Patent Application
20210330813
The instant application contains a Sequence Listing which is being submitted in ASCII format via EFS-WEB and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Mar. 4, 2021, is named 069818-0595SequenceListing.txt and is 44.9 KB.
C9ORF72 is a protein which in humans is encoded by the gene C9ORF72. The human C9ORF72 gene is located on the short (p) arm of chromosome 9 open reading frame 72, from base pair 27,546,542 to base pair 27,573,863. The protein is found in many regions of the brain, in the cytoplasm of neurons as well as in presynaptic terminals. Disease causing mutations, GGGGCC repeats, in the gene were linked to frontotemporal dementia (FTD) and of amyotrophic lateral sclerosis (ALS) and were first reported by DeJesus-Hernandez et al. (Neuron (2011) 72 (2): 245-56) and Renton et al. (Neuron (2011), 72 (2): 257-68.). The presence of the repeats results in the formation of RNA foci (sense, antisense) repeat-associated non-AUG (RAN) peptide translation (sense, antisense), which may underlie the cause of disease.
Hence, as the transcripts of the 5′-GGGGCC-3′ repeats, including corresponding antisense transcripts comprising 5′-GGCCCC-3′, are perceived to be the cause of the disease, approaches have been taken to sequence specifically target C9ORF72 transcripts. The paradigm underlying the approach involves a reduction of mutant protein and/or transcripts to thereby reduce the toxic effects resulting therefrom to achieve a reduction and/or delay of disease symptoms, or even to prevent disease symptoms altogether. Such approaches have focused largely on using synthetic oligonucleotides. Approaches for specific targeting of C9ORF72 transcripts utilising RNAi have also been proposed.
RNA interference (RNAi) is a naturally occurring mechanism that involves sequence specific down regulation of mRNA. The down regulation of mRNA results in a reduction of the amount of protein that is expressed. RNA interference is triggered by double stranded RNA. One of the strands of the double stranded RNA is substantially or completely complementary to its target, the mRNA. This strand is termed the guide strand. The mechanism of RNA interference involves the incorporation of the guide strand in the RNA-induced silencing complex (RISC). This complex is a multiple turnover complex that via complementary base paring binds to its target mRNA. Once bound to its target mRNA it can either cleave the mRNA or reduce translation efficiency. RNA interference has since its discovery been widely used to knock down specific target genes. The triggers for inducing RNA interference that have been employed involve the use of siRNAs or shRNAs. In addition, molecules that can naturally trigger RNAi, the so-called miRNAs, have been used to make artificial miRNAs that mimic their naturally occurring counterparts. These strategies have in common that they provide for substantially double stranded RNA molecules that are designed to target a gene of choice. RNAi based therapeutic approaches that utilise the sequence specific modality of RNAi are under development and several are currently in clinical trials.
The present invention provides for specific target RNA sequences that can in particular be applied in RNAi based gene therapy approaches. These specific target RNA sequences were found by selecting target RNA sequences that were conserved in the C9ORF72 target RNA sequence and were shown to provide for efficient silencing of expressed transcripts. Useful target RNA sequences were selected targeting intron 1, exon 2, exon 11 and the antisense transcripts encoded by the human C9ORF72 gene. Useful was also found to be the combination of targeting the antisense transcript and targeting the sense transcripts by selectively targeting a target RNA sequence in intron 1, exon 2, or exon 11.
The current invention relates to gene therapy, and in particular to the use of RNA interference in gene therapy for targeting RNA encoded by the human C9ORF72 gene. Expanded hexanucleotide repeats, (GGGGCCn), in the C9ORF72 gene have been associated with ALS and/or FTD. ALS and/or FTD are diseases of the central nervous system which affect millions of people and for which there is no cure. ALS is characterized by motor neuron degeneration, in the brain and in the spinal cord, that eventually results in respiratory failure, with a median survival of three years after onset. FTD is a common form of early onset dementia affecting younger people. C9ORF72 has been linked to behavioural variant FTD, which involves degeneration of the frontotemperal lobe. The C9ORF72 gene comprising the expanded hexanucleotide repeat, is estimated to be present in about 40% of familial ALS and in about 8-10% of sporadic ALS.
As depicted in
The current invention now provides for an expression cassette encoding a first RNA sequence and a second RNA sequence wherein the first and second RNA sequence are substantially complementary, wherein the first RNA sequence has a sequence length of at least 19 nucleotides and is substantially complementary to a target RNA sequence comprised in an RNA encoded by a human C9ORF72 gene (Reference Sequence: NG_031977.1)).
The first RNA sequence that is to be expressed in according with the invention is to be comprised, in whole or a substantial part thereof, in a guide strand, also referred to as antisense strand as it is complementary (“anti”) to the sense target RNA sequence, the sense target RNA sequence being comprised in an RNA encoded by a human C9ORF72 gene. The second RNA sequence, which is also referred to as “sense strand”, may have substantial sequence identity with or be identical with the target RNA sequence. The first and second RNA sequences are comprised in a double stranded RNA and are substantially complementary. The said double stranded RNA according to the invention is to induce RNA interference to thereby reduce expression of C9ORF72 transcripts, which may include knock down of 5′-GGGGCC-3′ repeat and/or 5′-GGCCCC-3′ repeat containing transcripts, but of non-disease associated C9ORF72 transcripts as well, knocking down both mutant and wild type gene expression. It is understood that substantially complementary in this context means that it is not required to have all the nucleotides of the first and second RNA sequences to be base paired, i.e. to be fully complementary, or all the nucleotides of the first RNA sequence and the target RNA sequence to be base paired. As long as the double stranded RNA is capable of inducing RNA interference to thereby sequence specifically target a sequence comprising the target RNA sequence, such substantial complementarity is contemplated in accordance with the invention.
In one embodiment the double stranded RNA according to the invention comprises a first RNA sequence and a second RNA sequence, wherein the first and second RNA sequence are substantially complementary, and wherein the first RNA sequence has a sequence length of at least 19 nucleotides and is substantially complementary to a target RNA sequence of an RNA encoded by a human C9ORF72 gene, which first RNA sequence is capable of inducing RNA interference to sequence specifically reduce expression of an RNA transcript comprising the target RNA sequence. In a further embodiment, said induction of RNA interference to reduce expression of an RNA transcript comprising the target RNA sequence means that it is to reduce C9ORF72 gene expression.
One can easily determine whether this is the case by using standard luciferase reporter assays and appropriate controls such as described in the examples and as known in the art (Zhuang et al. 2006 Methods Mol Biol. 2006; 342:181-7). For example, a luciferase reporter comprising a target RNA sequence can be used to show that the double stranded RNA according to the invention is capable of sequence specific knock down. Furthermore, such as shown i.a. in the example section, levels of C9ORF72 expression and/or of mutant C9ORF72 can be determined by detecting C9ORF72 RNA (nuclear and/or cytoplasmic), RNA foci, C9ORF72 protein, or C9ORF72 associated dipeptide repeat proteins (DeJesus-Hernandez et al. 2011 Neuron. 72 (2): 245-56) and Ash P E et al. 2013 Neuron. February 20; 77:639.
As said, the double stranded RNA is capable of inducing RNA interference. Double stranded RNA structures that are suitable for inducing RNAi are well known in the art. For example, a small interfering RNA (siRNA) can induce RNAi. An siRNA comprises two separate RNA strands, one strand comprising the first RNA sequence and the other strand comprising the second RNA sequence. An siRNA design that is often used involves 19 consecutive base pairs with a 3′ overhang. The first and/or second RNA sequence may comprise a 3′-overhang. The 3′-overhang preferably is a dinucleotide overhang on both strands of the siRNA. Such a design is based on observed Dicer processing of larger double stranded RNAs that results in siRNAs having these features. The 3′-overhang may be comprised in the first RNA sequence. The 3′-overhang may be in addition to the first RNA sequence. The length of the two strands of which an siRNA is composed may be 19, 20, 21, 22, 23, 24, 25, 26 or 27 nucleotides or more. Each of the two strands comprises the first and second RNA sequence. The strand comprising the first RNA sequence may also consist thereof. The strand comprising the first RNA sequence may also consist of the first RNA sequence and the overhang sequence.
siRNAs may also serve as Dicer substrates. For example, a Dicer substrate may be a 27-mer consisting of two strands of RNA that have 27 consecutive base pairs. The first RNA sequence is positioned at the 3′-end of the 27-mer duplex. At the 3′-ends, like with siRNAs, each or one of the strands may comprise a two nucleotide overhang. The 3′-overhang may be comprised in the first RNA sequence. The 3′-overhang may be in addition to the first RNA sequence. 5′ from the first RNA sequence, additional sequences may be included that are either complementary to the target RNA sequence adjacent or not. The other end of the siRNA dicer substrate is blunt ended. This dicer substrate design may result in a preference in processing by Dicer such that an siRNA can be formed like the siRNA design as described above, having 19 consecutive base pairs and 2 nucleotide overhangs at both 3′-ends. In any case, siRNAs, or the like, are composed of two separate RNA strands (Fire et al. 1998, Nature. 1998 Feb. 19; 391 (6669):806-1 1) each RNA strand comprising or consisting of the first and second RNA sequence in accordance with to the invention.
The first and second nucleotide sequences that are substantially complementary preferably do not form a double stranded RNA of 30 consecutive base pairs or longer, as these can trigger an innate immune response via the double-stranded RNA (dsRNA)-activated protein kinase pathway. Hence, the double stranded RNA is preferably less than 30 consecutive base pairs. Preferably, a pre-miRNA scaffold, a pri-miRNA scaffold, a shRNA, or an siRNA such as designed in accordance with the invention comprising the first and second RNA sequence as described herein does not comprise 30 consecutive base pairs.
