Patent Application
20070117771
This application is a continuation of international patent application no. PCT/EP2005/001970, filed Feb. 24, 2005, designating the United States of America, and published in German on Sep. 29, 2005 as WO 2005/090571, the entire disclosure of which is incorporated herein by reference. Priority is claimed based on Federal Republic of Germany patent application no. DE 10 2004 011 687.3, filed Mar. 10, 2004.
The invention relates to small, in particular interference-triggering, double-stranded RNA molecules (dsRNA), which are directed against members of the VGLUT family, and to host cells containing dsRNA according to the invention. dsRNA according to the invention and corresponding host cells are suitable as pharmaceutical compositions and for the production of pharmaceutical compositions, in particular for the treatment of pain and other diseases associated with VGLUT family members or the non-physiological expression thereof.
As defined by the International Association for the Study of Pain (IASP), pain is “an unpleasant sensory and emotional experience associated with acute or potential tissue damage, or described in terms of such damage” (Wall and Melzack, 1999). The organism reacts to a painful (nociceptive) stimulus with a complex reaction, in which sensory/discriminatory, cognitive, effective, autonomous and motor components participate. Whereas acute pain involves a physiological protective reaction and is vital to the survival of an individual, chronic pain, on the other hand, does not have a clear biological function. Nociceptive pain is triggered by noxious stimuli, such as heat, mechanical stimulation, protons or coldness, on specialized high-threshold sensory apparatus, the nociceptors, and is conveyed into the posterior horn of the spinal cord in the form of electrical activity by unmyelinated C-fibres or weakly myelinated Aδ fibres. The nociceptors are equipped with specific receptors and ion channels for this purpose (Scholz and Woolf, 2002).
Damaged or injured tissue, inflammation or tumour cells may be signals of nociceptors; they lead to the release of chemical mediators from inflamed cells, blood vessels and from the afferent terminals, which either themselves lead to activation of the nociceptors (for example by bradykinin) or alter the stimulus response behaviour of nociceptive afferent nerve fibres, for example by lowering the activation threshold (for example by prostaglandins, interleukins, NGF), and thus lead to sensitisation of the nociceptors (Sholz and Woolf, 2002).
The intensity of pain is coded by the number of impulses per unit time. In the case of nociceptive peripheral stimulation, the rapid synaptic response components (5 to 20 msec) and monosynaptic reflex responses in the spinal cord are brought about by AMP-kainate receptors, whereas NMDA and metabotrope glutamate receptors participate in particular in late, longer lasting (20 to 150 msec), polysynaptically mediated response components (Tölle, 1997).
The release of glutamate in the posterior horn of the spinal cord plays a decisive part in the creation of chronic pain (Baranauskas and Nistri, 1998; Zhuo, 2001). The release of glutamate leads to activation of the glutamate receptors (AMPA, kainite, mGlu-R, NMDA). Proteins which participate in the release of glutamate therefore represent interesting targets for pain research.
Glutamate is one of the most important excitatory neurotransmitters in the nervous system of vertebrates. The nonessential amino acid, glutamate, cannot breach the blood-nerve barrier and is therefore synthesized in the brain from glucose and a large number of other precursors.
The formation of glutamate in the excitatory nerve endings is catalysed by the enzyme, phosphate-activated glutaminase (PAG), from glutamine. The vesicular glutamate transporter (VGLUT) packs the glutamate in vesicles and releases the glutamate after arrival of a depolarized action potential by the influx of calcium from the vesicles into the synaptic cleft. From the synaptic cleft, the glutamate is transported in part by a plasma membrane transporter for excitatory amino acids (EAATs) back into the excitatory terminals, where it is packed in vesicles again.
Therefore, transporter proteins from two superfamilies participate in the transport of glutamate: plasma membrane transporters and the vesicular membrane transporters (Disbrow et al., 1982; Shioi et al., 1989; Tabb et al., 1992).
Three vesicular glutamate transporters, VGLUT1 (SLC17A6), VGLUT2 (SLC17A7) and VGLUT3 (SLC17A8), have been identified as members of the SLC-17 transporter family (type I phosphate/vesicular glutamate transporter), which induces the transport of organic anions, and have initially been identified as Na+-dependent phosphate transporters. VGLUT1, VGLUT2 and VGLUT3 bear the collective name of VGLUT. The proteins of the SLC17 family are expansive transmembrane proteins with 6 to 12 hypothetical transmembrane domains, the three aforementioned glutamate transporters occurring as the subfamily of the SLC17 family. The three aforementioned VGLUTs are highly homologous with one another in their amino acid sequence (Takamori et al., 2002). The cDNA sequence of human VGLUT2 appears under gene bank accession number NM—020346 in the databases. The amino acid sequence of human VGLUT2 appears under NP—065079 in the databases. The cDNA sequence of VGLUT2, rat, appears under NM—053427 in the databases. The amino acid sequence of VGLUT2, rat, appears under NP—445879 in the databases. The cDNA sequence of VGLUT2, mouse, appears under AN: BC038375 in the databases, the amino acid sequence of VGLUT2, mouse, appears under AAH38375 in the databases.
The cortical layers of the cerebrum have a pronounced mRNA expression level for VGLUT1, whereas VGLUT2 mRNA could be detected, in particular, in layer IV of the cortex. VGLUT3 expression is localized, for example, in the inhibitory cells in layer II of the parietal cortex, or in GAD-positive interneurons in the Stratum radiatum of CA1-CA3 of the hippocampus. Furthermore, VGLUT1 and VGLUT2 could only be detected in the nerve endings, whereas VGLUT3 was detected not only in the synaptic vesicles but also in vesicular structures of astrocytes and neuronal dendrites (Fremeau et al., 2002).
VGLUT1 and VGLUT2 are expressed in two separate populations in the spinal ganglion, a third subpopulation having coexpression for VGLUT1 and VGLUT2. VGLUT2-mRNA is expressed predominantly by small and medium DRG neurons, whereas VGLUT1-mRNA is expressed by medium and large DRG neurons (posterior root fibre ganglion). In isolated cases, VGLUT3-mRNA-expressing neurons can also appear in the spinal ganglion (Oliveira et al., 2003; Todd et al, 2003).
Both VGLUT1 and VGLUT2 can be detected at protein level in the grey matter of the spinal cord (Varoqui et al., 2002). The dominance of VGLUT2 in the superficial posterior horn is evidence of a prominent role in pain transmission. The dominance of VGLUT1 in the deep posterior horn is evidence of a role in proprioception. Therefore, VGLUT2, in particular, but also VGLUT1 is a pain target (Varoqui et al., 2002).
The effective treatment of pain is a great challenge in molecular medicine. Acute and transitory pain is an important signal from the body to protect people from serious damage by the environment or overloading of the body. On the other hand, chronic pain, which lasts longer than the cause of the pain and the expected duration of healing, has no known biological function and affects hundred of millions of humans throughout the world. Approximately 7.5 million people suffer from chronic pain in the Federal Republic of Germany alone. Unfortunately, the pharmacological treatment of chronic pain is still unsatisfactory and therefore remains a challenge in current medical research. Frequently, currently existing analgesics are not sufficiently effective and sometimes have serious side effects.
Due to their function and expression profile, VGLUT proteins in general, but VGLUT2 in particular, represent an interesting starting point as a target for new pain remedies (Varoqui et al., 2002).
Some substances which are capable of modulating the activity or expression of VGLUT are known, for example, from research into pain relief (Carrigan et al. 2002; Roseth et al., 1995; Roseth et al., 1998). However, they do not act subtype-specifically, and therapeutic formulations would be limited both by the availability of the inhibitors in the nervous system and at the synaptic vesicles and by the nonspecific effect on all three VGLUTs.
An object of the present invention is to provide further substances, which are capable of selectively and efficiently modulating the effect of the VGLUTs, for example VGLUT1, VGLUT2 and VGLUT3.
Another object of the invention is to provide VGLUT-modulating substance which optionally exhibit cell permeability.
A further object of the invention is to provide VGLUT-modulating substances which are usable for therapeutic purposes, in particular for the treatment of pain.
These and other objects are achieved by the present invention as described and claimed hereinafter. According to the invention, the object is achieved by VGLUT-specific dsRNAs that are capable of triggering the phenomenon of RNA interference.
Double-stranded RNA (dsRNA) according to the invention contains a sequence with the general structure 5′-(N17-25)-3′, wherein N is any base and represents nucleotides. The general structure consists of a double-stranded RNA with a macromolecule made up of ribonucleotides, wherein the ribonucleotide consists of a pentose (ribose), an organic base and a phosphate. The organic bases in the RNA consist of the purine bases, adenine (A) and guanine (G), and the pyrimidine bases, cytosine (C) and uracil (U). The dsRNA contains nucleotides with a directed structure with overhangs. Double-stranded RNAs according to the invention of this type can trigger the phenomenon of RNA interference (siRNAs).
The phenomenon of RNA interference as an immunological defence system was noticed during immunological research into higher eukaryotes.
The system was originally described in various species independently of one another, initially in C. elegans (Fire et al., 1998), before the underlying mechanisms could be identified as identical at specific levels of the processes: RNA-mediated virus resistance in plants (Lindbo and Dougherty, 1992), PTGS (post-transcriptional gene silencing) in plants (Napoli et al., 1990) and RNA interference in eukaryotes are accordingly based on a common mode of operation (Plasterk, 2002).
The in vitro technique of RNA interference (RNAi) is based on double-stranded RNA molecules (dsRNA) which trigger the sequence-specific suppression of gene expression (Zamore (2001) Nat. Struct. Biol. 9: 746-750; Sharp (2001) Genes Dev. 5:485-490: Hannon (2002) Nature 41: 244-251). The activation of protein kinase R and RNaseL brought about nonspecific effects such as an interferon response (Stark et al. (1998) Annu. Rev. Biochem, 67: 227-264; He und Katze (2002) Viral Immunol. 15: 95-119) during the transfection of mammalian cells with long dsRNA. These nonspecific effects are obviated by using smaller, for example 21 to 23-type so-called dsRNA (small interfering RNA), as nonspecific effects are not triggered by dsRNA that is smaller than 30 bp (Elbashir et al. (2001) Nature 411: 494-498). dsRNA molecules have also been used recently in vivo (McCaffrey et al. (2002), Nature 418: 38-39; Xia et al. (2002), Nature Biotech 20: 1006-1010; Brummelkamp et al. (2002), Cancer Cell 2: 243-247.
dsRNAs that are directed against members of the VGLUT family are disclosed in the context of the present invention. According to the invention, these dsRNAs may be of various categories. dsRNA according to the invention may exist in the form (i) of an siRNA, (ii) of a long dsRNA containing one or more identical or different siRNA(s) in the long dsRNA sequence, (iii) of an siRNA-based hairpin RNA or (iv) of a miRNA-based hairpin siRNA. All the aforementioned embodiments are covered by the term “dsRNA”.
Although (i) siRNA (typically chemically synthesized and then incorporated into the RISC complex intracellularly while bypassing the dicing step, so sequence-specific mRNA degradation (of the target sequence) takes place) is described in more detail hereinafter, (ii), in other words a long dsRNA (ds: double-stranded), is a precursor of siRNA (according to (i)), which is only processed into pure siRNA by a dicing step (enzyme: dicer). This precursor of siRNA, which is typically converted only intracellularly into mature siRNA, meets the requirements for use, for example, as a pharmaceutical composition or for production of a pharmaceutical composition for the treatment of the indications mentioned in the present application. In a preferred embodiment, a plurality of different siRNAs, which may differ in efficiency, are formed according to the invention in this respect, in other words from largerVGLUT-dsRNA molecules, in particularVGLUT1-, 2- or 3-specific dsRNA molecules (preferably >30 bp, more preferably >40 bp and even more preferably >50 bp), after dicer processing. Long (optionally hairpin-shaped) dsRNA molecules, which are transformed intracellularly into various siRNAs after dicer processing may also be expressed in a cell on a vector basis (apart from chemical synthesis) under the control of a Pol II promoter. The Pol II promoter allows inducible tissue- or cell type-specific expression (Kennerdell and Carthew, 2000). This form of application therefore allows inducible transient simultaneous expression of a large number of siRNAs, which originate from a precursor dsRNA. According to a further particularly preferred embodiment, dsRNA molecules according to the invention of this type may form a specific phenotype by genetic manipulation techniques such as homologous recombination of stem cells.
