Field of the invention

The present invention relates to the field of medicine. More in particular it relates to biotechnology and the use of synthetic antisense oligonucleotides for use in the treatment, prevention or delay of neurodegenerative disorders, preferably Huntington's disease.

Background of the invention

Huntington's disease (HD) is a progressive neurodegenerative genetic disorder that affects muscle movement and muscle coordination and leads to cognitive decline and dementia. It typically becomes noticeable in middle age. HD is the most common genetic cause of abnormal involuntary writhing movements called chorea and is much more common in people of Western Europe descent than in those from Asia or Africa, with an incidence of 4 to 15 in 100,000 people in Western Europe. The disease affects men and women equally and is caused by an autosomal dominant mutation of the Huntingtin {HTT) gene that encodes the Huntingtin (HTT) protein. A child of an affected parent has a 50% risk of inheriting the disease. Complications such as pneumonia, heart disease and physical injury from falls reduce life expectancy. Suicide is the cause of death in 9% of the cases, and death typically occurs fifteen to twenty years from when the disease is first detected. There is currently no cure for HD, and full time care is required in later stages of the disease.

HD is caused by a CAG trinucleotide repeat (TNR) expansion in the HTT gene. The expanded repeats are translated into an abnormally long polyglutamine tract close to the N- terminus of the Huntingtin protein. It has been suggested that this expansion mutation is associated with a deleterious gain-of-function and that the large Huntingtin protein is cleaved by proteases to produce a shorter N-terminal fragment containing the polyglutamine expansion that causes the protein to misfold and form aggregates (inclusions) in the nuclei of neuronal cells. It is likely that neurotoxicity is caused by the misfolded protein in its soluble form and/or in aggregates and/or in the process of aggregation. One potential mechanism of this neurotoxic process is thought to be caspase activation.

Proteolytic processing is a major form of post-translational modification that occurs when a protease cleaves one or more bonds in a target protein to modify its activity. This processing may lead to activation, inhibition, alteration or destruction of the protein's activity. The protease may remove a peptide segment from either end of a target protein, but it may also cleave internal bonds in the protein that lead to major changes in the structure and function of the protein. Proteolytic cleavage may have various (desired) functions under normal circumstances. For instance, proteolysis of precursor proteins regulates many cellular processes including gene expression, embryogenesis, the cell cycle, programmed cell death, intracellular protein targeting and endocrine/neural functions. In all of these processes, proteolytic cleavage of precursor proteins is necessary. In the case of Huntingtin its general function and the function of the cleavage of the N-terminus are not completely clear. The protein interacts with proteins involved in transcription, cell signaling, and intracellular transport. The absence of Huntingtin in animal models have shown that it is required for embryogenesis and it appears to play a part in preventing/regulating programmed cell death. It appears to control the production of brain-derived neurotrophic factor, a protein that protects neurons and regulates their creation. The gain-of-function that appears to cause HD is related to the number of CAG repeats present in the HTT gene. The presence of 26 or fewer repeats is not associated with disease. The presence of 27-35 repeats also generally is not associated with symptoms and disease but may increase the risk of further expansions in the offspring. The presence of 36-39 repeats may cause symptoms, generally later in life, while 40 or more repeats is said to be related to HD, although symptoms may appear much later than the determination of the number of repeats has been made. The art has described various strategies to inhibit proteases, for a variety of different therapeutic applications. As outlined above, proteases are responsible for the cleavage of the Huntingtin protein part that contains the TNR expansion, and for the treatment of HD it may be beneficial to inhibit such proteases. However, a problem with the use of protease inhibitors is that proteases generally have a wide range of targets in the human body and, associated therewith, a range of effects. Inhibiting a protease in the human body through the action of a protease inhibitor thus not only may inhibit the desired effect (prevention of Huntingtin cleavage), but typically also has a range of unwanted side-effects. It is therefore much more desired to target the HTT gene specifically, or its transcribed pre-mRNA, its matured mRNA or the encoded protein. One way of addressing this is through manipulation of the splicing machinery using specific antisense oligonucleotides (AONs) that specifically target the HTT pre-mRNA through interactions with their specific complementary sequences and influence the maturation from pre-mRNA into mRNA.

For the HTT gene - in view of what has been addressed above - it is preferred that the caspase-6 proteolytic cleavage site encoded by exon 12 is absent in the Huntingtin protein when the TNR expansion becomes too large and HD occurs, or is likely to occur (preferably when the number of CAG repeats is 36 or more). It is preferred that the coding region that codes for the proteolytic cleavage site is removed 'in frame', so as to allow incorporation of the normal downstream amino acid sequence into the mutant protein. The in frame removal may be achieved by providing a cell with an AON that enables skipping (a part) of exon 12 from the HTT pre-mRNA, that then matures into mRNA encoding for a functional HTT protein lacking the amino acids encoded by the skipped exon (part). Different isoforms of Huntingtin have in fact been discovered, one of which lacks the part of exon 12 that comprises the caspase-6 cleavage site (Ruzo et al. Discovery of novel isoforms of Huntingtin reveals a new hominid-specific exon. PLoS One. 2015. 10(5):e0127686.doi:10.1371 ). Ruzo and co-workers disclose the identification of ΗΤΤ-Δ12 (also referred therein as ΔΕχοη12), which is an isoform that indeed lacks a functional caspase-6 cleavage site. AONs can be used to stimulate the appearance of such alternatively spliced variants, and AONs were found and described in the art that stimulate the partial skip of exon 12, even before the ΗΤΤ-Δ12 isoform was identified as an isoform that appears in nature. US 9,61 1 ,471 , WO 2012/018257 and WO 2015/053624 disclose AONs that are applicable in a method of promoting the production of a human Huntingtin protein lacking the proteolytic caspase-6 cleavage site by inducing the appearance of the ΗΤΤ-Δ12 isoform, i.e. inducing the skip of nucleotides 207-341 of exon 12. Table 3 in US 9,61 1 ,471 discloses ten of such targeting AONs (hHTTEx12_1 to _10). Although a number of these known AONs are able to provide such specific skipping, there is always a need for improvement and more efficient and effective therapeutic compounds. Summary of the invention

