Nucleic acid molecules upregulating the translation of PRPF31 mRNA

Abstract

Nucleic acid molecule comprising : at least one target binding sequence comprising a sequence reverse complementary to a sequence of a pre-mRNA processing factor 31 ( PRPF31 ) mRNA; and at least one regulatory sequence comprising a SINE B2 element or a functionally active fragment of a SINE B2 element.

Description

[0001] NUCLEIC ACID MOLECULES UPREGULATING THE TRANSLATION OF

[0002] PRPF31 mRNA

[0003] Cross-Reference to Related Applications

[0004] This Patent Application claims priority from Italian Patent Application No . 102024000026616 filed on November 26 , 2024 , the entire disclosure of which is incorporated herein by reference .

[0005] Technical field of the invention

[0006] The present invention relates to a nucleic acid molecule comprising at least one target binding sequence comprising a sequence reverse complementary to a sequence of a pre-mRNA processing factor 31 mRNA, i . e . PRPF31 ( "pre-mRNA processing factor 31" ) and a regulatory sequence comprising a S INE element ( " short interspersed nuclear element" ) B2 or a functionally active fragment of a S INE B2 element . The present invention also relates to the medical uses of said molecule , in particular for the treatment of retinitis pigmentosa .

[0007] Background

[0008] Retinitis pigmentosa (RP ) is a genetic disease of the eye that af fects the pigmented epithelium and the retina . It is a type of progressive retinal dystrophy, that is , a group of hereditary disorders in which abnormalities of the photoreceptors ( cones and rods ) , of the retinal pigmented epithelium (RPE ) or of the retina itsel f lead to a gradual and progressive loss of vision .

[0009] Retinitis pigmentosa (RP ) type 11 (RP11 ) is a non- syndromic autosomal dominant retinal disease caused by null mutations in the pre-mRNA processing factor 31 gene encoding an essential , and ubiquitously expressed, component of the spliceosome (E. M. Brydon, R. Bronstein, A. Buskin, M. Lako, E. A. Pierce, and R. Fernandez-Godino, "AAV-Mediated Gene Augmentation Therapy Restores Critical Functions in Mutant PRPF31+ / - iPSC-Derived RPE Cells", Mol. Ther. - Methods Clin. Dev., vol. 15, pages 392-402, die. 2019, doi: 10.1016 / j . omtm.2019.10.014 ) . The total absence of PRPF31 is embryonically lethal, while haploinsuf f iciency (HI) is generally symptomatic and typically affects the process of ciliogenesis of the retinal pigmented epithelial (RPE) cells. The first manifestation is the degeneration of the rods and RPE but, as the disease progresses, the cones are also involved in this degenerative process. All this causes an early deterioration of the peripheral vision and a narrowing of the visual field, which makes RP one of the main causes of chronic visual impairment in developed countries .

[0010] Hundreds of mutations have been reported across the gene, most are loss-of-function (LOF) variants including frameshift, splice-site, nonsense or large indels, which lead to complete loss of protein expression by the mutated allele .

[0011] However, the inheritance of RP11 shows incomplete penetrance, that is, the presence of subjects carrying the mutation who are asymptomatic. Several studies have shown that this happens because the levels of the PRPF31 protein produced by the WT (wild-type) allele are higher in the asymptomatic carriers compared to the symptomatic subjects, ranging between 57% and 77% compared to healthy subjects. In addition, even among the same symptomatic subjects, it has been observed that the severity of the disease is reversely correlated to the level of expression of the WT allele (which ranges between individuals probably due to polymorphisms in the promoter of the PRPF31 gene that influence its transcription) (A . m . Rose and S . s . Bhattacharya, "Variant haploinsuf f iciency and phenotypic non-penetrance in PRPF31- associated retinitis pigmentosa" , Clin . Genet . , vol . 90 , file 2 , pages 118-126 , 2016 , doi : 10 . 1111 / cge . 12758 ) . These data suggest that a moderate increase in the expression of PRPF31 should be suf ficient to reverse the pathological phenotype , in particular, said increase should be around 30% . However, recent data have shown that even prolonged overexpression at non-physiological levels of PRPF31 protein causes cellular alterations similar to those observed in the opposite cases of haploinsuf f iciency (HI ) . These results therefore suggest that any long-term therapeutic intervention must regulate PRPF31 protein levels within a physiological range .

