Gene therapy

Abstract

The invention relates to guide polynucleotides and methods for targeting and editing a portion of the 5' UTR-encoding region of genes encoding VGSC alpha subunits (NaV) to abrogate or create an upstream open reading frame (uORF). The invention relates to guide polynucleotides and methods for targeting and editing a portion of the 5' splice acceptor site (SA) of an exon of a gene encoding a voltage-gated sodium channel (VGSC) alpha subunit (NaV). The invention also relates to use of the guide polynucleotides and methods for treating genetic disorders, in particular Dravet syndrome.

Description

[0001] GENE THERAPY

[0002] FIELD OF THE INVENTION

[0003] The invention relates to guide polynucleotides for targeting a portion of the 5’ UTR-encoding region of genes encoding voltage-gated sodium channel (VGSC) alpha subunits (Nav proteins). The invention also relates to editing systems and methods for editing the 5’ UTR- encoding region of genes encoding VGSC alpha subunits (Nav proteins) to abrogate or create upstream open reading frames (uORFs). In particular, the invention relates to guide polynucleotides for use in treating genetic disorders, particularly Dravet syndrome.

[0004] BACKGROUND TO THE INVENTION

[0005] Developmental and Epileptic Encephalopathies (DEE) are a group of severe genetic disorders that begin early in life, characterized by both seizures (often drug-resistant) and developmental impairment or regression. In these disorders, developmental outcomes can worsen as a consequence of seizure frequency and severity, but their primary cause is the underlying genetic defect. This suggests that efficient antiseizure medications can improve seizure control, but may have a limited impact on the developmental delay and that precision medicine approaches, removing the same causes of the diseases, need to be pursued to ameliorate both aspects. This possibility has been favored by the explosion of gene discovery: the advent of genome-wide screening technologies has dramatically increased the number of candidate genes for DEEs, explaining 20-25% of all cases with severe early-onset epilepsies that had otherwise no identifiable cause.

[0006] Variants in SC / V genes (SCN1 / 2 / 3 / 8) have been associated with the most severe DEEs. SCN genes encode for alpha subunits of voltage gated sodium channels (VGSCs), representing the main actors in the regulation of neuronal excitability.

[0007] Considering the relevant role exerted by VGSC for cell physiology, even loss of function (LOF) variants in a single allele of SCN genes dramatically affects channel activity and provokes severe defects at cellular level. The final outcome can be a spectrum of neurological disorders including DEEs (SCN1 / 2 / 3 / 8A), but also cardiac dysfunctions (SC / V5A), peripheral neuropathies (SCN9 / 10 / 11A) and skeletal muscle channelopathies (SCN4A).

[0008] Gene therapy is now becoming a real possibility for different genetic neuro-developmental disorders, including DEEs, thanks to the advancements in the generation of new adeno- associated viral vectors (AAV) tools and CRISPR / Cas9-based base editing tools. Different viral and non-viral gene / genetic therapeutic technologies are in preclinical phase for DEEs and specifically for DS. Some exploit high-cargo viral vectors to deliver a copy of Senia functional gene; others are based on the enhancement of Senia gene expression to boost the wild-type allele of the gene to rescue its haploinsufficiency (Mora-Jimenez et al. (2021) Mol Ther - Nucleic Acids. 25:585-602). Most of the strategies developed to rescue Senia haploinsufficiency are based on boosting gene transcription (Hsiao et al. (2016) EBioMedicine. 9:257-277; Colasante et al. dCas9-Based Senia Gene Activation Restores Inhibitory Interneuron Excitability and Attenuates Seizures in Dravet Syndrome Mice. Mol Ther. Published online 2019. doi:10.1016 / j.ymthe.2019.08.018; Yamagata et al. (2020) Neurobiol Dis. 141 ; Tanenhaus et al. (2022) Hum Gene Ther. 33:579-597) and enhancing the splicing of productive Senia mRNA (Lim et al. (2020) Nat Commun. 11 (1). doi:10.1038 / s41467-020- 17093-9; Han et al. (2020) Sei Transl Med. 12(558)). This second approach, already in clinical trial on DS patients, exploits antisense oligonucleotides (ASOs) to avoid the incorporation of a “poison exon” which normally addresses Senia mRNA to non-sense mediated decay (Carvill et al. (2018) Am J Hum Genet. 103:1022-1029). This post-transcriptional regulation has the main advantage that it increases Senia gene expression only in cell types in which Senia mRNA is already transcribed, while transcriptional enhancement approaches carry the risk of ectopic expression. However, in contrast to CRISPR / Cas9-mediated gene delivery, ASO- mediated splice-switching therapies require life-long administration to achieve a continued treatment effect.

[0009] Gene expression is a tightly regulated process and modifying it requires forcing changes in its regulative mechanisms. The second main event in gene expression, mRNA translation, is modulated by cis-acting elements in the 5' untranslated region (UTR) of the mRNA. One such element is the nucleotide sequence flanking the initiation codon (AUG), referred to as Kozak sequence. Sequence variability is present around AUG, giving rise to a plethora of suboptimal Kozak sequences able to influence the efficiency of protein translational. Kozak sequence optimization to efficiently tune protein translation has been recently exploited for different haploinsufficiency genes (Ambrosini et al. (2022) Nucleic Acids Res.50(18): 10756-10771). However, the effect can be quite limited or, in case where an AUG is already flanked by an optimal Kozak sequence, even entirely absent. Additionally, since this strategy targets codons both upstream and downstream of the AUG, it can introduce changes in N-terminal amino acids of the translated protein, affecting protein stability (Ambrosini (2022) Nucleic Acids Res.50(18): 10756-10771).

[0010] Thus, there remains a need for an approach which can effectively modify gene expression by altering mRNA translation without requiring continuous, life-long administration.

[0011] SUMMARY OF THE INVENTION The present applicants hypothesised that other elements in the 5’ UTR-encoding region of a gene can be interfered with in order to increase basal expression levels of said gene, particularly genes causing severe disorders. In particular, present applicants hypothesised that editing regulatory sequences in the 5’ UTR-encoding region of genes, such as SC / V gene family, could boost protein translation (of proteins such as Nav1.1 to Nav1.9).

[0012] Using a reporter system screening platform, the present applicants identified regulatory elements in the 5’ UTR of SCN1A / Scn7a mRNA impacting its translation. They tested different variants of SCN1A / Scn7a 5’ UTR harbouring distinct combinations of nucleotide conversions and identified variants which were able to significantly increase expression of a downstream gene in the reporter system over the expression levels observed with the unmodified SCN1A / Scn1a 5’ UTR. The applicants discovered that in all variants capable of increasing protein expression, the start codon (uATG) of an upstream open reading frame (uORF) was mutated.

[0013] Without wishing to be bound by theory, the applicants concluded that the observed increased protein expression was the result of the abolition of a uORF resulting from mutation of the ATG, benefiting translation from the ATG (mATG) of the main open reading frame (mORF).

[0014] The applicants subsequently hypothesised that changes to the SCN1A / Scn1a 5’ UTR could be made permanent by editing the 5’ UTR-encoding region of the SCN1A / Scn1a gene. To achieve this, the applicants used a novel gene therapy strategy exploiting permanent CRISPR- Cas9 base editing to boost protein translation of Navi .1 . Guide polynucleotides were designed to target sequences comprising uATGs of an identified uORF and adenine base editors were used to introduce A«T to G*C alterations in the uATG, abolishing the uORF. The applicants thus for the first time provide a system for permanently increasing Nav1.1 protein expression, which does not affect mRNA levels and does not require repeated or recurrent, life-long administration.

[0015] Additionally, the applicants were able to mutate more than one uATG within the target sequence of a single guide polynucleotide, abolishing more than one uORF and further improving protein expression. The applicants achieved this by precise, non-random editing of genomic DNA, avoiding common drawbacks of conventional CRISPR-Cas9 technology such as the introduction of small insertions / deletions (indels) or larger chromosomal modification / rearrangement.

[0016] Finally, the applicants provide evidence that they were able to edit post-mitotic cells with comparable efficacy to mitotic (e.g. HEK-293T) cells. Loss of function mutations in the SCN1A gene particularly impairs neural networks, resulting in the above-described neurological conditions. The present applicants set out to establish whether the endogenous Senia gene locus in mouse primary neurons, which are post-mitotic cells, could also be edited to mutate one or more uATGs. Surprisingly, the applicants found that they were able to mutate uATGs in post-mitotic cells with similar efficiency as mitotic cells.

[0017] The present applicants further found that the same regulative mechanism is conserved also for other SON genes, for example SCN2A, SCN3A, SCN5A, SCN8A, and that mutagenesis of the corresponding regions in their 5’UTR-encoding regions efficiently promoted gene expression in the reporter system. Consequently, the applicants designed and tested guide polynucleotides targeting a sequence located partially or fully in the 5’ UTR-encoding region of the endogenous Scn2a and Scn8a loci in mouse primary neurons and were able to mutate the conserved uATGs, abolishing the corresponding uORFs and enhancing Scn2a or Scn8a gene expression.

[0018] Overall, the present applicants provide evidence that protein levels of Nav1.1 and other VGSC alpha subunits (Nav proteins) can be augmented by introducing one or more nucleotide conversions in translation-regulative elements within the 5’ UTR-encoding region of the respective gene. This strategy represents a new way of rescuing haploinsufficiency in patients and offers a number of major advantages. Firstly, editing at the gene level removes the need for life-long, repeated / recurrent administration. Secondly, interspecies conservation of uORFs (e.g. between the uORFs of human SCN1A and mouse Senia) allows testing and application of gene editing in different organisms, accelerating application of this technology in the clinic. Thirdly, the gene editing strategy can be combined with other DNA / mRNA editing strategies, e.g. transcriptional or splicing enhancing therapies. Finally, while this technology is currently applied for gene expression enhancement and therefore to rescue gene haploinsufficiency for LOF mutations, the applicants also envisage using the gene editing strategy to introduce uORFs in the 5’ UTR-encoding region of a gene to reduce gene expression in the case of gain of function (GOF) mutations. This could, for example, be achieved using base editors to either introduce a C*G to T«A or A«T to G*C alteration to create one or more start codons upstream of the mATG (e.g. ATA or CTG trinucleotide modified to ATG by adenine or cytosine base editors, respectively).

[0019] Accordingly, one aspect of the invention discloses a guide polynucleotide comprising a sequence which binds to a target sequence comprising a portion of the 5’ UTR-encoding region of a gene encoding a voltage-gated sodium channel (VGSC) alpha subunit (Nav).

[0020] In preferred embodiments, the guide polynucleotide is a guide RNA. In some embodiments, the guide polynucleotide binds to a base editor or prime editor. In some embodiments, the guide polynucleotide binds to a base editor. In some embodiments, the base editor is an adenine base editor. In some embodiments, the base editor is a cytosine base editor. In some embodiments, the adenine base editor is ABE8e, preferably SpCas9- ABE8e or SpRYCas9-ABE8e, even more preferably SpCas9-ABE8e. In some embodiments, the adenine base editor is ABEmax, preferably SpRYCas9-ABEmax or SpCas9-ABEmax, even more preferably SpRYCas9-ABEmax.

[0021] In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is selected from any one of human SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN7A, SCN8A, SCN9A, SCN10A and SCN11A, or any one of mouse Senia, Scn2a, Scn3a, Scn4a, Scn5a, Scn7a, Scn8a, Scn9a, Scn10a and Scn11a. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is selected from any one of human SCN1A, SCN2A, SCN3A, SCN5A, SCN8A and SCN9A, or any one of mouse Senia, Scn2a, Scn3a, Scn5a, Scn8a and Scn9a, preferably any one of human SCN1A, SCN2A, SCN3A, SCN5A, SCN8A and SCN9A, even more preferably any one of human SCN1A, SCN2A and SCN8A. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is human SCN1A. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is human SCN2A. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is human SCN8a. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is mouse Senia. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is mouse Scn2a. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is mouse Scn8a.

[0022] In some embodiments, the target sequence comprises at least one start codon (uATG) of an upstream open reading frame (uORF). In some embodiments, the target sequence comprises at least two uATGs. In some embodiments, the at least two uATGs are in frame with each other. In some embodiments, the target sequence comprises at least three uATGs. In some embodiments, at least two of the at least three uATGs are in frame with each other. In some embodiments, the target sequence comprises two uATGs. In some embodiments, the two uATGs are in frame with each other. In some embodiments, the target sequence comprises three uATGs. In some embodiments, at least two of the at least three uATGs are in frame with each other.

[0023] In some embodiments, the sequence that binds to the target sequence is at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably 95%, most preferably 100% complementary to the target sequence. In some embodiments, the sequence that binds to the target sequence is 17-24 nucleotides long. In some embodiments, the sequence that binds to the target sequence is 18-22 nucleotides long. In some embodiments, the sequence that binds to the target sequence is

[0024] 19-21 nucleotides long. In some embodiments, the sequence that binds the target sequence is 17, 18, 19, 20, 21 , 22, 23 or 24 nucleotides long. In some embodiments, the sequence that binds the target sequence is at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23 or at least 24 nucleotides long. In some embodiments, the sequence that binds the target sequence is 18 nucleotides long. In some embodiments, the sequence that binds the target sequence is 19 nucleotides long. In some embodiments, the sequence that binds the target sequence is 20 nucleotides long. In some embodiments, the sequence that binds the target sequence is 21 nucleotides long. In some embodiments, the sequence that binds the target sequence is 22 nucleotides long.

[0025] In some embodiments, the sequence that binds the target sequence is the sequence set forth in any one of SEQ ID NOs: 15-28. In some embodiments, the sequence that binds the target sequence is the sequence of SEQ ID NO: 15 or SEQ ID NO: 16. In some embodiments, the sequence that binds the target sequence is the sequence set forth in any one of SEQ ID NOs: 17-22. In some embodiments, the sequence that binds the target sequence is the sequence set forth in any one of SEQ ID NOs: 23-28. In some embodiments, the sequence that binds the target sequence is the sequence set forth in SEQ ID NO: 15. In some embodiments, the sequence that binds the target sequence is the sequence set forth in SEQ ID NO: 16. In some embodiments, the sequence that binds the target sequence is the sequence set forth in SEQ ID NO: 17. In some embodiments, the sequence that binds the target sequence is the sequence set forth in SEQ ID NO: 18. In some embodiments, the sequence that binds the target sequence is the sequence set forth in SEQ ID NO: 24.

[0026] In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is human SCN1A and the sequence that binds the target sequence is the sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is mouse Senia and the sequence that binds the target sequence is the sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is human SCN2A and the sequence that binds the target sequence is the sequence set forth in SEQ ID NO: 17, SEQ ID NO: 18 or SEQ ID NO: 21. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is mouse Scn2a and the sequence that binds the target sequence is the sequence set forth in any one of SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO: 22. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is human SCN8A and the sequence that binds the target sequence is the sequence set forth in SEQ ID NO: 23, SEQ ID NO: 25 or SEQ ID NO: 27. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is mouse Scn8a and the sequence that binds the target sequence is the sequence set forth in SEQ ID NO: 24, SEQ ID NO: 26 or SEQ ID NO: 28.

[0027] In some embodiments, the target sequence is located between position -30 and position 10 in the gene encoding the VGSC alpha subunit (Nav) (position -1 is the position of the nucleotide immediately 5’ of the first nucleotide of the main open reading frame (mORF) and position 1 is the position of the first nucleotide of the mORF (the first nucleotide of the mORF is the first nucleotide of the start codon (mATG) of the mORF)). In some embodiments, the target sequence is located between position -30 and position 4. In some embodiments, the target sequence is located between position -30 and position 3. In some embodiments, the target sequence is located between position -30 and position 2. In some embodiments, the target sequence is located between position -30 and position 1. In some embodiments, the target sequence is located between position -18 and position 2. In some embodiments, the target sequence is located between position -19 and position 1. In some embodiments, the target sequence is located between position -14 and position 6. In some embodiments, the target sequence is located between position -13 and position 7. In some embodiments, the target sequence is located between position -10 and position 10. In some embodiments, the target sequence is located between position -11 and position 9. In some embodiments, the target sequence is located between position -19 and position 1. In some embodiments, the target sequence is located between position -13 and position 7. In some embodiments, the target sequence is located entirely in the 5’ UTR-encoding region of the gene encoding the VGSC alpha subunit (Nav).

[0028] In some embodiments, the target sequence is located between position -50 and position 10 in the gene encoding the VGSC alpha subunit (Nav). In some embodiments, the target sequence is located between position -45 and position 10 in the gene encoding the VGSC alpha subunit (Nav). In some embodiments, the target sequence is located between position -40 and position 10 in the gene encoding the VGSC alpha subunit (Nav). In some embodiments, the target sequence is located between position -35 and position 10 in the gene encoding the VGSC alpha subunit (Nav).

[0029] In some embodiments, the target sequence is located between position -50 and position 4 in the gene encoding the VGSC alpha subunit (Nav). In some embodiments, the target sequence is located between position -45 and position 4 in the gene encoding the VGSC alpha subunit (Nav). In some embodiments, the target sequence is located between position -40 and position 4 in the gene encoding the VGSC alpha subunit (Nav). In some embodiments, the target sequence is located between position -35 and position 4 in the gene encoding the VGSC alpha subunit (Nav).

[0030] In some embodiments, the target sequence is the sequence set forth in any one of SEQ ID

[0031] NOs: 1-14. In some embodiments, the target sequence is the sequence set forth in SEQ ID

[0032] NO: 1 or SEQ ID NO: 2. In some embodiment, the target sequence is the sequence set forth in any one of SEQ ID NOs: 3-8. In some embodiments, the target sequence is the sequence set forth in any one of SEQ ID NOs: 9-14. In some embodiments, the target sequence is the sequence set forth in SEQ ID NO: 1. In some embodiments, the target sequence is the sequence set forth in SEQ ID NO: 2. In some embodiments, the target sequence is the sequence set forth in SEQ ID NO: 3. In some embodiments, the target sequence is the sequence set forth in SEQ ID NO: 4. In some embodiments, the target sequence is the sequence set forth in SEQ ID NO: 10.

[0033] In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is human SCN1A and the target sequence is the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is mouse Senia and the target sequence is the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is human SCN2A and the target sequence is the sequence set forth in SEQ ID NO: 3, SEQ ID NO: 4 or SEQ ID NO: 7. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is mouse Scn2a and the target sequence is the sequence set forth in SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 8. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is human SCN8A and the target sequence is the sequence set forth in SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is mouse Scn8a and the target sequence is the sequence set forth in SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14.

[0034] The guide polynucleotide of the present invention may be suitable for use in a system for editing a 5’ UTR-encoding region of a gene encoding a VGSC alpha subunit (Nav). Editing of a region of an endogenous gene locus in a eukaryotic cell may require introduction of the guide polynucleotide into the cell. The guide polynucleotide can be directly introduced into the cell using an appropriate carrier or introduced in the form of a nucleic acid sequence encoding the guide polynucleotide and from which the guide polynucleotide is expressed after introduction of the nucleic acid into the cell.

[0035] Another aspect of the invention discloses a nucleic acid encoding the guide polynucleotide of the invention. Another aspect of the present invention provides a vector comprising the nucleic acid sequence of the present invention.

[0036] In some embodiments, the vector is a mammalian cell expression vector, a viral vector or a naked DNA vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector, such as an AAV6 vector. In some embodiments, the vector is a lentiviral (LV) vector, such as an integration-defective lentiviral (IDLV) vector.

[0037] Editing of the 5’ UTR-encoding region of a gene encoding a VGSC alpha subunit (Nav) may use a guide polynucleotide and an enzyme catalysing the editing reaction of the 5’ UTR- encoding region.

[0038] Another aspect of the present invention provides an editing system comprising (a) a guide polynucleotide or vector comprising a nucleic acid encoding a guide polynucleotide, and (b) a base editor or prime editor, polynucleotide encoding a base editor or prime editor or a vector comprising a nucleic acid encoding a base editor or portion thereof or a prime editor or portion thereof.

[0039] Another aspect of the present invention provides an editing system comprising (a) a guide polynucleotide or vector comprising a nucleic acid encoding a guide polynucleotide, and (b) a base editor, polynucleotide encoding a base editor or a vector comprising a nucleic acid encoding a base editor or portion thereof.

[0040] In some embodiments, the editing system comprises a guide polynucleotide. In some embodiments, the editing system comprises the guide polynucleotide of the present invention. In some embodiments, the editing system comprises a vector comprising a nucleic acid encoding a guide polynucleotide. In some embodiments, the editing system comprises a vector comprising a nucleic acid encoding a guide polynucleotide of the present invention. In some embodiments, the editing system comprises a base editor or prime editor. In some embodiments, the editing system comprises a base editor. In some embodiments, the editing system comprises a vector comprising a nucleic acid encoding a base editor or portion thereof or a prime editor or portion thereof. In some embodiments, the editing system comprises a vector comprising a nucleic acid encoding a base editor or portion thereof. In some embodiments, the editing system comprises a polynucleotide encoding a base editor or prime editor. In some embodiments, the editing system comprises a polynucleotide encoding a base editor.

[0041] In some embodiments, the editing system comprises a guide polynucleotide and a base editor or prime editor. In some embodiments, the editing system comprises a guide polynucleotide and a base editor. In some embodiments, the editing system comprises a guide polynucleotide of the present invention and a base editor or prime editor. In some embodiments, the editing system comprises a guide polynucleotide of the present invention and a base editor. In some embodiments, the editing system comprises a vector comprising a nucleic acid encoding a guide polynucleotide and a base editor or prime editor. In some embodiments, the editing system comprises a vector comprising a nucleic acid encoding a guide polynucleotide and a base editor. In some embodiments, the editing system comprises a vector comprising a nucleic acid encoding a guide polynucleotide of the present invention and a base editor or prime editor. In some embodiments, the editing system comprises a vector comprising a nucleic acid encoding a guide polynucleotide of the present invention and a base editor.

[0042] The base editor or prime editor (preferably base editor) may be in a complex with the guide polynucleotide (e.g. the guide polynucleotide and the base editor may together form a ribonucleoprotein (RNP)).

[0043] In some embodiments, the editing system comprises a guide polynucleotide and a polynucleotide encoding a base editor or prime editor. In some embodiments, the editing system comprises a guide polynucleotide and a polynucleotide encoding a base editor. In some embodiments, the editing system comprises a guide polynucleotide of the present invention and a polynucleotide encoding a base editor or prime editor. In some embodiments, the editing system comprises a guide polynucleotide of the present invention and a polynucleotide encoding a base editor. In some embodiments, the editing system comprises a vector comprising a nucleic acid encoding a guide polynucleotide and a polynucleotide encoding a base editor or prime editor. In some embodiments, the editing system comprises a vector comprising a nucleic acid encoding a guide polynucleotide and a polynucleotide encoding a base editor. In some embodiments, the editing system comprises a vector comprising a nucleic acid encoding a guide polynucleotide of the present invention and a polynucleotide encoding a base editor or prime editor. In some embodiments, the editing system comprises a vector comprising a nucleic acid encoding a guide polynucleotide of the present invention and a polynucleotide encoding a base editor.

[0044] In some embodiments, the editing system comprises a guide polynucleotide and a vector comprising a nucleic acid encoding a base editor or prime editor. In some embodiments, the editing system comprises a guide polynucleotide and a vector comprising a nucleic acid encoding a base editor. In some embodiments, the editing system comprises a guide polynucleotide of the present invention and a vector comprising a nucleic acid encoding a base editor or prime editor. In some embodiments, the editing system comprises a guide polynucleotide of the present invention and a vector comprising a nucleic acid encoding a base editor. In some embodiments, the editing system comprises a vector comprising a nucleic acid encoding a guide polynucleotide and a vector comprising a nucleic acid encoding a base editor or prime editor. In some embodiments, the editing system comprises a vector comprising a nucleic acid encoding a guide polynucleotide and a vector comprising a nucleic acid encoding a base editor. In some embodiments, the editing system comprises a vector comprising a nucleic acid encoding a guide polynucleotide of the present invention and a vector comprising a nucleic acid encoding a base editor or prime editor. In some embodiments, the editing system comprises a vector comprising a nucleic acid encoding a guide polynucleotide of the present invention and a vector comprising a nucleic acid encoding a base editor.

[0045] In some embodiments, the vector comprising the nucleic acid encoding the guide polynucleotide and the vector comprising the nucleic acid encoding the base editor or portion thereof or the prime editor or portion thereof are the same. In some embodiments, the vector comprising the nucleic acid encoding the guide polynucleotide further comprises a first portion of the base editor or prime editor and the editing system further comprises a second vector comprising a nucleic acid encoding a second portion of the base editor or prime editor. The vector and second vector may be such that the base editor or prime editor is functionally constituted when the vector and second vector are introduced into a cell.

[0046] The above-described guide polynucleotide and editing system can be, for example, used to edit the 5’ UTR-encoding region of a gene to abolish or create a uORF.

[0047] Another aspect of the present invention provides a method of editing a 5’ UTR-encoding region of a gene encoding a VGSC alpha subunit (Nav). The method may comprise contacting the 5’ UTR-encoding region with a guide polynucleotide and a base editor or prime editor. The guide polynucleotide may target the base editor or prime editor to effect an alteration to abolish or create a uORF.

[0048] Another aspect of the invention provides a method of editing a 5’ UTR-encoding region of a gene encoding a VGSC alpha subunit (Nav), the method comprising contacting the 5’ UTR- encoding region with (a) a guide polynucleotide, and (b) a base editor or prime editor (preferably a base editor), wherein the guide polynucleotide targets the base editor or prime editor to effect an alteration to abolish or create a uORF (preferably to abolish a uORF).

[0049] In some embodiments, the guide polynucleotide is the guide polynucleotide of the present invention. In some embodiments, the base editor or prime editor effects an alteration to abolish a uORF. Preferably, the base editor or prime editor is a base editor. Preferably, the base editor effects an alteration to abolish a uORF. In some embodiments, the method is an in vitro or ex vivo method. In some embodiments, the method comprises contacting a cell with the editing system of the present invention. In some embodiments, the cell expresses the guide polynucleotide from the vector comprising the nucleic acid encoding the guide polynucleotide and / or expresses the base editor or prime editor from the polynucleotide encoding the base editor or prime editor or the vector comprising the nucleic acid encoding the base editor or portion thereof or prime editor or portion thereof.

[0050] In some embodiments, the cell is contacted with the editing system of the present invention using any one of virus-mediated infection (e.g. with a recombinant viral vector), direct injection of nucleic acids, biolistic transformation, microinjection, electroporation, DEAE-dextran treatment, lipofection, nanoparticle-mediated transfection, or protein transduction domain (PTD) mediated transduction. In some embodiments, the cell is contacted with the editing system of the present invention using nanoparticle-mediated transfection.

[0051] Editing of the 5’ UTR-encoding region of a gene encoding a VGSC alpha subunit (Nav) can be, for example, used to compensate for a loss of function (LOF) or gain of function (GOF) mutation in said gene.

[0052] Another aspect of the present invention provides a cell genetically altered to abolish or create a uORF in the 5’ UTR-encoding region of a gene encoding a VGSC alpha subunit (Nav).

[0053] In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a mouse cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is mitotic cell. In some embodiments, the cell is a post-mitotic cell. In some embodiments, the cell is a neuron.

[0054] In some embodiments, the alteration abolishes or creates at least one uORF. In some embodiments, the alteration abolishes at least one uORF. In some embodiments, the alteration abolishes or creates at least two uORFs. In some embodiments, the alteration abolishes at least two uORFs. In some embodiments, the alteration comprises at least one A«T to G*C alteration. In some embodiments the alteration comprises at least two A«T to G*C alterations. In some embodiments, the at least one A«T to G*C alteration is in at least one start codon of the uORF. In some embodiments, the at least two A«T to G*C alterations are in at least two uORF start codons. In some embodiments, the at least one A«T to G*C alteration is between position -30 and position 10. In some embodiments, the at least one A«T to G*C alteration is between position -30 and position 4. In some embodiments, the at least one A«T to G*C alteration is between position -30 and position -4. In some embodiments, the alteration is between position -19 and position -4. In some embodiments, the alteration is between position -18 and position -4. In some embodiments, the alteration is between position -13 and position -4. In some embodiments, the at least one A«T to G*C alteration is between position -10 and position -4. In some embodiments, the at least one A«T to G*C alteration comprises an A«T to G*C alteration at position -30, -29, -28, -27, -26, -25, -24, -23, -22, -21 , -20, -19, - 18, -17, -16, 15, -14, -13, -12, -11 , -10, -9, -8, -7, -6, -5, -4, -3, -2, -1 , 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably at position -30, -29, -28, -27, -26, -25, -24, -23, -22, -21 , -20, -19, -18, -17, -16, 15, -14, -13, -12, -11 , -10, -9, -8, -7, -6, -5, or -4 . In some embodiments, the at least one A«T to G*C alteration comprises an A«T to G*C alteration at position -8 or position -17. In some embodiments, the at least two A«T to G*C alterations comprise two A«T to G*C alterations at positions selected from any one of positions -30, -29, -28, -27, -26, -25, -24, -23, -22, -21 , -20, -19, -18, -17, -16, 15, -14, -13, -12, -11 , -10, -9, -8, -7, -6, -5, -4, -3, -2, -1 , 1 , 2, 3, 4, 5, 6, 7, 8, 9 or 10, preferably any one of positions -30, -29, -28, -27, -26, -25, -24, -23, -22, -21 , -20, -19, -18, -17, -16, 15, -14, -13, -12, -11 , -10, -9, -8, -7, -6, -5, and -4. In some embodiments, the at least two A«T to G*C alterations comprise an A«T to G*C alteration at position -8 and an A«T to G*C alteration at position -17.

[0055] The guide polynucleotide of the present invention, the vector comprising the nucleic acid encoding the guide polynucleotide of the present invention and the editing system of the present invention can be, for example, used to treat a patient suffering from a genetic disorder, particularly one associated with an LOF or GOF mutation.

[0056] Another aspect of the present invention provides the polynucleotide of the present invention, the vector comprising the nucleic acid encoding the guide polynucleotide of the present invention or the editing system of the present invention for use in a method of treatment. In some embodiments, the method of treatment is a method of treating a genetic disorder. The guide polynucleotide or the vector may be, for example, administered in combination with any one of a base editor or prime editor (preferably a base editor), a polynucleotide encoding a base editor or prime editor (preferably a base editor) or a vector comprising a nucleic acid encoding a base editor or portion thereof or a prime editor or portion thereof (preferably a base editor or portion thereof).

[0057] In some embodiments, the guide polynucleotide, vector or editing system are administered in combination with (a) the guide polynucleotide of the present invention which binds to a target sequence comprising a portion of the 5’ splice acceptor site (SA) of an exon of a gene encoding a voltage-gated sodium channel (VGSC) alpha subunit (Nav); (b) the vector of the invention comprising the nucleic acid encoding the guide polynucleotide of the present invention which binds to a target sequence comprising a portion of the 5’ splice acceptor site (SA) of an exon of a gene encoding a voltage-gated sodium channel (VGSC) alpha subunit (Nav); or (c) the editing system of the invention comprising the guide polynucleotide of the present invention which binds to a target sequence comprising a portion of the 5’ splice acceptor site (SA) of an exon of a gene encoding a voltage-gated sodium channel (VGSC) alpha subunit (Nav)or comprising the vector comprising the nucleic acid encoding the guide polynucleotide of the present invention which binds to a target sequence comprising a portion of the 5’ splice acceptor site (SA) of an exon of a gene encoding a voltage-gated sodium channel (VGSC) alpha subunit (Nav).

[0058] The base editor (or portion thereof) or prime editor (or portion thereof) of the editing systems may be different or the same.

[0059] In some embodiments, one or both of the vectors are mammalian cell expression vector(s), viral vector(s) or a naked DNA vector(s). In some embodiments, one or both of the viral vectors are adeno-associated virus (AAV) vector(s), such as AAV9 or AAV6 vector(s). In some embodiments, the vector(s) are AAV9 vectors. In some embodiments, the vector(s) are lentiviral (LV) vector(s), such as an integration-defective lentiviral (IDLV) vector(s). In some embodiments, both of the vectors are AAV9 vectors.

[0060] In some embodiments, the genetic disorder is characterised by the presence of a LOF or GOF mutation (preferably a LOF mutation). In some embodiments, the LOF or GOF mutation (preferably LOF mutation) is in at least one allele of at least one gene encoding a VGSC alpha subunit (Nav). In some embodiments, the genetic disorder is characterised by the presence of a LOF or GOF mutation (preferably a LOF mutation). In some embodiments, the LOF mutation is in at least one allele of at least one gene encoding a VGSC alpha subunit (Nav).

[0061] In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is selected from any one of human SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN7A, SCN8A, SCN9A, SCN10A and SCN11A, or any one of mouse Senia, Scn2a, Scn3a, Scn4a, Scn5a, Scn7a, Scn8a, Scn9a, Scn10a and Scn11a. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is selected from any one of human SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SCN10A and SCN11A, or any one of mouse Senia, Scn2a, Scn3a, Scn4a, Scn5a, Scn8a, Scn9a, Scn10a and Scn11a. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is selected from any one of human SCN1A, SCN2A, SCN3A, SCN5A, SCN8A and SCN9A, or any one of mouse Senia, Scn2a, Scn3a, Scn5a, Scn8a and Scn9a, preferably any one of human SCN1A, SCN2A, SCN3A, SCN5A, SCN8A and SCN9A, even more preferably any one of human SCN1A, SCN2A and SCN5A. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is human SCN1A. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is human SCN2A. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is human SCN5A. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is human SCN8a. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is mouse Senia. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is mouse Scn2a. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is mouse Scn5a. In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is mouse Scn8a.

[0062] In some embodiments, the genetic disorder is associated with an LOF mutation in a gene encoding a VGSC alpha subunit (Nav). In some embodiments, the genetic disorder is associated with a GOF mutation in a gene encoding a VGSC alpha subunit (Nav). In some embodiments, the genetic disorder is a neurological disorder. In some embodiments, the genetic disorder belongs to any one of cardiac dysfunctions, peripheral neuropathies, skeletal muscle channelopathies, movement disorders, intellectual disability, familial hemiplegic migraine type 3, early infantile developmental encephalopathy with movement disorder, hyperkinetic movement disorder, severe-to-profound developmental delay, autism spectrum disorders (ASD), early infantile epilepsy, Brugada syndrome, long QT syndrome, dilated cardiomyopathy, or developmental and epileptic encephalopathies (DEE). In some embodiments, the genetic disorder is a neurological disorder. In some embodiments, the neurological disorder belongs to the group of DEEs. In some embodiments, the DEE is genetic epilepsy or Dravet syndrome. In some embodiments, the genetic disorder is Dravet syndrome.

[0063] In some embodiments of the present invention, the base editor or prime editor (preferably base editor) comprises a Cas9 protein. In some embodiments, the base editor or prime editor is a base editor comprising a Cas9 protein. In some embodiments, the base editor or prime editor is an adenine base editor or a cytosine base editor. In some embodiments, the base editor or prime editor is an adenine base editor. In some embodiments, the adenine base editor comprises a Cas9 protein (Cas9-ABE). In some embodiments, the Cas9-ABE is SpCas9-ABE or SpRYCas9-ABE. In some embodiments, the SpCas9-ABE is SpCas9-ABE8e or SpCas9-ABEmax. In some embodiments, the SpRYCas9-ABE is SpRYCas9-ABE8e or SpRYCas9-ABEmax. In some embodiments, the adenine base editor has the sequence set forth in SEQ ID NO: 29, SEQ ID NO: 30 or SEQ ID NO: 31.

[0064] In some embodiments, the base editor comprises or consists of an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 29. In some embodiments, the base editor comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0065] In some embodiments, the base editor comprises or consists of an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 30. In some embodiments, the base editor comprises or consists of the amino acid sequence of SEQ ID NO: 30.