The first and second RNA sequences can also be comprised in an shRNA. An shRNA may comprise or consist of from the 5′-end till the 3′-end, 5′-second RNA sequence-loop sequence-first RNA sequence-optional 2 nt overhang sequence-3′. Alternatively, a shRNA may comprise from 5′-first RNA sequence-loop sequence-second RNA sequence—optional 2 nt overhang sequence-3′. Such an RNA molecule forms intramolecular base pairs via the substantially complementary first and second RNA sequence. Suitable loop sequences are well known in the art (i.a. as shown in Dallas et al. 2012 Nucleic Acids Res. 2012 October; 40(18):9255-71 and Schopman et al., Antiviral Res. 2010 May; 86(2):204-11). The loop sequence may also be a stem-loop sequence, whereby the double stranded region of the shRNA is extended. Like the siRNA dicer substrate as described above, a shRNA can be processed by e.g. Dicer to provide for an siRNA having an siRNA design such as described above, having e.g. 19 consecutive base pairs and 2 nucleotide overhangs at both 3′-ends. In case the shRNA is to be processed by Dicer, it is preferred to have the first and second RNA sequence at the end of the shRNA, i.e. such that the putative strands of the siRNA are linked via a stem loop sequence: 5′-first RNA sequence-stem loop sequence-second RNA sequence-optional 2 nt overhang sequence-3′. Or, conversely, 5′-second RNA sequence-stem loop sequence-first RNA sequence-optional 2 nt overhang sequence-3′. Another shRNA design may be a shRNA structure that is processed by the RNAi machinery to provide for an activated RISC complex that does not require Dicer processing (Liu et al., Nucleic Acids Res. 2013, Apr. 1; 41(6):3723-33, incorporated herein by reference), so called AgoshRNAs, which are based on a structure very similar to the miR451 scaffold as described below. Such a shRNA structure comprises in its loop sequence part of the first RNA sequence. Such a shRNA structure may also consist of the first RNA sequence, followed immediately by the second RNA sequence.
A double stranded RNA according to the invention may also be incorporated in a pre-miRNA or pri-mi-RNA scaffold. MicroRNAs, i.e. miRNA, are guide strands that originate from double stranded RNA molecules that are endogenously expressed e.g. in mammalian cells. A miRNA is processed from a pre-miRNA precursor molecule, similar to the processing of a shRNA or an extended siRNA as described above, by the RNAi machinery and incorporated in an activated RNA-induced silencing complex (RISC) (Tijsterman M, Plasterk R H. Dicers at RISC; the mechanism of RNAi. Cell. 2004 Apr. 2; 1 17(1):1-3). A pre-miRNA is a hairpin RNA molecule that can be part of a larger RNA molecule (pri-miRNA), e.g. comprised in an intron, which is first processed by Drosha to form a pre-miRNA hairpin molecule. The pre-miRNA molecule is a shRNA-like molecule that can subsequently be processed by dicer to result in an siRNA-like double stranded RNA duplex. The miRNA, i.e. the guide strand, that is part of the double stranded RNA duplex is subsequently incorporated in RISC. An RNA molecule such as present in nature, i.e. a pri-miRNA, a pre-miRNA or a miRNA duplex, may be used as a scaffold for producing an artificial miRNA that specifically targets a gene of choice. Based on the predicted RNA structure of the RNA molecule as present in nature, e.g. as predicted using e.g. m-fold software using standard settings (Zuker. Nucleic Acids Res. 31 (13), 3406-3415, 2003), the natural miRNA sequence as it is present in the RNA structure (i.e. duplex, pre-miRNA or pri-miRNA), and the sequence present in the structure that is substantially complementary therewith are removed and replaced with a first RNA sequence and a second RNA sequence according to the invention. The first RNA sequence and the second RNA sequence are preferably selected such that the predicted secondary RNA structures that are formed, i.e. of the pre-miRNA, pri-miRNA and/or miRNA duplex, resemble the corresponding predicted original secondary structure of the natural RNA sequences. pre-miRNA, pri-miRNA and miRNA duplexes (that consist of two separate RNA strands that are hybridized via complementary base pairing) as found in nature often are not fully base paired, i.e. not all nucleotides that correspond with the first and second strand as defined above are base paired, and the first and second strand are often not of the same length. How to use miRNA precursor molecules as scaffolds for any selected target RNA sequence and substantially complementary first RNA sequence is described e.g. in Liu Y P Nucleic Acids Res. 2008 May; 36(9):281 1-24, which is incorporated herein by reference.
A pri-miRNA can be processed by the RNAi machinery of the cell. The pri-miRNA comprising flanking sequences at the 5′-end and the 3′-end of a pre-miRNA hairpin and/or shRNA like molecule. Such a pri-miRNA hairpin can be processed by Drosha to produce a pre-miRNA. The length of the flanking sequences can vary but may be around 80 nt in length (Zeng and Cullen, J Biol Chem. 2005 Jul. 29; 280(30):27595-603; Cullen, Mol Cell. 2004 Dec. 22; 16(6):861-5). The minimal length of the single-stranded flanks can easily be determined as when it becomes too short, the RNA molecule may lose its function because e.g. Drosha processing fails resulting in sequence specific inhibition being reduced or even absent. In one embodiment, the pri-miRNA scaffold carrying the first and second RNA sequence according to the invention has a 5′-sequence flank and a 3′ sequence flank relative to the predicted pre-miRNA structure of at least 5, at least 10, at least 15, at least 20, at least 30, at least 40, or at least 50 nucleotides. Preferably, the pri-miRNA derived flanking sequences (5′ and 3′) comprised in the miRNA scaffold are derived from the same naturally occurring pri-miRNA sequence. Preferably, pre-miRNA and/or the pri-miRNA derived flanking sequences (5′ and 3′) and/or loop sequences comprised in the miRNA scaffold are derived from the same naturally occurring pri-miRNA sequence, e.g. as shown and listed in table 16. As the (putative) guide strand RNA as comprised in the endogenous miRNA sequence can be replaced by a sequence including (or consisting of) the first RNA sequence, and the passenger strand sequence replaced by a sequence including (or consisting of) the second RNA sequence, it is understood that flanking sequences and/or loop sequences of the pri-miRNA or pre-miRNA sequences of the endogenous sequence may include minor sequence modifications such that the predicted structure of the scaffold miRNA sequence (e.g. M-fold predicted structure) is the same as the predicted structure of the endogenous miRNA sequence.
The first and second RNA sequence, which can form a double stranded RNA, of the invention are encoded by an expression cassette. It is understood that when the double stranded RNA is to be e.g. an siRNA, consisting of two RNA strands, that there may be two expression cassettes required. One encoding an RNA strand comprising the first RNA sequence, the other cassette encoding an RNA strand comprising the first RNA strand. When the double stranded RNA is comprised in a single RNA molecule, e.g. encoding a shRNA, pre-miRNA or pri-miRNA, one expression cassette may suffice. A pol II expression cassette may comprise a promoter sequence a sequence encoding the RNA to be expressed followed by a polyadenylation sequence. In case the double stranded RNA that is expressed comprises a pri-miRNA scaffold, the encoded RNA sequence may encode for intron sequences and exon sequences and 3′-UTR's and 3′-UTRs. A pol III expression cassette in general comprises a promoter sequence, followed by a sequence encoding the RNA (e.g. shRNA sequence, pre-miRNA, or a strand of the double stranded RNAs to be comprised in e.g. an siRNA or extended siRNA). A pol I expression cassette may comprise a pol I promoter, followed by the RNA encoding sequence and a 3′-Box. Expression cassettes for double stranded RNAs are well known in the art, and any type of expression cassette can suffice, e.g. one may use a pol III promoter, a pol II promoter or a pol I promoter (i.a. ter Brake et al., Mol Ther. 2008 March; 16(3):557-64, Maczuga et al., BMC Biotechnol. 2012 Jul. 24; 12:42).
As is clear from the above, the first and second RNA sequence that are comprised in a double stranded RNA can contain additional nucleotides and/or nucleotide sequences. The double stranded RNA may be comprised in a single RNA sequence or comprised in two separate RNA strands. Whatever design is used, it is designed such that from the first and second RNA sequence an antisense RNA molecule comprising the first RNA sequence, in whole or a substantial part thereof, of the invention can be processed by the RNAi machinery such that it is incorporated in the RISC complex to have its action, i.e. to induce RNAi against the RNA target sequence comprised in an RNA encoded by the C9ORF72 gene. The sequence comprising or consisting of the first RNA sequence, in whole or a substantial part thereof, being capable of sequence specifically targeting RNA encoded by a human C9ORF72 gene. Hence, as long as the double stranded RNA is capable of inducing RNAi, such a double stranded RNA is contemplated in the invention. In one embodiment, the double stranded RNA according to the invention is comprised in a pre-miRNA scaffold, a pri-miRNA scaffold, a shRNA, or an siRNA. Preferably the first and second RNA sequence as encoded by the expressed cassette are to be contained in a single transcript. It is understood that the expressed transcript in subsequent processing, i.e. cleavage, results in the single transcript being processed into multiple separate RNA molecules.