[0012] Currently, there are no therapies to treat this disease or prevent its progression . Among the known approaches for retinal diseases are a gene therapy treatment ( Luxturna ) for a retinitis , which is however caused by mutations in another gene (RPE 65 ) , and an RNA-based product of PYC Therapeutics which however targets another protein ( CNOT3 ) . The only alternative therapies for RP are limited to treating symptoms but are not curative , and because there is no cure , most people with this disease progress to the severe stage .

[0013] In consideration of the above , there is a need for new therapeutic approaches for retinitis pigmentosa, in particular type 11 . Speci fically, there is a need to find new therapies that do not increase the PRPF31 protein beyond physiological levels but still allow a suf ficient increase to reverse the pathological phenotype . Moreover, there is a need for new therapies that target PRPF31 expression in a highly gene-specific and tissue-specific manner by limiting the side effects and avoiding the ectopic expression of the gene of interest.

[0014] Recently, a new functional class of antisense IncRNAs (AS) has been identified, which increases the translation of mRNAs encoding partially overlapping proteins. These RNAs are also called SINEUP, since they require a SINE B2 element to increase translation and are shown in WO 2012 / 133947.

[0015] AS Uchll, an antisense IncRNA to the mouse orthologue of the human Uchll / PARK5 gene, can be considered the representative element of this new class of IncRNA, because it has been found to increase the synthesis of the UchLl protein acting at a post-transcriptional level. The activity of AS Uchll depends on the combination of two functional domains: at the 5' end, the overlapping region, referred to as the "binding domain" or "target binding sequence", dictates the specificity of AS Uchll towards Uchll mRNA; at the 3' end, the non-overlapping region contains an embedded reverse SINE B2 element, which acts as the "effector domain" (or "regulatory sequence") and triggers increased translation of the bound target mRNA.

[0016] More than 30 antisense IncRNAs promote the increase in translation of partially overlapping mRNAs. By substituting the binding domain, it is possible to redirect the activity of AS Uchll towards a chosen target mRNA.

[0017] Summary

[0018] It is a scope of the present invention to provide a nucleic acid molecule that sufficiently increases the PRPF31 protein to allow the reversal of the pathological phenotype of retinitis pigmentosa, in particular of type 11, while remaining within physiological ranges, targets PRPF31 expression in a highly gene-specific and tissue-specific manner and limits the side effects.

[0019] This scope is achieved by the nucleic acid molecule as defined in claim 1.

[0020] Other scopes of the present invention are to provide a vector as defined in claim 11, a composition as defined in claim 12, and uses as defined in claims 13 and 14.

[0021] Definitions

[0022] By "PRPF31 mRNA sequence" is meant a nucleotide sequence of any length of at least 40 nucleotides comprised in the mRNA of the corresponding PRPF31 gene.

[0023] The term "SINE B2 element" is defined in WO 2012 / 133947, in which specific examples are also provided (see the table beginning on page 69 of the PCT publication) . The term is intended to encompass both SINE B2 elements in forward orientation and in reverse orientation relative to the 5' to 3' orientation of the functional nucleic acid molecule.

[0024] The term "functionally active fragment of a SINE B2 element" means a portion of the sequence of a SINE B2 element that retains the efficacy of enhancing protein translation. This term also includes sequences that are mutated at one or more nucleotides relative to wild-type sequences, but retain enhancement efficiency. The term is intended to include both SINE B2 elements in forward orientation and in reverse orientation relative to the 5' to 3' orientation of the functional nucleic acid molecule.

[0025] The term "miniSINEUP" means a nucleic acid molecule consisting of a binding domain (sequence complementary to target mRNA, also called target binding sequence) , optionally a linker sequence, and any SINE or SINE-deriving sequence as effector domain (Zucchelli et al., Front Cell Neurosci., 9: 174, 2015.) . In the present invention, the terms "miniSINEUPs", "miniSINEUPs-PRPF31 " , "h-miniSINEUPs" , "h-miniSINEUPs-PRPF31 " are used as synonyms.

