WO2023219394A1 - Human smn1 protein variant and use thereof - Google Patents

Abstract

The present invention provides a mutated human SMN1 protein, a polynucleotide encoding same, and a pharmaceutical composition for treating spinal muscular atrophy, comprising a vector loading same. The composition lowers liver toxicity seen in existing SMN1 gene therapy through mutation of hSMN1, which can be used directly in humans, while increasing stability, making it possible to efficiently treat spinal muscular atrophy due to having a remarkable effect compared to the same dose and a long-lasting effect.

Description

Human SMN1 protein variants and uses thereof

It relates to a human SMN1 protein variant, a polynucleotide encoding the same, a vector loaded therewith, and a composition for treating spinal muscular atrophy containing the same.

Spinal Muscular Atrophy (SMA) is an autosomal recessive neuromuscular disorder caused by mutations in the Survival Motor Neuron 1 (SMN1) gene. Deficiency in the 294 amino acids encoded in the SMN protein is responsible for presenting a wide range of disease features. These include progressive muscle weakness and atrophy, respiratory failure and premature death resulting from degeneration of motor neurons within the spinal cord. Disease severity is inversely correlated with the copy number of the accessory gene by the ability of the paralogue gene, SMN2, to induce production of approximately 10-20% functional SMN.

Based on the current understanding of the underlying molecular basis of the disease, a number of therapeutic strategies are being considered aimed at increasing the level of functional SMN in more disease-prone cells. For example, Novartis' Zolgensma and Biogen's Spinraza are known as FDA-approved SMA treatments. In particular, in the case of Zolgensma, when administered to an individual, it can treat SMA or improve symptoms by inducing the expression of the SMN1 gene.

However, in the case of existing therapeutic pharmaceutical compositions, for example, siRNA such as Spinraza, not only multiple administrations are required for effective treatment, but the cost of one administration is very high. In addition, in the case of treatments that treat SMA with a single dose, such as Zolgensma, not only is the cost of a single dose very high, but some toxicities have been reported. Therefore, it is difficult to use it widely in SMA patients in infancy, and the economic burden is high.

Therefore, despite the development of such SMA treatments, there is a need to develop a treatment that is not only effective, but also has low treatment costs and is non-toxic. Accordingly, the present inventors studied this problem and developed a genetic variant that is not only more effective than the conventional Zolgensma, but also has lower toxicity.

One aspect of the present invention is a human SMN1 (Survival) in which lysine at positions 41, 51, 184, and/or 186 in an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 is replaced with another amino acid. of motor neuron 1) Provides protein variants.

Another aspect of the present invention provides a polynucleotide encoding the human SMN1 protein variant.

Another aspect of the present invention provides a vector loaded with the polynucleotide.

Another aspect of the present invention provides a pharmaceutical composition for treating or preventing spinal muscular atrophy (SMA) containing the vector as an active ingredient.

Another aspect of the present invention provides a method of treating or preventing spinal muscular atrophy comprising administering the pharmaceutical composition to a subject.

The present invention provides a human SMN1 protein variant, a polynucleotide encoding the same, a vector loaded therewith, and a pharmaceutical composition for treating spinal muscular atrophy containing the same. The pharmaceutical composition mutates hSMN1, which can be used directly in humans, to lower the liver toxicity seen in existing SMN1 gene treatments, while increasing stability, resulting in a remarkable effect compared to the same dose and good persistence of effect, effectively treating spinal muscular atrophy. can be treated.

Figure 1 is a graph showing the survival rate of mice according to date when AAV9-CMV-hSMN1, AAV9-CMV-hSMN1 ^K186R and their variants were injected.

Figure 2 is a graph showing the weight of mice according to the date when AAV9-CMV-hSMN1, AAV9-CMV-hSMN1 ^K186R and their variants were injected.

Figure 3 is an image showing the extent to which the AAV9-CMV-hSMN1 injected group and the AAV9-CMV-hSMN1 ^K186R injected group turn over in the righting reflex test.

Figure 4 is a graph showing the turning time in the righting reflex test of the AAV9-CMV-hSMN1 injection group and the AAV9-CMV-hSMN1 ^K186R injection group.

Figure 5 is an image showing the degree to which the AAV9-CMV-hSMN1 injected group and the AAV9-CMV-hSMN1 ^K186R injected group turned their bodies in the negative geotaxis test.

Figure 6 is a graph showing the success rate in the negative geotaxis test of the AAV9-CMV-hSMN1 injection group and the AAV9-CMV-hSMN1 ^K186R injection group.

Figure 7 is a graph showing the body turning time (seconds) in the negative geotaxis test of the AAV9-CMV-hSMN1 injection group and the AAV9-CMV-hSMN1 ^K186R injection group.

Figure 8 is an image showing the degree to which the AAV9-CMV-hSMN1 injected group and the AAV9-CMV-hSMN1 ^K186R injected group kicked their legs or crossed their legs in the tail suspension test.

Figure 9 is a graph showing the degree of hindlimb clasping observed in the tail suspension test in the AAV9-CMV-hSMN1 injection group and the AAV9-CMV-hSMN1 ^K186R injection group.

Figure 10 is a graph showing body weight changes in the AAV9-CMV-hSMN1 injection group and the AAV9-CMV-hSMN1 ^K186R injection group.

