Patent Application
20090307788
All patent and non-patent references cited in this application are hereby incorporated by reference in their entirety.
The present invention relates to ribozyme mediated stabilization of polynucleotides, such as ribonucleic acids (RNA). Stabilization of polynucleotides according to the present invention can be exploited in molecular biology, genetic engineering, genetics and disease treatment, prevention and/or alleviation. The invention exploits the fact that ribozyme GIR1 has been shown to stabilize polynucleotides.
RNA splicing is found in most prokaryotic and eukaryotic organisms and different RNA splicing mechanisms have evolved for different classes of genes (C. B. Burge, T. Tuschl, P. A. Sharp, in The RNA World, Second Edition, R. F. Gesteland, T. R. Cech, J. F. Atkins, Eds. [Cold Spring Harbor Laboratory (CSHL) Press, Cold Spring Harbor, N.Y. 1999] pp. 525-560; C. R. Trotta, J. Abelson, in The RNA World, Second Edition, R. F. Gesteland, T. R. Cech, J. F. Atkins, Eds. (CSHL Press, Cold Spring Harbor, N.Y., 1999) pp. 561-584B). Group I introns (T. R. Cech, in The RNA World, Second Edition, R. F. Gesteland, T. R. Cech, J. F. Atkins, Eds. (CSHL Press, Cold Spring Harbor, N.Y., 1999), pp. 321-349) carry out splicing in a structurally and chemically distinct way from that of group II introns and the spliceosomal introns found widespread in higher eukaryotes.
Group I introns are widespread in nature, but with a notable sporadic occurrence. Whereas organellar group I introns have been identified in rRNA, mRNA, and tRNA transcription units, the nuclear group I introns are confined to the rRNA transcription units (Johansen et al. 1996).
Group I introns have been studied from two different perspectives: (1) as a selfish genetic element, and (2) as a ribozyme responsible for its own splicing reaction.
Several observations support the notion of group I introns as selfish genetic elements. Group I introns catalyze their own excision, although secondarily recruited host factors are implicated in many instances (Lambowitz et al. 1999). The presence of a group I intron appears to have little effect on the host (see Nielsen and Engberg 1985 for an analysis of the Tetrahymena intron). Also, the mobility of group I introns within species is well documented and occurs by allelic homing, initiated by cleavage of the intron-lacking allele by an intron homing endonuclease (Lambowitz and Belfort 1993).
The GIR1 ribozyme is found in so-called “twin-ribozyme introns” in rDNA of isolates of the myxomycete Didymium and the amoebaflagellate Naegleria. It is structurally related to the group I splicing ribozymes. However, it catalyzes a cleavage reaction rather than splicing and is crucial in the formation of the 5′ end of an mRNA encoded within the intron.
The present invention in one aspect is directed to novel, recombinant polynucleotides comprising the GIR1 ribozyme, or a variant thereof as defined herein, vectors comprising such polynucleotides and recombinant host cells comprising such polynucleotides and/or such vectors.
GIR1 is a naturally occurring ribozyme (RNA enzyme) isolated from myxomycetes and amoebaeflagellates. It catalyses cleavage at an internal position and generate a 5′ fragment with a 3′OH and a 5′-fragment with a lariat cap. The lariat cap is a unique structure in which the first and the third nucleotide of the chain are connected with a 2′, 5′-phosphodiester bond.
In its natural setting, the group I introns of the twin-ribozyme type, the cleavage results in the release of a 3′-fragment that acts as an mRNA encoding a homing endonuclease. The lariat cap protects the mRNA against 5′-3′ exonucleases. In addition, it is possible that the lariat cap is involved in translation of the message.
Specific examples of GIR1 molecules are disclosed in Table 1 below:
Didymium iridis
Naegleria
jamiesoni GIR1
Below is provided a structure based alignment of DiGIR1 and NaGIR1 core sequences (excluding sequence originating from P2 and P2.1 as illustrated in
aatcggg ttgaacac ttaat tgggtt aaa acggtg gggg- acga tccc-
gatgggg ttcatacc ttaat ctgcc- aaa acggg- acctc tgtt gaggt
P10′ P15′ J15/3 P3′ J3/4 P4′ P5′ L5 P5″
cagactg cac ggccct gcct ctt- aggt gtgttcaa tga acagtcg
gagacta cac ggtag- acca attt tggt ggtatgaa tgg atagtcc
P7′ J7/3 P3″ P8′ L8 P8″ P15″ J15/7 P7″
The alignment shown is between GIR1 from Didymium iridis and Naegleria jamiesoni with annotation derived from structure modelling of the two. As with the closely related splicing ribozymes, the structure is more conserved than the sequence. In vitro mutagenesis has revealed that most of the paired (P) sequences and several tertiary interactions that are not described in the figure are necessary for activity. However, very few residues are obligatory at the sequence level. These include the G-binding site in P7 (in particular the pair G174:C215), G229 at the cleavage site, A231, and A153 that is involved in recognition of the G-U pair at the branch point. The nucleotides involved in the characteristic 2′, 5′ phosphodiester bond (C230 and U232) are not critical at the sequence level (H. Nielsen, unpublished). Sequences in bold represent the “core” of the ribozyme. These sequences appear to be more conserved than the remainder of GIR1 (43 of 61 identical residues in the present comprison).
The above polynucleotides, vectors and host cells have utility e.g. in the fields of genetics, recombinant DNA technology and applications thereof in e.g. development of novel and innovative methods for treating diseases associated with or caused by ribonucleotide instability.
In one aspect the invention is directed to a polynucleotide comprising a first and a second subsequence,
wherein the first subsequence comprises or encodes
wherein the first and second subsequences together are capable of forming a secondary and/or tertiary interaction resulting in modification and/or stabilization of the transcript of said second subsequence
wherein the first subsequence is not natively associated with the second subsequence.
In another aspect the present invention is directed to a polynucleotide comprising a first and a second subsequence,
wherein the first subsequence comprises or encodes
wherein the first and second subsequences together are capable of forming a secondary and/or tertiary interaction resulting in modification and/or stabilization of the transcript of said second subsequence
wherein the first subsequence is not natively associated with the second subsequence.
In yet another aspect the present invention is directed to a polynucleotide comprising a first and a second subsequence,
wherein the first subsequence comprises or encodes
wherein the first and second subsequences together are capable of forming a secondary and/or tertiary interaction resulting in modification and/or stabilization of a transcript of said second subsequence
wherein the first subsequence is not natively associated with the second subsequence.
In another aspect the present invention is directed to a polynucleotide comprising a first and a second subsequence,
wherein the first subsequence comprises or encodes
wherein the first and second subsequences together are capable of forming a secondary and/or tertiary interaction resulting in modification and/or stabilization of a transcript of said second subsequence,
wherein the first subsequence is not natively associated with the second subsequence.
Stringent conditions as used herein shall denote stringency as normally applied in connection with Southern blotting and hybridization as described e.g. by Southern E. M., 1975, J. Mol. Biol. 98:503-517. For such purposes it is routine practise to include steps of prehybridization and hybridization. Such steps are normally performed using solutions containing 6×SSPE, 5% Denhardt's, 0.5% SDS, 50% formamide, 100 μg/ml denaturated salmon testis DNA (incubation for 18 hrs at 42° C.), followed by washings with 2×SSC and 0.5% SDS (at room temperature and at 37° C.), and a washing with 0.1×SSC and 0.5% SDS (incubation at 68° C. for 30 min), as described by Sambrook et al., 1989, in “Molecular Cloning/A Laboratory Manual”, Cold Spring Harbor), which is incorporated herein by reference.
The above polynucleotides can be DNA (deoxyribonucleic acids) or RNA (ribonucleic acids) and the nucleotide residues can be natural and/or non-natural nucleotide residues preferably capable of being incorporated into a polynucleotide by polymerase mediated incorporation.