Modifications of siRNA according to (i), namely siRNA-based hairpin bends (iii), may also be used according to the invention. Hairpins of this type can preferably occur at one end, but optionally also at both ends of the siRNA double strand. There are typically at least 5, more preferably at least 8 and even more preferably at least 10 nucleotides, which, owing to modifications and/or in the absence of corresponding complementarity, do not form double-stranded interactions, but form a bend which initially bonds the two strands together covalently. An siRNA-based hairpin RNA of this type may be further processed into active siRNA by corresponding enzymes (for example dicers), for example intracellularly. Finally, (iv), miRNA-based hairpin RNAs directed against sequences of the VGLUT family, also form part of the present invention. These are imperfectly complementary siRNAs, preferably with at least one hairpin at the terminus (at the termini). Imperfectly complementary duplex strands of mRNA of this type comprise at least one defective conjugation, preferably between 1 and 4 defective conjugations in the duplex strand. The effect of miRNA-based hairpin RNA is based on the enzymatic processing thereof (for example by dicers) to miRNA(s), subsequent incorporation thereof into miRNPs and finally the translation inhibition thereof.
The respective length of the duplex strands in embodiments (i), (iii) and (iv) according to the invention does not differ and all embodiments can be chemically synthesized.
dsRNAs according to the invention preferably have the general structure 5′-(N19-25)-3′, more preferably 5′-(N19-24)-3′, even more preferably 5′-(N21-23)-3′, where N is any base. At least 90%, preferably 99% and, in particular 100% of the nucleotides of a dsRNA according to the invention may be complementary to a fragment of the (m)RNA sequence of a member of VGLUT family, in particularVGLUT1, VGLUT2 or VGLUT3. 90% complementary means that, for example, with a given length of 20 nucelotides of a dsRNA according to the invention, it is not complementary with the corresponding fragment on the (m)RNA in the case of at most 2 nucleotides. The sequence of the double-stranded RNA, with its general structure, is preferably completely complementary with a fragment of the (m)RNA of a member of the VGLUT family, in particularVGLUT1, VGLUT2 or VGLUT3.
VGLUT-dsRNA comprising the following sequence patterns are also preferred: AAN19TT, NAN19NN, NARN17YNN and/or NANN17YNN, wherein N represents any nucleotide, A represents adenosine, T represents thymidine, R represents purines (A or G) and Y represents pyrimidine bases (C or T).
A dsRNA according to the invention can basically be complementary with any desired fragment on the mRNA or the primary transcript of a member of the VGLUT family.
In a eukaryotic cell, the gene is transcribed over its entire length, including both introns and exons, into a long RNA molecule, the primary transcript, to produce an mRNA. The stability of the cellular mRNA is ensured by processing the primary transcript at the 5′ end with an addition of an untypical nucleotide having a methylated guanine and polyadenylation at the 3′ end. Before the RNA leaves the cell nucleus, the intron sequences are removed and the exons spliced together by RNA splicing.
Both the primary transcript and the processed mRNA may be target sequences for dsRNA according to the invention. The primary transcript and the mRNA are described hereinafter as (m)RNA for short.
Basically any 17 to 29, preferably 17 to 25, base pair long fragments occurring in the encoding region of the (m)RNA can serve as the target sequence for a dsRNA according to the invention. Target sequences for dsRNAs according to the invention which lie between position 70 and 1730 (calculated from the respective AUG starting triplet of the encoding region of the (m)RNA of human VGLUT2 or VGLUT3), preferably between 100 and 1500 and quite particularly preferably between 600 and 1200 are also particularly preferred. Therefore, 17 to 25, in particular 19 to 25 and quite particularly 21 to 23, base pair long fragments on the (m)RNA, of which the starting nucleotide corresponds to a nucleotide of a position 80 to 1600 (or the aforementioned further preferred regions) of the encoding region of the VGLUT2- or 3-(m)RNA and of which the terminal nucleotide lies 17 to 25, preferably 19 to 25 and quite particularly preferably 21 to 23 nucleotides further downstream from the respective initiating nucleotide, are preferred.
With respect to VGLUT1, target sequences of the encoding region are similarly particularly preferred, in particular target sequences lying between position 600 and 1200 of the encoding region (calculated from the AUG initiating triplet).
dsRNAs which are directed against regions in the encoding region (m)RNA of a member of the VGLUT family are particularly preferred. In particular, dsRNAs according to the invention of this type, which are located in the central area of the encoding region, preferably at least 50, 70, 100 nucleotides removed from the AUG initiating triplet of the (m)RNA or at least 50 nucleotides, preferably at least 70, and more preferably 100 nucleotides removed from the 3′-terminal encoding region of the (m)RNA, should be directed against VGLUT-(m)RNA fragments.
Particularly preferred are dsRNAs according to the invention, in particular siRNAs which are directed against fragments in the encoding region of the VGLUT1, 2 or 3 (m)RNA (or cDNA), which begin with the initiating sequence AA.
dsRNAs according to the invention, in particular siRNAs which are directed against fragments in the encoding region of the VGLUT1-(m)RNA (or cDNA) are more particularly preferred; dsRNAs according to the invention, in particular siRNAs, which are directed against the sequences AACGTGCGCAAGTTGATGAAC (SEQ ID NO: 14) or AAGTTGATGAACTGCGGAGGC (SEQ ID NO: 14), are additionally preferred.
With respect to dsRNAs according to the invention, in particular siRNAs against VGLUT2, dsRNAs, in particular siRNAs are more particularly preferred, which are complementary and therefore directed against (m)RNA-fragments (or cDNA) which, for example, comprise the sequence AATGCCTTTAGCTGGCATTCT (SEQ ID NO: 16), AATGGTCTGGTACATGTTTTG (SEQ ID NO: 17), AAAGTCCTGCAAAGCATCCTA (SEQ ID NO: 18), AAGAACGTAGGTACATAGAAG (SEQ ID NO: 20), AATTGTTGCAAACTTCTGCAG (SEQ ID NO: 21), AAATTAGCAAGGTTGGTATGC (SEQ ID NO: 22), AATTAGCAAGGTTGGTATGCT (SEQ ID NO: 23), AAGGTTGGTATGCTATCTGCT (SEQ ID NO: 24), AAGCAAGCAGATTCTTTCAAC (SEQ ID NO: 25), AATGGGCATTTCGAATGGTGT (SEQ ID NO: 27), AATAAGTCACGTGAAGAGTGG (SEQ ID NO: 28), AATATTTGCCTCAGGAGAGAA (SEQ ID NO: 31), AAGTCTTATGGTGCCACAACA (SEQ ID NO: 32), AAGACTCACATAGCTATAAGG (SEQ ID NO: 34), and preferably AAGTCCTGCAAAGCATCCTAC (SEQ ID NO: 19), AACCACTTGGATATCGCTCCA (SEQ ID NO: 26), AAGTCACGTGAAGAGTGGCAG (SEQ ID NO: 29), AAGAGTGGCAGTATGTCTTCC (SEQ ID NO: 30), and/or AATGGAGGTTGGCCTAGTGGT (SEQ ID NO: 33).
Quite particularly preferred are those dsRNAs according to the invention, in particular siRNAs, which are directed against fragments in the encoding region of the VGLUT3-(m)RNA (or cDNA), more preferably in turn those dsRNAs according to the invention, in particular siRNAs, which are directed against AATCTTGGAGTTGCCATTGTG (SEQ ID NO: 35), AATTCCAGGTGGTTTCATTTC (SEQ ID NO: 38), AACATCGACTCTGAACATGTT (SEQ ID NO: 39), AAGAGGTCTTTGGATTTGCAA (SEQ ID NO: 41), AATAAGTAAGGTGGGTCTCTT (SEQ ID NO: 42), AATCGTTGTACCTATTGGAGG (SEQ ID NO: 45), AAGAATGGCAGAATGTGTTCC (SEQ ID NO: 47), AATCATTGACCAGGACGAATT (SEQ ID NO: 48), AACTCAACCATGAGAGTTTTG (SEQ ID NO: 49), AAAGAAGATGTCTTATGGAGC (SEQ ID NO: 50), and/or AAGAGCTGACATCCTACCAGA (SEQ ID NO: 52), and preferably AACCGGAAATTCAGACAGCAC (SEQ ID NO: 36), AAACAGTGGGCCTTATCCATG (SEQ ID NO: 37), AAGGTTTAGTGGAGGGTGTGA (SEQ ID NO: 40), AAGTAAGGTGGGTCTCTTGTC (SEQ ID NO: 43), AAGGTGGGTCTCTTGTCAGCA (SEQ ID NO: 44), and/or AAGACCCGTGAAGAATGGCAG (SEQ ID NO: 46).
Preferably, (double-stranded) siRNAs according to the invention or suitable molecules of the other embodiments will comprise the sequence TT at the terminus of at least one strand, preferably in an overhanging manner relative to the terminus of the complementary other strand. The complementary other strand of the siRNA according to the invention then typically corresponds in its sequence at a terminus to the, for example aforementioned, sequences after AA (wherein T, in contrast to the foregoing target sequences, is replaced by U in the siRNA according to the invention) and at the other terminus typically has an overhanging TT (see also embodiment 4).
However, dsRNAs according to the invention could also be directed against nucleotide sequences on the VGLUT1, VGLUT2, VGLUT3-(m)RNA, which do not lie in the encoding region, in particular in the non-encoding 5′ region of the (m)RNA, of the regulating functions.
The boundaries at the 5′ end of the target sequence with, for example, AA of the nucleotide bonds are also reflected in the associated dsRNA according to the invention in a sequence 5′-AAN15-23 (with the strand 3′-TTN15-23 which is complementary therewith). A strand of the double-stranded RNA is therefore complementary with the primary or processed RNA transcript of the VGLUT1, 2 or 3 gene.
Preferably, however, effective blocking and cleavage of the (m)RNA of a member of the VGLUT family is achieved in particular in that certain rules of selection are adhered to when selecting the target sequence of dsRNAs according to the invention.
A particularly preferred embodiment of the present invention is a dsRNA which has a GC content of at least 30%, of 30 to 70% in a more preferred embodiment, and from 40% to 60% in a more preferred configuration, or even more preferably between 45% and 55%.
A further particularly preferred embodiment of the present invention is a target sequence which contains the same frequency of all nucleotides on the antisense strand. Finally, it is particularly preferred if 2′-deoxythymidine appears for the 2-nt 3′ overhang in an siRNA according to the invention or suitable dsRNA molecules of further embodiments, as it is thus protected from exonuclease activity. It is further preferred if the target sequence of a dsRNA according to the invention appears only once in the target genes or is also singular for the respective genome of the treated cells.
Combinations of the aforementioned properties in dsRNAs according to the invention are also particularly preferred.
dsRNAs according to the invention, which are not directed against binding points for proteins which bind to a VGLUT(m)RNA, are also quite particularly preferred. In particular, a dsRNA according to the invention should not be directed against those regions on a VGLUT-(m)RNA which relate, for example, to the 5′-UTR-region, the 3′-UTR-region (respective regions at which the splicing process takes place), an initiating codon and/or exon/exon transitions. It is also preferred if the target region on the (m)RNA, to which the dsRNA according to the invention binds, does not have monotonic or repetitive sequences, in particular fragments with poly-G-sequences. Target sequences in intron regions are also preferably avoided in the complementary dsRNA according to the invention, as RNAi is a cytoplasmatic process.
A modified nucleotide can preferably appear in a dsRNA according to the invention. According to the invention, the term “modified nucleotide” means that the respective nucleotide is chemically modified. By the term “chemical modification”, the person skilled in the art understands that the modified nucleotide is altered by replacement, attachment or removal of individual or a plurality of atoms or atom groups in comparison with naturally occurring nucleotides. At least one modified nucleotide in dsRNA according to the invention serves, on the one hand, for stability and, on the other hand, to prevent dissociation.
Preferably between 2 and 10, and quite particularly preferably between 2 and 5, nucleotides are modified.
The ends of the double-stranded RNA (dsRNA) can preferably be modified to counteract degradation in the cell or dissociation into the individual strands, in particular to prevent premature degradation by nucleases.
Dissociation of the individual strands of dsRNA, which is generally undesirable, occurs, in particular, when using low concentrations or short chain lengths. For particularly effective inhibition of dissociation, the nucleotide pair-mediated cohesion of the double-stranded structure of dsRNA according to the invention may be increased by at least one, preferably a plurality, in particular 2 to 5, chemical linkages. A dsRNA according to the invention, of which the dissociation is reduced, has higher stability to enzymatic and chemical degradation in the cell and in the organism or ex vivo.