The present invention relates to improved antisense oligonucleotides (AONs) for use in the treatment, prevention or delay of Huntington's disease (HD). The inventors of the present invention have sought for improved properties of AONs known from the art using computational analysis. The present invention specifically relates to an AON that is complementary to a sequence of SEQ ID NO:15, wherein the first 5' nucleotide of said AON is opposite position 31 in SEQ ID NO:15 and wherein the AON is 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 nucleotides in length. Preferably, said AON consists of a sequence selected from SEQ ID NO:2 to 12. More preferably, said AON consists of a sequence selected from SEQ ID NO:2 and 3. In yet another preferred aspect, the AON of the present invention is an oligoribonucleotide (RNA oligonucleotide) comprising at least one 2'-0 alkyl modification, such as a 2'-0-methyl modified sugar, and it is preferred that all nucleotides in said AON are then 2'-0-methyl modified. In another preferred aspect, the AON of the present invention is an oligoribonucleotide comprising at least one 2'-0-methoxyethyl modification, and it is preferred that all nucleotides in said AON are then 2'-0-methoxyethyl modified. In yet another preferred aspect, the AON according to the present invention comprises at least one phosphorothioate linkage, and it is preferred that all sequential nucleotides are interconnected by phosphorothioate linkages. The invention further relates to viral vectors and pharmaceutical compositions comprising an AON according to the present invention. The AONs of the present invention, as well as the viral vectors and/or pharmaceutical preparations are for use in the treatment, prevention or delay of Huntington's disease. The invention further relates to a method of promoting the production of a human Huntingtin protein lacking a proteolytic caspase 6 cleavage site in a human cell, the method comprising: providing a human cell that expresses a human Huntingtin protein comprising a caspase 6 proteolytic cleavage site from a pre-mRNA encoding said protein, with an AON, a viral vector, or a pharmaceutical composition according to the invention, wherein at least nucleotides 207 to 341 of exon 12 of the human HTT pre-mRNA are skipped. In another aspect, the invention relates to a method for the treatment, prevention or delay of Huntington's disease or a condition requiring the removal of a caspase 6 proteolytic cleavage site from human HTT pre-mRNA, of an individual in need thereof, said method comprising contacting a cell of said individual, preferably a neuronal cell, more preferably a brain cell, with an AON, a viral vector, or a pharmaceutical composition according to the invention.

Brief description of the drawings

Figure 1 shows the secondary structure prediction of exon 12 of the human HTT pre-mRNA. The position of the HD_AON12.1 known from the art is given, as well as the location of the elongated AONs of the present invention. The known AON is elongated at the 3' end to yield AONs with improved properties for skipping a part of exon 12 of human HTT pre-mRNA, wherein the skipped part of exon 12 encodes a caspase-6 site. This proteolytic cleavage site is preferably removed in the treatment, prevention or delay of Huntington's disease.

Figure 2 shows Bioanalyzer results of RT-PCR or HTT mRNA with primer set F2-R3 spanning the exon 12 deletion, on GM04857A Huntingtin's patient fibroblast cells treated with 8 nM, 16 nM and 32 nM of HD_AON12.1 , HD_AON29 and HD_AON30. The 262 bp wt HTT mRNA is observed in all samples, while the 127 bp Δ12 HTT mRNA is detected in all transfected samples and not present in the non-treated (NT) control.

Figure 3 shows a bar graph representing ΗΤΤ-Δ12 mRNA fractional abundance (%), calculated using Poisson statistics of the ddPCR analysis of GM04857A Huntingtin's patient fibroblast cells that were transfected with 8 nM, 16 nM and 32 nM HD_AON12.1 , HD_AON29 or HD_AON30. Increasing AON concentration resulted in a higher percentage of generated ΗΤΤ-Δ12 mRNA. Both HD_AON29 and HD_AON30 are more efficient in generating ΗΤΤ-Δ12 mRNA than HD_AON12.1 , at all three concentrations tested and most noticeable difference at 8 nM.

Figure 4 shows a western blot of GM04857A Huntingtin's patient fibroblast cells, transfected with 8 nM, 16 nM and 32 nM of HD_AON12.1 , HD_AON29 or HD_AON30, using antibodies against Huntingtin (MAB2166) and Vinculin (ab73412) as loading control. The wild type Huntingtin protein is observed as a 347 kDa band in all samples, while the modified ΗΤΤ-Δ12 Huntingtin protein (lacking the 45 amino acids due to the missing nucleotides 207-341 ) is detected as a 343 kDa band and is detected in all concentration ranges for HD_AON29 and HD_AON30 transfected samples, while in the samples transfected with HD_A0N12.1 , the band is visible only with the 16 nM and 32 nM oligo concentrations.

Detailed description

Skipping of nucleotides that code for the proteolytic cleavage site is typically achieved by skipping the exon that contains the nucleotides that code for the proteolytic cleavage site. The proteolytic cleavage site comprises the recognition sequence for the specific protease and the two amino acids between which the peptide linkage is cleaved by the protease. The proteolytic cleavage site can overlap the boundary of two adjacent exons or, if part of the exon is skipped, overlap the exon sequence that contains the cryptic splice acceptor/donor sequence. In this embodiment, it is preferred to skip the exon sequence that codes for the peptide linkage that is cleaved by the protease. Whether or not a recognition site for a protease is actually used in nature depends, not only on the presence of the recognition sequence itself, but also on the location of the site in the folded protein. An internally located recognition site is typically not used in nature. In the present invention, the proteolytic caspase-6 site in exon 12 of the human HTT gene is actually used in nature. Skipping of the exon (or part thereof) that contains the nucleotides that code for the proteolytic cleavage site is preferably achieved by means of an AON that is directed toward an exon internal sequence. An AON is said to be directed toward an exon internal sequence if the complementarity region that contains the sequence identity to the reverse complement of the target pre-mRNA is within the exon boundary.

US 9,61 1 ,471 discloses that AONs are directed toward a region delimited by nucleotides 207 until 341 of exon 12 of the human HTT gene. It was disclosed that the AON directed toward that preferred region induces the skipping of the 3' terminal 135 nucleotides of exon 12, thereby producing an in frame complete deletion of two active caspase 3 cleavage sites at amino acid 513 and 552, and removal of the first amino acid of an active caspase 6 cleavage site, partially located in exon 12 and partially in exon 13. AON HDEx12_1 (see Table 2 in US 9,61 1 ,471 ) activates a cryptic splice site at nucleotide 206 in exon 12, leading to the absence of the remainder of exon 12 from the formed mRNA. The present invention relates to further improvements of that earlierfinding, wherein the rationale behind the improvement was based on assessing the secondary structure of the HTT pre-mRNA as follows.

Rationale

The mechanism of action of an exon-skipping AON involves the second-order hybridization of the oligonucleotide to the target sequence on the pre-mRNA by canonical base pairing. The inventors of the present invention came to realize that the identification of putative exonic splicing enhancer sites (ESE), determined by Rescue-ESE or ESEfinder tools, was insufficient per se to find AONs with the highest exon-skipping activity for HTT pre-m RNA. In the lead optimization process, the inventors realized that additional hybridization thermodynamic considerations had to be taken into account to identify AONs that would outperform the AONs known from the art. These considerations relate predominantly to target accessibility, RNA binding affinity and propensity of candidate AONs to form self- intramolecular or self-intermolecular secondary structures. The first consideration, determination of the target accessibility, is important since the putative ESE sites can be masked in higher order stable secondary structure in the pre-mRNA, not very accessible for a second-order annealing to an AON (Shepard et al. 2008. RNA 14(8)1463-1469). The thermodynamic cost of disrupting the target structure must be compensated by the formation of the duplex target AON. The second consideration, the RNA binding affinity, can be related to the potency of an AON and is a function of its length, chemical modifications and GC content. However, there is a sweet-spot to achieve, because too much target affinity can actually be detrimental to the AON activity (Pedersen et al. 2014. Mol Ther Nucleic Acids 3(2):e149). Regarding the third consideration, the propensity of an AON to form self-structures (intra- or intermolecular) can exert a kinetic or thermodynamic competition with the formation of a duplex consisting of AON/pre-mRNA (Matveeva et al. 2003. Nucleic Acids Res 31 (17):4989-4994).