The second subsequence can be a DNA coding for an RNA (such as a coding RNA or non-coding RNA). The transcript can thus be in the form of mRNA; tRNA or rRNA (coding RNA), or the transcript can be in the form of a non-coding RNA having a (further) regulatory function in a biological cell. Examples of non-coding (regulatory) RNAs are cited herein below.
First and second subsequences are listed herein interchangably in both RNA and DNA annotation as is usual in the art.
The following table of features illustrates consensus sequences and constraints of GIR1 ribozymes and variants thereof. Reference is made to
Variants of GIR1 include ribozymes comprising the above-mentioned consensus sequences in combination with critical nucleotide residues and conserved sequences as indicated in Tables 1 and/or 2.
Further preferred GIR1 molecules according to the present invention are nucleotide sequences having greater than 80 percent sequence identity, and preferably greater than 90 percent sequence identity (such as greater than 91% sequence identity, for example greater than 92% sequence identity, such as greater than 93% sequence identity, for example greater than 94% sequence identity, such as greater than 95% sequence identity, for example greater than 96% sequence identity, such as greater than 97% sequence identity, for example greater than 98% sequence identity, such as greater than 99% sequence identity, for example greater than 99.5% sequence identity), to any of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:1A and SEQ ID NO:2A.
The present invention further includes the use of recombinant or synthetically or transgenically produced GIR1 molecules. In one embodiment, the GIR1 molecule is a homologue of GIR1.
There is also provided a recombinant polynucleotide molecule in the form of an expression vector comprising a recombinant polynucleotide according to the present invention. The vector can further comprise a replicon capable of directing extrachromosomal replication. The vector can also comprise an expression signal capable of directing the expression of the first and/or second subsequence either in vitro under suitable conditions, or in vivo in a host cell, The vector can also comprise a selection marker for suitable selection when transformed or transfected into a host cell.
In a further aspect there is provided a host organism or host cell transfected or transformed with the polynucleotide according to the invention or the vector according to the invention.
In one aspect there is provided a host cell or host organism transfected or transformed with
There is also provided a composition comprising the recombinant polynucleotide according to the invention, a composition comprising the vector according to the invention claim, and a host organism according to the invention, said composition further comprising a physiologically acceptable carrier.
The present invention also provides a method for stabilizing polynucleotides, such as RNAs, for example non-coding, regulatory RNAs having an affinity for the GIR1 ribozyme, or a variant thereof as defined herein. There is also provided a method for improving the production of polypeptides as a result of mRNA stabilization.
The invention in a further aspect is directed to a method for manipulating the phenotype of a biological cell, wherein said manipulation is achieved by modulation of GIR1 mediated polynucleotide stability in said biological cell. The.modulation can also be achieved by a GIR1 variant as defined herein below.
The types of cells which can be targeted includes mammalian cells, such as animal and human cells, higher eucaryotes, fungal calls, yeasts, as well as bacteria. Plant cells are also contemplated. Methods for introducing into the afore-mentioned cells a recombinant polynucleotide according to the present invention, a vector comprising such a polynucleotide and a recombinant host cell comprising such a polynucleotide and/or such a vector are well known in the art. Also, expression signals capable of directing consitutive or inducible expression of GIR1, or a GIR1 variant, are well known in the art.
In a further interesting aspect there is provided a method of treatment of an individual suffering from a disease caused by or associated with increased polynucleotide degradation, such as increased RNA degradation.
Non-Coding (Regulatory) RNA
Examples of further second subsequences according to the present invention are provided herein below.
A variety of RNAs do not function as mRNA, tRNAs or rRNAs. The latter class of RNAs can collectively be termed non-coding RNAs or regulatory RNAs. Such non-coding (regulatory) RNAs are present in many different biological cells and it is one object of the invention to stabilise such non-coding (regulatory) RNAs either in vivo or in vitro. ncRNAs may target RNA or DNA by direct base pairing, by mimicking the structure of other nucleic acids or as part of a larger RNA-protein complex. Non-coding RNAs (ncRNAs) have been referred to as small RNAs in bacteria (see Storz et al., 2002).
Second subsequences in the form of non-coding (regulatory) RNAs control and regulate a wide range of developmental and physiological pathways in animals, including hematopoietic differentiation, adipocyte differentiation and insulin secretion in mammals, and have been shown to be perturbed in cancer and other diseases. The extent of transcription -of non-coding sequences and the abundance of small RNAs suggests the existence of an extensive regulatory network on the basis of RNA signaling which may underpin the development and much of the phenotypic variation in mammals and other complex organisms and which may have different genetic signatures from sequences encoding proteins.
The sizes of ncRNAs varies depending on their function. For example, those associated with development in the nematode Caenorrhabditis elegans, Drosophila and mammals have been found to be 21 to 25 nucleotides in length. The translational regulators in bacteria are from 100 to 200 nucleotides in length and those e.g. involved in gene silencing in eukaryotes are larger than 10,000 nucleotides. All of the above are contemplated as second subsequences.
An example of second subsequences in the form of macro ncRNAs (i.e. larger than 10,000 nucleotides) include Xist and Air, which in mouse are approximately 18 and 108 Kb, respectively. Xist plays an essential role in mammals by associating with chromatin and causing widespread gene silencing on the inactive X chromosome, while Air is required for paternal silencing of the Igf2r/Slc22a2/Slc22a3 gene cluster. Apart from their extreme length, Xist and Air share two other important features: genomic imprinting and antisense transcription.
Genomic imprinting is a process by which certain genes are expressed differently according to whether they have been inherited from the maternal or paternal allele. Imprinting is critical for normal development, and loss of imprinting has been implicated in a variety of human diseases. ncRNAs have been discovered at many different imprinted loci and appear to be important in the imprinting process itself.
The other feature that Xist and Air have in common is that both are members of naturally occurring cis-antisense transcript pairs. Previous studies have indicated the existence of thousands of mammalian cis-antisense transcripts. These transcripts may regulate gene expression in a variety of ways including RNA interference, translational regulation; RNA editing, alternative splicing, and alternative polyadenylation, although the exact mechanisms by which antisense RNAs function are unknown. Mammalian cis-antisense transcripts constitute one example of second subsequences.
Mammalian cells harbor numerous small non-protein-coding RNAs. Examples of second subsequences of mammalian origin include small nucleolar RNAs (snoRNAs), microRNAs (miRNAs), short interfering RNAs (siRNAs), small nuclear RNAs (snRNAs) and small double-stranded RNAs, which regulate gene expression at many levels including chromatin architecture, RNA editing, RNA stability, translation, and quite possibly transcription and splicing.
ncRNAs have also been found to have a role in protein degradation and translocation. For example tRNAs in combination with spliceosomal snRNAs are housekeeping RNAs involved in mRNA splicing and translation. These RNAs are processed by multistep pathways from the introns and exons of longer primary transcripts, including protein-coding transcripts. Most show distinctive temporal- and tissue-specific expression patterns in different tissues, including embryonal stem cells and the brain, and some are imprinted.
mRNA Instability
It is one objective of the present invention to improve mRNA instability in cells and in vitro when such a stabilisation is desirabel. Messenger RNA (mRNA) expression in mammalian cells is highly regulated. Traditionally, emphasis has been placed on elucidating mechanisms by which genes are regulated at the transcriptional level; however, steady-state levels of mRNA is also dependent on its half-life or degradation rate.