The chemical linkage of the individual strands of a dsRNA according to the invention is advantageously formed by a covalent or ionic bond, hydrogen bridge bond, hydrophobic interaction, preferably van der Waals or stacking interactions or by metal ion coordination. According to a particularly advantageous configuration, it may be produced at least at one, preferably at both, ends. It has also proven to be advantageous that the chemical linkage is formed by means of one or more groups of compounds, the groups of compounds preferably being poly-(oxyphosphinicooxy-1,3-propane-diol) and/or polyethyleneglycol chains. The chemical linkage may also be formed by purine analogues used in the double-stranded structure, instead of purines. A further advantage is that the chemical linkage is formed by azabenzene units introduced in the double-stranded structure. It may also be formed by branched nucleotide analogues used in the double-stranded structure, instead of nucleotides.
It has proven advantageous to produce the chemical linkage using at least one of the following groups: methyl blue; bifunctional groups, preferably bis-(2-chorethyl)-amine; N-acetyl-N′-(p-glyoxybenzoyl)-cystamine; 4-thiouracil; psoralene. The chemical linkage may further be formed by thiophosphoryl groups arranged at the ends of the double-stranded region. The chemical linkage is preferably produced by triple helical bonds at the ends of the double-stranded region. The chemical linkage may advantageously be induced by ultraviolet light.
Modification of the nucleotides of the dsRNA leads to deactivation of a protein kinase (PKR) dependent on (double-stranded) RNA, in the cell. The PKR induces apoptosis. Advantageously, at least one 2′ hydroxy group of the nucleotides of the dsRNA in the double-stranded structure is replaced by a chemical group, preferably a 2′-amino or a 2′-methyl group. At least one nucleotide in at least one strand of the double-stranded structure may also be what is known as a locked nucleotide with a sugar ring which is preferably chemically modified by a 2′-O, 4′-C-methylene bridge. Advantageously, a plurality of nucleotides are locked nucleotides.
Modification of the nucleotides of dsRNA according to the invention affects, in particular, the dissociation of the nucleotides by reinforcing hydrogen bridge bonding. The stability of the nucleotides is increased and protected from an attack by RNAs.
A further method of preventing premature dissociation of dsRNA according to the invention in the cell involves the formation of the hairpin bend. In a preferred embodiment, a dsRNA according to the invention has a hairpin structure, to slow down the dissociation kinetics. With a structure of this type, a loop structure is preferably formed at the 5′- and/or 3′-end. A loop structure of this type does not have hydrogen bridges.
In addition, premature degradation may be prevented by modifying the backbone of the dsRNA according to the invention. dsRNA which is modified (for example, phosphorus thioate, 2′-O-methyl-RNA, LNA, LNA/DNA gapmers) and therefore has a longer half-life in vivo is particularly preferred.
A dsRNA according to the invention is preferably derived against the (m)RNA of the VGLUT family, in particular from VGLUT1, VGLUT2 and/or VGLUT3, from mammals, such as humans, monkeys, rats, dogs, cats, mice, rabbits, guinea pigs, hamsters, cattle, pigs, sheep and goats.
A dsRNA according to the invention preferably suppresses the expression of VGLUT1, VGLUT2 and/or VGLUT3 in the cell by at least 50%, 60%, 70%, particularly preferably to at least 90%; the dsRNAs according to the invention are therefore, in particular, suitable dsRNA molecules of the embodiments according to the invention, in other words (i) siRNA or (ii) long dsRNA or (iii) siRNA-based hairpin RNA or (iv) miRNA-based hairpin RNA, which can trigger the phenomenon of RNA interference. Suppression can be measured via a Northern blot, quantitative real time PCR or at protein level with VGLUT1-, VGLUT2-or VGLUT3-specific antibodies.
dsRNAs according to the invention, in particular human dsRNAs according to the invention, can have what are known as blunt ends, but also overhanging ends.
Overhanging ends can basically comprise at least two overhanging nucleotides, preferably 2 to 10, in particular 2 to 5, overhanging nucleotides at the 3′-terminus, optionally however also alternatively at the 5′-terminus.
dTdT at the respective 3′-terminus of the double-stranded dsRNA according to the invention, in particular siRNA, are preferred for the overhanging ends. As mentioned above, the overhanging nucleotides may be dT (deoxythymidine) or also uracil, but any overhanging ends can basically be attached to the dsRNA double strands according to the invention that are complementary with mRNA of VGLUT1, 2 or 3.
dsRNAs according to the invention, in particular siRNAs, may be directed against human VGLUT sequences or sequences of mammals, for example rats, pigs or mice or of domestic animals.
In the case of rat VGLUT sequences, preferred embodiments of the dsRNA according to the invention are directed against a target sequence of the VGLUT2-mRNA of the rat, which in a preferred embodiment is the (m)RNA-target sequence 5′-AAG GCU CCG CUA UGC GAC UGU-3′ (SEQ ID NO: 70) (the sequence corresponds at the level of the cDNA, although U is replaced by T). A particularly preferred dsRNA of the present invention is therefore a duplex molecule of which the sense strand has the sequence 5′-GGC UCC GCU AUG CGA CUG UTT-3′ (SEQ ID NO: 71) (i.e. with an overhanging TT at the 3′-terminus) and of which the antisense strand has the sequence 5′-ACA GUC GCA UAG CGG AGC CTT-3′ (SEQ ID NO: 72) (also with a TT which overhangs relative to the sense strand at the 3′-terminus). This ds molecule according to the invention is directed against the aforementioned fragment of the VGLUT2-mRNA. A further particularly preferred embodiment of the dsRNA according to the invention is directed against a different target sequence of the VGLUT2-mRNA (of the rat), namely against 5′-AAG CAG GAU AAC CGA GAG ACC-3′ (SEQ ID NO: 86). The two strands of a double-stranded siRNA according to the invention then typically contain the following sequences: 5′-r(GCAGGAUAACCGAGAGACC)dTT-3′ (SEQ ID NO: 87) (sense strand) and 5′-r(GGUCUCUCGGUUAUCCUGC)d(TT)-3′ (SEQ ID NO: 88) (antisense strand) or consist thereof.
The dsRNA is produced by processes known to the person skilled in the art by synthesizing nucleotides, in particular also oligonucleotides, for example by Merryfield synthesis, on an insoluble support (H. G. Gassen, Chemical and Enzymatic Synthesis of Gene Fragments (Verlag Chemie. Weinheim 1982)) or by a different method (Beyer/Walter, Lehrbruch der Organischen Chemie, 20th edition, (S. Hirzel Verlag, Stuttgart 1984), p. 816 ff.). VGLUT-mRNA may be obtained by hybridization using genome and cDNA databases. dsRNA molecules according to the invention, in particular siRNA molecules may, for example, be produced synthetically and optionally also obtained from various suppliers, for example IBA GmbH (Göttingen, Germany).
Double-stranded RNA according to the invention (dsRNA) may be enclosed in micellar structures which influence the separation of groups of substances in vitro and in vivo. The dsRNA preferably occurs in liposomes. Liposomes are artificial membranes, which are spherically closed in on themselves, of phospholipids in which hydrophilic substances are encapsulated in the aqueous interior and lipophilic substances may also be incorporated in the internal region of the lipid membrane. To be used for experimental or therapeutic purposes, liposomes have to be compatible with cells and tissues. The dsRNA, which is preferably present in the liposomes, may be modified by a peptide sequence, preferably by a lysine and arginine-rich sequence, for example a sequence of the viral TAT protein (for example containing AS 49-57) and then breach the cell membrane more easily as a transporter peptide.
The dsRNA can similarly be enclosed in viral natural capsids or in chemically or enzymatically produced artificial capsids or structures derived therefrom. The aforementioned features allow the dsRNA to be funnelled into predetermined target cells.
A further preferred subject of the present invention is a configuration of the VGLUT-dsRNAs according to the invention, which is an alternative to siRNA, namely as microRNAs (loc. cit.) with at least one hairpin bend, by means of which the two imperfectly complementary strands are covalently bound to one another (miRNA-based hairpin RNA) (cf. also Schwarz et al., 2002). These VGLUT-miRNAs according to the invention are transcribed by the cells themselves and, after sequence-specific binding to the mRNA, do not lead to mRNA degradation but merely induce translation repression. VGLUT-miRNAs according to the invention are transcribed as at least 50, preferably between 60 and 80, quite particularly preferably between 65 and 75 nucleotide-long precursors and form a characteristic “hairpin structure”. The enzyme dicer cuts, from these precursors in the cell, a 21 to 23 nucleotide-long double-stranded region that is unwound in further steps. Therefore, the mature miRNA can be incorporated, for example, into miRNP particles. These particles may then induce specific translation repression of the complementary mRNA. The degree of complementarity to the target mRNA decides whether the DNA duplex formed acts as miRNA or siRNA.
According to the invention, numerous vector systems allow the use of miRNAs for subsequent stable and regulated transcription of the corresponding VGLUT-siRNAs. The transcription of the miRNAs may be controlled by polymerase III promoters (for example HI or U6 promoters) and also by polymerase II promoters (Brummelkamp et al., 2002; Lee et al., 2002; Miyagishi and Taira, 2002). The sense and antisense strands of various promoters may be read off and accumulate in the cell to form 19-nt duplices with 4-nt overhangs (B) (Lee et al., 2002), or the expression of hairpin structures is used (Brummelkamp et al., 2002).
Viral vectors, for example retroviral or adenovirus-derived vectors, are preferably used for these vector systems. Viral vectors have very efficient targeted transduction of specific cells, including primary cells, and can therefore be used widely, for example, in pain therapy.
According to a further particularly advantageous embodiment, it is provided that the dsRNA is bound to at least one capsid protein which originates from a virus or is derived therefrom or from a synthetically produced viral capsid protein, associated therewith or surrounded thereby. The capsid protein may be derived from the polyoma virus. It may therefore be, for example, the virus protein 1 (VP1) and/or the virus protein 2 (VP2) of the polyoma virus. The use of such capsid proteins is known, for example, from DE 19618797 A1. The aforementioned features substantially simplify introduction of the dsRNA in to the cell.
In a preferred embodiment, the dsRNA according to the invention is expressed in that the first template (sense dsRNA) and the second template (antisense dsRNA) are under the control of two identical or different promoters. Expression takes place in vivo and is brought into the cells by vectors in the course of gene therapy.
The present invention further relates to a pharmaceutical composition containing at least one dsRNA according to the invention and/or a cell containing it, and optionally auxiliaries and/or additives.
Pharmaceutical composition: a substance corresponding to the definition in Article 1 §2 of the German law regulating the circulation of pharmaceutical compositions (AMG). In other words, substances or preparations of substances which are intended, by application onto or into the body of a human or animal,
The pharmaceutical compositions according to the invention may be administered as liquid pharmaceutical preparations in the form of injection solutions, drops or syrups, as semi-solid pharmaceutical preparations in the form of granules, tablets, pellets, patches, capsules, plasters or aerosols and contain, in addition to the at least one subject of the invention, optionally excipients, fillers, solvents, diluents, dyes and/or binders, depending on the galenical form. The choice of auxiliary agents and the quantities thereof to be used depend on whether the pharmaceutical preparation is to be applied orally, stemerally, intravenously, intraperitoneally, intradermally, intramuscularly, intranasally, buccally, rectally or topically, for example to infections of the skin, the mucous membranes or the eyes. Preparations in the form of tablets, dragees, capsules, granules, drops and syrups are suitable for oral application, solutions, suspensions, easily reconstitutable dry preparations and sprays are suitable for stemeral, topical and inhalative applications. Subjects according to the invention in a deposit, in dissolved form or in a plaster, optionally with the addition of agents to promote skin penetration, are suitable percutaneous application preparations. Orally or percutaneously applicable preparation forms can release the compounds according to the invention after a delay. The amount of active ingredient to be administered to the patient varies according to the weight of patient, the method of application, the indication and the severity of the disease. 2 to 500 mg/kg of at least one subject according to the invention are usually applied. If the pharmaceutical composition is to be used, in particular, for gene therapy, a physiological sodium chloride solution, stabilizers, proteinase, DNAse inhibitors etc., are recommended as suitable auxiliaries or additives.
The present invention further relates to host cells, except for human germ cells, and human embryonic stem cells, which are transformed by at least one dsRNA according to the invention. dsRNA molecules according to the invention may be introduced into the respective host cell by conventional methods, for example transformation, transfection, transduction, electroporation or particle gun. During transformation, at least two dsRNAs which are different from one another are introduced into the cell, one strand of each dsRNA being complementary, at least in certain fragments, with the (m)RNA of a member of the VGLUT family, in particular complementary to the (m)RNA of VGLUT1, VGLUT2 or VGLUT3. The region of the dsRNA complementary with the (m)RNA of VGLUT1, 2 or 3 contains less than 25 successive nucleotide pairs.