[0026] Brief description of the drawings

[0027] Figure 1A shows a Western Blot made on cell lysate of human-TERT-RPE-PRPF31 + / - cells after transfection with the two vectors pCS2-human-miniSINEUPl and pCS2-human- miniSINEUP2 and the vector used as negative control pCS2- miniABD. Western Blotting was done with anti-PRPF31 and anti- p-actin antibodies.

[0028] Figure IB shows a bar graph of the fold change of the amount of PRPF31 protein in h-TERT-RPE-PRPF31 + / - cells after transfection with the two vectors pCS2-h-miniSINEUPl and pCS2-h-miniSINEUP2 and the vector used as negative control pCS2-miniABD . For the calculation, the PRPF31 bands of Figure 1A were normalized first with the p-actin and then the fold change calculated by normalizing with the control.

[0029] Figure 1C shows a bar graph of RT-PCR analysis of PRPF31 mRNA expression in the transfected cells where 2A-AACt indicates the calculation method that allows to calculate the relative expression of the target gene in the experimental sample compared to the control sample. The change between samples, indicating h-TERT-RPE-PRPF31 + / - cells after transfection with the two vectors pCS2-h- miniSINEUPl and pCS2-h-miniSINEUP2 and the vector used as negative control pCS2-miniABD, is not significant. The PRPF31 transcripts were quantified by normalizing with L34 mRNA expression.

[0030] Figure 2A shows a Western Blot made on cell lysate of h-TERT-RPE-PRPF31 + / - cells after transfection with the two vectors pAAV-h-miniSINEUPl and pAAV-h-miniSINEUP2 and the vector used as negative control pAAV-miniABD . Western Blotting was done with anti-PRPF31 and anti-p-actin antibodies .

[0031] Figure 2B shows a bar graph of the fold change of the amount of PRPF31 protein in h-TERT-RPE-PRPF31 + / - cells after transfection with the two vectors pAAV-h-miniSINEUPl and pAAV-h-miniSINEUP2 and the vector used as negative control pAAV-miniABD. For the calculation, the PRPF31 bands of Figure 1A were normalized first with the p-actin and then the fold change calculated by normalizing with the control.

[0032] Figure 2C shows a bar graph of RT-PCR analysis of h- miniSINEUPl, h-miniSINEUP2 mRNA expression and control in transfected cells. The change between samples is not significant. The transcripts of miniSINEUPs were quantified by normalizing with L34 expression.

[0033] Figure 2D shows a bar graph of RT-PCR analysis of PRPF31 mRNA expression in h-TERT-RPE-PRPF31 + / - cells transfected with the two vectors pAAV-h-miniSINEUPl and pAAV-h- miniSINEUP2 and the vector used as a negative control pAAV- miniABD. The change between samples is not significant. The PRPF31 transcripts were quantified by normalizing with L34 expression .

[0034] Figure 3 shows immunostaining respectively for DAPI (lane 1) and for ARL13B (lane 2) in the 3 samples with: h- TERT-RPE-PRPF31 WT cells transfected with pAAV-mini ABD (A) , h-TERT-RPE-PRPF31 + / - cells transfected with pAAV-mini ABD (B) and h-TERT-RPE-PRPF31 + / - cells transfected with pAAV- h-miniSINEUP2 (C) . Bar scales: 25 pm.

[0035] Figure 4A shows a bar graph reporting the average cilia length (pm) in the three cell samples: h-TERT-RPE-PRPF31 WT transfected with pAAV-mini ABD, h-TERT-RPE-PRPF31 + / - cells transfected with pAAV-mini ABD and h-TERT-RPE-PRPF31 + / - cells transfected with pAAV-h-miniSINEUP2 measured by Image J. N=5.