Changes in mRNA stability play an important role in modulating the level of expression of many eukaryotic genes and different mechanisms have been proposed for the regulation of mRNA turnover (Cleveland and Yen, 1989, New Biol. 1:121; Mitchell and Tollervey, 2000, Curr. Opin. Genet. Dev. 10:193; Mitchell and Tollervey, 2001, Curr. Opin. Cell. Biol. 13:320; Ross, J. 1995, Microbiol. Rev. 59:423; Sachs, A. B., 1993, Cell 74:413; Staton et al. 2000, J. Mol. Endocrinology 25:17; Wilusz et al. 2001, Nat. Rev. Mol. Cell Biol. 2:237).
Regulation of mRNA stability is complex and the regulation can involve sequence elements in the mRNA itself, activation of nucleases, as well as the involvement of complex signal transduction pathway(s) that ultimately influence trans-acting factors' interaction with mRNA stability sequence determinants.
Recently, it has become increasingly apparent that the regulation of RNA half-life plays a critical role in the tight control of gene expression and that mRNA degradation is a highly controlled process. RNA instability allows for rapid up- or down-regulation of mRNA transcript levels upon changes in transcription rates.
A number of critical cellular factors, e.g. transcription factors such as c-myc, or gene products which are involved in the host immune response such as cytokines, are required to be present only transiently to perform their normal functions. Transient stabilization of the mRNAs which code for these factors permits accumulation and translation of these messages to express the desired cellular factors when required; whereas, under nonstabilized, normal conditions the rapid turnover rates of these mRNAs effectively limit and “switch off” expression of the cellular factors. Thus, aberrant mRNA turnover usually leads to altered protein levels, which can dramatically modify cellular properties. Dysregulation of mRNA stability has been associated with human diseases including cancer, inflammatory disease, and Alzheimer's disease.
The stabilization of mRNA appears to be a major regulatory mechanism involved in the expression of inflammatory cytokines, growth factors, and certain protooncogenes. In the diseased state, mRNA half-life and levels of disease-related factors are significantly increased due to mRNA stabilization (Ross, J. 1995, Microbiol. Rev. 59:423; Sachs, A. B., 1993, Cell 74:413; Staton et al. 2000, J. Mol. Endocrinology 25:17; Wilusz et al. 2001, Nat. Rev. Mol. Cell Biol. 2:237).
Transcription rates and mRNA stability are often tightly and coordinately regulated for transiently expressed genes such as c-myc and c-fos, and cytokines such as IL-1, IL-2, IL-3, TNF.alpha., and GM-CSF. In addition, abnormal regulation of mRNA stabilization can lead to unwanted build up of cellular factors leading to undesirable cell transformation, e.g. tumour formation, or inappropriate and tissue damaging inflammatory responses.
mRNA: Messenger RNA
rRNA(s): Ribosomal RNA
tRNA: Transfer RNA
miRNA(s): MicroRNA—putative translational regulatory gene family
ncRNA(s): Non-coding RNA—all RNAs other than mRNA
siRNA(s): Small interfering RNA—active molecules in RNA interference
snRNA(s): Small nuclear RNA—includes spliceosomal RNAs
snmRNA(s): Small non-mRNA—essentially synonymous with small ncRNAs
snoRNA(s): Small nucleolar RNA—most known snoRNAs are involved in rRNA modification
stRNA: Small temporal RNA—for example, lin-4 and let-7 in Caenorhabditis elegans tRNA Transfer RNA
Natural nucleotide: Any of the four deoxyribonucleotides, dA, dG, dT, and dC (constituents of DNA), and the four ribonucleotides, A, G, U, and C (constituents of RNA) are the natural nucleotides. Each natural nucleotide comprises or essentially consists of a sugar moiety (ribose or deoxyribose), a phosphate moiety, and a natural/standard base moiety. Natural nucleotides hybridize to complementary nucleotides in a number of ways. One way of hybridization is by means of the well-known rules of base pairing (Watson and Crick), where adenine (A) pairs with thymine (T) or uracil (U); and where guanine (G) pairs with cytosine (C), wherein corresponding base-pairs are part of complementary, anti-parallel nucleotide strands. The base pairing results in a specific hybridization between predetermined and complementary nucleotides. In nature, the specific interactibins leading to base pairing are governed by the size of the bases and the pattern of hydrogen bond donors and acceptors of the bases. A large purine base (A or G) pairs with a small pyrimidine base (T, U or C). Additionally, base pair recognition between bases is influenced by hydrogen bonds formed between the bases. In the geometry of the Watson-Crick base pair, a six membered ring (a-pyrimidine in natural oligonucleotides) is juxtaposed to a ring system composed of a fused, six membered ring and a five membered ring (a purine in natural oligonucleotides), with a middle hydrogen bond linking two ring atoms, and hydrogen bonds on either side joining functional groups appended to each of the rings, with donor groups paired with acceptor groups.
Base moiety: Nitrogeneous base moiety of a natural or non-natural nucleotide, or a derivative of such a nucleotide comprising alternative sugar or phosphate moieties. Base moieties include any moiety that is different from a naturally occurring moiety and capable of complementing one or more bases of the opposite nucleotide strad of a double helix.
Polynucleotide: A molecule comprising consecutively linked natural and/or non-natural nucleic acid residues. The polynucleotide can e.g. be an RNA or DNA molecule.
Isolated polynucleotide: Either (1) a DNA or RNA molecule that is separated from sequences with which it is immediately contiguous (in the 5′ and 3′ directions) in the naturally occurring genome of the organism from which it was derived or (2) a DNA or RNA molecule with an indicated sequence, but which has undergone some degree of purification relative to the genome and may retains some number of immediately contiguous genomic sequences. For example, such molecules include those present on an isolated restriction fragment or such molecules obtained by PCR amplification. DNA or RNA can be isolated and purified to any degree using methods well known in the art.
In accordance with the invention, the “isolated polynucleotide” may be inserted into or itself comprise a vector, such as a plasmid or virus vector, or be integrated into the genomic DNA of a prokaryote or eukaryote. With respect to RNA molecules of the invention, the term “isolated nucleic acid” primarily refers to an RNA molecule encoded by an isolated DNA molecule as defined above. But also includes RNA that has been isolated from a cellular source or RNA that has been chemically synthesized (and obtained at any level of purity). In these cases, the RNA molecule has been sufficiently separated from RNA molecules with which it would be associated in its natural state (i.e., in cells or tissues), such that it exists in a purified pure form, e.g., that the RNA is enriched in the mixture relative to its abundance as naturally produced.
RNA polynucLeotide: RNA molecule, such as mRNA, pre-mRNA, mature messenger RNA molecule, mRNA which was produced due to splicing of the pre-mRNA, ncRNA, small nucleolar RNAs (snoRNAs), microRNAs (miRNAs), short interfering RNAs (siRNAs), small nuclear RNAs (snRNAs) and small double-stranded RNAs that contains the same sequence information as the corresponding DNA molecule (albeit that U nucleotides replace T nucleotides) as the DNA molecule.
Ribose derivative: Non-natural ribose moiety forming part of a nucleoside capable of being enzymatically incorporated into a template or complementing template. Examples include e.g. derivatives distinguishing the ribose derivative from the riboses of natural ribonucleosides, including adenosine (A), guanosine (G), uridine (U) and cytidine (C). Further examples of ribose derivatives are described in e.g. U.S. Pat. No. 5,786,461.
Transcriptional product of a gene: A pre-messenger RNA molecule, pre-mRNA, that contains the same sequence information (albeit that U nucleotides replace T nucleotides) as the gene, or mature messenger RNA molecule, mRNA, which was produced due to splicing of the pre-mRNA, and is a template for translation of genetic information of the gene into a protein.
Translational product of a gene: A protein, which is encoded by a gene.
Polypeptide: A molecule comprising amino acid residues which do not contain linkages other than amide linkages between adjacent amino acid residues.
