Field of the invention

The present invention relates to the fields of medicine, particularly neurology and clinical genetics, molecular biology and biopharmacy. More specifically, the invention relates to nucleic acid molecules that upregulate the translation of STXBP1 mRNA and their use in the treatment of STXBP1 -related disorders.

Background of the invention

STXBP1 -related disorders comprise a spectrum of rare autosomal dominant neurodevelopmental conditions caused by mutations in the STXBP1 gene, together referred to as STXBP1 -encephalopathies, one of the early infantile epileptic encephalopathies.

The STXBP1 gene encodes syntaxin binding protein 1 , the presynaptic protein Munc18-1. STXBP1 -encephalopathies are caused by de novo heterozygous missense or truncating mutations or micro-deletions in the STXBP1 gene, first reported by Saitsu et al. (Nat Genet 2008; 40: 782- 8.), and include intellectual disability, epilepsy, autism spectrum disorders, and involuntary movements (spasms and jerks) (Hamdan et al., Ann Neurol 2009; 65: 748-53; de Rubeis et al., Nature 2014; 515: 209-15). The estimated incidence rate for STXBP1 -related disorders is approximately 1 in 30,000 (Lopez-Rivera et al., Brain. 2020;143(4):1099-1105).

Currently, there are no curative, disease-altering, or specific therapies available for individuals with STXBP1 encephalopathy. Medical management is principally symptomatic and supportive. There is therefore a need in the art for disease-altering and/or curative therapies for STXBP1 encephalopathy.

Recently, a novel functional class of antisense (AS) IncRNAs was identified, which increase translation of partially overlapping sense protein-coding mRNAs. These RNAs are also called SINEUPs, as they require a SINE B2 element to UP-regulate translation and are disclosed in WO 2012/133947 and WO 2019/150346.

AS Uchll, an IncRNA antisense to the mouse orthologue of human Uchl1/PARK5 gene, can be considered the representative member of this new class of IncRNAs, as it was found to increase UchLI protein synthesis acting at a post-transcriptional level. AS Uchll activity depends on the combination of two functional domains: at the 5’ end, the overlapping region, indicated as “binding domain” or “target binding sequence”, dictates AS Uchll specificity towards Uchll mRNA; at the 3’ end, the non-overlapping region contains an embedded inverted SINE B2 element, which acts as “effector domain” (or “effector domain sequence”) and triggers translation up-regulation of bound target mRNA.

More than 30 antisense IncRNAs promote translation up-regulation of partially overlapping mRNAs. By replacing the binding domain, it is possible to re-direct AS Uchll activity towards a target mRNA of choice. It is an object of the present invention to provide for nucleic acid molecules that upregulate the translation of STXBP1 mRNA, which are useful in the treatment of STXBP1 -related disorders.

Summary of the invention

In a first aspect, the invention relates to a nucleic acid molecule comprising: i) at least one target binding sequence comprising a sequence reverse complementary to an STXBP1 mRNA sequence; and, ii) at least one effector domain sequence comprising a SINE B2 element or a functionally active fragment of a SINE B2 element. Preferably, the nucleic acid molecule of the invention, when expressed in heterozygote STXBP1 ^+/_ iNeurons increases the steady state STXBP1 protein level between 1.1 to 5 fold as compared to the steady state STXBP1 protein level in corresponding heterozygote STXBP1 ^+/_ iNeurons expressing a corresponding effector domain without a target binding sequence.

In one embodiment, the nucleic acid molecule of the invention is a molecule wherein the at least one effector domain sequence comprises a sequence with at least 75% sequence identity with a sequence selected from the group consisting of SEQ ID NO:1 to SEQ ID NO:51 .

In one embodiment, the nucleic acid molecule of the invention is a molecule wherein the at least one target binding sequence comprises a sequence with complementarity to the STXBP1 mRNA transcript of between 10 and 250 nucleotides in length and which has an identity of at least 75% with a sequence reverse complementary to the nucleotide sequence of the STXBP1 mRNA transcript.

In one embodiment, the nucleic acid molecule of the invention is a molecule wherein the sequence with complementarity to the STXBP1 mRNA transcript comprises at least one of: a) a sequence with complementarity to the 5’-untranslated region of the STXBP1 mRNA sequence; b) a sequence with complementarity to the AUG translation initiation codon of the STXBP1 mRNA sequence; and, c) a sequence with complementarity to the protein coding sequence of the STXBP1 mRNA sequence, downstream of the AUG translation initiation codon. Preferably, the sequence with complementarity to the STXBP1 mRNA transcript comprises a sequence with at least 95% sequence identity to at least one of SEQ ID NO:57 and SEQ ID NO:58.

In one embodiment, the nucleic acid molecule of the invention is a molecule further comprising at least one linker sequence between the at least one target binding sequence and the at least one effector domain sequence.

In one embodiment, the nucleic acid molecule of the invention is a molecule wherein the molecule comprises a nucleotide sequence with at least 90% sequence identity to at least one of SEQ ID NO:59 and SEQ ID NO:60.

In a second aspect, the invention pertains to a DNA construct comprising a nucleotide sequence encoding the nucleic acid molecule of the invention as defined above. Preferably, the DNA construct is an expression construct wherein the nucleotide sequence is operably linked to expression control sequences comprising at least a promoter. The promoter preferably is an RNA polymerase III promoter, such as a (human) H1 promoter or a (human) U6 promoter, of which a promoter from a U6 snRNA gene is preferred. In one embodiment, the DNA and/or the expression construct is a viral gene therapy vector. Preferably, the viral gene therapy vector is an AAV vector.

In a third aspect, the invention relates to a composition comprising a nucleic acid molecule of the invention, or a DNA construct or expression construct comprising the nucleic acid molecule, wherein the composition optional comprises at least one pharmaceutically acceptable carrier or excipient.

In a fourth aspect, the invention relates to a nucleic acid molecule of the invention, a DNA construct or expression construct comprising the nucleic acid molecule, or a composition comprising the molecule or the construct, for use as a medicament.

In a fifth aspect, the invention pertains to a nucleic acid molecule of the invention, a DNA construct or expression construct comprising the nucleic acid molecule, or a composition comprising the molecule or the construct, for use in the prevention or treatment of an STXBP1- related disorder. Preferably the STXBP1 -related disorder to be prevented or treated is a disorder associated with an STXBP1 haploinsufficiency. In one embodiment, the STXBP1 -related disorder is a severe early onset epileptic encephalopathy or a non-syndromic epilepsy selected from the group consisting of: Ohtahara syndrome, West syndrome, Lennox-Gastaut syndrome, Dravet syndrome, early myoclonic encephalopathy, an unclassified early onset epileptic encephalopathy that is associated with STXBP1 haploinsufficiency, atypical Rett syndrome and a severe intellectual disability without epilepsy associated with STXBP1 haploinsufficiency.

Description of the invention

Definitions

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the method.

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 elements 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".

As used herein, the term "and/or" indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases.

As used herein, with "At least" a particular value means that particular value or more. For example, "at least 2" is understood to be the same as "2 or more" i.e. , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, ... ,etc. 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.

As used herein, "an effective amount" is meant the amount of an agent required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active agent(s) used to practice the present invention for therapeutic treatment of, for example an STXBP1- encephalopathy, varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount, which may be determined as genome copies per kilogram (GC/kg). Thus, in connection with the administration of a drug which, in the context of the current disclosure, is "effective against" a disease or condition indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in at least one disease sign or symptom, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disease or condition.