Figure 11 is a graph showing the survival rate (%) of the AAV9-CMV-hSMN1 injection group and the AAV9-CMV-hSMN1 ^K186R injection group.

Figure 12 is an image of liver tissue sampled from the AAV9-CMV-hSMN1 injection group and the AAV9-CMV-hSMN1 ^K186R injection group.

Figure 13 is a Western blot (14Dpi (Day post infection)) image showing the expression level of SMN and GAPDH proteins in the AAV9-CMV-hSMN1 injection group and the AAV9-CMV-hSMN1 ^K186R injection group.

Figure 14 is a Western blot (28Dpi) image showing the expression levels of SMN and GAPDH proteins in the AAV9-CMV-hSMN1 injection group and the AAV9-CMV-hSMN1 ^K186R injection group.

Figure 15 is a graph showing the quantitative qPCR analysis of the expression levels of SMN, IGF1, and IGFALS genes in the AAV9-CMV-hSMN1 injection group and the AAV9-CMV-hSMN1 ^K186R injection group.

Figure 16 is an image showing the results of liver tissue Ki67 and DAPI immunostaining obtained on day 12 of the AAV9-CMV-hSMN1 injection group and the AAV9-CMV-hSMN1 ^K186R injection group.

Figure 17 is an image showing the results of liver tissue Ki67 and DAPI immunostaining obtained on day 25 of the AAV9-CMV-hSMN1 injection group and the AAV9-CMV-hSMN1 ^K186R injection group.

Figure 18 is a graph showing the ratio of Ki67 positive cells in liver tissue of the AAV9-CMV-hSMN1 injection group and the AAV9-CMV-hSMN1 ^K186R injection group.

Human SMN1 protein variants

One aspect of the present invention is a human SMN1 (Survival) in which lysine at positions 41, 51, 184 and/or 186 in an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 is replaced with another amino acid. of motor neuron 1) Provides protein variants.

The term “human SMN1 (Survival of motor neuron 1) protein” used herein is a telomeric copy of the gene encoding the SMN protein. The centromeric copy is SMN2, and SMN1 and SMN2 are part of a 500 kbp retroduplication on chromosome 5q13. SMN1 and SMN2 encode the same protein. The structural difference between SMN1 and SMN2 is the presence of a single nucleotide in exon 7, the exon splice enhancer. SMM1 mutations are associated with spinal muscular atrophy (SMA), and SMN2 mutations alone do not cause the disease.

The human SMN1 protein may refer to a wild-type polypeptide form. The wild-type polypeptide may be isolated from nature, or may be produced recombinantly or synthetically. The wild-type polypeptide may include a naturally cleaved form, a naturally secreted form, or a naturally occurring mutant form of the SMN1 protein. The wild-type polypeptide of the human SMN 1 protein may include any one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6. Specifically, the wild-type polypeptide may include the amino acid sequence of SEQ ID NO: 6.

The human SMN1 protein may be a wild-type polypeptide of human SMN1 or an isoform thereof. In one embodiment, the isoform of the human SMN1 protein may have a similarity of about 90% or more to the wild-type polypeptide of SEQ ID NO: 6. Specifically, the isoform of the human SMN1 protein may be SMN1 isoform a, SMN1 isoform b, SMN1 isoform d, SMN1 isoform X1, SMN1 isoform X2, or analogs thereof.

The SMN1 isoform a may include the amino acid sequence of NCBI Reference Sequence NP_001284644.1. Specifically, the SMN1 isoform a may include the amino acid sequence of SEQ ID NO: 2.

The SMN1 isoform b may include the amino acid sequence of NCBI Reference Sequence NP_075012. Specifically, the SMN1 isoform b may include the amino acid sequence of SEQ ID NO: 3.

The SMN1 isoform d may include the amino acid sequence of NCBI Reference Sequence NP_000335.1. Specifically, the SMN1 isoform d may include the amino acid sequence of SEQ ID NO: 1.

The SMN1 isoform X1 may include the amino acid sequence of NCBI Reference Sequence XP_011541898.1. Specifically, the SMN1 isoform X1 may include the amino acid sequence of SEQ ID NO: 4.

The SMN1 isoform X2 may include the amino acid sequence of NCBI Reference Sequence XP_016865275.1. Specifically, the SMN1 isoform X2 may include the amino acid sequence of SEQ ID NO: 5.

Specifically, the isoform of the human SMN1 protein may include one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 5. Preferably, it may be SMN1 isoform d containing SEQ ID NO: 1 or a variant thereof.

The term “variant” used herein refers to a form in which one or more amino acids are deleted, added, or substituted in the entire amino acid sequence of a wild-type polypeptide. Specifically, the variant may be one in which one or more amino acid residues are added or deleted from the N-terminus or C-terminus of the wild-type amino acid sequence. Additionally, the variant may be one in which some amino acids of the wild-type amino acid sequence have been substituted.

The human SMN1 variant may have its ubiquitination inhibited compared to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6. Specifically, ubiquitination may be inhibited compared to the amino acid sequence of SEQ ID NO: 1.

As used herein, the term “ubiquitination” refers to a type of post-translational modification in which a ubiquitin protein is attached to a substrate protein. Ubiquitination generally means that glycine, the last amino acid of ubiquitin, binds to the lysine of the substrate protein and is degraded by the proteosome. Specifically, it may mean that an isopeptide bond is formed between the cariboxyl group of the ubiquitin glycine residue and the epsilon-amino group of the substrate protein.