In a preferred embodiment, the isolated nucleic acid is a polynucleotide or an expression vector and comprises a SNA or RNA sequence operably linked to a promoter or other regulatory sequences to control expression thereof. Expression vectors can encode one or more DNAs or RNAs and these can be coordinately or individually expressed, e.g., using one promoter or multiple promoters. Useful promoters and regulatory sequences for any of the expression vectors are well known to those of skill in the art.
Expression vectors are useful for any one of the following purposes: propagation of the DNA or RNA, purification of the DNA or RNA, or delivery and expression or transcription of the DNA or RNA in a subject. Expression vectors can be used for any cell type, including bacterial, yeast, fungi and mammalian systems, and include all types of vectors including viral vectors. Methods of making and using expression vectors, as well as selecting the appropriate host cell system are well known to those of skill in the art. Well-known promoters can be present, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, an arabinose-inducible promoter or a promoter system from phage lambda. The promoters typically control expression, optionally with an operator sequence and have ribosome binding site sequences for example, for initiating and completing transcription and translation. Among vectors preferred for use in bacteria include pQE70, pQE60 and pQE9, available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene Cloning Systems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia Biotech, Inc. Any expression vector is contemplated.
In one embodiment there are provided methods for stabilising polynucleotides and methods for improving the production of polypeptides as a result of said stabilisation. In a preferred embodiment the polynucleotide to be stabilized is an RNA polynucleotide.
The RNA polynucleotide to be stabilized can be any RNA, in particular a messenger RNA (mRNA), a ribosomal RNA (rRNA), a transfer RNA (tRNA), a nuclear RNA, a RNA-ribozyme, a small nucleolar RNA (snoRNA), a microRNA (miRNA), a short interfering RNA (siRNA), a small nuclear RNA (snRNA) and a small double-stranded RNA, an in vitro transcribed RNA or a chemically synthesised RNA, in which respect all said RNA molecules can be chemically modified. In that respect the RNA may have between 10 and 100,000 nucleotides, such as from 15 to 2500 nucleotides, for example from 20 to 1000 nucleotides.
Selected, but non-limited examples of second subsequences in the form of RNA polynucleotides of the invention are given in Table 3 below. It will be understood that such sequences, or a complementary strand thereof, can be operably linked to a first subsequence as defined herein elsewhere:
The below second subsequences can be accessed through the miRBASE: (http://microrna.sanger.ac.uk/sequences/)
Further non-limited examples of second subsequences in the form of RNA polynucleotides according to the present invention are listed in Table 4 below. It will be understood that such sequences, or a complementary strand thereof, can be operably linked to a first subsequence as defined herein elsewhere:
The sequences can be accessed through the miRBASE: (http://microrna.sanger.ac.uk/sequences/)
Further non-limited examples of second subsequences in the form of RNA polynucleotides according to the present invention are listed in Table 5 below. It will be understood that such sequences, or a complementary strand thereof, can be operably linked to a first subsequence as defined herein elsewhere.
The sequences can also be accessed through the mammalian noncoding RNA database (RNAdb):
(http://jsm-research.imb.uq.edu.au/rnadb/Database/default.aspx)
Homo sapiens PAR5 gene, complete
Homo
sapiens
H. sapiens predicted non coding
Homo
sapiens
Elephantidae gen. sp. H19 RNA
Elephantidae
Felis catus H19 RNA gene, partial
Felis catus
Lynx lynx H19 RNA gene, partial
Lynx lynx
Pongo pygmaeus H19 gene, partial
Pongo pygmaeus
Thomomys monticola H19 RNA
Thomomys
monticola
Homo sapiens steroid receptor RNA
Homo
sapiens
Homo sapiens steroid receptor RNA
Homo
sapiens
Homo sapiens steroid receptor RNA
Homo
sapiens
Homo sapiens steroid receptor RNA
Homo
sapiens
Homo sapiens miR-16-1 stem-loop
Homo
sapiens
Homo sapiens DLEU1 noncoding
Homo
sapiens
Homo sapiens DLEU2 noncoding
Homo
sapiens
Mus musculus makorin 1 pseudogene
Mus musculus
Homo sapiens testis-specific Testis
Homo
sapiens
Homo sapiens partial mRNA for
Homo
sapiens
Homo sapiens partial mRNA for
Homo
sapiens
Homo sapiens testis-specific Testis
Homo
sapiens
Homo sapiens non-coding RNA
Homo
sapiens
Homo sapiens non-coding RNA
Homo
sapiens
Homo sapiens non-coding RNA
Homo
sapiens
Homo sapiens non-coding RNA
Homo
sapiens
Homo sapiens non-coding RNA
Homo
sapiens
Homo sapiens PCGEM1 gene, non-
Homo
sapiens
Homo sapiens RNA for differentiation
Homo
sapiens
Homo sapiens BIC noncoding
Homo
sapiens
Mus musculus BIC noncoding
Mus musculus
Homo sapiens H19 gene, complete
Homo
sapiens
Homo sapiens H19 gene, complete
Homo
sapiens
Homo
sapiens
Mus musculus H19 fetal liver mRNA
Mus musculus
Ovis aries H19 gene, partial sequence
Ovis aries
Ovis aries H19 mRNA, partial sequence
Ovis aries
Ovis aries H19 gene, complete sequence
Ovis aries
Oryctolagus cuniculus H19/myoH
Oryctolagus
cuniculus
Peromyscus maniculatus bairdii H19
Peromyscus
maniculatus
Sus scrofa H19 gene, complete sequence
Sus scrofa
Homo sapiens LIT1 transcript
Homo
sapiens
Homo sapiens LIT1 transcript
Homo
sapiens
Homo sapiens LIT1 transcript
Homo
sapiens
Homo sapiens LIT1 transcript
Homo
sapiens
Homo sapiens LIT1 transcript
Homo
sapiens
Homo sapiens LIT1 transcript
Homo
sapiens
Homo sapiens LIT1 transcript
Homo
sapiens
Homo sapiens LIT1 transcript
Homo
sapiens
Homo sapiens LIT1 transcript
Homo
sapiens
Homo sapiens LIT1 transcript
Homo
sapiens
Mus musculus Peg8/lgf2as mRNA,
Mus musculus
Homo sapiens IPW mRNA sequence
Homo
sapiens
Homo sapiens hypoxia inducible
Homo
sapiens
Rattus
norvegicus
Mus musculus C57/Black6 BC1
Mus musculus
Mesocricetus auratus BC1 scRNA
Mesocricetus
auratus
Cavia porcellus Hartley BC1 scRNA
Cavia porcellus
Peromyscus maniculatus snRNA
Peromyscus
maniculatus
Peromyscus californicus snRNA
Peromyscus
californicus
Meriones unguiculatus snRNA (BC1
Meriones
unguiculatus
Aotus trivirgatus BC200 alpha
Aotus trivirgatus
Chlorocebus aethiops BC200 alpha
Cercopithecus
aethiops
Gorilla gorilla BC200 alpha scRNA
Gorilla gorilla
Homo
sapiens
Hylobates lar BC200 alpha scRNA
Hylobates lar
Macaca fascicularis BC200 alpha
Macaca fascicularis
Macaca mulatta BC200 alpha
Macaca mulatta
Pan paniscus BC200 alpha scRNA
Pan paniscus
Papio hamadryas BC200 alpha
Papio hamadryas
Pongo pygmaeus BC200 alpha
Pongo pygmaeus
Saguinus imperator BC200 alpha
Saguinus
imperator
Saguinus oedipus BC200 alpha
Saguinus
oedipus
Homo sapiens 1 DISC2 gene, complete
Homo
sapiens
Homo sapiens mitochondrial RNA-
Homo
sapiens
Homo sapiens RNase MRP RNA
Homo
sapiens
H. sapiens MRP RNA gene encoding
Homo
sapiens
B. taurus RNase MRP (RMRP) gene,
Bos taurus
Homo sapiens UBE3A antisense
Homo
sapiens
Mus musculus SJL/j viral integration
Mus musculus
Mus musculus SJL/j viral integration
Mus musculus
Mus musculus His-1 gene, exons 1,
Mus musculus
Mus musculus Tmevpg1, mRNA
Mus musculus
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens clone IMAGE:
Homo
sapiens
Homo sapiens clone IMAGE:
Homo
sapiens
Homo sapiens ST7 overlapping
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens miR-15a mature
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens mRNA for B-cell