Suitable host cells include any cells of a prokaryotic or eukaryotic nature, for example of bacteria, fungi, yeasts, vegetable or animal cells. Preferred host cells include bacterial cells such as Escherichia coli, Streptomyces, Bacillus or Pseudomonas, eukaryotic microorganisms such as Aspergillus or Saccharomyces cerevisiae or conventional baker's yeast (Stinchcomb et al. (1997) Nature 282: 39)
In a preferred embodiment, however, cells from multicellular organisms are selected for transformation by means of dsRNA constructs according to the invention. In principle, any higher eukaryotic cell culture is available as a host cell, although cells of mammals, for example monkeys, rats, hamsters, mice or humans, are quite particularly preferred. A large number of established cell lines is known to the person skilled in the art. The following cell lines are mentioned in a list, which is not exhaustive: 293T (embryonic renal cell line) (Grahan et al., J. Gen. Virol. 36:59 (1997), BHK (baby hamster renal cells), CHO (cells from hamster ovaries, Urlaub and Chasin, Proc. Natl. Accad. Sci. USA 77: 4216, (1980)), Hela (human carcinoma cells) and further cell lines—established in particular for laboratory use-, for example HEK293, SF9 or COS cells, wt-PC12 and DRG primary cultures. Quite particularly preferred are human cells, in particular neuronal stem cells and cells from the pain pathway, preferably primary sensory neurons. Human cells, in particular autologous cells from a patient, are suitable, after (in particular ex vivo) transformation with dsRNA molecules according to the invention, in other words after cell removal, optionally ex vivo expansion, transformation, selection and final retransplantation in the patient, quite particularly as pharmaceutical compositions for, for example, gene therapy.
A further preferred subject is also the use of at least one dsRNA according to the invention or pharmaceutical composition and/or of at least one cell according to the invention for producing a pharmaceutical preparation or pain remedy for the treatment of pain, in particular chronic pain, tactile allodynia, thermally triggered pain and/or inflammatory pain.
The subjects of the invention are suitable as pharmaceutical compositions, for example for nociception inhibition, for example by reducing the expression of at least one member of the VGLUT family, for example VGLUT1, -2 or -3, using dsRNA according to the invention.
Also disclosed is the use of at least one dsRNA according to the invention containing dsRNA according to the invention and/or a cell according to the invention for producing a pharmaceutical composition for the treatment of urinary incontinence; also of neurogenic bladder symptoms, pruritus, tumours, inflammation; in particular of VGLUT-associated inflammation with symptoms such as asthma; and of any disease symptoms associated with VGLUT family members.
The invention further relates to a process for the treatment, in particular pain treatment, of a non-human mammal or human, which requires the treatment of pain, in particular chronic pain, by administration of a pharmaceutical composition according to the invention, in particular those containing a dsRNA according to the invention. The invention further relates to corresponding processes for the treatment of pruritus and/or urinary incontinence.
A further preferred subject is also the use of at least one dsRNA according to the invention, in particular siRNA, and/or of a cell according to the invention for gene therapy, preferably in vivo or in vitro gene therapy. Gene therapy is understood to be a form of therapy during which, for example, an effector gene, usually a protein, and, in the present case, in particular dsRNA according to the invention, is expressed by the introduction of nucleic acids in cells.
A basic distinction is made between in vivo and in vitro processes. In the case of in vitro processes cells are removed from the organism and are transfected ex vivo with vectors and are then introduced back into the same organism or into another organism. With in vivo gene therapy, vectors, for example for combating tumours, are applied systemically (for example via the bloodstream) or directly into the tumour. According to a preferred embodiment, a vector is administered, which contains both the transcription element for the sense-dsRNA and the transcription element for the antisense-dsRNA under the control of suitable promoters. According to a further preferred embodiment, the two transcription elements may be located on different vectors.
A further preferred subject is also a diagnostic reagent containing at least one dsRNA and/or a cell according to the invention and optionally suitable additives. A diagnostic reagent herein denotes a compound or a process which may be used to diagnose a disease.
According to the invention, a further preferred subject is also a process for identifying pain-modulating substances. In a preferably upstream process step (a), over-expression of VGLUT, preferably VGLUT1, VGLUT2 or VGLUT3, takes place in a test cell. This over-expression in a test cell ensures that there is an increased concentration of VGLUT in these manipulated test cells, which are used for further examination, so the efficiency of potentially pain-modulating substances may be determined more accurately by means of scale expansion. However, in principle, cells that have not been manipulated in this manner, but nevertheless natively express VGLUT, may be used for the process according to the invention.
The preferably cultivatable cells, which may have been obtained by the placement upstream of process step (a), are subjected to the (in particular simultaneous) process steps (b) and (b′), namely (b) preferably genetic manipulation of at least one cell (test cell) with at least one dsRNA according to the invention and (b′) an (in particular simultaneous) comparative test (control test) with at least one identical cell (control cell). A comparative test of this type according to process step (b′) may follow different target directions, depending on the desired knowledge to be obtained. Various embodiments are therefore conceivable. The comparative test may thus, for example, be conducted with test cells that, in contrast to process step (b), are used without any genetic manipulation with dsRNA. Alternatively, however, control cells may also comprise an altered dsRNA, one not according to the invention, for example, or else be manipulated with a specific dsRNA that has a known effect on the VGLUT expression. Finally, process step (b′) may optionally also be omitted. In a process step (c), the test cells, which both express VGLUT and also, according to process step (b), comprise the substance to be tested, are incubated under suitable conditions. The test cells from process step (b) and the control cells according to process step (b′) are typically incubated simultaneously.
In a process step (d), for example, the binding of the test substance on the VGLUT-(m)RNA synthesized by the cells is then measured, preferably under suitable conditions. A preparation of the test cells manipulated with the test substance may, for example, be required for this purpose. Measurement of at least one of the functional parameters altered by the binding of the test substance, typically dsRNA, on the VGLUT-(m)RNA, for example, is, however, preferred. This altered parameter may, for example, be a quantifiable phenotype of the incubated cell that is adjusted by means of the binding of the test substance on VGLUT-(m)RNA, for example on the basis of the expression of the VGLUT protein suppressed by the binding. The measurement may also take place via immunofluorescence methods, for example, by means of which the concentration of VGLUT in the target cells is determined. However, the VGLUT that is over-expressed in the test cell by means of a process step (a) may (additionally) be configured with a reporter function. A fluorescence property connected to the over-expressed VGLUT by means of a corresponding gene construct (or the optional suppression of said property by means of the addition of a positively tested test substance according to the invention) would, for example, be directly measurable in the cell. Potentially pain-modulating substances are then identified, for example, via the extent of the difference between the measured value in the test cell and the measured value in the control cell, in a process step (e).
The dsRNA that is transferred into the test cells according to process step (b) or (b′) in the form of genetic manipulation, as a typical test substance of a process according to the invention, may also be transferred into the test cells via any alternative route. For example, the addition to the test cells may take place exogenously, optionally in conjunction with further chemical or physical measures known from the prior art, in order to ensure the absorption of the dsRNA into the cells, for example by means of electroporation, etc. Insofar as the dsRNA test substances applied exogenously to the test cells are unable per se to penetrate cellular membrane, their cellular membrane penetration capacity may also be increased by means of corresponding formulations, for example in liposomes or by coupling of known membrane penetration reinforcing agents, for example suitable polymers or transfection reagents.
The term pain-modulating refers to a potential regulating influence on the physiological occurrence of pain, in particular to an analgesic effect. The term substance covers any compound that is suitable as a pharmaceutical active ingredient, in particular therefore low-molecular active ingredients, but also others such as nucleic acids, fats, sugars, peptides or proteins such as antibodies. Incubation under suitable conditions herein means that the substance to be investigated can react with the cell or the corresponding preparation in an aqueous medium a defined time before measurement. The temperature of the aqueous medium may be controlled, for example at between 4° C. and 40° C., preferably at ambient temperature or at 37° C. The incubation time may be varied between a few seconds and a plurality of hours, depending on the interaction of the substance with the protein. However, times between 1 min and 60 min are preferred. The aqueous medium may contain suitable salts and/or buffer systems, so, for example, a pH between 6 and 8, preferably pH 7.0-7.5 prevails in the medium during incubation. Further suitable substances such as coenzymes, nutrients, etc. may be added to the medium. A person skilled in the art can easily determine suitable conditions as a function of the interaction of the substance to be investigated with the protein, on the basis of his experience, the literature or a few simple preliminary tests, in order thereby to obtain a measured value that is as clear as possible. A cell that has synthesized a protein is a cell which has already expressed this protein endogenously or a cell which has been genetically modified so it expresses this protein and accordingly contains the protein from the beginning of the process according to the invention. The cells may be cells from possibly immortalized cell lines or native cells originating from tissues and isolated from them, the cell assembly usually being dissolved. The preparation from these cells comprises, in particular, homogenates from the cells, the cytosol, a membrane fraction of the cells with membrane fragments, a suspension of isolated cell organelles, etc.
The criterion by which the process allows the discovery of useful substances is either the binding to the protein, which may be demonstrated, for example, by displacement of a known ligand or the extent of bound substance, or the alteration of a functional parameter by the interaction of the substance with the protein. This interaction may reside, in particular, in regulation, inhibition and/or activation of receptors, ion channels and/or enzymes. Altered functional parameters may be, for example, gene expression, ion milieu, the pH or the membrane potential, and the alteration of enzyme activity or the concentration of the second messenger. In the foregoing:
A further preferred embodiment of this process provides that the cell is genetically manipulated before process steps (b) and (b′).
A further preferred embodiment of this process provides that genetic manipulation allows the measurement of at least one of the functional parameters altered by the test substance.
A further preferred embodiment of this process provides that a form of a member of the VGLUT family, preferably VGLUT1, VGLUT2 or VGLUT3, which is not endogenously expressed in the cell, is expressed or a reporter gene is introduced by genetic manipulation.
A further preferred embodiment of this process provides that the bond is measured via the displacement of a known marked ligand of a member of the VGLUT family, preferably VGLUT1, VGLUT2 or VGLUT3.
A further preferred embodiment of this process provides that ≧8 hours, preferably ≧12 hours, in particular ≧24 hours elapse between the simultaneous process steps (b) and (b′) and process step (c).
The subjects according to the invention may be introduced into the cell in the above-described manner.
The invention will be described in further detail hereinafter with reference to the accompanying drawing figures, in which:
a shows siRNAs which have been produced in vitro: there are basically a plurality of ways of utilising RNAi technology: chemically synthesized siRNA may be used (see
(A) Chemically synthesized siRNA bypasses the dicing step, incorporates into the RISC and leads to sequence-specific mRNA degradation. (B) Long dsRNA is processed into active siRNAs by dicers. (C) Duplex hairpin RNA may be processed into active siRNAs by dicers. (D) Incomplete duplex hairpin RNA is processed into miRNAs by dicers, incorporated into miRNPs and leads to translation inhibition. Long dsRNA molecules (B), transfected in cells, are processed in short 19 to 21 bp siRNA molecules which lead to the degradation of complementary mRNA sequences. Chemically synthesizable single-stranded 21-mers imitate the siRNAs found in vivo and, after duplex formation, are used for relatively short transient RNAi effects in vitro and in vivo (Elbashir et al., 2001a; Holen et al., 2002; Yu et al., 2002). Four embodiments of dsRNA according to the invention against VGLUT family members are therefore described.
Plasmids which express dsRNA according to the invention, (in particular siRNA) (
(A) Long hairpin RNA expressed by RNA polymerase II leads, after dicer processing, to a plurality of siRNAs with a wide variety of sequence specificities.
(B) Tandem pol III promoters allow the expression of individual sense and antisense strands which accumulate in the cell to active siRNAs.
(C) An individual pol III promoter allows expression of a short hairpin-shaped (sh)RNA which is processed to active siRNA by dicers.
Using RNA polymerase III promoters such as U6 or H1, it is possible to express dsRNA and then siRNA molecules intracellularly and therefore to establish stable RNAi systems in mammalian cells (Brummelkamp et al., 2002; Lee et al., 2002; Miyagishi and Taira, 2002). Either the sense or antisense strands of various promoters may be read off and accumulate in the cell to 19-nt duplices with 4-nt overhangs, or the expression of hairpin structures is utilized. In both cases, effective, stable suppression of gene expression is achieved by the RISC-mediated RNAi process. Although the small size of a transcript which may be expressed by the pol III promoter does not initially impede siRNA technology, it restricts the number of different siRNAs which may be formed by a transcript (Myslinski, 2001).