In one embodiment, the human SMN1 variant may be one in which lysine (K) in one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 6 is replaced with another amino acid.

The substituted amino acids include alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), and histidine ( H), isoleucine (I), lysine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y), and valine ( It may be any one selected from the group consisting of V). Preferably, the substituted amino acid may be a non-polar amino acid.

In one embodiment, the human SMN1 variant may be one in which lysine (K) in an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 is replaced with alanine (A). Specifically, the lysine present at the C-terminus of one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 may be substituted with another amino acid.

Specifically, the 186th lysine (K) of one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 may be substituted with another amino acid. At this time, the lysine (K) may be replaced with alanine (A), arginine (R), serine (S), glutamine (Q), aspartic acid (D), or tyrosine (Y). At this time, the human SMN1 variant may include one amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 12 and 36 to 41.

Additionally, the 41st lysine (K) of one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 6 may be substituted with another amino acid. At this time, the lysine (K) may be replaced with alanine (A) or arginine (R). At this time, the human SMN1 variant may include one amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.

Additionally, the 51st lysine (K) of one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 6 may be substituted with another amino acid. At this time, the lysine (K) may be replaced with alanine (A) or arginine (R). At this time, the human SMN1 variant may include one amino acid sequence selected from the group consisting of SEQ ID NOs: 19 to 24.

Additionally, the 184th lysine (K) of one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 6 may be substituted with another amino acid. At this time, the lysine (K) may be replaced with alanine (A) or arginine (R). At this time, the human SMN1 variant may include one amino acid sequence selected from the group consisting of SEQ ID NOs: 25 to 30 and 41.

In the SMN1 protein variant, the lysine residue is replaced with a different amino acid, thereby relatively inhibiting ubiquitination, thereby increasing the stability and effectiveness of the variant protein, and reducing toxicity.

The human SMN1 variant may inhibit the degradation of SMN1 protein by replacing the lysine residue in the human SMN1 wild-type amino acid sequence with a different amino acid. Therefore, the stability of SMN1 protein may be improved and toxicity may be reduced.

Polynucleotides encoding protein variants

Another aspect of the invention provides polynucleotides encoding human SMN1 protein variants. At this time, the polynucleotide may be DNA or RNA.

Specifically, the polynucleotide encoding the human SMN1 protein may include one nucleic acid sequence selected from the group consisting of SEQ ID NO: 35 and 42 to 49.

At this time, the polynucleotide encoding the human SMN1 protein is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least one polynucleotide selected from the group consisting of SEQ ID NOs: 35 and 42 to 49. About 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least It may have an identity of about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%.

In one embodiment, the polypeptide encoded by the polynucleotide may include one amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 30 and 36 to 41.

At this time, the polypeptide of the human SMN1 variant is at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about a polypeptide encoding one selected from the group consisting of SEQ ID NOs: 7 to 30 and 36 to 41. 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about It may have an identity of 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%.

The polynucleotide may additionally include a nucleic acid encoding a signal sequence or leader sequence. The term “signal sequence” used herein refers to a signal peptide that directs secretion of a target protein. The signal peptide is cleaved after translation in the host cell. Specifically, the signal sequence is an amino acid sequence that initiates the movement of proteins through the ER (Endoplasmic reticulum) membrane.

The signal sequence has well-known characteristics in the art, and usually contains 16 to 30 amino acid residues, but may contain more or fewer amino acid residues. A typical signal peptide consists of three regions: a basic N-terminal region, a central hydrophobic region, and a more polar C-terminal region. The central hydrophobic region contains 4 to 12 hydrophobic residues that anchor the signal sequence throughout the membrane lipid bilayer while the immature polypeptide moves.

After initiation, the signal sequence is cleaved within the lumen of the ER by cellular enzymes commonly known as signal peptidases. At this time, the signal sequence may be a secretion signal sequence of tPa (Tissue Plasminogen Activation), HSV gDs (Signal sequence of Herpes simplex virus glycoprotein D), or growth hormone. Preferably, a secretion signal sequence used in higher eukaryotic cells, including mammals, can be used. In addition, the signal sequence can be used as a wild-type signal sequence, or by replacing it with a codon that is frequently expressed in host cells.

Vector loaded with polynucleotide

Another aspect of the present invention provides a vector loaded with the polynucleotide.

The vector can be introduced into a host cell and recombined and inserted into the host cell genome. Alternatively, the vector is understood as a nucleic acid vehicle containing a polynucleotide sequence capable of spontaneous replication as an episome.

The vector may be a linear nucleic acid, plasmid, phagemid, cosmid, RNA vector, viral vector, or analogues thereof. Specifically, the vector may be plasmid DNA, phage DNA, etc., commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), E. coli-derived plasmids (pYG601BR322, pBR325, pUC118, pUC119, etc.), Bacillus subtilis. plasmids (pUB110, pTP5, etc.), yeast-derived plasmids (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.).

Additionally, the viral vector may be a retrovirus, adenovirus vector, adeno-virus associated vector (AAV), lentivirus vector, insect virus vector (Baculovirus, etc.), or vaccinia vector.

In one embodiment, the viral vector may be an adeno-virus associated vector (AAV). The adeno-virus associated vector may include a serotype capsid of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAVrh8, AAV9, AAV10, AAVrh10, AAV11 or AAV12.