[0036] Figure 4B shows a bar graph reporting the average cilia length (pm) in the three cell samples: h-TERT-RPE-PRPF31 WT transfected with pAAV-mini ABD, h-TERT-RPE-PRPF31 + / - cells transfected with pAAV-mini ABD and h-TERT-RPE-PRPF31 + / - cells transfected with pAAV-h-miniSINEUPl measured by Image J . N=4.

[0037] Figure 5 shows a schematic representation of two miniSINEUPs binding to PRPF31 mRNA at position, respectively, -40 / +4 Ml-AUG and -40 / +4 M2-AUG where Ml and M2 indicate the first and second methionine of the PRPF31 sequence .

[0038] Figure 6A shows a microscope image of a retinal organoid (RO) whose cells were effectively transduced with the AAV2- SINEUP-PRPF31 vector as GFP staining (grey) is visible (reporter gene) .

[0039] Figure 6B shows the relative expression (2A-AACt) of PRPF31 mRNA in the healthy control (empty healthy control) ; in the untreated retinal organoid (deriving from a RP11 patient) (empty patient) ; in the retinal organoid (deriving from a RP11 patient) treated with AAV2-SINEUP-PRPF31 (AAV2- SINEUP2 patient) ; and in the retinal organoid (deriving from a RP11 patient) treated with AAV2-PRPF31 (AAV2-PRPF31 patient) .

[0040] Figure 6C shows the levels (fold change) of PRPF31 protein in the healthy control (empty healthy control) ; in the untreated retinal organoid (deriving from a patient with RP11) (empty patient) ; in the retinal organoid (deriving from a patient with RP11) treated with AAV2-SINEUP-PRPF31 (patient AAV2-SINEUP2 ) ; and in the retinal organoid (deriving from a patient with RP11) treated with AAV2-PRPF31 (patient AAV2-PRPF31) .

[0041] Figure 7 shows the result of the single-cell RNAseq analysis, in particular shows the number of significant "differentially expressed genes" (DEGs) in different cell types: AC = amacrine cells; HC = horizontal cells; MG = Muller glial cells; PBpre = photoreceptors / bipolar precursors; RGC = retinal ganglion cells; RPC = retinal progenitor cells.

[0042] Detailed description

[0043] The nucleic acid molecule according to the present invention comprises at least one target binding sequence comprising a sequence reverse complementary to a sequence of a PRPF31 mRNA and at least one regulatory sequence comprising a SINE B2 element or a functionally active fragment of a SINE B2 element.

[0044] Preferably the target binding sequence consists of the sequence reverse complementary to a sequence of a pre-mRNA processing factor 31 (PRPF31) mRNA. Furthermore, preferably, the at least one regulatory sequence consists of a SINE B2 element or a functionally active fragment of a SINE B2 element .

[0045] In a preferred embodiment, the nucleic acid molecule consists of the target binding sequence and the regulatory sequence .

[0046] As will be explained in more detail below, in an alternative preferred embodiment to the above, the nucleic acid molecule consists of target binding sequence, a linker and regulatory sequence. The nucleic acid molecule according to the present invention is a molecule capable of enhancing translation of a PRPF31 mRNA. In particular, the functional portion of the molecule is the regulatory sequence that has the efficacy of enhancing protein translation; the portion of the binding sequence, on the other hand, causes the molecule to bind specifically to the target of interest or, in the specific case, to the PRPF31 mRNA.

[0047] The SINE B2 element is preferably in a reverse orientation relative to the 5' to 3' orientation of the nucleic acid molecule, i.e. a reverse SINE B2 element. As mentioned in the definitions section, the reverse SINE B2 elements are shown and exemplified in WO 2012 / 133947.

[0048] Preferably, the at least one target binding sequence is at least 40 nucleotides long and comprises, preferably consists of, from 3' to 5' : a sequence reverse complementary to a number from 0 to 50 nucleotides of the 5' untranslated region (5' UTR) and from 0 to 10 nucleotides of the coding sequence (CDS) of the PRPF31 mRNA sequence. Alternatively, the target binding sequence is at least 40 nucleotides long and comprises, preferably consists of, from 3' to 5' : a sequence reverse complementary to a number from 0 to 50 nucleotides of the region upstream of an AUG site (start codon) and from 0 to 10 nucleotides of the region downstream of said AUG site which AUG site is comprised in the coding sequence (CDS) of the PRPF31 mRNA sequence.

