverse effects on surrounding host genes. The S / MAR element can circumvent these negative consequences.

[0034] Different nucleic acid formulations (mRNA or plasmid DNA, various promoters, S / MAR, etc.) allow for the adjustment of short or particularly long durations of action. A person skilled in the art is able to identify the suitable formulation for the respective application without having to inventively invent.

[0035] By releasing the protein directly at the site of action, the risk of overdose is greatly reduced and the immunogenic potential is lowered.

[0036] In a particularly preferred embodiment, the invention relates to nucleic acids encoding botulinum neurotoxin for the treatment of spasticity, dystonia, drooling, and / or hyperhidrosis. The nucleic acid of the invention can therefore also be used in Parkinson's disease, amyotrophic lateral sclerosis, or infantile cerebral palsy, since these conditions can be associated with impaired swallowing ability and thus, for example, can lead to drooling.

[0037] The nucleic acids of the invention can also be used in other diseases that are associated with movement disorders or impaired muscle activity.

[0038] It was completely unexpected that the invention can be successfully implemented both with and without a secretory signal. Transfection of the skeletal muscle cell without a secretory signal leads to intracellular expression of BoNT. Contrary to expectations, the BoNT moves from the cytoplasm into the synaptic cleft of the motor endplate and thus to its site of action, the presynaptic cholinergic nerve ending. In this way, a toxic systemic effect is avoided. A disadvantage of this method is that only the nerve endings near the transfected cells are paralyzed, and therefore, the uniform distribution of the transfection is a prerequisite for success. This disadvantage can be avoided by using a secretory signal; nevertheless, it is remarkable that BoNT also reaches its site of action without this signal. Depending on the indication, this embodiment can certainly be advantageous.

[0039] When using the secretory signal, BoNT does reach the extracellular space outside the neuromuscular synapse, but systemic distribution is significantly lower or even nonexistent, depending on the dosage. With standard, state-of-the-art BoNT protein administration, the fluid in the BoNT solution is rapidly absorbed, and BoNT is released into the circulation during this absorption. Because the application of nucleic acids and the expression of BoNT are temporally decoupled during transfection, the tissue around the application site has returned to normal by the time of secretion. There is no increased fluid in the extracellular space, and the BoNT migrates along the muscle membrane to the motor endplates, i.e., to its site of action.

[0040] A signal peptide of murine IgG (kappa) can be used as a secretory signal, for example. The signal sequence is preferentially inserted upstream of the luciferase gene (N-terminal). The sequence is (SEQ ID NO 6):

[0041] This is just one example, however. Different secretory signals can be used. The choice also depends on the area of ​​application, i.e., the cell to be transfected and the indication.

[0042] The invention can therefore be used with or without a secretory signal. When using the corresponding sequence without a secretory signal, it was already quite surprising that the BoNT could pass from the cytoplasm of a muscle cell into the synaptic cleft of the motor endplate. In the embodiment that includes a secretory signal, the decisive advantage over the prior art lies primarily in the lower systemic distribution at the same or higher dosage compared to the prior art.

[0043] Besides largely avoiding undesirable systemic effects, the risk of the patient developing antibodies against BoNT is also significantly reduced, since BoNT is only present extracellularly to a limited extent. It is mostly located either in the transfected cell that produces the BoNT or already in the target cell of the efferent nerve fiber.

[0044] Another advantage is the simpler and cheaper production. Significant cost savings can be achieved in manufacturing due to its standardization.

[0045] The complex and controversial activity determination is eliminated with this recombinant product, which represents a significant advantage over the prior art. Animal testing for batch release would no longer be necessary for the production of the substance according to the invention.

[0046] This opens up entirely new treatment options for patients. By selecting the appropriate transfection vector, the duration of expression can be controlled, and the proportion of transfected cells can also be varied. This allows for adjustments to the duration and intensity of the effect. This flexibility is a crucial advantage for therapeutic applications, as treatment can now be more precisely tailored to the individual patient.

[0047] For some therapeutic applications, partial paralysis is advantageous, such as in osteosynthesis, where it would be beneficial to reduce the muscle tone of the surrounding tissue during the initial healing phase. Such applications are now possible for the first time thanks to this invention.