The use of a substance as a medicament as described in this document can also be interpreted as the use of said substance in the manufacture of a medicament. Similarly, whenever a substance is used for treatment or as a medicament, it can also be used for the manufacture of a medicament for treatment. Products for use as a medicament described herein can be used in methods of treatments, wherein such methods of treatment comprise the administration of the product for use.

By “STXBP1 mRNA sequence” there is intended an mRNA sequence of any length of at least 10 nucleotides comprised in the mRNA of the corresponding STXBP1 gene.

The STXBP1 gene sequence is known in the art, for example see Gene ID: 6812 or Ensembl ID: ENSG00000136854. The STXBP1 gene encodes the syntaxin binding protein 1 or presynaptic protein Munc18-1 , the amino acid sequence of which is known in the art, for example see UniProt ID: P61764. Sequences of the cDNAs of the two major human STXBP1 isoforms STXBP1-201 and STXBP1-202 are given in SEQ ID NO.’s: 53 and 54, respectively.

The term “SINE” (Short Interspersed Nuclear Element) may be referred to as a non-LTR (long terminal repeat) retrotransposon, and is an interspersed repetitive sequence (a) which encodes a protein having neither reverse-transcription activity nor endonuclease activity or the like and (b) whose complete or incomplete copy sequences exist abundantly in genomes of living organisms.

The term “SINE B2 element” is defined in WO 2012/133947, where specific examples are also provided (see table starting on page 69 of the PCT publication). The term is intended to encompass both SINE B2 elements in direct orientation and in inverted orientation relative to the 5’ to 3’ orientation of the nucleic acid molecule of the invention. SINE B2 elements may be identified, for example, using programs like RepeatMask as published (Bedell et al. Bioinformatics. 2000 Nov; 16(11): 1040-1. MaskerAid: a performance enhancement to RepeatMasker). A sequence may be recognizable as a SINE B2 element by returning a hit in a Repbase database with respect to a consensus sequence of a SINE B2, with a Smith-Waterman (SW) score of over 225, which is the default cut-off in the RepeatMasker program. Generally, a SINE B2 element is not less than 20 bp and not more than 400 bp. Preferably, the SINE B2 is derived from tRNA.

By the term “functionally active fragment of a SINE B2 element” there is intended a portion of sequence of a SINE B2 element that retains protein translation enhancing efficiency. This term also includes sequences which are mutated in one or more nucleotides with respect to the wild- type sequences, but retain protein translation enhancing efficiency. The term is intended to encompass both SINE B2 elements in direct orientation and in inverted orientation relative to the 5’ to 3’ orientation of the nucleic acid molecule of the invention.

By the term “miniSINEUP” there is intended a nucleic acid molecule consisting of a binding domain (complementary sequence to target mRNA), a spacer sequence, and any SINE or SINE- derived sequence or IRES-derived sequence as the effector domain (Zucchelli et al.. Front Cell Neurosci., 9: 174, 2015).

By the term “microSINEUP” there is intended a nucleic acid molecule consisting of a binding domain (complementary sequence to target mRNA), a spacer sequence, and a functionally active fragment of the SINE or SINE-derived sequence or IRES-derived sequence. For example, the functionally active fragment may be a 77 bp sequence corresponding to nucleotides 44 to 120 of the 167 bp SINE B2 element in AS Uchll.

The terms “homology”, “sequence identity” and the like are used interchangeably herein. Sequence identity is herein defined as a relationship between two or more amino acid (polypeptide or protein) sequences or two or more nucleic acid (polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleic acid sequences, as the case may be, as determined by the match between strings of such sequences. "Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide. "Identity" and "similarity" can be readily calculated by known methods.

“Sequence identity” and “sequence similarity” can be determined by alignment of two peptide or two nucleotide sequences using global or local alignment algorithms, depending on the length of the two sequences. Sequences of similar lengths are preferably aligned using global alignment algorithms (e.g. Needleman Wunsch) which align the sequences optimally over the entire length, while sequences of substantially different lengths are preferably aligned using local alignment algorithms (e.g. Smith Waterman). Sequences may then be referred to as "substantially identical” or “essentially similar” when they (when optimally aligned by for example the programs GAP or BESTFIT using default parameters) share at least a certain minimal percentage of sequence identity (as defined below). GAP uses the Needleman and Wunsch global alignment algorithm to align two sequences over their entire length (full length), maximizing the number of matches and minimizing the number of gaps. A global alignment is suitably used to determine sequence identity when the two sequences have similar lengths. Generally, the GAP default parameters are used, with a gap creation penalty = 50 (nucleotides) / 8 (proteins) and gap extension penalty = 3 (nucleotides) / 2 (proteins). For nucleotides the default scoring matrix used is nwsgapdna and for proteins the default scoring matrix is Blosum62 (Henikoff & Henikoff, 1992, PNAS 89, 915-919). Sequence alignments and scores for percentage sequence identity may be determined using computer programs, such as the GCG Wisconsin Package, Version 10.3, available from Accelrys Inc., 9685 Scranton Road, San Diego, CA 92121-3752 USA, or using open source software, such as the program “needle” (using the global Needleman Wunsch algorithm) or “water” (using the local Smith Waterman algorithm) in EmbossWIN version 2.10.0, using the same parameters as for GAP above, or using the default settings (both for ‘needle’ and for ‘water’ and both for protein and for DNA alignments, the default Gap opening penalty is 10.0 and the default gap extension penalty is 0.5; default scoring matrices are Blossum62 for proteins and DNAFull for DNA). When sequences have a substantially different overall length, local alignments, such as those using the Smith Waterman algorithm, are preferred.

Alternatively, percentage similarity or identity may be determined by searching against public databases, using algorithms such as FASTA, BLAST, etc. Thus, the nucleic acid and protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the BLASTn and BLASTx programs (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403 — 10. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, wordlength = 12 to obtain nucleotide sequences homologous to oxidoreductase nucleic acid molecules of the invention. BLAST protein searches can be performed with the BLASTx program, score = 50, wordlength = 3 to obtain amino acid sequences homologous to protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17): 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., BLASTx and BLASTn) can be used. See the homepage of the National Center for Biotechnology Information at http://www.ncbi.nlm.nih.gov/.

A "nucleic acid construct" or "nucleic acid vector" is herein understood to mean a man-made nucleic acid molecule resulting from the use of recombinant DNA technology. The term "nucleic acid construct" therefore does not include naturally occurring nucleic acid molecules although a nucleic acid construct may comprise (parts of) naturally occurring nucleic acid molecules. A "vector" is a nucleic acid construct (typically DNA or RNA) that serves to transfer an exogenous nucleic acid sequence (i.e. DNA or RNA) into a host cell. A vector is preferably maintained in the host by at least one of autonomous replication and integration into the host cell’s genome. The terms "expression vector" or “expression construct" refer to nucleotide sequences that are capable of affecting expression of a gene in host cells or host organisms compatible with such sequences. These expression vectors typically include at least one “expression cassette” that is the functional unit capable of affecting expression of a sequence encoding a product to be expressed and wherein the coding sequence is operably linked to the appropriate expression control sequences, which at least comprises a suitable transcription regulatory sequence and optionally, 3' transcription termination signals. Additional factors necessary or helpful in affecting expression may also be present, such as expression enhancer elements. The expression vector will be introduced into a suitable host cell and be able to affect expression of the coding sequence in an in vitro cell culture of the host cell. A preferred expression vector will be suitable for expression of viral proteins and/or nucleic acids, particularly recombinant AAV proteins and/or nucleic acids.