[0049] More preferably, the at least one target binding sequence is 44 nucleotides long and consists of, from 3' to 5' : a sequence reverse complementary to 40 nucleotides of the 5' untranslated region (5' UTR) and to 4 nucleotides of the coding sequence (CDS) of the PRPF31 mRNA sequence; or a sequence reverse complementary to 40 nucleotides of the region upstream of an AUG site ( start codon) and to 4 nucleotides downstream of said AUG site which AUG site is comprised in the coding sequence ( CDS ) of the PRPF31 mRNA sequence .

[0050] AUG sites correspond to the methionines of the amino acid sequence of the PRPF31 protein . In particular, the molecule according to the invention is particularly suitable for binding to the first and second AUG sites of the coding region ( CDS ) of PRPF31 which, translated, correspond to the first (Ml ) and second (M2 ) methionine of its amino acid sequence . More particularly, miniS INEUPl binds the PRPF31 mRNA coding sequence to -40 / +4 nucleotides with respect to Ml , while miniS INEUP2 binds the PRPF31 mRNA coding sequence to -40 / +4 nucleotides with respect to M2 .

[0051] Preferably, the nucleic acid molecule according to the invention also comprises at least one linker sequence ( spacer sequence ) between the at least one target binding sequence and the at least one regulatory sequence . This linker sequence connects the target binding sequence and the regulatory sequence to each other and also acts as a spacer, allowing the miniS INEUP to be more flexible and therefore bind to the mRNA more easily . The sequence SEQ ID NO : 10 is a non-limiting example of the linker sequence .

[0052] The nucleic acid of the present invention is preferably RNA. However, the nucleic acid of the present invention may also be DNA.

[0053] In a preferred embodiment of the invention, the at least one regulatory sequence comprises , preferably consists of , a sequence with at least 90% homology to SEQ ID NO : 1 , preferably has a sequence SEQ ID NO : 1 . Preferably, the nucleic acid molecule according to the invention is SEQ ID NO: 2 (miniSINEUPl ) or SEQ ID NO: 3 (miniSINEUP2) .

[0054] According to the present invention, the nucleic acid molecule may also be DNA. Preferably said DNA molecule is SEQ ID NO: 4 or SEQ ID NO: 5.

[0055] The invention also relates to a vector comprising the above described DNA molecule. In particular, the following vectors have been used for the efficient expression of the nucleic acid molecules:

[0056] Viral vector:

[0057] Vector name: pAAV

[0058] Virus: Adeno-associated virus

[0059] Expression: CAG promoter / CMV enhancer

[0060] Terminator SV40 late poly (A)

[0061] Plasmid vector:

[0062] Vector name: pCS2

[0063] Expression: CMV promoter / CMV enhancer

[0064] Terminator SV40 late poly (A)

[0065] It should be noted that any promoter can be used in vectors and will act exactly like those mentioned above.

[0066] Preferably the vector according to the invention has a sequence which is SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

[0067] The present invention also relates to compositions comprising said nucleic acid molecules or vectors. Any composition included allows the release of said molecules. Examples of compositions may also be those which do not comprise viral or plasmid vectors, but which directly encapsulate the nucleic acid or DNA molecule, such as nanoparticles, lipid particles, micelles and the like. Said nucleic acid molecules, DNA molecules, vectors and / or compositions are used as medicaments, preferably for treating retinitis pigmentosa, more preferably retinitis pigmentosa of type 11 (RPE11) . EXAMPLES

[0068] Example 1 - human-minis INEUPs

[0069] Table 1 shows a summary of the h-miniSINEUPs-PRPF31 designed and tested in the experiments below. The binding domain (BD) is shown in italics, while the underlined section is the effector domain (ED) . The "CAT" sites, indicated in bold-cursive, represent the first and second methionine of PRPF31, respectively. The 19 nt linker (spacer) sequence between BD and ED is shown in bold. The numbering refers to the position according to the first or second methionine.