The present invention is characterized in more detail by the following practical examples.
A) Design of siRNA Molecules
The design of the siRNA molecules according to the invention used corresponded to particularly preferred embodiments.
B) Production of the siRNA
The siRNAs used were ordered for synthesis by Eurogentec (EGT), on the one hand, and were self-made using the siRNA construction kit from Ambion (AMB), on the other hand. The following siRNA molecules were ordered for synthesis at Eurogentec:
The following siRNA molecules were produced using the Silencer™ siRNA construction kit from Ambion:
A proportion of the self-produced siRNAs was labelled with the dye Cy3 using the Silencer siRNA labelling kit from Ambion. The aforementioned sequences were used for the in vitro experiments described hereinafter.
Production of siRNA Using Silencer siRNA Construction Kit (Ambion)
In order to produce efficient transcription patterns for siRNA synthesis, the sense and antisense oligonucleotides have to be converted in dsRNA using T7 promoter at the 5′ end. This is achieved by hybridizing the two oligonucleotides with the T7 promoter primer and lengthening them by a subsequent DNA polymerase reaction (cf.
The sense and antisense siRNA templates are transcribed in separate reaction mixtures for 2 hours. The mixtures are then blended and the common reaction mixture incubated overnight. The separated transcription mixtures prevent potential competition around the transcription reagents between the templates, as this could limit the synthesis of one of the two strands of siRNA. Hybridization of the two siRNA strands is simplified by mixing the transcription mixtures and continuous RNA synthesis thus permitted, increasing the yield of dsRNA. The siRNA obtained by in vitro transcription has, at the 5′ end, overhanging leader sequences which have to be removed before transfection. This leader sequence is digested by an individual strand-specific ribonuclease. The DNA template is removed by DNase digestion in the same reaction mixture (cf.
The resultant siRNA has to be cleaned up from the mixture of nucleotides, enzymes, short oligomers and salts, using RNA columns.
The siRNA purified in this way is eluted in nuclease-free water and is then available for transfection.
Procedure
A 100 μM solution of each siRNA ONV was produced from the 200 μM stock solution. The following respective reaction mixtures were produced for hybridization of the siRNA ONV with the T7 promoter primer for sense and antisense:
The mixtures were first heated to 70° C. for 5 min, then kept at ambient temperature for 5 min. The following reaction mix was then fed to the reaction mixtures, carefully mixed and incubated for 30 min at 37° C.:
For both DNA formulations, a respective transcription reaction mixture was produced at ambient temperature in order to synthesize the sense and antisense ssRNA strands. For this purpose, the following components were combined in the specified sequence, were carefully mixed, without pipetting, and were incubated for 2 hours at 37° C.:
After the 2 hours, two transcription mixtures were pipetted together and incubated overnight at 37° C.
The following reaction mixture was made up to digest the hybridized dsRNA with RNase and DNase and was added to the dsRNA by pipetting, carefully mixed and incubated at 37° C. for 2 hours:
400 μl siRNA binder buffer were then fed to nuclease digestion and incubated for 2 to 5 min at ambient temperature. The filter membrane also supplied was, in the meantime, moistened with 100 μl siRNA. The siRNA was applied to the moistened filter in the siRNA binder buffer and centrifuged for 1 min at 10,000 rpm. The flow was discarded and the filter membrane washed twice with 500 μl of the siRNA washing buffer in each case and centrifuged (2 min at 10,000 rpm). The purified siRNA was then eluted in 100 μl 75° C. hot nuclease-free water and centrifuged off into a clean receiver (2 min at 12,000 rpm). The siRNA was stored at −20° C. or −80° C. until use.
Synthesis at Eurogentec and siRNA Duplex Formation
The RNA oligonucleotides synthesized by Eurogentec were brought into a 50 μM solution by means of DEPC-treated H2O and aliquoted. 30 μl of the RNA oligonucleotide solutions belonging together were mixed in each case with 15 μl 5× annealing buffer (final concentration: 20 μM siRNA duplex; 50 mM tris pH 7.5-8.0; 100 mM NaCl in DEPC-H2O). The solution was heated for 1 to 2 min in a water bath at 90 to 95° C. and left to cool for 45 to 60 min at ambient temperature. The siRNA was stored at −20° C. until use.
Labelling of the siRNA with Cy3
The siRNAs synthesized by Eurogentec as well as the self-produced duplex siRNAs were used for siRNA labelling with the fluorescence dye Cy3. The following reaction mixture was produced and incubated for 1 hour at 37° C. in order to label 5 μg siRNA:
The Cy3-labelled siRNA was purified with ethanol precipitation. For this purpose, 0.1 volume 5 M NaCl and 2.5 volumes 100% ethanol were added to the reaction mixture, thoroughly mixed and stored for 60 min at −80° C. The precipitate was pelletized by centrifugation for 20 min (>8,000×g), the supernatant being carefully removed without destroying the pellet, and was finally washed with 175 μl 70% ethanol. After centrifuging off (5 min at >8,000×g), all the supernatant was removed, the pellet dried at ambient temperature for 5 to 10 min and finally dissolved in a corresponding amount of nuclease-free water (19.2 μl in this case).
In order to test the efficacy of the siRNAs produced, they were used in various in vitro models and protein expression was then determined by immunocytochemistry.
A) Optimization of the Transfection Conditions
Highly efficient siRNA gene suppression necessitates not only the actual effectiveness of the siRNA but also a high transfection rate of the respective cells. The cell density, the amount of transfection reagent and the concentration of the siRNA play an important part in it. While varying these various parameters, the transfection rate (RT=number of transfected cells/total number of cells) was determined using Cy3-labelled siRNA. Lipofectamine™ 2000 was used as the transfection reagent.
Trasfection of spinal ganglion cells in a primary culture proved extremely difficult. As shown in
The maximum transfection rates (RTmax≈80%) were achieved with cells in cell lines PC12 MR-A and PC12 MR-B, as shown, for example, in
Normal wt-PC12 cells may also be transfected well with siRNA.
B) Suppression of VGLUT Expression by siRNA in Various in vitro Models
1. Suppression of Endogenous VGLUT Expression in DRG Primary Cultures
The cells of DRG primary cultures were transfected with various siRNAs directly after dissociation of the ganglia and purification of the cell suspension with the transfection reagent LF2000™. The transfected cells were either seeded in normal culture dishes or cultivated in a 24-well plate on poly-L-lysine-coated cover slips.
DRG primary cultures, which had been transfected with Cy3-labelled siRNA (Cy3-si-rVGLUT2 166-186 AMB), were fixed 48 hours after transfection and characterized immunocytochemically with respect to theirVGLUT2 protein expression.
Approximately 2% transfected cells, which could be detected by their Cy3 dye, were on the siRNA-treated cover slips. None of these siRNA-transfected cells showed a clear protein signal for VGLUT1 after immunocytochemical labelling of the vesicular glutamate transporterVGLUT2. On the other hand, all VGLUT2-positive cells were without signals of the Cy3-labelled siRNA.
2. Suppression of Endogenous VGLUT Expression in Established Cell Lines
The differentiated cell lines PC12 MR-A and PC12 MR-B express the vesicular glutamate transporterVGLUT1 and VGLUT2.
Under optimized conditions, these cells were transfected with siRNA against VGLUT2 (si-rVGLUT2 100-120 EGT). As shown in
3. Suppression of VGLUT2 Expression in Transiently Transfected wt-PC12 Cells
Cells of the normal wt-PC12 cell line express only VGLUT1 and no VGLUT2. For this reason, these endocrine vesicle-producing cells are suitable as a model system for suppression experiments after transient VGLUT2 transfection. An EGFP vector, of which the gene product, the green fluorescent protein (GFP), may be detected directly by a fluorescence microscope was used to optimize DNA transfection. An EGFP transfection rate of ˜40% was achieved by optimising the DNA transfection conditions. A similar high transfection rate could also be achieved in the case of transfection with rVGLUT2 plasmids (
The siRNA suppression experiments were configured in different ways:
Co-transfection of siRNA cDNA vector
siRNA transfection 6 hours before cDNA
cDNA transfection 24 hours before siRNA transfection.
Co-transfection was initially carried out with rVGLUT2 plasmids and various siRNAs. Three siRNAs against VGLUT2 were used, as well as siRNAs against VGLUT1, VGLUT3 and a mismatch siRNA for specificity control. The cells were fixed 24 hours after transfection and the VGLUT2 protein expression detected by immunocytochemistry. Evaluation was carried out on a fluorescence microscope, the VGLUT2-positive cells being counted out both manually and by digital image analysis (MCID).
On the other hand, the siRNAs against VGLUT1 and against VGLUT3 and the mismatch siRNA do not significantly (p>0.05) influence the content of the VGLUT2-positive cells (A).
If the efficiency of the siRNAs used is calculated from these results as a percentage of the reduction of VGLUT2-positive cells, based on the VGLUT2 expression without the influence of siRNA treatment, the result shown in
It may be seen that the siRNAs directed against VGLUT2 have high efficiency in comparison with ineffective mismatch siRNA. They reduce the content of VGLUT2-expressing cells by 79 to 82%, the self-produced siRNAs acting more effectively in the concentrations used than the siRNA synthesized at Eurogentec. Whereas the siRNA against VGLUT3 and the mismatch siRNA do not exert a significant effect on VGLUT2 protein expression, the siRNA directed against VGLUT1 with an efficiency of ˜47% appears to act non-specifically on VGLUT2 expression. However, the siRNAs against VGLUT2 are significantly more efficient (p<0.001) than the siRNA against VGLUT1.
The transfection experiment has been modified hereinafter: the wt-PC12 cells were treated with the various siRNAs 6 hours before transfection with rVGLUT2 plasmid so the siRNAs were already in the cells at the moment of DNA transfection.
The influence of the existing VGLUT2 protein level on the suppression efficiency of the siRNAs was tested in a further experiment. This model corresponds rather to the endogenously VGLUT2-expresing cells and the situation in vivo. For this purpose the wt-PC12 cells were transfected with the rVGLUT2 plasmid 24 hours before the siRNA treatment. The proportion of VGLUT2-positive cells was determined after a further 24 hours.
The results of these experiments demonstrate the specific effectiveness of the siRNAs directed against VGLUT2 (si-rVGLUT2 100-120 EGT; si-rVGLUT2 100-120 AMB; si-rVGLUT2 166-186 AMB). There is no difference between the self-produced siRNA (si-rVGLUT2 100-120 AMB) and the siRNA synthesized by Eurogentec (si-rVGLUT2 100-120 EGT). The siRNA controls against VGLUT1 (si-rVGLUT1 739-759 EGT) and against VGLUT3 (si-rVGLUT3 220-240 EGT) do not lead to a reduction in the VGLUT2 protein level. What is known as the mismatch siRNA (si-rVGLUT2 MM EGT), which has the same nucleotides as the specific siRNA against VGLUT2, but in a random and therefore non-complementary arrangement, does not influence the VGLUT2 protein level either.
In these experiments, the efficiencies of the specific siRNAs lie between ˜15 and ˜80%. High siRNA efficiencies (78 to 82%) are achieved with simultaneous co-transfection of siRNA and DNA, whereas lower efficiencies (15 to 23%) are achieved in the suppression experiments with existing VGLUT2 protein levels at the moment of siRNA transfection. This shows that, firstly, the specific siRNAs highly efficiently reduce VGLUT2 protein formation and, secondly, the VGLUT2 proteins are very stable and have only a low turnover.
The spinal ganglia from neonatal rats were prepared and cultivated as described under the experimental conditions recited in more detail after the practical examples. 20 to 30 respective spinal ganglia from 8 to 16 neonatal rats were prepared for the cultures and the cells were purified. The proportions of neurons in the total number of cells was significantly increased relative to the standard procedure by purification over a BSA column, and by plating out the cell suspension onto poly-L-lysine-coated materials (Grothe and Unsicker, 1987). The neurons, which are much larger and therefore heavier than the non-neuronal cells, settle more rapidly on the coated support. The number of non-neuronal cells, which present predominantly as spindle-shaped cells with branches, is reduced after only 5 min by removing the supernatant. After 4 to 10 days in vitro, the cells were fixed and characterized immunocytochemically.
As the primary sensory nociceptive neurons would be of particular interest for the subsequent experiments, immunocytochemical labels indicated that this neuron population is present in the primary culture and may be cultivated in vitro for at least 8 days.