As used herein, the term "promoter" or "transcription regulatory sequence" refers to a nucleic acid fragment that functions to control the transcription of one or more coding sequences, and is located upstream with respect to the direction of transcription of the transcription initiation site of the coding sequence, and is structurally identified by the presence of a binding site for DNA- dependent RNA polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active in most tissues under most physiological and developmental conditions. An "inducible" promoter is a promoter that is physiologically or developmental^ regulated, e.g. by the application of a chemical inducer or biological entity.

The term "reporter" may be used interchangeably with marker, although it is mainly used to refer to visible markers, such as green fluorescent protein (GFP) or luciferase.

The terms "protein" or "polypeptide" are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3- dimensional structure or origin.

The term "gene" means a DNA fragment comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. an mRNA) in a cell, operably linked to suitable regulatory regions (e.g. a promoter). A gene will usually comprise several operably linked fragments, such as a promoter, a 5' leader sequence, a coding region and a 3'-nontranslated sequence (3'-end) comprising a polyadenylation site. "Expression of a gene" refers to the process wherein a DNA region which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA, which is biologically active, i.e. which is capable of being translated into a biologically active protein or peptide.

The term "homologous" when used to indicate the relation between a given (recombinant) nucleic acid or polypeptide molecule and a given host organism or host cell, is understood to mean that in nature the nucleic acid or polypeptide molecule is produced by a host cell or organisms of the same species, preferably of the same variety or strain. If homologous to a host cell, a nucleic acid sequence encoding a polypeptide will typically (but not necessarily) be operably linked to another (heterologous) promoter sequence and, if applicable, another (heterologous) secretory signal sequence and/or terminator sequence than in its natural environment. It is understood that the regulatory sequences, signal sequences, terminator sequences, etc. may also be homologous to the host cell. In this context, the use of only "homologous" sequence elements allows the construction of "self-cloned" genetically modified organisms (GMO's) (self-cloning is defined herein as in European Directive 98/81/EC Annex II). When used to indicate the relatedness of two nucleic acid sequences the term "homologous" means that one single-stranded nucleic acid sequence may hybridize to a complementary single-stranded nucleic acid sequence. The degree of hybridization may depend on a number of factors including the amount of identity between the sequences and the hybridization conditions such as temperature and salt concentration as discussed later.

The terms "heterologous" and "exogenous" when used with respect to a nucleic acid (DNA or RNA) or protein refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous and exogenous nucleic acids or proteins are not endogenous to the cell into which they are introduced but have been obtained from another cell or are synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins, i.e. exogenous proteins, that are not normally produced by the cell in which the DNA is transcribed or expressed. Similarly, exogenous RNA encodes for proteins not normally expressed in the cell in which the exogenous RNA is present. Heterologous/exogenous nucleic acids and proteins may also be referred to as foreign nucleic acids or proteins. Any nucleic acid or protein that one of skill in the art would recognize as foreign to the cell in which it is expressed is herein encompassed by the term heterologous or exogenous nucleic acid or protein. The terms heterologous and exogenous also apply to non-natural combinations of nucleic acid or amino acid sequences, i.e. combinations where at least two of the combined sequences are foreign with respect to each other.

As used herein, the term "non-naturally occurring" when used in reference to an organism means that the organism has at least one genetic alternation that is not normally found in a naturally occurring strain of the referenced species, including wild-type strains of the referenced species. Genetic alterations include, for example, modifications introducing expressible nucleic acids encoding proteins or enzymes, other nucleic acid additions, nucleic acid deletions, nucleic acid substitutions, or other functional disruption of the organism's genetic material. Such modifications include, for example, coding regions and functional fragments thereof for heterologous or homologous polypeptides for the referenced species. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a gene or operon. Genetic modifications to nucleic acid molecules encoding enzymes, or functional fragments thereof, can confer a biochemical reaction capability or a metabolic pathway capability to the non-naturally occurring organism that is altered from its naturally occurring state.

As used herein, the term “operably linked” refers to a linkage of polynucleotide (or polypeptide) elements in a functional relationship. A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a transcription regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. Operably linked means that the DNA sequences being linked are typically contiguous and, where necessary to join two protein encoding regions, contiguous and in reading frame.

An expression control sequence is "operably linked" to a nucleotide sequence when the expression control sequence controls and regulates the transcription and/or the translation of the nucleotide sequence. Thus, an expression control sequence can include promoters, enhancers, internal ribosome entry sites (IRES), transcription terminators, a start codon in front of a proteinencoding gene, splicing signal for introns, and stop codons.

The term "expression control sequence" is intended to include, at a minimum, a sequence whose presence is designed to influence expression, and can also include additional advantageous components. For example, leader sequences and fusion partner sequences are expression control sequences. The term can also include the design of the nucleic acid sequence such that undesirable, potential initiation codons in and out of frame, are removed from the sequence. It can also include the design of the nucleic acid sequence such that undesirable potential splice sites are removed. It includes sequences or polyadenylation sequences (pA) which direct the addition of a polyA tail, i.e., a string of adenine residues at the 3'-end of an mRNA, sequences referred to as polyA sequences. It also can be designed to enhance mRNA stability. Expression control sequences which affect the transcription and translation stability, e.g., promoters, as well as sequences which affect the translation, e.g., Kozak sequences, are known in insect cells. Expression control sequences can be of such nature as to modulate the nucleotide sequence to which it is operably linked such that lower expression levels or higher expression levels are achieved.

Detailed description of the invention

In a first aspect, the invention pertains to a nucleic acid molecule comprising at least one target binding sequence comprising a sequence complementary to an STXBP1 mRNA sequence and at least one effector domain sequence comprising an RNA comprising a SINE B2 element or a functionally active fragment of a SINE B2 element.

A nucleic acid molecule of the invention can modulate, in particular upregulate, protein translation of the mRNA target, e.g. the STXBP1 mRNA. However, compared to other methods for complementing genotypic insufficiencies, the upregulation by a molecule of the invention does not involve the risks associated with modification of the target gene by e.g. genome editing. Moreover, the nucleic acid molecules of the invention are highly specific to the target and only effective in those cells wherein the target mRNA is expressed, thereby reducing or preventing off-target side effects.

Effector domain sequences

In one embodiment, an effector domain sequence in a nucleic acid molecule of the invention has protein translation enhancing efficiency. The increase of the protein translation efficiency indicates that the efficiency is increased as compared to a case where a nucleic acid molecule of the invention is not present in a system. In one embodiment, expression of the protein encoded by the target mRNA is increased by at least 1.1 , 1.2, 1.5, 2, 3, 4 or 5 fold. In a further embodiment, expression of the protein encoded by the target mRNA is increased between 1 .1 to 5 fold, 1 .2 to 4 fold, 1.5 to 3 fold, such as between 1.5 and 2.6 fold. The increase in expression of the protein encoded by the target mRNA is herein understood as the increase in steady state protein level in a cell expressing a nucleic acid molecule of the invention as compared to the steady state protein level in a relevant control cell, as can be determined using e.g. an anti-STXBP1 antibody for measuring the amount of the STXBP1 protein expressed in the cells in a suitable immunoassay.