[0070] Example 2 - increase of endogenous PRPF31 protein levels after transfection with pCS2-miniSINEUPs-PRPF31 in h-TERT- RPE- PRPF31 + / - cells

[0071] To test the functionality of miniSINEUPs-PRPF31 in vitro, each of them was cloned into a vector prepared with plasmid pCS2 (obtained by modification of plasmid Addgene code 17095) . A third vector pCS2-mini ABD, control, comprising the effector domain only, was also prepared.

[0072] The h-TERT-RPE (human- telomerase-reverse- transcriptase-retinal pigment epithelium) cell line (obtained by modification of the commercial product (ATCC, CRL-4000) and whose reference publication is: Nazlamova, L., Thomas, N.S., Cheung, MK. et al. A CRISPR and high-content imaging assay compliant with ACMG / AMP guidelines for clinical variant interpretation in ciliopathies . Hum Genet 140, 593-607 (2021) . https: / / doi.org / 10.1007 / s00439- 020-02228-1) characterized by haploinsuf f iciency for the PRPF31 gene (PRPF31 + / -) was then transfected with said vectors. 48h after transfection the cells were harvested and lysed. Cell lysate was analysed by Western Blot with anti- PRPF31 and anti-p-actin (Fig. 1A) . The PRPF31 band was normalized with p-actin then, the fold change was calculated by normalizing with the control. The amount of PRPF31 protein in the 3 cell samples is shown in Figure IB.

[0073] The expression of PRPF31 mRNA was also analysed by RT- PCR analysis. The PRPF31 transcripts were quantified by normalizing with L34 expression. The results in Figure 1C show that the three samples do not vary significantly from each other.

[0074] The results of this experiment show that h-TERT-RPE- PRPF31 + / - cells transfected with pCS2 vectors comprising miniSINEUPl-2 both exhibit an increase in the amount of PRPF31 protein, while there is no significant effect on PRPF31 mRNA expression.

[0075] The experiment therefore shows that by means of the miniSINEUPs of the invention it is possible to advantageously increase the level of PRPF31 protein by acting on the regulation of the expression of its mRNA but leaving the expression of the transcripts themselves unchanged.

[0076] Example 3 - increase of the endogenous PRPF31 protein levels after transfection with pAAV-miniSINEUPs-PRPF31 in h- TERT-RPE-PRPF31 + / - cells

[0077] The miniSINEUPs of the invention were also tested with the vector pAAV (a modified version of an Addgene plasmid, code 28014, the modification of which is described in the publication: Espinoza, Stefano et al. "SINEUP Non-coding RNA Targeting GDNF Rescues Motor Deficits and Neurodegeneration in a Mouse Model of Parkinson's Disease" Molecular Therapy, Volume 28, Issue 2, 642 - 652) under the control of the CAG promoter (Cytomegalovirus enhancer, p-actin promoter) .

[0078] The same experiment referred to in example 2 was then repeated with the vectors pAAV-miniSINEUPs-PRPF31 and the respective control pAAV-mini ABD in h-TERT-RPE-PRPF31 + / - cells. In this second experiment, in addition to protein quantification and PRPF31 mRNA expression, the RNA expression of the miniSINEUPs used was also quantified. The results of the experiment in Figure 2 A-D show that the h- TERT-RPE-PRPF31 + / - cells transfected with pAAV vectors comprising miniSINEUPl-2 undergo an increase in the amount of PRPF31 protein compared to the control, while there is no significant effect on the amount of expression of either PRPF31 mRNA or miniSINEUPs RNA.

[0079] The experiment therefore shows the same advantageous result obtained in example 2 but with the use of a different vector, i.e. pAAV.

[0080] Example 4 - Restoration of the altered ciliogenesis in h-TERT-RPE-PRPF31 + / - cells transfected with pAAV- miniSINEUPs-PRPF31 vectors

[0081] It is known that haploinsuf f iciency of PRPF31 leads to an altered ciliogenesis and that overexpression of the WT allele of PRPF31 allows to reverse the pathological phenotype .