As used herein, the term “gene expression” or “expression” of a protein of interest is understood to mean transcription of a DNA sequence, translation of an mRNA transcript, and secretion of a fusion protein product or fragment thereof. A useful expression vector may be RcCMV (Invitrogen, Carlsbad) or variants thereof.

The expression vector may include a promoter to promote continuous transcription of the target gene in mammalian cells, or a bovine growth hormone polyadenylation signal sequence to increase the steady-state level of RNA after transcription. Specifically, the vector can additionally load a polynucleotide encoding the human β-glucuronidase promoter, chicken β-actin promoter, or CMV promoter.

At this time, the polynucleotide encoding the SMN1 gene variant may be operably linked to the promoter. In one embodiment, the promoter is capable of expressing a polynucleotide encoding a human SMN 1 variant in neurons of the spinal cord. In another embodiment, the promoter can express SMN1 in motor neurons of the spinal cord.

In one embodiment, a polynucleotide encoding a CMV promoter can be additionally loaded. In one embodiment, the CMV promoter may include the amino acid sequence of SEQ ID NO: 52.

In one embodiment, the vector is pAAV9-CMV-hSMN1 K186R, pAAV9-CMV-hSMN1 K186S, pAAV9-CMV-hSMN1 K186Q, pAAV9-CMV-hSMN1 K186D, pAAV9-CMV-K186A, pAAV9-CMV-hSMN1 K186Y, It may have the structure of pAAV9-CMV-hSMN1 K184R, pAAV9-CMV-K51R, or pAAV9-CMV-hSMN1 K41R.

Additionally, the vector may contain one or more polynucleotides selected from the group consisting of SEQ ID NOs: 33, 34, and 50 to 57.

Uses of SMN1 protein variants

Another aspect of the present invention is, remind Provided is a pharmaceutical composition for treating or preventing spinal muscular atrophy (SMA) containing a vector as an active ingredient.

Another aspect of the present invention is, The vector or A method of treating or preventing spinal muscular atrophy comprising administering the pharmaceutical composition to a subject is provided.

As used herein, the term “ Spinal Muscular Atrophy (SMA) ” is a type of degenerative neurological disease and is an autosomal recessive genetic disease caused by a mutation in the SMN1 gene, which encodes the SMN (survival motor neuron) protein. . A decrease in SMN protein causes functional impairment of motor neurons that exist between the spinal cord and the brainstem, resulting in the inability to receive signals commanding muscle movement, resulting in muscle weakness, muscle atrophy, and fasciculatory spasms.

The spinal muscular atrophy may be SMA type 1, SMA type 2, or SMA type 3. The SMA type 1 is characterized by onset less than 6 months after birth, making it impossible to sit without assistance, and is also called Werdnig-Hoffmann disease. Infants usually die within 2 years. SMA type 2 is characterized by onset 7 to 18 months after birth, and although it is possible to sit or stand without assistance, independent walking is impossible. Their characteristic is that they usually survive until infancy. The SMA type 3 is characterized by onset at more than 18 months after birth, and although independent walking is possible, gradual muscle weakness occurs in infancy.

In some embodiments, the subject may be a primate, and may specifically be a pediatric or adult subject. In one embodiment, the child is 2 months old, 3 months old, 4 months old, 5 months old, 6 months old, 7 months old, 8 months old, 9 months old, 10 months old, 11 months old, 12 months old, 13 months old, 14 months old, 15 months old, 16 months, 17 months, 18 months, 1 year, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14 years. , is under any of the following: 15, 16, 17, or 18 years of age. In some embodiments, the human subject is over 18 years of age.

The pharmaceutical composition has improved liver toxicity and is therefore suitable for administration to infants aged 1 to 18 months after birth, and the effect may last for a long time even after a single administration.

When the pharmaceutical composition is administered to an individual in an effective amount, it can be transduced into motor neurons at the site of administration in a vertebrate. In one embodiment, about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70% of the motor neurons. Approximately any excess of %, 75% or 100% may be transduced. In one embodiment, about 5%, 10%, 15%, 20%, 25%, 30%, 35% of motor neurons throughout the spinal cord (e.g., throughout the lumbar, chest, and neck regions) , 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75% or 100%. If a variant of SMN1 is expressed after administration of the pharmaceutical composition, an individual with SMA symptoms may be treated or the symptoms may be alleviated.

The preferred dosage of the pharmaceutical composition varies depending on the patient's condition and weight, degree of disease, drug form, administration route and period, but can be appropriately selected by a person skilled in the art. In the pharmaceutical composition for treating or preventing SMA of the present invention, the active ingredient is used in any amount (effective amount) depending on the use, formulation, purpose of formulation, etc., as long as it exhibits SMA treatment activity or, in particular, can exhibit a therapeutic effect on SMA. It may be included, and a typical effective amount will be determined within the range of 0.001% by weight to 20.0% by weight based on the total weight of the composition. Here, “effective amount” refers to the amount of active ingredient that can improve the condition of the disease or induce a treatment effect, especially improvement of the condition or treatment effect of SMA. Such effective amounts can be determined experimentally within the scope of the ordinary ability of those skilled in the art.

As used herein, the term “treatment” may be used to include both therapeutic treatment and preventive treatment. At this time, prevention can be used to mean alleviating or reducing the pathological condition or disease of an individual. In one embodiment, the term “treatment” includes any form of administration or application to treat a disease in mammals, including humans. The term also includes inhibiting or slowing the progression of a disease; Restoring or repairing damaged or missing function, thereby partially or completely relieving a disease; or stimulating inefficient processes; It includes the meaning of alleviating serious diseases.

