Methods for the treatment of spinal muscular atrophy

Abstract

The present invention relates to systems and methods for increasing the expression of SMN protein based on gene editing technology in the treatment of spinal muscular atrophy (SMA).

Description

[0001] METHODS FOR THE TREATMENT OF SPINAL MUSCULAR ATROPHY

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to systems and methods for increasing the expression of SMN protein based on gene editing technology in the treatment of spinal muscular atrophy (SMA).

[0004] STATE OF THE ART

[0005] Spinal muscular atrophy is a neurodegenerative disorder characterized by the loss of alpha motor neurons that occurs mainly in infancy. It is characterized by progressive weakness and atrophy of voluntary muscles in the body. There are 2 genes associated with SMA disease, SMN1 and SMN2, and among these genes, the SMN1 gene expresses the functional Survival Motor Neuron (SMN) protein at a rate of about 100%. The 20 kb SMN1 gene, consisting of 9 exons, encodes a 38 kDa protein of 294 amino acids. SMA is mainly caused by low levels of SMN protein resulting from homozygous deletion on the SMN1 gene or mutation of the SMN1 gene.

[0006] In humans, the SMN protein is also encoded by the SMN2 gene. The severity of SMA symptoms caused by mutations and homozygous deletions in the SMN1 gene varies according to the SMN2 gene copy number, which expresses about 10% of the functional SMN protein. The SMN2 gene, compared to the SMN1 gene, has a point mutation within exon 7, leading to an altered splicing mechanism with most SMN2 mRNAs lacking exon 7. The resulting full-length and non-functional SMN protein has an unstable structure. The wild type SMN1 gene produces high levels of SMN protein. However, homozygous mutations in the SMN1 gene result in low levels of functional SMN protein produced by the SMN1 and SMN2 gene and lead to SMA.

[0007] SMA disease is divided into 5 groups according to the severity of the disease, age of onset and the movement capabilities of the patients.

[0008] In type 0 SMA, symptoms become apparent before birth and infants die within weeks. Type I SMA is the most severe type of SMA disease. The disease is symptomatic before 6 months of age. The main symptoms of Type I SMA patients are reduced movement, lack of head control, and frequent respiratory tract infections. As a result of these infections, the lung capacity of infants decreases, and they must receive respiration support in the future. This type is the most common cause of infant death worldwide due to affected respiratory muscles and respiratory tract infections.

[0009] In Type II SMA patients, although the infant’s development is normal before the 6th month, the symptoms are observed after the 6th month. These patients have head control and can usually sit up. However, most patients are unable to stand and walk without support. Type II SMA patients, who may also have spinal curvatures, are highly susceptible to respiratory tract infections.

[0010] In Type III SMA patients, the first symptoms begin after the 18th month. As the disease progresses, because of loss of strength in the gluteal and leg muscles, falling is common and walking becomes difficult. It can be observed that spinal curvatures become more pronounced.

[0011] In Type IV SMA patients, symptoms appear in adulthood. It is less common than other types and the progression of the disease is slow. Adult SMA patients are usually able to walk. Muscles close to the torso are more affected.

[0012] After the discovery of the CRISPR / Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR-associated nuclease 9) system in 2012, gene editing technologies for many neurodegenerative diseases, including SMA, have become one of today’s major topics. CRISPR / Cas9 technology can catalyze the formation of doublestrand breaks in a desired site of the genome via the endonuclease Cas9 and guide RNA (sgRNA). Genome editing is carried out using these breaks and DNA repair mechanisms. CRISPR / Cas9 genome design technology, with a wide area of use, is utilized in areas such as the creation of disease models, gene splicing and silencing (suppression), and epigenetic regulation, and appears in the treatment of SMA.

[0013] Recent studies have led to the development of “search-and-replace” genome editing technology, which allows alterations to be made only on a single base. This new CRISPR technology, called “Prime Editing (PE)”, which consists of a combination of two important proteins (Cas9 and reverse transcriptase) and pegRNA, is a versatile and efficient technology that allows genetic editing on human cells.