[0066] In some embodiments, the base editor comprises or consists of an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 31. In some embodiments, the base editor comprises or consists of the amino acid sequence of SEQ ID NO: 31.

[0067] The present applicants have also developed methods to identify candidates in the 5’ UTR- encoding region of a gene which, if altered, affect protein expression levels.

[0068] Another aspect of the present invention discloses a method of identifying a variant of a portion of the 5’ UTR-encoding region of a gene encoding a VGSC alpha subunit (Nav), wherein the variant comprises an alteration in at least one nucleotide capable of increasing or decreasing the expression levels of the gene. The method may comprise the steps of (a) synthesising a nucleic acid comprising a portion of the 5’ UTR-encoding region of the gene and at least one nucleic acid comprising a variant of the portion of the 5’ UTR-encoding region comprising a sequence alteration in at least one nucleotide, b) cloning each of the nucleic acids into an expression vector comprising a bicistronic expression cassette allowing simultaneous expression of a first marker and a second marker, wherein expression of the first marker is dependent on the sequence of the nucleic acid cloned into the expression vector, c) introducing each of the expression vectors into a cell for expression of the first marker and the second marker, d) measuring the expression of the first marker and the second marker in the cell, e) determining the expression levels of the first marker relative to those of the second marker, f) identifying at least one nucleic acid comprising a variant of the portion of the 5’ UTR- encoding region, wherein the presence of said nucleic acid in the expression vector increases or decreases the relative expression levels of the first marker compared to the nucleic acid comprising the unaltered portion of the 5’ UTR-encoding region, and g) determining the nature of the sequence alteration in the at least one nucleotide.

[0069] Another aspect of the present invention discloses a method of generating a guide polynucleotide, for example a guide polynucleotide of the invention, the method comprising the steps of: (a) determining a target sequence comprising a portion of the 5’ UTR-encoding region of a gene encoding a VGSC alpha subunit (Nav), wherein a variant of said portion of the 5’ UTR-encoding region has been identified using the method according to the present invention, and b) synthesising the guide polynucleotide which binds to the determined target sequence. The present applicants further hypothesised that preventing the integration of a “poison exon” in mRNA splice variants of genes causing severe disorders such as DEE, in particular genes of the SC / V gene family such as SCN1A, could also boost protein translation (of proteins such as Nav1.1 to Nav1.9).

[0070] “Poison exons” are exons the integration of which in splice variants of a gene transcript causes the introduction of a premature STOP codon that directs the transcript toward nonsense- mediated decay (NMD), effectively reducing the amount of functional protein produced. The inclusion of a poison exon in a gene transcript can be increased in gene variants comprising pathogenic variations within or near the poison exon.

[0071] The present applicants hypothesized that the presence of a premature STOP codon caused by the integration of the poison exon in the transcript could be avoided on the gene level by preventing the splice event resulting in the integration of the poison exon in splice variants of the transcript. To this end, the present applicants have designed a gene editing strategy to disrupt the splice acceptor site located 5’, i.e. upstream, of the poison exon.

[0072] Thus, another aspect of the present invention discloses a guide polynucleotide comprising a sequence which binds to a target sequence comprising a portion of the 5’ splice acceptor site (SA) of an exon of a gene encoding a voltage-gated sodium channel (VGSC) alpha subunit (Nav).

[0073] In preferred embodiments, the guide polynucleotide is a guide RNA.

[0074] In some embodiments, the target sequence is located between position -30 and position +30 with respect to the exon, position -1 being the position of the nucleotide immediately 5’ of the first nucleotide of the exon. In some embodiments, the target sequence comprises the 5’ SA.

[0075] In some embodiments, the gene encoding a VGSC alpha subunit (Nav) is selected from any one of human SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN7A, SCN8A, SCN9A, SCN10A and SCN11A, or any one of mouse Senia, Scn2a, Scn3a, Scn4a, Scn5a, Scn7a, Scn8a, Scn9a, Scn10a and Scn11a. In some embodiments, the gene is selected from any one of human SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN7A, SCN8A, SCN9A, SCN10A and SCN11A. In some embodiments, the gene is human SCN1A.

[0076] In some embodiments, the exon is an exon located within an intron. In some embodiments, the exon is a poison exon. In some embodiments, the exon is an exon located within intron 1 , 20 or 22 of the gene, preferably intron 1 , 20 or 22 of human SCN1A or mouse Senia, more preferably intron 1 , 20 or 22 of human SCN1A. In some embodiments, the exon is an exon located in intron 20, preferably in intron 20 of human SCN1A or mouse Senia, more preferably in intron 20 of human SCN1A. In some embodiments, the poison exon is exon 1 N located in intron 1 , exon 20N located in intron 20 or exon 22N located in intron 22, preferably exon 1 N located in intron 1 of human SCN1A or mouse SCN1A, exon 20N located in intron 20 of human SCN1A or mouse Senia, or exon 22N located in intron 22 of human SCN1A or mouse Senia, more preferably exon 1 N located in intron 1 of human SCN1A, exon 20N located in intron 20 of human SCN1A, or exon 22N located in intron 22 of human SCN1A. In some embodiments, the poison exon is exon 20N located in intron 20, preferably exon 20N located in intron 20 of human SCN1A or mouse Senia. In some embodiments, the exon is exon 20N of human SCN1A.

[0077] In some embodiments, the sequence which binds to the target sequence comprises any one of the sequences set forth in SEQ ID NOs: 44-52. In some embodiments, the sequence which binds to the target sequence consists of any one of the sequences set forth in SEQ ID NOs: 44-52. In some embodiments, the sequence which binds to the target sequence comprises any one of the sequences set forth in SEQ ID NOs: 44-48. In some embodiments, the sequence which binds to the target sequence consists of any one of the sequences set forth in SEQ ID NOs: 44-48. In some embodiments, the sequence which binds to the target sequence consists of the sequence set forth in SEQ ID NO: 44.

[0078] Another aspect of the invention discloses a nucleic acid encoding the guide polynucleotide of the invention. In some embodiments, the nucleic acid is a DNA. In some embodiments, the nucleic acid is an RNA.

[0079] Another aspect of the present invention provides a vector comprising the nucleic acid sequence of the present invention. In some embodiments, the vector further comprises a nucleic acid encoding a base editor or a portion thereof or a prime editor or portion thereof. In some embodiments, the vector comprises the nucleic acid of the present invention and a first portion of an adenine base editor, for example SpRYCas9-ABE8e or SpCas9-ABE8e. In some embodiments, the vector is suitable for use with a second vector comprising a second portion of the adenine base editor.

[0080] In some embodiments, the vector is a mammalian cell expression vector, a viral vector or a naked DNA vector. In some embodiments, the viral vector is an adeno-associated virus (AAV) vector, such as an AAV6 or AAV9 vector. In some embodiments, the viral vector is AAV9 vector. In some embodiments, the vector is an AAV9 vector. In some embodiments, the vector is a lentiviral (LV) vector, such as an integration-defective lentiviral (IDLV) vector.

[0081] Another aspect of the present invention provides an editing system comprising: (a) the guide polynucleotide or vector the present invention; and (b) a base editor or prime editor, a polynucleotide encoding a base editor or prime editor or a vector comprising a nucleic acid encoding a base editor or portion thereof or a prime editor or portion thereof.

[0082] The editing system may be in the form of two vectors, preferably two AAV vectors. In some embodiments, one or both of the vectors are mammalian cell expression vector(s), viral vector(s) or a naked DNA vector(s). In some embodiments, one or both of the viral vectors are adeno-associated virus (AAV) vector(s), such as AAV6 or AAV9 vector(s). In some embodiments, the vector(s) are AAV9 vectors. In some embodiments, the vector(s) are lentiviral (LV) vector(s), such as an integration-defective lentiviral (IDLV) vector(s). In some embodiments, both of the two vectors are AAV6 or AAV9 vectors. In some embodiments, both of the two vectors are AAV9 vectors.

[0083] In some embodiments, a first vector comprises a nucleic acid encoding the guide polynucleotide of the present invention, a nucleic acid encoding a first portion of a base or prime editor and a second vector comprises a nucleic acid encoding a second portion of the base or prime editor.

[0084] In some embodiments, the two portions of the base or prime editor can, when expressed, be assembled to the base or prime editor. In some embodiments, the two portions can be assembled using a split intein system. In some embodiments, one of the two vectors additionally comprises a nucleic acid encoding a N-intein or C-intein and the other vector comprises a nucleic acid encoding the other of the N-intein or C-intein.

[0085] Another aspect of the present invention provides a method of editing a 5’ splice acceptor site (SA) of an exon of a gene encoding a VGSC alpha subunit (Nav), the method comprising contacting the 5’ SA with (a) a guide polynucleotide, optionally a guide polynucleotide of the present invention; and (b) a base editor or prime editor, wherein the guide polynucleotide targets the base editor or prime editor to effect an alteration to disrupt the 5’ SA.

[0086] In some embodiments, the exon is an exon located within an intron. In some embodiments, the exon is a poison exon. In some embodiments, the exon is an exon located within intron 1 , 20 or 22 of the gene, preferably intron 1 , 20 or 22 of human SCN1A or mouse Senia, more preferably intron 1 , 20 or 22 of human SCN1A. In some embodiments, the exon is an exon located in intron 20, preferably in intron 20 of human SCN1 A or mouse Senia, more preferably in intron 20 of human SCN1A. In some embodiments, the poison exon is exon 1 N located in intron 1 , exon 20N located in intron 20 or exon 22N located in intron 22, preferably exon 1 N located in intron 1 of human SCN1A or mouse Senia, exon 20N located in intron 20 of human SCN1A or mouse Senia, or exon 22N located in intron 22 of human SCN1A or mouse Senia, more preferably exon 1 N located in intron 1 of human SCN1A, exon 20N located in intron 20 of human SCN1A, or exon 22N located in intron 22 of human SCN1A. In some embodiments, the poison exon is exon 20N located in intron 20, preferably exon 20N located in intron 20 of human SCN1A or mouse Senia. In some embodiments, the exon is exon 20N of human SCN1A.

[0087] In some embodiments, the method further comprises contacting a cell with the editing system of the present invention. In some embodiments, the contacting comprises using any one of virus-mediated infection with recombinant viral vectors, direct injection of nucleic acids, biolistic transformation, microinjection, electroporation, DEAE-dextran treatment, lipofection, nanoparticle-mediated transfection, virus-like particle delivery, or protein transduction domain (PTD) mediated transduction. In some embodiments, the method comprises contacting using nanoparticle-mediated transfection, preferably of a ribonucleoprotein (RNP) comprising the guide polynucleotide and the base or prime editor.

[0088] In some embodiments, the alteration comprises at least one A«T to G*C alteration, optionally wherein the at least one A«T to G*C alteration is in the 5’ SA. In some embodiments, the at least one A«T to G*C alteration is at position -2 with respect to the exon, position -1 being the position of the nucleotide immediately 5’ of the first nucleotide of the exon.

[0089] The present applicants also hypothesized that, in order to increase basal expression levels of the genes of interest, in particular genes of the SCN gene family such as SCN1A, one may combine editing one or more uATGs of the 5’ UTR of a gene with editing of the 5’ SA of an exon of said gene.

[0090] Accordingly, another aspect of the present invention provides a combination of (a) a guide polynucleotide of the present invention that binds to a target sequence comprising a portion of the 5’ UTR-encoding region of a gene encoding a Nav, the vector comprising a nucleic acid encoding said guide polynucleotide, or the editing system comprising said guide polynucleotide or comprising the vector comprising a nucleic acid encoding said guide polynucleotide; and (b) a guide polynucleotide of the present invention which binds to a target sequence comprising a portion of the 5’ splice acceptor site (SA) of an exon of a gene encoding a Nav, the vector comprising a nucleic acid encoding said guide polynucleotide, or the editing system comprising said guide polynucleotide or comprising the vector comprising a nucleic acid encoding said guide polynucleotide.

[0091] The base editor (or portion thereof) or prime editor (or portion thereof) of the editing system of (a) may be different to the base editor (or portion thereof) or prime editor (or portion thereof) of the editing system of (b). The base editor (or portion thereof) or prime editor (or portion thereof) of the editing system of (a) may be the same as the base editor (or portion thereof) or prime editor (or portion thereof) of the editing system of (b).

[0092] A guide polynucleotide, nucleic acid, vector, editing system or combination as described above may be used to treat a genetic disorder, particularly one associated with an LOF or GOF mutation.

[0093] Thus, another aspect of the present invention provides the guide polynucleotide of the present invention which binds to a target sequence comprising a portion of the 5’ splice acceptor site (SA) of an exon of a gene encoding a Nav; the vector comprising the nucleic acid encoding said guide polynucleotide; the editing system comprising said guide polynucleotide or comprising a vector comprising the nucleic acid encoding said guide polynucleotide; or the combination of the present invention for use in a method of treatment. In some embodiments, the method of treatment is a method of treating a genetic disorder. The guide polynucleotide, vector, editing system or combination may be, for example, administered in combination with any one of a base editor or prime editor (preferably a base editor), a polynucleotide encoding a base editor or prime editor (preferably a base editor) or a vector comprising a nucleic acid encoding a base editor or portion thereof or a prime editor or portion thereof (preferably a base editor or portion thereof).

[0094] In some embodiments, the base editor or prime editor is a base editor comprising a Cas9 protein, optionally an adenine base editor, further optionally wherein the adenine base editor has the sequence set forth in SEQ ID NO: 29 [SpRYCas9ABEmax], SEQ ID NO: 30 [SpRYCas9-ABE8e] or SEQ ID NO: 31 [SpCas9-ABE8e],

[0095] In some embodiments, the vector is an AAV vector. In some embodiments, the AAV vector is an AAV6 or AAV9 vector. In some embodiments, the AAV vector is an AAV9 vector.

[0096] In some embodiments, the guide polynucleotide or vector are administered in combination with the guide polynucleotide of the present invention that binds to a target sequence comprising a portion of the 5’ UTR-encoding region of a gene encoding a Nav; the vector comprising the nucleic acid encoding said guide polynucleotide; or the editing system comprising said guide polynucleotide or comprising the vector comprising the nucleic acid encoding said guide polynucleotide.

[0097] In some embodiments, one or both of the vectors are mammalian cell expression vector(s), viral vector(s) or a naked DNA vector(s). In some embodiments, one or both of the viral vectors are adeno-associated virus (AAV) vector(s), such AAV6 or AAV9 vector(s). In some embodiments, the vector(s) are AAV9 vectors. In some embodiments, the vector(s) are lentiviral (LV) vector(s), such as an integration-defective lentiviral (IDLV) vector(s). In some embodiments, both of the two vectors are AAV6 or AAV9 vectors. In some embodiments, both of the two vectors AAV9 vectors.

[0098] In some embodiments, the genetic disorder is characterized by the presence of a loss of function (LOF) or gain of function (GOF) mutation in at least one allele of at least one gene encoding a VGSC alpha subunit (Nav), optionally wherein the gene is selected from human SCN1A, SCN2A, SCN3A, SCN5A, SCN8A and SCN9A, more optionally wherein the gene is human SCN1A.

[0099] In some embodiments, the genetic disorder belongs to the group of developmental and epileptic encephalopathies (DEE), optionally wherein the DEE is Dravet syndrome. In some embodiments, the genetic disorder is Dravet syndrome.

[0100] In some embodiments of any of the vectors of the present invention for use in a method of treatment, the vector further comprises a nucleic acid encoding a first of two portions of a base or prime editor and is administered in combination with a second vector comprising a nucleic acid encoding a second of two portions of the base or prime editor. In some embodiments, one or both of the vectors are viral vectors. In some embodiments, the viral vector is, or the viral vectors are, AAV vector(s). In some embodiments, the AAV vector is, or the AAV vectors are, AAV6 or AAV9 vector(s). In some embodiments, the AAV vector is, or the AAV vectors are, AAV9 vector(s).

[0101] In some embodiments, the two portions of the base or prime editor can, when expressed, be assembled to the base or prime editor. In some embodiments, the two portions can be assembled using a split intein system. In some embodiments, one of the two vectors additionally comprises a nucleic acid encoding a N-intein or C-intein and the other vector comprises a nucleic acid encoding the other of the N-intein or C-intein not comprised in the first vector.

[0102] In some embodiments of any of the vectors of the present invention for use in a method of treatment, the vector is administered systemically, intranasally, or by intraparenchymal, intracerebroventricular, intracoronary, intramuscular, intramyocardial, perineural, intraneural, subcutaneous, intradermal, intrathecal or epidural injection.

[0103] In some embodiments, the vector is administered to a human.

[0104] In some embodiments, the amount of vector administered is provided as total viral genomes (vg) or as vg / kg. In particular, where the vector is administered locally, e.g. by intraparenchymal injection, the amount of vector administered may be provided as vg, while the amount of vector for systemic, e.g. intravenous, administration may be provided as vg / kg.

[0105] In some embodiments, the vector is administered at an amount of 1x 1012to 1x 1015vg / kg, preferably 1x 1013to 1x 1015vg / kg, more preferably 1x 1013to 1x 1014vg / kg. In some embodiments, the vector is administered at a total amount of 1x 1011to 1x 1015vg. In some embodiments, the vector is administered at a total amount of 1x 1011to 1x 1013vg, preferably at a total amount of 1x 1012to 1x 1013vg. In some embodiments, the vector is administered at a total amount of 1x 1013to 1x 1015vg, preferably at a total amount of 1x 1013to 1x 1014vg.

[0106] In some embodiments, the vector is administered systemically. In some embodiments, the vector is administered at an amount of 1x 1013to 1x 1014vg / kg. In some embodiments, the vector is administered intravenously.

[0107] In some embodiments, the vector is administered by intraparenchymal injection. In some embodiments, the vector is administered at a total amount of 1x 1011to 1x 1013vg. In some embodiments, the vector is administered by intracerebral injection in the hippocampus, cerebral cortex, thalamus, cerebellum or striatum. The Nav may for example be Nav1.1, Nav1 .2, Nav1 .3 or Nav1 .6.

[0108] In some embodiments, the vector is administered by intrathecal injection. In some embodiments, the vector is administered at a total amount of 1x 1011to 1x 1013vg.

[0109] In some embodiments, the vector is administered by intracerebroventricular injection. In some embodiments, the vector is administered to a human at a total amount from 1x 1013to 1x 1015vg. The Nav may for example be Nav1.1 , Nav1.2, Nav1.3 or Nav1.6.

[0110] In some embodiments, the vector is administered by intramuscular injection. In some embodiments, the vector is administered to a human at a total amount from 1x 1013to 1x 1014vg. The Nav may for example be Navi .4.

[0111] In some embodiments, the vector is administered by intracoronary injection. In some embodiments, the vector is administered to a human at a total amount from 1x 1012to 1x 1014vg. The Nav may for example be Navi .5.

[0112] In some embodiments, the vector is administered by intramyocardial injection. In some embodiments, the vector is administered to a human at a total amount from 1x 1012to 1x 1014vg. The Nav may for example be Navi .5. In some embodiments, the vector is administered by perineural injection. In some embodiments, the vector is administered to a human at a total amount from 1x 1012to 1x 1013vg. The Nav may for example be Navi .7.

[0113] In some embodiments, the vector is administered by intraneural injection. In some embodiments, the vector is administered to a human at a total amount from 1x 1012to 1x 1013vg. The Nav may for example be Navi .7.

[0114] In some embodiments, the vector is administered by subcutaneous injection. In some embodiments, the vector is administered to a human at a total amount from 1x 1012to 1x 1013vg. The Nav may for example be Navi .7.

[0115] In some embodiments, the vector is administered by intradermal injection. In some embodiments, the vector is administered to a human at a total amount from 1x 1012to 1x 1013vg. The Nav may for example be Navi .7.

[0116] In some embodiments, the vector is administered by epidural injection. In some embodiments, the vector is administered to a human at a total amount from 1x 1012to 1x 1013vg. The Nav may for example be Navi .7.

[0117] The guide polynucleotides, nucleic acids, editing systems and combinations of the present invention may be comprised in a vector, such as the vectors of the present invention. AAV vectors, such as an AAV6 or AAV9 vector, are particularly suitable for therapeutic delivery, for example for use in the methods of treatment disclosed herein.

[0118] Another aspect of the invention provides a method of producing a viral vector, in particular an AAV vector, e.g. an AAV6 or AAV9 vector, of the present invention.

[0119] DESCRIPTION OF THE DRAWINGS

[0120] Figure 1

[0121] (A-D) Reporter assay screen of SCN1A Kozak sequence mutations identified variants able to significantly enhance translation efficiency. (A) Schematic representation of reporter assay used for identification of variants. Variants carrying mutations within SCN1A Kozak sequence were cloned in place of EGFP canonical Kozak in a bicistronic reporter plasmid expressing EGFP and mCherry, separated by an internal ribosomal entry site (IRES). Plasmids harboring Kozak variants were lipofected into HEK-293T cells. 3 days following transfection, flow cytometry analysis was performed. (B) Flow cytometry gating strategy for the analysis of EGFP mean fluorescence intensity (MFI) in mCherry-positive HEK-293T cell population lipofected with SCN1A Kozak variants. MFI quantification allowed selection of the most promising candidates as measured by their ability to enhance EGFP intensity compared to the wild-type (wt) Kozak sequence. (C) Table of screened Kozak variants harboring distinct combination of A>G or C>T mutations, starting from the wt SCN1A sequence. The start codon of the SCN1A coding sequence (CDS) is highlighted in grey. (D) Quantification of translation enhancement determined as relative EGFP / mCherry expression using flow cytometry analysis. Plotted data indicate the fold change increase of the variants over the wt Kozak sequence (indicated by the dashed line). Data are shown as means ± SEM from at least n = 3 biological replicates. Statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test.

[0122] Figure 2

[0123] (A-E) Whole 5’ UTR reporter assay screen identified variants able to enhance translation efficiency due to mutation of conserved upstream “ATG”s (uATGs) of a non-CDS upstream ORFs (uORFs). (A) Schematic representation of endogenous human-conserved SCN1A / Scn1a genomic context around mATG. Sequences for mORF and uORFs are highlighted in a zoomed-in view of the 5’ UTR region proximal to the mORF. uORF-1 and uORF-2 are in-frame with each other but out-of-frame with the mORF. The uORFs share a stop codon in exon 2. (B) Schematic representation of RiboSeq-available data integration and analysis workflow via the Ribo-uORF online tool (https: / / rnainformatics.org.cn / RiboUORF). Candidate uORFs, starting at two alternative uATGs conserved among murine and human SCN1A transcripts, were identified as actively translated, supported by Ribo-sequencing experiments in mouse and human brain tissues and cells. (C) Table of screened 5’ UTR variants harboring distinct combination of A>G conversions. ATG codons are highlighted. (D) Organization of mouse Senia mutually exclusive non-coding exons (exon A and exon B) upstream of exon 1 leading to the generation of alternative 5' UTRs (referred to as “Distal" and “Proximal", respectively). Exons are depicted as boxes, and introns as dashed lines. The localization of in-frame uATGs, the stop codon of their respective uORFs and the mORF ATG are highlighted in the scheme. The size in base pairs (bp) of the exons is also indicated. Variants carrying mutations within Senia the alternative 5' UTRs were cloned in place of the canonical EGFP Kozak sequence into the bicistronic reporter vector used for the experiments in Figure 1 , as illustrated. (E) Quantification of translation enhancement determined as relative EGFP / mCherry expression for both Distal and Proximal 5’UTR variants. Data are shown as means ± SEM from n = 4 biological replicates. The numbers indicate the fold change increase of the variants over the respective wt sequence (indicated by the dashed line). Statistical significance was assessed using one-way ANOVA followed by Sidak’s multiple comparisons test, [ns: p-value > 0.05; *: p-value < 0.05; **: p-value < 0.01 ; ****: p-value < 0.0001],

[0124] Figure 3

[0125] (A-C) uATG-3 disruption further increased mORF translation. (A) Schematic representation of endogenous Senia genomic context around mATG. Sequences for mORF and uORFs are highlighted in a zoomed-in view of the genomic region surrounding mATG. uORF-3 is out-of- frame with uORF-1 / 2 and terminates before the start of mORF. (B) Table of screened 5’ UTR variants harboring distinct combinations of A>G conversions and respective distinct uATG elimination. ATG codons are highlighted. (C) Quantification of translation enhancement determined as relative EGFP / mCherry expression for both Distal and Proximal 5’ UTR variants. Data are shown as means ± SEM from n = 4 biological replicates. The numbers indicate the fold change increase of the variants over the respective wt sequence (indicated by the dashed line). Statistical significance was assessed using one-way ANOVA followed by Sidak’s multiple comparisons test, [ns: p-value > 0.05; **: p-value < 0.01 ; ****: p-value < 0.0001],

[0126] Figure 4

[0127] (A-E) CRISPR / Cas9-based base editing achieved conversion of adenines of uATGs in HEK- 293T cells. (A) Schematic representation of the endogenous, conserved sequence in the 5’ UTR-encoding region in SCN1A / Scn1a targeted by selected SpRYCas9-ABE sgRNAs. The uATGs and mATG are highlighted. (B) Schematic depicting the experimental workflow and timeline of the base editing strategy. HEK-293T cells were co-lipofected with a sgRNA and either SpRYCas9-ABEmax or SpRYCas9-ABE8e plasmids. One day after transfection, blasticine selection of successfully transfected cells was started and continued for 5 days when genomic DNA was extracted for downstream assessments. (C) Schematic representation showing a qualitative assessment of base editing activity as measured by the amounts off cut and uncut fragments from BtsCI restriction enzyme digest of a restriction fragment length polymorphism (RFLP). The conservation or alteration of the BtsCI restriction site allowed the preliminary evaluation of editing activity. Following BtsCI digestion of purification of the PCR amplicons, fragments were run on an electrophoresis gel. Uncut (UF) and cut (CF) fragments are indicated by arrows. (D) Quantification of base editing efficiency by deep sequencing of both SpRYCas9-ABE8e and SpRYCas9-ABEmax with the selected sgRNA-11 as percentage (%) of alleles harboring at least one of the two A>G target edits (left) or both of them (right). Numbers are defined as % of Illumina reads harboring either target edit, insertions / deletions (indels), wild-type non-modified sequence or other non-target edits. Data are shown as means ± SEM from n = 2 biological replicates. (E) Distribution of A>G conversions (%) among the sgRNA-11 protospacer sequence with either SpRYCas9-ABE8e or SpRYCas9-ABEmax. Target adenines are indicated by arrows. Data are shown as means ± SEM from n = 2 biological replicates.

[0128] Figure 5

[0129] Abrogation of Senia uORF regulation by base editing of uATGs in mouse primary neurons resulted in Nav1.1 protein upregulation. (A) Schematic depicting the experimental timeline of base editing and analysing mouse neurons: cortical neurons were dissected from E18.5 mice and, following either three or one days in culture (i.e. days in vitro, DIV), were transduced with AAVs or lentivirus vectors (LV) carrying a base editors and sgRNA. At DIV14, genomic DNA, membrane-associated proteins and RNA were extracted for deep sequencing, evaluating Nav1.1 levels and assessing Senia transcript levels, respectively, and immunofluorescence was performed to determine transduction efficiency. (B) AAV and LV designs used for base editing testing in cortical neurons and their corresponding lengths in kilobase pairs (kb, excluding ITRs / LTRs). Constructs are not necessarily depicted to scale. (C) Base editing efficiency as assessed by deep sequencing of cortical neurons transduced with LV- SpRYCas9-ABE8e and sgRNA-11. Quantification is shown as percentage (%) of alleles harboring at least one of the two (left) or both 2 A>G target edits (right). Numbers are defined as % of sequencing reads harboring either target edit, insertions / deletions (indels), wild-type non-modified sequence or other non-target edits. Data are shown as means ± SEM from n = 3 biological replicates. (D) Distribution of A>G conversions (%) wihtin the sgRNA-11 protospacer sequence in cortical neurons transduced with LV-SpRYCas9-ABE8e and sgRNA- 11. Target adenines are indicated by arrows. Data are shown as means ± SEM from n = 3 biological replicates. (E) Nav1.1 and Calnexin protein levels of cortical neurons transduced with either LV-SpRYCas9-ABE8e + sgRNA-11 or LV-SpRYCas9-ABE8e alone were determined by Western blotting. Western blotting images (left) are representative of three independent experiments. Quantification of protein levels, assessed by relative band densitometry of Nav1.1 / Calnexin bands, is shown on the right. (F) Relative expression levels of Senia transcript, assessed by qRT-PCR, in cortical neurons either not transduced, transduced with LV-SpRYCas9-ABE8e alone or in combination with LV-sgRNA-11. Senia mRNA expression levels are presented as 2-ddct, where dCt is calculated for each transcript as: Ct (Senia) - Ct (murine 18S rRNA as normalizer) and ddCt as: dCt (normalized Senia) - dCt (LV-ABE8e-SpRY alone as control). 2-ddCt represents the fold change of expression in respect to the control (LV-ABE8e-SpRY alone). Each value represents the mean ± SEM of at least n=4 biological replicates. Statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test, [ns: p-value > 0.05],

[0130] Figure 6

[0131] (A-E) SpCas9-ABE8e-mediated base editing of uATGs in mouse primary neurons provided more efficient editing and upregulation of Nav1.1 compared to SpRY-ABE8e (A) Schematic representation of the conserved 5’ UTR-encoding region in SCN1A / Scn1a targeted by either SpRYCas9-ABE sgRNA-11 (SpRY_sgRNA11 or sgRNA-11) or SpCas9-ABE sgRNA-1 (Sp_sgRNA1 or sgRNA-1). The uATGs and mATG are highlighted. (B) Results from BtsCI restriction enzyme of RFLP evidencing SpRYCas9- and SpCas9- ABE8e activity in DIV14 neurons. UF and CF are shown. (C) Quantification of base editing efficiency by deep sequencing of DIV14 cortical neurons transduced with LV-SpCas9-ABE8e and sgRNA-1. Quantification is shown as percentage (%) of alleles harboring at least one of the two (left) or both 2 A>G target edits (right). Data are shown as means ± SEM from n = 2 biological replicates. (D) Distribution of A>G conversions (%) within the sgRNA-1 protospacer sequence of DIV14 neurons transduced with LV-SpCas9-ABE8e and sgRNA-1. Target adenines are indicated by arrows. Data are shown as means ± SEM from n = 3 biological replicates. (E) Nav1.1 and Calnexin protein levels of DIV14 cortical neurons either not transduced or transduced with LV-SpCas9-ABE8 and sgRNA-1 or LV-SpCas9-ABE8e alone were determined by Western blotting. Western blotting images (left) are representative of two independent experiments. Quantification of protein levels, assessed by relative band densitometry of Nav1.1 / Calnexin bands, is shown on the right. Statistical significance was assessed using one-way ANOVA followed by Dunnett’s multiple comparisons test. [**: p-value < 0.01].

[0132] Figure 7

[0133] (A,B) uORF-mediated regulation of mORF is a translation regulatory mechanism shared by other SCN paralog genes. (A) Table of screened 5’ UTR variants conserved between human and mouse and harboring distinct combination of A>G conversions abrogating uATGs for SCN2A, SCN3A, SCN5A, SCN8A and SCN9A genes. ATG codons are highlighted. (B) Top panel: schematic representation of reporter assay previosuly described in Figure 1. Plasmids harboring either uATG variants or wt sequences were lipofected into HEK-293T cells and 3 days later flow cytometry analysis was performed. Bottom panel: quantification of translation enhancement determined as relative EGFP / mCherry expression. Data are shown as means ± SEM from n = 3 biological replicates. Numbers indicate the fold change increase of the variants over the respective wt sequence (indicated by the dashed line). Statistical significance was assessed using one-way ANOVA followed by Sidak’s multiple comparisons test, [ns: p- value > 0.05; ****: p-value < 0.0001],

[0134] Figure 8

[0135] (A-F) Base editing strategy for Scn2a and Scn8a uATGs in mouse primary neurons efficiently installed the correct variant abrogating uORFs. (A) Schematic representation of endogenous human and mouse SCN2A / Scn2a and SCN8A / Scn8a genomic context around the start of the CDS. Sequences for mORF and uORFs are highlighted below in a zoomed-in view of the genomic region surrounding the mATG. sgRNAs used for mutagenizing uATGs are also shown for both mouse and human gene loci. B-F) Quantification of base editing efficiency in primary neurons transduced with LV-SpRYCas9-ABE8e in combination with sgRNA-2A1 (B,C), sgRNA-2A2 (B,D) or sgRNA-8a2 (E,F). Quantification is shown as percentage (%) of alleles harboring target edit. Plotted data are defined as % of sequencing reads harboring either target edit, insertions / deletions (indels), wild-type non-modified sequence or other nontarget edits. (C,D,F) Distribution of A>G conversions (%) within the sgRNA-2A1 (C), sgRNA- 2A2 (D), and sgRNA-8a2 (F) protospacer sequences in DIV10 neurons transduced with LV- SpRY-ABE8e and respective sgRNA. Target adenines are indicated by arrows.

[0136] Figure 9

[0137] (A-K) Disruption of upstream ATGs (uATGs) in the 5’ UTR, which are conserved between human SCN1A and mouse Senia, increases the expression of Senia isoform 1 and isoform 2. (A, B) Schematic representations of gene structure, transcript diversity, and upstream open reading frames (uORFs) organization for both human (A) and mouse (B) SCN1A gene. Top panel shows alternative splicing patterns of gene mRNA, showing multiple transcripts isoform produced from variable exon usage. The main coding sequence (CDS) is indicated between the translation start codon (ATG) and stop codon (*), with 5' and 3' untranslated regions (UTRs) flanking the CDS. 5’IITR and 3’IITR (darker) and gene coding sequence (CDS; lighter) are highlighted. Low panel represents enlarged view of 5' region, detailing the different uORFs (uORF-1 to uORF-4) and the main open reading frame (mORF). Positions of upstream ATG codons (uATGs), main ATG (mATG) and uORF stop codons are marked. (C) LICSC Genome Browser conservation analysis of 100 vertebrate species across the SCN1A uORFs and mORF translation start sites (uATG and mATG, respectively). Top panel shows PhyloP-based sequence logo illustrating evolutionary nucleotide conservation. Bottom panel shows multisequence alignment across representative species. Sequence gap, misalignments, and discontinuities are also highlighted as explained in the legend. (D, E) Ribosome profiling (Ribo- seq) of elongating ribosomes (footprints) and RNA sequencing (RNA-seq) coverage traces across the uORFs and the mORF start CDS of the human SCN1A (D) and mouse SCN1A (E) genes. (F) Schematic depicting the experimental setting: uATG variants of SCN1A isoform 1 (inclusion of exon A) or isoform 2 (inclusion of exon B) 5’llTRs were cloned in frame to GFP in GFP-IRES-mCherry reporter plasmid. Next, constructs were lipofected in HEK-293T cells and 3 days following transfection, flow citometry analysis was performed. Localization of uATGs and mATG are displayed. Table shows 5’IITR variants harboring distinct combination of A>G conversions. ATG triplets are highlighted. (G) Quantification of translation enhancement determined as GFP / mCherry expression for both SCN1A isoform 1 and isoform 2 5’IITR variants. Data are shown as means ± SEM from n = 4 biological replicates. Numbers indicate the fold change increase of the variants over the respective WT sequence (indicated by the dashed line). Statistical significance was assessed using unpaired t-test [ns: p-value > 0.05; *: p-value < 0.05; **: p-value < 0.01 ; *** p-value < 0.001], (H, I) Schematics of nucleotide sequence of SCN1A 5’IITR and starting of CDS for both human (H) and mouse (I) gene loci. All uORFs and mORF are highlighted with respective nucleotide sequence and directionality (indicated by “>”). (J) Ribo-sequencing of elongating ribosomes (footprints) and RNA- sequencing coverage traces across the mouse SCN1A exon 1 , 2 and 3. (K) Magnification of Ribo-seq and RNA-seq traces in correspondence of STOP codon associated to uORF-1 / 2 in SCN1A exon 2.