[0048] Due to its reduced systemic availability, not only is the toxicity lower, but also the immunogenicity of BoNT. This is a major advantage for patients with chronic diseases who require continuous therapy.

[0049] All known serotypes of BoNT, or the corresponding nucleic acids, can be used for the invention. That is, the invention relates primarily to nucleic acids for encoding BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F, and BoNT / G. Nucleic acids for encoding BoNT / A, BoNT / B, BoNT / E, and BoNT / F are preferred. Nucleic acids for encoding BoNT / A, BoNT / B, and BoNT / E are particularly preferred.

[0050] In addition to using different transfection vectors, the gene sequence of naturally occurring BoNT serotypes can be modified to achieve altered protein properties, such as changes in duration of action, a different target cell population, a different effect of the translocated light chain, or increased release from muscle cells. Increased release from the muscle cell can be achieved, for example, by inserting a secretory signal.

[0051] The protease required to activate the expressed neurotoxin can be additionally expressed, or it can be depleted after the in vivo Transfection is achieved by local injection of a small dose. Suitable proteases include, for example, thrombin or Stuart-Prower factor / factor Xa, which are available as finished pharmaceutical products (e.g., Recothrom®, Coagadex®). For this purpose, a recognition motif for the respective protease (thrombin: LVPRGS, factor Xa: LVPRGS) is inserted into the BoNT sequence between the light and heavy chains in the linker region. This approach further focuses the BoNT effect on the injected area or muscle compartment and thus avoids systemic or distant adverse effects of the neurotoxin.

[0052] For this embodiment, a nucleic acid comprising a sequence that codes for SEQ ID NO 4 is particularly suitable.

[0053] Codon optimization of the BoNT sequence for expression in human cells is also preferred, which can improve the efficiency of expression.

[0054] Preferably, the nucleic acid comprises a sequence that encodes one of the following protein sequences: SEQ ID No 1 (BoNT / A Hall Strain): SEQ ID No. 2 (BoNT / E): SEQ ID No 3 (BoNT / B): SEQ ID NO 4 (BoNT / A Hall Strain with protease recognition motif):

[0055] Also preferred are nucleic acids comprising a nucleic acid sequence encoding a protein with up to 80% homologous sequence, particularly preferably 90%, most preferably 95%, to SEQ ID No 1, SEQ ID NO 2 or SEQ ID NO 3.

[0056] It is further preferred that the nucleic acid of the invention comprises the nucleic acid sequence SEQ ID NO 5 or is complementary to it, or the corresponding RNA sequence. Preferably, the sequences are 80% homologous, particularly preferably 90% homologous, and most preferably 95% homologous. SEQ ID NO 5:

[0057] The present invention also relates to a pharmaceutical composition comprising a nucleic acid according to the invention and a pharmaceutically acceptable carrier and / or excipient.

[0058] The invention also relates, in a preferred embodiment, to a kit comprising a nucleic acid according to the invention and auxiliary materials and optionally a protease.

[0059] Preferably, the pharmaceutical composition is designed for application into a muscle, skin, or gland.

[0060] The teaching practiced in accordance with the application is characterized above all by the following features: Departure from the usual technical approach, new task, existence of a long-unresolved urgent need for the solution of the problem solved by the invention, development of scientific technology went in a different direction.

[0061] In particular, the advantageous embodiments of the invention have at least one or more of the aforementioned advantages. Examples

[0062] The invention is illustrated below by means of some examples and figures, which, however, should not be understood as limiting the scope of protection.

[0063] For our example, we used mRNA from the BoNT / A Hall strain encoding a sequence according to SEQ ID No. 1. This was initially injected intramuscularly into the tibialis muscle of mice at a concentration of 0.4 mg / ml without any further additives. Only very mild paralysis was observed (Digit Abduction Score below 1).