[0014] The prime editing method can induce any small genetic alteration, including alteration such as insertions, deletions, and their combinations, without requiring donor DNA or double-strand cuts. The prime editing system consists of Cas9-nickase, reverse transcriptase (RT), and prime-editing guide RNA (pegRNA). The Cas9 enzyme has been modified to bind to only one strand of DNA and cut the desired part. The RT enzyme is responsible for creating new DNA by copying the RNA template. pegRNAs consist of two important structures, i.e. the part that binds to the DNA strand and the part consisting of RNA bases that will encode the desired alteration in the strand, and they act as a guide to lead the Cas9-RT prime editor complex to the strand to be cut.

[0015] The next-generation CRISPR-based Prime editing (PE) technology, which enables various precise genomic alterations without the need for double-strand breaks or donor DNA sequences, includes a catalytically altered Cas9 nicase, a multifunctional Prime editing guide RNA (pegRNA) that binds to the target site and acts as a template for reverse transcription (RT). The prepared pegRNA sequences are like standard single guide RNAs (sgRNAs) but have a customized extension at the 3' end. The 3' extension consists of an RT template encoding the desired editing and a primer binding site (PBS) that allows prime editors to bind to the targeted site in the genome. Working on these principles, the CRISPR-PE system reduces the risk of in-del in the genome by 270 times compared to HDR-based CRISPR technology and reduces the off-target activity rate (Anzalone et aL, 2019). In contrast to CRISPR-Base Editing (BE) technology, which corrects 4 different transitions (point) mutations without DNA strand breaks, the CRISPR- PE strategy precisely edits 12 different transition (point) mutations (Kantor et aL, 2020). Not only does this increase the reliability of genetic therapy studies that can be performed with the CRISPR-PE method, but it is also possible to cure point mutation-induced disease (ClinVar), which covers 50% of the more than 75,000 known disease-related genetic variants in humans.

[0016] The most important advantage of the CRISPR / PE method over CRISPR / Cas9 technology is its high target sensitivity and low off-target effect since it cuts only one strand of DNA. In the conventional CRISPR / Cas9 method, both strands of DNA are cut, and then new bases are added through the cell’s repair mechanism. In prime editing technology, only one strand of the DNA helix is cut, and the other strand is edited according to the chain in which the alteration was made.

[0017] Some of the approaches described to treat or cure SMA have been studied. For example, it has been described in the literature that Zolgensma®, developed using the AAV virus that is not permanently integrated into the cell genome of the functional SMN1 gene, is not reliable in maintaining high and stable SMN1 gene expression (Lin et al., 2020). (Long version of the reference: Lin, X., Chen, H., Lu, Y.Q., Hong, S., Hu, X., Gao, Y., Lai, L.L., Li, J.J., Wang, Z., Ying, W., Ma, L., Wang, N., Zuo, E., Yang, H., Chen, W.J. 2020. "Base editing-mediated splicing correction therapy for spinal muscular atrophy”, Cell Res 30, 548-550.)

[0018] Patent application no. WO 2009 / 151546A2 describes methods for treating SMA by enhancing the incorporation of exon 7 of SMN2 into mRNA copied from the SMN2 gene.

[0019] Functional alteration of the mutated SMN1 gene has also been tested and the efficacy of this gene therapy has been demonstrated (Mendell, J.R., Al-Zaidy, S., Shell, R., Arnold, W.D., Rodino-Klapac, L. R., Prior, T.W., Lowes, L., Alfano, L., Berry, K., Church, K., Kissel, J.T., Nagendran, S., L'ltalien, J., Sproule, D. M., Wells, C., Cardenas, J. A., Heitzer, M. D., Kaspar, A., Corcoran, S., Braun, L., Likhite S, Miranda, C., Meyer, K., Foust, K.D., Burghes, A.H.M., Kaspar, B.K. 2017. "Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy”, The New England journal of medicine, 377(18), 1713-1722).

[0020] The antisense oligonucleotide (ASO) was used to modify the pre-mRNA splicing of the SMN2 gene and thus the production of the full-length SMN protein was promoted. (Finkel RS et al, Nusinersen versus Sham Control in Infantile-Onset Spinal Muscular Atrophy, N Engl J Med (2017) 377: 1723-32; U.S. Patent No. 7838657).