[0138] Figure 10

[0139] (A-l) Cas9-guided adenine base editors are more efficient at disrupting uATGs than Cas9 alone. (A, B) Scheme of SpRY / Sp-ABE strategy (A) and Sp-Cas9 NHEJ-mediated strategy (B) to edit or delete uATGs, respectively. SpRY-sgRNA-11 and Sp-sgRNA-1 spacer sequences with position of target adenines (As) are shown. (C) Timeline of editing experiment in HEK-293T cells: one day after plating HEK-293T cells were transduced with lentiviral vectors (LV) harboring ABE or Cas9 and sgRNA. 24h later, blasticidine selection started and continued for 5 days, when genomic DNA was extracted for deep sequencing. (D) Quantification of editing efficiency is shown as percentage (%) of sequencing reads harboring at least one uATG edit (left) or both uATG edits (right) for both SpRY-ABE and Sp-ABE in presence or absence of relative sgRNA in transduced cells. Sequencing reads are grouped as harboring target edit, insertions / deletions (indels), wild-type non-modified sequence or other non-target edits. Data are shown as means ± SEM from n = 3 biological replicates. Reads with at least one uATG edit: SpRY-ABE Ctrl: 0%±0%, SpRY-ABE + sg11 : 25.92% ± 3.85%, Sp-ABE Ctrl: 0%±0%, Sp-ABE + sg1 : 44.35% ± 0.04%. Reads with both uATG edits: SpRY-ABE Ctrl: 0%±0%, SpRY-ABE + sg11 : 8.15% ± 1.49%, Sp-ABE Ctrl: 0%±0%, Sp-ABE + sg1 : 4.65% ± 0.13%. (E, F) SpRY / Sp-ABE editing efficiency in HEK-293T. Distribution of A>G conversions (%) among sgRNA-11 (E) and sgRNA-1 (F) sequence in transduced HEK- 293T cells. Target adenines are marked by triangles and shown in bold. Data are shown as means ± SEM from n = 3 biological replicates. (G-l) SpCas9 NHEJ-mediated editing efficiency in HEK-293T. Pie charts show percentages of reads with at least one uATG (G) or both uATG disrupted (H), and mATG elimination (I). Percentages of unedited reads, insertions-carrying reads or other unwanted edits are also shown.

[0140] Figure 11

[0141] (A-J) SpRY-ABE + sgRNA-11 (sg11) and Sp-ABE + sgRNA-1 (sg1) are able to disrupt uATG- 1 and uATG-2 of Senia in Ctrl and Dravet mouse neurons. (A) Schematic representation of adenine base editing sgRNAs tested to install SCN1A / Scn1a uATG>uGTG edits. SpRY- sgRNA-11 and Sp-sgRNA-1 sequences with position of target adenines (As) are shown. (B) Maps of AAV constructs used for the in vitro and in vivo delivery of split-intein Sp / SpRY-ABE8e and relative sgRNA sequence. N-ABE split expresses TadA-8e deaminase domain fused to the N-terminal part of nSpRY / nSpCas9 as wells as the sgRNA cassette under the human U6 RNA pol.lll promoter. C-ABE split co-expresses the C-terminal part of nSpRY / nSpCas9 and P2A-tagRFP. Both split-intein constructs are under the control of CbH strong promoter. NLS: nuclear localization signal, intein: Npu N / C intein. (C) Schematic depicting the experimental timeline: cortical and hippocampal neurons were derived from E17.5-18.5 mice, either Ctrl or Dravet, and, following three days in culture were transduced with AAVs carrying base editors and sgRNA. At day 21 , genomic DNA was extracted for deep sequencing and membrane- associated protein lysates were obtain for NaV1.1 levels assessment. (D) Quantification of editing efficiency is shown as percentage (%) of sequencing reads harboring at least one uATG edit (left) or both uATG edits (right) for both SpRY-ABE and Sp-ABE in presence or absence of relative sgRNA in mouse neurons. Sequencing reads are grouped as harboring target edit, insertions / deletions (indels), wild-type non-modified sequence or other non-target edits. Data are shown as means ± SEM from n = 3 biological replicates. Reads with at least one uATG edit: SpRY-ABE Ctrl: 0%±0%, SpRY-ABE + sg11 : 18.19%±1.14%, Sp-ABE Ctrl: 0%±0%, Sp-ABE + sg1 : 61.18%±1.37%. Reads with both uATG edits: SpRY-ABE Ctrl: 0%±0%, SpRY-ABE + sg11 : 9.26%±0.80%, Sp-ABE Ctrl: 0%±0%, Sp-ABE + sg1 : 12.95%±0.17%. (E, F) Distribution of A>G conversions (%) among the sgRNA-11 (E) and sgRNA-1 (F) protospacer sequences in transduced mouse primary neurons. Target adenines, belonging to uATGs, are marked by triangles and shown in bold. Data are shown as means ± SEM from n = 3 independent biological replicates. (G) Representative peaks (left) and relative quantification (right) obtained by Jess Automated Western Blot analysis of transduced mouse neurons, either Ctrl or Dravet untreated (Ctrl) or treated with either SpRY-ABE sg11 or Sp- ABE sg1. NaV1.1 protein quantification is determined by fold change of peak area over Ctrl untreated neurons (first bar). Data are shown as means ± SEM from at least n=5 biological replicates. Statistical significance was assessed using one-way ANOVA with Dunnett’s multiple comparison test [ns: p-value > 0.05; *: p-value < 0.05; *** p-value < 0.001], (H, I) Inputoutput (IO) curve showing the number of action potentials (AP) fired in response to 500ms current injection steps of increasing intensity (0 to 800 pA; delta = 20pA) by Scn1aSTOP / + pyramidal neurons (PYR, H) and inhibitory neurons (IN, I) in the Ctrl condition compared to neurons transduced with the ABE_uORF (PYR *** P = 0,0005, mixed-effects analysis via REML; IN *** P = 0,0010, mixed-effects analysis via REML, n indicated on each graph). Maximal AP number extrapolated from IO curve (PYR *P = 0,0140, unpaired t test; IN ** P = 0,0074, Mann-Whitney test) are shown on the right. (J) Representative Western Blot (WB) of NaV1.1 and Calnexin (Calnx) protein levels in Ctrl and Dravet mouse-derived neurons, untreated or treated with either SpRY-ABE + sg11 or Sp-ABE + sg1. Predicted molecular weights (kDa) are also highlighted. (K) Schematic depicting experimental workflow of mouse neurons patched for electrophysiological recordings at day 14-16. (L) Representative pictures of a transduced (GFP+) pyramidal neuron patched for electrophysiological recordings at day 14-16, visualized through epifluorescence (left) and infrared-differential interference contrast (IR-DIC, right) under a 40X objective (scale bar = 20pm). (M) Representative voltage traces of the AP fired in response to 500 and 800 pA current stimulation steps by Scn1aSTOP / + pyramidal neurons of the ABE_Ctrl condition (left, gray) or transduced with the ABE_uORF vectors (right, light blue). (N) Box plots of membrane excitability parameters showing no changes in resting membrane potential (P = 0,7220) or threshold potential (P = 0,7053), but a significant reduction in the rheobase (* P = 0,0421) and current threshold density (* P = 0,0346) in Scn1aSTOP / + pyramidal neurons upon transduction with ABE_uORF compared to control (n = 9 cells ABE_Ctrl and 12 cells ABE_uORF; all comparisons by unpaired t test). (O) Representative pictures of a transduced (GFP+) inhibitory neuron patched for electrophysiological recordings at days 14-16, visualized through epifluorescence (left) and infrared-differential interference contrast (IR-DIC, right) under a 40X objective (scale bar = 20pm). (P) Representative voltage traces of the AP fired in response to 500 and 800 pA current stimulation steps by Scn1aSTOP / + INs of the ABE_Ctrl condition (left, gray) or transduced with the ABE_uORF vectors (right, light blue). (Q) Box plots of membrane excitability parameters showing no changes in resting membrane potential (P = 0, 9845) or threshold potential (P = 0,3977), but a significant reduction in the rheobase (** P = 0,0093) and current threshold density (** P = 0,0069) in Scn1aSTOP / + INs upon transduction with the base editing system compared to control (n = 18 cells ABE_Ctrl and 12 cells ABE_uORF; all comparisons by unpaired t test). Figure 12

[0142] (A-D) Sp-ABE + sg1 (ABE_uORF) is able to disrupt uATG-1 and uATG-2 in human neurons.

[0143] (A) Timeline of NPC differentiation protocol to obtain human neurons and ABE editing. NPCs were exposed to SHH / CHIR, plated on day 7, and differentiated with BDNF / GDNF. ABE transduction occurred on day 21 , followed by analysis (deep sequencing / IF) at days 35-40.

[0144] (B) Immunofluorescence (IF) panels illustrate GABAergic neurons, with co-localization of Map2 and Hoechst, Map2 and Gaba or Map2 and Cas9 and co-localization of Gaba and Cas9. Relative quantification of Gaba+ / Map2+, Cas9+ / Map2+ and Cas9+ / Gaba+ is shown on the bottom-right. Scale bar: 10 urn. (C) Quantification of editing efficiency is shown as percentage (%) of sequencing reads harboring at least 1 uATG edit (left) or both uATG edits (right). Reads with at least one uATG edit: Sp-ABE Ctrl: 0%±0%, Sp-ABE + sg1 : 41.63% ± 2.14%. Reads with both uATG edits: Sp-ABE Ctrl: 0%±0%, Sp-ABE + sg1 : 6.20% ± 0.44%. Data are shown as means ± SEM from n = 3 biological replicates. (D) Distribution of A>G conversions (%) among the sgRNA-1 protospacer sequence in transduced human neurons. Target adenines are marked by triangles and shown in bold. Data are shown as means ± SEM from n = 3 biological replicates.

[0145] Figure 13

[0146] (A-D) Both SpRY-ABE + sg 11 and Sp-ABE + sg1 show limited off-target effects (A) Schematic representation showing workflow for off-target editing activity determination at predicted off- target (OT) sites in mouse neurons. OT sites were in silico nominated using CRISPOR algorithm based on the alignment of the candidate sgRNA sequence to the whole genome. Including criteria for the search were: <3 mismatches, exonic and with a cfd>0. Next, OT sites were PCR-amplified from genomic DNA of transduced murine neurons to insert Illumina adapters and NGS allowed quantification of OT editing activity. (B) Percentage of sequencing reads carrying A>G substitutions in the editing window of CRISPOR nominated OT sites in transduced mouse neurons. OT editing % of neurons treated with Ctrl ABE are labeled with hollow circles, and those treated by ABE + sg are labeled with solid circles. (C) Table shows Senia (inclusion of exon A) or isoform 2 (inclusion of exon B) 5’IITR sequences harboring distinct combination of A>G conversions in uATGs (ATG triplets are highlighted) and in other As representing bystander edits (i.e. unwanted conversions in the quantification window, highlighted in the scheme with lighter grey). Variants were cloned in GFP-IRES-mCherry reporter vector and screened in HEK-293T cells as previously described. (D) Quantification of translation enhancement determined as GFP / mCherry expression for Senia bystanders- carrying variants. Data are shown as means ± SEM from n = 4 biological replicates. Numbers indicate the fold change increase of the variants over Senia wild-type 5’IITR sequence (indicated by the dashed line). Statistical significance was assessed using unpaired t-test [ns: p-value > 0.05; *: p-value < 0.05],

[0147] Figure 14

[0148] (A-H) Disruption of uATG-3 increases Senia isoform 1 and isoform 2 expression alone and in combination with disruption of uATG-1 and uATG-2. (A) Schematic depicting the experimental setting for testing uATG3 variants: uATG variants of Senia isoform 1 (inclusion of exon A) or isoform 2 (inclusion of exon B) 5’llTRs were cloned in GFP-IRES-mCherry reporter vector and screened in HEK-293T cells as previously described. Table shows tested variants carrying wild-type Senia 5’IITR sequence or distinct combination of A>G conversions in in uATG-1 , uATG-2 and uATG-3. All uATGs and mATG are highlighted. B) Quantification of translation enhancement determined as GFP / mCherry expression for Senia variants. Data are shown as means ± SEM from n = 4 biological replicates. Numbers indicate the fold change increase of the variants over Senia wild-type 5’IITR sequence (indicated by the dashed line). Statistical significance was assessed using unpaired t-test [ns: p-value > 0.05; *: p-value < 0.05; **: p- value < 0.01 ; *** p-value < 0.001 ; **** p-value < 0.0001], C) Schematic representation of Sp- ABE sgRNAs tested in transduced mouse neurons to install Senia uATG-3 edits (T>C on forward strand). Sequences of Sp-sgRNA-1 , used to edit uATG-1 and uATG-2 and Sp-sgRNA- 3.1 , 3.2 and 3.3, used to edit uATG-3, are shown. D-E-F) Representative Sanger sequencing chromatograms showing A>G conversions at the target site following ABE delivery in mouse neurons with sgRNA-3.1 (D), 3.2 (E) or 3.3 (F). Editing quantification was made using BEAT tool. Target adenine and corresponding base conversion are highlighted. G) Distribution of NGS A>G conversions (%) among sgRNA-3.1 sequence in mouse neurons transduced with Ctrl ABE, Sp-ABE + sg3.1. Target adenine is marked by triangles and shown in bold. H) Quantification of editing efficiency is shown as percentage (%) of NGS reads harboring uATG- 3 edit Ctrl and Sp-ABE + sg3.1. Data are shown as means ± SEM from n = 3 biological replicates.

[0149] Figure 15

[0150] (A-K) AAV -based delivery of ABE_uORF is able to increase Nav1.1 levels in the cerebral context and hippocampus of Dravet mice. (A) Schematic of experimental design: neonatal (P0-P1) control and Dravet mice received intracerebroventricular (ICV) injection of dual AAV vectors (low dose: 2x109 vg / AAV / mouse) encoding split Sp-ABE8e (N-ABE and C-ABE) and sgRNA-1 . Mice were monitored for survival, and at postnatal day 30 (P30), underwent thermal seizure induction increasing body temperature of 0.5 °C every 30 s, followed by brain collection for deep sequencing and protein analysis. (B) Survival curve up to P30 of Ctrl and Dravet mice, treated with Ctrl ABE without sgRNA-1 (ABE_Ctrl) or ABE + sgRNA-1 (ABE_uORF) with relative sample size shown. (C) Percentage of Ctrl and Dravet mice, both treated with ABE_Ctrl or ABE_uORF remaining seizure-free after the thermal induction protocol at P30. (D, E) Distribution of A>G conversions (%) among sgRNA-1 sequence (D) and quantification of editing efficiency (% of reads harboring at least one uATG edited) (E) in cerebral cortex of ABE_uORF and ABE_Ctrl treated Dravet mice. T arget adenines are marked by triangles and shown in bold. Data are shown as means ± SEM from n = 3 biological replicates. Reads with at least one uATG edit: ABE_Ctrl: 0.09%±0.09%, ABE_uORF: 17.11 % ± 4.02%. (F, G) Representative WB of NaV1.1 cortical protein levels of Ctrl and Dravet mice treated with ABE_Ctrl or ABE_uORF (F) and their relative band densitometry quantification normalized over Calnx (G). Data are shown as means ± SEM from at least n=4 biological replicates. Statistical significance was assessed using one-way ANOVA with Sidak’s multiple comparison test [**: p-value < 0.01 ; *** p-value < 0.001], (H, I) Distribution of A>G conversions (%) among sgRNA-1 sequence (H) and quantification of editing efficiency (% of reads harboring at least one uATG edited) (I) in hippocampus of ABE_uORF and ABE_Ctrl treated Dravet mice. Target adenines are marked by triangles and shown in bold. Data are shown as means ± SEM from n = 3 biological replicates. Reads with at least one uATG edit: ABE_Ctrl: 0.09% ± 0.09%, ABE_uORF: 5.81 % ± 1.45%. (J, K) Representative WB of NaV1.1 hippocampal protein levels of Ctrl and Dravet mice treated with ABE_Ctrl or ABE_uORF (J) and their relative band densitometry quantification normalized over Calnx (K). Data are shown as means ± SEM from at least n=4 biological replicates. Statistical significance was assessed using one-way ANOVA with Sidak’s multiple comparison test [*: p-value < 0.05; *** p-value < 0.001],

[0151] Figure 16

[0152] (A-M) Increasing the dose of AAV-based delivery of ABE_uORF further increases Nav1.1 levels in the cerebral context and hippocampus of Dravet mice and increases the survival of Dravet mice. (A) Schematic of experimental design: neonatal (P0-P1) control and Dravet mice received intracerebroventricular (ICV) injection of dual AAV vectors (high dose: 1x1010 vg / AAV / mouse) encoding split Sp-ABE8e (N-ABE and C-ABE) and sgRNA-1. Mice were monitored for survival, and at P30, underwent thermal seizure induction increasing body temperature of 0.5 °C every 30 s, followed by brain collection for immunofluorescence (IF), deep sequencing, and protein analysis. (B) Representative IF of sagittal brain section from Dravet mouse injected with ABE_uORF AAVs. RFP expression represents transduction efficiency of C-ABE AAV across multiple brain regions (Visual cortex (Vis), dentate gyrus (DG), CA1-CA3 hippocampal subfields, thalamus (Th), hypothalamus (Hyp), piriform cortex (Pyr), auditory cortex (AuD), ectorhinal cortex (Ect), and entorhinal cortex (Ent). (B'-B'") Higher magnification representative fields of of hippocampal CA1 (B'), auditory cortex (AuD, B"), and entorhinal cortex (Ent, B"') regions. Nuclei are counterstained with Hoechst. (C) Survival curve up to P30 of Ctrl and Dravet mice, treated with ABE_Ctrl or ABE_uORF with relative sample size shown. [**** p-value < 0.0001 ; Log-rank Mantel-Cox test], (D) Percentage of Ctrl and Dravet mice, both treated with ABE_Ctrl or ABE_uORF remaining seizure-free after the thermal induction protocol at P30 [**** p-value < 0.0001 ; Log-rank Mantel-Cox test], (E) Pie chart showing the proportion (%) of Dravet mice treated with ABE_Ctrl or ABE_uORF experiencing seizures (grey) or being seizure free (white) after being subjected to thermal induction protocol. Sample size is also shown. (F, G) Distribution of A>G conversions (%) among sgRNA-1 sequence (F) and quantification of editing efficiency (% of reads harboring at least one uATG edited) (G) in cerebral cortex of ABE_uORF and ABE_Ctrl treated Dravet mice. Target adenines are marked by triangles and shown in bold. Data are shown as means ± SEM from n = 3 biological replicates. Reads with at least one uATG edit: ABE_Ctrl: 0%±0%, ABE_uORF: 29.13% ± 1.67%. (H, I) Representative WB of NaV1.1 cortical protein levels of Ctrl and Dravet mice treated with ABE_Ctrl or ABE_uORF (H) and their relative band densitometry quantification normalized over Calnx (I). Data are shown as means ± SEM from at least n=5 biological replicates. Statistical significance was assessed using one-way ANOVA with Sidak’s multiple comparison test [*: p-value < 0.05; **: p-value < 0.01 ; **** p-value < 0.0001], (J, K) Distribution of A>G conversions (%) among sgRNA-1 sequence (J) and quantification of editing efficiency (% of reads harboring at least one uATG edited) (K) in hippocampus of ABE_uORF and ABE_Ctrl treated Dravet mice. Target adenines are marked by triangles and shown in bold. Data are shown as means ± SEM from n = 3 biological replicates. Reads with at least one uATG edit: ABE_Ctrl: 0.09% ± 0.09%, ABE_uORF: 7.84% ± 1.08%. (L, M) Representative WB of NaV1.1 hippocampal protein levels of Ctrl and Dravet mice treated with ABE_Ctrl or ABE_uORF (L) and their relative band densitometry quantification normalized over Calnx (M). Data are shown as means ± SEM from at least n=5 biological replicates. Statistical significance was assessed using one-way ANOVA with Sidak’s multiple comparison test [ns: p-value > 0.05; *: p-value < 0.05; *** p-value < 0.001],

[0153] Figure 17

[0154] (A-M) Disruption of uATG s in the 5’ UTR of SCN2A, SCN3A, SCN5A, SCN8A and SCN9A also expression levels of the respective genes. (A) MUSCLE multiple sequence alignment of uATG-containing 5’UTR regions across SCN paralogs (SCN2A, SCN3A, SCN5A, SCN8A, SCN9A) in human (top) and mouse (bottom). The consensus sequence logo visualizes nucleotide conservation across aligned sequences; the height of each base indicates its degree of conservation at that position. uATGs and mATG are also highlighted. (B) Table shows human / mouse 100% conserved 5’IITR sequences harboring distinct combination of A>G conversions in uATGs (ATG triplets are highlighted). Variants were cloned in GFP-IRES- mCherry reporter vector and screened in HEK-293T cells as previously described. (C) Quantification of translation enhancement determined as GFP / mCherry expression for SON paralogs. Data are shown as means ± SEM from n = 3 biological replicates. Numbers indicate the fold change increase of the variants over the respective wild-type sequence (indicated by the dashed line). Statistical significance was assessed using unpaired t-test [ns: p-value > 0.05; *: p-value < 0.05; **: p-value < 0.01 ; *** p-value < 0.001], (D) Schematic depicting SpRY- ABE sgRNA-2A1 designed to install Scn2a uATG>uGTG edit in mouse primary neurons. Protospacer sequence is 100% conserved in human and mouse. Position of target A (uATG) and mATG A in the sgRNA protospacer are also shown. (E, F) Quantification of editing efficiency (% of reads) (E) and distribution of A>G conversions (%) among sgRNA-2A1 sequence (F) of Ctrl and SpRY-ABE + sg2A1 treated mouse neurons. Target adenine is marked by triangle and shown in bold. Data are shown as means ± SEM from n = 3 biological replicates. Reads with uATG edit: Ctrl: 0.28% ± 0.28%, SpRY-ABE + sg2A1 : 28.71 % ± 0.39%. (G, H) Representative WB of NaV1 .2 protein levels of Ctrl mouse neurons treated with Ctrl or SpRY-ABE + sg2A1 (G) and their relative band densitometry quantification normalized over Calnx (H). Data are shown as means ± SEM from at least n=4 biological replicates. Statistical significance was assessed using unpaired t-test [*** p-value < 0.001], (I) Schematic depicting Sp-ABE sgRNA-8a3 designed to install Scn8a uATG>uGTG edit in mouse primary neurons. Protospacer sequence is mouse-specific. Position of target A (uATG) and mATG A in the sgRNA protospacer are also shown. (J, K) Quantification of editing efficiency (% of reads) (J) and distribution of A>G conversions (%) among sgRNA-8a3 sequence (K) of Ctrl and Sp-ABE + sg8a3 treated mouse neurons. Target adenine is marked by triangle and shown in bold. Data are shown as means ± SEM from n = 3 biological replicates. Reads with uATG edit: Ctrl: 0.28% ± 0.28%, Sp-ABE + sg8a3: 27.87% ± 1.54%. %. (L, M) Representative WB of NaV1.6 protein levels of Ctrl mouse neurons treated with Ctrl or Sp-ABE + sg8a3 (L) and their relative band densitometry quantification normalized over Calnx (M). Data are shown as means ± SEM from n=3 biological replicates. Statistical significance was assessed using unpaired t-test [*: p-value < 0.05],

[0155] Figure 18

[0156] (A-H) Cas9-guided adenine base editors and uATG-targeting guide RNAs can effectively disrupt uATGs in human SCN2A and SCN8A and mouse Scn2a and Scn8a. (A) Schematic representation of SCN2A human target site and tested adenine base editing sgRNAs. uORF and mORF are also highlighted in the scheme. (B) Representative Sanger sequencing chromatograms showing A>G conversions at the SCN2A target site following ABE delivery in HEK-293T cells. Editing quantification was made using BEAT tool. (C) Schematic representation of Scn2a mouse target site and tested adenine base editing sgRNAs. uORF and mORF are also highlighted in the scheme. Sp or SpRY-ABE8e associated with each sgRNA are also indicated. (D) Representative Sanger sequencing chromatograms showing A>G conversions at the Scn2a target site following ABE delivery in murine primary neurons. Editing quantification was made using BEAT tool. (E) Schematic representation of SCN8A human target site and tested adenine base editing sgRNAs. uORF and mORF are also highlighted in the scheme. (F) Representative Sanger sequencing chromatograms showing A>G conversions at the SCN2A target site following ABE delivery in HEK-293T cells. Editing quantification was made using BEAT tool. (G) Schematic representation of Scn8a mouse target site and tested adenine base editing sgRNAs. uORF and mORF are also highlighted in the scheme. Sp or SpRY-ABE8e associated with each sgRNA are also indicated. (H) Representative Sanger sequencing chromatograms showing A>G conversions at the Scn8a target site following ABE delivery in murine primary neurons. Editing quantification was made using BEAT tool.

[0157] Figure 19

[0158] (A-J) Disruption of exon 20N splice acceptor site boosts increases productive Senia mRNA levels. (A) Human SCN1A locus: schematic representation of exon 20, exon 21 , and the intronic region containing the poison exon (20N). Candidate sgRNAs targeting exon 20N splice acceptor site (arrows) and intronic regions surrounding exon 20N are shown. (B) Mouse Senia locus: corresponding schematic of the orthologous region in the mouse genome, including exon 20N. sgRNAs were designed to target murine exon 20N splice acceptor site with sgRNA- ex20N_1 sequence being 100% human-mouse conserved. Dashed lines represent exon junctions. Canonical AG-GT splice sites and the location of exon 20N AG splice acceptor site (SA) are also indicated. (C) Schematic representation of ABE-mediated splicing modulation of exon 20N inclusion in SCN1A / Scn1a mRNA. Inclusion of poison exon 20N between exons 20 and 21 results in nonproductive splicing leading to transcript degradation by NMD. Adenine base editing at exon 20N SA prevents its inclusion, thereby restoring productive splicing between exon 20 and exon 21. Schematics of the spliced transcript isoforms corresponding to nonproductive (with exon 20N) and productive (without exon 20N) are also shown. Nucleotide lengths obtained by RT-PCR of each splice isoform are also indicated. (D-G) Representative Sanger sequencing chromatograms showing A>G conversions at the target site following ABE delivery in either HEK-293T cells (D, E, F) and mouse neurons (G). Editing quantification was made using BEAT tool. (H) TBE-PAGE of murine RT-PCR products of Senia productive and nonproductive transcript. Total RNA was extracted from DIVIO transduced mouse neurons and RT-PCR was performed using primers flanking Senia exon 20N to detect the nonproductive isoform (upper band, 562 bp) and the productive isoform lacking exon 20N (lower band, 498 bp). (I, J) Percentage of exon 20N inclusion (I) and productive isoform expression (J) in Senia transcripts, quantified from the RT-PCR products shown in Figure 10H. Band intensities were measured by densitometry, and values are plotted as the proportion of either the exon 20N-containing (nonproductive) or the productive isoform relative to total Senia transcript (sum of both isoforms).

[0159] DETAILED DESCRIPTION OF THE INVENTION

[0160] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise.

[0161] The terms "comprising", "comprises" and "comprised of' as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps. The terms "comprising", "comprises" and "comprised of” also include the term "consisting of”.

[0162] The term “subject” as used herein refers to either a human or non-human animal. Examples of non-human animals include vertebrates, for example mammals, such as non-human primates (particularly higher primates), dogs, rodents (e.g. mice, rats or guinea pigs), pigs and cats. The non-human animal may be a companion animal. Preferably, the subject is human.

[0163] The term “expression”, as used herein, refers to the production of a functional end-product (e.g. a guide polynucleotide or a protein).

[0164] The terms “5’ untranslated region” and “5’ UTR” refers to RNA sequences upstream of a coding sequence on an mRNA transcript which includes regulatory cis-acting elements which affect mRNA translation.

[0165] The term “RNA transcript” or “mRNA” transcript refers to the product resulting from RNA polymerase-catalysed transcription of a DNA sequence. The term “messenger RNA” or “mRNA” refers to RNA that can be translated into a protein in a cell.

[0166] The term “open reading frame” refers to a continuous gene or mRNA transcript sequence between a start (e.g. ATG) and stop codon, which, if in the form of an RNA template, can be translated into a polypeptide by the ribosome. It begins with a start codon (canonically, ATG in DNA or AUG in RNA, which codes for methionine, but also non-canonical start codons) and ends with a stop codon (such as TAA, TAG, or TGA in DNA or UAA, UAG, UGA in RNA).

[0167] A “deaminase” is an enzyme or enzyme domain that catalyses a deamination reaction. For example, deamination of adenine with an adenine deaminase results in the formation of hypoxanthine. Hypoxanthine selectively forms a base pair with cytosine instead of thymine. This results in a post-replicative transition mutation, such that the original A«T nucleotide pair is altered into a G*C nucleotide pair.

[0168] “Complementarity” of a sequence to another sequence is measured as a percentage of the number of nucleotides in the latter sequence which can be paired with a complementary nucleotide in the former sequence. Accordingly, if a sequence that binds a target sequence is 80% complementary to the target sequence, this is to be understood as 80% of the nucleotides of the target sequence being paired with a complementary nucleotide in the sequence that binds the targets sequence. A sequence being referred to as “complementary” to another sequence without reference to a percentage value of complementarity, or any other indication that the other sequence cannot be bound by the first sequence in such a way that every nucleotide in the other sequence is paired with a complementary nucleotide in the first sequence, is to be understood as the first sequence being 100% complementary to the other sequence.

[0169] Numeric ranges are inclusive of the numbers defining the range. Unless otherwise indicated, any nucleic acid sequences are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively. Where an element of the invention is described herein as being “between” two end points (e.g. “positions”) denoting a range, this is to be understood as including the end points of that range. For example, where the description refers to the target sequence being located between position -30 and position 4, this may mean the target sequence could range from position -30 to position 4.

[0170] GUIDE POLYNUCLEOTIDES

[0171] In one aspect, the present invention provides a guide polynucleotide comprising a sequence which binds to a target sequence comprising a portion of the 5’ UTR-encoding region of a gene encoding a VGSC alpha subunit (Nav).

[0172] As used herein, a “guide polynucleotide” may confer target sequence specificity to a RNA- guided nuclease. Guide polynucleotides may be non-coding short RNA sequences (guide RNAs or gRNAs) comprising a sequence which bind to a target DNA sequence. For example, in the CRISPR / Cas9 system, the guide polynucleotide binds to the Cas9 enzyme and the gRNA sequence guides the resulting complex via base-pairing to a specific location on the DNA.

[0173] The term “guide polynucleotide” encompasses any suitable RNA molecule that can be used with any RNA-guided nuclease, RNA-guided nickase or catalytically inactivated RNA-guided nuclease, and not only those RNAs that are compatible with a particular nuclease such as Cas9.

[0174] The guide polynucleotide may comprise a trans-activating CRISPR RNA (tracrRNA) that provides the stem loop structure and a target-specific CRISPR RNA (crRNA) comprising the sequence that binds the target sequence, also called the spacer sequence. The tracrRNA and crRNA may be annealed, for example by heating them at 95°C for 5 minutes and letting them slowly cool down to room temperature for 10 minutes. Alternatively, the guide RNA may be a single guide RNA (sgRNA), for example that consists of both the crRNA and tracrRNA as a single construct.

[0175] The guide polynucleotide may comprise of a 3’-end, which forms a scaffold for nuclease binding, and a 5'-end which is programmable to bind a target sequence. For example, the targeting specificity of CRISPR-Cas9 may be determined by the 17-24 bp spacer sequence at the 5' end of the guide RNA. The desired target sequence typically precedes a protospacer adjacent motif (PAM) which is a short DNA sequence usually 2-6 bp in length that follows the DNA region targeted by the CRISPR system, such as a base editor or prime editor comprising an inactive Cas9 enzyme or a Cas9 nickase. The PAM is required for a base editor or prime editor to catalyse a reaction and, in the case of base editors, is typically found 13-17 bp downstream from the nucleotide position desired for alteration.

[0176] Numerous tools exist for designing guide polynucleotides (e.g. Cui, Y., et al., 2018. Interdisciplinary Sciences: Computational Life Sciences, 10(2), pp.455-465). For example, COSMID is a web-based tool for identifying and validating guide RNAs (Cradick TJ, et al. Mol Ther - Nucleic Acids. 2014;3(12):e214). Other examples include Hwang et al. (2018) BMC Bioinformatics 19, 542; and algorithms, such as Mandana Arbab et al. "Determinants of Base Editing Outcomes from Target Library Analysis and Machine Learning." Cell, 2020.

[0177] In some embodiments, the target sequence is 17, 18, 19, 20, 21 , 22, 23 or 24 nucleotides long. In some embodiments, the target sequence is at least 17, at least 18, at least 19, at least 20, at least 21 , at least 22, at least 23 or at least 24 nucleotides long. In some embodiments, the target sequence is 18 nucleotides long. In some embodiments, the target sequence is 19 nucleotides long. In some embodiments, the target sequence is 20 nucleotides long. In some embodiments, the target sequence is 21 nucleotides long. In some embodiments, the target sequence is 22 nucleotides long. In some embodiments, that target sequence is located in the endogenous locus of human SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN7A, SCN8A, SCN9A, SCN10A or SCN11A. In some embodiments, the target sequence is located in the endogenous locus of mouse Senia, Scn2a, Scn3a, Scn4a, Scn5a, Scn7a, Scn8a, Scn9a, ScnIOa or Scn11a. Human SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A,

[0178] SCN10A and SCN11A share strong sequence conservation with their mouse orthologues.

[0179] Accordingly, in some embodiments, the target sequence in human SCN1A is identical to the target sequence in mouse Senia, the target sequence in human SCN2A is identical to the target sequence in mouse Scn2a, the target sequence in human SCN3A is identical to the target sequence in mouse Scn3a, the target sequence in human SCN4A is identical to the target sequence in mouse Scn4a, the target sequence in human SCN5A is identical to the target sequence in mouse Scn5a, the target sequence in human SCN7A is identical to the target sequence in mouse Scn7a, the target sequence in human SCN8A is identical to the target sequence in mouse Scn8a, the target sequence in human SCN9A is identical to the target sequence in mouse Scn9a, the target sequence in human SCN10A is identical to the target sequence in mouse ScnIOa, or the target sequence in human SCN11A is identical to the target sequence in mouse Scn11a.