After identification of oligo HD_AON12.1 (SEQ ID NO:1 ), which is an AON capable of mediating the skipping of a part of exon 12 in HTT pre-mRNA (see WO 2012/018257), the inventors hypothesized that optimization of the length of this oligo (a 20-mer) could lead to a AON with potentially higher exon-skipping activity. The inventors hypothesized that a shorter oligonucleotide would potentially have a lower RNA binding affinity and a higher propensity for off-targets. On the other hand, an AON that is too long can have a detrimental high affinity, can be more difficult for cellular uptake and have an intrinsic structural bias for self- hybridization (Khatsenko et al. 2000. Antisense Nucleic Acid Drug Dev 10(1 ):35-44). Following these guidelines, the sequence of HD_AON12.1 (SEQ ID NO:1 ) was optimized to identify AONs with a higher exon-skipping ability. For this purpose, the inventors used several publicly available software tools to determine the optimal length of an optimized version of AON HD_AON12.1 , according to the aforementioned considerations. Firstly, the inventors generated a computational model for the human HTT exon 12 pre-mRNA with the ViennaRNA software package (Lorenz et al. 201 1 . Algorithms for Molecular Biology 6:26). Predicting the secondary structure of large RNAs such as the full-length HTT pre-mRNA can be computationally very laborious and not very accurate. Following this rational, the inventors decided to consider only folding predictions using the exon 12 primary nucleotide sequence as input. As an additional consideration, large RNA can exist as ensembles of multiple stable structural conformations. Because of the computational complexity that derives from this fact, the inventors decided to focus solely on the predicted most stable structure, in the modelling analysis. The minimum free energy (MFE) secondary structure of the most stable predicted folding was determined using the primary sequence of the 341 nucleotide-long exon 12 region of the HTTgene, which is a domain containing 57% GC content. The free Gibbs energy value was determined with RNAfold to be -1 17.3 kcal/mol and from the total 341 nucleotides, 208 are predicted to be base-paired (190 Watson-Crick and 18 wobble-type). The prediction showed that the HD_AON12.1 target sequence partially consists of a 6 single-stranded nucleotides part of an internal loop in the secondary structure. This MFE secondary structure, specifically the CT secondary structure file generated, was used as input in the OligoWalk module (Lu and Mathews. 2008. Nucleic Acids Res 36(1 1 ):3738-3745) that is part of RNAStructure, a publicly available software package (Mathews 2014. Curr Protoc Bioinformatics 46:12). As for user-defined specifications for running this software, the computation form exon 12 HTT pre-mRNA was performed in its slowest mode (i.e. refolding the whole RNA input sequence for each candidate AON), selecting a oligo concentration of 1 μΜ by default, an oligomer chemistry of "RNA" and AON lengths between 18 and 35 nucleotides. The goal was to determine with single-nucleotide resolution the best flanking sequence of the HD_AON12.1 target domain that would allow optimizing its length towards its 5'-end, 3'-end or both. Such can be achieved by computing the free Gibbs energy (AG ) of the formation of a duplex consisting of a candidate AON and the HTT pre-mRNA. Notably, a more negative value than what is obtained for HD_AON12.1 would eventually mean a superior RNA binding affinity and as a consequence, an improved exon-skipping activity.

The outcome of the theoretical prediction outlined above showed that extending the 3'- end sequence of HD_AON12.1 would lead to AONs that would stabilize the target HTT exon 12 pre-mRNA sequences by more than -35 kcal/mol (see Figure 1 ). Additionally, OligoWalk was used to compute the propensity of the candidate AONs to form either self-intramolecular or self-intermolecular secondary structures. This can be determined theoretically by computing the corresponding AG°37-values for secondary structure formation. The more close to zero these values will be, less likely it will be for potential AON self-structures to compete with the AON/pre-mRNA duplex formation. In essence, the AONs predicted to form a self- structure with a theoretical AG -value lower (more negative) than -5 kcal/mol and -15 kcal/mol (for intra- and intermolecular self-structures, respectively) were discarded. A phenotype screening was performed with the AON sequences HD_AON29 (SEQ ID NO:2) and HD_AON30 (SEQ ID NO:3) and these were shown to have improved properties over the known HD_AON12.1 (SEQ ID NO:1 ), confirming the rationale behind the design of the new AONs. Definitions

The term 'exon skipping' is herein defined as the process wherein a mature mRNA appears from a pre-mRNA that does not contain one or more particular exons (in the current case exon 12 or a part thereof, preferably the exon 12 part comprising nucleotides 207 to 341 of exon 12 of the human HTT gene). Exon 12, or the skipped part thereof, would normally be present in the mature mRNA when no exon skipping occurs, such as for instance in a wild type situation. Inducing such exon skipping is achieved by providing a cell expressing the pre- mRNA of the HTT gene comprising the HD causing variant, with a molecule capable of interfering at sequences such as, for example, a (cryptic) splice donor and/or a (cryptic) splice acceptor sequence required for allowing the enzymatic process of splicing.

The term 'pre-mRNA' refers to a non-processed or partly processed precursor mRNA that is synthesized from a DNA template of a cell by transcription, such as in the nucleus. Within the context of the invention, inducing and/or promoting skipping of an exon sequence that codes for a proteolytic cleavage site, as indicated herein, means that at least 1 %, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more of the mRNA encoding the protein in a cell will not contain the skipped sequence [modified/(modified+unmodified) RNA]. This is preferably assessed by PCR as described for instance in US 9,61 1 ,471 .

The term 'antisense oligonucleotide' (herein generally abbreviated to AON) is understood to refer to a nucleotide sequence which is substantially complementary to a target nucleotide sequence in a pre-mRNA molecule, heterogeneous nuclear RNA (hnRNA) or mRNA molecule. The degree of complementarity (or substantial complementarity) of the antisense sequence is preferably such that a molecule comprising the antisense sequence can form a stable double stranded hybrid with the target nucleotide sequence in the RNA molecule under physiological conditions. The terms 'antisense oligonucleotide', ΆΟΝ', 'oligonucleotide' and 'oligo' are often and also herein used interchangeably and are understood to refer to an oligonucleotide comprising an antisense sequence in respect of the target (pre-) mRNA sequence.

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

The word 'about or 'approximately when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 0.1 % of the value. The term 'substantially complementary used in the context of the invention indicates that some mismatches in the antisense sequence are allowed as long as the functionality, i.e. skipping of the HTT exon 12, or part thereof as outlined herein, is still acceptable. Preferably, the complementarity is from 90% to 100%. In general this allows for 1 or 2 mismatches in an AON of 20 nucleotides or 1 , 2, 3 or 4 mismatches in an AON of 40 nucleotides, or 1 , 2, 3, 4, 5, or 6 mismatches in an AON of 60 nucleotides, etc.

'Prevention, treatment or delay of HD' is herein preferably defined as preventing, halting, ceasing the progression of, or reversing partial or complete occurrence of the disorder caused by CAG trinucleotide repeat expansions (as outlined herein) in the human HTT gene.