[0021] Antisense oligonucleotide-mediated splicing correction of SMA has been approved by the U.S. Food and Drug Administration (FDA). However, the prepared ASO is degraded intracellularly, the expression of functional SMN protein is short-term and continuous ASO supplementation is required. The existing ASO therapies do not allow for an effective treatment opportunity due to the weak hydrogen bond interaction with the target sequence and the resulting unstable complementary structure (dimer structure). It is also a very expensive treatment method and has many side effects.

[0022] Spinraza (NURSINERSEN), one of the ASO therapies for the SMA disease, increases exon 7 incorporation in the SMN2 gene with 18 nucleobase DNA-ASO products designed with a 2'- O- methoxyethyl modification (MOE) and a phosphorothioate structure provided to the patient. However, this method does not offer a comfortable treatment model since it requires repeated doses to maintain high and long-term SMN protein expression as the duplex structure formed with the target SMN2-pre-mRNA sequence is not highly stable (Neil and Bisaccia, 2019).

[0023] There is an ongoing need to develop new treatment methods for SMA, as the existing treatment methods for SMA do not offer an effective and long-term stable therapy for SMA.

[0024] With the present invention, three different genetic therapy plans are described that aim to keep SMN protein expression levels high and stable in the long term.

[0025] DESCRIPTION OF THE DRAWINGS

[0026] Fig. 1 : Flow Cytometry Analysis, a. 48th hour analysis of cell viability of SMA Fibroblast cells in flow cytometry treated with untreated control, PEI transfected control, positive control (InM), MERLiNA (InM) and GENLiNA (InM) b. 48th hour analysis of SMN expression in flow cytometry of SMN antibody staining of SMA fibroblast cells treated with untreated control, PEI transfected control, positive control (InM), MERLiNA (InM) and GENLiNA (InM). c. 48th hour flow cytometry analysis of Annexin V-PI antibody staining of SMA fibroblast cells treated with untreated control, PEI transfected control, positive control (InM), MERLiNA (InM) and GENLiNA (InM). d. 48th hour 5x phase images of SMA fibroblast cells under fluorescent microscope, treated with untreated control, PEI transfected control, positive control (InM), MERLiNA (InM) and GENLiNA (InM).

[0027] Fig. 2: Upregulation of SMN protein expression determined as Viability, a. 24th hour analysis of SMN expression by flow cytometry of negative control SMA fibroblast cells, PEI transfected controls, and positive control ASO-treated SMN antibody stainings at concentrations of 200nM, lOOnM, 50nM, lOnM, 3nM and InM. b. Bar graph showing, 24th hour analysis of cell viability by flow cytometry of negative control SMA fibroblast cells, PEI transfected controls, and positive control ASO-treated SMN antibody stainings at concentrations of 200nM, lOOnM, 50nM, lOnM, 3nM and InM. (P < 0.05 is significant NS= Non-Significant.)

[0028] Fig.3:Upregulation of SMN protein expression determined as Viability. a. Bar graph showing, 48th hour analysis of cell viability of SMA Fibroblast cells in Untreated Control, PEI Control, Positive Control, MERLiNA and GENLiNA. b. Bar graph showing, 48th hour analysis of SMN expression of SMA Fibroblast cells in Untreated Control, PEI Control, Positive Control, MERLiNA and GENLiNA.

[0029] (Black is control, red is Positive Control, blue is GENLiNA, green is MERLiNA). (P < 0.05 is significant).

[0030] Fig. 4: Apoptotic effect at 48th hour after transfection with ASO sequence in primary SMA fibroblast cell line. a. Bar graph showing the proportion of Annexin- V negative PI negative viable cells after annexin-V / PI staining, b. Bar graph showing the proportion of Annexin-V positive PI negative cells in the early apoptosis stage, c. Bar graph showing the proportion of Annexin-V positive PI positive cells in the late apoptosis stage. (Black is control, red is Positive Control, blue is GENLiNA, green is MERLiNA) (P < 0.05 is significant).