When the pharmaceutical composition is prepared as a parenteral formulation, it can be formulated in the form of injections, transdermal administration, nasal inhalation, and suppositories along with a suitable carrier according to methods known in the art. When formulated as an injection, sterilized water, ethanol, polyols such as glycerol or propylene glycol, or mixtures thereof can be used as suitable carriers.

The preferred dosage of the pharmaceutical composition may vary depending on the patient's condition, weight, gender, age, and severity of the patient. Administration can be done once a day or divided into several times. These dosages should not be construed as limiting the scope of the present invention in any respect.

The subjects to which the pharmaceutical composition can be applied (prescribed) are mammals and humans, and humans are particularly preferred. In addition to the active ingredients, the pharmaceutical composition of the present application may further include any compounds or natural extracts known to have a SMA treatment effect.

Hereinafter, the present invention will be described in more detail by the following examples. However, the following examples are only for illustrating the present invention and do not limit the scope of the present invention.

Example 1. Preparation of viruses containing hSMN1 variants

Example 1.1. DNA transfection

Table 1 below shows the amino acid sequences of hSMN1 and hSMN1 K186R used in the experiment. hSMN1 K186S, hSMN1 K186Q, hSMN1 K186D, hSMN1 K186A, hSMN1 K186Y, hSMN1 K184R, hSMN1 K51R and hSMN1 K41R, respectively, replacing the 184th, 186th, 41st or 51st amino acid lysine (K) of SEQ ID NO: 1 with each amino acid. It was manufactured.

	Amino acid sequence
hSMN1 (SEQ ID NO: 1)	MAMSSGGSGGGVPEQEDSVLFRRGTGQSDDSDIWDDTALIKAYDKAVASFKHALKNG DICETSGKPKTTPKRKPAKKNKSQKKNTAASLQQWKVGDKCSAIWSEDGCIYPATIAS IDFKRETCVVVYTGYGNREEQNLSDLLSPICEVANNIEQNAQENENESQVSTDESENS RSPGNKSDNIKP K SAPWNSFLPPPPPMPGPRLGPGKPGLKFNGPPPPPPPPPPHLLSC WLPPFPSGPPIIPPPPPICPDSLDDADALGSMLISWYMSGYHTGYYMGFRQNQKEGRC SHSLN
hSMN1 K186R (SEQ ID NO: 7)	MAMSSGGSGGGVPEQEDSVLFRRGTGQSDDSDIWDDTALIKAYDKAVASFKHALKNG DICETSGKPKTTPKRKPAKKNKSQKKNTAASLQQWKVGDKCSAIWSEDGCIYPATIAS IDFKRETCVVVYTGYGNREEQNLSDLLSPICEVANNIEQNAQENENESQVSTDESENS RSPGNKSDNIKP R SAPWNSFLPPPPPMPGPRLGPGKPGLKFNGPPPPPPPPPPHLLSC WLPPFPSGPPIIPPPPPICPDSLDDADALGSMLISWYMSGYHTGYYMGFRQNQKEGRC SHSLN

Table 2 below shows the nucleic acid sequence of hSMN1 used in the experiment.

mRNA sequence

Human SMN1 (SEQ ID NO: 35)

atggcgatgagcagcggcggcagtggtggcggcgtcccggagcaggaggattccgtgctgttccggcgcggcacaggccagagcgatgattctgacatttgggatgatacagcactgataaaagcatatgataaagctgtggcttcatttaagcatgctctaaagaatggtgacatttgtgaaacttcgggtaaaccaaaaaccacacctaaaagaaaacctgctaagaagaataaaagccaaaagaagaatactgcagcttccttacaacagtggaaagttggggacaaatgttctgccatttggtcagaagacggttgcatttacccagctaccattgcttcaattgattttaagagagaaacctgtgttgtggtttacactggatatggaaatagagaggagcaaaatctgtccgatctactttccccaatctgtgaagtagctaataatatagaacaaaatgctcaagagaatgaaaatgaaagccaagtttcaacagatgaaagtgagaactccaggtctcctggaaataaatcagataacatcaagcccaaatctgctccatggaactcttttctccctccaccaccccccatgccagggccaagactgggaccaggaaagccaggtctaaaattcaatggcccaccaccgccaccgccaccaccaccaccccacttactatcatgctggctgcctccatttccttctggaccaccaataattcccccaccacctcccatatgtccagattctcttgatgatgctgatgctttgggaagtatgttaatttcatggtacatgagtggctatcatactggctattatatgggtttcagacaaaatcaaaaagaaggaaggtgctcacattccttaaattaa