The present invention relates to an antisense oligonucleotide (AON) that is complementary to a sequence of SEQ ID NO:15, wherein the first 5' nucleotide of said AON is opposite position 31 in SEQ ID NO:15 and wherein the AON is 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 nucleotides in length. Preferably, said AON consists of a sequence selected from SEQ ID NO:2 to 12. More preferably, said AON consists of a sequence selected from SEQ ID NO:2 and 3. In another preferred aspect, the AON according to the present invention is an oligoribonucleotide (RNA oligonucleotide) comprising at least one 2'-0 alkyl modification, such as a 2'-0-methyl modified sugar; and it is even more preferred that then all nucleotides in said AON are 2'-0-methyl modified. In another preferred aspect, the AON according to the present invention is an oligoribonucleotide comprising at least one 2'-0-methoxyethyl modification; and it is even more preferred that then all nucleotides in said AON are 2'-0- methoxyethyl modified. In another preferred aspect, the AON of the present invention comprises at least one phosphorothioate linkage. It is even more preferred that all sequential nucleotides within the AON are interconnected by phosphorothioate linkages. The invention also relates to a viral vector expressing an AON according to the invention. Clearly, the viral vector cannot encode chemically modified AONs, but can comprise a sequence encoding the sequence of the AONs. The invention furthermore relates to a pharmaceutical composition comprising an AON according to the invention, or a viral vector according to the invention, and a pharmaceutically acceptable carrier, wherein the pharmaceutical composition is preferably suitable for administration toward the human brain. The invention also relates to an AON, a viral vector, or a pharmaceutical composition according to the invention, for use as a medicament, preferably for use in the treatment, prevention or delay of Huntington's disease. In yet another embodiment, the invention relates to a use of an AON, a viral vector, or a pharmaceutical composition according to the invention, for the treatment, prevention or delay of Huntington's disease. In yet a further embodiment, the invention relates to a use of an AON, a viral vector, or a pharmaceutical composition according to the invention, for the manufacturing of a medicament for the treatment, prevention or delay of Huntington's disease. In another embodiment, the invention relates to a method of promoting the production of a human Huntingtin protein lacking a proteolytic caspase 6 cleavage site in a human cell, the method comprising: providing a human cell that expresses a human Huntingtin protein comprising a caspase 6 proteolytic cleavage site from a pre-mRNA encoding said protein, with an AON according to the invention, a viral vector according to the invention, or a pharmaceutical composition according to the invention, wherein at least nucleotides 207 to 341 of exon 12 of the human HTT pre-mRNA are skipped. In yet another embodiment, the invention relates to a method for the treatment, prevention or delay of Huntington's disease or a condition requiring the removal of a caspase 6 proteolytic cleavage site from human HTT pre-mRNA, of an individual in need thereof, said method comprising contacting a cell of said individual, preferably a neuronal cell, more preferably a brain cell, with an AON according to the invention, a viral vector according to the invention, or a pharmaceutical composition according to the invention. Preferred embodiments

In a preferred embodiment, the invention relates to an AON that is complementary in full or in part to an internal sequence of exon 12 of the human HTT pre-mRNA, wherein said internal sequence comprises the sequence of SEQ ID NO:15:

5'-CUGACCCUGCCAUGGACCUGAAUGAUGGGAC-3'

In a preferred aspect, the AON according to the invention comprises the sequence of

HD_AON 12.1 (a 20-mer; SEQ ID NO:1 ) and wherein said AON is elongated up to 1 1 nucleotides at the 3' end (see Figure 1 ), and is selected from any of the following AON sequences:

5'-GUCCCAUCAUUCAGGUCCAUG-3' (21 -mer; SEQ ID NO:4)

5'-GUCCCAUCAUUCAGGUCCAUGG-3' (22-mer; SEQ ID NO:5)

5'-GUCCCAUCAUUCAGGUCCAUGGC-3' (23-mer; SEQ ID NO:6)

5'-GUCCCAUCAUUCAGGUCCAUGGCA-3' (24-mer; HD_AON29; SEQ ID NO:2)

5'-GUCCCAUCAUUCAGGUCCAUGGCAG-3' (25-mer; SEQ ID NO:7)

5'-GUCCCAUCAUUCAGGUCCAUGGCAGG-3' (26-mer; SEQ ID NO:8)

5'-GUCCCAUCAUUCAGGUCCAUGGCAGGG-3' (27-mer; SEQ ID NO:9)

5'-GUCCCAUCAUUCAGGUCCAUGGCAGGGU-3' (28-mer; HD_AON30: SEQ ID NO:3) 5'-GUCCCAUCAUUCAGGUCCAUGGCAGGGUC-3' (29-mer; SEQ ID NO:10)

5'-GUCCCAUCAUUCAGGUCCAUGGCAGGGUCA-3' (30-mer; SEQ ID NO:1 1 )

5'-GUCCCAUCAUUCAGGUCCAUGGCAGGGUCAG-3' (31 -mer; SEQ ID NO:12) In a preferred embodiment, the length of the complementary part of the AON of the present invention is at least 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 nucleotides. An AON of the invention is preferably an isolated single stranded molecule in the absence of its (target) counterpart sequence, and that does not self-hybridize. An AON of the invention is preferably complementary to, or under physiological conditions binds to SEQ ID NO:15 within exon 12 of the human HTT pre-mRNA. A preferred AON of the invention consists of 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 nucleotides.

It is preferred that an AON of the invention comprises one or more residues that are modified to increase nuclease resistance, and/or to increase the affinity of the AON for the target sequence. Therefore, in a preferred embodiment, the AON sequence comprises at least one nucleotide analogue or equivalent, wherein a nucleotide analogue or equivalent is defined as a residue having a modified base, and/or a modified backbone, and/or a non-natural internucleoside linkage, or a combination of these modifications.

In a preferred embodiment, the nucleotide analogue or equivalent comprises a modified backbone. Examples of such backbones are provided by morpholino backbones, carbamate backbones, siloxane backbones, sulfide, sulfoxide and sulfone backbones, formacetyl and thioformacetyl backbones, methyleneformacetyl backbones, riboacetyl backbones, alkene containing backbones, sulfamate, sulfonate and sulfonamide backbones, methyleneimino and methylenehydrazino backbones, and amide backbones. Phosphorodiamidate morpholino oligo's are modified backbone oligonucleotides that have previously been investigated as antisense agents. Morpholino oligonucleotides have an uncharged backbone in which the deoxyribose sugar of DNA is replaced by a six membered ring and the phosphodiester linkage is replaced by a phosphorodiamidate linkage. Morpholino oligonucleotides are resistant to enzymatic degradation and appear to function as antisense agents by arresting translation or interfering with pre-mRNA splicing rather than by activating RNase H. Morpholino oligonucleotides have been successfully delivered to tissue culture cells by methods that physically disrupt the cell membrane, and one study comparing several of these methods found that scrape loading was the most efficient method of delivery; however, because the morpholino backbone is uncharged, cationic lipids are not effective mediators of morpholino oligonucleotide uptake in cells. A recent report demonstrated triplex formation by a morpholino oligonucleotide and, because of the non-ionic backbone, these studies showed that the morpholino oligonucleotide was capable of triplex formation in the absence of magnesium.