[0031] Fig. 5: Mitochondrial activity (MTT) analysis. (Black is control, red is Positive Control, blue is GENLiNA, green is MERLiNA).

[0032] Fig. 6: Titration results of GFP virus produced with FUG-E, FUG-B and VSV-G envelope plasmids performed in the Jurkat cell line. A) Negative Control B) Viability of cells in different conditions C) Titration results of GFP virus produced with FUG-E, FUG-B and VSV-G envelope plasmids.

[0033] Fig. 7: Titration results of GFP virus produced with FUG-E, FUG-B and VSV-G envelope plasmids performed on the HEK293T cell line (lOpL, 3pL, and IpL). A) Negative control B) Titration result of the GFP virus produced with the VSV-G envelope plasmid. C) Titration result of the GFP virus produced with the FUG-B envelope plasmid. D) Titration result of the GFP virus produced with the FUG-E envelope plasmid.

[0034] Fig. 8: Titration results of GFP virus produced with FUG-E, FUG-B and VSV-G envelope plasmids performed on the HEK293T cell line (40pl, 20pl, lOpl and 3 pl). A) Negative control B) Titration result of the GFP virus produced with the VSV-G envelope plasmid. C) Titration result of the GFP virus produced with the FUG-B envelope plasmid. D) Titration result of the GFP virus produced with the FUG-E envelope plasmid.

[0035] Fig. 9: Titration results of GFP virus produced with FUG-E, FUG-B and VSV-G envelope plasmids performed on the Al 72 cell line (lOpl, 3 pl, and 1 pl). A) Negative control B) Titration result of the GFP virus produced with the VSV-G envelope plasmid.

[0036] C) Titration result of the GFP virus produced with the FUG-B envelope plasmid. D) Titration result of the GFP virus produced with the FUG-E envelope plasmid.

[0037] Fig. 10: Titration results of GFP virus produced with FUG-E, FUG-B and VSV-G envelope plasmids performed on the Al 72 cell line (40pl, 20pl, lOpl and 3 pl). A) Negative control B) Titration result of the GFP virus produced with the VSV-G envelope plasmid. C) Titration result of the GFP virus produced with the FUG-B envelope plasmid.

[0038] D) Titration result of the GFP virus produced with the FUG-E envelope plasmid.

[0039] Fig. 11: Titration results of GFP virus produced with FUG-E, FUG-B and VSV-G envelope plasmids performed on the SH-SY5Y cell line (40pl, 20pl, lOpl and 3 pl). A) Negative control B) Titration result of the GFP virus produced with the VSV-G envelope plasmid. C) Titration result of the GFP virus produced with the FUG-B envelope plasmid. D) Titration result of the GFP virus produced with the FUG-E envelope plasmid.

[0040] Fig. 12: Titration results of GFP virus produced with FUG-E, FUG-B and VSV-G envelope plasmids in SMA Type 1, SMA Type 2, WT fibroblast cell lines (lOpl). A) Negative control B) Titration result of the GFP virus produced with the VSV-G envelope plasmid. C) Titration result of the GFP virus produced with the FUG-B envelope plasmid. D) Titration result of the GFP virus produced with the FUG-E envelope plasmid.

[0041] Fig. 13: Bar graphs of GFP expression in Jurkat, HEK293T, A172 and SH-SY5Y cell lines by FUG-E, FUG-B neural lentiviruses compared to VSV-G control. A) Neural lentivirus GFP expression in Jurkat cell line B) Neural lentivirus GFP expression in HEK293T cell line C) A172 D) Neural lentivirus GFP expression in SH-SY5Y cell line. p<0.05. NS = Non-Significant.

[0042] Fig. 14: Bar graphs containing MFI values of GFP expression in A172, SH-SY5Y and HEK293T cell lines of FUG-E, FUG-B neural lentiviruses compared to VSVG control. A) A172 B) SH-SY5Y C) HEK293T MFI values of GFP expression. p<0.05. NS = Nonsignificant.