In one embodiment, the increase of the protein translation efficiency achieved by a nucleic acid molecule of the invention can be assayed in in vitro brain cells, preferably human brain cells, and/or in an in vitro human STXBP1 syndrome cell model as described in the Examples herein. More specifically, the increase of the protein translation efficiency achieved by a nucleic acid molecule of the invention can be assayed by expression of a nucleic acid molecule of the invention in heterozygote STXBP1 ^+/_ iNeurons and comparing the steady state STXBP1 protein level to the steady state STXBP1 protein level in corresponding control heterozygote STXBP1 ^+/_ iNeurons expressing a corresponding effector domain without target binding sequence, or that do not express a nucleic acid molecule of the invention. Alternatively, the increase of the protein translation efficiency achieved by a nucleic acid molecule of the invention can be assayed by expression in isogenic iNeuron lines carrying one a disease mutant in one allele. Suitable disease mutants are S241fs or D207G and they may be generated using genome editing techniques know in the art, such as CRISPR/Cas9. Generation of hetero zygote iNeurons, expression of nucleic acid molecule of the invention (and negative controls), and measurements of steady state STXBP1 protein levels can be performed as out lined in the Examples herein.

In one embodiment, the effector domain sequence is located 3’ of the target binding sequence. The effector domain sequence may be in a direct or inverted orientation relative to the 5’ to 3’ orientation of the nucleic acid molecule of the invention. Reference to “direct” refers to the situation in which the effector domain sequence is embedded (inserted) with the same 5’ to 3’ orientation as the nucleic acid molecule of the invention. Instead, “inverted” refers to the situation in which the effector domain sequence is 3’ to 5’ oriented relative to the nucleic acid molecule of the invention.

Preferably, the at least one effector domain sequence comprises a sequence with at least 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1-51. In one embodiment, the at least one effector domain sequence consists of a sequence with at least 75% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity, even more preferably 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1-51.

In one embodiment, the effector domain sequence comprises a SINE B2 element or a functionally active fragment of a SINE B2 element. The SINE B2 element is preferably in an inverted orientation relative to the 5’ to 3’ orientation of the nucleic acid molecule of the invention, i.e. an inverted SINE B2 element. As mentioned in the definitions section, inverted SINE B2 elements are disclosed and exemplified in WO 2012/133947.

SEQ ID NO: 1 (the inverted SINE B2 element in AS Uchll) and SEQ ID NO: 2 (the 77 nucleotide variant of the inverted SINE B2 element in AS Uchll that includes nucleotides 44 to 120) are particularly preferred. Preferably, the at least one effector domain sequence comprises a sequence with at least 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% sequence identity with a sequence of SEQ ID NO: 1 or 2. Other inverted SINE B2 elements and functionally active fragments of inverted SINE B2 elements are SEQ ID NO: 3 to SEQ ID NO: 51 . Experimental data showing the protein translation enhancing efficiency of these sequences is not explicitly shown in the present patent application, but is disclosed in a previous patent application in the name of the same applicant. SEQ ID NO: 3 to SEQ ID NO: 51 can therefore be used as effector domain sequences in molecules according to the present invention.

SEQ ID NO:3 to SEQ ID NO:6, SEQ ID NO:8 to SEQ ID NO:11 , SEQ ID NO:18, SEQ ID NO:43 to SEQ ID NO:51 are functionally active fragments of inverted SINE B2 transposable element derived from AS Uchll. The use of functional fragments reduces the size of the effector domain sequence which is advantageous if used in an expression vector (e.g. viral vectors which may be size-limited) because this provides more space for the target sequence and/or expression elements.

SEQ ID NQ: 7 is a full length 183 nt inverted SINE B2 transposable element derived from AS Uchll. SEQ ID NQ: 12 to SEQ ID NQ: 17, SEQ ID NQ: 19 and SEQ ID NQ: 20, SEQ ID NO: 39 to SEQ ID NQ: 42 are mutated functionally active fragments of inverted SINE B2 transposable element derived from AS Uchll.

SEQ ID NQ: 21 to SEQ ID NQ: 25 and SEQ ID NQ: 28 to SEQ ID NQ: 38 are different SINE B2 transposable elements. SEQ ID NQ: 26 and SEQ ID NQ: 27 are sequences in which multiple inverted SINE B2 transposable element have been inserted.

Target binding sequences

In WO 2012/133947 it was already shown that the target binding sequence needs to have only about 60% similarity with a sequence reverse complementary to the target mRNA. As a matter of fact, the target binding sequence can even display a large number of mismatches and retain activity.

The target binding sequence comprises a sequence which is sufficient in length and in complementarity to bind specifically to the STXBP1 mRNA transcript. Therefore, in one embodiment, the target binding sequence comprises a sequence with complementarity to the STXBP1 mRNA transcript that is at least 10, 11 , 12, 13, 14, 15, 16, 17 or 18 nucleotides long. In a preferred embodiment however, the target binding sequence comprises a sequence with complementarity to the STXBP1 mRNA transcript that is at least 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 55, 60, 65, 70 or 80 nucleotides long. Furthermore, the sequence with complementarity to the STXBP1 mRNA transcript comprised in the target binding sequence can be less than 250 nucleotides long, preferably less than 200 nucleotides long, less than 150 nucleotides long, less than 100 nucleotides long, less than 80 nucleotides long, less than 60 nucleotides long or less than 50 nucleotides long. In one embodiment, the sequence with complementarity to the STXBP1 mRNA transcript comprised in the target binding sequence is between 10 and 250 nucleotides in length, preferably between 12 and 150, more preferably between 14 and 100 and most preferably between 16 and 60 nucleotides long. The sequence with complementarity to the STXBP1 mRNA transcript comprised in the target binding sequence, with a length as specified above, has an identity of at least 60, 65, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99 or 100% with a sequence reverse complementary to the nucleotide sequence of the STXBP1 mRNA transcript.

In one embodiment, the target binding sequence comprises a sequence with complementarity to the STXBP1 mRNA transcript comprises at least one of: a) a sequence with complementarity to the 5’-untranslated region (5’ UTR) of the STXBP1 mRNA sequence; b) a sequence with complementarity to the AUG translation initiation codon of the STXBP1 mRNA sequence; and, c) a sequence with complementarity to the protein coding sequence of the STXBP1 mRNA sequence, downstream of the AUG translation initiation codon. In one embodiment, the target binding sequence comprises a sequence with complementarity to the STXBP1 mRNA transcript comprises: a) a sequence with complementarity to the 5’-untranslated region (5’ UTR) of the STXBP1 mRNA sequence; b) a sequence with complementarity to the AUG translation initiation codon of the STXBP1 mRNA sequence; and, c) a sequence with complementarity to the protein coding sequence of the STXBP1 mRNA sequence, downstream of the AUG translation initiation codon. In one embodiment, sequence with complementarity to the 5’ UTR of the STXBP1 mRNA sequence has a length of at least 10, 11 , 12, 13, 14, 15, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 or 40 nucleotides. In one embodiment, sequence with complementarity to the protein coding sequence of the STXBP1 mRNA sequence, downstream of the AUG translation initiation codon has a length of at least 1 , 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18, 20, 25, 30, 35, 40, 50, 60, 80, 100, 150 or 200 nucleotides.