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens clone IMAGE: 782833
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens ST7 overlapping
Homo
sapiens
Homo sapiens ST7 overlapping
Homo
sapiens
Homo sapiens ST7 overlapping
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens clone IMAGE:
Homo
sapiens
Homo sapiens clone IMAGE:
Homo
sapiens
Homo sapiens clone IMAGE:
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens partial BCMS gene
Homo
sapiens
Homo sapiens ST7OT1 mRNA, non-
Homo
sapiens
Homo sapiens ST7 overlapping
Homo
sapiens
Homo sapiens ST7 overlapping
Homo
sapiens
Homo sapiens metastasis associated
Homo
sapiens
Homo sapiens metastasis associated
Homo
sapiens
Homo
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo
sapiens
Homo sapiens PAR1 gene, complete
Homo
sapiens
Homo sapiens SZ-1 mRNA
Homo
sapiens
Homo sapiens telomerase RNA
Homo
sapiens
Homo sapiens noncoding RNA
Homo
sapiens
Homo sapiens miR-16 mature
Homo
sapiens
Homo sapiens AAA1 variant IB
Homo
sapiens
Homo sapiens non-coding RNA in
Homo
sapiens
Homo sapiens SCA8 mRNA, repeat
Homo
sapiens
Homo sapiens maternally expressed
Homo
sapiens
Mus musculus RNA component of
Mus musculus
Homo sapiens AAA1 variant II
Homo
sapiens
Homo sapiens AAA1 variant III
Homo
sapiens
Homo sapiens AAA1 variant IV
Homo
sapiens
Homo sapiens AAA1 variant IX
Homo
sapiens
Homo sapiens AAA1 variant V
Homo
sapiens
Homo sapiens AAA1 variant VI
Homo
sapiens
Homo sapiens AAA1 variant VII
Homo
sapiens
Homo sapiens AAA1 variant VIII
Homo
sapiens
Further non-limited examples of second subsequences in the form of bacterial RNA polynucleotides according to the present invention are listed in Table 6 below. It will be understood that such sequences, or a complementary strand thereof, can be operably linked to a first subsequence as defined herein elsewhere.
Salmonella typhe
Escherichia coli
Salmonella enter
Pseudomonas aer
Escherichia coli
Escherichia coli
Salmonella typheri
Escherichia coli
Salmonella typhe
Klebsiella pneum
Serratia marcesc
Salmonella enter
Escherichia coli
Escherichia coli
Escherichia coli
Salmonella typhe
Salmonella enteric
Escherichia coli
Escherichia coli
Salmonella enteric
Escherichia coli
indicates data missing or illegible when filed
Further non-limited examples of second subsequences in the form of plant RNA polynucleotides according to the present invention are listed in Table 7 below. It will be understood that such sequences, or a complementary strand thereof, can be operably linked to a first subsequence as defined herein elsewhere.
Arabidopsis thaliana
Nicotiana tabacum
Solanum tuberosum
Solanum tuberosum
Solanum tuberosum
Arabidopsis thaliana
Cucumis sativus
Glycine max
Medicago truncatula
Lotus japonicus
Citrullus lanatus
Lycopersicon esculentum
Gossypium hirsutum
Zea mays
Oryza sativa
Arabidopsis thaliana
Medicago truncatula
Arabidopsis thaliana
Lycopersicon esculentum
Lotus japonicus
Glycine max
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Arabidopsis thaliana
Brassica oleracea
Zea mays
Brassica oleracea
Zea mays
Further non-limited examples of second subsequences in the form of yeast RNA polynucleotides according to the present invention are listed in Table 8 below. It will be understood that such sequences, or a complementary strand thereof, can be operably linked to a first subsequence as defined herein elsewhere.
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
Saccharomyces
cerevisiae
It will be understood that in preferred embodiments, mammalian second subsequences are expressed in mammalian cells, human second subsequences are expressed in human cells, fungal second subsequences are expressed in fungal cells, yeast second subsequences are expressed in yeast cells, and bacterial second subsequences are expressed in bacterial cells.
Also, It will be understood that in preferred embodiments, mammalian second subsequences are cloned in vectors capable of being transformed or transfected into mammalian cells prior to expression, human second subsequences are cloned in vectors capable of being transformed or transfected into human cells prior to expression, fungal second subsequences are cloned in vectors capable of being transformed or transfected into fungal cells prior to expression, yeast second subsequences are cloned in vectors capable of being transformed or transfected into yeast cells prior to expression, and bacterial second subsequences are cloned in vectors capable of being transformed or transfected into bacterial cells prior to expression.
In all of the above cases the expression of the first subsequence and the second subsequence is directed by an expression signal capable of directing said expression in the host cell in question under appropriate cultivation conditions.
Gene Therapy
Having identified RNA instability or a decrease in the RNA level, for example due to decreased transcription, as the cause of a disease it is also rendered possible in accordance with the present invention to provide a genetic therapy for subjects being diagnosed as having.a-predisposition for or suffering from a disease associated With RNA instability or a decrease in RNA level, said therapy comprising administering to said subject a therapeutically effective amount of a gene therapy vector.
The gene therapy vectors comprise a sequence coding for the RNA associated with the disease and/or a polynucleotide sequence comprising GIR1 or a variant thereof. In particular the invention relates to a gene therapy vector comprising i) a first DNA or RNA subsequence selected from the group consisting of SEQ ID NO 1, SEQ ID NO:2; SEQ ID NO:1A and SEQ ID NO:2A, or a variant or a fragment thereof, or the complementary strand thereof, and a second subsequence selected from the group consisting of second subsequences listed in Table 3, second subsequences listed in Table 4, second subsequences listed in Table 5, second subsequences listed in Table 6 and second subsequences listed in Table 7, or a variant or a fragment thereof, or the complementary strand of any of said sequences.
Various different methods of gene therapy can be used for treating subjects suffering from a disease as defined in the present invention. The person skilled in the art will be well aware of such methods.
Other types of gene therapy include the use of retrovirus (RNA-virus). Retrovirus can be used to target many cells and integrate stably into the genome. Adenovirus and adeno-associated virus can also be used. A suitable retrovirus or adenovirus for this purpose comprises an expression construct comprising a sequence coding for the RNA associated with the disease and/or a polynucleotide sequence comprising GIR1 or a variant thereof under the control of a constitutive promoter or a regulatable promoter such as a repressible and/or inducible promoter or a promoter comprising both repressible and inducible elements. The construct comprising a sequence coding for the RNA associated with the disease and/or a sequence comprising GIR1, or a variant thereof, may be inserted into the appropriate cells within a patient, using vectors that include, but are not limited to adenovirus, adeno-associated virus, and retrovirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes.
Described below are- methods and compositions whereby a disorder associated with RNA instability may be treated. In particular diseases associated with RNA instability selected from the group consisting of but not limited to: Cancer, such as for example chronic lymphocytic leukemia, ovarian cancer, breast cancer and melanoma; Cachexia and a-thalessemia.