[0043] Fig. 15: MTT analysis performed on HEK293T and SH-SY5Y cell lines transduced with FUG-E, FUG-B neural lentiviruses and control VSV-G GFP. A) HEK293T B) Bar graphs created after MTT in the SH-SY5Y cell line. p<0.05. NS = Non-Significant.

[0044] Fig. 16: Bar graphs of GFP expression in SMA Type 1, SMA Type 2 and WT fibroblast cell lines of FUG-E, FUG-B neural lentiviruses compared to VSV-G control. p<0.05. NS = Non-Significant.

[0045] Fig. 17: Flow cytometry analyzes taken on the post infection (PI) day 24 after transduction with pegRNA 1-2-3, control group PE and GFP in primary SMA Type 1 and SMA Type 2 PE(+) fibroblast cell lines. SMA Type 1 and Type 2 PE(+) pegRNA 1-2-3 and PE(+) and GFP control A) flow cytometry results showing cell viabilities B) flow cytometry results showing SMN expression C) GFP and SMN positive cell populations flow cytometry results shown.

[0046] Fig. 18: Flow cytometry analyzes taken on the post infection (PI) day 42 after transduction with pegRNA 1-2-3, control group PE and GFP in primary SMA Type 1 and SMA Type 2 PE(+) fibroblast cell lines. SMA Type 1 and Type 2 PE(+) pegRNA 1-2-3 and PE(+) and GFP control A) flow cytometry results showing cell viabilities B) flow cytometry results showing SMN expression C) GFP and SMN positive cell populations flow cytometry results shown.

[0047] Fig. 19: In primary SMA Type 1 and SMA Type 2 PE(+) fibroblast cell lines, GFP MFI, viability, SMN expression and FITC / APC values were obtained on days PI 24 and 42 after transduction with pegRNA 1-2-3, control group PE and GFP. SMA Type 1 and Type 2 PE(+) pegRNA 1-2-3 and PE(+) and GFP control A) Bar graphs of GFP MFI values B) Bar graphs of viability rates C) Bar graphs of SMN expressions. D) Bar graphs created for FITC / APC values. ** Purple 24 day, pink 42 day.

[0048] Fig. 20: Bar graphs created for delta GFP MFI values on days PI 3-11-35 and 54 after transduction with pegRNA 1-2-3, control group PE and GFP in primary SMA Type 1 and SMA Type 2 PE(+) fibroblast cell lines. A) Bar graphs of GFP MFI values on varying PI days in SMA Type 1 pegRNAl-2-3. B) Bar graphs of GFP MFI values on varying PI days in SMA Type 2 pegRNA 1-2-3.

[0049] DETAILED DESCRIPTION OF THE INVENTION

[0050] The main object of the present invention is to provide safe, effective and economical methods based on gene editing technology for the treatment of spinal muscular atrophy (SMA).

[0051] Another object of the present invention is to increase the level of functional SMN protein expressed from the SMN2 gene and to ensure its long-term stability.

[0052] According to an embodiment of the present invention, there is provided a method for increasing the expression of functional SMN protein for the treatment of spinal muscular atrophy, characterized in that it comprises the process steps of:

[0053] - Transfer of a cDNA copy of the Cas9-RT prime editor complex and pegRNA sequences with the pU6 promoter into the motor neuron cells,

[0054] - Permanent single base modification of A — > G at position cDNA.845, A→C at position cDNA 841, A→G at position cDNA 888, A→C at position cDNA 885, T→G at position cDNA 878, T — > C at position cDNA 840 and G — > C at position cDNA.859 of the SMN2 gene,

[0055] - Further, from the double base modifications in the SMN2 gene, combined base modification of T→G and A→G at positions cDNA 840 and 845, combined base modifications of T— >C and A→G at positions cDNA 874 and 888, C→G and A→G at positions cDNA 844 and 845, T→A and A→G at positions cDNA 840 and 845, T→C and G→C at positions cDNA 840 and 859,

[0056] - In addition to the SMN2 exon 7 site, a permanent base exchange of G→A at position 100 in the intron 7 site and deletion of bases at positions 10, 290, and 290-295 in intron 7.