In one embodiment, the target binding sequence comprises a sequence with complementarity to the STXBP1 mRNA transcript comprising a sequence with at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to at least one of SEQ ID NO:57 and SEQ ID NO:58. In a preferred embodiment, the target binding sequence comprises a sequence with complementarity to the STXBP1 mRNA transcript that consists of at least one of SEQ ID NO: 57 and SEQ ID NO: 58.

Structural features

In one embodiment, a nucleic acid molecule of the invention is an RNA molecule. The RNA molecule of the invention can be produced by transcription of nucleotide sequence coding for the nucleic acid molecule of the invention in a suitable expression system, e.g. host cell. Alternatively, the RNA molecule of the invention can be produced by chemical synthesis.

In another embodiment, a nucleic acid molecule of the invention can be a DNA molecule. Although a DNA molecule of the invention in itself does not have the ability to modulate, in particular upregulate, protein translation of the target STXBP1 mRNA, the DNA molecule of the invention is useful as a sequence encoding the active RNA molecule of the invention, thus allowing it expression in a target cell wherein the protein translation of the target STXBP1 mRNA is to be upregulated. A DNA molecule of the invention can be double or single stranded. A DNA molecule of the invention can thus be part of a DNA construct or expression construct of the invention as detailed below. A DNA molecule of the invention can be produced by practice of conventional techniques in molecular biology that are well-known to those of skill in the art and are discussed, for example, in the following literature references: Sambrook et al., Molecular Cloning. A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y., 1989; Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1987 and periodic updates; and the series Methods in Enzymology, Academic Press, San Diego.

In one embodiment, a nucleic acid molecule of the invention can comprise more than one effector domain sequence, which can be the same sequence repeated more than once, or a different regulatory sequence (i.e. a different SINE B2 element/functionally active fragment of a SINE B2 element).

In one embodiment, in a nucleic acid molecule of the invention, the at least one target binding sequence and the at least one effector domain sequence can be connected by at least one spacer/linker sequence. In case of multiple sequences, several spacer/linker sequences can be inserted in-between the sequences.

SEC ID NO: 52 is a non-limiting example of a spacer/linker sequence that can be used in a nucleic acid molecule of the invention.

In one embodiment, a nucleic acid molecule of the invention is a circular molecule. This conformation leads to a much more stable molecule that is degraded with greater difficulty within the cell (exonucleases cannot degrade circular molecules) and therefore remains active for a longer time.

Furthermore, the nucleic acid molecule of the invention may optionally comprise a non-coding 3’ tail sequence, which e.g. includes restriction sites useful for cloning the molecule in appropriate plasmids.

In one embodiment, a nucleic acid molecule of the invention comprises or consists of a nucleotide sequence with at least 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99 or 100% sequence identity to at least one of SEQ ID NO:59 and SEQ ID NO:60.

DNA constructs and expression constructs

In a further aspect, the invention pertains to a nucleic acid construct comprising a nucleotide sequence encoding a nucleic acid molecule of the invention as herein defined above. The nucleic acid construct is typically a DNA or RNA construct, preferably a DNA construct.

In one embodiment, the nucleic acid construct is an expression construct wherein the nucleotide sequence, encoding a nucleic acid molecule of the invention, is operably linked to expression control sequences. The expression control sequences at least comprise a transcription regulatory sequence or promoter to drive transcription of the nucleotide sequence encoding a nucleic acid molecule of the invention, thus producing an RNA molecule of the invention. The expression control sequences may further comprise 3' transcription termination signals and/or expression enhancer elements. Suitable promoters include e.g. strong constitutive promoters such as the CMV, CAG and PGK promoters. In one embodiment, the promoter is a neurospecific promoter, such as a Neuron-Specific Enolase (NSE) promoter, a human synapsin 1 promoter and a CaMKII kinase promoter. In a preferred embodiment, the promoter is an RNA polymerase III promoter such as a promoter from a U6 snRNA gene or the H1 promoter, preferably a primate or human U6 or H1 promoter.

Exemplary expression constructs (or vectors) are known in the art and may include, for example, plasmid vectors, viral vectors (for example adenovirus, adeno-associated virus, retrovirus or lentivirus vectors), phage vectors, cosmid vectors and the like.

In one embodiment, the expression construct for expression of the nucleotide sequence encoding a nucleic acid molecule of the invention is a viral gene therapy vector. As provided herein, viral vector methods can include the use of either DNA or RNA viral vectors. Examples of appropriate viral vectors can include adenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, herpes simplex virus (HSV), or murine Maloney-based viral vectors.

In one embodiment, the viral gene therapy vector is an AAV vector. Preferably the AAV vector that is used is an AAV vector of serotype 5. AAV of serotype 5 may be in particularly useful for transducing neurons as shown in the examples. The production of AAV vectors comprising any expression cassette of interest is well described in: W02007/046703, W02007/148971 , W02009/014445, W02009/104964, WO2011/122950, and WO2013/036118, 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.

Compositions

In another aspect, the invention relates to a composition comprising a nucleic acid molecule of the invention as herein defined above or a composition comprising a DNA construct encoding the nucleic acid molecule of the invention as herein defined above. Thus in one embodiment, the composition comprises as active ingredient a nucleic acid molecule of the invention as such, or a non-viral DNA construct encoding the nucleic acid molecule of the invention. In such compositions the active ingredient can suitably be formulated by encapsulation in nanoparticles such as liposomes and lipid particles. Liposomal suspensions, including targeted liposomes can be prepared according to methods known to those skilled in the art, for example, as described in US 4,522, 811 or US 2011305751 , incorporated herein by reference.

In another embodiment, the composition comprises as active ingredient a viral gene therapy vector for expression of the nucleic acid molecule of the invention. These can be prepared according to methods known to those skilled in the art.

In one embodiment, the composition is a pharmaceutical composition comprising in addition to the active ingredient (i.e. a nucleic acid molecule of the invention as such, a non-viral DNA construct encoding the nucleic acid molecule or a viral vector for expression of the nucleic acid molecule) at least one pharmaceutically acceptable carrier or excipient.

The term "pharmaceutically acceptable carrier", as used herein, is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration (see e.g. “Handbook of Pharmaceutical Excipients”, Rowe et al eds. 7 ^th edition, 2012, www.pharmpress.com). The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3- pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g. Zn-protein complexes); and/or nonionic surfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

Thus, in a particular embodiment, the pharmaceutical composition of the invention may be in a form suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in the appropriate unit dosage form. Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CremophorEM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol or sodium chloride in the composition.

Methods and therapeutic use

According to a further aspect of the invention, there is provided a method for enhancing protein translation of STXBP1 mRNA in a cell comprising administering to the cell a nucleic acid molecule of the invention, a DNA construct encoding the nucleic acid molecule or composition as defined herein. Preferably the cell is a mammalian cell, such as a human or a mouse cell.

According to a further aspect of the invention, there is provided a method for increasing the protein synthesis efficiency of STXBP1 in a cell comprising administering to the cell a nucleic acid molecule of the invention, a DNA construct encoding the nucleic acid molecule or composition as defined herein.