To prepare a mutant human SMN1 gene loaded into an adeno-associated virus (AAV) vector with a CMV promoter, 2 μg of DNA (pAAV9-CMV-hSMN1 (SEQ ID NO: 33), pAAV9-CMV-hSMN1 was added to HEK-293T cells. K186R (SEQ ID NO: 34), pAAV9-CMV-hSMN1 K186S (SEQ ID NO: 50), pAAV9-CMV-hSMN1 K186Q (SEQ ID NO: 51), pAAV9-CMV-hSMN1 K186D (SEQ ID NO: 52), pAAV9-CMV-K186A (SEQ ID NO: No. 53), pAAV9-CMV-hSMN1 K186Y (SEQ ID No. 54), pAAV9-CMV-hSMN1 K184R (SEQ ID No. 55), pAAV9-CMV-K51R (SEQ ID No. 56) or pAAV9-CMV-hSMN1 K41R (SEQ ID No. 57) Triple transfection with 8 μg each of Rep2/Cap9 plasmid (Cell biolabs, VPK-418 plasmid), (addgene, 112865_Rep2/Cap9) and 4 μg of pHelper virus plasmid (addgene, 112867_pAdDeltaF6). ). At this time, 1 ㎕/㎍ of polyethylenimine (PEI) and Opti-MEM ^TM were used. For transfection, 42 100 mm dishes were used. 20 hours after transfection, it was replaced with new medium, and 48 hours later. It was further cultured for a while.

Example 1.2. AAV production and purification

48 hours after transfection, the cells in each dish were scraped with a scraper, collected in a 50 ml tube, and centrifuged at 420xg, 10 minutes, 4°C. The supernatant was discarded, the pellet was resuspended in lysis buffer (5 M NaCl, 1 M Tris HCl, ultrapure water), and the freeze-thaw process was performed three times. In the above process, the freezing and thawing process was repeated using liquid nitrogen and a water bath. After performing the above process three times, centrifugation was performed at 1,167xg, 15 minutes, and 4°C. Afterwards, the supernatant was transferred to another 50 ml tube, treated with 50 U/ml of benzonase, and maintained at 37°C for a total of 30 minutes with agitation once every 10 minutes.

Afterwards, centrifugation was performed at 13,490 While the fraction was stored at 4°C, iodixanol was added to an ultracentrifuge tube with the composition shown in Table 3 below to form an iodixanol gradient of 15%, 25%, 40%, and 60%. Sanol was added. Table 3 below shows the components of the composition.

composition name		ingredient
15% Iodixanol	12.5 ml of Optiprep density gradient medium
	10ml of 5M NaCl
	10ml of 5xPBS-MK
	17.5 ml of Ultrapure water
25% Iodixanol	20.8 ml of Optiprep density gradient medium
	10ml of 5xPBS-MK
	19.2 ml of Ultrapure water
	100 μL of phenol red
40% Iodixanol	33.3 ml of Optiprep density gradient medium
	10ml of 5xPBS-MK
	6.7 ml of Ultrapure water
60% Iodixanol	50 ml of Optiprep density gradient medium
	100 μL of phenol red

After that, add 8 ml of 15% iodixanol to the ultracentrifuge tube first, then add 5.5 ml of 25%, 5 ml of 40%, and 4.5 ml of 60% to the bottom of the tube in the same order, so that a higher concentration of iodixanol is added to the bottom. The iodixanol fraction was positioned. Crude lysate stored at 4°C was added to the top of this iodixanol gradient and ultracentrifuged at 301,580xg for 2 hours at 12°C. Afterwards, the needle was inserted into 40% iodixanol containing AAV, extracted, transferred to a new tube, and stored at 4°C.

Example 1.3. Desalting and concentration of AAV

5 ml of 1xPBS-MK was placed in a centrifugal filter tube and centrifuged at 4,000xg, 5 minutes, 4°C to perform a pre-rinse process. Meanwhile, 1xPBS was added to the AAV fraction stored at 4°C. -5 ml of MK was added. After emptying the waste tube and reconnecting the filter, the AAV fraction mixed with 1xPBS-MK was added and centrifuged at 4,000xg for 1 hour at 4°C.

After further centrifugation until about 1 ml of fraction remains on the filter, empty the waste tube, add 13 ml of 1xPBS-MK on the filter, and centrifuge again at 4,000xg, 1 hour, 4°C to perform desalination. did. Dechlorination was repeated three or more times. When desalting was completed, 13 ml of 1xPBS-MK mixed with 0.01% surfactant was administered and centrifuged at 4,000xg, 1 hour, 4°C until the final volume reached 200 ㎕. Afterwards, the AAV fraction remaining on the filter was transferred to a new tube and stored at 4°C to obtain an AAV vector carrying the SMN1 gene.

Example 1.4. Titer confirmation of AAV

To analyze the expression level of the SMN1 gene, quantitative PCR (qPCR) was performed. Specifically, quantitative PCR (qPCR) was performed using 1 uM forward primer, 1 uM reverse primer, serially diluted template DNA, and SYBR Green qPCR mix. Table 4 below shows the sequences of primers used in qPCR.

direction	primer sequence	sequence number
Forward primer	5'-CCCACTTGGCAGTACATCAA-3'	31
Reverse primer	5'-GCCAAGTAGGAAAGTCCCAT-3'	32

To create a standard curve, the transgene expressing plasmid was linearized by treating it with a restriction enzyme. Next, it was purified by electrophoresis on a 2% agarose gel. This DNA was diluted to 1x10 ⁹ vector genomes (VG)/㎕, and was finally diluted 1/10 times from 1x10 ⁷ to 1x10 ¹ for the standard curve. In the case of genes present in AAV, first, 2 ㎕ of AAV vector stock was mixed with 198 ㎕ of DNase1 buffer and 2 ㎕ of DNase1 (1 U/㎕) to create a concentration of 1x10 ^-2 , and then maintained at 37°C for 30 minutes. After DNase1 was inactivated at high temperature, 2 ㎕ of proteinase K (10 mg/ml) was added, maintained at 50°C for 1 hour, and then inactivated again at high temperature. Next, the sample for the AAV genome was diluted 1/10 times from the final 1x10 ^-3 to 1x10 ^-5 . qPCR mix was added to the samples for the standard curve and AAV genome, and qPCR was performed under the conditions shown in Table 5 below.