[0057] According to another embodiment of the present invention, the transfer of a cDNA copy of the Cas9-RT prime editor complex and pegRNA sequences with the pU6 promoter into neuron cells is carried out using a neural lentivirus.

[0058] According to an embodiment of the present invention, the permanent single base exchanges of A — > G at position cDNA.845, T — > C at position cDNA.840 and cDNA.859 of the SMN2 gene, A — > G at position cDNA.845, A→C at position cDNA 841 , A→G at position cDNA 888, A→C at position cDNA 885, T→G at position cDNA 878, T — > C at position cDNA 840 and G — > C at position cDNA.859 of the SMN2 gene, combined (double) base exchanges of T— >G and A→G at positions cDNA 840 and 845 in the SMN2 gene, combined base exchanges of T→C and A→G at positions cDNA 874 and 888, C→G and A→G at positions cDNA 844 and 845, T→A and A→G at positions cDNA 840 and 845, permanent base exchanges of G→A at position 100 in the SMN2 intron 7 site and deletion of bases at positions 10, 290, and 290-295 in intron 7, which are carried out in the second step of the method, are carried out with the CRISPR-PE method.

[0059] The present invention aims to increase the expression level of the SMN protein expressed from the wild-type SMN2 gene from the level of 10% to the level of 90% and to keep this increased expression level stable in the long term.

[0060] According to an embodiment of the present invention, 33 different SMN2 gene-targeted pegRNA sequences and the CRISPR-PE system designed to perform efficient base exchange are produced by packaging with neural lentiviral vectors specific for neuron cells. 33 different pegRNA sequences designed for prime editors to find the target site and perform base modification are transferred with neural lentivirus-B2 and neural lentivirus- E vector systems. The cDNA copy encoding the CRISPR-PE protein with promoters specific for different neuron cell types and pegRNA sequences with the pU6 promoter are transferred with neural lentiviruses enabling transfer to specific neuron types and having high transduction capacity, thereby enabling targeted nucleotide exchanges to be performed effectively. The neural lentivirus systems that will perform the CRISPR-PE base modification are tested on cells to determine the optimal pegRNA sequence and neural lentiviral vector system. The pegRNA sequences are provided in Table 1 -2 and the distance of the single guide RNA sequences to the cutting site (distance between the pegRNA sequence and the nucleotide to be exchanged) was calculated using the Benchling program. (Table 1 and Table 2). Table 1: 33 different SMN2 gene-targeted pegRNA sequences designed for efficient base exchange.

[0061]

[0062]

[0063] Table 2: Additionally, 14 different SMN2 gene-targeted pegRNA designed designed for efficient base exchange.

[0064] According to an embodiment of the present invention, there is a method for increasing the expression of functional SMN protein for the treatment of spinal muscular atrophy, characterized in that it comprises the process of transferring the functional SMN1 gene and / or CRISPR-PE designs into motor neuron cells using neural lentiviruses.

[0065] Various viral and non-viral vector systems are used in gene therapy, each with its advantages and disadvantages. In the present invention, lentivirus vector systems are used, which are specialized diploid retroviruses that can also infect non-dividing cells, with advantages such as intact transfer and high levels of gene expression, efficient transduction into cells, low immunogenicity, and toxicity.

[0066] According to another embodiment of the present invention, the transfer of the functional SMN1 gene and / or CRISPR-PE designs into motor neuron cells is carried out using neural lentivirus-B2 and neural lentivirus-E vector systems.

[0067] Unlike Zolgensma® therapy, which transfers the SMN1 gene with AAV9 vectors, which are not expected to keep the expression levels stable in the long term since they do not integrate into the genome and do not perform gene transfer in a motor neuron-specific manner, the neural lentiviruses according to the invention are new generation vectors with promoters and envelope proteins that provide motor neuron cell-specific expression.

[0068] According to an embodiment of the present invention, the FUG-E and FUG-B2 capsids, which confer high transduction capability to these vectors, are combined with promoters such as hSYN, Hb9, Hb9-CMVmp (438 bp) and hb9-CMV (313 bp), each of which have been observed to be highly effective in different studies, to produce vectors that are expected to be much more effective than previous studies.