[0082] To verify the efficacy of the miniSINEUPs in reversing the pathological phenotype caused by altered ciliogenesis, h-TERT-RPE-PRPF31 + / - cells were transfected respectively with: pAAV-miniABD as negative control, pAAV-mini SINEUP1 and pAAV-mini SINEUP2. h-TERT-RPE-PRPF31 WT cells were transfected only with pAAV-mini ABD (positive control) . After transfection, cells were immunostained with the markers DAPI for nuclei and ARL13B for cilia (Fig. 3; staining data for miniSINEUP2 only) .

[0083] As shown in the results in Figure 4A (miniSINEUP2 ) and 4B (miniSINEUPl ) , the average cilia length in h-TERT-RPE- PRPF31 + / - cells treated with pAAV-mini ABD is signi ficantly reduced compared to the cilia of the h-TERT-RPE-PRPF31 WT cells treated with the same pAAV-mini ABD control vector . However, the h-TERT-RPE-PRPF31 + / - cells treated with pAAV- miniS INEUPs-PRPF31 showed a signi ficant increase in average cilia length compared to the corresponding h-TERT-RPE-PRPF31 + / - cells treated with the pAAV-mini ABD control .

[0084] The experiment therefore shows that it is advantageously possible to reverse the pathological phenotype , in terms of altered ciliogenesis , in haploinsuf f icient cells for the PRPF31 gene after trans fection with the vector comprising the miniS INEUPs of the invention .

[0085] Example 5 - AAV2-S INEUP-PRPF31 restores transcriptional dysregulation in the retinal organoids

[0086] For the present experiment , retinal organoids (RO) were generated from RP11 patients and healthy controls and transduced with either AAV2 -S INEUP-PRPF31 or AAV2-PRPF31 (which mimics a standard gene therapy approach) .

[0087] The vectors express the S INEUP or transgene under the CAG promoter and include a GFP gene as a reporter . Infection was performed at day 40 and sampling was performed at day 70 .

[0088] As shown in Figure 6A, which shows staining of GFP, AAV2 ef fectively transduced RO cel ls . Furthermore , as shown in Figure 6B and 6C, AAV2-S INEUP-PRPF31 restored the physiological levels of the PRPF31 protein without af fecting PRPF31 mRNA, thereby avoiding the side ef fects that can result from dysregulations at both the mRNA and protein levels . In contrast, AAV2-PRPF31 showed an increase in PRPF31 protein but beyond physiological (WT ) levels and, moreover, acting at the mRNA level .

[0089] Subsequently, a single-cell RNA- seq analysis was performed on the transduced ROs , to understand the ef fect of the overexpression of S INEUP and PRPF31 in each cell type of the ROs . As shown in Figure 7 , in most cell types , AAV2 - S INEUP-PRPF31 reduces the number of "differentially expressed genes" ( DEG) which are dysregulated in RP11 - derived ROs . Instead, it was observed that AAV2 -PRPF31 does not restore DEGs in the rods ( one of the most af fected cell types in RP11 ) and, indeed, increases DEGs in some cell types . These data therefore show a detrimental ef fect of the overexpression of PRPF31 beyond physiological levels , as occurs , for example , with a normal gene therapy approach .

[0090] The experiment therefore shows how the S INEUP approach compared to a normal gene therapy approach works better in that it allows to increase PRPF31 protein levels in a precise manner, that is , within physiological levels , by acting directly on protein levels and not on its mRNA.

[0091] Advantages

[0092] The miniS INEUPs of the invention allow to increase the amount of PRPF31 protein inside h-TERT-RPE-PRPF31 + / - cells with haploinsuf f iciency for the PRPF31 gene which, therefore , reproduce the pathological phenotype characteristic of retinitis pigmentosa, in particular of type 11 .

[0093] The increase in PRPF31 protein levels thanks to miniS INEUPs leads to the advantageous reversal of the pathological phenotype in terms of altered ciliogenesis , as shown in the examples .