It is further preferred that the linkage between the residues in a backbone do not include a phosphorus atom, such as a linkage that is formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. A preferred nucleotide analogue or equivalent comprises a Peptide Nucleic Acid (PNA), having a modified polyamide backbone (Nielsen, et al. (1991 ) Science 254, 1497-1500). PNA- based molecules are true mimics of DNA molecules in terms of base-pair recognition. The backbone of the PNA is composed of N-(2-aminoethyl)- glycine units linked by peptide bonds, wherein the nucleobases are linked to the backbone by methylene carbonyl bonds. An alternative backbone comprises a one-carbon extended pyrrolidine PNA monomer (Govindaraju and Kumar (2005) Chem Commun 495-497). Since the backbone of a PNA molecule contains no charged phosphate groups, PNA-RNA hybrids are usually more stable than RNA-RNA or RNA-DNA hybrids, respectively (Egholm et al. (1993) Nature 365:566-568). A further preferred backbone comprises a morpholino nucleotide analog or equivalent, in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring. A most preferred nucleotide analog or equivalent comprises a phosphorodiamidate morpholino oligomer (PMO), in which the ribose or deoxyribose sugar is replaced by a 6-membered morpholino ring, and the anionic phosphodiester linkage between adjacent morpholino rings is replaced by a non-ionic phosphorodiamidate linkage.

In yet a further embodiment, a nucleotide analogue or equivalent of the invention comprises a substitution of one of the non-bridging oxygens in the phosphodiester linkage. This modification slightly destabilizes base-pairing but adds significant resistance to nuclease degradation. A preferred nucleotide analogue or equivalent comprises phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, H- phosphonate, methyl and other alkyl phosphonate including 3'-alkylene phosphonate, 5'- alkylene phosphonate and chiral phosphonate, phosphinate, phosphoramidate including 3'- amino phosphoramidate and aminoalkylphosphoramidate, thionophosphoramidate, thionoalkylphosphonate, thionoalkylphosphotriester, selenophosphate or boranophosphate. In an especially preferred embodiment all internucleotide linkages comprise a phosphorothioate modification.

A further preferred nucleotide analogue or equivalent of the invention comprises one or more sugar moieties that are mono- or disubstituted at the 2', 3' and/or 5' position. Examples are -OH; -F; substituted or unsubstituted, linear or branched lower (CI-C10) alkyl, alkenyl, alkynyl, alkaryl, allyl, or aralkyl, that may be interrupted by one or more heteroatoms; 0-, S-, or N-alkyl; 0-, S-, or N-alkenyl; 0-, S-or N-alkynyl; 0-, S-, or N- allyl; O-alkyl-O-alkyl, -methoxy, -aminopropoxy; methoxyethoxy; dimethylamino oxyethoxy; and -dimethylaminoethoxyethoxy. A particularly preferred embodiment relates to an AON that comprises a sugar moiety carrying a 2'-0-methyl (2'-OMe) modification, and more preferably wherein all nucleotides in the AON carry a 2'-0-methyl modification. In yet another preferred embodiment, the AON according to the invention carries a 2'-0-methoxyethyl (2'-0-MOE) modification, and more preferably wherein all nucleotides in the AON carry a 2'-0-methoxyethyl modification. It is understood by a skilled person that it is not necessary for all positions in an AON to be modified uniformly. In addition, more than one of the aforementioned analogues or equivalents may be incorporated in a single AON or even at a single position within an AON. In certain embodiments, an AON of the invention has at least two different types of analogues or equivalents. A preferred exon skipping AON according to the invention relates to a 2'-0-alkyl phosphorothioate modified antisense oligonucleotide. An effective AON according to the invention relates to an AON carrying a 2'-0-methyl modification with a (preferably full) phosphorothioate backbone. A highly preferred AON according to the present invention relates to an AON carrying a 2'-0- methoxyethyl modification with a (preferably full) phosphorothioate backbone.

The sugar moiety can be a pyranose or derivative thereof, or a deoxypyranose or derivative thereof, preferably ribose or derivative thereof, or deoxyribose or derivative thereof.

A preferred derivatized sugar moiety comprises a Locked Nucleic Acid (LNA), in which the 2'-carbon atom is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bicyclic sugar moiety. A preferred LNA comprises 2'-0, 4'-C-ethylene-bridged nucleic acid (Morita et al. 2001 . Nucleic Acid Res Supplement No.1 :241 -242). These substitutions render the nucleotide analogue or equivalent RNase H and nuclease resistant and increase the affinity for the target RNA.

In another embodiment, a nucleotide analogue or equivalent of the invention comprises one or more base modifications or substitutions. Modified bases comprise synthetic and natural bases such as inosine, xanthine, hypoxanthine and other -aza, deaza, -hydroxy, -halo, -thio, thiol, -alkyl, -alkenyl, -alkynyl, thioalkyl derivatives of pyrimidine and purine bases that are or will be known in the art.

It will also be understood by a skilled person that different AONs can be combined for efficiently skipping exon 12 or a part thereof from the human HTT pre-mRNA. In a preferred embodiment, a combination of at least two AONs are used in a method of the invention, such as 2, 3, 4, or 5 different AONs. Hence, the invention also relates to a set of AONs comprising at least one AON according to the present invention.

An AON can be linked to a moiety that enhances uptake of the AON in cells, preferably neuronal cells. Examples of such moieties are cholesterols, carbohydrates, vitamins, biotin, lipids, phospholipids, cell-penetrating peptides including but not limited to antennapedia, TAT, transportan and positively charged amino acids such as oligoarginine, poly-arginine, oligolysine or polylysine, antigen-binding domains such as provided by an antibody, a Fab fragment of an antibody (e.g. Brainshuttle), or a single chain antigen binding domain such as a cameloid single domain antigen-binding domain.

An AON according to the invention may be indirectly administrated using suitable means known in the art. It may for example be provided to an individual or a cell, tissue or organ of said individual in the form of an expression vector wherein the expression vector encodes a transcript comprising said oligonucleotide. The expression vector is preferably introduced into a cell, tissue, organ or individual via a gene delivery vehicle. In a preferred embodiment, there is provided a viral-based expression vector comprising an expression cassette or a transcription cassette that drives expression or transcription of an AON as identified herein. Accordingly, the invention provides a viral vector expressing an AON according to the invention when placed under conditions conducive to expression of the AON. Expression may be driven by a polymerase ll-promoter (Pol II) such as a U7 promoter or a polymerase III (Pol III) promoter, such as a U6 RNA promoter. A preferred delivery vehicle is a viral vector such as an adeno associated virus vector (AAV), or a retroviral vector such as a lentivirus vector and the like. Also, plasmids, artificial chromosomes, plasmids usable for targeted homologous recombination and integration in the human genome of cells may be suitably applied for delivery of an oligonucleotide as defined herein. Preferred for the current invention are those vectors wherein transcription is driven from Pol III promoters, and/or wherein transcripts are in the form fusions with U1 or U7 transcripts, which yield good results for delivering small transcripts. It is within the skill of the artisan to design suitable transcripts. Preferred are Pol III driven transcripts, preferably, in the form of a fusion transcript with an U1 or U7 transcript. Such fusions may be generated as described (Gorman et al. 1998. Stable alteration of pre- mRNA splicing patterns by modified U7 small nuclear RNAs. Proc Natl Acad Sci U S A 95(9):4929-34; Suter et al. 1999. Double-target antisense U7 snRNAs promote efficient skipping of an aberrant exon in three human beta-thalassemic mutations. Hum Mol Genet 8(13):2415-23).