[0069] According to this embodiment of the present invention, the designated motor neuron cellspecific Hb9 promoter, CMV-HB9 (313 bp) and CMVmp-Hb9 (413 bp) promoters which are expected to be specific for motor neuron cells, the hSYN1 promoter which is specific for neuron cells, and other promoters (CMV, PGK) which are expected to be specific for neuron cells after combining with neuron-specific capsids, are individually packaged with functional synthetic SMN1 gene copies and neuron-specific neural lentivirus-B2 and neural lentivirus-E vector systems to produce the CRISPR-PE systems.

[0070] In this way, it is aimed to increase the expression level of the SMN protein to the level of 90% and to keep this increased expression level stable in the long term with the effect of the SMN1 gene transferred by the neural lentivirus vectors carrying the neuronspecific capsid and motor neuron / neuron-specific promoter sequences or with the help of the CRISPR-PE system to be expressed. The designed neural lentivirus systems are indicated in Fig. 2.

[0071] According to another embodiment of the invention, there is a method for increasing the expression of functional SMN protein for the treatment of spinal muscular atrophy, characterized in that it comprises the use of modified ASO sequences specific for the ISS-N1 and ISS-N2 site (Intronic Splicing Silencer site) in intron 7 of the SMN2 gene.

[0072] According to this embodiment of the present invention, the modified ASO sequences for the ISS-N1 and ISS-N2 sites in intron 7 of the SMN2 gene are the XNA / DNA-ASO and XNA-ASO sequences.

[0073] In addition to DNA molecules, Xeno nucleic acid (XNA) molecules are also used in XNA / DNA-ASO sequences designed to overcome the stability problem in the existing DNA-ASO products.

[0074] XNA sequences retain the natural nucleobases and phosphodiester linkages of DNA and RNA sequences and form a series of artificial genetic polymers but have a different sugar chemistry than DNA and RNA molecules. This sugar structure is an RNA base analog produced by methylene bridges between 4’- carbon and 2’ oxygen in the furanose structure.

[0075] According to this embodiment of the present invention, new chemical modifications are added to increase the base pairing strength of the prepared XNA / DNA-ASO and XNA- ASO sequences specific for the ISS-N1 and ISS-N2 site in intron 7 of the SMN2 gene with the target pre-mRNA sequence compared to the original Watson-Crick base pairing.

[0076] In this way, the present invention offers a more effective and long-term treatment compared to the existing DNA-ASO products (e.g. Spinraza) that perform the original Watson-Crick base pairing.

[0077] Antisense Oligonucleotides (ASO), which have a length of 20-25 bases, are complementary to the target mRNA. ASOs, which hybridize by selective Watson-Crick base pairing with the complementary RNA, are designed to modulate the splicing activity (pre-mRNA processing) in the context of the present invention, i.e. to increase the level of SMN protein expressed from the SMN2 gene.

[0078] According to this embodiment of the present invention, a more compact structure is created with the target SMN2 pre-mRNA sequence by increasing the strength of hydrogen bonds between the weak adenine-thymine and guanine-cytosine bases with the designed XNA / DNA-ASO mixmer sequences and XNA-ASO sequences by adding new chemical modifications. Likewise, the bonds formed by increasing the hydrogen bond strength between guanine and cytosine will not break for a long time. This compact structure is intended to result in low toxicity in tissues with a small number of doses as it lasts for a long time, without the need for repeated doses for an effective treatment.

[0079] XNA / DNA-ASO mixmer sequences phosphorothioated with different amounts and having high stability designed to provide the compact structure were grouped according to their length. The motif and length of each group of XNA / DNA-ASO mixmer sequences containing XNA and DNA nucleobases; the number of XNA and DNA nucleobases used in the XNA / DNA-ASO mixmer sequences and their ratio in the sequence were considered to increase the stability of XNA / DNA-ASO mixmer sequences. The designed XNA / DNA-ASO mixmers are provided in Table 3-4.