step	condition
Step1	95℃, 5 min, 1 cycle
Step2	95℃, 10 min, 1 cycle
Step3	95℃, 10 s / 60℃ 40 s / 72℃, 1 s, 40 cycles
Step4	40℃, 10 s, 1 cycle

After qPCR, the concentration of the AAV vector was converted based on the Ct value of the standard curve, and ultimately 3x10 ¹³ VG/ml of AAV was obtained.

Example 2. Injection of AAV

AAV containing SMN1 obtained in Example 1 or AAV containing mutant SMN1 was injected into newborn type 1.5 SMA mice.

SMA model mice were purchased from Jackson lab, SMA type 1 (Smn-/-; SMN2+/+) and SMA type 2 (Smn-/-; SMN△7+/+; SMN2+/+) mice, respectively, and the two were crossed. Thus, SMA type 1.5 (Smn-/-; SMN△7+/-; SMN2+/+) mouse was produced and used in the experiment.

The injection process is as follows. Cut off part of the toe of a newborn baby, place it in a tube containing 50mM NaOH, and leave it at 95°C for about 30 minutes. After confirming that the tissue had dissolved, PCR was performed using primers that could confirm mouse SMN deletion. As a result, AAV injection was performed on the individual identified as a mutant. AAV injection was performed within 24 hours of birth, and was administered intravenously using a 1 ml, 31 G, 8 mm insulin syringe under ice anesthesia and facial fixation.

The experimental group was AAV9-SMN, AAV9-SMN ^K186R , AAV9-SMN ^K186S , AAV9-SMN ^K186Q , AAV9-SMN ^K186D , AAV9-SMN ^K186A , AAV9-SMN ^K186Y , AAV9-SMN ^K41R , AAV9-SMN ^K51R , AAV9-SMN ^K184 . R was injected at an amount of 1.2 x 10 ¹¹ VG/g (body weight). After injection, the animal was kept at 37°C for more than 30 minutes, and after confirming that the animal had recovered from anesthesia normally, it was transferred to a cage with its mother.

Afterwards, the mice injected with AAV were maintained under the care of their mothers in their birth cages, and their survival and weight were recorded daily.

As a result, as shown in Figure 1, the experimental group injected with a variant of the 186th amino acid (K186R, K186S, K186Q, K186D, K186A, K186Y, indicated in the form of a triangle) had an increased survival period like the wild-type control group (indicated in the form of a circle). It survived for more than 50 days. However, other mutant groups (K41R, K51R, K184R, indicated in squares) injected with variants at the 184th, 41st, and 51st amino acids died relatively early at 4 weeks.

These results mean that functional differences in SMN are not caused even if the 41st, 51st, and 184th lysines are substituted, but the effect increases when the 186th lysine is substituted with various amino acids.

Additionally, as shown in Figure 2, it was confirmed that the K186R, K186S, K186Q, K186D, K186A, K186Y, K41R, K51R, and K184R groups all weighed more than the control group. In particular, it was confirmed that the weight of mice significantly increased when other mutant groups (K186S, K186Q, K186D, K186A, K186Y) were injected.

Experimental Example 1. Righting reflex test in animal model

Three motor function tests were performed: righting reflex, negative geotaxis, and tail suspension (Hindlimb clasping). The righting reflex was performed from about 5 days after birth (Postnatal day, PND), and negative geotaxis and tail suspension (Hindlimb clasping) were performed from about 20 days after birth.

The righting reflex is an experiment to check whether a rat can turn over and stand upright using its dorsal muscles. It is reported that newborn rats are unable to turn over, and SMA model rats are also unable to turn over. It is done. The judgment was made based on cases where the right back was completely flat on the ground and cases where the left back was completely flat on the ground. Each animal was observed for 30 seconds and the time it took to turn over was recorded. If the animal was unable to turn over, it was recorded as 30 seconds. Function was evaluated by calculating the average time measured three times each. After about two weeks or more, when all the rats quickly turned over and woke up, measurement of the righting reflex was stopped.

As a result, as shown in Figure 3, it was confirmed that the body turned over correctly when AAV9-CMV-hSMN1 ^K186R was injected. In addition, as shown in Figure 4, in the case of SMA model mice injected with AAV9-CMV-hSMN1, they began to turn over from about 11 days after birth, and the speed of turning over gradually became faster, but even at PND14, it took about 10 seconds to turn over. It has been done. However, when AAV9-CMV-hSMN1 ^K186R was injected, it was confirmed that the baby began to turn over in about 25 seconds from the 9th day after birth, and it was confirmed that the body could be turned over in 1 to 2 seconds at about 13 days of age.

Experimental Example 2. Negative geotaxis test

Negative geotaxis is an experiment that measures how a rat instinctively tries to climb up by turning its body upward on a tilted slope. It is known that such behavior is possible when the body has overall balance and uses its muscles accordingly. When rats were placed with their heads facing downward at an incline of approximately 50 degrees, how quickly they turned their bodies in the opposite direction was measured daily for each experimental group. The degree of body rotation was judged to be valid only when the body was fully turned 180 degrees opposite to the initial downward direction. The test was repeated 10 times, and if the body turned, the time taken to turn was recorded. If the body could not be turned, it was recorded as 30 seconds, and the time taken to turn and the number of complete turns were recorded.