The methods described herein may comprise transfecting into a cell the nucleic acid molecule of the invention, the DNA construct encoding the nucleic acid molecule or expression construct as defined herein. The nucleic acid molecule of the invention, the DNA or expression construct can be administered to target cells using methods known in the art and include, for example, microinjection, lipofection, electroporation, using calcium phosphate, self-infection by the vector or transduction of a virus. The target cell to be treated may comprise a reduced amount of STXBP1. In one embodiment, the level of STXBP1 in the cell is lower than the level of STXBP1 in a normal cell (i.e. a cell comprising a normal phenotype with functional copies of the STXBP1 gene). For example, the level of STXBP1 in the cell may be less than 70% of the level of STXBP1 in a normal cell, such as less than 60% or less than 50% of the level of STXBP1 in a normal cell. In a further embodiment, the level of STXBP1 in the cell is about 50% of the level of STXBP1 in a normal cell.

In a further embodiment, the cell is STXBP1 haploinsufficient, i.e. wherein the presence of a variant allele in a heterozygous combination results in the amount of product generated by the single wild-type gene is not sufficient for complete or normal function. Generally, haploinsufficiency is a condition that arises when the normal phenotype requires the protein product of both alleles, and reduction to 50% or less of gene function results in an abnormal phenotype.

Methods of the invention result in increased levels of STXBP1 in a cell and therefore find use, for example, in methods of treatment for diseases which are associated with STXBP1 defects (i.e. reduced STXBP1 levels and/or loss-of-function mutations of the STXBP1 gene). Methods of the invention find particular use in diseases caused by a quantitative decrease in the predetermined, normal protein level. Methods of the invention can be performed in vitro, ex vivo or in vivo.

In one aspect the invention relates to a nucleic acid molecule of the invention, a DNA construct encoding the nucleic acid molecule, or a composition comprising the nucleic acid molecule or the DNA construct as defined herein, for use as a medicament.

In another aspect the invention pertains to a method of treating an STXBP1 -related disorder, the method comprising administering a therapeutically effective amount of a nucleic acid molecule of the invention, a DNA construct encoding the nucleic acid molecule, or a composition comprising the nucleic acid molecule or the DNA construct as defined herein.

Thus in yet another aspect, the invention relates to a nucleic acid molecule of the invention, a DNA construct encoding the nucleic acid molecule, or a composition comprising the nucleic acid molecule or the DNA construct as defined herein, for use in the prevention and/or treatment of an STXBP1 -related disorder.

In one embodiment, the STXBP1 -related disorder to be prevented and/or treated in accordance with the invention is disorder associated with STXBP1 haploinsufficiency.

In one embodiment, the STXBP1 -related disorder to be prevented and/or treated in accordance with the invention is an STXBP1 -related disorder associated with STXBP1 haploinsufficiency that is a severe early onset epileptic encephalopathy or a non-syndromic epilepsy.

In one embodiment, the severe early onset epileptic encephalopathy is one or more of Ohtahara syndrome, West syndrome, Lennox-Gastaut syndrome, Dravet syndrome, early myoclonic encephalopathy and an unclassified early onset epileptic encephalopathy that is associated with STXBP1 haploinsufficiency.

In one embodiment, the non-syndromic epilepsy is one or more of atypical Rett syndrome and severe intellectual disability without epilepsy associated with STXBP1 haploinsufficiency.

In some embodiments of the invention, the methods provided herein comprise direct administration of a composition comprising a nucleic acid molecule of the invention or a DNA construct encoding the nucleic acid molecule into a region in the nervous system (e.g., a region in the brain or spinal cord) of a subject. In some embodiments, the composition is delivered locally to a region in the nervous system. In one embodiment, the composition is administered to the subject by stereotaxic or convection enhanced delivery to a brain region (e.g., striatum). Using stereotaxic positioning system, one skilled in the art would be able to locate a specific brain region (e.g., striatum) that is to be administered with the composition. Such methods and devices can be readily used for the delivery of the composition as provided herein to a subject. In other embodiments, the composition is administered systemically to the subject, e.g. by intravenous injection.

In one embodiment, when using a gene therapy vector (e.g. an AAV vector) for expression of a molecule of the invention, the composition comprising the vector can be administered by direct infusion of a vector of the invention into the brain. Said direct infusion in further embodiments comprising an intrathecal infusion of the vector into the cerebrospinal fluid. Such an intrathecal infusion represents an efficient way to deliver the gene therapy vector to the CNS and to target the neurons. Preferably striatal and cortical structures are targeted via intrastriatal convection enhanced diffusion (CED) delivery of AAV vectors through injections into the striatum. More preferably, for a larger coverage of the CNS, injections are into the striatum and into the thalamus as well. Hence, gene therapy vectors of the invention are delivered intrastriatally, or delivered intrastriatally and intrathalamically through convection enhanced diffusion (CED) injections in the striatum, or the striatum and the thalamus. Such injections are preferably carried out through MRI- guided injections.

A composition provided herein can be administered to a subject in a dosage volume of about 0.0005, 0.001 , 0.002, 0.005, 0.01 , 0.02, 0.05, 0.1 , 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.7, 0.8, 0.9, 1 .0 ml_, or more. The composition can be administered as a 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more dose- course regimen. Sometimes, the composition can be administered as a 2, 3, or 4 dose-course regimen. Sometimes the composition can be administered as a 1 dose-course regimen.

The administration of the first and second dose (e.g. of an AAV vector encoding a nucleic acid molecule of the invention) of the 2 dose-course regimen can be separated by about 0 day, 1 day, 2 days, 5 days, 7 days, 14 days, 21 days, 30 days, 2 months, 4 months, 6 months, 9 months, 1 year, 1.5 years, 2 years, 3 years, 4 years, 5 years, 10 years, 20 years, or more. A composition described herein can be administered to a subject once a day, once a week, once two weeks, once a month, a year, twice a year, three times a year, every 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, or more years.

Sometimes, the composition can be administered to a subject every 2, 3, 4, 5, 6, 7, or more years. Sometimes, the composition can be administered to a subject once.

The instant invention provides two main advantage over other approaches for the compensation of STXBP1 haploinsufficiency. First, this invention ensures selective upregulation of STXBP1 expression. While other approaches currently underway can also increase expression of haploinsufficiency genes (e.g. viral vector whole gene therapy), the nucleic acid molecules of the invention upregulate expression exclusively where and when the STXBP1 gene is naturally active. Second, the instant invention requires shorter DNA fragments to be introduced into haploinsufficient cells than other approaches currently underway wherein the entire STXBP1 gene or cDNA is to be delivered. This greatly facilitates delivery opportunities.

The present invention has been described above with reference to a number of exemplary embodiments as shown in the drawings. Modifications and alternative implementations of some parts or elements are possible, and are included in the scope of protection as defined in the appended claims.

Description of the figures

Figure 1. STXBPI-SINEUPs increase STXBP1 protein levels in wild-type human neurons.

A. Schematic presentation of STXBPI-SINEUPs and STXBP1 mRNA. B. Schematic presentation of human iNeuron induction and culture protocol. Neurons were infected with lentivirus expressing STXBP1-SINEUP at DIV18 and harvested at DIV28.

C. Example image of iNeurons at DIV28 stained for STXBP1 (green), the dendritic marker MAP2 (blue) and mCherry (Red). D. Western blot showing technical duplicates of cell lysates from neurons infected with DBD, STXBP1- SINEUP_002 or STXBP1-SINEUP_003 at DIV18 and harvested at DIV28. g-tubulin and mCherry were used as loading and infection controls, respectively.