Delivery of an AON to cells of the brain can be achieved by various means. For instance, they can be delivered directly to the brain via intracerebral inoculation, intraparenchymal infusion, intrathecally or intraventricularly. Alternatively, the AON can be coupled to a single domain antibody or the variable domain thereof that has the capacity to pass the Blood Brain Barrier. Nanotechnology has also been used to deliver oligonucleotides to the brain, e.g. a nanogel consisting of cross-linked PEG and polyethylenimine. Encapsulation of AON in liposomes is also well known to one of skill in the art.

The AON of the present invention may be delivered as such (naked). However, an AON of the present invention may also be encoded by a viral vector. Typically, this is in the form of an RNA transcript that comprises the sequence of an oligonucleotide according to the invention in a part of the transcript. An AAV vector according to the invention is a recombinant AAV vector and refers to an AAV vector comprising part of an AAV genome comprising an encoded AON according to the invention encapsulated in a protein shell of capsid protein derived from an AAV serotype as depicted elsewhere herein. Part of an AAV genome may contain the inverted terminal repeats (ITR) derived from an adeno-associated virus serotype, such as AAV1 , AAV2, AAV3, AAV4, AAV5, AAV8, AAV9 and others. Protein shell comprised of capsid protein may be derived from an AAV serotype such as AAV1 , 2, 3, 4, 5, 8, 9 and others. A protein shell may also be named a capsid protein shell. AAV vector may have one or preferably all wild type AAV genes deleted, but may still comprise functional ITR nucleic acid sequences. Functional ITR sequences are necessary for the replication, rescue and packaging of AAV virions. The ITR sequences may be wild type sequences or may have at least 80%, 85%, 90%, 95, or 100% sequence identity with wild type sequences or may be altered by for example in insertion, mutation, deletion or substitution of nucleotides, as long as they remain functional. In this context, functionality refers to the ability to direct packaging of the genome into the capsid shell and then allow for expression in the host cell to be infected or target cell. In the context of the invention a capsid protein shell may be of a different serotype than the AAV vector genome ITR. An AAV vector according to present the invention may thus be composed of a capsid protein shell, i.e. the icosahedral capsid, which comprises capsid proteins (VP1 , VP2, and/or VP3) of one AAV serotype, e.g. AAV serotype 2, whereas the ITRs sequences contained in that AAV5 vector may be any of the AAV serotypes described above, including an AAV2 vector. An "AAV2 vector" thus comprises a capsid protein shell of AAV serotype 2, while e.g. an "AAV5 vector" comprises a capsid protein shell of AAV serotype 5, whereby either may encapsulate any AAV vector genome ITR according to the invention. Preferably, a recombinant AAV vector according to the invention comprises a capsid protein shell of AAV serotype 2, 5, 8 or AAV serotype 9 wherein the AAV genome or ITRs present in said AAV vector are derived from AAV serotype 2, 5, 8 or AAV serotype 9; such AAV vector is referred to as an AAV2/2, AAV 2/5, AAV2/8, AAV2/9, AAV5/2, AAV5/5, AAV5/8, AAV 5/9, AAV8/2, AAV 8/5, AAV8/8, AAV8/9, AAV9/2, AAV9/5, AAV9/8, or an AAV9/9 vector.

More preferably, a recombinant AAV vector according to the invention comprises a capsid protein shell of AAV serotype 2 and the AAV genome or ITRs present in said vector are derived from AAV serotype 5; such vector is referred to as an AAV 2/5 vector. More preferably, a recombinant AAV vector according to the invention comprises a capsid protein shell of AAV serotype 2 and the AAV genome or ITRs present in said vector are derived from AAV serotype 8; such vector is referred to as an AAV 2/8 vector. More preferably, a recombinant AAV vector according to the invention comprises a capsid protein shell of AAV serotype 2 and the AAV genome or ITRs present in said vector are derived from AAV serotype 9; such vector is referred to as an AAV 2/9 vector. More preferably, a recombinant AAV vector according to the invention comprises a capsid protein shell of AAV serotype 2 and the AAV genome or ITRs present in said vector are derived from AAV serotype 2; such vector is referred to as an AAV 2/2 vector. A nucleic acid molecule encoding an exon 12 skipping AON (or partial exon 12 skipping AON) according to the invention represented by a nucleic acid sequence of choice is preferably inserted between the AAV genome or ITR sequences as identified above, for example an expression construct comprising an expression regulatory element operably linked to a coding sequence and a 3' termination sequence. 'AAV helper functions' generally refers to the corresponding AAV functions required for AAV replication and packaging supplied to the AAV vector in trans. AAV helper functions complement the AAV functions which are missing in the AAV vector, but they lack AAV ITRs (which are provided by the AAV vector genome). AAV helper functions include the two major ORFs of AAV, namely the rep coding region and the cap coding region orfunctional substantially identical sequences thereof. Rep and Cap regions are well known in the art. The AAV helper functions can be supplied on an AAV helper construct, which may be a plasmid. Introduction of the helper construct into the host cell can occur e.g. by transformation, transfection, or transduction prior to or concurrently with the introduction of the AAV genome present in the AAV vector as identified herein. The AAV helper constructs of the invention may thus be chosen such that they produce the desired combination of serotypes for the AAV vector's capsid protein shell on the one hand and for the AAV genome present in said AAV vector replication and packaging on the other hand. The 'AAV helper virus' provides additional functions required for AAV replication and packaging. Suitable AAV helper viruses include adenoviruses, herpes simplex viruses (such as HSV types 1 and 2) and vaccinia viruses. The additional functions provided by the helper virus can also be introduced into the host cell via vectors, as described in US 6,531 ,456. Preferably, an AAV genome as present in a recombinant AAV vector according to the invention does not comprise any nucleotide sequences encoding viral proteins, such as the rep (replication) or cap (capsid) genes of AAV. An AAV genome may further comprise a marker or reporter gene, such as a gene for example encoding an antibiotic resistance gene, a fluorescent protein (e.g. gfp) or a gene encoding a chemically, enzymatically or otherwise detectable and/or selectable product (e.g. lacZ, aph, etc.) known in the art. A preferred AAV vector is preferably an AAV2/5, AAV2/8, AAV2/9 or AAV2/2 vector, expressing an AON according to the invention that comprises, or preferably consists of, a sequence that is complementary or substantially complementary to a nucleotide sequence of exon 12 of the human Htt pre-mRNA. A further preferred AAV vector according to the invention is an AAV vector, preferably an AAV2/5, AAV2/8, AAV2/9 or AAV2/2 vector, expressing an AON according to the invention that comprises, or preferably consists of any one of SEQ ID NO:2 or 3.