Protein expression behaves in a corresponding manner for VGLUT1 (not shown): VGLUT1 is expressed in a subpopulation of PGP9.5-positive neurons and does not occur in Schwann cells.
The expression of the vesicular glutamate transporters VGLUT1 and VGLUT2 was then investigated in peptidergic CGRP-positive neurons (
Double immunofluorescence for VGLUT2 and TRVP1 was carried out in order to investigate VGLUT2 expression in polymodal nociceptors. As shown in
Bennett's pain model of the rat was used for this purpose. The analgesic effect of the siRNA according to the invention was investigated in vivo in the rat model. An SiRNA directed against the target sequence of VGLUT2 AAGCAGGATAACCGAGAGACC was used as the active component for this purpose. The two strands of this double-stranded siRNA have the following sequences: r(GCAGGAUAACCGAGAGACC)dTT and r(GGUCUCUCGGUUAUCCUGC)d (TT).
The control siRNA is directed against the following target sequence (AACGACTAGCAAAGCGAGCCA) (no VGLUT2 sequence). The strands of the double-stranded control siRNA each have the following sequences: r(GGACUAGCAAAGCGAGCCA)d(TT) and r(UGGCUCGCUUUGCUAGUCC)d(TT).
All the aforementioned sequences were chemically synthesized (Xeragon, Germantown, SA) and their purity checked by MALDI-TOF analysis. The sequences were dissolved in 0.9% NaCl solution.
Neuropathic pain occurs inter alia after damage to peripheral or central nerves and may accordingly be induced and observed by intentional lesions to individual nerves in animal experiments. Bennett's nerve lesion (Bennett and Xie 1988) Pain 33: 87-107) is one animal model. In Bennett's model, the sciatic nerve is provided unilaterally with loose ligatures. The development of signs of neuropathic pain is observed and may be quantified by thermal or mechanical allodynia.
For this purpose, five male Sprague-Dawley rats (Janvier, France) weighing 140 to 160 g were initially anaesthetized with Pentobarbital (50 mg per kg body weight of the rat Nembutal®, i.p. Sanofi, Wirtschaftsgenossenschaft deutscher Tierärzte eG, Hanover, Germany). One-sided multiple ligatures were then formed on the right-hand main sciatic nerve of the rat. For this purpose the sciatic nerve was exposed halfway along the femur and four loose ligatures (softcat® chrom USP 4/0, metric2, Braun Melsungen, Germany) were bound round the sciatic nerve in such a way that the epineural circulation of blood was not interrupted. The date of the operation was day 1. Measurements were taken I week after ligature of the nerve.
The allodynia was tested on a metal plate of which the temperature was controlled to 4° C. by means of a water bath. To check the allodynia, the rats were placed on the cold metal plate, which was located in a plastic cage. The frequency with which the animals flinched violently from the cooled metal plate with their damaged paw was then counted over a period of 2 minutes prior to application of a solution (preliminary value). The solutions containing 3.16 μg (5 μl) of siRNA according to the invention in 15 μl NaCl or 3.16 μg (5 μl) control RNA (sense strand of the siRNA) in 15 μl NaCl i.t. or NaCl solutions (5 μl) were then applied for control purposes after a single acute intrathecal application under ether narcosis, and the number of retraction reactions was again counted for 2 min after 60 min in each case (test value). The measurements were taken on 4 successive days at respective intervals of 24 hours (days 2-5) for 3 different respective doses (0.001, 0.01 and 0.1 μg/animal). Animals to which pure NaCl solution was applied were used in the experiments both with siRNA and with control RNA as a comparison group.
The siRNA according to the invention against VGLUT2 showed a pronounced analgesic effect in this pain model, namely clear inhibition of cold allodynia without dose dependency with the best effect with the lowest dose group (1 ng/animal). With the highest dose, the animals showed increased spontaneous activity both in the control group and in the verum group. This could be the reason for the weaker effect in the high dose group. There were no further side effects (cf.
The materials and methods used and mentioned in the aforementioned practical examples and specific experimental conditions are described in more detail hereinafter:
1. Materials Used
1.1 Experimental Animals
All adult experimental animals were male or female Wistar rats (300 g) and were obtained from Charles River (Sulzfeld) and from the German Experimental Animal Institute (Hanover). The animals were kept in a 12 h/12 h day/night rhythm with free access to food and water. At least 4 days, in which the health of the animals was monitored, elapsed between supply of the animals and the beginning of the experiment. The neonatal rats originated from an individual breed of male and female Wistar rats which were covered at regular intervals. All neonatal animals were used for the production of primary cultures from P0 (postnatal day 0) to P5.
1.2 Cell Lines
wt-PC12
The immortal tumour cell line PC12 was isolated from a tumour of the adrenal marrow of the rat in 1976 (Greene and Tischler, 1976). The cells grow in a non-adherent manner, lead to transplantable tumours in rats and react reversibly to NGF (nerve growth factor) with the formation of neuron-like projections. The PC12 cell line was made available to Grünenthal's laboratory (Aachen), which, in turn, obtained the cell line from ATCC. The cells were cultivated in a modified DMEM medium. These PC12 cells are designated hereinafter as wtPC12 (wild type) for better distinction.
PC12 MR-A and PC12 MR-B
These two cell lines are mutated or differentiated variants of the wt-PC12 cells which were originally obtained from Dr. Reiner Westermann (Institute for Anatomy and Cell Biology, Marburg) from ATCC. In contrast to their wt mutants, the cells grew adherently, and were flat and spindle-shaped and formed projections without the addition of NGF. References in the literature lead to the assumption that these variants could form a glutamatergic phenotype (Jimenez et al, 2003; Zheng et al, 1996).
1.5 Chemicals
Any chemicals not listed in detail here were obtained from Fluka (Buchs, Switzerland), GibcoBRL (Eggenstein), Merck(Darmstadt), Riedel de Haen (Seelze), Roche (Basel, Switzerland), Roth (Karlsruhe), Serva (Heidelberg) and Sigma (Deisenhofen), unless stated otherwise in the method section.
1.6 Buffer and Solutions
1.7 Media and Media Additives
I. Basic Media
All media were obtained from GibcoBRL (Eggenstein):
DMEM (Dulbecco's modified Eagle Medium) with a high glucose content
II. Media Additives
Composition of the Growth Media
Medium for PC12 cells: RPMI with
10% Horse serum
5% FCS
1% Glutamine (200 mM)
1% Antibiotic mix (100×)
Medium for spinal ganglion cells: DMEM with
10% FCS
1% Antibiotic mix (100×)
1% Glutamine
10 μM Cytosine arabinoside
25 ng/ml NGF
1.8 Enzymes and other proteins
1.9 Nucleic Acids and Vectors
1.10 Oligonucleotides
All oligonucleotides were designed with the oligo 4.0 programme itself, checked for undesirable sequence homology using BLAST and the synthesis ordered from MWG-Biotech(Ebersberg).
1.11 Kits
1.12 Antibodies and Detection Systems
The various cell types were detected with primary antibodies against generally recognized, readily characterized epitopes (labels) in the respective cell type. These are PGP9.5 for neurons, GFAP for astrocytes and S100 for oligodendrocytes. In addition to these, further antibodies against specific proteins were used. The optimum concentrations of the primary antibodies were titrated out in each case. Table 1 gives an overview of the primary antibodies used and also the working dilutions thereof.
The primary antibodies were then detected by fluorochrome-coupled secondary antibodies or fluorochrome-coupled streptavidin (Table 2) which interacted with species-specific biotinylated antibodies. Cy3 (red fluorescence) or Alexa 488 (green fluorescence) were used as fluorochromes.
The biotinylated Isolectin B4 (Sigma, Deisenhofen) was also used in a concentration of 1:20.
2. Methods
2.1 Removal and Treatment of Tissue
The animals were killed by CO2 inhalation and subsequent decapitation. The tissue required for the various purposes was then removed in different ways:
Nucleic Acid Extraction:
After removal, the tissue was spread over ice, placed in a cryotube and, after shock-freezing in liquid nitrogen, the tissue was initially stored on dry ice and then at −70° C.
Primary Culture:
For the application of primary cultures, the tissue (spinal ganglia) was immediately removed on self-produced ice-cooled preparation dishes and initially collected in 1× CMF medium for further treatment. The calcium-free and magnesium-free 1× CMF medium consisted of 10% HBSS (10×), 1% antibiotic mix and 0.2% phenol red (0.5%). The pH was titrated with bicarbonate (7.5%) and could be detected by the cherry-red color of the indicator.
2.2 Application of Primary Cultures
For application of the primary cultures, neonatal rats from stages P0 to P5 were used for the spinal ganglion cell culture and P0 to P3 for the cerebellum cultures. The animals were disinfected with alcohol and killed by decapitation with sterile shears.
2.2.1 Neuronal Primary Cultures of Spinal Ganglia
The spinal ganglia were prepared by C-Grothe's modified procedure (Grothe and Unsicker, 1987).
Dissection of the Spinal Ganglia
The body of the killed rat was fixed ventrally on a cork board and the vertebral column exposed by removing the skin and the muscles at the back of the neck and shoulders. The vertebral column was then opened from the caudal end to the cranial end and the bone marrow displayed. In the juvenile animals, the bone marrow was left in the spinal canal and the opened vertebral column removed in its entirety for fixing in a cooled preparation dish. Only then was the bone marrow carefully removed in steps and the spinal ganglia taken from the exposed intervertebral holes.
The dissected spinal ganglia were collected in a cooled Petri dish with 1× CMF medium until fine preparation. The spinal ganglion was cleaned of nerves, connective tissue and blood residues under the binocular device, and was then transferred into an ice-cooled tube filled with 1× CMF medium.
Dissociation and Purification of the Spinal Ganglion Cells
After removal of the medium, the spinal ganglia were incubated for 30 to 45 min at 37° C. and 5% CO2 for chemical dissociation with an enzyme mixture of 0.075% collagenase and 0.15% dispase in CMF medium. Adhesion of the ganglia was to be prevented by repeated shaking during incubation. Half of the enzyme mixture was removed on completion of incubation and the same volume of a 0.25% trypsin solution fed to the remaining residue. After incubation for 15 to 25 min at 37° C. and 5% CO2, the enzyme solution was removed to 300 μl and the ganglia in this volume were mechanically dissociated. Three siliconized sterile Pasteur pipette orifices of different sizes, produced under the Bunsen burner flame were used for this purpose. By increasingly reducing the orifice diameter, a uniform cell suspension of the spinal ganglia could be produced. The cell suspension was finally centrifuged for 5 min at 1000 rpm, the supernatant discarded and the cell pellet resuspended in ˜1 ml CMF. To concentrate the culture with neuronal cells, the cell suspension was coated over a 20% BSA column (20% BSA w/v in 0.5 ml CMF medium) and then centrifuged for 5 min at 1000 rpm. The medium was suction filtered to ˜100 μl and resuspended in 1 ml fresh medium. The later culture medium (DMEM/FCS) was used for this purpose.
Plating Out of the Cells
After determining the number of cells, the spinal ganglion cells were distributed over the culture vessels, according to the subsequent use, and cultivated at 37° C. in 5% CO2. A glucose-rich DMEM medium with various additives was used for cultivation purposes (exact composition described under the heading: Cell Culture). In addition to glutamine and an antibiotic mix, the nerve growth factor NGF 7S was supplied in a concentration of 25 ng/ml, as the survival and the differentiation of the neonatal neurons of the spinal ganglion are NGF-dependent. In addition, the mitose inhibitor, cytosine arabinoside, was added in a concentration of 10 μM to the medium to prevent proliferation of the non-neuronal cells. The plated out spinal ganglion cells could be cultivated without difficulty for up to 2 weeks by changing the medium every 2 to 3 days.
2.3 Eukaryotic Cell Culture
Cultivation and Passaging of Eukaryotic Cells
All the aforementioned cell lines work best in their respective growth medium at 37° C. in the gasification incubator at 95% relative atmospheric humidity and 5% CO2. Media and solutions were heated to 37° C. prior to use.
The adherently growing cell lines (F-11, wt-PC12 MR-A, PC12 MR-B) were routinely split every 4 to 6 days. For this purpose, they were treated with trypsin/EDTA solution (0.05% trypsin, 0.02% EDTA in PBS) for a few minutes at 37° C. (microscopic control). Trypsin is a proteolytic enzyme which hydrolyses peptide bonds of cell/cell bonds in which the carbonyl group is taken from the lysine or arginine. Slight traces of medium can impair the effect of trypsin, which is why the cells had to be washed with phosphate-buffered calcium-free and magnesium-free salt solution (PBS) prior to the trypsin treatment. The trypsin used could be inactivated again by addition of medium with addition of FCS. The chelator EDTA binds calcium which is required by some cell/cell bonds.