[0180] T arget sequences of guide polynucleotides may also be referred to as protospacer sequences. Exemplary protospacer sequences of SCN1A / Scn1a, SCN2A / Scn2a and SCN8A / Scn8a are shown below. Where the target sequence is identical between the human and mouse orthologue, the title of the sequence indicates both genes:

[0181] SCN1A / Scn1a protospacer sequence 11 (SEQ ID NO: 1)

[0182] ATCTTGTCATCCTGCACATT

[0183] SCN1A / Scn1a protospacer sequence 1 (SEQ ID NO: 2)

[0184] TCTTGTCATCCTGCACATTT

[0185] SCN2A / Scn2a protospacer sequence 2A 1 (SEQ ID NO: 3)

[0186] TGCCATCTTTTCATCCTGCT

[0187] SCN2A / Scn2a protospacer sequence 2A2 (SEQ ID NO: 4)

[0188] GTGCCATCTTTTCATCCTGC

[0189] Scn2a protospacer sequence 2a3 (SEQ ID NO: 5) ATTGTGCCATCTTTTCATCC

[0190] Scn2a protospacer sequence 2a4 (SEQ ID NO: 6)

[0191] TTGTGCCATCTTTTCATCCT

[0192] SCN2A protospacer sequence 2A5 (SEQ ID NO: 7)

[0193] TCTTTTCATCCTGCTCCTTT

[0194] Scn2a protospacer sequence 2a6 (SEQ ID NO: 8)

[0195] TCTTTTCATCCTGCTTCTTT

[0196] SON 8 A protospacer sequence 8A1 (SEQ ID NO: 9)

[0197] CTGCCATCTTCTCATCCTGC

[0198] Scn8a protospacer sequence 8a2 (SEQ ID NO: 10)

[0199] CTGCCATCTTCTCATTCTGC

[0200] SCN8A protospacer sequence 8A2 (SEQ ID NO: 11)

[0201] CGCTGCCATCTTCTCATCCT

[0202] Scn8a protospacer sequence 8a3 (SEQ ID NO: 12)

[0203] CGCTGCCATCTTCTCATTCT

[0204] SCN8A protospacer sequence 8A3 (SEQ ID NO: 13)

[0205] GCGCTGCCATCTTCTCATCC

[0206] Scn8a protospacer sequence 8a4 (SEQ ID NO: 14)

[0207] GCGCTGCCATCTTCTCATTC

[0208] Thus, in some embodiments, the gene encoding the VGSC alpha subunit (Nav) is human SCN1A and the target sequence is the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the gene encoding the VGSC alpha subunit (Nav) is mouse Senia and the target sequence is the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2. In some embodiments, the gene encoding the VGSC alpha subunit (Nav) is human SCN2A and the target sequence is the sequence set forth in any one of SEQ ID NO: 3, SEQ ID NO: 4, or SEQ ID NO: 7. In some embodiments, the gene encoding the VGSC alpha subunit (Nav) is mouse Scn2a and the target sequence is the sequence set forth in any one of SEQ ID NOs: 3-6 or SEQ ID NO: 8. In some embodiments, the gene encoding the VGSC alpha subunit (Nav) is human SCN8A and the target sequence is the sequence set forth in any one of SEQ ID NO: 9, SEQ ID NO: 11 or SEQ ID NO: 13. In some embodiments, the gene encoding the VGSC alpha subunit (Nav) is mouse Scn8a and the target sequence is the sequence set forth in any one of SEQ ID NO: 10, SEQ ID NO: 12 or SEQ ID NO: 14.

[0209] In some embodiments, the sequence binding to the target sequence is 20 nucleotides long. The sequence of a guide polynucleotide that binds the target or protospacer sequence may also be referred to as spacer sequence. In some embodiments, the spacer sequence is 100% complementary to the target or protospacer sequence. For example, the spacer sequences of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 , SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25 are 100% complementary to the protospacer sequences of SEQ ID NO: 1 , SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, respectively.

[0210] The sequences of SEQ ID NOs: 15-28 are provided below:

[0211] Spacer sequence of sqRNA-11 (SEQ ID NO: 15)

[0212] AATGTGCAGGATGACAAGAT

[0213] Spacer sequence ofsqRNA-1 (SEQ ID NO: 16)

[0214] AAATGTGCAGGATGACAAGA

[0215] Spacer sequence of sqRNA-2A1 (SEQ ID NO: 17)

[0216] AGCAGGATGAAAAGATGGCA

[0217] Spacer sequence of sqRNA-2A2 (SEQ ID NO: 18)

[0218] GCAGGATGAAAAGATGGCAC

[0219] Spacer sequence ofsqRNA-2a3 (SEQ ID NO: 19)

[0220] GGATGAAAAGATGGCACAAT

[0221] Spacer sequence ofsqRNA-2a4 (SEQ ID NO: 20) AGGATGAAAAGATGGCACAA

[0222] Spacer sequence ofsqRNA-2A5 (SEQ ID NO: 21)

[0223] AAAGGAGCAGGATGAAAAGA

[0224] Spacer sequence ofs RNA-2a6 (SEQ ID NO: 22)

[0225] AAAGAAGCAGGATGAAAAGA

[0226] Spacer sequence of sqRNA-8A1 (SEQ ID NO: 23)

[0227] GCAGGATGAGAAGATGGCAG

[0228] Spacer sequence ofsqRNA-8a2 (SEQ ID NO: 24)

[0229] GCAGAATGAGAAGATGGCAG

[0230] Spacer sequence of sqRNA-8A2 (SEQ ID NO: 25)

[0231] AGGATGAGAAGATGGCAGCG

[0232] Spacer sequence ofsqRNA-8a3 (SEQ ID NO: 26)

[0233] AGAATGAGAAGATGGCAGCG

[0234] Spacer sequence of sqRNA-8A3 (SEQ ID NO: 27)

[0235] GGATGAGAAGATGGCAGCGC

[0236] Spacer sequence ofsqRNA-8a4 (SEQ ID NO: 28)

[0237] GAATGAGAAGATGGCAGCGC

[0238] In some embodiments, the sequence binding to the target sequence comprises or consists of any one of the sequences set forth in SEQ ID NOs: 29-43 below.

[0239] Spacer sequence ofsqRNA-3. 1 (SEQ ID NO: 29)

[0240] CTCTTCATGTGAGATTCCTA

[0241] Spacer sequence ofsqRNA-3.2 (SEQ ID NO: 30)

[0242] TTCATGTGAGATTCCTAAGG

[0243] Spacer sequence ofsqRNA-3.3 (SEQ ID NO: 31) TCATGTGAGATTCCTAAGGA

[0244] Spacer sequence ofsqRNA-3.4 (SEQ ID NO: 32)

[0245] ATTCTGCATATGAAATTCCT

[0246] Spacer sequence ofs RNA-3.5 (SEQ ID NO: 33)

[0247] TTCTGCATATGAAATTCCTA

[0248] Spacer sequence ofsqRNA-3.6 (SEQ ID NO: 34)

[0249] CTGCATATGAAATTCCTAAA

[0250] Spacer sequence ofsqRNA-4. 1 (SEQ ID NO: 35)

[0251] AAATGGTAATTAAAATGTGC

[0252] Spacer sequence ofsqRNA-4.2 (SEQ ID NO: 36)

[0253] AATAAATGGTAATTAAAATG

[0254] Spacer sequence ofsqRNA-4.3 (SEQ ID NO: 37)

[0255] AGAATAAATGGTAATTAAAA

[0256] Spacer sequence of sqRNA-3A1 (SEQ ID NO: 38)

[0257] AAATGTGCAGGATGAAAAGA

[0258] Spacer sequence of sqRNA-3A2 (SEQ ID NO: 39)

[0259] AATGTGCAGGATGAAAAGAT

[0260] Spacer sequence of sqRNA-5A1 (SEQ ID NO: 40)

[0261] CCAGAAGCAGGATGAGAAGA

[0262] Spacer sequence of sqRNA-5A2 (SEQ ID NO: 41)

[0263] AGCAGGATGAGAAGATGGCA

[0264] Spacer sequence ofsqRNA-5A3 human (SEQ ID NO: 42)

[0265] CTCATCCTGCTTCTGGGCAC Spacer sequence ofsqRNA-5A3 mouse (SEQ ID NO: 43)

[0266] CATCCTGCTTCTGGGGGCAC

[0267] In another aspect, the present invention provides a guide polynucleotide comprising a sequence which binds to a target sequence comprising a portion of the 5’ splice acceptor site (SA) of an exon of a gene encoding a voltage-gated sodium channel (VGSC) alpha subunit (Nav).

[0268] In some embodiments, the sequence binding to the target sequence is 20 nucleotides long. The sequence of the guide polynucleotide that binds the target sequence may also be referred to as spacer sequence. In some embodiments, the spacer sequence is 100% complementary to the target or protospacer sequence. In some embodiments, the spacer sequence comprises or consists of any one of the sequences detailed below:

[0269] Spacer sequence of sqRNA-ex20N 1 human / mouse (SEQ ID NO: 44)

[0270] AGGATAATCTTGCTCCAACT

[0271] Spacer sequence of sqRNA-ex20N 2 human (SEQ ID NO: 45)

[0272] TATTTTGTATAGGATAATCT

[0273] Spacer sequence of sqRNA-ex20N 3 human (SEQ ID NO: 46)

[0274] AAGATTATCCTATACAAAAT

[0275] Spacer sequence ofsqRNA-ex20N 4 human (SEQ ID NO: 47)

[0276] TTTGTATAGGATAATCTTGC

[0277] Spacer sequence ofsqRNA-ex20N 5 human (SEQ ID NO: 48)

[0278] GTATAGGATAATCTTGCTCC

[0279] Spacer sequence of sqRNA-ex20N 2 mouse (SEQ ID NO: 49)

[0280] TATTTTATATAGGATAATCT

[0281] Spacer sequence of sqRNA-ex20N 3 mouse (SEQ ID NO: 50)

[0282] AAGATTATCCTATATAAAAT

[0283] Spacer sequence of sqRNA-ex20N 4 mouse (SEQ ID NO: 51) TTTATATAGGATAATCTTGC

[0284] Spacer sequence of sqRNA-ex20N 5 mouse (SEQ ID NO: 52)

[0285] ATATAGGATAATCTTGCTCC

[0286] In some embodiments relating to any aspect of the present invention, the guide polynucleotide is suitable for use with a specific adenine base editor, for example any one of SpRYCas9- ABEmax, SpCas9-ABEmax, SpRYCas9-ABE8e and SpCas9-ABE8e. In some embodiments, the guide polynucleotide is suitable for use with SpRYCas9-ABE8e. In some embodiments, the guide polynucleotide is suitable for use with SpCas9-ABE8e.

[0287] In some embodiments, the target or protospacer sequence precedes a protospacer-adjacent motif (PAM) sequence on the opposite strand of the DNA strand comprising the target or protospacer sequence.

[0288] ABOLISHING A uORF

[0289] The present invention discloses a method of editing a 5’ UTR-encoding region of a gene encoding a VGSC alpha subunit (Nav) comprising contacting the 5’ UTR-encoding region with a guide polypeptide and a base editor or prime editor. In some embodiments the guide polypeptide targets the base editor to affect an alteration to abolish a upstream open reading frame (uORF). In some embodiments, the alteration comprises at least a A«T to G*C alteration. In some embodiments, the at least one A«T to G*C alteration is in at least one start codon of the uORF.

[0290] Altering the start codon, for example by changing its A«T nucleotide pair to a G*C nucleotide pair, prevents the initiation complex of translation from recognizing its respective ORF, freeing the initiation complex up to initiate translation of another ORF, such as the main open reading frame (mORF). In a gene comprising a mORF and several uORFs, abolishing a uORF by preventing initiation of its translation is therefore able to shift translation in favour of the remaining ORFs. The applicants have surprisingly found that in a gene comprising multiple uORFs, altering the start codon of a single uORF can significantly increase expression levels of the protein encoded by the mORF. In some embodiments, abolishing a uORF by altering the start codon improves expression of the mORF of the gene encoding a VGSC alpha subunit (Nav) by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or 100% or more. In some embodiments, abolishing a uORF improves expression of the mORF of the gene encoding a VGSC alpha subunit (Nav) by 20% or more. In some embodiments, abolishing a uORF improves expression of the mORF of the gene encoding a VGSC alpha subunit (Nav) by 30% or more. In some embodiments, abolishing a uORF improves expression of the mORF of the gene encoding a VGSC alpha subunit (Nav) by 40% or more. In some embodiments, abolishing a uORF improves expression of the mORF of the gene encoding a VGSC alpha subunit (Nav) by 50% or more. In some embodiments, abolishing a uORF improves expression of the mORF of the gene encoding a VGSC alpha subunit (Nav) by 60% or more. In some embodiments, abolishing a uORF improves expression of the mORF of the gene encoding a VGSC alpha subunit (Nav) by 70% or more. In some embodiments, abolishing a uORF improves expression of the mORF of the gene encoding a VGSC alpha subunit (Nav) by 80% or more. In some embodiments, abolishing a uORF improves expression of the mORF of the gene encoding a VGSC alpha subunit (Nav) by 90% or more.

[0291] Levels of protein expression in a cell population can be assessed with any one of a number of well-established methods with which the skilled person is familiar. For example, in the case of membrane-bound proteins such as a VGSC alpha subunit (Nav), cells can be homogenised using a membrane protein extraction kit (e.g. the Mem-PER Plus Membrane Protein Extraction Kit of Thermo Fisher Scientific). Western blot analysis may then be carried out on the membrane protein-enriched cell lysate using a polyacrylamide gel (e.g. a NuPage 4%-8% gradient gel of Thermo Fisher Scientific) and antibodies against the protein of interest and a control protein ubiquitously expressed in the cell population (e.g. Calnexin or beta-tubulin). Relative protein expression levels between cell populations may be calculated from quantification of, for example, signal intensity of a chemiluminescent signal for the protein of interest, which is proportional to the amount of antibody bound and therefore the amount of the protein of interest in the cell lysate. Quantification can be carried out using any of a number of available software tools.

[0292] Increasing protein expression from the remaining functional gene by abolishing a uORF may therefore compensate for the loss of one allele, without causing overexpression of the relevant protein, which in turn may be associated with certain conditions. Therefore, in some embodiments, the gene encoding a VGSC alpha subunit (Nav) is human SCN1A.

[0293] Since this approach enhances protein expression by abolishing a uORF rather than by improving the translation initiation site of the mORF, it does not require that the sequence immediately around the start codon of the mORF, i.e. the Kozak sequence, can be optimized. As the latter might not always be the case, the approach presented by the present applicants constitutes a reliable way of significantly increasing protein expression without affecting mRNA levels. Further, abolishing a uORF comprising a uATG within a certain distance from the mATG may be particularly effective in improving expression of the mORF. Therefore, in some embodiments, the adenine of the at least one start codon of the uORF is located between position -30 and position -1 in the gene encoding a VGSC alpha subunit (Nav), position -1 being the position of the nucleotide immediately 5’ of the first nucleotide of the main open reading frame (mORF). In some embodiments, the adenine of the at least one start codon of the uORF is located between position -25 and position -1. In some embodiments, the adenine of the at least one start codon of the uORF is located between position -20 and position -1 .

[0294] Additionally, depending on the nature of the Kozak sequence immediately surrounding the mATG, the sequence immediately upstream of the mATG may, for example, not comprise a start codon of a uORF. Accordingly, in some embodiments the adenine of the at least one start codon of the uORF is located between position -30 and position -4. In some embodiments, the adenine of the at least one start codon of the uORF is located between position -30 and position -5. In some embodiments, the adenine of the at least one start codon of the uORF is located between position -30 and position -6. In some embodiments, the adenine of the at least one start codon of the uORF is located between position -30 and position -7. In some embodiments, the adenine of the at least one start codon of the uORF is located between position -30 and position -8.

[0295] In some embodiments, the adenine of the at least one start codon of the uORF is located between position -20 and position -8. In some embodiments, the adenine of the at least one start codon of the uORF is located between position -17 and position -8. In some embodiments, the alteration comprises at least two A«T to G*C alterations in at least two uORF start codons. In some embodiments, the at least two A«T to G*C alterations are at position - 17 and position -8.

[0296] DISRUPTING A SPLICE ACCEPTOR SITE

[0297] The present invention discloses a method of editing a 5’ splice acceptor site (SA) of an exon of a gene encoding a VGSC alpha subunit (Nav) comprising contacting the 5’ SA with a guide polynucleotide and a base or prime editor. In some embodiments, the guide polypeptide targets the base or prime editor to affect an alteration to disrupt the 5’ SA. In some embodiments, the alteration affected by the prime or base editor comprises at least a A«T to G*C alteration.

[0298] The term “disrupt” in the context of the method of editing a 5’ SA may mean that the edited 5’ SA, when transcribed, cannot serve as an acceptor site for a splicing event. Affecting an alteration disrupting the 5’ SA, for example by changing its A«T nucleotide pair to a G*C nucleotide pair, may prevent the spliceosome from recognising the 5’ SA. In the case of an exon located within an intron, the spliceosome may skip over the exon, the 5’ SA of which has been disrupted, to instead act on the 5’ SA of the subsequent exon, which is located at the end or 3’, i.e. downstream, of the intron in which the skipped exon is present. Thus, an alteration disrupting the 5’ SA of an exon located within an intron may cause the exon to be skipped, i.e. to not be integrated in the final transcript.

[0299] The present applicants have found that disrupting the 5’ SA of an exon within an intron, particularly of a poison exon, can significantly increase expression levels of the protein encoded by the gene which comprises said exon. In some embodiments, disrupting the 5’ SA improves expression of a Nav protein by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or 100% or more. In some embodiments, disrupting the 5’ SA improves expression of a Nav protein by 20% or more. In some embodiments, disrupting the 5’ SA improves expression of a Nav protein by 30% or more. In some embodiments, disrupting the 5’ SA improves expression of a Nav protein by 40% or more. In some embodiments, disrupting the 5’ SA improves expression of a Nav protein by 50% or more. In some embodiments, disrupting the 5’ SA improves expression of a Nav protein by 60% or more. In some embodiments, disrupting the 5’ SA improves expression of a Nav protein by 70% or more. In some embodiments, disrupting the 5’ SA improves expression of a Nav protein by 80% or more. In some embodiments, disrupting the 5’ SA improves expression of a Nav protein by 90% or more.

[0300] The term “poison exons” refers to exons the integration of which in splice variants of a gene transcript causes the introduction of a premature STOP codon that directs the transcript toward nonsense-mediated decay (NMD), effectively reducing the amount of functional protein produced. The inclusion of a poison exon in a gene transcript can be increased in gene variants comprising pathogenic variations within or near the poison exon.

[0301] Where one allele of a gene is lost, causing haplo-insufficiency, increasing protein expression from the remaining functional allele by disrupting the 5’ SA of an exon within an intron, particularly of a poison exon, can therefore help compensate for the loss of protein expression caused by the haplo-insufficiency without causing overexpression of the relevant protein, which in turn may be associated with certain conditions. Therefore, in some embodiments, the gene encoding a VGSC alpha subunit (Nav) is human SCN1A.

[0302] Affecting a A«T to G*C alteration at certain positions of the 5’ SA may be particularly successful in disrupting the 5’ SA. Thus, in some embodiments, the alteration comprises at least one A«T to G*C alteration at the -2 position with respect to the exon, the position -1 being the position of the nucleotide immediately 5’ of the first nucleotide of the exon. In a preferred embodiment, the alteration edits a “A«T G*C” to a “G*C G*C”.

[0303] VECTORS

[0304] In one aspect, the present invention discloses vectors comprising a nucleic acid encoding the guide polynucleotide. In another aspect, the present invention discloses an editing system. In some embodiments, the editing system comprises a vector comprising a nucleic acid encoding the guide polynucleotide. In some embodiments, the editing system comprises a vector comprising a nucleic acid encoding a base editor or portion thereof or a prime editor or portion thereof.

[0305] A vector is a tool that allows or facilitates the transfer of an entity from one environment to another. In accordance with the invention, and by way of example, some vectors used in recombinant nucleic acid techniques allow entities, such as a nucleic acid (e.g. a heterologous DNA segment, such as a heterologous cDNA segment), to be transferred into a target cell. The vector may serve the purpose of maintaining the heterologous nucleic acid (DNA or RNA) within the cell, facilitating the replication of the vector comprising a nucleic acid or facilitating the expression of the RNA or protein encoded by the nucleic acid. Vectors may be non-viral or viral. Examples of vectors used in recombinant nucleic acid techniques include, but are not limited to, plasmids, mRNA molecules (e.g. in vitro transcribed mRNAs), chromosomes, artificial chromosomes and viruses. The vector may also be, for example, a naked nucleic acid (e.g. DNA). In its simplest form, the vector may itself be a nucleotide of interest.

[0306] The vector may be used to deliver the guide polynucleotide into a cell. The vector may be used for expression of the guide polynucleotide in a cell. The vector may be used to deliver the base editor or a portion thereof or the prime editor or a portion thereof into a cell. The vector may be used for expression of the base editor or portion thereof or expression of the prime editor or portion thereof.

[0307] The vector or vectors may be suitable for editing a genome using the guide polynucleotide of the present invention and a base editor or prime editor. The base editor or prime editor may be encoded on the same vector as the guide polynucleotide. The base editor or prime editor and the guide polynucleotide may be encoded on different vectors.

[0308] The guide polynucleotide and a first portion of the base editor or prime editor may be encoded on a first vector and a second portion of the base editor or prime editor may be encoded on a second vector. When expressed in a cell, the first and the second portion of the base editor or prime editor may be assembled to the base editor or prime editor, for example using a split intein system comprising an N-intein and C-intein. The first vector may comprise an N-intein at the C-terminus of the first portion of the base editor or prime editor and the second vector may comprise a C-intein at the N-terminus of the second portion of the base editor or prime editor. When introduced into the cell, the N-intein and C-intein associate with each other followed by intein-excision and ligation of the first and second portion of the base editor and prime editor.

[0309] The vector may be capable of transducing mammalian cells, for example human cells or mouse cells. The vector may be capable of transducing neurons, particularly human or mouse neurons. The vector may be capable of transducing mouse or human primary neurons, particularly human primary neurons.

[0310] The vectors used in the invention may be, for example, plasmid, mRNA or virus vectors and may include a promoter for the expression of a polynucleotide and optionally a regulator of the promoter.

[0311] Vectors comprising nucleic acids encoding guide polynucleotides of the present invention may be introduced into cells using a variety of techniques known in the art, such as transfection, transformation and transduction. Several such techniques are known in the art, for example virus-mediated infection with recombinant viral vectors, such as retroviral, lentiviral (e.g. integration-defective lentiviral), adenoviral, adeno-associated viral, baculoviral and herpes simplex viral vectors, direct injection of nucleic acids, biolistic transformation, microinjection, electroporation, DEAE-dextran treatment, lipofection, nanoparticle-mediated transfection, or protein transduction domain (PTD) mediated transduction.

[0312] Non-viral delivery systems include but are not limited to DNA transfection methods. Here, transfection includes a process using a non-viral vector to deliver a gene to a target cell. Typical transfection methods include electroporation, DNA biolistics, lipid-mediated transfection, compacted DNA-mediated transfection, liposomes, immunoliposomes, lipofectin, cationic agent-mediated transfection, cationic facial amphiphiles (CFAs) (Nat. Biotechnol. (1996) 14: 556) and combinations thereof.

[0313] Transfection of cells with mRNA vectors can be achieved, for example, using nanoparticles, such as liposomes.

[0314] In some embodiments, the vector is comprised in a nanoparticle. In some embodiments, the nanoparticle is a polymeric nanoparticle, inorganic nanoparticle or lipid nanoparticle. In some embodiments, the nanoparticle is a liposome. The nanoparticle may be targeted to a specific cell type(s) using one or more ligand displayed on its surface.

[0315] In one embodiment, the polynucleotide is an mRNA. The mRNA may be comprised in a nanoparticle.

[0316] Viral vectors

[0317] In preferred embodiments, the vector is a viral vector. The viral vector may be, for example, a retroviral, lentiviral, adeno-associated viral (AAV) or adenoviral vector. In some embodiments, the vector is a lentiviral vector. In some embodiments, the vector is an AAV vector.

[0318] The vector may be in the form of a viral vector particle. Suitably, the viral vector is in the form of an AAV vector particle. Suitably, the viral vector is in the form of a lentiviral vector particle, for example an IDLV vector particle.

[0319] Methods of preparing and modifying viral vectors and viral vector particles, such as those derived from AAV, are well known in the art. Suitable methods are described in Ayuso, E., et al., 2010. Current gene therapy, 10(6), pp.423-436, Merten, O.W., et al., 2016. Molecular Therapy-Methods & Clinical Development, 3, p.16017; and Nadeau, I. and Kamen, A., 2003. Biotechnology advances, 20(7-8), pp.475-489.

[0320] Adeno-associated viral (AAV) vectors

[0321] The vector of the present invention may be an adeno-associated viral (AAV) vector. Optionally, the vector is an AAV9 vector. Optionally, the vector is an AAV6 vector. The vector may be an AAV9 vector or a derivative thereof. The vector of the present invention may be in the form of an AAV vector particle. Optionally, the vector is in the form of an AAV6 vector particle. The vector may be in the form of an AAV9 vector particle.

[0322] The AAV vector or AAV vector particle may comprise an AAV genome or a fragment or derivative thereof. An AAV genome is a polynucleotide sequence, which may encode functions needed for production of an AAV particle. These functions include those operating in the replication and packaging cycle of AAV in a host cell, including encapsidation of the AAV genome into an AAV particle. Naturally occurring AAVs are replication-deficient and rely on the provision of helper functions in trans for completion of a replication and packaging cycle. Accordingly, the AAV genome of the AAV vector of the invention is typically replicationdeficient. The AAV genome may be in single-stranded form, either positive or negative-sense, or alternatively in double-stranded form. The use of a double-stranded form allows bypass of the DNA replication step in the target cell and so can accelerate transgene expression.

[0323] The AAV genome may be from any naturally derived serotype, isolate or clade of AAV. AAV serotypes include AAV1 , AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 and AAV11. The AAV vector of the invention may be an AAV6 serotype. The AAV vector of the invention may be an AAV9 serotype.

[0324] The AAV genome used in the AAV vector of the invention may therefore be the full genome of a naturally occurring AAV. For example, a vector comprising a full AAV genome may be used to prepare an AAV vector or vector particle in vitro. However, while such a vector may in principle be administered to patients, this will rarely be done in practice. Preferably, the AAV genome will be derivatised for the purpose of administration to patients. Such derivatisation is standard in the art and the invention encompasses the use of any known derivative of an AAV genome, and derivatives which could be generated by applying techniques known in the art. Derivatisation of the AAV genome and of the AAV capsid are reviewed in Coura and Nardi (2007) Virology Journal 4: 99.

[0325] Derivatives of an AAV genome include any truncated or modified forms of an AAV genome which allow for expression of a transgene from an AAV in vivo. Typically, it is possible to truncate the AAV genome significantly to include minimal viral sequence yet retain the above function. This is preferred for safety reasons to reduce the risk of recombination of the vector with wild-type virus, and also to avoid triggering a cellular immune response by the presence of viral gene proteins in the target cell.

[0326] Typically, a derivative will include at least one inverted terminal repeat sequence (ITR), preferably more than one ITR, such as two ITRs or more. One or more of the ITRs may be derived from AAV genomes having different serotypes, or may be a chimeric or mutant ITR. A preferred mutant ITR is one having a deletion of a trs (terminal resolution site). This deletion allows for continued replication of the genome to generate a single-stranded genome which contains both coding and complementary sequences, i.e. a self-complementary AAV genome. This allows for bypass of DNA replication in the target cell, and so enables accelerated transgene expression.

[0327] In some embodiments, the AAV comprises at least one, such as two, AAV serotype 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11 ITRs. The one or more ITRs will preferably flank the nucleotide sequence of interest (which may also be referred to as a transgene) at either end. The inclusion of one or more ITRs is preferred to aid concatamer formation in the nucleus of a host cell, for example following the conversion of single-stranded vector DNA into double-stranded DNA by the action of host cell DNA polymerases. The formation of such episomal concatamers protects the vector construct during the life of the host cell, thereby allowing for prolonged expression of the transgene in vivo.

[0328] In preferred embodiments, ITR elements will be the only sequences retained from the native AAV genome in the derivative. Thus, a derivative will preferably not include the rep and / or cap genes of the native genome and any other sequences of the native genome. This is preferred for the reasons described above, and also to reduce the possibility of integration of the vector into the host cell genome. Additionally, reducing the size of the AAV genome allows for increased flexibility in incorporating other sequence elements (such as regulatory elements) within the vector in addition to the transgene.

[0329] The following portions could therefore be removed in a derivative of the invention: one inverted terminal repeat (ITR) sequence, the replication (rep) and capsid (cap) genes. However, in some embodiments, derivatives may additionally include one or more rep and / or cap genes or other viral sequences of an AAV genome. Naturally occurring AAV integrates with a high frequency at a specific site on human chromosome 19, and shows a negligible frequency of random integration, such that retention of an integrative capacity in the vector may be tolerated in a therapeutic setting.

[0330] The invention additionally encompasses the provision of sequences of an AAV genome in a different order and configuration to that of a native AAV genome. The invention also encompasses the replacement of one or more AAV sequences or genes with sequences from another virus or with chimeric genes composed of sequences from more than one virus. Such chimeric genes may be composed of sequences from two or more related viral proteins of different viral species.

[0331] The AAV particles of the invention include transcapsidated forms wherein an AAV genome or derivative having an ITR of one serotype is packaged in the capsid of a different serotype. The AAV particles of the invention also include mosaic forms wherein a mixture of capsid proteins from two or more different serotypes makes up the viral capsid. The AAV particle also includes chemically modified forms bearing ligands adsorbed to the capsid surface. For example, such ligands may include antibodies for targeting a particular cell surface receptor. The AAV vector may be adapted to cross the blood-brain barrier (e.g. the human bloodbrain barrier). AAV vectos adapted to cross the blood-brain barrier are known in the art and include, for example, AAV9 variants, such as BI-hTFR1 and derivatives thereof. The AAV vector may be an AAV bector that binds Tfr1 receptor (e.g. on endothelial cells). Such vectors are known in the art, for example as disclosed in Huang et al. (2024) Science 384: 1220-1227.

[0332] The present invention also relates to a method of producing a viral vector, in particular an AAV vector, e.g AAV9 or AAV6, of the present invention. Such a method can be any method known in the art and in particular the method as set out in Rothgangl et al. Treatment of a metabolic liver disease in mice with a transient prime editing approach. Nat. Biomed. Eng (2025). https: / / doi.org / 10.1038 / s41551-025-01399-4.

[0333] AAV vectors (e.g. AAV9 or AAV2 / 9 vectors) may be produced by a method comprising cotransfection of packaging, capsid and helper plasmids into cells. The method may further comprise: incubation of the cells; lysis of the cells; precipitation of the lysate; resuspension of the precipitate; purification (e.g. by gradient centrifugation); and / or concentration.

[0334] By way of example, pseudo-typed AAV9 vectors (AAV2 / 9) may be produced by co-transfection of packaging, capsid and helper plasmids (e.g. Addgene numbers 112865 and 112867) into cells, which may be incubated for 5 days until collection. The cells may be pelleted by low- speed centrifugation (e.g. 1 ,500g, 15 min). The medium may then be decanted, and the resulting cell pellet resuspended in resuspension buffer (e.g. 150 mM NaCI, 50 mM Tris-HCI), transferred into a Lysis Kit tube (e.g from Precellys Evolution) and mechanically disrupted. Subsequently, the samples may be lysed with benzonase, for example at 37 °C for 30 min, followed by centrifugation, for example at 5,000g for 1 h at 4 °C. The supernatant from this centrifugation step may then be combined with the supernatant from the previous step and precipitated using PEG8000 and NaCI solutions (1-2 days, 4 °C). The precipitated material may then be pelleted by medium-speed centrifugation (5,000g, 1 h). The pellet may be resuspended in resuspension buffer (final volume of 6 ml), and 1 .5 ml of 5 M NaCI added. The AAV particles may further be purified by gradient centrifugation, for example using OptiPrep. Finally, the purified AAVs may be concentrated, for example using Vivaspin 20 centrifugal concentrators (VWR).

[0335] Physical titres (vector genomes per millilitre) may be determined, for example using a Qubit 3.0 Fluorometer. The Qubit Fluorometer 3.0 (Life Technologies) may be used to measure the concentrations (ng ml-1) of the extracted genomes by denaturation at 95°C for 5 min, after which the readings may be converted to vector genomes per millilitre using the genome’s molecular mass and Avogadro’s constant. The identity of the packaged genomes of each AAV vector may be confirmed by Sanger DNA sequencing, for example by testing 500 ng of denatured AAV using an AAV-genome-specific sequencing primer. AAV2 / 9 viruses may be stored at -80 °C until use and diluted with phosphate-buffered saline if necessary.

[0336] Retroviral and lentiviral vectors

[0337] The vector of the present invention may be a retroviral vector or a lentiviral vector. The vector of the present invention may be a retroviral vector particle or a lentiviral vector particle.

[0338] A retroviral vector may be derived from or may be derivable from any suitable retrovirus. A large number of different retroviruses have been identified. Examples include murine leukaemia virus (MLV), human T-cell leukaemia virus (HTLV), mouse mammary tumour virus (MMTV), Rous sarcoma virus (RSV), Fujinami sarcoma virus (FuSV), Moloney murine leukaemia virus (Mo-MLV), FBR murine osteosarcoma virus (FBR MSV), Moloney murine sarcoma virus (Mo-MSV), Abelson murine leukaemia virus (A-MLV), avian myelocytomatosis virus-29 (MC29) and avian erythroblastosis virus (AEV).

[0339] Retroviruses may be broadly divided into two categories, “simple” and “complex”. Retroviruses may be even further divided into seven groups. Five of these groups represent retroviruses with oncogenic potential. The remaining two groups are the lentiviruses and the spumaviruses.

[0340] The basic structure of retrovirus and lentivirus genomes share many common features such as a 5’ LTR and a 3’ LTR. Between or within these are located a packaging signal to enable the genome to be packaged, a primer binding site, integration sites to enable integration into a host cell genome, and gag, pol and env genes encoding the packaging components - these are polypeptides required for the assembly of viral particles. Lentiviruses have additional features, such as rev and RRE sequences in HIV, which enable the efficient export of RNA transcripts of the integrated provirus from the nucleus to the cytoplasm of an infected target cell.

[0341] In the provirus, these genes are flanked at both ends by regions called long terminal repeats (LTRs). The LTRs are responsible for proviral integration and transcription. LTRs also serve as enhancer-promoter sequences and can control the expression of the viral genes.

[0342] The LTRs themselves are identical sequences that can be divided into three elements: U3, R and U5. U3 is derived from the sequence unique to the 3’ end of the RNA. R is derived from a sequence repeated at both ends of the RNA. U5 is derived from the sequence unique to the 5’ end of the RNA. The sizes of the three elements can vary considerably among different retroviruses. In a defective retroviral vector genome gag, pol and env may be absent or not functional.

[0343] In a typical retroviral vector, at least part of one or more protein coding regions essential for replication may be removed from the virus. This makes the viral vector replication-defective. Portions of the viral genome may also be replaced by a library encoding candidate modulating moieties operably linked to a regulatory control region and a reporter moiety in the vector genome in order to generate a vector comprising candidate modulating moieties which is capable of transducing a target host cell and / or integrating its genome into a host genome.

[0344] Lentivirus vectors are part of the larger group of retroviral vectors. In brief, lentiviruses can be divided into primate and non-primate groups. Examples of primate lentiviruses include but are not limited to human immunodeficiency virus (HIV), the causative agent of human acquired immunodeficiency syndrome (AIDS); and simian immunodeficiency virus (SI ). Examples of non-primate lentiviruses include the prototype “slow virus” visna / maedi virus (VMV), as well as the related caprine arthritis-encephalitis virus (CAEV), equine infectious anaemia virus (EIAV), and the more recently described feline immunodeficiency virus (FIV) and bovine immunodeficiency virus (BIV).

[0345] The lentivirus family differs from retroviruses in that lentiviruses have the capability to infect both dividing and non-dividing cells. In contrast, other retroviruses, such as MLV, are unable to infect non-dividing or slowly dividing cells such as those that make up, for example, muscle, brain, lung and liver tissue.

[0346] A lentiviral vector, as used herein, is a vector which comprises at least one component part derivable from a lentivirus. Suitably, that component part is involved in the biological mechanisms by which the vector infects cells, expresses genes or is replicated.

[0347] The lentiviral vector may be a “primate” vector. The lentiviral vector may be a “non-primate” vector (i.e. derived from a virus which does not primarily infect primates, especially humans). Examples of non-primate lentiviruses may be any member of the family of lentiviridae which does not naturally infect a primate.

[0348] As examples of lentivirus-based vectors, HIV-1- and HIV-2-based vectors are described below.