Improvements in means for providing an individual or a cell, tissue, organ of said individual with an AON according to the invention, are anticipated considering the progress that has already thus far been achieved. Such future improvements may of course be incorporated to achieve the mentioned effect on restructuring of mRNA using a method of the invention. An AON according to the invention can be delivered as is to an individual, a cell, tissue or organ of said individual. When administering an AON according to the invention, it is preferred that the AON is dissolved in a solution that is compatible with the delivery method. A preferred delivery method for an AON or a plasmid for AON expression is a viral vector, a nanoparticle or a microparticle. Nanoparticles and microparticles that may be used for in vivo AON delivery are well known in the art. Alternatively, a plasmid can be provided by transfection using known transfection reagents. For intravenous, subcutaneous, intramuscular, intrathecal and/or intraventricular administration it is preferred that the solution is a physiological salt solution. Particularly preferred in the invention is the use of an excipient or transfection reagents that will aid in delivery of each of the constituents as defined herein to a cell and/or into a cell (preferably a neuronal cell). Preferred are excipients or transfection reagents capable of forming complexes, nanoparticles, micelles, vesicles and/or liposomes that deliver each constituent as defined herein, complexed or trapped in a vesicle or liposome through a cell membrane. Many of these excipients are known in the art. Suitable excipients or transfection reagents comprise polyethylenimine (PEI; ExGen500 (MBI Fermentas)), LipofectAMINE™ 2000 (Invitrogen) or derivatives thereof, or similar cationic polymers, including polypropyleneimine or polyethylenimine copolymers (PECs) and derivatives, synthetic amphiphils (SAINT-18), lipofectinTM, DOTAP and/or viral capsid proteins that are capable of self-assembly into particles that can deliver each constituent as defined herein to a cell, preferably a neuronal cell. Such excipients have been shown to efficiently deliver an AON to a wide variety of cultured cells. Their high transfection potential is combined with an excepted low to moderate toxicity in terms of overall cell survival. The ease of structural modification can be used to allow further modifications and the analysis of their further (in vivo) nucleic acid transfer characteristics and toxicity. Lipofectin represents an example of a liposomal transfection agent. It consists of two lipid components, a cationic lipid N-[1 -(2,3 dioleoyloxy)propyl]-N, N, N- trimethylammonium chloride (DOTMA) (cp. DOTAP which is the methylsulfate salt) and a neutral lipid dioleoylphosphatidyl ethanolamine (DOPE). The neutral component mediates the intracellular release. Another group of delivery systems are polymeric nanoparticles. Polycations such as diethylamino ethylaminoethyl (DEAE)-dextran, which are well known as DNA transfection reagent can be combined with butylcyanoacrylate (PBCA) and hexylcyanoacrylate (PHCA) to formulate cationic nanoparticles that can deliver AONs across cell membranes into cells. In addition to these common nanoparticle materials, the cationic peptide protamine offers an alternative approach to formulate an oligonucleotide with colloids. This colloidal nanoparticle system can form so called proticles, which can be prepared by a simple self-assembly process to package and mediate intracellular release of an AON. The skilled person may select and adapt any of the above or other commercially available alternative excipients and delivery systems to package and deliver an AON for use in the current invention to deliver it for the prevention, treatment or delay of HD.

In addition, an AON according to the invention could be covalently or non-covalently linked to a targeting ligand specifically designed to facilitate the uptake into the cell, cytoplasm and/or its nucleus. Such ligand could comprise (i) a compound (including but not limited to peptide(-like) structures) recognizing cell, tissue or organ specific elements facilitating cellular uptake and/or (ii) a chemical compound able to facilitate the uptake in to cells and/or the intracellular release of an oligonucleotide from vesicles, e.g. endosomes or lysosomes. Therefore, in a preferred embodiment, an AON according to the invention is formulated in a composition or a medicament or a composition, which is provided with at least an excipient and/or a targeting ligand for delivery and/or a delivery device thereof to a cell and/or enhancing its intracellular delivery.

It is to be understood that if a composition comprises an additional constituent such as an adjunct compound as later defined herein, each constituent of the composition may not be formulated in one single combination or composition or preparation. Depending on their identity, the skilled person will know which type of formulation is the most appropriate for each constituent as defined herein. In a preferred embodiment, the invention provides a composition or a preparation which is in the form of a kit of parts comprising an AON according to the invention and a further adjunct compound as later defined herein. If required, an AON according to the invention or a vector, preferably a viral vector, expressing an AON according to the invention can be incorporated into a pharmaceutically active mixture by adding a pharmaceutically acceptable carrier. Accordingly, the invention also provides a composition, preferably a pharmaceutical composition, comprising an AON according to the invention, or a viral vector according to the invention and a pharmaceutically acceptable excipient. Such a pharmaceutical composition may comprise any pharmaceutically acceptable excipient, including a carrier, filler, preservative, adjuvant, solubilizer and/or diluent. Such pharmaceutically acceptable carrier, filler, preservative, adjuvant, solubilizer and/or diluent may for instance be found in Remington (Remington. 2000. The Science and Practice of Pharmacy, 20th Edition. Baltimore, MD: Lippincott Williams Wilkins). Each feature of said composition has earlier been defined herein.

A treatment in a use or in a method according to the invention is at least once, lasts one week, one month, several months, 1 , 2, 3, 4, 5, 6 years or longer, such as lifelong. Each AON or equivalent thereof as defined herein for use according to the invention may be suitable for direct administration to a cell, tissue and/or an organ in vivo of individuals already affected by HD, or at risk of developing HD, and may be administered directly in vivo, ex vivo or in vitro. The frequency of administration of an AON, composition, compound or adjunct compound of the invention may depend on several parameters such as the severity of the disease, the age of the patient, the mutation of the patient (the number of CAG repeats), the number of AONs (i.e. dose), the formulation of said AON, the route of administration and so forth. The frequency may vary between daily, weekly, at least once in two weeks, or three weeks or four weeks or five weeks or a longer time period. Dose ranges of an AON according to the invention are preferably designed on the basis of rising dose studies in clinical trials (in vivo use) for which rigorous protocol requirements exist. An AON as defined herein, may be used at a dose which is ranged from 0.01 and 20 mg/kg, preferably from 0.05 and 20 mg/kg body weight. A suitable intracranial dose would be between about 1 mg and about 20 mg per dose, preferably in the range of 5 and 12 mg, such as about 5, 6, 7, 8, 9, 10, 1 1 , or 12 mg. In a preferred embodiment, a concentration of an AON as defined herein, is ranged from 0.1 nM and 1 μΜ for therapeutic administration. More preferably, the concentration used is ranged from 1 to 400 nM, even more preferably from 10 to 200 nM, even more preferably from 50 to 100 nM. In that respect, a 10kDa AON in 1 liter cranium is approximately 100 nM. If several AONs are used, this concentration or dose may refer to the total concentration or dose of AONs or the concentration or dose of each AON added. In a preferred embodiment, a viral vector, preferably an AAV vector as described earlier herein, as delivery vehicle for a molecule according to the invention, is administered in a dose ranging from 1 x10 ⁹ to 1 x10 ¹⁷ virus particles per injection, more preferably from 1 x10 ¹⁰ to 1 x10 ¹² virus particles per injection. The ranges of concentration or dose of AONs as given above are preferred concentrations or doses for in vivo, in vitro or ex vivo uses. The skilled person will understand that depending on the AONs used, the target cell to be treated, the gene target and its expression levels, the medium used and the transfection and incubation conditions, the concentration or dose of AONs used may further vary and may need to be optimized any further.