The term complementary is herein defined herein as nucleotides of a nucleic acid sequence that can bind to another nucleic acid sequence through hydrogen bonds, i.e. nucleotides that are capable of base pairing. Ribonucleotides, the building blocks of RNA are composed of monomers (nucleotides) containing a sugar, phosphate and a base that is either a purine (guanine, adenine) or pyrimidine (uracil, cytosine). Complementary RNA strands form double stranded RNA. A double stranded RNA may be formed from two separate complementary RNA strands or the two complementary RNA strands may be comprised in one RNA strand. In complementary RNA strands, the nucleotides cytosine and guanine (C and G) can form a base pair, guanine and uracil (G and U), and uracil and adenine (U and A) can form a base pair as well. The term substantial complementarity means that is not required to have the first and second RNA sequence to be fully complementary, or to have the first RNA sequence and target RNA sequence or sequences of RNA encoded by a human C9ORF72 gene to be fully complementary.
The substantial complementarity between the first RNA sequence and the target RNA sequence preferably consists of at most two mismatched nucleotides, more preferably having one mismatched nucleotide, most preferably having no mismatches. It is understood that one mismatched nucleotide means that over the entire length of the first RNA sequence when base paired with the target RNA sequence one nucleotide does not base pair with the target RNA sequence. Having no mismatches means that all nucleotides of the first RNA sequence base pair with the target RNA sequence, having 2 mismatches means two nucleotides of the first RNA sequence do not base pair with the target RNA sequence. The first RNA sequence may also comprise additional nucleotides that do not have complementarity to the target RNA sequence, and may be longer than e.g. 21 nucleotides, in such a scenario, the substantial complementarity is determined over the entire length of the target RNA sequence. This means that the target RNA sequence in this embodiment has either no, one or two mismatches over its entire length when base paired with the first RNA sequence.
As shown in the example section, double stranded RNAs comprising a first nucleotide sequence length of 21 and 22 nucleotides were tested. These first RNA sequences had no mismatches and were fully complementary with the target RNA sequence. Having a few mismatches between the first nucleotide sequence and the target RNA sequence may however be allowed according to the invention, as long as the double stranded RNA according to the invention is capable of reducing expression of transcripts comprising the target RNA sequence, such as a luciferase reporter or e.g. a transcript comprising the target RNA sequence. In this embodiment, substantial complementarity between the first RNA sequence and the target RNA sequence consists of having no, one or two mismatches over the entire length of either the first RNA sequence or the target RNA sequence encoded by an RNA of the human C90RF72, whichever is the shortest.
As said, a mismatch according to the invention means that a nucleotide of the first RNA sequence does not base pair with the target RNA sequence encoded by an RNA of the human C90RF72. Nucleotides that do not base pair are A and A, G and G, C and C, U and U, A and C, C and U, or A and G. A mismatch may also result from a deletion of a nucleotide, or an insertion of a nucleotide. When the mismatch is a deletion in the first RNA sequence, this means that a nucleotide of the target RNA sequence is not base paired with the first RNA sequence when compared with the entire length of the first RNA sequence. Nucleotides that can base pair are A-U, G-C and G-U. A G-U base pair is also referred to as a G-U wobble, or wobble base pair. In one embodiment the number of G-U base pairs between the first RNA sequence and the target RNA sequence is 0, 1 or 2. In one embodiment, there are no mismatches between the first RNA sequence and the target RNA sequence and a G-U base pair or G-U pairs are allowed. Preferably, there may be no G-U base pairs between the first RNA sequence and the target RNA sequence, or the first RNA sequence and the target RNA sequence only have base pairs that are A-U or G-C. Preferably, there are no G-U base pairs and no mismatches between the first RNA sequence and the target RNA sequence. The first RNA sequence of the double stranded RNA according to invention preferably is fully complementary to the target RNA sequence, said complementarity consisting of G-U, G-C and A-U base pairs. The first RNA sequence of the double stranded RNA according to invention more preferably is fully complementary to the target RNA sequence, said complementarity consisting of G-C and A-U base pairs.
In one embodiment the first RNA sequence and the target RNA sequence have at least 15, 16, 17, 18, or 19 nucleotides that base pair. Preferably the first RNA sequence and the target RNA sequence are substantially complementary, said complementarity comprising at least 19 base pairs. In another embodiment, the first RNA sequence has at least 8, 9, 10, 11, 12, 13 or 14 consecutive nucleotides that base pair with consecutive nucleotides of the target RNA sequence. In another embodiment, the first RNA sequence has at least 19 consecutive nucleotides that base pair with consecutive nucleotides of the target RNA sequence. In another embodiment the first RNA sequence comprises at least 19 consecutive nucleotides that base pair with 19 consecutive nucleotides of the target RNA sequence. In still another embodiment, the first RNA sequence has at least 17 nucleotides that base pair with the target RNA sequence and has at least 15 consecutive nucleotides that base pair with consecutive nucleotides of the target RNA sequence. The sequence length of the first nucleotide is preferably at most 21, 22, 23, 24, 25, 26, or 27 nucleotides. In another embodiment, the first RNA sequence has at least 20 consecutive nucleotides that base pair with 20 consecutive nucleotides of the target RNA sequence. In another embodiment the first RNA sequence comprises at least 21 consecutive nucleotides that base pair with 21 consecutive nucleotides of the target RNA sequence.
As said, it may be not required to have full complementarity (i.e. full base pairing (no mismatches) and no G-U base pairs) between the first nucleotide sequence and the target RNA sequence as such a first nucleotide sequence can still allow for sufficient suppression of gene expression. Also, not having full complementarity may be contemplated for example to avoid or reduce off-target RNA sequence specific gene suppression while maintaining sequence specific inhibition of transcripts comprising the target RNA sequence. However, it may be preferred to have full complementarity as it may result in more potent inhibition. Without being bound by theory, having full complementarity between the first RNA sequence and the target RNA sequence may allow for the activated RISC complex comprising said first RNA sequence (or a substantial part thereof) to cleave its target RNA sequence, whereas having mismatches may hamper cleavage and can result in mainly allowing inhibition of translation, of which the latter may result in less potent inhibition.
With regard to the second RNA sequence, the second RNA sequence is substantially complementary with the first RNA sequence. The second RNA sequence combined with the first RNA sequence forms a double stranded RNA. As said, this is to form a suitable substrate for the RNA interference machinery such that a guide sequence derived from the first RNA sequence is comprised in the RISC complex in order to sequence specifically inhibit expression of its target, i.e. RNA encoded by a human C9ORF72 gene. The sequence of the second RNA sequence has sequence similarity with the target RNA sequence. However, the substantial complementarity if the second RNA sequence with the first RNA sequence may be selected to have less substantial complementarity as compared with the substantial complementarity between the first RNA sequence and the target RNA sequence. Hence, the second RNA sequence may comprise 0, 1, 2, 3, 4, or more mismatches, 0, 1, 2, 3, or more G-U wobble base pairs, and may comprise insertions of 0, 1, 2, 3, 4, nucleotides and/or deletions of 0, 1, 2, 3, 4, nucleotides. Preferably the first RNA sequence and the second RNA sequence are substantially complementary, said complementarity comprising 0, 1, 2 or 3 G-U base pairs and/or wherein said complementarity comprises at least 17 base pairs. These mismatches, G-U wobble base pairs, insertions and deletions, are with regard to the first RNA sequence, i.e. the double stranded region that is formed between the first and second RNA sequence. As long as the first and second RNA sequence can substantially base pair, and are capable of inducing sequence specific inhibition of an RNA encoded by a human C9ORF72 gene, such substantial complementarity is allowed according to the invention. It is also understood that substantially complementarity between the first RNA sequence and the second RNA sequence may depend on the double stranded RNA design of choice. It may depend for example on the miRNA scaffold that is chosen for in which the double stranded RNA is to be incorporated.
As is clear from the above, the substantial complementarity between the first RNA sequence and the second RNA sequence, may comprise mismatches, deletions and/or insertions relative to a first and second RNA sequence being fully complementary (i.e. fully base paired). In one embodiment, the first and second RNA sequences have at least 11 consecutive base pairs. Hence, at least 11 consecutive nucleotides of the first RNA sequence and at least 11 consecutive nucleotides of the second RNA sequence are fully complementary. In another embodiment the first and second RNA sequence have at least 15 nucleotides that base pair. Said base pairing between at least 15 nucleotides of the first RNA sequence and at least 15 nucleotides of the second RNA sequence may consist of G-U, G-C and A-U base pairs, or may consist of G-C and A-U base pairs. In another embodiment, the first and second RNA sequence have at least 15 nucleotides that base pair and have at least 11 consecutive base pairs. In another embodiment, the first RNA sequence and the second RNA sequence are substantially complementary, wherein said complementarity comprises at least 17 base pairs. Said 17 base pairs may preferably be 17 consecutive base pairs, said base pairing consisting of G-U, G-C and A-U base pairs or consisting of G-C and A-U base pairs.
As said, the current invention now provides for an expression cassette encoding a first RNA sequence and a second RNA sequence wherein the first and second RNA sequence are substantially complementary, wherein the first RNA sequence has a sequence length of at least 19 nucleotides and is substantially complementary to a target RNA sequence comprised in an RNA encoded by a human C9ORF72 gene. As shown in the examples, suitable target RNA sequences in accordance with the invention are provided (see e.g. table 14). Hence, in one embodiment, an expression cassette is provided encoding a first RNA sequence and a second RNA sequence wherein the first and second RNA sequence are substantially complementary, wherein the first RNA sequence has a sequence length of at least 19 nucleotides and is substantially complementary to a target RNA sequence selected from the group listed in table 14 comprised in an RNA encoded by a human C9ORF72 gene.
Preferably, the target RNA sequence in accordance with the invention is selected from the group listed in table 1 below, consisting of SEQ ID NOs. 2, 4, 15, 21, 31, 32, and 46.