Once the cells had dissolved, they were transferred with serum-containing medium from the culture vessel into a Falcon tube. After centrifuging and suction filtering the old medium, the cell pellets were resuspended in 5 ml fresh medium and plated out into the respective culture vessels in their growth medium, depending on the subsequent use.
The wt-PC12 cells are suspension cells. For passaging, the culture vessels were placed obliquely so the wt-PC12 cells growing in grape-like heaps sedimented gradually in a corner of the vessel. The supernatant, which also contained the lighter cell debris, was removed and the cells transferred into a Falcon tube with fresh medium. The suspension was then centrifuged for 5 min at 1000 rpm, the supernatant was removed and the cells were plated out in fresh growth medium.
Freezing of Eukaryotic Cells
The cells were frozen in DMSO-containing medium in liquid nitrogen for long-term storage, in order to protect them from genetic modification and to minimize the risk of contamination. Without the addition of reagents, which act as cryoprotection for the cells, most mammalian cells die when frozen. The mortality of cells is minimized by DMSO in the medium, as the freezing point is lowered and the cooling process therefore decelerated.
After separation with trypsin/EDTA solution, the cells were initially washed with PBS and the number of cells determined using the Neubauer counting chamber. The amount of cells to be frozen was placed in a Falcon tube, and the supernatant discarded after centrifugation at 1000 rpm. The cell pellet was finally resuspended in the freezing medium (90% growth medium, 10% DMSO) and aliquoted in Nunc tubes with 2 ml in each case (for example with 2 million cells). The tubes were frozen at −80° C. for several hours in a cryofreezing unit with a cooling rate of 1° C. per minute and were then stored in liquid nitrogen.
Revitalization of Eukaryotic Cells
In some cases, cells were recultivated. For this purpose, they were heated rapidly to 37° C. in a water bath after removal from the nitrogen tank (−196° C.). The cell suspension was removed, transferred into 5 ml preheated medium and sedimented in the centrifuge (3 min, 200×g). The medium was suction filtered, the target pellet resuspended in fresh medium and transferred into a cell culture dish. On the next day, the cells were washed with PPS and supplied with fresh medium.
Determination of Number of Cells
A Neubauer counting chamber was used to determine the number of cells. For this purpose, a drop of the cell suspension was applied to the Neubauer counting chamber and the number of cells (Z=cells/0.01 mm2) counted out under the microscope from four corner squares and the number of cells per ml determined (number of cells=Z×2500) (Amiri et al.).
Mycoplasm Test
All cell lines were tested at regular intervals for mycoplasm contamination. Mycoplasms are obligate parasitic bacteria. They are wall-less, very small intracellular parasites and cannot propagate independently of the host cell. As they have only one cell membrane, but no bacterial wall of murein, they do not have a fixed form and are insensitive to penicillin. Their size varies between 0.22 and 2 μm. Filtration through a membrane with a pore size of 0.1 μm allows separation of mycoplasm. Contamination with mycoplasm may be detected most rapidly by staining the mycoplasm DNA with the fluorochrome DAPI (4-6-diamidino-2-phenylindol-di-hydrochloride) which binds specifically to DNA. In the case of mycoplasm contamination of cell cultures, individual fluorescing points are found in the cytoplasm and sometimes also in the intercellular space.
For the mycoplasm test, a corresponding amount of DAPI stock solution (1 mg/ml, 10 mg DAPI dissolved in 10 ml water, aliquoted and stored at −20° C.) was diluted with methanol to a working concentration of 1 μg/ml (stable for about 6 months at 4° C.). To stain the cells, the cells cultivated on cover slips or Petri dishes were washed once with the working solution and incubated for 15 min at ambient temperature with the working solution. The solution was washed once with methanol and the cover slips were embedded in a drop of glycerine or PBS then evaluated under the fluorescence microscope.
Coating of Culture Vessels
For certain experiments, the culture vessels or cover slips were coated to assist adhesion of the cells. As coating materials, poly-L-lysine (0.1 mg/ml) was used for wt-PC12 cells or poly-D-lysine (0.5 mg/ml) for the cerebellum culture. The culture vessels were incubated for at least 2 hours at ambient temperature with poly-L-lysine and poly-D-lysine, then washed twice with H2O and coated with H2O and stored at 4° C. until use. Whereas the poly-L-lysine coated materials were used in the moist state, the poly-D-lysine coated materials were not used until they had been dried under the sterile bench.
Transfection in Eukaryotic Cells
Transfection is understood to be the introduction of extraneous DNA or RNA into eukaryotic cells by physical or chemical methods. The most important chemical method for the transfer of nucleic acids is lipofection: reagents from cationic lipids form small (100-400 mm) unilamellar liposomes under optimum conditions in aqueous solution. The surface of these liposomes is positively charged and is electrostatically attracted both by the phosphate backbone of the nucleic acids and by the negatively charged cell membrane (Gareis et al., 1991; Gershon et al., 1993; Smith et al., 1993). The nucleic acids are not enclosed within the liposomes, but bind spontaneously to the positively charged liposomes and form DNA/RNA lipid complexes (Felgner et al., 1987). There are indications that the complexes are incorporated via the endosomal or lysosomal pathway (Coonrod et al., 1997).
Transfection of DNA by Liptofectamine™ 2000
For the transfection of DNA in eukaryotic cells, the cationic lipid reagent Liptofectamine™ 2000 (LF2000™) was predominantly used, while adhering to the following general recommendations from the manufacturer, Invitrogen:
A DNA to LF2000™ ratio of 1:2 to 1:3 was recommended for producing DNA-LF2000™ complexes. A cell density of 90 to 95% should exist at the moment of transfection, to achieve high efficiency and a high expression level. Antibiotics were not added during transfection, as they would trigger cell death. Transfection under various conditions was carried out with an EGFP vector to optimize the efficiency of transfection. To measure the efficiency of transfection, the protein expression of the green fluorescing protein GFP was determined semi-quantitatively using a fluorescence microscope.
Tranfection of DNA in wt-PC12 Cells
2.5×105 wt-PC12 cells were plated out in a 24-well plate on poly-L-lysine-coated cover slips in 0.5 ml medium on the day before transfection. The medium was the normal growth medium for wt-PC12 cells, but without the addition of antibiotics. 0.8-1 μg DNA was dissolved in 50 μl OPTI-MEM® for each well to be transfected. In addition, 1-2 μl LF2000™ were diluted in 50 μl OPTI-MEM® for each well and incubated for 5 min at ambient temperature. The dissolved DNA was now mixed with the diluted LF2000™ solution and incubated for 20 min at ambient temperature to form the DNA-LF2000™ complexes, and 100 μl of the complexes then placed directly in the corresponding wells, which were mixed by careful swinging. The cells were cultivated for a further 24-72 hours in the same medium under normal conditions until analysis of the expression.
Transfection of DNA in Spinal Ganglion Cells
For transfection, 5×105 die cells of the freshly purified DRG suspension were used, which were plated out in a 24-well plate on poly-L-lysine-coated cover slips in 0.5 ml growth medium without the addition of antibiotics. 0.8-1 μg DNA was dissolved in 50 μl OPTI-MEM® for each well to be transfected. In addition, 1-2 μl LF2000™ were diluted in 50 μl OPTI-MEM® for each well and incubated for 5 min at ambient temperature. The dissolved DNA was now mixed with the diluted LF2000™ solution and incubated for 20 min at ambient temperature to form the DNA-LF2000™ complexes, and 100 μl of the complexes then placed directly in the corresponding wells, which were mixed by careful swinging. The cells were cultivated for a further 24-72 hours in the same medium under normal conditions until analysis of the expression.
Transfection of siRNA
The transfection of siRNA in eukaryotic cells was carried out using Lipofectamine™ 2000. LF2000™ has already been successfully used for RNAi experiments in mammalian cells by other groups (Gitlin et al., 2002; Yu et al., 2002). As with the transfection of DNA, the optimum transfection conditions had to be determined experimentally. Cy3-labelled siRNA was used for this purpose.
Transfection of siRNA in wt-PC12 Cells
2.5×105 wt-PC12 cells were plated out in a 24-well plate on poly-L-lysine-coated cover slips in 0.5 ml medium on the day before transfection. The medium was the normal growth medium for wt-PC12 cells, but without the addition of antibiotics. 20-100 pmol siRNA were dissolved in 50 μl OPTI-MEM® for each well to be transfected. In addition, 1-2 μl LF2000™ were diluted in 50 μl OPTI-MEM® for each well and incubated for 5 min at ambient temperature. The dissolved siRNA was now mixed with the diluted LF2000™ solution and incubated for 20 min at ambient temperature to form the DNA-LF2000™ complexes, and 100 μl of the complexes then placed directly in the corresponding wells, which were mixed by careful swinging. The cells were cultivated for a further 24-72 hours in the same medium under normal conditions until analysis of the expression.
Transfection of siRNA in Cells of the PC12 MR-A and PC12 MR-B Lines
2.5×105 PC12 cells were plated out in a 24-well plate on cover slips in 0.5 ml medium on the day before transfection. The medium was the normal growth medium for PC12 MR-A/B cells, but without the addition of antibiotics. 20-200 pmol siRNA were dissolved in 50 μl OPTI-MEM® for each well to be transfected. In addition, 1-2 μl LF2000™ were diluted in 50 μl OPTI-MEM® for each well and incubated for 5 min at ambient temperature. The dissolved siRNA was now mixed with the diluted LF2000™ solution and incubated for 20 min at ambient temperature to form the DNA-LF2000™ complexes, and 100 μl of the complexes then placed directly in the corresponding wells, which were mixed by careful swinging. The cells were cultivated for a further 24-72 hours in the same medium under normal conditions until analysis of the expression.
Transfection of siRNA in Cells of the DRG Primary Cultures
For transfection, 5×105 die cells of the freshly purified DRG suspension were used, which were plated out in a 24-well plate on poly-L-lysine-coated cover slips in 0.5 ml growth medium without the addition of antibiotics. 20-200 pmol siRNA were dissolved in 50 μl OPTI-MEM® for each well to be transfected. In addition, 1-2 μl LF2000™ were diluted in 50 μl OPTI-MEM® for each well and incubated for 5 min at ambient temperature. The dissolved siRNA was now mixed with the diluted LF2000™ solution and incubated for 20 min at ambient temperature to form the DNA-LF2000™ complexes, and 100 μl of the complexes then placed directly in the corresponding wells, which were mixed by careful swinging. The cells were cultivated for a further 24-72 hours in the same medium under normal conditions until analysis of the expression.
Toxicity Study
An assay with trypan blue was carried out to investigate the cytotoxic influence of the transfection reagent LF2000™ on the cells. For this purpose, 3.0×104 cells were plated out in a 24-well plate 24 hours before transfection. Transfection was carried out with siRNA negative controls and different amounts of transfection reagents. All cells were washed with PBS 48 hours after transfection and stained with 10% trypan blue. The survival rate was calculated as follows:
Vitality rate=(total number of cells−number of stained cells)/total number of cells×100.
Fixing of Cells
The cells were fixed either for 20 min at ambient temperature in PBS with 3-4% (v/v) paraformaldehyde or for 15 min at ambient temperature with methanol (−20% ° C).
2.4 Microbiology
Competent bacteria were used to amplify double-stranded DNA fragments. For this purpose, the DNA to be amplified was incorporated into plasmids with selection labels and transformed into bacteria. Bacteria were plated out on selective agar plates and corresponding clones were removed and cultured in antibiotic-containing LB medium. The plasmids with insert were later isolated from the propagated bacteria and further processed.
Production of Transformation-Competent Cells
Treatment with ice-cold magnesium and calcium chloride solution enables the bacteria to absorb extraneous DNA spontaneously (transformation). The E. coli strains DH5α and XL-1 blue were used to produce the competent bacterial cells. For this purpose, a cell culture was placed in TYM medium (LB-medium+10 mM MgSO4) and incubated until an adequate density (OD600 nm=0.4-0.6) was achieved. The cells should be in the log phase. After brief centrifugation and pouring off the supernatant, the pellet was resuspended in 40 ml Tfb1-buffer (30 mM KAc, 50 nM MgCl2×2H2O, 100 mM KCl, 10 mM CaCl2×2H2O, 15% glycerol) per 100 ml culture. After 45 min incubation on ice, the cells were pelletized again and incorporated in 1/10 volume Tfb2-buffer (10 mM Na-MOPS, 10 mM KCl, 75 mM CaCl2×2H2O, 15% glycerol). The competent cells were stored as 100 μl aliquots at −70° C. until use.