Gene replacement therapy techniques should be capable delivering a sequence coding for the RNA associated with the disease and/or GIR1 or a variant thereof to cells transcribing the corresponding RNA within patients. Thus, in one embodiment, techniques that are well known to those of skill in the art (see, e.g., PCT Publication No. WO89/10134, published Apr. 25, 1988) can be used to enable the sequence coding for the RNA associated with the disease and/or GIR1 or a variant thereof to be uptaken by the cells. Viral vectors may advantageously be used for the purpose. Also included are methods using liposomes either in vivo ex vivo or in vitro, wherein the sense or antisense DNA sequence coding for the RNA associated with the disease and/or GIR1 or a variant thereof is delivered to the cytoplasm and nucleus of target cells. Liposomes can deliver the sense or antisense DNA sequence coding for the RNA associated with the disease and/or GIR1 or a variant thereof to humans and the lungs or skin through intrathecal delivery either as part of a viral vector or as DNA conjugated with nuclear localizing proteins or other proteins that increase take up into the cell nucleus.
In another embodiment, techniques for delivery involve direct administration of such sense or antisense DNA sequence coding for the RNA associated with the disease and/or GIR1 or a variant thereof to the site of the cells in which the sense or antisense DNA sequence coding for the RNA associated with the disease and/or GIR1 or a variant thereof are to be expressed.
Treatment of Cachexia
Muscle wasting (cachexia) is a consequence of chronic diseases, such as cancer, and is associated with degradation of muscle proteins such as MyoD. Cachexia is a condition that leads to the alteration of several physiological and behavioral attributes, ranging from fatigue and fever to excessive weight loss. The detrimental effects of cachexia occur as a consequence of excessive wasting of skeletal muscle tissue. It is well established that muscle atrophy requires the activation of transcription factors such as NF-κB and Foxo-3, leading to the rapid decrease of MyoD mRNA. three highly conserved muscle-specific microRNAs, miR-1, miR-133 and miR-206, are robustly induced during the myoblast-myotube transition, both in primary human myoblasts and in the mouse mesenchymal C(2)C(12) stem cell line. MyoD binds to regions upstream of these microRNAs and, therefore, are likely to regulate their expression.
Thus in one embodiment the RNA to be stabilized is MyoD mRNA or a variant thereof.
Treatment of α-Thalassemia
Globin mRNA is particularly stable. Three C-rich elements located in the 39UTR of α-globin mRNA are targets for binding of the a-complex, a group of proteins predominantly containing the PCBPs, which maintain stability. An α-globin gene variant, a constant spring, or acs, is the most common cause of nondeletional a-thalassemia worldwide. This variant contains a stop codon. mutation that allows read through of translation into the 39UTR, and this is associated with a major decrease in mRNA half-life, which is associated with a-thalassemia.
Thus in one embodiment the polynucleotide to be stabilized is α-globin mRNA or a variant thereof.
Treatment of Cancer
A number of miRNAs are associated with cancer diseases. For example a high portion of miRNA containing genes exhibit copy number alterations in ovarian cancer, breast cancer, and melanoma and these copy changes correlate with miRNA expression. For example the miRNA mir-320 is located in regions with DNA copy number loss in all of the three cancer types. A notable mir-320 target predicted by two independent programs is methyl CpG binding protein 2 (MECP2), which is overexpressed in breast cancer and serves as an oncogene promoting cell proliferation. Also mir-218-1 is located within the tumor suppressor gene SLIT2 (human homologue of Drosophila Slit2), which is frequently inactivated in breast, lung, and colorectal cancer because of allelic loss. It has been shown that there is a copy number loss of the region containing mir-218-1 ovarian cancers, breast cancers, and melanoma lines.
Treatment of Chronic Lymphocytic Leukemia
Chronic lymphocytic leukemia is the most common form of adult leukemia in the Western world. To miRNAs miR15 and miR16 lie within a small regionof chromosome 13q14 that is deleted in more than 65% of CLL and that allelic loss in this region correlates with down-regulation of both miR-15 and miR-16 expression suggest that these genes represent the targets of inactivation by allelic loss in CLL.
Thus in one embodiment the polynucleotide to be stabilized is mir-15 miRNA or a variant thereof. In another embodiment the polynucleotide to be stabilized is mir-16 miRNA or a variant thereof.
Compositions
Compositions or pharmaceutical compositions or formulations for use in the present invention include a preparation of a recombinant polynucleotide or a vector or a host cell according to the invention in combination with, preferably dissolved in, a pharmaceutically acceptable carrier, preferably an aqueous carrier or diluent. The composition may be a solid, a liquid, a gel or an aerosol. A variety of aqueous carriers may be used, such as 0.9% saline, buffered saline, physiologically compatible buffers and the like. The compositions may be sterilized by conventional techniques well known to those skilled in the art. The resulting aqueous solutions may be packaged for use or filtered under aseptic conditions and freeze-dried, the freeze-dried preparation being dissolved in a sterile aqueous solution prior to administration.
The compositions may contain pharmaceutically acceptable auxiliary substances or adjuvants, including, without limitation, pH adjusting and buffering agents and/or tonicity adjusting agents, such as, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc. The formulations may contain pharmaceutically acceptable carriers and excipients including microspheres, liposomes, microcapsules, nanoparticles or the like. Conventional liposomes are typically composed of phospholipids (neutral or negatively charged) and/or cholesterol. The liposomes are vesicular structures based on lipid bilayers surrounding aqueous compartments. They can vary in their physiochemical properties such as size, lipid composition, surface charge and number and fluidity of the phospholipids bilayers. The most frequently used lipid for liposome formation are: 1,2-Dilauroyl-sn-Glycero-3-Phosphocholine (DLPC), 1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine (DMPC), 1,2-Dipalmitoyl-sn-Glycero-3-Phosphocholine (DPPC), 1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC), 1,2-Dioleoyl-sn-Glycero-3-Phosphocholine (DOPC), 1,2-Dimyristoyl-sn-Glycero-3-Phosphoethanolamine (DMPE), 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine (DPPE), 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine (DOPE), 1,2-Dimyristoyl-sn-Glycero-3-Phosphate (Monosodium Salt) (DMPA), 1,2-Dipalmitoyl-sn-Glycero-3-Phosphate (Monosodium Salt) (DPPA), 1,2-Dioleoyl-sn-Glycero-3-Phosphate (Monosodium Salt) (DOPA), 1,2-Dimyristoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium Salt) (DMPG), 1,2-Dipalmitoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium Salt) (DPPG), 1,2-Dioleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (Sodium Salt) (DOPG), 1,2-Dimyristoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium Salt) (DMPS), 1,2-Dipalmitoyl-sn-Glycero-3-[Phospho-L-Serine) (Sodium Salt) (DPPS), 1,2-Dioleoyl-sn-Glycero-3-[Phospho-L-Serine] (Sodium Salt) (DOPS), 1,2-Dioleoyl-sn-Glycero-3-Phosphoethanolamine-N-(glutaryl) (Sodium Salt) and 1,1′,2,2′-Tetramyristoyl Cardiolipin (Ammonium Salt). Formulations composed of DPPC in combination with other lipids or modifiers of liposomes are preferred e.g. in combination with cholesterol and/or phosphatidylcholine.
Long-circulating liposomes are characterized by their ability to extravasate at body sites where the permeability of the vascular wall is increased. The most popular way of producing long-circulating liposomes is to attach hydrophilic polymer polyethylene glycol (PEG) covalently to the outer surface of the liposome. Some of the preferred lipids are: 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-2000] (Ammonium Salt), 1,2-Dipalmitoyl-sn-Glycero-3-Phosphoethanolamine-N-[Methoxy(Polyethylene glycol)-5000] (Ammonium Salt), 1,2-Dioleoyl-3-Trimethylammonium-Propane (Chloride Salt) (DOTAP).