E. STXBP1 expression levels expressed as fold-change compared to neurons expressing DBD and normalized to y-tubulin (n=4). Data are plotted as mean + SEM. (_002 1.65 + 0.28; _003 1.56 + 0.21 -fold ** p < 0.01 , oneway ANOVA.

F. Average mRNA levels for the effector domain and STXBP1 assessed using qPCR shows similar mRNA levels (n=4).

Figure 2. STXBPI-SINEUPs increase STXBP1 protein levels in human cell models for STXBP1 haploinsufficiency.

A. STXBP1 protein levels in isogenic wild type (WT), STXBP1+/- (HZ) and S241fs and D207G mutant iNeurons.

B. STXBPI-SINEUPs increase STXBP1 protein levels in STXBP1 HZ iNeurons compared to non- infected (Nl) or DBD-expressing iNeurons. (_002 1 .73 + 0.30; _003 1 .74 + 0.50-fold ** p < 0.01 , * p < 0.05, n = 3).

C. STXBPI-SINEUPs increase STXBP1 protein levels in S241fs iNeurons compared to DBD- expressing S241fs iNeurons (_002 2.59 + 0.69; _003 1.53 + 0.36-fold ** p < 0.01 , * p < 0.05, n = 4).

D. STXBPI-SINEUPs increase STXBP1 protein levels in D207G iNeurons compared to DBD- expressing D207G iNeurons (_002 1 .73 + 0.1 ; _003 1 .24 + 0.15-fold ** p < 0.01 , ns p > 0.05, n =

4).

Figure 3. STXBP1-SINEUP RNA expressed from three different promoters (U6, H1 and CMV, as indicated) and subcellular localization of the expressed RNAs (Cyt = cytosol; Nucl = nucleus).

Examples Example 1

1. Materials and Methods 1.1 STXBP1 SINEUP sequences

Table 1. Overview of STXBP1 -SINEUP BD overlap with human STXBP1 (top) and STXBP1- SINEUP BD sequence (bottom). Start codon in bold. Adenosine (a) of start codon is +1.

1.2 Generation of STXBP1-SINEUP vectors

For expression in human neurons STXBP1-SINEUP targeting sequences 002 and 003 (SEQ ID No.’s: 57 and 58, resp.) and DBD sequences (Table 1) with SINEB2 effector domains (Fig. 1A, SEQ ID No.’s: 59 and 60, resp.) were cloned into pLL3.7 Lenti vectors downstream of a U6 RNA promoter. These vectors also expressed mCherry downstream of a human synapsin promoter to quantify infection efficiency.

1.3 Generation of human iPSC-derived neurons

Gene-edited iPSC line BIONi010-C-13 with a doxycycline inducible NGN2 cassette in the AAVS1 safe harbour locus was purchased from EBiSC (www.ebisc.org). Mutations in STXBP1 were engineered using CRISPR-cas9 technology generating STXBP1+/- and two disease-mutant (S241fs, D207G) iPSCs on isogenic background. iPSCs were cultured in Essential 8 medium (StemCell) on geltrex (ThermoFischer) coating and passaged using Trypsin/EDTA. For differentiation of human neurons, 1 x 10 ⁶ iPSCs were dissociated with accutase cell detachment solution (Stemcell technologies) and seeded on geltrex-coated 10 cm petri dishes in Essential 8 (ThermoFisher A1517001) supplemented with Rock inhibitor Y27632 (TetuBio; T1725). One day after seeding, the medium was switched to N2 medium (DMEM/F12 ThermoFisher 10565018, 1% N2 supplement; ThermoFisher 17502048) supplemented with doxycycline to induce NGN2 expression and refreshed every day for 4 days. On day 5, the cells were dissociated with accutase and re-plated on PO/laminin-coated 6 wells plates with NB/B27 medium (neurobasal medium (ThermoFisher 21103049), 2% B27 supplement ThermoFisher 0080085SA), 1 ml GlutaMax (Thermofisher 35050061), 1-2 pg/ml laminin, 10ng/ml BDNF (Preprotech 450-02), GDNF (Preprotech 450-10), CTNF (Stem Cell Tech 78010.1) supplemented with doxycycline (Sigma D9891). Neurons were refreshed 2 times a week (half medium).

1.4 Infection of human NGN2-neurons

NGN2-neurons were plated onto 6 well plates coated with polyornithine-laminin (2 x 10 ⁵ cells per well) and infected at 18 DIV with the appropriate SINEUP-expressing lentiviral particles (20 ul per well). Half of the medium was refreshed every 72h and cells were collected 10 days after infection. RNA and protein were obtained from the same infection in each replica. 1.5 Western blot

Human NGN2-neurons were collected 10 days post infection by scraping in PBS with protease inhibitor E-64d (E8640, Sigma-Aldrich). After 5-min centrifugation at 3000 RCF, cell pellets were dissolved in 70 pi 1X Laemmli sample buffer, homogenized by passage through a 20-gauge needle, boiled and loaded 10 pl/each sample on 10% SDS-PAGE gel. Proteins were transferred to nitrocellulose membrane 0.2 pm (#1620112, Bio-Rad) using Trans-Blot ^R Turbo™ Transfer System, with High MW protocol (2.5 A, up to 25 V; 10 min). Membranes were blocked with 2% protease- free Bovine Serum Albumin (268131000, ACROS Organics™) in PBS-0.05% Tween solution for 1 h at RT and incubated with primary antibodies, diluted in the blocking buffer. The following antibodies were used: Rabbit STXBP1 1 :5000 (HPA023483, Sigma-Aldrich) for 2 hours at RT, followed by 30 minutes incubation at RT with IRDyeR 680LT IRDyeR 800CW Goat anti-Rabbit IgG 1 :5000 (926- 32211 , LI-COR) secondary antibodies; Monoclonal Mouse Anti-g-Tubulin 1 :1000 (T5326, Sigma- Aldrich), overnight at 4°C, followed by 30 minutes incubation at room temperature with IRDyeR 680LT Goat anti-Mouse IgG secondary antibody 1 :5000 (926-68020, LI-COR); Polyclonal Rabbit Anti-mCherry 1 :10000 (GTX128508, GeneTex), followed by 30 minutes incubation at RT with IRDyeR 800CW Goat anti-Rabbit IgG 1 :5000 (926-32211 , LI-COR) secondary antibody. Western blot images were acquired using LI-COR Odyssey Fc according to the manufacturer’s instructions. Densitometric analysis was performed using Image Studio™ Software. 1.6 RNA isolation, Reverse Transcription (RT) and quantitative RT-PCR (qRT-PCR)

Total RNA was extracted from 1/3 of cell pellets from each well using RNeasy Micro Kit (74004, Qiagen), including DNAse treatment to avoid DNA contamination, following manufacturer's instructions. A total of 50 ng RNA was subjected to reverse-transcription using iScript™cDNA Synthesis Kit (Bio-Rad, Cat. No. 1708890), according to manufacturer's instructions. qRT-PCR was carried out using SYBR green fluorescent dye (iQ SYBR Green Super Mix, Bio-Rad, Cat. No. 1708884) real-time thermal cycler (CFX-96, Bio-Rad) and the primers in Table 2. The reactions were performed on diluted cDNA (1 :5). Relative expression was calculated using the 2-AACt method (Pfaffl, 2001 , Nucleic Acids Res. 29(9):e45. doi: 10.1093/nar/29.9.e45) by normalizing data to the geometric mean of housekeeping transcripts (GAPDH and RLP13A) using the CFX Manager 3.0 software (BioRad).