[0349] The HIV-1 vector contains cis-acting elements that are also found in simple retroviruses. It has been shown that sequences that extend into the gag open reading frame are important for packaging of HIV-1. Therefore, HIV-1 vectors often contain the relevant portion of gag in which the translational initiation codon has been mutated. In addition, most HIV-1 vectors also contain a portion of the env gene that includes the RRE. Rev binds to RRE, which permits the transport of full-length or singly spliced mRNAs from the nucleus to the cytoplasm. In the absence of Rev and / or RRE, full-length HIV-1 RNAs accumulate in the nucleus. Alternatively, a constitutive transport element from certain simple retroviruses such as Mason-Pfizer monkey virus can be used to relieve the requirement for Rev and RRE. Efficient transcription from the HIV-1 LTR promoter requires the viral protein Tat.

[0350] Most HIV-2-based vectors are structurally very similar to HIV-1 vectors. Similar to HIV-1-based vectors, HIV-2 vectors also require RRE for efficient transport of the full-length or singly spliced viral RNAs.

[0351] Optionally, the viral vector used in the present invention has a minimal viral genome.

[0352] By “minimal viral genome” it is to be understood that the viral vector has been manipulated so as to remove the non-essential elements and to retain the essential elements in order to provide the required functionality to infect, transduce and deliver a nucleotide sequence of interest to a target host cell. Further details of this strategy can be found in WO 1998 / 017815.

[0353] Optionally, the plasmid vector used to produce the viral genome within a host cell / packaging cell will have sufficient lentiviral genetic information to allow packaging of an RNA genome, in the presence of packaging components, into a viral particle which is capable of infecting a target cell, but is incapable of independent replication to produce infectious viral particles within the final target cell. Optionally, the vector lacks a functional gag-pol and / or env gene and / or other genes essential for replication.

[0354] However, the plasmid vector used to produce the viral genome within a host cell / packaging cell will also include transcriptional regulatory control sequences operably linked to the lentiviral genome to direct transcription of the genome in a host cell / packaging cell. These regulatory sequences may be the natural sequences associated with the transcribed viral sequence (i.e. the 5’ U3 region), or they may be a heterologous promoter, such as another viral promoter (e.g. the CMV promoter).

[0355] The vectors may be self-inactivating (SIN) vectors in which the viral enhancer and promoter sequences have been deleted. SIN vectors can be generated and transduce non-dividing cells in vivo with an efficacy similar to that of wild-type vectors. The transcriptional inactivation of the long terminal repeat (LTR) in the SIN provirus should prevent mobilisation by replication- competent virus. This should also enable the regulated expression of genes from internal promoters by eliminating any cis-acting effects of the LTR.

[0356] The vectors may be integration-defective. Integration defective lentiviral vectors (IDLVs) can be produced, for example, either by packaging the vector with catalytically inactive integrase (such as an HIV integrase bearing the D64V mutation in the catalytic site) or by modifying or deleting essential att sequences from the vector LTR, or by a combination of the above.

[0357] Adenoviral vectors

[0358] The vector of the present invention may be an adenoviral vector. The vector of the present invention may be an adenoviral vector particle.

[0359] The adenovirus is a double-stranded, linear DNA virus that does not go through an RNA intermediate. There are over 50 different human serotypes of adenovirus divided into 6 subgroups based on the genetic sequence homology. The natural targets of adenovirus are the respiratory and gastrointestinal epithelia, generally giving rise to only mild symptoms. Serotypes 2 and 5 (with 95% sequence homology) are most commonly used in adenoviral vector systems and are normally associated with upper respiratory tract infections in the young.

[0360] Adenoviruses have been used as vectors for gene therapy and for expression of heterologous genes. The large (36 kb) genome can accommodate up to 8 kb of foreign insert DNA and is able to replicate efficiently in complementing cell lines to produce very high titres of up to 1012. Adenovirus is thus one of the best systems to study the expression of genes in primary non- replicative cells.

[0361] The expression of viral or foreign genes from the adenovirus genome does not require a replicating cell. Adenoviral vectors enter cells by receptor mediated endocytosis. Once inside the cell, adenovirus vectors rarely integrate into the host chromosome. Instead, they function episomally (independently from the host genome) as a linear genome in the host nucleus. Hence the use of recombinant adenovirus alleviates the problems associated with random integration into the host genome.

[0362] Herpes simplex viral vector

[0363] The vector of the present invention may be a herpes simplex viral vector. The vector of the present invention may be a herpes simplex viral vector particle.

[0364] Herpes simplex virus (HSV) is a neurotropic DNA virus with favorable properties as a gene delivery vector. HSV is highly infectious, so HSV vectors are efficient vehicles for the delivery of exogenous genetic material to cells. Viral replication is readily disrupted by null mutations in immediate early genes that in vitro can be complemented in trans, enabling straightforward production of high-titre pure preparations of non-pathogenic vector. The genome is large (152 Kb) and many of the viral genes are dispensable for replication in vitro, allowing their replacement with large or multiple transgenes. Latent infection with wild-type virus results in episomal viral persistence in sensory neuronal nuclei for the duration of the host lifetime. The vectors are non-pathogenic, unable to reactivate and persist long-term. The latency active promoter complex can be exploited in vector design to achieve long-term stable transgene expression in the nervous system. HSV vectors transduce a broad range of tissues because of the wide expression pattern of the cellular receptors recognized by the virus. Increasing understanding of the processes involved in cellular entry has allowed targeting the tropism of HSV vectors.

[0365] Vaccinia virus vectors

[0366] The vector of the present invention may be a vaccinia viral vector. The vector of the present invention may be a vaccinia viral vector particle.

[0367] Vaccinia virus is a large enveloped virus that has an approximately 190 kb linear, doublestranded DNA genome. Vaccinia virus can accommodate up to approximately 25 kb of foreign DNA, which also makes it useful for the delivery of large genes.

[0368] A number of attenuated vaccinia virus strains are known in the art that are suitable for gene therapy applications, for example the MVA and NYVAC strains.

[0369] EDITING SYSTEMS

[0370] Another aspect of the present invention provides an editing system comprising (a) a guide polynucleotide or vector comprising a nucleic acid encoding a guide polynucleotide, and (b) a base editor, polynucleotide encoding a base editor or a vector comprising a nucleic acid encoding a base editor or portion thereof.

[0371] In some embodiments, the editing system comprises a guide polynucleotide and a base editor.

[0372] The base editor or prime editor (preferably base editor) may be in a complex with the guide polynucleotide (e.g. the guide polynucleotide and the base editor may together form a ribonucleoprotein (RNP)). A RNP may be formed by any method known in the art, for example by incubating a base editor with a guide polynucleotide for 5-30 minutes at room temperature.

[0373] BASE EDITORS AND PRIME EDITORS

[0374] The editing system provided herein may comprise the guide polynucleotide or vector comprising a nucleic acid encoding the guide polynucleotide of the invention and a base editor or prime editor (preferably a base editor), a polynucleotide encoding a base editor or prime editor (preferably a base editor) or a vector comprising a nucleic acid encoding a base editor or portion thereof or a prime editor or portion thereof (preferably a base editor or portion thereof).

[0375] Base editors and prime editors are known in the art. Base editors provide a different approach to genome editing over conventional CRISPR / Cas9 technology by catalysing nucleotide alterations without generating double-strand DNA breaks, requiring a donor template or inducing stochastic insertions or deletions. Most base editors are fusion proteins of a catalytically inactive Cas9 enzyme (dCas9) or portion thereof or Cas9 nickase or portion thereof, and an enzyme or portion thereof (e.g. an enzymatic domain) with nucleotide-altering activity. The enzyme or portion thereof with nucleotide-altering activity can catalyse a nucleotide transition, i.e. an interchange of purines or pyrimidines such as a change from an adenine to a guanosine, or a nucleotide transversion, i.e. an interchange between a purine and pyrimidine such as from an adenine to a cytosine. Examples of base editors are adenine base editors, which catalyse an A«T to G*C nucleotide alteration, cytosine base editors, which catalyse a C*G to T«A nucleotide alteration and guanosine base editors, which can catalyse a G*C to T«A alteration or a G*C to C*G alteration. Examples of enzymes or portions thereof with nucleotide-altering activity are deaminases, such as adenine deaminases, which catalyse an A«T to G*C nucleotide alteration or transition, and cytosine deaminases, which catalyse a OG to T *A nucleotide alteration or transition. Another example of an enzyme or portion thereof with nucleotide-altering activity is N-methylpurine DNA glycosylase protein (MPG), which can catalyse a G*C to T«A alteration or transversion or a G*C to OG alteration or transversion.

[0376] Thus, in some embodiments, the base editor comprises a polynucleotide binding domain and a nucleotide-altering domain (e.g. a deaminase domain) for altering one or more nucleotides. In some embodiments, the base editor is an adenine base editor (ABE). In some embodiments, the adenine base editor is ABE8e. In some embodiments, the adenine base editor is ABEmax. In some embodiments, the base editor is a cytosine base editor ( BE). In some embodiments the nucleotide-altering domain is a deaminase domain. In some embodiments the polynucleotide binding domain is a polynucleotide DNA binding domain.

[0377] In some embodiments, the deaminase domain is a cytosine deaminase. In preferred embodiments, the deaminase domain is an adenine deaminase.

[0378] In some embodiments, the base editor comprises more than one nucleotide-altering domain. For example, the base editor may comprise more than one deaminase. In some embodiments, the base editor may comprise one or more cytosine deaminase and / or one or more adenine deaminases. In some embodiments, a particular guide polynucleotide of the invention may be utilized to target different deaminases to a target nucleic acid sequence. In some embodiments, a pair of guide polynucleotides of the invention may be utilized to target different deaminases to a target nucleic acid sequence.

[0379] The nucleotide-altering domain and the polynucleotide binding domain of the base editor may be associated with each other covalently or noncovalently. In some embodiments, a deaminase domain can be targeted to a target sequence by a polynucleotide binding domain. In some embodiments, a polynucleotide binding domain can be fused or linked to a deaminase domain.

[0380] In some embodiments, a polynucleotide binding domain can target a deaminase domain to a target by noncovalently interacting with or associating with the deaminase domain. For example, in some embodiments, the nucleotide-altering domain, e.g. the deaminase domain, can comprise an additional heterologous portion or domain that is capable of interacting with, associating with, or capable of forming a complex with an additional heterologous portion or domain that is part of a polynucleotide binding domain. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polypeptide. In some embodiments, the additional heterologous portion may be capable of binding to, interacting with, associating with, or forming a complex with a polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a guide polynucleotide. In some embodiments, the additional heterologous portion may be capable of binding to a polypeptide linker. In some embodiments, the additional heterologous portion may be capable of binding to a polynucleotide linker. The additional heterologous portion may be a protein domain. In some embodiments, the additional heterologous portion may be a K Homology (KH) domain, a MS2 coat protein domain, a PP7 coat protein domain, a SfMu Com coat protein domain, a steril alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or a RNA recognition motif.

[0381] Prime editors have more extensive nucleotide-altering abilities than base editors and can additionally catalyse the short, targeted nucleotide insertions or deletions.

[0382] Prime editors are most commonly fusion proteins of a catalytically inactive Cas9 enzyme (dCas9) or portion thereof or Cas9 nickase or portion thereof and an enzyme or portion thereof (e.g. an enzymatic domain) with reverse transcriptase activity.

[0383] Thus, in some embodiments, the prime editor comprises a polynucleotide binding domain and a nucleotide-altering domain for altering one or more nucleotides or catalysing short nucleotide insertions or deletions. The nucleotide-altering domain and the polynucleotide binding domain of the base editor or prime editor may be associated with each other covalently. For example, in some embodiments, the nucleotide-altering domain, e.g. the deaminase domain, is covalently bound to a Cas9 enzyme or Cas9 RNA binding domain. In one embodiment, the Cas9 is catalytically inactive Cas9 (dCas9) or a Cas9 nickase (nCas9). In some embodiments, the dCas9, nCas9 or Cas9 RNA binding domain may be capable of binding to a guide polynucleotide. In some embodiments, the dCas9, nCas9 or Cas9 RNA binding domain may bind to a linker sequence in the guide polynucleotide. In some embodiments, the Cas9 or Cas9 RNA binding domain may be of Streptococcus pyogenes Cas9 (SpCas9). In some embodiments, the Cas9 may be SpCas9. In some embodiments, the Cas9 may be a variant of Streptococcus pyogenes Cas9, such as SpRYCas9, SpGCas9, SpCas9-VQR, SpCas9-VRQR, SpCas9-VRER, SpCas9-NG, SpCas9-NRCH, SpCas9-NRRH, or iSpyMac. In some embodiments, the Cas9 may be SpRYCas9. In some embodiments, the Cas9 may be a circularly permutated variant of SpCas9 (CP-SpCas9), such as CP1041-SpCas9, CP1041-SpCas9-NG. In some embodiments, the Cas9 or Cas9 RNA binding domain may be of Staphylococcus aureus Cas9 (SaCas9). In some embodiments, the Cas9 may be SaCas9. In some embodiments, the Cas9 or Cas9 RNA binding domain may be of Neisseria meningitidis Cas9 (NmeCas9). In some embodiments, the Cas9 may be NmeCas9. In some embodiments, the Cas9 or Cas9 RNA binding domain may be of Campylobacter jejuni Cas9 (CjCas9). In some embodiments, the Cas9 may be CjCas9. In some embodiments, the Cas9 may be a variant of CjCas9, such as evoCjCas9. In some embodiments, the nucleotide-altering domain, e.g. the deaminase domain, is covalently bound to Cpf1.

[0384] Examples of adenine base editors comprising an adenine deaminase domain are SpRYCas9- ABEmax, SpRYCas9-ABE8e and SpCas9-ABE8e, the sequences of which are shown below:

[0385] SpRYCas9-ABEmax(SEQ ID NO: 29)

[0386] SEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIM ALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLH HPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTDSGGSSGGSSGSETPGT SESATPESSGGSSGGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEG WNRAIGLHDPTAHAEIMALRQSGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRWF GVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQS STDSGGSSGGSSGSETPGTSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKV PSKKFKVLGNTDRHSIKKNLIGALLFDSGETAERTRLKRTARRRYTRRKNRICYLQEIFSNEM AKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRL IYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLS KSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLA

[0387] QIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLP

[0388] EKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFD

[0389] NGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKS

[0390] EETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVT

[0391] EGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGT

[0392] YHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYT

[0393] GWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDS

[0394] LHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERM

[0395] KRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVP

[0396] QSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNLTKAE

[0397] RGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFR

[0398] KDFQFYKVREINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEI

[0399] GKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQV

[0400] NIVKKTEVQTGGFSKESIRPKRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLWAKVEKGK

[0401] SKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASA

[0402] KQLQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKR

[0403] VILADANLDKVLSAYNKHRDKPI REQAEN II HLFTLTRLGAPRAFKYFDTTIDPKQYRSTKEVL

[0404] DATLIHQSITGLYETRIDLSQLGGD

[0405] SpRYCas9-ABE8e (SEQ ID NO: 30)

[0406] SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA

[0407] LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRWFGVRNSKRGAAGSLMNVLN

[0408] YPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSINSGGSSGGSSGSETPG

[0409] TSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN

[0410] LIGALLFDSGETAERTRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE

[0411] DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGD

[0412] LNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG

[0413] LFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDA

[0414] ILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYI

[0415] DGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQ

[0416] EDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEWDKGASA

[0417] QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD

[0418] LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENE

[0419] DI LEDI VLTLTLFEDREM I EERLKTYAH LFDDKVM KQLKRRRYTGWGRLSRKLI NGI RDKQSG

[0420] KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQ TVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK

[0421] NRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVE

[0422] TRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESIRP KRNSDKLIARKKDWDPKKYGGFLWPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF

[0423] EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAKQLQKGNELALPSKYVNFLY LASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK PI REQAEN 11 H LFTLTRLGAPRAFKYFDTTI DPKQYRSTKEVLDATLI HQSITGLYETRI DLSQL GGD

[0424] SpCas9-ABE8e (SEQ ID NO: 31)

[0425] SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMA LRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRWFGVRNSKRGAAGSLMNVLN YPGMNHRVEITEGILADECAALLCDFYRMPRQVFNAQKKAQSSINSGGSSGGSSGSETPG TSESATPESSGGSSGGSDKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKN

[0426] LIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEE DKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGD LNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNG LFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDA I LLSDI LRVNTEITKAPLSASM I KRYDEH HQDLTLLKALVRQQLPEKYKEI FFDQSKNGYAGYI DGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQ

[0427] EDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEWDKGASA QSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENE DI LEDI VLTLTLFEDREM I EERLKTYAH LFDDKVM KQLKRRRYTGWGRLSRKLI NGI RDKQSG

[0428] KTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQ TVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPV ENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDK NRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVE

[0429] TRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILP KRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSF

[0430] EKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLY LASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDK PIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQL GGD

[0431] In some embodiments, the base editor comprises or consists of an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 29. In some embodiments, the base editor comprises or consists of the amino acid sequence of SEQ ID NO: 29.

[0432] In some embodiments, the base editor comprises or consists of an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 30. In some embodiments, the base editor comprises or consists of the amino acid sequence of SEQ ID NO: 30.

[0433] In some embodiments, the base editor comprises or consists of an amino acid sequence that has at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 31. In some embodiments, the base editor comprises or consists of the amino acid sequence of SEQ ID NO: 31.

[0434] VGSC ALPHA SUBUNIT (Nav)

[0435] The voltage-gated sodium channel (VGSC) alpha subunit (Nav) is a transmembrane protein that forms the ion-conducting pore of the sodium channel with four domains of six transmembrane segments. It forms a complex with one or more small auxiliary beta subunits with a single transmembrane domain which modulate the function and localization of the alpha subunit. They open during membrane depolarization to allow sodium inward current and the consequent generation of the action potential; subsequently, they enter an inactive state, which can be partial, resulting in a persistent current.

[0436] In humans, voltage-gated sodium channels (VGSCs) can have one of nine different sodium channel alpha subunits, Nav1.1-Nav1.9, which have been attributed both distinct and overlapping roles in human physiology, for example the generation and propagation of action potentials in neurons and muscle cells. Consequently, GOF and, in particular, LOF mutations in the genes encoding the VGSC alpha subunits (SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SCN10A and SCN11A) are associated with a number of genetic disorders, particularly Developmental and Epileptic Encephalopathies (DEE), such as Dravet syndrome, which is caused by a LOF mutation in SCN1A.

[0437] In humans, SCN1A codes for Nav1.1, SCN2A codes for Nav1.2, SCN3A codes for Nav1.3, SCN4A codes for Navi .4, SCN5A codes for Navi .5, SCN8A codes for Navi .6, SCN9A codes for Nav1.7, SCN10A codes for Nav1.8 and SCN11A codes for Nav1.9. The protein sequences of Nav1.1-Nav1.9 are well known. For example, the canonical sequence of human Nav1.1 is provided below:

[0438] Amino acid sequence of Nay1.1 (SEQ ID NO: 32)

[0439] MEQTVLVPPGPDSFNFFTRESLAAIERRIAEEKAKNPKPDKKDDDENGPKPNSDLEAGKNL

[0440] PFIYGDIPPEMVSEPLEDLDPYYINKKTFIVLNKGKAIFRFSATSALYILTPFNPLRKIAIKILVHS

[0441] LFSMLIMCTILTNCVFMTMSNPPDWTKNVEYTFTGIYTFESLIKIIARGFCLEDFTFLRDPWN

[0442] WLDFTVITFAYVTEFVDLGNVSALRTFRVLRALKTISVIPGLKTIVGALIQSVKKLSDVMILTVF

[0443] CLSVFALIGLQLFMGNLRNKCIQWPPTNASLEEHSIEKNITVNYNGTLINETVFEFDWKSYIQ

[0444] DSRYHYFLEGFLDALLCGNSSDAGQCPEGYMCVKAGRNPNYGYTSFDTFSWAFLSLFRLM

[0445] TQDFWENLYQLTLRAAGKTYMIFFVLVIFLGSFYLINLILAVVAMAYEEQNQATLEEAEQKEA

[0446] EFQQMIEQLKKQQEAAQQAATATASEHSREPSAAGRLSDSSSEASKLSSKSAKERRNRRK

[0447] KRKQKEQSGGEEKDEDEFQKSESEDSIRRKGFRFSIEGNRLTYEKRYSSPHQSLLSIRGSL

[0448] FSPRRNSRTSLFSFRGRAKDVGSENDFADDEHSTFEDNESRRDSLFVPRRHGERRNSNLS

[0449] QTSRSSRMLAVFPANGKMHSTVDCNGVVSLVGGPSVPTSPVGQLLPEVIIDKPATDDNGTT

[0450] TETEMRKRRSSSFHVSMDFLEDPSQRQRAMSIASILTNTVEELEESRQKCPPCWYKFSNIF

[0451] LIWDCSPYWLKVKHVVNLVVMDPFVDLAITICIVLNTLFMAMEHYPMTDHFNNVLTVGNLVF

[0452] TGIFTAEMFLKIIAMDPYYYFQEGWNIFDGFIVTLSLVELGLANVEGLSVLRSFRLLRVFKLAK

[0453] SWPTLNMLIKIIGNSVGALGNLTLVLAIIVFIFAVVGMQLFGKSYKDCVCKIASDCQLPRWHM

[0454] NDFFHSFLIVFRVLCGEWIETMWDCMEVAGQAMCLTVFMMVMVIGNLWLNLFLALLLSSF

[0455] SADNLAATDDDNEMNNLQIAVDRMHKGVAYVKRKIYEFIQQSFIRKQKILDEIKPLDDLNNKK

[0456] DSCMSNHTAEIGKDLDYLKDVNGTTSGIGTGSSVEKYIIDESDYMSFINNPSLTVTVPIAVGE

[0457] SDFENLNTEDFSSESDLEESKEKLNESSSSSEGSTVDIGAPVEEQPWEPEETLEPEACFTE

[0458] GCVQRFKCCQINVEEGRGKQWWNLRRTCFRIVEHNWFETFIVFMILLSSGALAFEDIYIDQR

[0459] KTIKTMLEYADKVFTYIFILEMLLKWVAYGYQTYFTNAWCWLDFLIVDVSLVSLTANALGYSE

[0460] LGAIKSLRTLRALRPLRALSRFEGMRWVNALLGAIPSIMNVLLVCLIFWLIFSIMGVNLFAGK

[0461] FYHCINTTTGDRFDIEDVNNHTDCLKLIERNETARWKNVKVNFDNVGFGYLSLLQVATFKG

[0462] WMDIMYAAVDSRNVELQPKYEESLYMYLYFVIFIIFGSFFTLNLFIGVIIDNFNQQKKKFGGQ

[0463] DIFMTEEQKKYYNAMKKLGSKKPQKPIPRPGNKFQGMVFDFVTRQVFDISIMILICLNMVTM

[0464] MVETDDQSEYVTTILSRINLVFIVLFTGECVLKLISLRHYYFTIGWNIFDFVWILSIVGMFLAEL

[0465] IEKYFVSPTLFRVIRLARIGRILRLIKGAKGIRTLLFALMMSLPALFNIGLLLFLVMFIYAIFGMSN

[0466] FAYVKREVGIDDMFNFETFGNSMICLFQITTSAGWDGLLAPILNSKPPDCDPNKVNPGSSVK

[0467] GDCGNPSVGIFFFVSYIIISFLVWNMYIAVILENFSVATEESAEPLSEDDFEMFYEVWEKFDP

[0468] DATQFMEFEKLSQFAAALEPPLNLPQPNKLQLIAMDLPMVSGDRIHCLDILFAFTKRVLGES

[0469] GEMDALRIQMEERFMASNPSKVSYQPITTTLKRKQEEVSAVIIQRAYRRHLLKRTVKQASFT YNKNKIKGGANLLIKEDMIIDRINENSITEKTDLTMSTAACPPSYDRVTKPIVEKHEQEGKDEK

[0470] AKGK.

[0471] The Uniprot protein sequence references for the canonical sequences of Nav1.1-Nav1.9 are P35498-1 (Nav1.1), Q99250-1 (Nav1.2), Q9NY46-1 (Nav1.3), P35499 (Nav1.4), Q14524- 1 (Nav1.5), Q9UQD0-1 (Nav1.6), Q15858-1 (Nav1.7), Q9Y5Y9 (Nav1.8) and Q9U 133-1 (Nav1.9). Orthologues of human Nav1.1-Nav1.9 are expressed in all mammalian species. For example, in mice Nav1.1-Nav1.9 are encoded by Senia, Scn2a, Scn3a, Scn4, Scn5, Scn8a, Scn9a, Sen 10a and Scn11a.

[0472] The tenth member of the VGSC alpha subunit family, Navx, is not voltage-gated and allows Na+to pass through the ion-conducting pore along a concentration gradient. In humans, Navx is encoded by SCN7A. In mice, Navx is encoded by Scn7a.

[0473] METHOD OF TREATMENT

[0474] Considering the relevant role exerted by VGSCs for cell physiology, even loss of function (LOF) variants in a single allele of SCN genes may affect channel activity and provoke severe defects at cellular level. The final outcome can be a spectrum of neurological disorders including DEEs (SCN1 / 2 / 3 / 8A), but also cardiac dysfunctions (SC / V5A), peripheral neuropathies (SCN9 / 10 / 11A) and skeletal muscle channelopathies (SCN4A).

[0475] LOF mutations in SCN1A manifest with variable phenotypes, ranging from milder presentations such as genetic epilepsy with febrile seizures to Dravet syndrome (DS), considered one of the most severe DEEs. DS starts with drug resistant epilepsy starting in the first year of life with febrile or hyperthermic seizures followed by afebrile seizures, cognitive and behavioral deficits, ataxia, and high mortality for sudden unexplained death in epilepsy (SUDEP). Gain-of-function (GOF) in SCN1A are associated with familial hemiplegic migraine type 3 (FHM3), an inherited form of migraine with aura. Other SCN1A GOF mutations cause early infantile developmental encephalopathy with movement disorder (EIDEE / MD) characterized by seizure onset within the first 3 months of life, hyperkinetic movement disorder, intellectual disability, and severe-to-profound developmental delay.

[0476] Mutations in SCN2 / 3 / 8A are clinically heterogenous, causing different forms of epilepsy ranging from self-limited infantile epilepsy to DEEs including early infantile DEE (Ohtahara syndrome) and infantile spasms syndrome. LOF mutations in SCN2A result in either later- onset epilepsies, not responding to sodium channel blockers, or autism spectrum disorders (ASD) and intellectual disability (ID) without epilepsy, while GoF variants cause early-infantile epilepsies of variable severity, with seizure onset typically occurring before age 3 months, responding pretty well to anti-seizure medications (ASMs). Similarly, LOF variants in SCN8A cause severe intellectual disability without seizures while clinical manifestations of GOF mutation range from movement disorders or intellectual disability only to severe DEE.

[0477] SCN5A gene encodes cardiac sodium channel alpha-subunit Nav1.5, which determines cardiac excitability and conduction of electrical stimuli through the heart. LOF mutations in SCN5A cause Brugada syndrome, while GOF mutations in SCN5A lead to more sodium influx into cardiomyocytes through aberrant channel gating and cause long QT syndrome. In addition, both loss- and gain-of-function mutations may cause dilated cardiomyopathy, which is an arrhythmogenic disease with gross structural defects of the left ventricle.

[0478] Accordingly, one aspect of the present invention discloses a method of treating a genetic disorder comprising administering to a subject a therapeutically effective amount of the guide polynucleotide or vector of the present invention, preferably in combination with a base editor or prime editor (preferably base editor), polynucleotide encoding a base editor or prime editor (preferably base editor) or a vector comprising a nucleic acid encoding a base editor or prime editor (preferably base editor).

[0479] The different components of the combination (e.g. the guide polynucleotide, vector and / or base editor) may be, for example, administered simultaneously, sequentially or separately.

[0480] The term “combination”, or terms “in combination”, “used in combination with” or “combined preparation” as used herein may refer to the combined administration of two or more agents simultaneously, sequentially or separately.

[0481] The term “simultaneous” as used herein means that the agents are administered concurrently, i.e. at the same time.

[0482] The term “sequential” as used herein means that the agents are administered one after the other.

[0483] The term “separate” as used herein means that the agents are administered independently of each other but within a time interval that allows the agents to show a combined, preferably synergistic, effect. Thus, administration “separately” may permit one agent to be administered, for example, within 1 minute, 5 minutes or 10 minutes after the other.

[0484] In a related aspect of the invention, there is disclosed a method of treating a genetic disorder comprising administering to a subject a therapeutically effective amount of the editing system or kit of the present invention. In some embodiments, the genetic disorder is associated with an LOF mutation in a gene encoding a VGSC alpha subunit (Nav). In some embodiments, the genetic disorder is associated with a GOF mutation in a gene encoding a VGSC alpha subunit (Nav). In some embodiments, the genetic disorder is a neurological disorder. In some embodiments, the genetic disorder belongs to any one of cardiac dysfunctions, peripheral neuropathies, skeletal muscle channelopathies, movement disorders, intellectual disability, familial hemiplegic migraine type 3, early infantile developmental encephalopathy with movement disorder, hyperkinetic movement disorder, severe-to-profound developmental delay, autism spectrum disorders (ASD), early infantile epilepsy, Brugada syndrome, long QT syndrome, dilated cardiomyopathy, or developmental and epileptic encephalopathies (DEE). In some embodiments, the genetic disorder is a neurological disorder. In some embodiments, the neurological disorder belongs to the group of DEEs. In some embodiments, the DEE is genetic epilepsy or Dravet syndrome. In some embodiments, the genetic disorder is Dravet syndrome.

[0485] In some embodiments, the subject may have an LOF mutation in at least one allele of SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN7A, SCN8A, SCN9A, SCN10A, or SCN11A. In some embodiments, the subject may have an LOF mutation in at least one allele of SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN8A, SCN9A, SCN10A and SCN11A. In some embodiments, the subject may have an LOF mutation in at least one allele of SCN1A, SCN2A, SCN3A, SCN5A, SCN8A, and SCN9A, preferably any one of SCN1A, SCN2A and SCN8A. In some embodiments, the subject may have an LOF mutation in at least one allele of SCN1A.

[0486] PHARMACEUTICAL COMPOSITION

[0487] The guide polynucleotides, vectors and editing systems of the invention may be formulated for administration to subjects with a pharmaceutically acceptable carrier, diluent or excipient. Suitable carriers and diluents include isotonic saline solutions, for example phosphate- buffered saline, and potentially contain human serum albumin.

[0488] Handling of the cell therapy products is preferably performed in compliance with FACT-JACIE International Standards for cellular therapy.

[0489] A pharmaceutical composition may be a composition that comprises or consists of a therapeutically effective amount of a pharmaceutically active agent. A pharmaceutical composition preferably includes a pharmaceutically acceptable carrier, diluent or excipient (including combinations thereof).

[0490] By “pharmaceutically acceptable” it is included that the formulation is sterile and pyrogen free. The carrier, diluent, and / or excipient must be “acceptable” in the sense of being compatible with the enveloped viral particle and not deleterious to the recipients thereof. Typically, the carriers, diluents and excipients will be saline or infusion media which will be sterile and pyrogen free, however other acceptable carriers, diluents and excipients may be used.

[0491] Acceptable carriers, diluents, and excipients for therapeutic use are well known in the pharmaceutical art. The choice of pharmaceutical carrier, excipient or diluent can be selected with regard to the intended route of administration and standard pharmaceutical practice. The pharmaceutical compositions may comprise as (or in addition to) the carrier, excipient or diluent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilising agent(s).

[0492] Examples of pharmaceutically acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.

[0493] The guide polynucleotides, vectors, editing systems or pharmaceutical compositions according to the present invention may be administered in a manner appropriate for treating and / or preventing the diseases described herein. Suitable administration routes will be known to the skilled person.

[0494] The quantity and frequency of administration may be determined by the skilled person, for example depending by such factors as the condition of the subject, and the type and severity of the subject's disease. The pharmaceutical composition may be formulated accordingly.

[0495] The guide polynucleotides, vectors, editing systems or pharmaceutical compositions according to the present invention may be administered parenterally, (e.g. intravenous, intraarterial, intramuscular, intrathecal, subcutaneous), or by infusion techniques. The guide polynucleotides, vectors, editing systems or pharmaceutical compositions may be administered in the form of a sterile aqueous solution which may contain other substances, for example enough salts or glucose to make the solution isotonic with blood. The aqueous solution may be suitably buffered (preferably to a pH of from 3 to 9). The pharmaceutical composition may be formulated accordingly. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well-known to the skilled person.

[0496] The guide polynucleotides, vectors, editing systems or pharmaceutical compositions according to the present invention may be administered systemically, for example by intravenous injection or intraperitoneal injection. In some embodiments, the guide polynucleotide, vector, editing system or pharmaceutical composition according to the present invention is administered by intravenous injection. The pharmaceutical composition may be formulated accordingly.

[0497] For example, viral (e.g. AAV) vectors may be administered locally or systemically. Ribonucleoproteins (RNPs) may, for example, be administered locally.

[0498] The guide polynucleotides, vectors or editing systems may, for example, be administered by in utero electroporation (e.g. in mice). This may aid prevention of symptoms, for example before the onset of seizures.

[0499] Preferably, the guide polynucleotides, vectors, editing systems or pharmaceutical compositions according to the present invention are administered locally, preferably by intracerebroventricular injection (ICV). For example, local administration may be by stereotaxic injection in the hippocampus and / or cortex. In mice, administration may be carried out between postnatal days 1-2.

[0500] The guide polynucleotide, vector, editing system or pharmaceutical composition according to the present invention may be administered locally, for example by direct injection. The pharmaceutical composition may be formulated accordingly.

[0501] The pharmaceutical compositions may comprise enveloped viral particles or cells of the invention in infusion media, for example sterile isotonic solution. The pharmaceutical composition may be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0502] The guide polynucleotide, vector, editing system or pharmaceutical composition may be administered in a single or in multiple doses. Suitably, the guide polynucleotide, vector, editing system or pharmaceutical composition may be administered in a single, one off dose. The pharmaceutical composition may be formulated accordingly.

[0503] The guide polynucleotide, vector, editing system or pharmaceutical composition may be administered at varying doses (e.g. measured in Transducing Units (TU) per kg). The physician in any event may determine the actual dosage which will be most suitable for any individual subject and the dosage may, for example, vary with the age, weight and response of the particular subject. The pharmaceutical composition may be formulated accordingly.

[0504] The guide polynucleotide, vector, editing system or pharmaceutical composition may be administered to any subject in need thereof. The subject may be a mammal (e.g. a human). The guide polynucleotide, vector or editing system of the invention may be used to transduce cells ex vivo. The transduced cells may subsequently be administered to a subject.

[0505] In another aspect, the invention provides a method of transducing a cell, comprising contacting the cell with the guide polynucleotide, vector or editing system of the invention. The method may be, for example, an in vitro or ex vivo method.

[0506] VARIANTS, DERIVATIVES, ANALOGUES AND FRAGMENTS

[0507] In addition to the specific polypeptides and polynucleotides mentioned herein, the invention also encompasses variants, derivatives and fragments thereof.

[0508] In the context of the invention, a “variant” of any given sequence is a sequence in which the specific sequence of residues (whether amino acid or nucleic acid residues) has been modified in such a manner that the polypeptide or polynucleotide in question retains at least one or all of its endogenous functions. A variant sequence can be obtained by addition, deletion, substitution, modification, replacement and / or variation of at least one residue present in the naturally occurring polypeptide or polynucleotide.

[0509] The term “derivative” as used herein in relation to proteins or polypeptides of the invention includes any substitution of, variation of, modification of, replacement of, deletion of and / or addition of one (or more) amino acid residues from or to the sequence, providing that the resultant protein or polypeptide retains at least one or all of its endogenous functions.

[0510] Typically, amino acid substitutions may be made, for example from 1 , 2 or 3, to 10 or 20 substitutions, provided that the modified sequence retains the required activity or ability. Amino acid substitutions may include the use of non-naturally occurring analogues.