Possible lipids applicable for liposomes are supplied by Avanti, Polar Lipids, Inc, Alabaster, Ala. Additionally, the liposome suspension may include lipid-protective agents which protect lipids against free-radical and lipid-peroxidative damage on storage. Lipophilic free-radical quenchers, such as alpha-tocopherol and water-soluble iron-specific chelators, such as ferrioxianine, are preferred.
A variety of methods are available for preparing liposomes, as described in, e.g., Szoka et al., Ann. Rev. Biophys. Bioeng. 9:467 (1980), U.S. Pat. Nos. 4,235;871, 4,501,728 and 4,837,028, all of which are incorporated herein by reference. Another method produces multilamellar vesicles of heterogeneous sizes. In this method, the vesicle-forming lipids are dissolved in a suitable organic solvent or solvent system and dried under vacuum or an inert gas to form a thin lipid film. If desired, the film may be redissolved in a suitable solvent, such as tertiary butano, and then lyophilized to form a more homogeneous lipid mixture which is in a more easily hydrated powder-like form. This film is covered with an aqueous solution of the targeted drug and the targeting component and allowed to hydrate, typically over a 15-60 minute period with agitation. The size distribution of the resulting multilamellar vesicles can be shifted toward smaller sizes by hydrating the lipids under more vigorous agitation conditions or by adding solubilizing detergents such as deoxycholate.
Micelles are formed by surfactants (molecules that contain a hydrophobic portion and one or more ionic or otherwise strongly hydrophilic groups) in aqueous solution.
Common surfactants well known to one of skill in the art can be used in the micelles of the present invention. Suitable surfactants include sodium laureate, sodium oleate, sodium lauryl sulfate, octaoxyethylene glycol monododecyl ether, octoxynol 9 and PLURONIC F-127 (Wyandotte Chemicals Corp.). Preferred surfactants are nonionic polyoxyethylene and polyoxypropylene detergents compatible with IV injection such as, TWEEN-80, PLURONIC F-68, n-octyl-beta-D-glucopyranoside, and the like. In addition, phospholipids, such as those described for use in the production of liposomes, may also be used for micelle formation.
In some cases, it will be advantageous to include a compound, which promotes delivery of the active substance to its target. For example a ligand which is capable of binding to a receptor present on the target tissue(s) and/or the target cell(s) can be included.
Dosing Regimes
The preparations are administered in a manner compatible With the dosage formulation, and in such amount as will be therapeutically effective. The quantity to be administered depends on the subject to be treated, including, e.g. the weight and age of the subject, the disease to be treated and the stage of disease. Suitable dosage ranges are per kilo body weight normally of the order of several hundred pg active ingredient per administration with a preferred range of from about 0.1 μg to 10000 μg per kilo body weight. Using monomeric forms of the compounds, the suitable dosages are often in the range of from 0.1 μg to 5000 μg per kilo body weight, such as in the range of from about 0.1 μg to 3000 μg per kilo body weight, and especially in the range of from about 0.1 μg to 1000 μg per kilo body weight. Using multimeric forms of the compounds, the suitable dosages are often in the range of from 0.1 μg to 1000 μg per kilo body weight, such as in the range of from about 0.1 μg to 750 μg per kilo body weight, and especially in the range of from about 0.1 μg to 500 μg per kilo body weight such as in the range of from about 0.1 μg to 250 μg per kilo body weight. A preferred dosage would be from about 0.1 to about 5.0 mg, preferably from about 0.3 mg to about 3.0 mg, such as from about 0.5 to about 1.5 mg and especially in the range from 0.8 to 1.0 mg per administration. Administration may be performed once or may be followed by subsequent administrations. The dosage will also depend on the route of administration and will vary with the age, sex and weight of the subject to be treated. A preferred dosage of multimeric forms would be in the interval 1 mg to 70 mg per 70 kilo body weight.
Suitable daily dosage ranges are per kilo body weight per day normally of the order of several hundred pg active ingredient per day with a preferred range of from about 0.1 μg to 10000 μg per kilo body weight per day. Using monomeric forms of the compounds, the suitable dosages are often in the range of from 0.1 μg to 5000 μg per kilo body weight per day, such as in the range of from about 0.1 μg to 3000 μg per kilo body weight per day, and especially in the range of from about 0.1 μg to 1000 μg per kilo body weight per day. Using multimeric forms of the compounds, the suitable dosages are often in the range of from 0.1 μg to 1000 μg per kilo body weight per day, such as in the range of from about 0.1 μg to 750 μg per kilo body weight per day, and especially in the range of from about 0.1 μg to 500 μg per kilo body weight per day, such as in the range of from about 0.1 μg to 250 μg per kilo body weight per day. A preferred dosage would be from about 0.1 to about 100 μg, preferably from about 0.1 μg to about 50 μg, such as from about 0.3 to about 30 μg and especially in the range from 1.0 to 10 μg per kilo body weight per day. Administration may be performed once or may be followed by subsequent administrations. The dosage will also depend on the route of administration and will vary with the age, sex and weight of the subject to be treated. A preferred dosage of multimeric forms would be in the interval 1 mg to 70 mg per 70 kilo body weight per day.
Medical Packaging
The compounds used in the invention may be administered alone or in combination with pharmaceutically acceptable carriers or excipients, in either single or multiple doses. The formulations may conveniently be presented in unit dosage form by methods known to those skilled in the art.
It is preferred that the compounds according to the invention are provided in a kit. Such a kit typically contains an active compound in dosage forms for administration. A dosage form contains a sufficient amount of active compound such that a desirable effect can be obtained when administered to a subject.
Thus, it is preferred that the medical packaging comprises an amount of dosage units corresponding to the relevant dosage regimen. Accordingly, in one embodiment, the medical packaging comprises a composition comprising a compound as defined above or a pharmaceutically acceptable salt thereof and pharmaceutically acceptable carriers, vehicles and/or excipients, said packaging comprising from 1 to 7 dosage units, thereby having dosage units for one or more days, or from 7 to 21 dosage units, or multiples thereof, thereby having dosage units for one week of administration or several weeks of administration.
The dosage units can be as defined above. The medical packaging may be in any suitable form for systemic or local administration. In a preferred embodiment the packaging is in the form of a vial, ampule, tube, blister pack, cartridge or capsule.
When the medical packaging comprises more than one dosage unit, it is preferred that the medical packaging is provided with a mechanism to adjust each administration to one dosage unit only.
Preferably, a kit contains instructions indicating the use of the dosage form to achieve a desirable affect and the amount of dosage form to be taken over a specified time period. Accordingly, in one embodiment the medical packaging comprises instructions for administering the composition.
The following examples illustrate embodiments of the present invention and shall not be construed as a narrowing of the protection sought.
Reference is made to Science, vol. 309, September 2005.
Templates, In Vitro Transcription and Cleavage Analysis:
Templates for in vitro transcription were made by standard PCR using Pfu DNA polymerase (Stratagene) and pDi162SG1 (C. Einvik, H. Nielsen, E. Westhof, F. Michel, S. Johansen, RNA 4, 530 (1998)) as template. The oligonucleotide primers were C289 (5′-AAT TTA ATA CGA CTC ACT ATA GGT TGG GTT GGG MG TAT CAT) and OP233 (5′-GAT TGT CTT GGG ATA CCG) for 166.22, and C294 (5′-AAT TTA ATA CGA CTC ACT ATA GGG MG TAT CAT) and OP233 for 157.22. The PCR products were purified using a commercial kit (GenElute PCR Clean-up kit, Sigma) and transcribed by T7 RNA polymerase (Fermentas) in a 50-μl reaction according to the manufacturer's recommendations. For radioactive labeling of the RNA, 1 μl of [α-32P]UTP (3000 Ci/mmol; Amersham Biosciences) was included in the transcription reaction. Transcripts were purified by phenol:chloroform:isoamylalcohol (25:24:1) extraction and ethanol precipitated. Cleavage experiments were carried out as described in C. Einvik, H. Nielsen, R. Nour, S. Johansen, Nucl. Acids Res. 28, 2194 (2000).