Table 2. STXBP1 qPCR primers and effector domain qPCR primers.

1.7 Statistical analysis In all experiments, the significance of differences between groups was evaluated by one-way ANOVA followed by Dunnett's post-test. Quantitative data are presented as mean + SEM of three or four independent experiments.

1.8 Subcellular fractionation and RNA extraction

For subcellular fractionation and RNA preparation 250,000 iNeurons were collected at DIV42 by centrifugation and washed once with PBS. The cell pellet was resuspended in 100 ul of ice-cold NP-40 lysis buffer (10 mM Tris-HCI pH 7.5, 0.15% NP40, 150 mM NaCI). The lysate was layered on 2.5 volumes of a sucrose buffer and centrifuged for 10 min at 13,000 rpm at 4°C. The supernatant (cytoplasmic fraction) was collected and supplemented with 350 ul of RLY Buffer with beta- mercapto-ethanol (BioLine). The nuclei pellet was gently rinsed with ice-cold 1X PBS and resuspended in 200pl ice-cold glycerol buffer (20 mM Tris-HCI pH 7.9, 75 mM NaCI, 0.5 mM EDTA, 0.85 mM DTT, 0.125 mM PMSF, 50% glycerol) by gently flicking the tube. An equal volume of ice- cold nuclei lysis buffer (10 mM HEPES pH 7.6, 1 mM DTT, 7.5 mM MgCI2, 0.2 mM EDTA, 0.3 M NaCI, 1 M UREA, 1 % NP-40) was added and gently vortexed twice for 2 sec, incubated for 2 min on ice, and supplemented with 350 ul of RLY Buffer with beta-mercapto-ethanol (BioLine). RNA purification from RLY-dissolved samples was performed using Isolate II RNA Mini Kit (Bioline). 1.9 RT-PCR on subcellular fractions

About 100 ng of total RNA was used to synthetize the first-strand cDNA using iScript cDNA Synthesis Kit. The gene expression levels were detected by using SYBR Green qPCR Mastermix and performing real-time PCR on the QuantStudio Real-time PCR system.

Differential expression was determined by the 2-DDCT method using GAPDH, RLP13A and beta-actin as the internal control for cytoplasmic fraction, and GAPDH, U6 and U2snRNA for the nucleoplasm fraction.

2. Results

STXBP1 syndrome is a rare early onset neurodevelopmental disorder caused by de novo heterozygous mutations in the STXBP1 gene and characterized by (severe) developmental delay, intellectual disability, epilepsy and spasms. The heterozygous mutations lead to a reduced cellular protein level, which are not enough for normal brain development (haploinsufficiency). The invention described here compensates for the reduced expression of STXBP1 protein and restores normal cellular STXBP1 levels and can be used as a therapeutic approach to treat STXBP1 haploinsufficiency.

The invention uses short interspersed nuclear elements (SINE) domains derived from natural long noncoding RNAs (Carrieri et al., 2012, Nature 15;491 (7424):454-7; Vassetzky and Kramerov 2013, Nucleic Acids Res.41 (Database issue):D83-9. doi: 10.1093/nar/gks1263) linked to binding domains designed for complementarity to STXBP1 mRNA for compensation of STXBP1 haploinsufficiency. 2.1 Design of STXBP1 SINEUP sequences

Based on 5' UTR and transcription start site (TSS) sequence for human STXBP1 , identified using FANTOM5 (https ://fantom.gsc.riken.j p/5/) and Zenbu Genome Browser, two SINEUPs with different binding domains (BDs) for STXBP1 mRNA were generated: STXBP1_002 BD -40/+4, STXBP1_003 BD -14/+4, antisense to STXBP1 mRNA, and cloned into expression vectors. As negative control, a SINEUP lacking the BD region (SINEUP-ABD), was used.

2.2 STXBP1 -SINEUPs increase STXBP1 protein levels in human IPSC-derived neurons Two short STXBP1-SINEUP constructs (stxbp1_002; -40/+4) and (stxbp1_003; -14/+4) were selected for the evaluation of STXBP1 protein upregulation in human induced pluripotent stem cell (IPSC)-derived neurons (iNeurons). STXBP1 -SINEUP and DBD constructs were cloned into a modified pLL3.7 lentiviral vector expressing the SINEUP downstream of a U6 promoter and mCherry from a synapsin promoter. mCherry co-expression was used to assess infection efficiency and to quantify STXBP1 protein levels in the infected cells, expressing mCherry. iNeurons were infected with lentiviral particles, titrated to infect >90% of neurons, at DIV18 and harvested at DIV28 (Fig. 1 A-C). STXBP1 protein levels in STXBP1-SINEUP_002 and _003 expressing neurons were upregulated 1 .65 and 1 .56-fold, respectively, compared with DBD expressing neurons (Fig. 1 D,E). STXBP1 -SINEUP infection did not change STXBP1 mRNA levels, indicating that STXBP1- SINEUPs increase protein levels post-transcriptionally (Fig. 1 F). Together these data show that STXBP1 -SINEUPs efficiently increase STXBP1 protein levels in human iNeurons.

2.3 STXBP1 -SINEUPs increase STXBP1 protein levels in human cell models for STXBP1 haploinsufficiency STXBP1 syndrome is a rare early onset neurodevelopmental disorder caused by de novo heterozygous mutations in the STXBP1 gene. The heterozygous mutations lead to a reduced cellular protein level, which are not enough for normal brain development (haploinsufficiency). To test if STXBP1 -SINEUPS also increase STXBP1 protein levels in neurons with reduced levels of STXBP1 , SINEUPs were infected in STXBP1 heterozygote (STXBP1 +/-) iNeurons and two isogenic lines with CRISPR/Cas9-generated disease mutants (S241fs and D207G) with + 50% reduced STXBP1 levels compared to wild type (Fig. 2A). Using a similar strategy as described for wild-type iNeurons, STXBP1 levels were measured using Western blot ten days post-infection with STXBP1- SINEUP_002 and _003. STXBP1 -SINEUPs increased STXBP1 expression 1.5 to 2.2-fold compared to expression levels in non-infected (Nl) or DBD expressing HZ or disease-mutant iNeurons (Fig. 2B-D). STXBP1-SINEUP_002 was more efficient compared to STXBP1- SINEUP_003 in increasing STXBP1 protein levels in disease-mutant iNeurons (Fig. 2C,D). Hence, STXBP1 -SINEUPs efficiently increase STXBP1 protein levels in human cell models for STXBP1 haploinsufficiency. 2.4 Sineup RNA expression and localization To test the efficiency of different RNA promoters, SINEUP vectors were generated driven by U6, H1 and CMV RNA promoters. Human iNeurons were infected at DIV24 with lentiviral particles expressing these SINEUP vectors and cells harvested ten days post infection. Nuclei and cytoplasmic fraction were collected and subjected to qPCR analysis using SINEUP-specific primers. All three promoters efficiently supported RNA expression (Figure 3). SINEUP RNA was present in both the nuclear fraction and cytosolic fraction confirming efficient nuclear export of SINEUP RNA. All promoters were equally efficient in expressing (range 1.52-2.01 fold compared to controls) and targeting SINEUP RNA.