Table 1. SEQ ID NOs. 2, 4 and 31 correspond with target RNA sequences of transcripts encoded by the human C9ORF72 gene of intron 1; SEQ ID NO. 15 and 21 corresponds with a target RNA sequence in antisense transcripts encoded by the human C9ORF72 gene; SEQ ID NO. 32 corresponds with a target RNA sequence of transcripts encoded by the human C9ORF72 gene of exon 2; SEQ ID NO. 46 corresponds with a target RNA sequence of transcripts encoded by the human C9ORF72 gene of exon 11.
For these target RNA sequences it was found that surprisingly highly advantageous suitable first and second RNA sequences could be made in accordance with the invention to provide for an expression cassette encoding said first RNA sequence and said second RNA sequence, wherein the first and second RNA sequence are substantially complementary, wherein the first RNA sequence has a sequence length of at least 19 nucleotides and is substantially complementary to one of said target RNA sequences to highly efficiently induce RNAi to reduce C9ORF72 gene expression. It is understood that the reduction of gene expression may include a reduction of transcripts that encode hexanucleotide repeat sequences as well.
As shown in the examples, the first and second RNA sequence of the invention, may be preferably incorporated in a pre-miRNA or a pri-miRNA scaffold derived from miR101 or a pri-miRNA or pre-miRNA scaffold derived from miR451. Pri-miRNA scaffolds for miR451 and miR101 are depicted in
As is shown in the examples, a first RNA sequence of 21 (for a miR101 scaffold) or 22 nucleotides (e.g. for a miR451) in length may be selected and incorporated in a miRNA scaffold. Such a miRNA scaffold sequence is subsequently processed by the RNAi machinery as present in the cell. When reference is made to miRNA scaffold it is understood to comprise pri-miRNA structures or pre-miRNA structures. As shown in the examples, such miRNA scaffolds, when processed in a cell, result in guide sequences comprising the first RNA sequence, or a substantial part thereof, in the range of 18-23 nucleotides in length for the 101 scaffold and in the range of 21-30 nucleotides in length for the 451 scaffold. Such guide strands being capable of reducing C9ORF72 transcript expression by targeting the selected target sequences. As is clear from the above, and as shown in the examples, the first RNA sequence as it is encoded by the expression cassette of the invention, is comprised in part or in whole, in a guide strand when it has been processed by the RNAi machinery of the cell. Hence, the guide strand that is to be generated from the RNA encoded by the expression cassette, comprising the first RNA sequence and the second RNA sequence is to comprise at least 18 nucleotides of the first RNA sequence. Preferably, such a guide strand comprises at least 19 nucleotides, 20 nucleotides, 21 nucleotides, or 22 nucleotides. A guide strand can comprise the first RNA sequence also as a whole. In selecting a miRNA scaffold, the first RNA sequence can be selected such that it is to replace the original guide strand. As shown in the example section, this does not necessarily mean that guide strand produced from such an artificial scaffold are identical in length as the first RNA sequence selected, nor that the first RNA sequence is in its entirety to be found in the guide strand that is produced.
A miRNA 451 scaffold, as shown in the examples, and as shown in
A miRNA 101 scaffold, as shown in the examples and in
In one embodiment, an expression cassette according to the invention is provided, wherein said first RNA sequence is substantially complementary to a target RNA sequence comprised in antisense RNA transcripts encoded by the human C9ORF72 gene. Preferably said first RNA sequence is substantially complementary to SEQ ID NO. 15 or 21. More preferably said first RNA sequence has a length of 19, 20, 21, or 22 nucleotides. More preferably said first RNA sequence is fully complementary over its entire length with said first RNA target sequence. Most preferably said first RNA sequence has a length of 19, 20, 21, or 22 nucleotides, wherein said first RNA sequence is fully complementary over its entire length with said first RNA target sequence. Said first RNA strand can be SEQ ID NO. 68 or 74.
As said, such a first RNA sequence is to be combined with a second RNA sequence. As described herein, the skilled person is well capable of designing and selecting a suitable second RNA sequence in order to provide for a first and second RNA sequence that can induce RNA interference when expressed in a cell. Suitable second RNA sequences that can be contemplated are listed below.
Said first RNA sequence is preferably comprised in a miRNA scaffold, more preferably a miR101 scaffold or a miR451 scaffold, such as shown in the examples. A suitable scaffold comprising a first and second RNA sequence in accordance with the invention can be a sequence such as SEQ ID NO. 119 or 120.
Such first RNA sequences as described above can be comprised in expression cassettes, such as e.g. depicted in
Such first and second RNA sequences as described above can be comprised in expression cassettes, such as e.g. depicted in
Accordingly, targeting these target RNA sequences, utilizing such first and second RNA sequences, was found to be in particular useful for reducing expression of antisense RNA transcripts encoded by the human C9ORF72 gene.
In another embodiment, an expression cassette according to the invention is provided, wherein said first RNA sequence is substantially complementary to a target RNA sequence comprised in exon 2 containing RNA transcripts encoded by the human C9ORF72 gene. Preferably said first RNA sequence is substantially complementary to SEQ ID NO. 32. More preferably said first RNA sequence has a length of 19, 20, 21, or 22 nucleotides. More preferably said first RNA sequence is fully complementary over its entire length with said first RNA target sequence. Most preferably said first RNA sequence has a length of 19, 20, 21, or 22 nucleotides, wherein said first RNA sequence is fully complementary over its entire length with said first RNA target sequence. Preferably, said first RNA strand is selected from either SEQ ID NO. 86 or 91.
Such a first RNA sequence is to be combined with a second RNA sequence. As described herein, the skilled person is well capable of designing and selecting a suitable second RNA sequence in order to provide for a first and second RNA sequence that can induce RNA interference when expressed in a cell. Suitable second RNA sequences that can be contemplated are listed below.
Said first RNA sequence is preferably comprised in a miRNA scaffold, more preferably a miR101 scaffold or a miR451 scaffold, such as shown in the examples. A suitable scaffold comprising a first and second RNA sequence in accordance with the invention can be a sequence such as SEQ ID NO. 123 or 124.
Such first RNA sequences as described above can be comprised in expression cassettes, such as e.g. depicted in
Such first and second RNA sequences as described above can be comprised in expression cassettes, such as e.g. depicted in
Accordingly, targeting these target RNA sequences, utilizing such first and second RNA sequences, was found to be in particular useful for reducing expression of exon 2 containing RNA transcripts encoded by the human C9ORF72 gene.
In another embodiment, an expression cassette according to the invention is provided, wherein said first RNA sequence is substantially complementary to a target RNA sequence comprised in intron 1 containing RNA transcripts encoded by the human C9ORF72 gene. Preferably said first RNA sequence is substantially complementary to SEQ ID NO. 2, SEQ ID NO. 4 or SEQ ID NO. 31. More preferably said first RNA sequence has a length of 19, 20, 21, or 22 nucleotides. More preferably said first RNA sequence is fully complementary over its entire length with said first RNA target sequence. Most preferably said first RNA sequence has a length of 19, 20, 21, or 22 nucleotides, wherein said first RNA sequence is fully complementary over its entire length with said first RNA target sequence. Preferably, said first RNA strand is selected from the SEQ ID NO. 52, 59 and 85.
Such a first RNA sequence is to be combined with a second RNA sequence. As described herein, the skilled person is well capable of designing and selecting a suitable second RNA sequence in order to provide for a first and second RNA sequence that can induce RNA interference when expressed in a cell. Suitable second RNA sequences that can be contemplated are listed below.
Said first RNA sequence is preferably comprised in a miRNA scaffold, more preferably a miR101 scaffold or a miR451 scaffold, such as shown in the examples. A suitable scaffold comprising a first and second RNA sequence in accordance with the invention can be a sequence such as SEQ ID NO. 128, 129 or 130.
Such first RNA sequences as described above can be comprised in expression cassettes, such as e.g. depicted in
Accordingly, targeting these target RNA sequences, utilizing such first and second RNA sequences, was found to be in particular useful for reducing expression of intron 1 containing RNA transcripts encoded by the human C9ORF72 gene.
In another embodiment, an expression cassette according to the invention is provided, wherein said first RNA sequence is substantially complementary to a target RNA sequence comprised in exon 11 containing RNA transcripts encoded by the human C9ORF72 gene. Preferably said first RNA sequence is substantially complementary to SEQ ID NO. 46. More preferably said first RNA sequence has a length of 19, 20, 21, or 22 nucleotides. More preferably said first RNA sequence is fully complementary over its entire length with said first RNA target sequence. Most preferably said first RNA sequence has a length of 19, 20, 21, or 22 nucleotides, wherein said first RNA sequence is fully complementary over its entire length with said first RNA target sequence. Preferably, said first RNA sequence is either SEQ ID NO. 104 or 109.
Such a first RNA sequence is to be combined with a second RNA sequence. As described herein, the skilled person is well capable of designing and selecting a suitable second RNA sequence in order to provide for a first and second RNA sequence that can induce RNA interference when expressed in a cell. Suitable second RNA sequences that can be contemplated are listed below.
Said first RNA sequence is preferably comprised in a miRNA scaffold, more preferably a miR101 scaffold or a miR451 scaffold, such as shown in the examples. A suitable scaffold comprising a first and second RNA sequence in accordance with the invention can be a sequence such as SEQ ID NO. 133 or 134.
Such first RNA sequences as described above can be comprised in expression cassettes, such as e.g. depicted in
Such first and second RNA sequences as described above can be comprised in expression cassettes, such as e.g. depicted in
Accordingly, targeting these target RNA sequences, utilizing such first and second RNA sequences, was found to be in particular useful for reducing expression of exon 11 containing RNA transcripts encoded by the human C9ORF72 gene.