Briefly, the RNA was renatured at 45° C. for 5 min in acetate buffer (pH=5.5) containing 1 M KCl and 25 mM MgCl2. Then the reaction was started by addition of 4 vols. of 47.5 mM Hepes-KOH (pH=7.5) containing 1 M KCl and 25 mM MgCl2 and time samples withdrawn at the appropriate times. The kinetic analysis was performed as described in C. Einvik, H. Nielsen, R. Nour, S. Johansen, Nucl. Acids Res. 28, 2194 (2000) except in that Sigmaplot 8.0 was used in data treatment.
RNA Purification From Gels, 3′-End Labeling, and Primer Extension Analysis:
RNA was purified from polyacrylamide gels by overnight elution at 4° C. in 250 mM sodium acetate (pH=5.2), 1 mM EDTA mixed with 1 vol. of phenol see J. Kjems, J. Egebjerg, J. Christiansen, Analysis of RNA-Protein Complexes in Vitro, (Elsevier Science Ltd, Amsterdam, 1998). pCp was made from Cp and [γ-32P]ATP (6000 Ci/mmol; Amersham Biosciences) using T4 polynucleotide kinase (Fermentas) see J. Kjems, J. Egebjerg, J. Christiansen, Analysis of RNA-Protein Complexes in Vitro, (Elsevier Science Ltd, Amsterdam, 1998). The [32P]pCp was used without further purification to 3′-end label RNA using T4 RNA ligase (Amersham Biosciences) see J. Kjems, J. Egebjerg, J. Christiansen, Analysis of RNA-Protein Complexes in Vitro, (Elsevier Science Ltd, Amsterdam, 1998). The 3′-end labeled RNA was gel-purified before use. Primer extension analysis of cleavage reactions were performed as described (C. Einvik, H. Nielsen, E. Westhof, F. Michel, S. Johansen, RNA 4, 530 (1998) using C291 (5′-GAT TGT CTT GGG AT) as primer.
Ligation Experiments and β-Elimination:
Ligation experiments were performed by mixing gel purified RNAs in dH2O followed by addition of 1 vol. of a 2×reaction buffer (2 M KCl, 50 mM MgCl2, 95 mM Hepes-KOH, pH=7.5) at 45° C. Time samples were withdrawn and stopped by pipetting into denaturing (7 M urea) loading buffer. β-elimination of gel purified 166 RNA was carried out by oxidation in 20 mM sodium periodate followed by aniline cleavage as described (N. K. Tanner, T. R. Cech, Biochemistry 26, 3330 (1987). The RNA was gel-purified before subsequent ligation experiments.
Enzymatic 5′-End Analysis and Alkaline Ladders:
For analysis of the 5′-end, RNAs were initially 3′-end labeled by [32P]pCp and gel purified. Aliquots of the RNA were then subjected to enzymatic analysis using shrimp alkaline phosphatase (SAP; Fermentas) and T4 polynucleotide kinase (Fermentas) or to partial alkaline hydrolysis by boiling in 50 mM NaHCO3/Na2CO3, pH 9.0 (3). The samples were analyzed on 10% denaturing (7 M urea) polyacrylamide gels. A partial RNase T1 (Sigma) digest was used as a size marker.
Analysis of Branch Nucleotides:
The structure analysis of the branch nucleotides were performed on gel purified 3′-fragments isolated from cleavage reactions with body-labeled RNA. Aliquots of the RNA were subjected to enzymatic analysis using mung bean nuclease (Stratagene) and calf intestinal phosphatase (New England Biolab) according to the manufacturers' recommendations. In double digestions, 1 vol. of a 2×reaction buffer (200 mM Tris (pH=9.0), 20 mM MgCl2, 1 mM ZnCl2, 10 mM spermidine) was added to the mung bean nuclease digest and incubation continued in the presence of CIP. The samples were analyzed on 20% denaturing (7 M urea) polyacrylamide gels. A partial alkaline hydrolysis reaction was used as a size marker. Digestion with snake venom phosphodiesterase (Crotalus atrox venom; Pharmacia) was in 100 mM Tris-HCl (pH=8.9), 100 mM NaCl, 14 mM MgCl2 at 25° C. for 30 min. TLC analyses were performed on PEI-cellulose plates using 0.9 M Acetic acid/0.3 M LiCl as running buffer. In preparative experiments, the material was scaped of the plate and the nucleotides eluted in 2 M NH4OH. In the experiments on characterization of the lariat circle (
Cleavage Experiment With Deoxy-Substituted RNA Oligos:
The deoxy-substituted oligonucleotides were purchased from Dharmacon. The ribozyme version used in cleavage experiments with these oligos was made by PCR using C294 and C421 (5′-TCG GM CGA CTG TTC ATT GM C). The cleavage experiments were carried out as described above.
Individual RNA species described in the document are named according to the number of nucleotides included. For example, 166.22 refers to a GIR1 ribozyme including 166 nt upstream of the IPS (internal processing site), and 22 nt down-stream of the IPS. Parentheses are used to describe the origin of a particular RNA species. (166)22 means a 22-nt fragment isolated from cleavage of a 166.22 precursor RNA. Nucleotide numbering is according to the position in the full-length intron (The sequence of Dir.S956 intron has acc. no. X71792 in Genbank).
The cleavage analysis shown in
The reaction with 157.22 is dominated by the forward transesterification. In the mung bean nuclease analysis of branched nucleotides (
The group I twin-ribozyme intron found in the extrachromosomal ribosomal DNA (rDNA) of the myxomycete Didymium iridis (Dir.S956-1) consists of two self-catalytic units, a conventional group I splicing ribozyme (GIR2) and a group I-like cleavage ribozyme (GIR1) (
Primer extension analyses have led to the suggestion of two cleavage sites located three nucleotides apart (5, 8) referred to as IPS1 (internal processing site 1), and IPS2, respectively (
A primer extension stop at IPS1 accumulates over time in 166.22 and a stop at IPS2 accumulates in 157.22 (
The 5′ ends of the two 22-nt RNAs were analyzed by treatment of 3′ end-labeled RNA with modifying enzymes (
Branches in RNA are resistant to digestion with various RNases including mung bean nuclease (13). A resistant fragment was found in mung bean nuclease digests of bodylabeled (157)22 RNA but not (166)22 RNA (
Only the dU232 substrate did not support cleavage (
Individual RNA species described are named according to the number of nucleotides included. For example, 166.22 refers to a GIR1 ribozyme including 166 nt up-stream of the IPS (internal processing site), and 22 nt downstream of the IPS. Parentheses are used to describe the origin of a particular RNA species. (166)22 means a 22-nt fragment isolated from cleavage of a 166.22 precursor RNA. Nucleotide numbering is according to the position in the full-length intron.
The cleavage analysis shown in
The constructs described in
Cells containing the different constructs were plated on LB/Amp plates without or with the inducer arabinose. On the ara+ plate, bright fluorescence is observed with the pBAD-GFP construct, medium fluorescence with the GIR1wtGFP and GIR1 P7−GFP constructs, and no fluorescence with the GIR1 invGFP construct, as expected (
| Number | Date | Country | Kind |
|---|---|---|---|
| PA 2006 00833 | Jun 2006 | DK | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/DK2007/000296 | 6/19/2007 | WO | 00 | 12/17/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60814564 | Jun 2006 | US |