[0511] Polypeptides used in the invention may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent polypeptide. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity and / or the amphipathic nature of the residues as long as the endogenous function is retained. For example, negatively charged amino acids include aspartic acid and glutamic acid; positively charged amino acids include lysine and arginine; and amino acids with uncharged polar head groups having similar hydrophilicity values include asparagine, glutamine, serine, threonine and tyrosine.

[0512] Conservative substitutions may be made, for example according to the table below. Amino acids in the same block in the second column and in the same line in the third column may be substituted for each other:

[0513] The effect of additions, deletions, substitutions, modifications, replacements and / or variations may be predicted using any suitable prediction tool, for example SIFT (Vaser et al. (2016) Nature Protocols 11 : 1-9), PolyPhen-2 (Adzhubei et al. (2013) Current Protocols in Human Genetics 76: 7-20), CADD (Rentzsch et al. (2021) Genome Medicine 13: 1-12), REVEL (loannidis et al. (2016) The American Journal of Human Genetics 99: 877-885), MetaLR (Dong et al. (2015) Human Molecular Genetics 24: 2125-2137) and / or MutationAssessor (Reva et al. (2011) Nucleic Acids Research 39: e118-e118) or based on clinical data, for example ClinVar (Landrum et al. (2016) Nucleic Acids Research 44: D862-D868). Suitable additions, deletions, substitutions, modifications, replacements and / or variations may be considered tolerated, benign and / or likely benign.

[0514] Typically, a variant may have a certain identity with the wild type amino acid sequence or the wild type nucleotide sequence.

[0515] In the present context, a variant sequence is taken to include an amino acid sequence which may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical, suitably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the subject sequence. Although a variant can also be considered in terms of similarity (i.e. amino acid residues having similar chemical properties / functions), in the context of the present invention it is preferred to express in terms of sequence identity.

[0516] In the present context, a variant sequence is taken to include a nucleotide sequence which may be at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85% or at least 90% identical, suitably at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the subject sequence. Although a variant can also be considered in terms of similarity, in the context of the present invention it is preferred to express it in terms of sequence identity.

[0517] Suitably, reference to a sequence which has a percent identity to any one of the SEQ ID NOs described herein refers to a sequence that has the stated percent identity over the entire length of the SEQ ID NO referred to. Sequence identity comparisons can be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate percent identity between two or more sequences.

[0518] Percent identity may be calculated over contiguous sequences, i.e. one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an “ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.

[0519] Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion in the amino acid or nucleotide sequence may cause the following residues or codons to be put out of alignment, thus potentially resulting in a large reduction in percent identity when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without penalising unduly the overall identity score. This is achieved by inserting “gaps” in the sequence alignment to try to maximise local identity.

[0520] However, these more complex methods assign “gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids or nucleotides, a sequence alignment with as few gaps as possible, reflecting higher relatedness between the two compared sequences, will achieve a higher score than one with many gaps. “Affine gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties will produce optimised alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.

[0521] Calculation of maximum percent identity therefore firstly requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (Devereux et al. (1984) Nucleic Acids Research 12: 387-395). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package (Altschul, et al. (1990) Journal of Molecular Biology 215: 403-410), BLAST 2 (Tatusova et al. (1999) FEMS Microbiology Letters 174: 247-250), FASTA (Pearson et al. (1988) PNAS 85: 2444-2448), EMBOSS Needle (Madeira et al. (2019) Nucleic Acids Research 47: W636-W641) and the GENEWORKS suite of comparison tools. For some applications, it is preferred to use EMBOSS Needle.

[0522] Although the final percent identity can be measured, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix.

[0523] Once the software has produced an optimal alignment, it is possible to calculate percent sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result. The percent sequence identity may be calculated as the number of identical residues as a percentage of the total residues in the SEQ ID NO referred to.

[0524] “Fragments” are also variants and the term typically refers to a selected region of the polypeptide or polynucleotide that is of interest either functionally or, for example, in an assay. “Fragment” thus refers to an amino acid or nucleic acid sequence that is a portion of a full- length polypeptide or polynucleotide.

[0525] Such variants, derivatives and fragments may be prepared using standard recombinant DNA techniques, such as site-directed mutagenesis. Where insertions are to be made, synthetic DNA encoding the insertion together with 5’ and 3’ flanking regions corresponding to the naturally-occurring sequence either side of the insertion site may be made. The flanking regions will contain convenient restriction sites corresponding to sites in the naturally- occurring sequence so that the sequence may be cut with the appropriate enzyme(s) and the synthetic DNA ligated into the cut. The DNA is then expressed in accordance with the invention to make the encoded polypeptide. These methods are only illustrative of the numerous standard techniques known in the art for manipulation of DNA sequences and other known techniques may also be used.

[0526] The skilled person will understand that they can combine all features of the invention disclosed herein without departing from the scope of the invention as disclosed.

[0527] Preferred features and embodiments of the invention will now be described by way of nonlimiting examples.

[0528] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, biochemistry, molecular biology, microbiology and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press; Ausubel, F.M. et al. (1995 and periodic supplements) Current Protocols in Molecular Biology, Ch. 9, 13 and 16, John Wiley & Sons; Roe, B., Crabtree, J. and Kahn, A. (1996) DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons; Polak, J.M. and McGee, J.O’D. (1990) In Situ Hybridization: Principles and Practice, Oxford University Press; Gait, M.J. (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; and Lilley, D.M. and Dahlberg, J.E. (1992) Methods in Enzymology: DNA Structures Part A: Synthesis and Physical Analysis of DNA, Academic Press. Each of these general texts is herein incorporated by reference.

[0529] EXAMPLES

[0530] EXAMPLE 1

[0531] RESULTS

[0532] Optimisation of SCN1A Kozak sequence increases expression in EGFP reporter system

[0533] To test the hypothesis that an enhancement in the translation efficiency of mRNA molecules can be achieved by optimising the SCN1A / Scn1a gene Kozak sequence, we designed and generated a reporter system to facilitate the screening of different Kozak sequence variants. The reporter plasmid contains a bicistronic expression cassette, granting the simultaneous expression of EGFP and mCherry reporters from the same promoter - Eflalpha (Figure 1A). In this setting, EGFP expression relied on the SCN1A / Scn1a wt and variant Kozak sequences we cloned into the reporter vector in place of the EGFP canonical Kozak sequence, while mCherry expression depended on a fixed Kozak sequence contained in the internal ribosome entry site (IRES), and was used as a normalizer for EGFP expression (Figure 1A). Reporter plasmids carrying the different Kozak variants were lipofected into HEK-293T cells and their ability to drive the translation of EGFP in comparison to the Senia wild-type (wt) Kozak sequence was evaluated by flow cytometry analysis (Figure 1 B). In a first screening, 13 distinct Kozak variants were designed from the SCN1A / Scn1a wt Kozak sequence, which is conserved between the mouse and human genome, and the variants and wt sequence cloned into the reporter vector (Figure 1C). Those variants have been designed on the basis of the possible point mutations that could be inserted by recently developed base editing tools, including both adenine base editors (ABEs) and cytosine base editors (CBEs). In all mCherry positive cells, the ratio between the intensities of EGFP and mCherry fluorophores was determined for each variant and expressed as fold change with respect to the SCN1A / Scn1a wt Kozak sequence (Figure 1 D). Interestingly, three out of 13 variants (variant 1-2-14) significantly increased basal expression levels of EGFP (Figure 1 D), indicating that they provided increased translation efficacy of EGFP protein in comparison to the SCN1A / Scn1a wild-type Kozak sequence.

[0534] Removal of upstream open reading frames (uORFs) enhances main ORF (mORF) expression of SCN1A / Scn1a gene

[0535] We noticed that the three Kozak variants that efficiently increased the basal levels of EGFP carried an A to G mutation in a putative ATG codon upstream of the mORF (Figure 1 D), converting it in a GTG codon. This observation opened the possibility that the 5’ UTR-encoding region of the SCN1A / Scn1a gene contains uORFs that cooperate in the regulation of gene expression by modulating SCN1A / Scn1a mRNA translation efficiency. Indeed, uORFs are another class of major gene expression regulatory elements: half of eukaryotic mRNAs feature uORFs which may promote or inhibit the translation of the mORFs. They are mostly conserved among species, suggesting evolutionary selection of functional uORFs.

[0536] We observed that the 26-nucleotide sequence (100 % conserved between murine and human gene) immediately upstream of the ATG of the mORF in SCN1A / Scn1a contains two ATG codons (Figure 2A) that are in frame with each other, but out of frame with the main ATG (mATG). They both represent starting codons (uATGs) of two overlapping uORFs (uORF-1 and uORF-2); nevertheless, they can also be considered as two alternative start codons (uATGs) of an uORF whose stop codon falls at the beginning of exon 2 in the SCN1A / Scn1a coding sequence (CDS) (Figure 2A), and thus overlaps with the mORF. Interestingly, this uORF is present in the two main isoforms of the murine Senia transcript and, most importantly, is conserved in the human SCN1A transcript. It is likely that start codons in such close proximity to the start codon of the mORF compete with the start codon of the mORF for translation initiation and that translation of uORFs initiated by these competing start codons may negatively regulate mORF translation.

[0537] Preliminary inspection of the 5’ UTR-encoding region of both human and mouse SCN1A / Scn1a evidenced several candidate uORFs belonging to the different mRNA isoforms with half of them being actively translated uORFs, as supported by Ribo-sequencing (Ribo- seq) data (https: / / rnainformatics.org.cn / RiboUORF / search_result.php) (Figure 2B). Interestingly, the 5’ UTR-encoding region of human SCN1A contains 12 predicted uORFs, but we decided to focus on the two uATGs most proximal to the mATG we identified, as they are the only uATGs conserved between the human and mouse SCN1A / Scn1a transcripts, both characterized by a moderate Kozak strength (Figure 2B). The issue of sequence conservation between human and mouse genome is not secondary, considering that it offers the opportunity to test a technology suitable to treat human disorders in cellular and mouse models of disease.

[0538] Given that the presence of nearby secondary structure in the mRNA can affect uATG codon recognition by the translation complex, we performed a new screening strategy by cloning both the entire sequence of either one of the two alternative mouse 5’ UTRs (portion of Exonl before mATG plus ExonA or ExonB) (Figure 2D) and their variants carrying point mutations in one or both of the above identified proximal uATGs (uATG-1 and uATG-2) (Figure 2C) into our bicistronic reporter vector. Flow cytometry analysis confirmed that variants carrying mutations in one or both of the uATGs increased the basal expression of the reporter (Figure 2D). Interestingly, fold changes in EGFP expression were higher when cloning the entire mouse Senia 5’ UTRs (both distal and proximal) compared to the short 5’UTR (conserved 26 nucleotides) (Figure 2D). Specifically, a 2-3 fold increase in EGFP expression was achieved in both proximal and distal variants 15 and 17, carrying both uATGs mutated to GTG. Interestingly, 5’ UTRs variants carrying a mutation in only one of the two uATGs (variants 14 and 16) were also able to induce a detectable increase in the basal expression of the EGFP reporter (Figure 2D). Conversely, variant 23 carrying only the A to G conversion of an adenine located between the two uATGs did not significantly affect the EGFP reporter expression (Figure 2D).

[0539] In close proximity and upstream of uATG-1 and uATG-2 we found a third starting codon (uATG3), of a small uORF terminating before uATG-1 (Figure 3A). To assess its impact on mORF translation, we performed a new screening with 5’ UTR variant 24, in which only uATG- 3 has been mutated, and variant 25, carrying mutations in all three uATGs (Figure 3B). Interestingly, mutation of uATG-3 alone does not impact EGFP translation efficiency; conversely, its mutation concomitant with uATG-1 and uATG-2 provides a small but not significant trend towards a further increase of EGFP translation efficiency (Figure 3C).

[0540] DISCUSSION

[0541] The data described here shows the establishment of a new platform for the identification of target nucleotides in the 5’ UTR-coding region of a gene of interest for increasing protein translation without affecting transcription levels of said gene. The basis of this platform is the discovery that optimising the Kozak sequence surrounding the start codon of the mORF can improve translation of the relevant mRNA. We confirmed these findings by showing that optimised variants of the Kozak sequence surrounding the start codon of the Senia gene mORF increased EGFP expression over the Senia wt Kozak sequence (Figure 1 D). Surprisingly, we found that the variants showing the strongest improvement in EGFP expression levels (variants 1 , 2 and 14) all shared a mutation at position -8 of the Kozak sequence (8 positions upstream of the mORF start codon), which is only of limited importance for strength of the Kozak sequence (Figure 10). We discovered that the mutated nucleotide belongs to a start codon of an uORF, which is conserved in the human and mouse SCN1a / Scn1a transcript (Figure 2B). By mutating this start codon (uATG-2) together with another conserved start codon (uATG-1) upstream of and in frame with uATG-1 , we were able to increase EGFP expression far beyond that achieved with Kozak sequence optimisation (compare var 1 , 2 and 14 in Figure 1 D with v15 and 17 in Figure 2E; see Figure 20 for an explanation of the mutations introduced in variants 15 and 17).

[0542] In conclusion, the outcomes shown here demonstrate that the reporter system screening platform previously used to identify improvements in translation from Kozak sequence optimisation can be adapted to identify the usefulness of mutations in other cis-acting elements within the 5’ UTR, particularly mutations in starts codons of uORFs, for improving translation of a transcript of interest.

[0543] METHODS

[0544] Bicistronic reporter generation and variants cloning

[0545] EGFP / mCherry bicistronic reporter encoding construct - pLV-eF1as-EGFP-IRES-mCherry - was designed to allow the simultaneous expression of both EGFP and mCherry under the control of the same eF1a core promoter. To allow controlled expression of EGFP, its Kozak sequence was designed to be easily exchanged with a Kozak sequence variant-containing short or whole 5’ UTR using restriction-digestion (Nhel-HF, BamHI-HF; New England Biolabs). mCherry expression, instead, was controlled by an afixed Kozak sequence contained into the internal ribosome entry site (IRES), and hence was used as a normalizer for EGFP expression. For short variants, oligonucleotide pairs (Microsynth) encoding the sequence and including overhangs for ligation into the Nhel-HF, BamHI-HF sites (New England Biolabs) were annealed and ligated into the pLV-eF1as-EGFP-IRES-mCherry plasmid predigested with Nhel-HF, BamHI-HF restriction enzymes. Briefly 1 pl of forward and 1 pl of reverse primer (100 pM) were mixed with 1 pl of 10x T4 PNK Ligase Buffer and 0.5 pl of T4-PNK enzyme (New England Biolabs) in a 10 pl reaction. Ligation was performed in a thermocycler using the following program: 37 °C, 30 min; 95 °C, 5 min; ramp-down to 25 °C with 5°C / min; 4 °C, hold. Annealed oligonucleotides were then diluted 1 :100 and 2 pl of this dilution was incubated for ligation. For longer variants, containing Distal or Proximal whole Senia 5’ UTR (Figure 2D), the region of interest was amplified from mouse cortex cDNA with GoTaq G2 DNA Polymerase (Promega) reaction using primers including overhangs for ligation into Nhel-HF, BamHI-HF sites. Digested PCR products were then inserted into predigested pLV-eF1as-EGFP-IRES- mCherry vector (Nhel-HF, BamHI-HF). Tested variants are listed in Figures 1 , 2 and 3.

[0546] Lipofection of HEK-293T cells

[0547] HEK-293T cells were cultured in alpha-MEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma- Aldrich), 1% non-essential amino acids (Gibco), 1% sodium pyruvate (Sigma-Aldrich), 1% glutamine (Sigma-Aldrich), and 1% penicillin / streptomycin (Sigma- Aldrich). Cells were maintained at 37 °C and 5% CO2 at a confluency below 90%, passaged every 2-3 days using 0.25% Trypsin (Sigma-Aldrich). HEK-293T cells were seeded (1x105 cells / well) into 24-well suspension culture plates in 300 ul of medium. Cells were transfected with 1 pl of Lipofectamine 3000 (Thermo Fisher Scientific) with 500 ng of plasmid per well. 3 days after transfection, cells were analysed at flow cytometry.

[0548] Flow cytometry and reporter assay data analysis

[0549] After 3 days from the lipofection of variants cloned in EGFP / mCherry reporter plasmid, cells were resuspended in PBS and kept on ice until analysis. For each sample, 80’000 events were counted on CytoflexS (Beckman Coulter) using the CytExpert (Beckman Coulter). Data analysis was carried out using FlowJo software with the following gating strategy: first, single cells were gated by plotting FSC-H vs. FSC-A. From this population, to identify cells of interest based on size and granularity, FSC vs. SSC were plotted. From the gated population, mCherry+ population was isolated by plotting ECD-A vs. FSC-A. Finally, from mCherry+ population, by gating FITC-A vs. FSC-A, we isolated GFP+ cells. Gating strategy is shown in Figure 1 B. From both mCherry+ and mCherry+>GFP+ populations we determined the median fluorescence intensity (MFI) and, to measure the fold increase of each variant in respect to the wild-type sequence, we calculated the ratio between mCherry+>GFP+ MFI / mCherry+ MFI. This ratio for every variant was then further divided by the ratio of wild-type sequence and plotted as fold change.

[0550] EXAMPLE 2

[0551] RESULTS

[0552] Base editing strategy efficiently installs point mutations in uATGs of SCN1A / Scn1a gene in HEK-293T cells Following the findings in Example 1 , we developed a system to edit in the 5’ UTR-encoding region in the SCN1A / Scn1a endogenous gene locus in a cell using an adenine BE (ABE)- based strategy. Given their close proximity to one another, we decided to focus on an editing strategy for the concomitant mutation of uATG-1 and uATG-2. The complexity of the strategy is that the two adenines to be edited are interspaced by 8 nucleotides and thus the selected sgRNAs have to be able to target both with a reasonable degree of efficiency. To avoid further constrains, SpRYCas9-based BEs (ABEmax and ABE8e) were initially selected because of their high flexibility in the PAM site. Three sgRNAs (SpRY-sgRNA-11 , SpRY-sgRNA-15, SpRY-sgRNA-16) were designed to target both uATG-1 and uATG-2 (Figure 4A) and were tested for their editing efficiency together with two different BEs in HEK-293T cells using lipofection (Figure 4B). To easily assess editing efficiency, we exploited the generation of a restriction fragment length polymorphism (RFLP) resulting from mutating uATG-2. The mutation of the adenine in uATG-2 removes a BstCI restriction site from the locus (Figure 4C). Thus, BstCI restriction analysis of a carefully designed PCR amplicon allowed us to easily discriminate between edited (uncut fragment, UF) and unedited (cut fragment, CF) locus (Figure 4C-D). SpRY-sgRNA-11 , or in short sgRNA-11 , (targeting the first A in position 2 and the second A in position 11) in combination with ABE8e was able to provide a detectable uncut PCR amplicon (645 bps) indicating that editing occurred (Figure 4D). Deep sequencing analysis of the PCR amplicons highlighted that in around 25% of SCN1A alleles at least one of the two uATGs was edited, while more than 7% of the alleles carried edits in both uATGs (Figure 4D). The efficiency in the A to G conversion of the first A (the A of uATG-1) was around 10%, while that of the second A (the A of uATG-2) was around 25% of the total of the alleles (Figure 4E).

[0553] Base editing strategy efficiently installs point mutations in uATGs in mouse primary neurons

[0554] To assess the effect of our base editing strategy in a physiological cell type in which the Senia gene is expressed, we used cortical I hippocampal primary neurons derived from mouse embryos (Figure 5A). Adeno-associated viral (AAV) or lentiviral (LV) vectors were chosen to deliver ABE8e and sgRNA11 in neurons (Figure 5C). In order to circumvent the limited cargo capacity of adeno-associated viral (AAVs) vectors, we exploited an intein-split base editor which allows the splitting of the gene encoding the BE protein into two parts carried by two different AAVs and is assembled by intein-mediated trans-splicing in vivo (Figure 5C). Two weeks after neuron plating, we performed molecular and biochemical analysis to assess the editing efficiency and NaV1.1 protein level. Deep sequencing analysis of a PCR amplicon spanning the edited genomic region of the Senia gene revealed that in around 20% of Senia alleles at least one of the two uATGs was edited and that around 9% of the alleles carried edits in both uATGs (Figure 5D). More specifically, the efficiency in the A to G conversion of the first A (the A of uATG-1) was around 12%, while of the second A (the A of uATG-2) was around 20% over the total of the alleles (Figure 5E). Sequencing data reveal that our base editing strategy has similar efficiency in both proliferating and post mitotic cells, despite the latter being generally more resistant to editing. In addition, Western Blot analysis performed at 14 days after dissection of the primary neurons from mice (14 days in vitro', DIV14) showed increased Nav1.1 protein levels in membrane-enriched protein lysates of samples treated with sgRNA-11 and ABE8e compared to samples treated with ABE8e only (Figure 5F). At the same time, no change was observed at mRNA level (Figure 5G).

[0555] SpRYCas9-based BEs are considered to have more off-target activity in comparison to SpCas9-based BEs. Therefore, after assessing that a BE-based strategy is suitable for our purpose of enhancing NaV1.1 expression by removing the start codons of uORFs, we aimed at also testing SpCas9-BEs. We designed a SpCas9-based sgRNA (Sp-sgRNA-1 or sgRNA- 1) with classic NGG PAM (Figure 6A), targeting the first A (the A of uATG-1) in position 3 and the second A (the A of uATG-2) in position 12, and compared its editing efficacy to SpRY- sgRNA-11 in primary neurons. According to the BtsCI-based PCR amplicon restriction (see Example 1 above), which is a read-out of uATG-2 editing, the two sgRNAs had comparable editing efficiencies at the Senia gene locus (Figure 6B). Deep sequencing analysis of a PCR amplicon spanning the edited genomic region of Senia gene showed that in cells transduced with sgRNA-1 in combination with SpCas9-ABE8e the on-target editing efficiency is much higher than that of sgRNA-11 in combination with SpRYCas9-ABE8e. Transduction with sgRNA-1 and SpCas9-ABE8e resulted in around 60% of Senia alleles being edited in at least one of the two uATGs edited and around 13% of the alleles carrying edits in both uATGs (Figure 6C). The efficiency in the A to G conversion of the first A (the A of uATG-1) was around 60%, while that of the second A (the A of uATG-2) was around 20% over the total of the alleles (Figure 6D). In addition, Western Blot analysis performed at DIV14 showed a significant 1.3- fold increase in the level of Nav1.1 in membrane enriched protein lysates of neurons transduced with sgRNA-1 and SpCas9-ABE8e-treated as compared to neurons transduced with SpCas9-ABE8e only or not transduced (Figure 6E).

[0556] Interfering with uORF effects expression in other SCN genes

[0557] As aforementioned in the Introduction, the SCN gene family includes several paralog genes, whose LOF and GOF mutations can cause other neurological and non-neurological disorders, depending on the organ in which each specific channel is mainly expressed. Considering the high level of homology among the different SCN genes, we hypothesised that they could also share the same uORF-mediated regulation of mRNA translation that we observed in SCN1A / Scn1a. We inspected the portion of the 5’ UTR-encoding regions conserved between human and mouse SCN2A / Scn2a, SCN3A / Scn3a, SCN5A / Scn5a, SCN8A / Scn8a and SCN9A / Scn9a genes and found that they also presented candidate uORFs that could affect mORF expression (Figure 7A). Specifically, SCN2A / Scn2a, SCN8A / Scn8a and SCN9A / Scn9a presented a single uATG, while SCN3A / Scn3a showed a 5’ UTR extremely similar to SCN1A / Scn1a, with two different uATGs in frame with each other and out of frame with the mATG (Figure 7A). Similarly to what we had previously done for SCN1A / Scn1a, we designed variants of the 5’ regions carrying mutations in the adenines of the identified uATGs and cloned them into our bicistronic reporter vector for HEK-293T cell transfection and flow cytometry analysis. We found that all tested variants significantly enhanced EGFP expression levels in comparison to the wild-type region, except for SCN8A / Scn8a where no difference was reported (Figure 7B). However, the short length of the tested 5’ region is likely the reason for the absence of an effect in SCN8A / Scn8a (Figure 7A) and we expect to see an effect if a longer 5’ region or the entire 5’ UTR is to assess the variant.

[0558] Following these encouraging results, we developed a base editing strategy particular for those genes whose LOF mutation cause severe neurological syndrome, namely SCN2A / Scn2a and SCN8A / Scn8a. We designed different sgRNAs compatible with SpRYCas9-ABE8e, some of which target a 5’ region conserved between human and mouse and some of which are species-specific (Figure 8A), and cloned them into LV vectors for testing in primary neurons. Deep sequencing analysis confirmed the results of Sanger sequencing data, showing that sgRNA-2a1 and sgRNA-2a2 converted their target adenine in Scn2a gene locus in almost 30% of alleles (Figure 8B-D). For sgRNA-8a2, the on-target editing efficiency on the adenine of the mATG-proximal uATG in the5’ UTR-encoding region in Scn8a was around 20% (Figure 8E-F). Importantly, the three sgRNAs had no editing effect on the mATG of either Scn2a or Scn8a (Figure 8C, F).

[0559] SUMMARY

[0560] In conclusion, here we provide evidence that a combination of a gRNA and a BE can effectively target the 5’ UTR-encoding region in a gene in its endogenous gene locus and perform several base conversions with a good degree of efficiency to modulate cis-acting elements in the 5’ UTR-encoding region of the gene. Specifically, we identified at least two sgRNAs and at least two different BEs that in combination can efficiently edit the two adenines of uATG-1 and uATG-2 to remove the two mATG-proximal uORFs in the endogenous SCN1A / Scn1a gene locus (Figures 4D, 5C and 6C). We showed that this approach is able to modulate protein levels by acting on translation efficiency, for example of Nav1.1 protein in this case, without affecting mRNA levels (Figure 5G). Further, we found that the same combination of sgRNA and BE could achieve comparable editing efficiency in both mitotic (HEK-293T) and postmitotic (mouse primary neurons) cells, highlighting the systems suitability for the treatment of genetic disorders characterised by LOF or GOF mutations affecting particularly post-mitotic cells (Figure 4D and Figure 5C). Finally, we were able to established that one or both of uATG-1 and uATG-2 of SCN1A / Scn1a are conserved across the 5’ UTRs of human and mouse SCN2A / Scn2a, SCN3A / Scn3a, SCN5A / Scn5a, SCN8A / Scn8a and SCN9A / Scn9a (Figure 7A), that mutations of the conserved uATGs in these genes also enhanced protein expression (Figure 7B) and that the conserved uATGs of at least SCN2A / Scn2a and SCN8A / Scn8a could also effectively be edited using a system combining a sgRNA and a CRISPR / Cas9-based base editor (Figure 8B, E).

[0561] METHODS

[0562] Base editing constructs cloning

[0563] For base editing experiments, sgRNAs were designed against uATGs upstream of mATG of either the Scn1a / SCN1A, Scn2a / SCN2A or Scn8a / SCN8A locus. Nucleic acid (Microsynth) pairs encoding the 20 nt sgRNA sequences and including overhangs for ligation into the Bsmbl site (New England Biolabs) were annealed and ligated as previously described into the LentiGuide-U6-filler-gRNA scaffold plasmid pre-digested with BsmBI restriction enzyme, thus removing the filler sequence.

[0564] For the lentiviral construct containing SpCas9-ABE8e (LV-ef1as-SpCas9-ABE8e), the backbone was generated from LV-ef1aL-SpCas9-ABE8.20 (digested with BamHI-HF, Xmal; New England Biolabs). The relative insert containing the Tad8e domain was amplified from the LV-ef1aL-SpRY-ABE8e construct with Phusion High-Fidelity DNA Polymerase (New England Biolabs) and using primers including overhangs for ligation into BamHI-HI and Xmal sites.

[0565] For AAV constructs of SpCas9-ABE8e, we generated both AAV-Sp-ABE8e-C-int-split (AAV- Cbh_NLS-SpRY(574-1368)-NLS-P2A-RFP-W3-bGH) and AAV-Sp-ABE8e-N-int-split (AAV- Cbh-ABE8e-SpRY(1-573)-N-lnt-NLS-W3-bGH-hU6sgRNA). To generate the C-split construct, we restriction-digested AAV-Cbh_NLS-SpRY(574-1368)-NLS-P2A-RFP-W3-bGH for the backbone (EcoRI-HF, EcoRV-HF; New England Biolabs) and exchanged the region of interest with the insert digested from LV-ef1as-SpCas9-ABE8e (EcoRI-HF, EcoRV-HF). For the N-split construct, the backbone was generated from restriction-digestion of AAV-Cbh- ABE8e-Sp(1-573)-N-lnt-NLS-W3-bGH-hU6sgRNA (Sacl-HF; Pstl-HF; New England Biolabs). The relative insert was amplified from the LV-ef1as-SpCas9-ABE8e construct with Phusion High-Fidelity DNA Polymerase (New England Biolabs) and using primers including overhangs for ligation into Sacl-HF and Pstl-HF. Both AAV-Cbh_NLS-SpRY(574-1368)-NLS-P2A-RFP- W3-bGH and AAV-Cbh-ABE8e-SpRY(1-573)-N-lnt-NLS-W3-bGH-hU6sgRNA were gifts from Lucas Kissling and Gerald Schwank.

[0566] Unless otherwise stated, all plasmids were ligated by incubation for 1 h @ room temperature (RT) with 50 ng of backbone and 5 pl of Ligation Mix (Takara Bio) in a 10 pl reaction. The ligated reaction was then transformed into Stable Chemically Competent E. coli and spread on Luria-Bertani agar plates. Correct cloning of the plasmids was confirmed by Sanger sequencing (Microsynth). Plasmids were isolated using the NucleoBond Xtra Midi Kit (Macherey-Nagel).

[0567] Lipofection of HEK-293T cells

[0568] HEK-293T cells were cultured in alpha-MEM (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Sigma- Aldrich), 1% non-essential amino acids (Gibco), 1% sodium pyruvate (Sigma-Aldrich), 1% glutamine (Sigma-Aldrich), and 1% penicillin / streptomycin (Sigma- Aldrich). Cells were maintained at 37 °C and 5% CO2 at a confluency below 90%, passaged every 2-3 days using 0.25% Trypsin (Sigma-Aldrich). HEK-293T cells were seeded (1x10s cells / well) into 24-well suspension culture plates in 300 pl of medium. One day after seeding, cells were transfected using 1 pl of Lipofectamine 3000 (Thermo Fisher Scientific) with 375 ng of base editor plasmid and 125 ng of sgRNA plasmid per well. After 24 hrs, the medium was removed and replaced with fresh medium supplemented with 2.5 pg / pL Blasticidine. Antibiotic selection of successfully transfected cells was continued for 5 days, at the end of which genomic DNA was extracted for downstream assessments. pT2-CMV-ABE8e-SpRY-Blast and pT2-CMV-ABEmax-SpRY-Blast plasmids gifted from Lucas Kissling and Gerald Schwank, were used (see above).

[0569] Lentiviral vectors production

[0570] Lentiviral replication-incompetent, VSVg-coated lentiviral particles were packaged in H EK- 2937 cells. Cells were transfected with 30 pg of vector and packaging constructs, according to a conventional CaCh transfection protocol. After 30 hrs, medium was collected, filtered through 0.44 pm cellulose acetate and centrifuged at 20000 rpm for 2 hrs at 20 °C in order to concentrate the virus.

[0571] AAV production and purification

[0572] AAV replication-incompetent, recombinant viral particles were produced in HEK-293T cells, cultured in Dulbecco Modified Eagle Medium - high glucose (Sigma-Aldrich) containing 10% fetal bovine serum (Sigma-Aldrich), 1% non-essential amino acids (Gibco), 1% sodium pyruvate (Sigma-Aldrich), 1 % glutamine (Sigma-Aldrich) and 1 % penicillin / streptomycin (Sigma-Aldrich). Cells were split every 3-4 days using Trypsin 0.25% (Sigma-Aldrich). The viral particles were produced by co-transfecting HEK-293T cells with three plasmid - a transgene-containing plasmid, packaging plasmid for rep and cap genes and pHelper (Agilent) for the three adenoviral helper genes - using polyethylenimine (PEI) (Polyscience). The cells and supernatant were harvested at 120 hrs. Cells were lysed in hypertonic buffer (40 mM Tris, 500 mM NaCI, 2 mM MgCh, pH=8) containing 100 ll / rnl Salt Active Nuclease (SAN, Arcticzymes) for 1 hr at 37 °C. The viral particles present in the supernatant were concentrated by precipitation with 8% PEG8000 (Polyethylene glycol 8000, Sigma-Aldrich) and then added to supernatant for an additional incubation of 30 min at 37 °C. In order to clarify the cell lysate, cellular debris was separated by centrifugation (4000 g, 30 min). The viral phase was isolated with an iodixanol step gradient (15%, 25%, 40%, 60% Optiprep, Sigma-Aldrich) in the 40% fraction of the gradient and subsequently concentrated in PBS (Phosphate Buffer Saline) with 100K cut-off concentrator (Amicon Ultral 5, MERCK-Millipore). Virus titers were determined using AAVpro® Titration Kit Ver2 (TaKaRa).

[0573] Derivation of primary neurons

[0574] Primary cultures of mouse embryonic hippocampal neurons were prepared from embryonic day 18.5 (E18.5) from CD1 wt pregnant females. After dissection, cortices ad hippocampi were enzymatically digested with 0.025% trypsin (Gibco) in Hank’s balanced salt solution (HBSS; Euroclone) for 20 min at 37 °C. Then HBSS with trypsin was removed, and the hippocampi were washed with plating medium (neurobasal medium [Gibco] supplemented with 2% B27, 3.3 mM glucose, 1 % glutamine, and penicillin / streptomycin) and mechanically dissociated with a P1000 pipette to obtain a homogeneous cell suspension. Cells were then plated on plates coated with poly-L-lysine (PLL; 0.1 mg / mL) and coverslips. LV and AAV (see above) infections were performed at DIV 1 or DIV3 respectively, and neurons were collected at DIV 14 for genomic DNA or RNA extraction and western blot analysis.

[0575] Western Blot

[0576] Primary neurons were homogenized using the Mem-PER Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions to enrich for the membrane-bound proteins. Western blot analysis was performed on NuPage 4%-8% gradient gels (Thermo Fisher Scientific) using primary antibodies against the following proteins: anti- Nav1.1 (1 :200, Alomone lab) and anti-Calnexin (1 :2,000, Sigma).

[0577] RNA isolation and qRT-PCR Total RNA was extracted using NucleoZOL reagent (Macherey-Nagel) according to the manufacturer’s instructions. For qRT-PCR, cDNA synthesis was obtained using the ImProm- II Reverse Transcription System (Promega), and then qRT-PCR was performed in triplicate with custom-designed oligos using Titan HotTaq EvaGreen qPCR Mix (no ROX) (BIOATLAS). Analysis of relative expression was performed using the AACt method. Briefly, ACt was calculated for each transcript as: Ct (transcript) - Ct (murine 18S rRNA as normalizer) and AACt as: ACt (treated sample) - ACt (control sample).

[0578] Genomic DNA preparation

[0579] Cells were harvested after been detached from transfection / infection plate using Trypsin and centrifuged 1000 x g T. Each pellet was then resuspended in 200 pl / each of PBS and genomic DNA was extracted using QIAamp DNA Mini kit (Qiagen) following the protocol according to the manufacturer’s instructions.

[0580] Restriction fragment length polymorphism (RFLP)

[0581] RFLP allows the rapid and cost-ineffective assessment of base editing efficiency based on the use of a restriction enzyme which is able to cut, or not cut, the DNA only if an edit has been installed (and thus the restriction site is either inserted or deleted). Purified amplicons were checked on electrophoresis gel and digested with pre-selected enzyme (BtsCI, New England Biolabs) in a 20 pl reaction for 2 hr @ 50 °C. To qualitatively assess base editing efficiency, the pattern of digestion (uncut and cut fragments) was then checked on a 2% Agarose electrophoretic gel and imaged using Gel Doc (Biorad).