As described above, and as shown in the examples, these target sequences were found to be in particular suitable for reducing C9ORF72 gene expression via an RNAi approach that utilizes an expression cassette encoding a first RNA sequence and a second RNA sequence wherein the first and second RNA sequence are substantially complementary, wherein the first RNA sequence has a sequence length of at least 19 nucleotides and is substantially complementarity to a target RNA sequence comprised in an RNA encoded by a human C9ORF72 gene.
Moreover, and in further embodiments, one or more expression cassettes are provided for combined targeting of said target RNA sequences. In particularly useful was found to be the combination of targeting sense and antisense transcripts. Hence, combined targeting of RNA target sequences comprised in antisense RNA encoded by a human C9ORF72 gene, with targeting of RNA target sequences comprised in either an exon 2, exon 11 or intron 1 containing RNA encoded by the human C9ORF72 gene is contemplated in the invention. Such combined targeting is to reduce hexanucleotide repeat containing transcripts and/or expressed DPR polypeptides even further.
Combined targeting of RNA target sequences can be obtained by providing two separate expression cassettes. Examples of expression cassettes for a miR451 scaffold and a miR101 scaffold are depicted in
Hence, in one embodiment, one or more expression cassettes are provided for combined targeting of SEQ ID NO.15, with one of the target RNA sequences selected from the group consisting of SEQ ID NO. 2, 4, 31, 32 and 46. In another embodiment, one or more expression cassettes in accordance with the invention are provided for combined targeting of SEQ ID NO. 21 with one of the target RNA sequences selected from the group consisting of SEQ ID NO. 2, 4, 31, 32 and 46.
Preferably a pol II promoter is used, such as a CAG promoter (i.a. Miyazaki et al. Gene. 79 (2): 269-77; Niwa, Gene. 108 (2): 193-9), a PGK promoter, or a CMV promoter (Such as depicted e.g. in FIG. 2 of WO2016102664, which is herein incorporated by references). As ALS and/or FTD affects neurons, it may in particularly be useful to use a neurospecific promoter. Examples of suitable neurospecific promoters are Neuron-Specific Enolase (NSE), human synapsin 1, caMK kinase and tubuline (Hioki et al. Gene Ther. 2007 June; 14(11):872-82). Other suitable promoters that can be contemplated are inducible promoters, i.e. a promoter that initiates transcription only when the host cell is exposed to some particular stimulus.
Said expression cassettes according to the invention can be transferred to a cell, using e.g. transfection methods. Any suitable means may suffice to transfer an expression cassette according to the invention. Preferably, gene therapy vectors are used that stably transfer the expression cassette to the cells such that stable expression of the double stranded RNAs that induce sequence specific inhibition of the C9ORF72 gene as described above can be achieved. Suitable vectors may be lentiviral vectors, retrotransposon based vector systems, or AAV vectors. It is understood that as e.g. lentiviral vectors carry an RNA genome, the RNA genome will encode for the said expression cassette such that after transduction of a cell, the said DNA sequence and said expression cassette is formed. Preferably a viral vector is used such as AAV. Preferably the AAV vector that is used is an AAV vector of serotype 5. AAV of serotype 5 (also referred to as AAV5) may be in particularly useful for transducing human neurons and human astrocytes such as shown in the examples. Thus, AAV5 can efficiently transduce different human cell types of the CNS including FBN, dopaminergic neurons, motor neurons and astrocytes and is therefore a suitable vector candidate to deliver therapeutic genes to the CNS to treat neurogenerative diseases, including but not limited to the treatment of ALS and/or FTD via targeting e.g. C9ORF72 as described herein. The production of AAV vectors comprising any expression cassette of interest is well described in; WO2007/046703, WO2007/148971, WO2009/014445, WO2009/104964, WO201 1/122950, WO2013/0361 18, which are incorporated herein in its entirety.
AAV sequences that may be used in the present invention for the production of AAV vectors, e.g. produced in insect or mammalian cell lines, can be derived from the genome of any AAV serotype. Generally, the AAV serotypes have genomic sequences of significant homology at the amino acid and the nucleic acid levels, provide an identical set of genetic functions, produce virions which are essentially physically and functionally equivalent, and replicate and assemble by practically identical mechanisms. For the genomic sequence of the various AAV serotypes and an overview of the genomic similarities see e.g. GenBank Accession number U89790; GenBank Accession number J01901; GenBank Accession number AF043303; GenBank Accession number AF085716; Chlorini et al. (1997, J. Vir. 71: 6823-33); Srivastava et al. (1983, J. Vir. 45:555-64); Chlorini et al. (1999, J. Vir. 73:1309-1319); Rutledge et al. (1998, J. Vir. 72:309-319); and Wu et al. (2000, J. Vir. 74: 8635-47). AAV serotypes 1, 2, 3, 4 and 5 are preferred source of AAV nucleotide sequences for use in the context of the present invention. Preferably the AAV ITR sequences for use in the context of the present invention are derived from AAV1, AAV2, and/or AAV5. Likewise, the Rep52, Rep40, Rep78 and/or Rep68 coding sequences are preferably derived from AAV1, AAV2 and AAV5. The sequences coding for the VP1, VP2, and VP3 capsid proteins for use in the context of the present invention may however be taken from any of the known 42 serotypes, more preferably from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9 or newly developed AAV-like particles obtained by e.g. capsid shuffling techniques and AAV capsid libraries. AAV capsids may consist of VP1, VP2 and VP3, but may also consist of VP1 and VP3.
In another embodiment, a host cell is provided comprising the said DNA sequence or said expression cassette according to the invention. For example, the said expression cassette or DNA sequence may be comprised in a plasmid contained in bacteria. Said expression cassette or DNA sequence may also be comprised in a production cell that produces e.g. a viral vector. Said expression cassette may also be provided in a baculovirus vector.
As shown in the example section, and as explained above, the double stranded RNA according to the invention, the DNA sequence according to invention, the expression cassette according to the invention and the gene therapy vector according to the invention are for use in a medical treatment, in particular for use in the treatment of ALS and/or FTD disease.
Said first and second RNA sequences in accordance with the invention, when expressed in a cell preferably reduce expression of RNA encoded by a human C9ORF72 gene both in the cell nucleus as in the cytoplasm. In particularly it was found that miRNA-scaffolds such as based on miR101 and miR451 were in particular useful as scaffolds for inducing sequence specific knock down of a selected target RNA sequence comprised in nuclear RNA targets, e.g. comprised in intronic sequences. It is understood that such uses may not necessarily be restricted to targeting C9ORF72 transcripts in the nucleus, but may be in general useful against other nuclear transcripts as well. In a further embodiment, such nuclear RNA targeting preferably comprises nuclear RNAs expressed in the CNS, preferably in neuronal cells, most preferably in human neuronal cells.
Accordingly said gene therapy vector when said first and second RNA sequences are expressed in a cell can advantageously reduce expression of C9 RAN protein levels. Furthermore, said first and second RNA sequences when expressed in a cell can also advantageously reduce expression of G4C2 foci and/or G2C4 foci.
The invention also provides for a medical treatment, said medical treatment when using an AAV vector (or likewise for any suitable gene therapy vector) comprising delivery of AAV vector in accordance with the invention to the CNS. Preferably, said medical treatment, utilizing a gene therapy vector in accordance with the invention, comprises transfer of the vector to a motorneuron, as affected cells in the CNS include motorneurons. Preferably, said medical treatment may also comprise transfer of the vector to a human frontal brain neuron and/or anterior brain neuron. Preferably, said gene therapy vector is administered to the spinal cord. In another embodiment, said gene therapy vector in accordance with the invention is administered to the frontal lobe and/or anterior temporal lobe.
Said delivery to the CNS may comprise intraparenchymal injections (Samaranch et al., Gene Ther. 2017 April; 24(4):253-261). Said delivery may also comprise delivery to the cerebrospinal fluid upon which affected CNS regions may be effectively transduced as the vector via the cerebrospinal fluid can reach affected areas in the disease, such as cortical areas and the spinal cord, via diffusion. Said delivery in further embodiments may thus comprise intrathecal (e.g. WO2015060722; Bailey et al., Mol Ther Methods Clin Dev. 2018 Feb. 15; 9:160-171) or subpial (Miyanohara et al., Mol Ther Methods Clin Dev. 2016 Jul. 13; 3:16046.) injections of the vector. Said delivery may also comprise intracerebroventricular (ICV) or intrastriatal injections. Preferably, the delivery does not comprise intraparenchymal injections, as such delivery routes may have a risk of inducing injury. Said delivery may also comprise a combination of said delivery methods. For example, intrathecal or subpial injection may be combined within tracerebroventricular and/or intrastriatal injections. Intrathecal or subpial injection may also be combined with intraparenchymal injections.
Such delivery methods representing an efficient way to deliver the gene therapy vector to the CNS, including affected cortical and spinal cord regions and to target the neurons. Such injections are preferably carried out through MRI-guided injections. Said methods of treatments are in particular useful for human subjects having ALS and/or FTD. It is understood that the treatment of ALS and/or FTD involves human subjects having ALS and/or FTD including human subjects having a genetic predisposition of developing ALS and/or FTD that do not yet show signs of the disease. Hence, the treatment of human subjects with ALS and/or FTD includes the treatment of any human subject carrying an C9ORF72 allele with hexanucleotide repeats.