[0080] Table 3: The designed XNA / DNA-ASO mixmer sequences specific for the ISS-N1 and ISS-N2 site in intron 7 of the SMN2 gene

[0081] Table 4: Additionally, XNA-DNA-ASO mixmer sequences targeted at SMN2 gene intron 7 region.

Claims

CLAIMS1. A method for increasing the expression of functional SMN protein for the treatment of spinal muscular atrophy, characterized in that it comprises the process steps of:- Transfer of a cDNA copy of the Cas9-RT prime editor complex and pegRNA sequences with the pU6 promoter into the motor neuron cells,- Permanent single base modification of A — > G at position cDNA.845, A→C at position cDNA 841 , A→G at position cDNA 888, A→C at position cDNA 885, T→G at position cDNA 878, T — > C at position cDNA 840 and G — > C at position cDNA.859 of the SMN2 gene,- Further, from the double base modifications in the SMN2 gene, combined base modification of T→G and A→G at positions cDNA 840 and 845, combined base modifications of T→C and A→G at positions cDNA 874 and 888, C→G and A→G at positions cDNA 844 and 845, T→A and A→G at positions cDNA 840 and 845, T→C and G→C at positions 840 and 859,- In addition to the SMN2 exon 7 site, a permanent base exchange of G→A at position 100 in the intron 7 site and deletion of bases at positions 10, 290, and 290-295 in intron 7.

2. The method according to claim 1 , is characterized in that the transfer of the cDNA copy of the Cas9-RT prime editor complex and pegRNA sequences with the pU6 promoter into neuron cells, which is carried out in the first step of the method, is carried out using a neural lentivirus.

3. The method according to claim 1 , is characterized in that the permanent single base exchanges of A — > G at position cDNA.845, T — > C at position cDNA.840 and cDNA.859 of the SMN2 gene, A — > G at position cDNA.845, A→C at position cDNA 841 , A→G at position cDNA 888, A→C at position cDNA 885, T→G at position cDNA 878, T — > C at position cDNA 840 and G — > C at position cDNA.859 of the SMN2 gene, combined (double) base exchanges of T→G and A→G at positions cDNA 840 and 845 in the SMN2 gene, combined base exchanges of T→C and A→G at position cDNA 874 and 888, C→G and A→G at position cDNA 844 and 845, T→A and A→G at positions cDNA 840 and 845, T→C and G→C at positions 840 and 859, permanent base exchanges of G→A at position 100 inthe SMN2 intron 7 site and deletion of bases at positions 10, 290, and 290-295 in intron 7, which are carried out in the second step of the method, are carried out with the CRISPR-PE method.

4. A method for increasing the expression of functional SMN protein for the treatment of spinal muscular atrophy, characterized in that it comprises the transfer of the functional SMN1 gene and / or CRISPR-PE designs into motor neuron cells using neural lentiviruses.

5. The method according to claim 4, is characterized in that the transfer of the functional SMN1 gene and / or CRISPR-PE designs into degenerating motor neuron cells is carried out using neural lentivirus-B2 and neural lentivirus-E vector systems. The neural lentiviruses will be produced by combining the lentiviral vectors carrying the FUG-E and FUG-B2 capsids with hSYN, Hb9, Hb9-CMVmp (438 bp), CMV, CAG, and hb9-CMV (313 bp) promoter sequences. It was aimed to perform targeted nucleotide exchanges effectively by transferring the cDNA copy encoding the CRISPR-PE protein having motor neuron cell-specific promoters and the pegRNA sequences having the pU6 promoter with neural lentiviruses allowing specific transfer to motor neuron cells and having high transmission activity.

6. A method for increasing the expression of functional SMN protein for the treatment of spinal muscular atrophy, characterized in that it comprises the use of modified ASO sequences for the ISS-N1 and ISS-N2 sites in intron 7 of the SMN2 gene.

7. The method according to claim 6, is characterized in that the modified ASO sequences for the ISS-N1 and ISS-N2 site in intron 7 of the SMN2 gene are the XNA / DNA-ASO mixmer and XNA-ASO sequences.

Images