[0582] Deep sequencing and data analysis

[0583] Next-generation sequencing (NGS) preparation of DNA was performed as previously described. In short, the first PCR was performed to amplify genomic sites of interest with primers containing Illumina forward and reverse adaptor sequences using Phusion High- Fidelity DNA Polymerase (New England Biolabs). 50-150 ng of the extracted genomic DNA per replicate was used in a 50 pL PCR reaction. The PCR program was set as follows: 98°C, 30 s; 98°C, 10 s; 58-60°C, 30 s; 72°C, 1.30 min (repeat 38-40x); 72°C, 10 min, 12 °C, hold. The PCR products were gel-purified with a Wizard SV Gel and PCR Clean-Up System (Promega). Amplicons were quantified using the Qubit instrument (Thermo Fisher Scientific). Next, a second PCR was performed to add barcodes with primers containing unique sets of p5 / p7 Illumina barcodes and the library prepared by Azenta Amplicon-EZ (GeneWitz). All experiments were sequenced paired-end on the MiSeq system (50.000 reads per sample). FASTQ reads were analyzed using CRISPResso. Statistics

[0584] Values are expressed as mean ± standard error of the mean (SEM) as indicated. All statistical analysis was carried out in GraphPad Prism 8.0, using one-way ANOVA, P-values below 0.05 were considered significant. In multi-group comparisons, multiple testing correction for pairwise tests among groups was applied using either Dunnett’s or Sidak’s corrections.

[0585] EXAMPLE 3

[0586] RESULTS

[0587] Removal of upstream Open Reading Frames (uORFs) regulating SCN1A / Scn1a gene expression enhances main ORF (mORF) expression

[0588] Previously identified Kozak variants, sharing an A-to-G mutation in a putative ATG codon upstream of the mORF, raised the possibility that the 5’ UTR of the SCN1A / Scn1a gene contains uORFs before the mORF that cooperate in the regulation of gene expression by modulating mRNA translation efficiency. Due to alternative splicing of untranslated exons, 5' UTRs of human SCN1A and murine Senia differ among their various mRNA isoforms, except for the portion in exon 1 , that is common to all isoforms (Figure 9A, B). Preliminary inspection of this shared region evidenced the presence of four candidate uATGs in human gene, with three of them conserved also in the mouse gene (Figure 9A, B, H, I). uATG-1 , uATG-2 and the human specific uATG-4 are in-frame with each other but out-of-frame to the mATG. Their STOP codons fall at the beginning of the second exon of the mORFs, which makes them candidate start codons of overlapping uORFs (Figure 9A, B, H, I). uATGs are putative start codons of candidate short uORFs with STOP codon falling before mATGs (Figure 9A, B, H, I). For comparative evolutionary analysis, conservation of upstream open reading frames (uORFs) and the main coding sequence (mORF) in the SCN1A 5' untranslated region (5'UTR) was assessed using UCSC Genome Browser (https: / / genome.ucsc.edu). The PhyloP 100- way vertebrate alignment track was used to visualize base-wise conservation across species. This analysis revealed a high degree of evolutionary conservation, peaking at 100% for uATG- 2 and its upstream CAGG sequence in all species analyzed until Lamprey, which is considered the most ancient vertebrate (Figure 9C). A high level of conservation is also evident for uATG- 2 and uATG-3, while it is reduced for uATG-4 (Figure 9C).

[0589] Analysis of publicly available ribosome profiling (Ribo-seq) data of elongating ribosomes from human neural progenitor cells (NPCs) and mouse hippocampal tissues, displayed ribosome footprint peaks before SCN1A / Scn1a mATG, thus supporting the effective translation of uORFs in both human and murine settings (Figure 9D, E). In addition, the higher quality of murine data, allowed to detect a clear drop in the Ribo-seq signal in the exon 2 in correspondence of the STOP codon associated to uORF-1 / 2 (Figure 9J), while no drop in the Ribo-seq signal is present in exon 3 (Figure 9J).

[0590] High conservation usually correlates with functional relevance; therefore, we initially focused on uORF-1 and uORF-2 and hypothesized that abrogating their expression could increase SCN1A / Scn1a gene expression. To test this, we exploited again the EGFP / mCherry bicistronic reporter platform. Given that secondary structures in the 5’ UTR of mRNAs can affect uORF translation efficiency, we cloned the entire alternative murine 5’ UTRs (portion of exonl before mATG plus either ExonA or ExonB, belonging to isoform 1 and isoform 2, respectively) and their variants carrying point mutations removing either one or both uATGs (Figure 9F). Flow cytometry analysis confirmed that variants carrying mutations in at least one uATG (variants 14 and 16) increased the basal expression of the reporter, with higher increase achieved when both uATGs were mutated (variants 15 and 17) (Figure 9G). Conversely, the only A to G conversion of the conserved adenine between the two uATGs (variant 23) did not significantly affect the EGFP reporter expression (Figure 9G).

[0591] Base editing (BE) strategy efficiently install point mutations removing uATGs of SCN1A / Scn1a gene in HEK-293T cells

[0592] As previously described, we first set the technology to install permanent editing in SCN1A / Scn1a 5’IITR in the gene locus by adenine BE (ABE) based strategy using previously identified sgRNA-1 (sg-1) and sgRNA-11 (sg-11). To induce A to G transitions abrogating uATGs, those sgRNAs were tested in combination with ABE8e (ABE) editor (Figure 10A), recognized for its broader editing window in comparison to ABEmax. In parallel, we evaluated sg1 in combination with SpCas9 nuclease, hypothesizing that NHEJ-driven indels abrogating uATGs and altering their regulatory context might result in a comparable enhancement of SCN1A gene expression (Figure 10B). In this setting, SpRY / Sp-ABEs and SpCas9 alone (Ctrl) or associated with sgRNAs were delivered by lentiviral vectors (LVs) in HEK-293T cells, processed after three days of selection for genomic extraction and deep sequencing of PCR product spanning the region of interest (Figure 10C). Deep sequencing analysis evidenced that both SpRY- and Sp-ABEs with their relative sgRNAs could induce target edits, although with different efficiencies. In details, HEK-293T exposed to Sp-ABE with sg1 presented at least one uATG edited in 44% of total reads, in comparison to 26% of SpRY-ABE treated samples (Figure 10D, left panel). The percentage of reads carrying both uATG edited dropped to 8% and 4% in SpRY-ABE with sg11 and Sp-ABE with sg1 , respectively (Figure 10D, right panel). Analysis of the distribution of A>G conversions across the protospacer regions indicated that sg11 was less efficient than sg1 in editing uATG-2 (around 12% versus 50%), but more efficient in editing uATG-1 (25% versus 6%) (Figure 10E, F). Interestingly, with both sgRNAs also the adenine belonging to the ultra-conserved CAGG region before uATG-2 was efficiently edited (Figure 10E, F).

[0593] The approach based on sg1 associated with SpCas9 resulted in lower editing efficiency and posed a higher risk of undesired outcomes. In fact, around 5% of reads presented at least one uATG disrupted (Figure 10G) and only in less than 1% of reads both uATGs were eliminated (Figure 10H). Importantly, around 12% of reads presented disruption of the main ATG of SCN1A (Figure 101). For this reason, we selected ABEs with both sg1 and sg11 for further testing in relevant cell types expressing SCN1A / Scn1a, specifically mouse and human neurons (Figure 11).

[0594] SUMMARY

[0595] In conclusion, we identified ultra-conserved overlapping and non-overlapping uORFs in the 5’ UTR of SCN1A / Scn1a mRNA and showed that their abrogation in a reporter system can alter the translation efficiency of the mORF, enhancing its expression. Using a system of a SpCas9- based ABE8e and a sgRNA (ABE_uORF), we were able to abrogate two of the uORFs (uORF- 1 and uORF-2) alone or together in HEK-293T cells.

[0596] METHODS

[0597] Multiple sequence alignment of 5’ UTR

[0598] For comparative evolutionary analysis, conservation of upstream open reading frames (uORFs) and the main coding sequence (mORF) in the SCN1A 5' untranslated region (5' UTR) was assessed using the UCSC Genome Browser (https: / / genome.ucsc.edu). The PhyloP 100-way vertebrate alignment track was used to visualize base-wise conservation across species. Multispecies alignments were examined to assess conservation of start codons and surrounding sequence for annotated and predicted uORFs, and the SCN1A main ATG. uORF start codons were annotated relative to the human reference genome (hg38) and compared across representative vertebrates. Sequence logos and alignment columns were extracted directly from the UCSC display using the Multiz Alignments of 100 Vertebrates track.

[0599] 5’ UTR nucleotide sequences of human SCN1A, SCN2A, SCN3A, SCN5A, SCN8A and SCN9A and their murine orthologs (Scn1a-Scn9a) were aligned using MUSCLE algorithm (v3.8.1551) implemented within SnapGene software (https: / / www.snapgene.com). Alignments were performed to compare conservation of upstream AUG codons (uATGs) and surrounding sequence context across human and mouse paralogs. Predicted uORFs ATG (uATG-1 to uATG-4) and the main ATG (mATG) were annotated based on the SCN1A transcript and aligned against corresponding positions in related sodium channel gene family members. Sequence logos were generated from the aligned regions to highlight conservation and sequence features across paralogs and species.

[0600] Utilization of Ribo- and RNA- sequencing data

[0601] Publicly available Ribo-seq and RNA-seq datasets were retrieved from NCBI GEO and are listed in Figure 9D, E, J. FASTQ file download from NCBI SRA and initial quality control (QC) filtering were performed with fastq-dump v2.11.0 (https: / / trace.ncbi. nlm.nih.gov / Traces / sra / sra.cgi?view=toolkit_doc&f=fastq-dump). Additional quality checks and adaptor trimming were performed with FastQC vO.11.9 (https: / / www.bioinformatics.babraham.ac.uk / projects / fastqc / ) and Trimmomatic v0.39 (Bolger et al. A flexible trimmer for Illumina sequence data. Bioinformatics. 2014;30(15):2114-2120. doi:10.1093 / bioinformatics / btu170) . Read mapping to the human and mouse reference genomes (GRCh38 and GRCm39 primary genome assemblies), finalized with GENCODE gene annotations (Frankish et al. GENCODE reference annotation for the human and mouse genomes. Nucleic Acids Res. 2019;47(D1):D766-D773. doi:10.1093 / nar / gky955), was performed with STAR v2.7.9a (Dobin et al. STAR: Ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29(1):15-21. doi:10.1093 / bioinformatics / bts635). Post-mapping QC was performed with MultiQC v1.10.1(Ewels et al. Summarize analysis results for multiple tools and samples in a single report. Bioinformatics. 2016;32(19):3047-3048. doi:10.1093 / bioinformatics / btw354). The bigwig coverage tracks were computed with bamCoverage (Ramirez et al. deepTools2: a next generation web server for deep-sequencing data analysis. Nucleic Acids Res. 2016;44(W1):W160-W165. doi:10.1093 / NAR / GKW257). Strand-specific and normalized coverage tracks were generated and visualized in Integrative Genomics Viewer (IGV) (Thorvaldsdottir et al. Integrative Genomics Viewer (IGV): High- performance genomics data visualization and exploration. Brief Bioinform. 2013;14(2):178- 192. doi:10.1093 / bib / bbs017) to assess ribosome occupancy and transcript expression across the SCN1A / Scn1a locus. Ribo-seq and RNA-seq tracks were scaled uniformly across samples to enable direct comparison.

[0602] Transduction of HEK-293T cells for editing experiments

[0603] For base editing experiments, one day after seeding, HEK-293T cells were transduced using lentiviral vectors carrying ABE and sgRNA (3:1 ratio). After 24 hrs, the medium was removed and replaced with fresh medium supplemented with 2.5 pg / pL Blasticidine. Antibiotic selection of successfully transfected cells was continued for 5 days, at the end of which genomic DNA was extracted for downstream assessments. Differentiation of human neurons from iPSC-derived NPCs

[0604] NPCs were generated as previously described with appropriated optimization (Marchetto et al., 2010. A model for neural development and treatment of Rett syndrome using human induced pluripotent stem cells. Cell. 2010 Nov 12;143(4):527-39. doi: 10.1016 / j.cell.2010.10.016). For neuronal differentiation, NPCs were dissociated with Accutase and plated on matrigel-coated 6-well plates (1 * 30.0000 cells per well) in NPC medium. Two days after, the differentiation medium containing Neurobasal (ThermoFisher Scientific), Pen / Strep (1 %), B27 (1 :50), SU5402 (Sigma-Aldrich, 10 pM), PD0325901 (Sigma- Aldrich, 8 pM), DAPT (Sigma-Aldrich, 10 pM) was added and kept for 2 days. Differentiation medium was replaced every day with a fresh one on days 1 and 2. At day 3, cells were detached by Accutase solution incubation at 37 °C for 10 min to obtain a single-cell suspension. Cells were centrifuged, counted, and seeded at a density of 55.000 cells / cm2onto poly-L-lysine / laminin / fibronectin (all from Sigma-Aldrich, 100 pg / ml, 2 pg / ml, 2 pg / ml)-coated plates in neuronal maturation medium supplemented with ROCK inhibitor Y27632 (10 pM) for the first 24 h. Neuronal maturation medium was composed by Neurobasal (ThermoFisher Scientific) supplemented with B27 (1 :50), 2 mM glutamine, 1 % Pen / Strept, BDNF (Peprotech, 20 ng / ml), ascorbic acid (Sigma-Aldrich, 100 nM), Laminin (1 pg / pl), DAPT (10 pM), dbcAMP (Selleckchem, 250 pM). The culture medium was replaced the next day to remove the ROCK inhibitor, and then half of the medium was replaced with a fresh neuronal maturation medium twice a week.

[0605] EXAMPLE 4

[0606] RESULTS

[0607] Base editing strategy efficiently installs point mutations removing uATGs and increases basal Nav1.1 protein level in mouse primary neurons

[0608] To assess the effect of our base editing (BE) strategy in a physiological cell type in which Senia gene is expressed, we moved to assessing the editing efficiency of Senia uATGs in cortical / hippocampal primary neurons derived from mouse embryos (Figure 11). To deliver ABEs in those cells, adeno associated viral vectors 9 (AAV9) were chosen and, to circumvent their limited cargo capacity, intein-split ABEs were employed with one AAV vector carrying the N-terminal part of ABE and sgRNA cassette (N-ABE), while the second AAV carrying the C-terminal part of the editor and a red fluorescent protein (RFP) reporter (C-ABE) (Figure 11 B). Mouse primary neurons were derived from embryonic cerebral cortex and hippocampus, transduced by N-ABE and C-ABE AAVs (or C-ABE alone in control condition) 3 days after plating and harvested at day 21 for molecular and biochemical analysis to assess uATG editing efficiency and Nav1.1 protein level. Deep sequencing analysis of the PCR amplicon spanning the edited genomic region of Senia gene revealed that, in mouse post-mitotic cells, Sp-ABE with sg1 outperformed SpRY-ABE with sg11 , yielding higher percentages of reads carrying target mutations (around 60% versus 20% with at least one uATG mutated and around 12% versus 9% carrying both uATGs mutated, respectively) (Figure 11 D). Editing efficiency of Sp-ABE with sg1 was higher in comparison to SpRY-ABE with sg11 also when we looked at the distribution of A>G conversions across the protospacer sequences of the two sgRNAs (Figure 11 E, F).

[0609] To assess if uATGs editing achieved by the two sgRNAs was sufficient to shift ribosome preference to the mORF translation and ultimately increase Nav1.1 protein level, we performed Western Blot and Jess Automated Western Blot analysis in primary neurons derived from Dravet embryos and relative controls (Figure 11C, G, J). Quantification of Jess revealed that the combination of Sp-ABE with sg1 induced a significant increase in Nav1.1 protein level in Dravet (Scn1aSTOP / +)neurons in comparison to C-ABE transduced control, that restored protein to levels comparable to Ctrl neurons (Scn1a+ / +); instead, only a trend toward the increase was detected in sg11 SpRY-ABE treated samples (Figure 11G, J). Given the effect on Nav1.1 protein level, we named ABE_uORF the condition in which Sp-ABE was combined with sg1 and ABE_Ctrl the condition in which samples were treated only with C- ABE.

[0610] To assess the effect of increased Nav1.1 protein level on the activity of Dravet neurons, we performed whole-cell patch clamp electrophysiology of both pyramidal neurons and GABAergic INs in primary cultures of Dravet neurons at 14-16 days in vitro treated with ABE_uORF and compared their excitability to ABE_Ctrl Dravet neurons (Figure 11C, L, M, P). Both pyramidal and GABAergic INs from ABE_uORF treated Dravet neurons were substantially more excitable than their respective counter parts in the ABE_Ctrl condition, as measured by input-output curves generated in response to square-wave current injections of ascending amplitude and the maximal number of action potentials (AP) generated (Figure 11H, I). While resting membrane potential and threshold potential did not change in both Dravet pyramidal cells and INs upon ABE_uORF, we observed a significant reduction in the rheobase and current threshold density in both ABE_uORF Dravet pyramidal cells and INs in comparison (Figure 11 N, O, Q, R).

[0611] Base editing strategy proves to be effective in human NPC-derived neurons

[0612] We finally tested ABE_uORF in human neurons differentiated from human induced pluripotent stem cell (hiPSCs)-derived from Neural Progenitor Cells (NPCs), with a protocol yielding 80% of GABAergic neurons, the neuronal subtype expressing higher level of Nav1.1 (Figure 12A, B). Cas9 immunofluorescence signal was detected in around 40% of Map2+ cells and 50% of total GABA+ neurons, indicating good level of transduction in human neurons (Figure 12B). Sequencing analysis of human neurons exposed to ABE_uORF tool evidenced that 40% of the reads carried at least one uATG mutated and 8% of reads carried both target edits (Figure 12C). Also in human context, higher level of editing was detected at adenine at position 3 in the protospacer sequence (A3) belonging to uATG-2 (Figure 12D).

[0613] Base editing shows negligible off-target activity and bystander editing effect

[0614] Next, we performed analysis to detect off-target activity of our ABE tools on a selection of CRISPOR (CRISPOR.tefor.net) nominated candidate off-target sites (OTs). OTs presenting no more than 3 mismatches within target protospacer regions and falling in exons were selected, resulting in a total of six OTs for sg11 and two for sg1 (Figure 13A, B). We assessed editing at each of these candidate sites by deep sequencing of neurons exposed to SpRY- ABE+sg11 or Sp-ABE+sg1 , that revealed no editing activity for both sgRNA-ABE combinations at most of their respective OTs. Neglectable editing activity (around 2%) was detected at OT 1 of sg1 , corresponding to the 5’ UTR of Scn3a gene, a paralog gene of Senia which is, however, mainly expressed during embryonic / fetal development (Figure 13B).

[0615] To determine the effect of bystander editing - undesired modifications at non-target adenines in the protospacer - on mORF translation, we exploited again our EGFP / mCherry reporter platform to screen 5’ UTR of both murine Senia isoforms. In this case, 5' UTR variants carried A>G conversions at non-target adenines together with the target uATG edits (Figure 13C). We selected these variants based on NGS analysis, which identified them as the most abundant events, and cloned them into a reporter to assess their effect on EGFP / mCherry fluorescence. Interestingly, these variants showed only minor changes, with no significant differences in EGFP / mCherry expression, when compared to the ideal editing configuration, with only one or both uATGs edited (Figure 13D).

[0616] SUMMARY

[0617] In conclusion, we demonstrated that ABE_uORF editing leads to functional effects in human and mouse neurons, with increase in the firing activity of both human pyramidal neurons and human GABAergic INs. Our data suggest thatABEs efficiently edit SCN1A / Scn 1a uATGs with minimal unintended editing outcomes and no detected off-target edits of anticipated consequence in the CNS. Altogether, these findings demonstrate targeted and efficient activity of ABE_uORF in both mouse and human neurons, supporting its therapeutic potential for future translational applications. METHODS

[0618] Base editing cloning for whole-cell patch clamp and poison exon SA base editing

[0619] For whole-cell patch clamp recordings, C-split construct harboring GFP instead of RFP as reporter cassette was also generated by exchanging P2A-RFP with T2A-GFP using EcoRI and Bglll enzymes.

[0620] For base editing experiment, sgRNAs were designed against splicing acceptor of exon 20N (“poison exon”) of Scn1a / SCN1A locus. SgRNAs were cloned in LentiGuide-U6-filler-gRNA scaffold plasmid as previously described.

[0621] Derivation of primary neurons for whole-cell patch clamp

[0622] Primary cultures of mouse embryonic hippocampal neurons were prepared from embryonic day 18.5 (E18.5) from wild-type or Dravet pregnant females. Neurons were derived as previously described from cortices ad hippocampi and 200.000 cells / well were plated on plates coated with poly-L-lysine (PLL; 0.1 mg / mL) and coverslips cover slips in Neurobasal™ medium [Gibco] (supplemented with 2% B27, 3.3 mM glucose, 1 % glutamine, and penicillin / streptomycin). AAVs transduction was performed at day 3, and 50% of medium was changed every 3-4 days with BrainPhys™ Neuronal Culture Medium (supplemented with NeuroCult™ SM1 Neuronal Supplement and penicillin / streptomycin) (STEMCELL Technologies).

[0623] Whole-cell patch clamp recordings

[0624] At day 13-15, mouse primary neurons were placed in the recording chamber beneath a 40X water immersion lens and visualized using both epifluorescence and infrared differential interference contrast (IR-DIC) video microscopy. Transduced cells were identified thanks to the expression of GFP reporter, expressed in the C-split AAV of base editor. Both excitatory (pyramidal) and inhibitory (IN) neurons were recorded, identified by the shape of their somata (pyramidal vs big, round or bipolar) and confirmed by typical electrophysiological parameters including action potential (AP) frequency and amplitude adaptation as well as slow afterhyperpolarization (AHP > 10ms) for pyramidal neurons vs little or no frequency adaption and fast AHP (< 5 ms) for fast-spiking INs. Whole-cell patch-clamp recordings were performed at room temperature (23-25°C) in continuous superfusion with ACSF (in mM: 140 NaCI, 10 glucose, 10 HEPES, 4 KCI, 1 MgCI2 and 2 CaCI2 (pH 7.4)), in the presence of CNQX (20pM) and picrotoxin (30|JM) to block synaptic transmission. For current clamp (CC) recordings patch electrodes (2.5-3 MQ) contained (in mM): 125 K-gluconate, 5 NaCI, 5 KCI, 5 EGTA, 10 HEPES, 2MgCI2, 3 ATP-Mg2, 0.3 GTP-Na, pH 7.3. Resting membrane potential (Vm) was recorded for at least 2 minutes before holding the potential at -70 mV for following measurements. Input-output (IO) curves were obtained by injecting 500ms-current steps of increasing amplitude from -100 to 800 pA (A = 20pA). An AP was defined by a rising slope > 15 mV / ms and a peak exceeding -10 mV. Neurons with unstable or depolarized (> -50 mV) Vm and / or with a holding current > 200 pA when held at -70 mV were discarded. Recordings were acquired using a Multiclamp 700B Amplifier (Molecular Devices), low-pass filtered at 10 kHz, and digitized at 50 kHz using a Digidata 1550 D / A converter (Molecular Devices) controlled by the pCLAMPH software (Molecular Devices). Access resistance (Ra) was monitored continuously during the recording and cells with a Ra > 15 MQ, or with a variation greater than 20% were excluded from analysis.

[0625] Electrophysiology analysis

[0626] All CC recordings were analysed using Clampfit 11.2 (Molecular Devices). Passive membrane properties were derived from the voltage response to the hyperpolarizing current steps of the current step protocol (average of five steps), as previously described (Colasante G, Lignani G, Brusco S, et al. dCas9-Based Senia Gene Activation Restores Inhibitory Interneuron Excitability and Attenuates Seizures in Dravet Syndrome Mice. Mol Ther. 2020;28(1):235-253. doi:10.1016 / j.ymthe.2019.08.018). The voltage threshold for firing was determined through the phase-plane plot (plot of the time derivative of voltage (dV / dt) versus voltage) of the first AP elicited by the current step protocol (Bean, 2007). The threshold was defined as the first voltage value at which dV / dt exceeded 10 mV / ms. Rheobase was determined as the amplitude of the first current step at which an AP was fired, and current threshold density was calculated by dividing the rheobase by cell capacitance. Data are presented as means ± SEM in IO curves while other parameters are reported as box plots displaying mean (+), median (internal horizontal line), first and third quartiles (upper and lower box edges) and minimal and max values (whiskers) of the data distribution. Circles represent individual data points from each cell. Statistical analysis was performed using Prism 8.0 (GraphPad) and is reported in each figure legend. Normal distribution of experimental data was assessed using D’Agostino- Pearson’s normality test, while ROUT test was used to detect outliers. Unpaired t test was used to compare means of normally distributed sample groups. When data were not normally distributed, Mann-Whitney non-parametric test was used. IO curves were analysed through a mixed-effects model via restricted maximum likelihood (REML) estimation followed by Sidak’s post-hoc comparison. Statistical significance was reached when p-value < 0.05.

[0627] Immunostaining For immunostaining cells, cells on slides were fixed with paraformaldehyde 4% / PBS for 20 minutes. After washing in PBS, cells were blocked in a solution containing 10% donkey serum, 0.1 % TritonX-100 in PBS and then incubated with primary antibodies diluted in the same blocking solution.

[0628] Automated Capillary Western Blot (Jess)

[0629] Capillary Western Blot experiments were performed using the Jess Simple Western system with a 66 to 440-kDa fluorescence separation module (Bio-Techne, Abingdon, UK) according to the manufacturer’s instructions. Primary antibodies anti-Nav1.1 (1 :25 Alomone lab) was used for relative quantification. RePlex Module (cat. No. RP-001) and Total protein detection module for chemiluminescence-based assays (cat. No. DM-TP01) were used to remove primary and secondary antibodies and perform total protein quantification, respectively. Secondary antibody aRb-HRP (Bio-Techne, Abingdon, UK) was used as per the manufacturer’s instructions (i.e., undiluted). The results were analyzed using Compass for Simple Western software (version 6.3.0).

[0630] For tissue immunostaining, mice were anesthetized and perfused with paraformaldehyde 4% / PBS. Brains were extracted and post-fixed overnight in paraformaldehyde 4% / PBS, then cryoprotected in sucrose 30% / PBS and frozen in isopentane (Sigma). 50 pm-thick coronal brain sections were cut with a cryostat, and free-floating sections were used for immunofluorescence analyses. Briefly, after a quick wash in PBS, brain sections were blocked in a solution containing 10% donkey serum, 0.3% TritonX-100 in PBS and then incubated with primary antibodies diluted in the same blocking solution. The following primary antibodies were used and nuclei were counterstained with Hoechst (ThermoFisher):

[0631] Primary antibody Dilution Company

[0632] Anti-Cas9 (Mouse) 1 :200 Abeam

[0633] Anti-GABA (Rabbit) 1 :500-1000 Sigma-Aldrich

[0634] Anti-MAP2 (Chicken) 1 :500 Abeam

[0635] Anti-RFP (Rabbit) 1 :500 MBL

[0636] Genomic DNA preparation for deep sequencing from tissues

[0637] To extract gDNA from tissues (cortex and hippocampus), NucleoSpin DNA Lipid Tissue kit (Macherey-Nagel) was used according to manufacturer’s instructions. Target site was then PCR-amplified using primers harboring Illumina adapters and sequenced as previously described.

[0638] Sanger sequencing Genomic sites were amplified using Phusion High-Fidelity DNA Polymerase (New England Biolabs) with the following PCR program: 98°C, 30 s; 98°C, 10 s; 58-60°C, 30 s; 72°C, 1.30 min (repeat 38-40x); 72°C, 10 min, 12 °C, hold. PCR products, purified as previously described, were then sequenced with the respective in-sequence primers via Sanger sequencing (Microsynth). Editing efficiency was determined by BEAT analysis (https: / / hanlab.cc / beat / ) (Xu et al. A Python Program to Quantify Base Editing from Sanger Sequencing. Cris J. 2019;2(4):223-229. doi:10.1089 / crispr.2019.0017).

[0639] Off-target editing activity analysis

[0640] SgRNA-dependent off-target sites were in silico predicted using CRISPOR (https: / / crispor.gi.ucsc.edu / ). Including criteria for candidate off-targets search were: up to 3 mismatch from sgRNA sequence, belonging to exonic sequences and with a CFD specificity score > 0. Predicted sites are listed in Figure 13A. Preparation of samples for NGS was performed as previously described using off-target sites specific primers.

[0641] To assess the impact of unwanted conversions in the target sequence (i.e. bystander edits) on fluorescence intensity, and thus on translation efficiency, variants harboring one or more bystander edits in the target region were cloned in the reporter vector as previously described and screened by flow cytometry.

[0642] EXAMPLE 5

[0643] RESULTS

[0644] Efficient ABE-mediated disruption of uATG-3

[0645] Upstream to uATG-1 and uATG-2 in SCN1A / Scn1a gene locus, we identified a third uATG-3, candidate starting codon of a small uORF terminating before uATG-2 (Figure 9 and Figure 14). While this uORF is present in both human and mice, the specific sequence is not conserved. To assess its impact on mORF translation, and determine if its abrogation could have additive effect on uATG-1 and uATG-2 removal, we performed a new screening with 5’ UTR variant 24, in which only uATG-3 has been abolished, and variant 25, carrying all the three uATGs mutated (Figure 14A). Interestingly, mutation of uATG-3 already increases EGFP translation efficiency; conversely, its mutation concomitant with uATG-1 / uATG-2 provides significant further increase of EGFP translation efficiency (Figure 14B). To test the effect of uATG-3 abrogation in the Senia locus, we designed three specific sgRNAs targeting uATG-3 in mouse genome (Figure 14C). Sanger sequencing analysis in primary neurons treated with Sp-ABE8e showed comparable editing efficiency at the target adenine among the three sgRNA tested (Figure 14D, E, F). Sg3.1 was then selected and tested again in primary neurons. The editing of uATG-3 adenine (belonging to “TAG” on the opposite strand) was detected in around 60% of total reads in Sp-ABE+ sg3.1 samples (Figure 14G, H).

[0646] SUMMARY

[0647] Considering the results obtained in the reporter, our findings show that the efficient editing of uATG-3 itself can provide an effect on Nav1.1 protein level. In addition, they show that the simultaneous editing of uATG-1 , uATG-2 and uATG-3 further increases protein expression, highlighting benefits for combinatorial strategies.

[0648] EXAMPLE 6

[0649] In vivo delivery of ABE_uORF in Dravet mouse model ameliorates SUDEP and thermal induced seizures by increasing Nav1.1 gene dosage

[0650] To test the therapeutic benefit of this novel treatment in a DS model, we delivered our dual- AAV9 ABE_uORF or ABE_Ctrl in vivo by intracerebroventricular (ICV) injection into neonatal (postnatal day 0-1 , P0-P1) mice. We tested two viral doses, 2x109total viral genomes (vg) (Figure 15) and 1x101° total vg (Figure 16), to correlate editing efficiency with specific phenotypic effects. We measured the effect of the treatment on two hallmark phenotypes of Dravet mouse models - SUDEP and susceptibility to thermal-induced seizures - which are also characteristic of disease in humans.

[0651] After AAV injection in neonatal mice, we monitored Scn1a+I+(Ctrl) and Scn7aSTOP / +(Dravet) mice treated either with ABE_Ctrl or ABE_uORF for SUDEP up to P30; in addition, to test whether ABE_uORF can ameliorate susceptibility to thermal-induced seizures, at P30, we elevated the core body temperature of Dravet mice treated with Ctrl or ABE_uORF and monitored seizure activity up to 42.5 °C. Then, mice were euthanized, brain dissected and cortical and hippocampal tissues processed for molecular and biochemical analysis, to assess uORF editing efficiency and Nav1.1 protein level (Figure 15A, Figure 16A). Sen 7aSTOP / +mouse model displays a strong SUDEP phenotype, with around 70% of mice dying between P18 and P30. This rate of mortality was maintained in ABE_Ctrl treated Dravet mice, with 9 out of 15 mice (60%) (2x109vg dose cohort: Figure 16B) and 11 out of 16 mice (68.8%) (1x101° dose cohort: Figure 16C) dead before P30.

[0652] Starting with mice receiving 2x109total vg, a trend toward the amelioration of SUDEP phenotype was observed in ABE_uORF treated Dravet mice, with first mice dying at P22 and a total of 55% (11 / 20) deaths before P30 (Figure 15B). No effect of ABE_uORF treatment was observed on Ctrl group of mice (Scn1a+I+) (Figure 15B). When surviving mice were subjected to a body temperature increase session, no difference in the percentage of mice developing a tonic-clonic seizure was observed between the two groups, when the temperature was increased up to 42.5 °C (Figure 15C). Molecular analysis performed on the cerebral cortices and hippocampi of ABE_uORF treated Dravet mice evidenced an efficiency of around 18% of uATGs on-target editing in the cerebral cortex (Figure 15D, E) and around 8% in the hippocampus (Figure 15H, I). Despite the low rate of editing, Western blot analysis of contralateral cortices and hippocampi showed a non-significant trend toward the increase of Nav1.1 protein level in comparison to ABE_Ctrl group in both cortex (Figure 15F, G) and hippocampus (Figure 15J, K).

[0653] We proceeded to test the efficacy of ABE_uORF treatment with a higher viral dose (1x1O10total vg) using the same experimental paradigm as the previous experiment (Figure 16A). Immunofluorescence analysis of injected brains highlighted that higher viral dose ensured diffuse level of transduction in multiple areas of the cerebral cortex (including visual, auditory and entorhinal cortex), of the hippocampus (including CA1 , CA2 and CA3), and of the thalamus (Figure 16B-B”). In contrast to the previous dose cohort, in this case a dramatic reduction in the SLIDEP rate was observed in the ABE_uORF treated group, with only 21% (3 / 14) of mice dying and first deaths occurring at P23 (Figure 16C). When looking at susceptibility to thermal induced seizures, 5 out of 5 (100%) of them had a tonic clonic seizure between 41.5 and 42.5°C, while only 4 out of 11 (36%) of ABE_uORF treated Dravet mice experienced a seizure.

[0654] Also, in this group of animals, we performed molecular and biochemical analysis to determine the level of uORF editing underlying this significant phenotypic recovery. In the cerebral cortex, the editing efficiency at A3, representing target adenine of uATG-2, was around 33% and similar level of editing was detected at A9 of sg1 protospacer; instead, the editing rate at A12, constituting target adenine of uATG-1 , dropped to 7% (Figure 16F, G). Lower editing was observed in the hippocampus on both all the three adenines (Figure 16J, K). Importantly, in this case WB analysis evidenced a significant increase in the level of Nav1.1 protein in ABE_uORF treated Dravet mice in comparison to ABE_Ctrl, in both the cerebral cortex and hippocampus (Figure 161, M).

[0655] SUMMARY

[0656] Collectively, these findings establish that higher dosage of ABE_uORF treatment reduces premature death and susceptibility to thermal induced seizures in DS mice, by significantly increasing Nav1.1 protein levels.

[0657] METHODS Mice

[0658] Mice were maintained at San Raffaele Scientific Institute Institutional mouse facility (Milan, Italy) housed under 12 h dark-light cycle, with a relative humidity of 50-60%, a temperature of 25 °C and fed ad libitum. Scn1aSiopl+knock-in mice (Valassina N, Brusco S, Salamone A, et al. Senia gene reactivation after symptom onset rescues pathological phenotypes in a mouse model of Dravet syndrome. Nat Commun. 2022;13(1). doi:10.1038 / s41467-021-27837-w) were maintained in a Sv129 background and crossed with C57BL / 6 J to generate F1 mice for the study. The experiments were designed to achieve a power > 0.8 with an a = 0.05 and the 3Rs guidelines for animal welfare were followed. All procedures were performed according to protocols approved by the internal IACUC and reported to the Italian Ministry of Health according to the European Communities Council Directive 2010 / 63 / EU. Each animal was considered as an experimental unit in survival and thermoinduction assay.