The human C9ORF72 gene consists of 12 exons that can be transcribed in three different transcript variants (V1, V2, V3) (
Notably, we found that C9ORF72 is expressed at higher levels (˜2 fold) in the cerebellum compared to cortex in both C9ORF72-ALS patients and controls. It was also found that C9ORF72 mRNA expression is consistently reduced in both cerebellum and cortex of C9ORF72ALS patients. In both cerebellum and cortex, the relative expression of V1, V2 and V3 in patients and controls were found to be similar, suggesting that the reduction of C9ORF72 mRNA levels in C9-ALS patients is not variant specific.
From a wild-type RNA, the C9ORF72 Intron 1 should be spliced out and degraded. In a G4C2 repeat containing transcript defective splicing may result in accumulation of transcripts. Read alignments from C9-ALS patients and controls were compared to investigate the sequence conservation of intronic and exonic regions of C9ORF72 RNA transcripts (
Design of miRNAs Targeting Conserved C9ORF72 Regions
We selected target sites for a total silencing approach in exon 2 and 11, as target sites in these regions are present in all sense transcripts, spliced and unspliced (see
Table 14. C1-C11 and C22-C31 correspond with target RNA sequences of transcripts encoded by the human C9ORF72 gene of intron 1; C12-C21 correspond with target RNA sequences in antisense transcripts encoded by the human C9ORF72 gene; C32-C45 correspond with target RNA sequences of transcripts encoded by the human C9ORF72 gene of exon 2; C46-050 correspond with target RNA sequences of transcripts encoded by the human C9ORF72 gene of exon 11. Selected target RNA sequences listed are either 21 or 22 nucleotides in length, or both. For a target RNA sequence either a miRNA scaffold was designed based on a 21 nucleotide target RNA sequence (101-scaffold) or a 22 nucleotide target RNA sequence (451 scaffold). For C2, C4, C5, C8-C29, C32, C34-C36, C46, C49, and C50 both a scaffold based on miR101 and miR451 was designed, wherein the target RNA sequence for the 101 scaffold represents the first 5′ 21 nucleotides listed. The first RNA sequences that were used to replace the endogenous guide strand sequence in the miRNA scaffolds are listed below. The miC sequences were incorporated into human pri-miRNA miR-101-1 or miR-451 scaffold sequences. 200 nt 5′ and 3′ flanking regions were included and the mfold program (http://unafold.rna.albany.edu/?q=mfold) was used with standard settings to determine whether the miC candidates are folded into the secondary structures as depicted in
Table 15. C1-C11 and C22-C31 correspond with first RNA sequences targeting the human C9ORF72 gene of intron 1; C12-C21 correspond with first RNA sequences targeting antisense transcripts encoded by the human C9ORF72 gene; C32-C45 correspond with first RNA sequences targeting transcripts encoded by the human C9ORF72 gene of exon 2; C46-050 correspond with first RNA sequences targeting transcripts encoded by the human C9ORF72 gene of exon 11. Selected first RNA sequences listed are either 21 or 22 nucleotides in length. miRNA scaffold design was based on a 21 nucleotide target RNA sequence (101-scaffold) or a 22 nucleotide target RNA sequence (451 scaffold). For C2, C4, C5, C8-C29, C32, C34-C36, C46, C49, and C50 both a scaffold based on 101 and 451 was designed (See i.a.
In Vitro Testing of miC-101 and miC-451 Constructs on Reporter Systems
To test the efficacy of the miC candidates, we designed Luc reporters bearing complementary C9ORF72 target regions fused to the renilla luciferase (RL) gene (
We first performed a prescreening for all the miC expression constructs by co-transfection with the corresponding Luc reporters in a 1:1 ratio. Of the miC variants designed to target the sense intronic transcripts, miC2_101 and miC4_101 showed a moderate knockdown (˜40%) and miC31_101 showed a strong knockdown (˜80%) (
Our data indicate that intron 1 is a difficult target region as all miC candidates in the highly structured repeat region between exon 1a and 1b did not induce a knockdown as strong as observed for the other target regions. The most potent knock down was observed with miC candidates downstream of exon 1b and in exonic regions 2 and 11. Nevertheless, a moderate knockdown of the G4C2 repeat containing sequences can be sufficient, miC2_101 and miC4_101 are considered suitable candidates to target the repeat containing transcripts in C9-ALS patients.
Bidirectional targeting, i.e. targeting of both sense and antisense transcripts as expressed from C9ORF72, is possible by introduction of e.g. a second miRNA scaffold (see
We next investigated whether the selected miC candidates reduce the endogenous levels of C9ORF72 in HEK293T cells. RNA was isolated from cells using standard procedures, DNA removed using DNase and endogenous levels of C9ORF72 transcipts was determined as described previously and gene expression levels were normalized to GADPH (Liu et al. (2017). Cell Chem. Biol. 24: 141-148).
Cells were transfected with the selected miC candidates and endogenous levels of C9ORF72 mRNA were determined 2 days post-transfection by RT-qPCR. As HEK293T cells lack the G4C2 expansion linked to the C9ORF72 pathology we first determined the expression of total C9ORF72 and intronic C9ORF72 mRNA. We found abundant expression of total C9ORF72, while the intronic C9ORF72 was detectable but at very low levels (
Different Processing Pattern from miR-101 and miR-451 Scaffolds
To assess the processing of the miC candidates, we analyzed the mature miC lengths and sequence composition of the guide and passenger strands by next-generation sequencing (NGS) for small transcriptome analysis (
miR-101 is processed into a miRNA duplex, first by Drosha cleavage at 3′end and then by Dicer cleavage at the hairpin structure. The miRNA duplex is then separated and one of the strands is incorporated into the RISC while the other strand is degraded. The miC-101 candidates were processed into a 20-23 nt long mature miRNA, (
The processing of the miC-451 candidates did not produce passenger strands but often generated longer guide strands than the predicted 22 nt by miRbase based on the wt miRNA structure. Drosha cleavage sites at 5′ end of the mature miC-451 are precise but the trimming of the 3′ ends of the mature miC-451 by PARN varies between candidates leading to a variety of mature lengths. miC39_451, miC43_451 and miC49_451 processing generated most often mature lengths between 21-26 nt long and processing of miC38_451 and miC50_451 often resulted in mature length longer than 27 nt (
Overall, we demonstrated that expressing different C9ORF72 target sequences from miR-101 scaffold yields a differential processing of mature guide and passenger strands. Using the miC-451 no passenger strands were detected.
miC-101 and miC-451 are Active in the Nucleus
pre-miRNAs are transported from the nucleus to cytoplasm for further processing and incorporation into the RNA-induced silencing complex (RISC). However, because C9ORF72 related ALS and FTD are characterized by accumulation of the G4C2 containing transcripts in the nucleus, active mature miC in the cell nucleus is useful from a therapeutic perspective. Based on the efficacy in vitro, we selected miC2_101 and miC4_101 as the promising candidates to target only the intronic transcripts. Similarly, miC32_101, miC46_101, miC49_451 and miC50_451 were selected for a total silencing approach based on their strong silencing efficacy in vitro. Human embryonic kidney (HEK)293T were transfected with the different miC candidates, nuclear and cytoplasmic fractions were separated and expression of the mature miC2, miC4, miC32, miC46, miC49 and miC50 in nucleus and cytoplasm was evaluated. We detected mature miC in both nucleus and cytoplasmic fractions for all miC candidates but the expression levels in nucleus was consistently ˜5 fold lower compared to cytoplasm (
Next, we evaluated the silencing efficacy of the miC candidates in nucleus and cytoplasm by measuring the endogenous levels of total C9ORF72 mRNA. miC32_101, miC46_101 and miC49_451 caused a reduction of total C9ORF72 mRNA in both nucleus and cytoplasm (
A hallmark of the RNA mediated toxicity in ALS/FTD is the formation of toxic RNA foci by the repeat containing transcripts. We generated a cell model that develops nuclear RNA foci using methods as described previously (Su et al. (2014). Neuron 83: 1043-1050; Steptoet al. (2014) Acta Neuropathol. 127: 377-389). We expressed constructs consisting of (G4C2)44 or (G4C2)3 including 150 nt 5′ and 50 nt 3′ flanking regions linked to C9ORF72 exon 2 in HEK293T cells. Nuclear RNA foci were visualized by fluorescence in situ hybridization (FISH) using a TYE563-(C4G2)3 locked nucleic acid (LNA) probe. Using a green fluorescence protein (GFP) construct the transfection efficiency was determined to be ˜95-100% (data not shown). We observed sense RNA foci at 2 days post-transfection in ˜40% of (G4C2)44 cells, but antisense RNA foci were not detected (data not shown). RNA foci were primarily present in the nucleus. Control cells expressing a shorter (G4C2)3 repeat did not accumulate RNA foci. To evaluate whether the foci were RNA specific, transfected cells were treated with RNAse or DNase. Almost all observed foci were degraded by RNAse but not DNAse, confirming that the observed foci are primarily composed of RNA.
miC4_101 and miC32_101 were evaluated for efficacy on RNA foci formation by cotransfection. Both miC candidates significantly decreased the percentage of (G4C2)44 foci-positive cells by ˜50% after 24 hours. miScr served as control and had did not reduce the amount of foci in the cells. This confirms that our miC candidates are functional in reducing RNA foci in the cell nucleus and may reduce RNA foci in human patients as well.