[0094] The same examples also show that the scope of the invention is achieved regardless of the vector used . Furthermore , both miniS INEUPs showed ef fective results , demonstrating that the speci fic binding site on the target mRNA is non-discriminating and that , however, it can be both the first AUG (Ml ) and the second (M2 ) . Declaration pursuant to Art . 170bis (paragraphs 2 to 4 ) of the Intellectual Property Code

[0095] The biological material underlying the invention derives from cloned synthetic molecules obtained using information obtained from online sequence databases . The biological material of human origin used in the invention has been purchased in accordance with the applicable legal provisions . In particular, the cell lines have been regularly purchased from the sources cited in the above description . In addition, any biological material containing microorganisms or genetically modi fied organisms used in the invention has been treated according to the statutory provisions related to such modi fications .

Claims

CLAIMS1. A nucleic acid molecule comprising:- at least one target binding sequence comprising a sequence reverse complementary to an mRNA sequence of a pre- mRNA processing factor 31 (PRPF31) ; and- at least one regulatory sequence comprising a SINE B2 element or a functionally active fragment of a SINE B2 element .

2. The nucleic acid molecule according to claim 1, wherein the at least one target binding sequence is at least 40 nucleotides long and comprises from 3' to 5' :- a sequence reverse complementary to a number from 0 to 50 nucleotides of the 5' untranslated region (5' UTR) and from 0 to 10 nucleotides of the coding sequence (CDS) of the PRPF31 mRNA sequence; or- a sequence reverse complementary to a number from 0 to 50 nucleotides of the upstream region of an AUG site (start codon) and from 0 to 10 nucleotides of the downstream region of said AUG site which AUG site is comprised in the coding sequence (CDS) of the PRPF31 mRNA sequence.

3. The nucleic acid molecule according to claim 2, wherein the at least one target binding sequence is 44 nucleotides long and consists of, from 3' to 5' :- a sequence reverse complementary to 40 nucleotides of the 5' untranslated region (5' UTR) and to 4 nucleotides of the coding sequence (CDS) of the PRPF31 mRNA sequence; or- a sequence reverse complementary to 40 nucleotides from the upstream region of an AUG site (start codon) and to 4 nucleotides downstream of said AUG site which AUG site is comprised in the coding sequence (CDS) of the PRPF31 mRNA sequence .

4. The nucleic acid molecule according to any one of claims 1 to 3, further comprising at least one linker sequence between the at least one target binding sequence and the at least one regulatory sequence.

5. The nucleic acid molecule according to claim 4, wherein the linker sequence is 10 to 30 nucleotides long, preferably 15 to 25, more preferably 19 nucleotides.

6. The nucleic acid molecule according to any one of the preceding claims, wherein the nucleic acid is RNA.

7. The nucleic acid molecule according to any one of the preceding claims, wherein the at least one regulatory sequence comprises a sequence with at least 90% homology to SEQ ID NO: 1, preferably has a sequence SEQ ID NO: 1.

8. The nucleic acid molecule according to any one of the preceding claims, wherein said molecule comprises SEQ ID NO: 2 or SEQ ID NO: 3, preferably has a sequence SEQ ID NO: 2 or SEQ ID NO: 3.

9. The nucleic acid molecule according to any one of claims 1 to 5, wherein the nucleic acid is DNA.

10. The nucleic acid molecule according to claim 9, comprising SEQ ID NO: 4 or SEQ ID NO: 5, preferably having sequence SEQ ID NO: 4 or SEQ ID NO: 5.

11. A viral or plasmid vector comprising the DNA molecule according to claim 10, preferably having sequence SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 or SEQ ID NO: 9.

12. A composition comprising the nucleic acid molecule according to any one of claims 1 to 10 or the vector according to claim 11.

13. The nucleic acid molecule according to any one of claims 1 to 10, the vector according to claim 11 or the composition according to claim 12 for use as a medicament.14 . Nucleic acid molecule or vector or composition for use according to claim 13 in the treatment of retinitis pigmentosa, preferably retinitis pigmentosa type 11 (RP11 ) .

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