[0064] In further experiments, various transfection vectors were tested, including a polymer-based transfection reagent (Viromer®) from the manufacturer Lipocalyx. This was prepared with the BoNT mRNA solution according to the manufacturer's (Lipocalyx) instructions. A clear BoNT-mediated paralysis was observed. Figure 1 : Figure 1 shows a bioluminescence image showing luciferase expression 6 days after application of plasmid DNA (pEPI-1-luc) at different dosages, concentrations and volumes. Figure 2 : Figure 2This image shows immunohistochemical staining of expressed luciferase in tissue after injection of plasmid DNA into mouse gastrocnemius muscle. It is clearly evident that expression is limited to muscle cells and no luciferase is detected in the extracellular space. Figures 3 and 4 The Digit Abduction Score is used to assess BoNT-induced paralysis in a mouse model. This involves utilizing the mouse's toe abduction reflex. Following injection into the gastrocnemius and / or tibialis muscles, mice are unable to abduct their toes, or can only do so to a limited extent, depending on the severity of the paralysis. The paralysis is then classified according to a scoring system by Aoki et al., ranging from 0 (no paralysis) to 4 (complete paralysis). Figure 3In this study, BoNT-A encoding RNA was applied to the gastrocnemius and tibialis muscles of mice using a transfection vector. One group received only the transfection vector as a control. Another control group received a BoNT antitoxin beforehand. Of the BoNT-RNA-treated groups, one received a single treatment and the other received two treatments 24 hours apart. Both control groups showed no paralysis, while the treated groups exhibited clear, though not complete, paralysis. Figure 4 : Comparison of a non-paralyzed and a BoNT / A-RNA-treated hind limb of a mouse.

Claims

1. A nucleic acid for use as a drug and / or in therapeutic methods comprising a botulinum neurotoxin-encoding nucleic acid, wherein the BoNT-encoding nucleic acid sequence is modified such that the encoded protein between the light and heavy chains in the linker region comprises a recognition motif for the respective protease, preferably LVPRGS or LVPRGS.

2. The nucleic acid according to claim 1 for the treatment of spasticity, dystonia, saliva leakage, movement disorders, impaired muscle activity, osteosynthesis and / or hyperhydrosis.

3. The nucleic acid for use / treatment according to claim 1 or 2, wherein the nucleic acid comprises a sequence according to SEQ ID NO 5 or a sequence 80%, preferably 90%, particularly preferred 95% homologous thereto.

4. The nucleic acid for use / treatment according to any one or more of the preceding claims, wherein the nucleic acid encodes a protein which comprises one of the sequences selected from the group comprising SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3 and SEQ ID NO 4, or 80%, preferably 90%, particularly preferred 95% homologous thereto.

5. The nucleic acid for use / treatment according to any one or more of the preceding claims for direct application in the organ to be treated and / or muscle.

6. The nucleic acid for use / treatment according to any one or more of the preceding claims for the transfection of skeletal muscle cells, smooth muscle cells, smooth muscle cells of the glands or the skin and / or other skin cells.

7. The nucleic acid for use / treatment according to any one or more of the preceding claims, wherein said nucleic acid is a DNA or RNA.

8. The nucleic acid for use / treatment according to any one or more of the preceding claims, wherein the BoNT-encoding nucleic acid sequence encodes BoNT / A, BoNT / B, BoNT / C, BoNT / D, BoNT / E, BoNT / F or BoNT / G.

9. The nucleic acid for use / treatment according to any one or more of the preceding claims additionally comprising a secretory signal-encoding sequence.

10. The nucleic acid for use / treatment according to any one or more of the preceding claims additionally comprising a S / MAR element.

11. The nucleic acid for use / treatment according to any one or more of the preceding claims additionally comprising a necessary protease-encoding sequence.

12. A use of a nucleic acid comprising a botulinum neurotoxin-encoding nucleic acid for non-therapeutic cosmetic purposes, wherein the nucleic acid is one of the nucleic acids according to claims 1 to 11.

13. The use of claim 12, wherein said botulinum neurotoxin expressed in vivo is activated by a simultaneously or subsequently injected protease.

14. A pharmaceutical composition comprising a nucleic acid according to claims 1 to 11 and a pharmaceutically acceptable carrier and / or excipient.