Transformation of E. coli
Competent bacteria (approximately 108 clones per μg plasmid DNA) were reacted with plasmid DNA, and incubated for 45 min on ice and just 1 min at 42° C. LB medium (Sambrook et al., 1989) was then added and the mixture incubated for a further 30 min at 37° C. while shaking. The bacteria were separated on selective agar plates (Sambrook et al., 1989) and incubated overnight at 37° C. Precultures (5 ml LB medium with a suitable antibiotic) were inoculated with separated colonies and shaken for several hours at 37° C. Preparatory cultures (100 ml LB with a suitable antibiotic) were inoculated 1:1000 with the precultures and incubated overnight at 37° C. while shaking.
Analytical Plasmid Isolation
In order to isolate the plasmids from the bacteria, the bacteria have to be lysed. The plasmids are then separated by centrifugation of proteins and genomic bacteria-DNA. The plasmids were purified by column chromatography with commercial kits from Qiagen. The bacteria were pelletized by centrifugation for 5 min at 5000×g, the supernatant was discarded and the pellet resuspended in 250 μl buffer P1. The bacteria were lysed by addition of 250 μl buffer P2 and the solution was neutralized by a further 350 μl buffer N3. The mixture was then centrifuged for 10 min (the centrifugation steps were carried out at maximum speed, unless otherwise stated), the supernatant being carefully removed and transferred into a QIAprep column. After 1 min centrifugation, the column was again washed with 750 μl buffer PE and in turn centrifuged for 1 min. The plasmid DNA was eluted into a clean receiver by addition of 30-50 μl H2O onto the column and subsequent centrifugation for 1 min. For quality control, 5 to 10 ml of this mixture were analysed by restriction digestion and subsequent agarose gel electrophoresis.
Quantitative Plasmid Isolation
Relatively large amounts of plasmid DNA with a high degree of purity for carrying out transfections were isolated using the Qiagen Plasmid Maxi Kit. For this purpose, a preculture was prepared during the day, i.e. a bacterial colony was inoculated in 3 ml ampicillin-containing medium and cultivated for approximately 7 hours in the shaking incubator at 37° C. This preculture was then transferred into 500 ml LB medium and shaken overnight at 37° C. The bacterial suspension was centrifuged off at 4° C. at 3000 rpm the next morning. The resultant bacterial pellet was used for plasmid isolation following the manufacturer's directions.
After absorption of the DNA pellets in sterile H2O, the concentration was determined platemetrically and the DNA quality checked by restriction digestion and subsequent agarose gel electrophoresis.
2.5 Molecular Biological Methods
2.5.1 Nucleic Acids
Purification of DNA
Nucleic acids may be concentrated from dilute aqueous solutions or purified from non-precipitable substances by salt formation and subsequent alcohol precipitation or by purification using silica get columns (QIAGEN). An advantage of the first method is, for example, the possibility of concentrating the nucleic acid during absorption in the eluate after drying. Advantages of purification using columns include the ease of handling.
Precipitation of DNA
1:10 volumes of 3 M sodium acetate (pH 5.2) and 3 volumes of 100% ethanol mixture were added to the DNA solution and stored at −20° C. for at least 4 hours. The mixture was then centrifuged for 30 min at +4° C. with 13,000×g, the supernatant was discarded and the pellet resuspended by addition of 500 μl 70% ethanol (−20° C.). After a further centrifugation step for 10 min (10 min, 13.000×g, 4° C.), the supernatant was discarded and the pellet dried for 10 min in the precooled cold trap and finally dissolved in a corresponding amount of water.
Purification of the PCR Amplificate DNA with the Qiagen PCR Purification Kit
The PCR mixture was diluted in 5 volumes of buffer PB and transferred onto the column after thorough mixing and centrifuged for 1 min. 500 μl of washing buffer PE were applied to the column and again centrifuged. The bound DNA amplificates were eluted in a clean receiver by addition of 30-50 μl water onto the column and by centrifugation.
Purification of Nucleic Acids from Agarose Gels with the Qiagen Gel Extraction Kit
The agarose gel strips with the nucleic acid to be isolated were cut out, weighed and a corresponding amount of buffer QX1 (3 volumes of the gel weight) was added. The gel was dissolved by 10 min incubation at 50° C., the solution transferred onto a column and centrifuged. After addition of 750 μl washing buffer PE and subsequent centrifugation, the bound nucleic acid was eluted into a clean receiver by addition of 30-50 μl water onto the column and subsequent centrifugation.
Isolation and Purification of RNA
RNA was extracted from tissue and cells for analysis of expression by means of RT-PCR. Extraction was carried out either by the TRIzol chloroform method with corresponding RNA isolation kits from Qiagen or Roche.
RNA Extraction Using TRIzol Reagent
During purification of RNA from tissue, the pieces of tissue (<100 mg) were weighed out and homogenized with a glass pot after addition of 1 ml TRIzol reagent. During RNA purification from cultivated cells, 1 ml TRIzol reagent for 3.5 cm2 culture dish monolayer or 1 ml TRIzol reagent for centrifuged suspension culture (5-10×106 cells) was applied directly to the cells and homogenized by pipetting on and off. The TRIzol mixture was incubated for approximately 5 min at ambient temperature. The mixture was shaken vigorously after addition of 20 μl chloroform, incubated for a further 5 min and finally centrifuged (20 min, 12000×g, 4° C.), so three phases could form: (a) RNA in the upper aqueous phase, (b) DNA in the middle interphase and (c) protein in the lower organic phase.
The aqueous phase was carefully removed, without touching the interphase, and reacted with 500 μl 100% isopropanol. Brief vortexing was followed by incubation for 10 min and subsequent centrifugation (10 min, 16000×g, 4° C.). The supernatant was discarded and the pellet washed with 1 ml 70% ethanol (−20° C.) and re-pelletized (10 min, 12000×g, 4° C.). The supernatant was discarded and the pellet dried in the precooled cold trap. The RNA was finally dissolved in the desired volume of water and stored at −80° C.
RNA Extraction Using Qiagen RNeasy Mini Kit
350 μl buffer RLT were added to the cell pellet (approximately 5×106 cells) and the cell pellet homogenized by Qiagen shredder columns. After addition of 350 μl 70% ethanol and vigorous shaking, the suspension was applied to the column and centrifuged (30 sec at 8000×g). The column was washed by addition of 700 μl buffer RW1 and centrifugation (30 sec at 8000×g). Washing was carried out twice by addition of 500 μl buffer RPE in each case and centrifugation (30 sec, 8000×g). Washing was then carried out twice by addition of 500 μl buffer RPE and centrifugation (30 sec, 8000×g) in each case. The column was subsequently dried by centrifugation (2 min, 12000 rpm). The bound RNA was eluted by addition of 30 to 50 μl water and centrifugation (1 min at 8000×g).
DNase-I-Treatment
To avoid possible DNA contamination of the purified RNA, the RNA solution was treated enzymatically with DNase-I. For this purpose, 4 μl DNase-I and 6 μl 10× transcription buffer were added to 50 μl RNA and incubated for 30 to 45 min at 37° C. RNA purification was then carried out using QIAGEN RNeasy mini columns.
Restriction Digestion
Restriction digestion was used to check whether the desired insert is also contained in a plasmid. This may be cut out by the digestion and identified by means of its size. Restriction digestion was carried out using enzymes and reaction buffers from GincoBRL/Eggenstein and Boehringer/Mannheim. 1 μl plasmid DNA was digested in a total volume of 20 μl. The incubation time was 1 to 2 hours at a temperature corresponding to the optimum temperature for the enzyme.
Quantification of Nucleic Acids
The concentration was detected using UV spectroplatemeters. The concentration was calculated from the absorption at the specific wavelength I (for RNA, oligonucleotides and DNA 260 nm).
A=d×e×c (A absorption; d layer thickness; e material constant; c concentration). An OD260 of 1 corresponds to a concentration of: 50 μg/ml DNA, 40 μg/ml RNA or 30 μg/ml oligonucleotide. Measurement was carried out in quartz vessels which were thoroughly washed with autoclaved water between measurements.
DNA Oligonucleotide Starting Materials for siRNA Production
The oligonucleotide starting materials (ONV) were diluted in nuclease-free water to a final concentration of 200 μm, and the absorption A was measured at 260 nm of a 1:250 dilution. By means of the measured absorption, the ONV concentration B was calculated in μg/ml and B′ in μm:
A×5000=B[μg/ml]
(5000=250 fold dilution×20 μg oligo/ml/absorption unit)
B[μg/ml]/9.7=B′[μm]
(29 nt×0.333 μg/nmol for each nt=9.7 μg/nmol)
siRNA
To quantify the siRNA solutions, the absorption A was measured at 260 nm of a 1:25 dilution. The siRNA concentration B in μg/ml and B′ in μM was calculated from the measured absorption:
A×1000=B[μg/ml]
(1000=25 fold dilution×40 μg siRNA/ml/absorption unit)
B[μg/ml]/14=B′[μm]
(21 nt×2 strands=42 nt×0.333 μg/nmol for each nt=14 μg/nmol)
Quality Check by Agarose Gel Electrophoresis
1 to 2 percent TAE agarose gels with 0.5 μg/ml ethidium bromide were used to separated plasmid DNA, restriction-digested plasmid DNA, PCR products and RNA. Electrophoresis was carried out in 1× TAE buffer (cf. 2.1.3). The samples and 1 μg size label (“100 bb DNA ladder” “1 kb DNA ladder”) were reacted with ⅙ volume sample buffer and separated at 10 V/cm. Using the intercalating dye, ethidium bromide, the DNA and RNA could be made visible under UV light and documented plategraphically. To avoid degeneration during the electrophoresis of RNA, both the chamber and the comb were cleaned with ethanol and the flow buffer freshly prepared with autoclaved DEPC-H2O.
2.5.2 Reverse Transcription Polymerase Chain Reaction (RT-PCR)
During reverse transcription, RNA is transcribed into cDNA. Oligo(dT)15-18 primers, which bind specifically to the poly-A-tail of the mRNA, for example, are used as a base for the reverse transcriptase. Depending on the starting material, 0.5-2.5 μg RNA (dissolved in 11 μl water) were mixed with 1 μl 100 nm oligo(dT) 15-18 primer and denatured for 10 min at 70° C. After cooling to 4° C., 4 μl 5× RT buffer, 2 μl 100 nM DTT, 1 μl 10 mM dNTP-Mix, 1 μl RNAsin and 1 μl Superscript™ II (200 U/μl) were added and incubation was carried out at 42° C. for 1 hours. The cDNA was stored at −20° C. until use.
2.5.3 Polymerase Chain Reaction
The polymerase chain reaction (PCR) developed in 1987 enables nucleotide sequences determined in vitro to be enzymatically copied a million times (Saiki et al., 1988). This procedure, known as amplification, also enables very small amounts of DNA to be analysed. The following reaction mixture was laid out for a PCR reaction (25 μl):
The optimum MgCl2 concentration and the number of amplification cycles were determined individually for each primer pair (sequence, position and size of the amplicon are shown under (0)). Before the start, denaturation was carried out for 1 min at 94° C. and the duration of the individual steps of a cycle was 20 sec in each case. The thermocycler (PTC-200 (MJ Research)) was used.
Standard PCR:
2.5.4 Ligation
Ligation was carried out using the T4-DNA ligase (Promega). The T4-DNA ligase is an enzyme which covalently bonds 3′- and 5′- ends of linear DNA as a function of energy (ATP). Much less energy is consumed with ‘cohesive-ended’ overhangs than with ‘blunt-ended’ overhangs. Ligation was therefore carried out for 1 hour at ambient temperature in the case of cohesive ends and overnight at 4° C. in the case of PCR products or DNA without overhangs. The pGEM-T vector was used as the plasmid.
The foregoing description and examples have been set forth merely to illustrate the invention and are not intended to be limiting. Since modifications of the described embodiments incorporating the spirit and substance of the invention may occur to persons skilled in the art, the invention should be construed broadly to include all variations within the scope of the appended claims and equivalents thereof.
(All documents cited in the present patent application also form part of the disclosure of the present invention.)
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2004 011 687.3 | Mar 2004 | DE | national |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP05/01970 | Feb 2005 | US |
| Child | 11518284 | Sep 2006 | US |