To further investigate the silencing of C9ORF72 in context of a gene therapy approach for ALS and FTD, we selected miC32_101 as a candidate to target total C9ORF72 based on the strong efficacy and low frequency of passenger strand formation. miC46_101 was selected as a candidate as well based on silencing efficacy, but its high amount of passenger strand observed could make it less suitable to treat patients which would indicate to useC46 candidate in a miR451 scaffold instead. Nevertheless, both candidate expression cassettes were incorporated in an AAV5 vector as previously described (Miniarikova et al. Mol Ther. 2018 Apr. 4; 26(4):947-962). Increasing doses of AAV5-miC32_101 and AAV5-miC46_101 were used to transduce HEK293T cells. Expression of the mature miC32 and miC46 was verified using TaqMan. Cells transduced with AAV5-GFP served as control for the transduction efficiency. At 3 days post-transduction, AAV5-GFP transduced ˜80% of HEK293T cells. The mature guide strand expression of miC32 and miC46 was expressed at a dose-dependent manner and resulted in a dose dependent reduction of total C9ORF72 expression at a maximum of ˜40-50%. The levels of mature miC32_101 and miC46_101 produced in transduced cells correlated well with C9ORF72 silencing. Hence, these results support further proof of concept studies in animal models of C9ORF72-ALS leading towards a miRNA-based gene therapy to treat ALS and FTD.
Overall, the combined results described above indicate that miRNAs targeting C9ORF72 could be used as therapeutics to reduce the gain of toxicity caused by the G4C2 expanded repeat of C9ORF72. We demonstrated the feasibility of different targeting approaches by miC to silence the sense, antisense or both transcripts of C9ORF72. In addition, the processing of miC in the miR-101 and miR-451 was demonstrated and both scaffolds produced mature miC that were functional in the cell nucleus and cytoplasm. Silencing of C9ORF72 was also demonstrated by miC delivered by AAV5 confirming suitability of further development of a miRNA-based gene therapy for ALS and FTD.
The main areas affected in ALS patients are motor neurons in the brain and spinal cord, whereas neurons in the frontal and temporal lobes of the brain are mainly affected in patients with FTD. About 15% of patients develop both ALS and FTD, where different types of neurons in the brain and spinal cord are affected. There are multiple supporting evidences of other cell types of the CNS such as astrocytes, microglia, and oligodendrocytes also contributing to progression of the diseases. For example, astrocytes carrying the C9ORF72 hexanucleotide expansion showed toxicity toward motor neurons, supporting their role in ALS pathogenesis. Thus, may be advantageous if delivery of therapeutics to the CNS to treat ALS and/or FTD can target a large variety of neuronal and non-neuronal cell types. We generated and characterized different human derived iPSC-neurons and astrocytes to validate the transduction of AAV5 in different cell types of the CNS (
Human control (ND42245) and Frontotemporal Dementia (ND42765) iPSC cells derived from fibroblast were ordered from Coriell biorepository and was cultured on Matrigel (corning)-coated 6 wells plates in mTeSR1 (STEMCELL). For embryoid body-based neural induction, iPS cells were seeded on AggreWel1800 plates and cultured in STEMdiff Neural Induction Medium (STEMCELL) for 5 days with daily medium changes. Embroid bodies were harvested and plated on 6 wells plates coated with poly-D-lysine (Sigma-Aldrich) and laminin (Sigm-Aldrich) in STEMdiff Neural Induction Medium for 7 days with daily medium changes. Rosettes were selected with rosette selection medium and plated on poly-D-lysine and laminin coated 6-wells plates in STEMdiff Neural Induction Medium for 24 hours. For differentiation into FBN, STEMdiff Neural Induction Medium was replaced for STEMdiff Neuron Differentiation medium (STEMCELL) and neuroprogenitor cells were differentiated for 5 days. For differentiation into astrocytes, neuroprogenitor cells were differentiated in STEMdiff astrocyte differentiation medium. The neuroprogenitor cells were then plated on poly-D-lysine and laminin coated plates in STEMdiff Neuron Maturation medium (STEMCELL) for one week or STEMdiff Astrocytes Maturation medium for 3 weeks. The mature FBN and astrocytes were stored in liquid nitrogen in neuroprogenitor freezing medium (STEMCELL). Cryopreserved non-diseased mature dopaminergic neurons (iCELL Dopaneurons, 01279, Cat # C1028, Lot #102477) were ordered at FUJIFILM Cellular Dynamics, Inc. Cryopreserved non-diseased mature motor neurons (cat #40HU-005, lot #400089) were ordered at iXCells Biotechnologies. AAV5 GFP was produced and characterized as described. For transductions with AAV, FBN, DPN and MN were plated in 24-wells plates at 0.3*106 cells per well. Astrocytes were plated at 0.1*106 cells per well in STEMdiff Astrocyte Maturation medium (STEMCELL) on matrigel coated plates. FBN were plated in STEMdiff Neuron Maturation medium (STEMCELL) on poly-D-lysine and laminin coated plates. Dopaminergic neurons were plated in iCell Neural Base medium FUJIFILM Cellular Dynamics, Inc) according to the manufacturer description on poly-D-lysine and laminin coated plates. Motor neurons were plated in motor neuron maintenance medium according to the manufacturer description matrigel coated plates. After 1 week of acclimation, cells were transduced with AAV5 for 1-2 weeks.
Induced pluripotent stem cells were induced into neural progenitor state and differentiated into frontal brain like neurons (FBN) or astrocytes. Immunohistochemistry was performed and about 60% of FBN were ß-tubulin III positive and glial fibrillary acidic protein (GFAP) negative implicating a successful differentiation rate into mature neurons. Similarly, mature astrocytes were ˜90% GFAP positive, confirming a successful differentiation. Mature dopaminergic neurons representing neurons in the mid brain region were obtained from cellular dynamic international (CDI) and were ˜90% tyroxine hydroxylase positive. Mature motor neurons were ordered at ixcellsbiotechnolgy and ˜85% were choline acetyltransferase (CHAT) positive. Transduction with AAV5-GFP showed that ˜90% of all the different neuronal cell types were GFP positive (
C9ORF72 Levels is Reduced in Cells from FTD Patient
Induced pluripotent stem cells from a healthy control person (ND42245) and a FTD patient (ND42765) were differentiated into FBN (FTD-FBN) and astrocytes (
Lowering of C9ORF72 in iPSC-Neurons by AAV5-miC
miC32 (SEQ ID NO. 86) and miC46 (SEQ ID NO. 104) were designed in exon 2 and exon 11 respectively, aiming for a total silencing of C9ORF72 mRNA, targeting all sense C9ORF72 transcripts, whether or not they contain the G4C2repeat (
Additionally, we investigated silencing of C9ORF72 in motor neurons, which is highly affected in ALS. Control (healthy) mature motor neurons where obtained from IXcells and acclimatized for 1 week. Total and intronic C9ORF72 was detected in non-transduced motor neurons but the intronic C9ORF72 expression was very low and slightly above the detection limit (data not shown). After acclimatization, motor neurons were transduced with AAV5-miC32 and AAV5-miC46 for 2 weeks. We found high expression of the mature miC32 and miC46 after transductions confirming that human motor neurons are transduced by AAV5 (
Accumulation of the G4C2 containing transcripts in the cell nucleus appears to contribute to the progression of both ALS and FTD. These transcripts form RNA foci in the cell nucleus that sequester RNA binding proteins and inhibit their function, or are transported to the cytoplasm for RAN translation into toxic DPRs. Thus, efficacy in the cell nucleus by miC may contribute in a therapeutic approach that targets i.a. RNA mediated toxicity in ALS and FTD. We now evaluated whether transduction of iPSC neurons by AAV5-miC is sufficient to express the mature miC and reduce C9ORF72 levels in nucleus.
FTD-FBNs were transduced with AAV5-miC32 and AAV5-miC46 for one week and RNA was isolated from nuclear and cytoplasmic fractions. The percentage of RNA transcript in nucleus and cytoplasm was calculated. About 80% of total C9ORF72 mRNA was detected in the nucleus and ˜20% was measured in the cytoplasm of non-transduced FBN. Whereas, the sense intronic transcripts was predominately (˜95%) expressed in nucleus of FTD-FBNs (
Having established the efficacy of AAV5-miC in different cells, including human neuronal cell types, we next evaluated their efficacy in vivo in the Tg(C9ORF72_3) line 112 mice (O'Rourke et al. (2015) Neuron 88: 892-901). This mouse model contains several tandem copies of the human C9ORF72 with repeat sizes ranging from 100-1000 repeats. Although the progressive neurodegeneration seen in ALS and FTD patients is not recapitulated, the Tg(C9ORF72_3) line 112 mice exhibit some of the pathological features such as RNA foci (starting at ˜3 months of age) and poly GP protein (starting at ˜6-20 months of age). Three months old mice were injected bilaterally in the striatum with AAV5-GFP, AAV5-miC32 and AAV5-miC46. Mice were sacrificed 6 weeks post injection to determine the genomic copy distribution of AAV5, mature miC expression, C9ORF72 lowering and the effect on RNA foci formation. A widespread distribution of AAV5 to the cortex, striatum and midbrain was observed after injections in the striatum (
We further investigated the miC processing in the mouse model. After transcription of the miC construct, the primary miR-101 is processed by Drosha cleavage at 3′ end and then by Dicer cleavage at the hairpin structure into a miRNA duplex. The miRNA duplex is then separated, and the guide strand is usually incorporated into the RNA-induced silencing complex (RISC) while in most cases the passenger strand is degraded. The processing of the miC32 and miC46 was analyzed by next-generation sequencing (NGS) for small transcriptome to determine the ratio of guide and passenger strands that are produced. small transcriptome analysis was performed on RNA isolated from striatum of 4 mice that was injected with AAV5-miC32 or AAV5-miC46. For each sample, we obtained between 15-30 million small RNA reads that were subsequently adaptor-trimmed and aligned against the corresponding reference sequence. All reads shorter than 10 nucleotide (nt), longer than 45 nt, or represented less than 10 times were excluded from the analysis. miC32 was processed into predominantly strands comprising a first RNA sequence, an RNA sequence that has complementarity to the C9orf72 target sequence (˜87%) of 19-20 nucleotide (nt) long (also referred to as guide strand), with low percentages of the sense strand, also referred to as passenger strand (˜13%). However, miC46 processing yielded higher amounts of sense strands (“passenger strand”) (˜82%) of between 19-22 nt long and low amounts (˜18%) of RNA strands targeting C9orf72 (
RNA foci formation formed by the repeat containing transcripts is considered a hallmark of the RNA mediated toxicity in ALS/FTD. Fluorescence in situ hybridization (FISH) using a TYE563-(C4G2)3 locked nucleic acid (LNA) probe RNA FISH showed that ˜60-80% of cells in cortex, hippocampus and cerebellum of the Tg(C9ORF72_3) line 112 mice contained RNA foci (
To conclude, we demonstrated that AAV5 can transduce different cell types of the CNS relevant for ALS/FTD treatment and that miC candidates targeting C9ORF72 are effective in a mouse model for ALS/FTD. Furthermore, we show that total C9ORF72 mRNA and sense intronic C9ORF72 transcripts can be lowered in both nucleus and cytoplasm.