An AON according to the invention, or a viral vector according to the invention, or a composition according to the invention for use according to the invention may be suitable for administration to a cell, tissue and/or an organ in vivo of individuals already affected or at risk of developing HD, and may be administered in vivo, ex vivo or in vitro. Said AON according to the invention, or viral vector according to the invention, or composition according to the invention may be directly or indirectly administered to a cell, tissue and/or an organ in vivo of an individual already affected by HD or at risk of HD, and may be administered directly or indirectly in vivo, ex vivo or in vitro.

Examples

Example 1. Efficacy of AONs with extended 3' ends in mediating HTT exon 12 mRNA partial skipping

The antisense oligonucleotides HD_AON29 (SEQ ID NO:2) and HD_AON30 (SEQ ID

NO:3) were compared with the known oligo HD_AON12.1 (SEQ ID NO:1 ) for their ability to induce skip of part of HTT exon 12 or, in other words, the stimulation of the expression/appearance of the ΗΤΤ-Δ12 isoform as described by Ruzo et al. Sequences of the AONs with their specific modifications are provided in Table 1 . As outlined above, it was envisioned that the two novel 3' extended oligonucleotides might have improved skipping abilities. Experiments were performed in a dose dependent manner of 8 nM, 16 nM and 32 nM for each of the oligonucleotides. Negative controls were a non-transfected sample (NT) in which no AONs were transfected into the cells, and a water (H2O) control. Table 1. Sequences and modifications of antisense oligonucleotides (AONs) tested for partial skip of HTT exon 12. Upper case is RNA; m = 2'-0-methyl modification; * = phosphorothioate (PS) linkage; underlined are extended sequence in comparison to HD_AON12.1.

GM04857A HD patient fibroblast cells (Coriell Institute for Medical Research) were seeded in 6-well plates and transfected after 24 h with either 8 nM, 16 nM or 32 nM of each of the three AONs HD_AON29, HD_AON30 and HD_AON12.1 . RNA analysis was performed 24 h after transfection. Protein analysis was performed 72 h after transfection (see example 2). The transfection reagent used was Lipofectamine 2000 (Invitrogen), and the ratio RNA:transfection reagent was 1 :5. PBS (Gibco) was used as diluent. For RNA analysis, transfected cells were harvested and RNA was isolated with the ReliPrepTM RNA Cell Miniprep System (Promega) according to manufacturer's instructions. After isolation, RNA concentration in each sample was measured with NanoDrop 2000 (Thermo Fisher Scientific). After all samples were measured for RNA content, cDNA synthesis followed with 500 ng RNA of each sample, using the Thermo Scientific Maxima Reverse Transcriptase with oligo(dT) (Thermo Scientific) according to manufacturer's instructions. Two types of PCR analyses were performed: reverse transcriptase PCR (RT-PCR) followed by microfluidic electrophoresis, and droplet digital PCR (ddPCR). The PCR primers that were used were F2: 5'- ACAAGCTCTGCCACTGATG-3' (SEQ ID NO:13) and R3: 5'-GGCTGTCCAATCTGCAG-3' (SEQ ID NO:14). With RT-PCR the partially skipped exon 12 (ΗΤΤ-Δ12) mRNA can be detected next to the wild type HTT mRNA. The assay was performed with the AmpliTaq Gold ® DNA Polymerase kit (Thermo Fisher Scientific) according to manufacturer's instructions. For each sample, 3 μΙ cDNA was used. After the RT-PCR run was finished, samples were loaded on a 1000bp DNA chip and ran on Agilent 2100 BioAnalyzer. Results of these experiments (Figure 2) indicate that at low concentrations of transfected AONs (8 nM) a significant amount of wild type H77 mRNA is still present when the known HD_AON12.1 oligo is being transfected. However, at this low concentration almost all wild type HTT mRNA including nucleotides 207-341 of exon 12 has disappeared when the cells were transfected with the oligonucleotides that comprise the sequence of HD_AON 12.1 but that are elongated at the 3' end. This shows that at low concentrations the elongated versions are significantly more effective in inducing this skip and inducing the appearance of the ΗΤΤ-Δ12 isoform, than the oligonucleotide from the art.

Then, the droplet digital PCR (ddPCR) method with EvaGreen dye was used for quantification of the fractional abundance of skipped ΗΤΤ-Δ12 mRNA. The standard Bio-Rad protocol was used for this purpose. Each sample was diluted 5x in order to remove the influence of the cDNA mixture components, and tested in triplicate. For each sample, 2 μΙ of template cDNA and 10 μΙ ddPCR EvaGreen Supermix (Bio-Rad) was used, together with 0.2 μΙ of 10 μΜ Primers (F2, R3) and 7.6 μΙ H2O. The results are shown in Figure 3. These indicate that the lower the concentration, the higher the efficiency in inducing the partial skip of exon 12. This is clearly important, because it may be that in vivo concentrations of oligonucleotides during treatment, or the number of oligonucleotides that act within the treated cell may be relatively low. Hence, the higher the efficiency at lower concentrations as determined here, the better. Clearly, at low concentrations the new oligonucleotides HD_AON29 and HD_AON30 outperform the known HD_AON12.1 , indicating that elongating the sequence at the 3' end of the oligonucleotide is beneficial for the treatment HD.

Example 2. Efficacy of new AONs in generating exon 12 modified Huntingtin protein

The transfected GM04857A cells of example 1 were harvested 72 h post-transfection and protein lysates were made. First, cells were washed twice (per well) with 1 ml ice-cold 1x PBS (Gibco) and 100 μΙ cold M-PER Mammalian Protein Extraction Reagent (ThermoFisher Scientific) with protease inhibitor was added to each sample well. After 5 min incubation on ice, the lysates were collected, transferred to a micro-centrifuge tube and agitated for 30 min at 4 °C. Finally, samples were centrifuged at 10,000 x g for 20 min to pellet the cell debris and the supernatant was stored at -20 °C until further use. After the lysate samples were prepared, a BCA assay was performed (Pierce) following the protocol from the manufacturer to determine the total protein concentration of each sample. Next, 12 μg total protein of each sample was mixed with 1 x NuPAGE loading buffer (Thermo Fisher Scientific) + 50 mM DTT and loaded on an 3-8 % criterion XT Precast Gel (Bio-Rad) for SDS-PAGE. Running buffer contained XT Tricine (Bio-Rad). Western blot was treated by overnight incubation at 4 °C with primary antibodies diluted in Odyssey Blocking Buffer PBS: anti-Huntingtin (mouse, MAB2166, EMD Millipore) 1 :2000 and anti-Vinculin (rabbit, ab73412, Abeam) 1 :1000 as a loading control. Next day membrane was washed with PBS-Tween 0.1 % and incubated for 1 h at RT with secondary antibodies: 800 CW goat anti-Rb 1 :10000 (LI-COR) and 680 RD 1 :10 000 (LI-COR). After incubation, membrane was washed 5 x 20 min with PBS-Tween 0.1 %, followed by 2 x 20 ml PBS. Finally membrane was dried and then scanned using an Odyssey CLx Imaging System (LI-COR). Results are provided in Figure 4 and show that the appearance of the protein band that represents the ΗΤΤ-Δ12 version, lacking the 45 amino acids in comparison to the wild type protein does occur when 8 nM of the oligonucleotides HD_AON29 and HD_AON30 were transfected, whereas this mutant version does not occur at this concentration when the known AON is used, again showing the higher efficiency of the AONs that are elongated at the 3' end.