[0659] Mouse genotyping

[0660] Mouse genomic DNA was isolated from tail biopsies following the tail DNA purification protocol: the biopsies were digested overnight at 55°C with gentle shaking in 450pl solution formed by Tail Lysis buffer (20% SDS, 1 M Tris HCI pH 8.0, 0.5M EDTA pH 8.0, 5M NaCI) and proteinase K (200 pg / ml). After lysis, samples were centrifuged (13,000 rpm, 10 min) to pellet mouse tail impurities and the supernatant was transferred to a new tube. 1 :1 isopropanol was added to the solution, mixing vigorously to allow DNA precipitation and a second centrifugation was then performed (13000 rpm, 2 min) to pellet the genomic DNA. The supernatant was discarded, and the pellet washed with 150 pl of 70% ethanol by centrifugation (13000 rpm, 2 min). The supernatant was again removed, the samples were air-dried for 15-20 minutes and at the end the genomic DNA was resuspended in sterile water. PCR thermocycler program was: 95°C, 3 min; 95°C, 30 s; 60°C, 30 s; 72°C, 45 s (repeat 34x); 72°C, 5 min, 12 °C, hold.

[0661] AAV delivery in neonatal mice

[0662] AAV delivery of ABE in perinatal pups was performed by unilateral intracerebroventricular injection (ICV) of either 2 x 109vg / AAV / mouse or 1 x 101° vg / AAV / mouse in a total volume of 5 ul of PBS. Pups were anesthetized directly on ice for 30-60 seconds and, following injection, the animals were placed on a warm pad for at least 2-3 min to facilitate recovery and rewarming.

[0663] Survival monitoring and seizure thermal induction

[0664] Mice were monitored daily for survival evaluation up to P30 without knowing the genotype and treatment. Survival data for each genotype were analyzed, and Kaplan-Meier survival curves were generated using GraphPad Prism (GraphPad Software, San Diego, CA, USA). For seizure thermal induction, we adopted a previously published protocol (Oakley et al. Temperature- and age-dependent seizures in a mouse model of severe myoclonic epilepsy in infancy. Proc Natl Acad Sci USA. 2009 Mar 10;106(10):3994-9. doi: 10.1073 / pnas.0813330106) with some modifications. Briefly, mice were placed in a glass becker and heated with an infrared heat lamp (HL-1 , Phisitemp) to gradually increase the body temperature, controlled by a TCAT-2DF thermo controller (Phisitemp). Mouse rectal temperature was continuously controlled by using a RET-4 probe (Phisitemp). Seizures were identified by visual inspection or EEG recording and video analysis. Mice were recorded at baseline for 10 minutes, then seizures were evoked by increasing the body temperature by 0.5 °C every 30 s. The heating lamp was the switched-off to allow recovery and mice were then monitored until temperature returned to the baseline or death occurred. Threshold temperature was considered.

[0665] EXAMPLE 7

[0666] RESULTS

[0667] Interfering with uORF mediated regulation in other SC / V gene

[0668] As previously noted, SC / V gene family includes several paralog genes, whose LOF mutations can cause other neurological and non-neurological disorders, depending on the organ in which each specific channel is enriched. Considering the high level of homology among the different SC / V genes, we checked whether they also share the uORF-mediated regulation on mRNA translation that we identified in SCN1A / Scn1a. We performed a MUSCLE alignment of human and mouse 5’UTRs of SCN2A / Scn2a, SCN3A / Scn3a, SCN5A / Scn5a, SCN8A / Scn8a and SCN9A / Scn9a genes and found that they also presented uATGs (Figure 17A). Specifically, SCN2A / Scn2a, SCN5A / Scn5a, SCN8A / Scn8a and SCN9A / Scn9a genes presented only uATG-1 , while SCN3A / Scn3a showed a 5’UTR extremely similar to SCN1A / Scn1a, with uATG-1 and uATG-2 in frame each other and uATG-3 (Figure 17A). To determine the effect of those uATGs on the mORF translation, we designed oligonucleotides carrying the human / mouse conserved 5’UTR region of each SCN paralog and corresponding variants mutating the adenines of identified uATGs and cloned them in our bicistronic reporter vector for HEK-293T cell transfection and flow cytometry analysis (Figure 17B). We found that all the variants provided a significant enhancement in EGFP levels in comparison to the wild-type oligonucleotide, except for SCN8A / Scn8a, where no significant difference was reported (Figure 17C). We imputed this negative result to the absence of the ultra-conserved CAGG / CAAG region immediately upstream to ATG1 in SCN8A / Scn8a oligonucleotides (Figure 17B).

[0669] Nevertheless, we proceeded to set ABE strategies to mutagenize uATG for SCN2A / Scn2a and SCN8A / Scn8a, whose haploinsufficiency causes severe neurological syndromes, characterized by autistic traits with or without epilepsy. We designed different sgRNAs working with both SpRY- and Sp-ABE8e, some of which targeting a conserved sequence between human and mouse and some others being species-specific (Figure 17 and Figure 18); we next cloned them in LV vectors for testing in primary neurons. Based on Sanger sequencing data (Figure 18), we selected sg2A1 (Figure 17D) and sg8a3 (Figure 171) for NGS assessment of editing efficiency. Deep sequencing analysis confirmed Sanger sequencing results, showing that sg2A1 associated to SpRY-ABE induced more than 30% of editing efficiency on its target adenine in Scn2a gene locus (Figure 17E, F), and with the same editing efficiency it induced mutation of A? adenine in the protospacer with consequent disruption of CAGG conserved sequence immediately upstream to uATG-2, and no effect on the mATG (Figure 17F). This editing efficiency was sufficient to induce a 1.8 fold increase in Nav1.2 protein level in comparison to control neurons, as assessed by WB analysis on neurons at day 21 (Figure 17G, H). Similarly, for Sp-ABE with sg8a3 the on-target editing efficiency was around 30% (Figure 17J, K), with also 5% of undesired editing of the mATG (A12) in this case. However, also in this case the Sp-ABE with sg8a3 induced a detectable and significant 1.4 fold increase in the Navi .6 protein in primary neurons in comparison to control cultures (Figure 17L, M).

[0670] SUMMARY

[0671] Altogether these data suggest that, similarly to SCN1A gene, other paralog genes share the same evolutionary conserved uORF-mediated mechanism of regulation repressing the translation of the mORF. Interfering with this mechanism by ABE-mediated strategies can efficiently alter their gene dosage and represent novel therapeutic strategies for SC / V-related haploinsufficiency conditions.

[0672] METHODS

[0673] Western Blot for Nav1.2 and Nav1.6

[0674] Primary neurons and brain tissue were dissected and homogenized using the Mem-PER Plus Membrane Protein Extraction Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions to enrich for the membrane-bound proteins. Protein extracts were quantified using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) following manufacturer’s instructions. Western blot analysis was performed on NuPage 3%-8% gradient gels (Thermo Fisher Scientific) and subsequently were transferred to a nitrocellulose membrane. Primary antibodies for Nav1.2 (rabbit, 1 :200, #ASC-002 Alomone lab), Nav1.6 (rabbit, 1 :200, #ASC- 009 Alomone lab), Calnexin (1 :2000, Sigma-Aldrich) were used. Anti-rabbit HRP (1 :5000, Dako) was used as secondary antibody. The densitometric analyses of the corresponding protein bands were performed by Imaged by normalizing Navon Calnexin signal.

[0675] EXAMPLE 8

[0676] RESULTS

[0677] Base editing approach to remove splicing acceptor of exon 20N (poison exon) in SCN1A / Scn1a gene

[0678] Gene expression control is a complex process, which involves multiple sequential steps. Thus, targeting multiple steps can further contribute to the restoration of the physiological gene dosage at single cell level. Although in vivo treatment of Dravet mice with ABE_uORF resulted in a marked improvement of the disease phenotype, it did not lead to a complete recovery. This observation suggests that targeting a single level of the gene expression cascade may not be sufficient to fully restore normal functionality. For this reason, we considered that a promising strategy could be to implement combinatorial interventions, acting at distinct molecular levels on different control step of gene expression, to ultimately_achieve a more comprehensive correction of the phenotype.

[0679] SCN1A gene contains a non-coding exon within intron 20, known as exon 20N, that functions as a so-called “poison exon” (Carvill GL, Engel KL, Ramamurthy A, et al. Aberrant Inclusion of a Poison Exon Causes Dravet Syndrome and Related SCN1A-Associated Genetic Epilepsies. Am J Hum Genet. 2018; 103(6): 1022-1029. doi:10.1016 / j.ajhg.2018.10.023). When this exon is included in the transcript, it introduces a premature STOP codon that directs the mRNA toward nonsense-mediated decay (NMD), effectively reducing the amount of functional Nav1.1 protein produced. Pathogenic variants located within or near exon 20N can disrupt normal splicing control and increase its inclusion, leading to SCN1A haploinsufficiency and epileptic phenotypes such as Dravet syndrome (Carvill et al. 2018). Importantly, therapeutic strategies using antisense oligonucleotides (for example, Zorevunersen by Stoke Therapeutic) have been developed to inhibit 20N incorporation and restore normal SCN1A expression, showing promising preclinical and early clinical results (Han et al. Antisense oligonucleotides increase Senia expression and reduce seizures and SUDEP incidence in a mouse model of Dravet syndrome. Sci Transl Med. 2020; 12(558). doi:10.1126 / SCITRANSLMED.AAZ6100). In this regard, we designed a base editing strategy to specifically target and abrogate the splicing acceptor site (SA) of exon 20N, thereby preventing its inclusion and restoring productive splicing between exon 20 and 21 (Figure 19A, B, C). To this aim, we first designed sgRNAs targeting mouse and human exon 20N SAs to install editing of “AG” (canonical SA) to “GG” (disrupted SA) (Figure 19A, B), coupled with either Sp or SpRY-based ABE8e. SpRY / Sp-ABEs alone or associated with sgRNAs were delivered by lentiviral vectors (LVs) in HEK-293T cells, harvested after three days of selection for DNA extraction. Since sgRNA_ex20N-1 , harboring NGG PAM, was the only candidate that presented complete sequence conservation between human and mouse (Figure 19A, B) and demonstrated efficient (around 30%) on-target editing activity in HEK-293T cells (Figure 19D, E, F), we selected it for subsequent testing in mouse primary neurons. Cortical / hippocampal primary neurons derived from mouse embryos were transduced with LV vectors to deliver Sp-ABE8e and sgRNA_ex20N-1. Two weeks after neuron plating, genomic DNA was extracted and target edited amplicon was PCR-amplified. Sanger sequencing of target amplicon revealed even higher editing efficiency of target Ai belonging to exon 20N SA being edited in 45% of NGS reads (Figure 19G). Since Senia mRNA is moderately expressed in day 14 in vitro mouse primary neurons, we could simultaneously quantify exon 20N inclusion and productive Senia mRNA by RT-PCR analysis, using primers flanking exon 20N. Resulting RT-PCR products differed in size depending on whether exon 20N was incorporated or not, allowing clear discrimination between nonproductive and productive splice isoforms (in mouse: nonproductive: 562 bp; productive: 498 bp) (Figure 19C). Densitometric measurement of the RT-PCR products from neurons transduced with ABE and sgRNA_ex20N-1 showed enrichment of productive mRNA (lower band) and reduction of nonproductive mRNA (upper band) in respect to ABE Ctrl treated neurons (Figure 19H, I, J).

[0680] SUMMARY

[0681] Together our results demonstrate that base editing targeting Senia exon 20N splicing acceptor effectively increases the abundance of productive transcripts by abrogating poison exon inclusion. Since this approach complements the ABE_uORF strategy by acting on distinct layers of gene expression - splicing regulation and protein translation - combining the two strategies is anticipated to result in a more sustained and robust therapeutic effect than either intervention alone.

[0682] METHODS

[0683] Total RNA isolation and retrotranscription for splicing assay Total RNA was extracted using NucleoZOL reagent (Macherey-Nagel) according to the manufacturer’s instructions and as previously described. Complementary DNA (cDNA) was synthesized using the ImProm-ll reverse transcriptase kit (Promega). From neurons, 300 ng of RNA template was used and mixed with random primers. The mixture was incubated at 70°C for 5 min and quickly chilled to 4°C. Master mix containing MgCh, deoxynucleotide triphosphates (dNTPs), and ImProm-ll reverse transcriptase was added to the RNA and oligo(dT) mixture, and the reaction was carried out as follows: 25°C annealing for 5 min, 42°C extension for 60 min, 70°C heat inactivation for 15 min, followed by 4°C hold.

[0684] Reverse transcription polymerase chain reaction (RT-PCR)

[0685] PCRs were prepared by mixing the following reagents: 1 * GoTaq G2 polymerase (Promega), 5x Green GoTaq Buffer, forward and reverse primers (0.4 pM each), cDNA template (2 pl), and nuclease-free H2O in a total volume of 25 pl. The PCR cycle conditions were 95°C for 2 min for 1 cycle; 95°C for 30 s, 56°C for 30 s, 72°C for 75 s for 40, 72°C for 5 min 4°C hold. PCR products were separated on a 5% tris-borate EDTA (TBE) polyacrylamide gel by electrophoresis. The gel was stained with SYBR Safe Dye for 20 min and scanned using a Gel Doc XR+ gel imaging system (BioRad). The following primers from Zhou Han et al. 20202were used: mouse Senia transcript (exons 21 to 24), 5'-

[0686] CAGTTTAACAGCAAATGCCTTGGGTT-3' (forward) and 5'-

[0687] AAGTACAAATACATGTACAGGCTTTCCTCATACTTA-3' (reverse). Predicted molecular weights of the RT-PCR products for Senia mRNA'. productive transcript (containing exons 21 , 22, 23, and 24): 498 bp; nonproductive transcript (containing exons 21 , 21 N, 22, 23, and 24): 562 bp. Band intensities were measured by densitometry, and values are plotted as the proportion of either the exon 20N-containing (nonproductive) or the productive isoform relative to total Senia transcript (sum of both isoforms).

[0688] All publications mentioned in the above specification are herein incorporated by reference. Various modifications and variations of the disclosed guide polynucleotide, vector, editing system, method of the present invention and the disclosed guide polynucleotide, vector or editing system for use according to the present invention will be apparent to the skilled person without departing from the scope and spirit of the invention. Although the invention has been disclosed in connection with specific preferred embodiments, it should be understood that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the disclosed modes for carrying out the invention, which are obvious to the skilled person are intended to be within the scope of the following claims. Various features and embodiments of the present invention will now be described with reference to the following numbered paragraphs (paras):

[0689] 1. A guide polynucleotide comprising a sequence which binds to a target sequence comprising a portion of the 5’ UTR-encoding region of a gene encoding a voltage-gated sodium channel (VGSC) alpha subunit (Nav).

[0690] 2. The guide polynucleotide of para 1 , wherein the gene encoding a VGSC alpha subunit (Nav) is selected from any one of human SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN7A, SCN8A, SCN9A, SCN10A, and SCN11A, or any one of mouse Senia, Scn2a, Scn3a, Scn4a, Scn5a, Scn7a, Scn8a, Scn9a, Scn10a and Scn11a.

[0691] 3. The guide polynucleotide of para 1 or para 2, wherein the gene encoding a VGSC alpha subunit (Nav) is any one of human SCN1A, SCN2A, SCN3A, SCN5A, SCN8A and SCN9A, or any one of mouse Senia, Scn2a, Scn3a, Scn5a, Scn8a and Scn9a.

[0692] 4. The guide polynucleotide of any one of paras 1-3, wherein the gene encoding a VGSC alpha subunit (Nav) is human SCN1A or mouse Senia, optionally wherein the gene encoding a VGSC alpha subunit (Nav) is human SCN1A.

[0693] 5. The guide polynucleotide of any one of paras 1-4, wherein the target sequence comprises at least one start codon of an upstream open reading frame (uORF).

[0694] 6. The guide polynucleotide of para 5, wherein the target sequence comprises at least one two upstream open reading frame (uORF) start codons.

[0695] 7. The guide polynucleotide of any one of paras 1-6, wherein the sequence that binds to the target sequence is 17-24 nucleotides long.

[0696] 8. The guide polynucleotide of para 7, wherein the sequence that binds the target sequence is 19-21 nucleotides long.

[0697] 9. The guide polynucleotide of para 7 or para 8, wherein the sequence that binds the target sequence is 20 nucleotides long.

[0698] 10. The guide polynucleotide of any one of paras 1-9, wherein the target sequence is located between position -30 and position 10 in the gene, position -1 being the position of the nucleotide immediately 5’ of the first nucleotide of the main open reading frame (mORF) and position 1 being the position of the first nucleotide of the mORF. 11 . The guide polynucleotide of para 10, wherein the target sequence is located between position -18 and position 2, between position -19 and position 1 , between position -14 and position 6, between position -13 and position 7, between position -10 and position 10, between position -11 and position 9, or between position -13 and position 7

[0699] 12. The guide polynucleotide of para 11 , wherein the target sequence is located between position -18 and position 2.

[0700] 13. The guide polynucleotide of para 11 , wherein the target sequence is located between position -19 and position 1.

[0701] 14. The guide polynucleotide of any one of paras 1-11 , wherein

[0702] (a) the gene encoding a VGSC alpha subunit (Nav) is human SCN1A and the target sequence is the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2;

[0703] (b) the gene encoding a VGSC alpha subunit (Nav) is human SCN2A and the target sequence is any one of the sequences set forth in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 7; or

[0704] (c) the gene encoding a VGSC alpha subunit (Nav) is human SCN8A and the target sequence is the sequence set forth in any one of the sequences set forth in SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13.

[0705] 15. The guide polynucleotide of any one of paras 1-11 , wherein

[0706] (a) the gene encoding a VGSC alpha subunit (Nav) is mouse Senia and the target sequence is the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2;

[0707] (b) the gene encoding a VGSC alpha subunit (Nav) is mouse Scn2a and the target sequence is any one of the sequences set forth in SEQ ID NOs: 3-6 and SEQ ID NO: 8; or

[0708] (c) the gene encoding a VGSC alpha subunit (Nav) is mouse Scn8a and the target sequence is any one of the sequences set forth in SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14.

[0709] 16. A nucleic acid encoding the guide polynucleotide of any one of paras 1-15.

[0710] 17. A vector comprising the nucleic acid of para 16, optionally wherein the vector is a mammalian cell expression vector, a viral vector or a naked DNA vector. 18. The vector of para 17, wherein the vector further comprises a nucleic acid encoding a base editor or portion thereof or a prime editor or portion thereof.

[0711] 19. The vector of para 17 or para 18, wherein the viral vector is an AAV vector or a lentiviral vector.

[0712] 20. An editing system comprising

[0713] (a) the guide polynucleotide of any one of paras 1-15 or the vector of any one of paras 17-19 and

[0714] (b) a base editor or prime editor, a polynucleotide encoding a base editor or prime editor or a vector comprising a nucleic acid encoding a base editor or portion thereof or a prime editor or portion thereof.

[0715] 21 . The editing system of para 20, comprising a guide polynucleotide of any one of paras 1-15.

[0716] 22. The editing system of para 20 or para 21 , comprising a base editor or prime editor.

[0717] 23. The editing system of para 22, comprising a base editor.

[0718] 24. The editing system of para 23, wherein the base editor is an adenine base editor.

[0719] 25. The editing system of any one of paras 20-24, wherein the vector comprising a nucleic acid encoding a base editor or portion thereof or a prime editor or portion thereof is a mammalian cell expression vector, a viral vector or a naked DNA vector, optionally wherein the viral vector is an AAV vector or a lentiviral vector.

[0720] 26. A kit comprising

[0721] (a) the guide polynucleotide of any one of paras 1-15 or the vector of any one of paras 17-19; and

[0722] (b) a base editor, a polynucleotide encoding a base editor or a vector comprising a nucleic acid encoding a base editor.

[0723] 27. A method of editing a 5’ UTR-encoding region of a gene encoding a VGSC alpha subunit (Nav), the method comprising contacting the 5’ UTR-encoding region with

[0724] (a) a guide polynucleotide, optionally a guide polynucleotide of any one of paras 1-15, and (b) a base editor or prime editor, wherein the guide polynucleotide targets the base editor or prime editor to effect an alteration to abolish or create a uORF.

[0725] 28. The method of para 27, wherein the guide polynucleotide targets the base editor or prime editor to effect an alteration to abolish a uORF.

[0726] 29. The method of para 27 or para 28, further comprising contacting a cell with the editing system of any one of paras 20-25.

[0727] 30. The method of any one of paras 27-29, wherein the cell is contacted with the editing system using any one of virus-mediated infection with recombinant viral vectors, direct injection of nucleic acids, biolistic transformation, microinjection, electroporation, DEAE- dextran treatment, lipofection, nanoparticle-mediated transfection, or protein transduction domain (PTD) mediated transduction.

[0728] 31 . A cell genetically altered to abolish or create a uORF in the 5’ UTR-encoding region of a gene encoding a VGSC alpha subunit (Nav).

[0729] 32. The cell of para 31 , wherein the cell is a eukaryotic cell.

[0730] 33. The cell of para 32, wherein the cell is a human or mouse cell.

[0731] 34. The cell of para 32 or para 33, wherein the cell is a neuron, preferably a primary neuron.

[0732] 35. The method of any one of paras 27-30 or the cell of any one of paras 31-34, wherein the alteration comprises at least one A«T to G*C alteration, optionally wherein the at least one A«T to G’C alteration is in at least one start codon of the uORF.

[0733] 36. The method or the cell of para 35, wherein the at least one A«T to G*C alteration is between position -30 and position 4, position -1 being the position of the nucleotide immediately 5’ of the first nucleotide of the main open reading frame (mORF), optionally at position -8 and / or position -17.

[0734] 37. The method or cell of para 36, wherein the alteration comprises at least two A«T to G’C alterations.

[0735] 38. The method or cell of para 36 or para 37, wherein the at least one A«T to G*C alteration is at position -8 or -17 or the at least two A«T to G*C alterations are at position -8 and -17. 39. The guide polynucleotide of any one of paras 1-15 or the vector of any one of paras 17-19 for use in a method of treating a genetic disorder, wherein the guide polynucleotide or vector is administered in combination with any one of a base editor or prime editor, a polynucleotide encoding a base editor or prime editor or a vector comprising a nucleic acid encoding a base editor or portion thereof or a prime editor or portion thereof.

[0736] 40. The editing system of any one of paras 20-25 for use in a method of treating a genetic disorder.

[0737] 41 . The guide polynucleotide or vector for use of para 39 or the editing system for use of para 40, wherein the genetic disorder is characterized by the presence of a loss of function (LOF) or gain of function (GOF) mutation in at least one allele of at least one gene encoding a VGSC alpha subunit (Nav), optionally wherein the gene is selected from human SCN1A, SCN2A, SCN3A, SCN5A, SCN8A and SCN9A, more optionally wherein the gene is human SCN1A.

[0738] 42. The guide polynucleotide or vector for use of para 39 or para 41 or the editing system for use of para 40 or para 41 , wherein the genetic disorder belongs to the group of developmental and epileptic encephalopathies (DEE), optionally wherein the DEE is Dravet syndrome.

[0739] 43. The guide polynucleotide of any one of para 1-15, the editing system of any one of para 19-24, the method of any one of par...

Claims

CLAIMS1. A guide polynucleotide comprising a sequence which binds to a target sequence comprising a portion of the 5’ UTR-encoding region of a gene encoding a voltage-gated sodium channel (VGSC) alpha subunit (Nav).

2. The guide polynucleotide according to claim 1 , wherein the gene encoding a VGSC alpha subunit (Nav) is selected from any one of human SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN7A, SCN8A, SCN9A, SCN10A and SCN11A, or any one of mouse Senia, Scn2a, Scn3a, Scn4a, Scn5a, Scn7a, Scn8a, Scn9a, Scn10a and Scn11a, optionally wherein the gene is human SCN1A.

3. The guide polynucleotide according to claim 1 or claim 2, wherein the target sequence comprises at least one start codon of an upstream open reading frame (uORF).

4. The guide polynucleotide according to any one of claims 1-3, wherein the sequence that binds to the target sequence is between 17-24 nucleotides long.

5. The guide polynucleotide according to any of claims 1-4, wherein the target sequence is located between position -30 and position 10 in the gene, position -1 being the position of the nucleotide immediately 5’ of the first nucleotide of the main open reading frame (mORF) and position 1 being the position of the first nucleotide of the mORF.

6. The guide polynucleotide according to any one of claims 1-5, wherein(a) the gene encoding a VGSC alpha subunit (Nav) is human SCN1A and the target sequence is the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2;(b) the gene encoding a VGSC alpha subunit (Nav) is human SCN2A and the target sequence is any one of the sequences set forth in SEQ ID NO: 3, SEQ ID NO: 4 and SEQ ID NO: 7; or(c) the gene encoding a VGSC alpha subunit (Nav) is human SCN8A and the target sequence is the sequence set forth in any one of the sequences set forth in SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13.

7. The guide polynucleotide according to any one of claims 1-5, wherein(a) the gene encoding a VGSC alpha subunit (Nav) is mouse Senia and the target sequence is the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 2;(b) the gene encoding a VGSC alpha subunit (Nav) is mouse Scn2a and the target sequence is any one of the sequences set forth in SEQ ID NOs: 3-6 and SEQ ID NO: 8; or(c) the gene encoding a VGSC alpha subunit (Nav) is mouse Scn8a and the target sequence is any one of the sequences set forth in SEQ ID NO: 10, SEQ ID NO: 12 and SEQ ID NO: 14.

8. A nucleic acid encoding the guide polynucleotide according to any one of claims 1-7.

9. A vector comprising the nucleic acid of claim 8, optionally wherein the vector is a mammalian cell expression vector, a viral vector or a naked DNA vector.

10. The vector according to claim 9, wherein the vector further comprises a nucleic acid encoding a base editor or portion thereof or a prime editor or portion thereof.11 . The vector according to claim 9 or claim 10, wherein the viral vector is an AAV vector or a lentiviral vector.

12. An editing system comprising(a) the guide polynucleotide according to any one of claims 1-7 or the vector according to any one of claims 8-11 , and(b) a base editor or prime editor, a polynucleotide encoding a base editor or prime editor or a vector comprising a nucleic acid encoding a base editor or portion thereof or a prime editor or portion thereof.

13. The editing system according to claim 12, wherein the vector comprising a nucleic acid encoding a base editor or portion thereof or a prime editor or portion thereof is a mammalian cell expression vector, a viral vector or a naked DNA vector, optionally wherein the viral vector is an AAV vector or a lentiviral vector.

14. A method of editing a 5’ UTR-encoding region of a gene encoding a VGSC alpha subunit (Nav), the method comprising contacting the 5’ UTR-encoding region with(a) a guide polynucleotide, optionally a guide polynucleotide according to any one of claims 1-7, and(b) a base editor or prime editor,wherein the guide polynucleotide targets the base editor or prime editor to effect an alteration to abolish or create a uORF.

15. The method according to claim 14 further comprising contacting a cell with the editing system of any one of claims 8-11 .

16. The method according to claim 14 or claim 15, wherein the cell is contacted with the editing system using any one of virus-mediated infection with recombinant viral vectors, direct injection of nucleic acids, biolistic transformation, microinjection, electroporation, DEAE- dextran treatment, lipofection, nanoparticle-mediated transfection, or protein transduction domain (PTD) mediated transduction.

17. A cell genetically altered to abolish or create a uORF in the 5’ UTR-encoding region of a gene encoding a VGSC alpha subunit (Nav).

18. The method according to any one of claims 14-16 or the cell according to claim 17, wherein the alteration comprises at least one A«T to G*C alteration, optionally wherein the at least one A«T to G*C alteration is in at least one start codon of the uORF.

19. The method or the cell according to claim 18, wherein the at least one A«T to G*C alteration is between position -30 and position -4, position -1 being the position of the nucleotide immediately 5’ of the first nucleotide of the main open reading frame (mORF), optionally at position -8 and / or position -17.

20. The guide polynucleotide according to any one of claims 1-7 or the vector according to any one of claims 8-11 for use in a method of treating a genetic disorder, wherein the guide polynucleotide or vector is administered in combination with any one of a base editor or prime editor, a polynucleotide encoding a base editor or prime editor or a vector comprising a nucleic acid encoding a base editor or portion thereof or a prime editor or portion thereof.21 . The guide polynucleotide or vector for use according to claim 20, wherein the genetic disorder is characterized by the presence of a loss of function (LOF) or gain of function (GOF) mutation in at least one allele of at least one gene encoding a VGSC alpha subunit (Nav), optionally wherein the gene is selected from human SCN1A, SCN2A, SCN3A, SCN5A, SCN8A and SCN9A, more optionally wherein the gene is human SCN1A.

22. The guide polynucleotide or vector for use according to claim 20 or claim 21 , wherein the genetic disorder belongs to the group of developmental and epileptic encephalopathies (DEE), optionally wherein the DEE is Dravet syndrome.11823. The guide polynucleotide according to any one of claims 1-7, the editing system according to claim 12 or claim 13, the method according to any one of claims 14-16, or the guide polynucleotide or vector for use according to any one of claims 20-22, wherein the base editor or prime editor is a base editor comprising a Cas9 protein.

24. The guide polynucleotide, editing system, method or the guide polynucleotide or vector for use according to claim 23, wherein the base editor comprising a Cas9 protein is an adenine base editor.

25. The guide polynucleotide, editing system, method or the guide polynucleotide or vector for use according to claim 24, wherein the adenine base editor has the sequence set forth in SEQ ID NO: 29 [SpRYCas9ABEmax], SEQ ID NO: 30 [SpRYCas9-ABE8e] or SEQ ID NO: 31 [SpCas9-ABE8e],26. A guide polynucleotide comprising a sequence which binds to a target sequence comprising a portion of the 5’ splice acceptor site (SA) of an exon of a gene encoding a voltage-gated sodium channel (VGSC) alpha subunit (Nav).

27. The guide polynucleotide according to claim 26, wherein the target sequence is located between position -30 and position +30 with respect to the exon, position -1 being the position of the nucleotide immediately 5’ of the first nucleotide of the exon, optionally wherein the target sequence comprises the SA.

28. The guide polynucleotide according to claim 26 or claim 27, wherein the gene encoding a VGSC alpha subunit (Nav) is selected from any one of human SCN1A, SCN2A, SCN3A, SCN4A, SCN5A, SCN7A, SCN8A, SCN9A, SCN10A and SCN11A, or any one of mouse Senia, Scn2a, Scn3a, Scn4a, Scn5a, Scn7a, Scn8a, Scn9a, Scn10a and Scn11a, optionally wherein the gene is human SCN1A29. The guide polynucleotide according to claim 28, wherein the exon is an exon located within intron 20, optionally wherein the exon is exon 20N.

30. The guide polynucleotide according to any one of claims 26 to 30, wherein the sequence which binds to the target sequence comprises any one of the sequences set forth in SEQ ID NOs: 44-52.31 . A nucleic acid encoding the guide polynucleotide according to any one of claims 26-30.11932. A vector comprising the nucleic acid of claim 31 and optionally a nucleic acid encoding a base editor or portion thereof or a prime editor or portion thereof.

33. An editing system comprising(a) the guide polynucleotide according to any one of claims 26-30 or the vector according to claim 32, and(b) a base editor or prime editor, a polynucleotide encoding a base editor or prime editor or a vector comprising a nucleic acid encoding a base editor or portion thereof or a prime editor or portion thereof.

34. The vector according to claim 32 or the editing system according to claim 33, wherein the vector is a mammalian cell expression vector, a viral vector or a naked DNA vector, optionally wherein the viral vector is an AAV vector or a lentiviral vector.

35. A method of editing a 5’ splice acceptor site (SA) of an exon of a gene encoding a VGSC alpha subunit (Nav), the method comprising contacting the 5’ SA with(a) a guide polynucleotide, optionally a guide polynucleotide according to any one of claims 26-30, and(b) a base editor or prime editor, wherein the guide polynucleotide targets the base editor or prime editor to effect an alteration to disrupt the 5’ SA, optionally wherein the 5’ SA is the 5’ SA of an exon located within intron 20.

36. The method according to claim 35 further comprising contacting a cell with the editing system of any one of claim 33 or claim 34, optionally using any one of virus-mediated infection with recombinant viral vectors, direct injection of nucleic acids, biolistic transformation, microinjection, electroporation, DEAE-dextran treatment, lipofection, nanoparticle-mediated transfection, virus-like particle delivery or protein transduction domain (PTD) mediated transduction.

37. The method according to claim 35 or claim 36, wherein the alteration comprises at least one A«T to G*C alteration, optionally wherein the at least one A«T to G*C alteration is in the 5’ SA.12038. The method according to claim 37, wherein at least one A«T to G*C alteration is at position -2 with respect to the exon, position -1 being the position of the nucleotide immediately 5’ of the first nucleotide of the exon.

39. A combination of(a) a guide polynucleotide of any one of claims 1-7, a vector of any one of claims 9-11 , or an editing system of claim 12 or claim 13; and(b) a guide polynucleotide of any one of claims 26-30, a vector of claim 32 or claim 34, or an editing system of claim 33 or claim 34.

40. The guide polynucleotide according to any one of claims 26-30, the vector according to claim 32 or claim 34, or the combination according to claim 39 for use in a method of treating a genetic disorder, wherein the guide polynucleotide or vector is administered in combination with any one of a base editor or prime editor, a polynucleotide encoding a base editor or prime editor or a vector comprising a nucleic acid encoding a base editor or portion thereof or a prime editor or portion thereof.

41. The guide polynucleotide or vector for use according to any one of claims 20-25, wherein the guide polynucleotide or vector is administered in combination with the guide polynucleotide of any one of claims 26-30, the vector of claim 32 or claim 34, or the editing system of claim 33 or claim 34.

42. The guide polynucleotide, vector or combination for use according to claim 40 or claim 41 , wherein the genetic disorder is characterized by the presence of a loss of function (LOF) or gain of function (GOF) mutation in at least one allele of at least one gene encoding a VGSC alpha subunit (Nav), optionally wherein the gene is selected from human SCN1A, SCN2A, SCN3A, SCN5A, SCN8A and SCN9A, more optionally wherein the gene is human SCN1A.

43. The guide polynucleotide, vector or combination for use according to any one of claims 40-42, wherein the genetic disorder belongs to the group of developmental and epileptic encephalopathies (DEE), optionally wherein the DEE is Dravet syndrome.

44. The vector according to claim 32 or claim 34, the editing system according to claim 33 or claim 34, the method according to any one of claims 35-38, the combination according to claim 39, or the guide polynucleotide, vector or combination for use according to any one of claims 40-43, wherein the base editor or prime editor is a base editor comprising a Cas9 protein, optionally an adenine base editor, further optionally wherein the adenine base editor121has the sequence set forth in SEQ ID NO: 29 [SpRYCas9ABEmax], SEQ ID NO: 30 [SpRYCas9-ABE8e] or SEQ ID NO: 31 [SpCas9-ABE8e],45. The vector for use according to any one of claims 20-25, 40 or 42-44, wherein the vector is an AAV vector, preferably an AAV9 vector.

46. The vector for use according to any one of claims 20-25 or 45, wherein the vector is administered systemically, optionally wherein the vector is administered to a human at an amount from 1x 1013to 1x 1014vg / kg.

47. The vector for use according to any one of claims 20-25 or 45, wherein the vector is administered by intraparenchymal injection, optionally wherein the vector is administered to a human at a total amount from 1x 1011to 1x 1013vg.

48. The vector for use according to claim 47, wherein the vector is administered by intracerebral injection in the hippocampus, cerebral cortex, thalamus, cerebellum or striatum.

49. The vector for use according to any one of claims 20-25 or 45, wherein the vector is administered by intracerebroventricular injection, optionally wherein the vector is administered to a human at a total amount from 1x 1013to 1x 1015vg.122

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