1. An expression cassette encoding a double stranded RNA comprising a first RNA sequence and a second RNA sequence wherein the first and second RNA sequence are substantially complementary, wherein the first RNA sequence has a sequence length of at least 19 nucleotides and is substantially complementarity to a target RNA sequence comprised in an RNA encoded by a human C9orf72 gene.
2. An expression cassette according to embodiment 1, wherein said first and second RNA sequence are comprised in a pre-miRNA scaffold, a pri-miRNA scaffold or a shRNA.
3. An expression cassette according to embodiment 1 or 2, wherein said first and second RNA sequence are comprised in a pre-miRNA scaffold or a pri-miRNA scaffold from miR101 or miR451.
4. An expression cassette according to any one of embodiments 1-3, wherein said first RNA sequence is comprised in a guide sequence.
5. An expression cassette according to any one of embodiments 1-4, wherein said first RNA sequence and said second RNA sequence when expressed in a cell are processed by the cell to produce a guide sequence comprising the first RNA sequence.
6. An expression cassette according to any one of embodiments 1-5, wherein said first RNA sequence is substantially complementary to a target RNA sequence comprised in antisense RNA transcripts encoded by the human C9orf72 gene.
7. An expression cassette according to embodiment 6, wherein said first RNA sequence is substantially complementary to SEQ ID NO. 15 or SEQ ID NO. 21.
8. An expression cassette according to embodiment 7 wherein the first RNA sequence is of SEQ ID. 68 or SEQ ID NO. 74.
9. An expression cassette according to embodiment 8 wherein the first RNA sequence and second RNA sequence are selected from the group consisting of the combinations of SEQ ID NOs. 68 and 117 or 153 and SEQ ID NOs. 74 and 118 or 154.
10. An expression cassette according to embodiment 9, wherein said encoded RNA comprises an RNA sequence selected from the group consisting of SEQ ID NOs. 119, 120, 139, 140, 155 and 156.
11. An expression cassette according to any one of embodiments 1-5, wherein said first RNA sequence is substantially complementary to exon 2 sequence comprising RNA transcripts encoded by the human C9orf72 gene.
12. An expression cassette according to embodiment 11, wherein said first RNA sequence is substantially complementary to SEQ ID NO. 32.
13. An expression cassette according to embodiment 12 wherein the first RNA sequence is SEQ ID NO. 86 or SEQ ID NO. 91.
14. An expression cassette according to embodiment 13 wherein the first RNA sequence and second RNA sequence are selected from the group consisting of the combinations of SEQ ID NOs. 86 and 121, and SEQ ID NOs. 91 and 122 or 157.
15. An expression cassette according to embodiment 14, wherein said encoded RNA comprises an RNA sequence selected from the group consisting of SEQ ID NOs. 123, 124, 141, 142 and 158.
16. An expression cassette according to any one of embodiments 1-5, wherein said first RNA sequence is substantially complementary to intron 1 sequence comprising RNA transcripts encoded by the human C9orf72 gene.
17. An expression cassette according to embodiment 16, wherein said first RNA sequence is substantially complementary to SEQ ID NO. 2, SEQ ID NO. 4 or SEQ ID NO. 31.
18. An expression cassette according to embodiment 17 wherein the first RNA sequence is selected from the group consisting of SEQ ID NO. 52, SEQ ID NO. 54, SEQ ID NO. 58, SEQ ID NO. 59 and SEQ ID NO. 85.
19. An expression cassette according to embodiment 16 wherein the first RNA sequence and second RNA sequence are selected from the group consisting of the combinations of SEQ ID NO. 52 and 125; SEQ ID NO. 58 and SEQ ID NO. 126 or 159; SEQ ID NO. 54 and SEQ ID NO. 127, SEQ ID NO. 59 and SEQ ID NO128 and 160; and, SEQ ID NO. 85 and 129.
20. An expression cassette according to embodiment 19, wherein said encoded RNA comprises an RNA sequence selected from the group consisting of SEQ ID NOs 130, 131, 132, 133, 134, 143, 144, 145, 146, 147, 161 and 162.
21. An expression cassette according to any one of embodiments 1-5, wherein said first RNA sequence is substantially complementary to exon 11 sequence comprising RNA transcripts encoded by the human C9orf72 gene.
22. An expression cassette according to embodiment 21, wherein said first RNA sequence is substantially complementary to SEQ ID NO. 46.
23. An expression cassette according to embodiment 22 wherein the first RNA sequence is SEQ ID NO. 104 or SEQ ID NO. 109.
24. An expression cassette according to embodiment 23 wherein the first RNA sequence and second RNA sequence are selected from the group consisting of the combinations of SEQ ID NOs. 104 and 135, and, SEQ ID NOs. 109 and 136 or 163.
25. An expression cassette according to embodiment 24, wherein said encoded RNA comprises an RNA sequence selected from the group consisting of SEQ ID NOs. 137, 138, 148, 149 and 164.
26. An expression cassette comprising a combination of a first and second RNA sequence as defined in any one of embodiments 6-10 and a first and second RNA sequence as defined in any one of embodiments 11-15.
27. An expression cassette comprising a combination of a first and second RNA sequence as defined in any one of 6-10 and a first and second RNA sequence as defined in any one of embodiments 16-20.
28. An expression cassette comprising a combination of a first and second RNA sequence as defined in any one of embodiments 6-10 and a first and second RNA sequence as defined in any one of embodiments 21-25.
29. An expression cassette according to any one of embodiments 1-28, wherein the expression cassette comprises a PGK promoter, a CMV promoter, a neurospecific promoter or a CBA promoter operably linked to said first RNA sequence and said second RNA sequence.
30. A gene therapy vector comprising the expression cassette according to any one of embodiments 1-29.
31. A gene therapy vector according to embodiment 30, wherein the gene therapy vector is an AAV vector.
32. A gene therapy vector according to embodiment 30, wherein the gene therapy vector is an AAV vector of serotype 5.
33. A gene therapy vector according to any one of embodiments 30-32, for use in a medical treatment.
34. A gene therapy vector according according to embodiment 33, for use in the treatment of ALS and/or FTD.
35. A gene therapy vector according to embodiment 33 or embodiment 34, wherein said first and second RNA sequences when expressed in a cell reduce expression of RNA encoded by a human C9orf72 gene both in the cell nucleus as in the cytoplasm.
36. A gene therapy vector according to any one of embodiments 33-35, wherein said gene therapy vector reduces wherein said first and second RNA sequences when expressed in a cell reduce expression of C9 RAN protein levels.
37. A gene therapy vector according to any one of embodiments 33-36, wherein said first and second RNA sequences when expressed in a cell reduce expression of G4C2 foci and/or G2C4 foci.
38. A gene therapy vector according to any one of embodiments 33-37, wherein said medical treatment comprises transfer of the vector to a motorneuron.
39. A gene therapy vector according to any one of embodiments 33-384, wherein said medical treatment comprises transfer of the vector to a human frontal brain neuron and/or anterior brain neuron.
40. A gene therapy vector according to any one of embodiments 33-39, wherein said gene therapy vector is administered to the spinal cord.
41. A gene therapy vector according to any one of embodiments 33-40, wherein said gene therapy vector is administered to the frontal lobe and/or anterior temporal lobe.
42. A gene therapy vector according to any one of embodiments 33-41, wherein said vector is administered by intraparenchymal injection.
About 20% of cells in the cortex had 0 RNA foci (white bar) per cell in the Tg(C9ORF72_3) line 112 mice treated with mock. After treatment with AAV5-miC32 and AAV5-miC46, this was increased to ˜50% of cells with no RNA foci. About 23% of Cells had 1-5 RNA foci (grey bar) in mice treated with mock. After treatment with AAV5-miC32 and AAV5-miC46, about 35% had 1-5 RNA foci. About 60% of Cells had more that 5 RNA foci (black bar) in mice treated with mock. After treatment with AAV5-miC32 and AAV5-miC46, this was decreased to ˜10-15%.
| Number | Date | Country | Kind |
|---|---|---|---|
| 18194026.3 | Sep 2018 | EP | regional |
This application is a continuation of International Application No. PCT/EP2019/074198 filed Sep. 11, 2019 and claims the benefit of priority to European Patent Application No. 18194026.3 filed Sep. 12, 2018, the entire contents of which are hereby incorporated by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/EP2019/074198 | Sep 2019 | US |
| Child | 17196531 | US |