As a result, as shown in Figure 5, it was confirmed that when AAV9-CMV-hSMN1 ^K186R was injected, the body turned over 180 degrees. In addition, as shown in Figure 6, in the case of SMA model mice injected with AAV9-CMV-hSMN1, the success rate decreased to 50% 22 days after injection, but when AAV9-CMV-hSMN1 ^K186R was injected, the injection It was confirmed that the success rate was maintained at 100% even until the 23rd day. Additionally, as shown in Figure 7, when AAV9-CMV-hSMN1 ^K186R was injected, it took less than 5 seconds to turn over after 21 days after birth, but when AAV9-CMV-hSMN1 was injected, it took 10 seconds on day 21. Above, after the 22nd, it took about 25 seconds or more.

Experimental Example 3. Tail suspension test

The tail suspension (Hindlimb clasping) test measures whether a rat moves its hind limbs toward its stomach or kicks its legs upward when its tail is suspended. At this time, if there is a problem with the muscles or nerves, the characteristic of crossing the legs rather than kicking them appears. The hindlimb clasping phenomenon described above is observed in model mice for various diseases such as SMA and ALS.

The time for hanging the tail for a maximum of 30 seconds and crossing the legs for 30 seconds was measured. Measurements were made a total of three times per time, and after performing for 30 seconds, the test was allowed to rest for about a minute and then repeated twice more, recording the average time. Behavioral differences were analyzed by comparing the time spent crossing the legs.

As a result, as shown in Figure 8, it was confirmed that mice injected with AAV9-CMV-hSMN1 ^K186R kicked their legs without crossing them. Additionally, as shown in Figure 9, in the case of the SMA model mouse injected with AAV9-CMV-hSMN1, the legs were crossed for more than 20 seconds out of the 30 seconds measured, and kicking the legs upward was rarely observed. In contrast, when AAV9-CMV-hSMN1 ^K186R was injected, the leg crossing time was about 3 to 4 seconds out of 30 seconds, and it was confirmed that the leg was kicked upward for most of the measurement time.

Experimental Example 4. Confirmation of body weight and viability of animal model

In mice injected with the SMA gene as in Example 2, differences in body weight were observed for each experimental group after PND10 to PND14, and the greatest difference was observed in motor function tests after PND23.

As a result of measuring body weight in mice injected with the SMA gene as in Example 2, as shown in FIG. 10, there was no difference in body weight for each experimental group until PND10, but thereafter, body weight for each experimental group was observed. A difference in weight appeared. Specifically, it was confirmed that the body weight of mice injected with AAV9-CMV-hSMN1 ^K186R increased significantly compared to mice injected with AAV9-CMV-hSMN1.

In addition, as shown in Figure 11, negative control Type 1.5 SMA mice that were not treated with anything died quickly, but those injected with AAV9-CMV-hSMN1 were observed to survive for about 4 weeks and then die. In contrast, when AAV9-CMV-hSMN1 ^K186R was injected, survival was observed for more than 260 days.

Experimental Example 5. Liver tissue analysis by experimental group

Experimental Example 5.1. Experimental group sampling

As in Experimental Example 4, mice injected with AAV9-CMV-hSMN1 and AAV9-CMV-hSMN1 ^K186R were obtained at PND14, when body weight begins to differ, and at PND28, when the greatest difference in exercise ability is observed. To compare the CNS and the periphery, the brain, spinal cord, liver, and muscle were each removed and placed in RIPA and Trizol for sampling. The muscle used was the gastrocnemius muscle (GA). As a result, as shown in FIG. 12, liver tissue of mice in the experimental group was obtained, and analysis was performed using each tissue sample as follows.

Experimental Example 5.2. western blot

To confirm the expression level of SMN protein in the brain, spinal cord, liver, and gastrocnemius muscle of the experimental group rats, Western blot was performed as follows.

A 10% polyacrylamide gel was used for protein electrophoresis, and blotting was performed using SMN (ms, 1:2,000) and GAPDH (Rb, 1:1,000) on a PVDF membrane.

As a result, as shown in Figures 13 and 14, no significant tendency was observed in the brain, spinal cord, and muscles, but excessive accumulation of SMN was observed in the liver when AAV9-CMV-hSMN1 was injected.

Experimental Example 5.3. RNA extraction and qRT-PCR

To confirm the expression levels of SMN, IGF1, and IGFALS mRNA in the liver tissue of mice in the experimental group, qRT-PCR was performed as follows.

The tissue was placed in Trizol reagent and homogenized to lyse the cells, then chloroform was added and incubated at room temperature for 10 minutes. Afterwards, the supernatant was separated by centrifugation at 12,000 rpm at 4°C for 15 minutes, transferred to a new tube, and then processed with isopropanol and 75% ethanol to generate an RNA pellet. The resulting pellet was dissolved in DEPC-dH2O and the RNA concentration was quantified. Based on the quantitative values, cDNA was synthesized using an equal amount of RNA and ReverTra Ace reagent, and qRT-PCR was performed using this. GAPDH was used as a reference gene.

As a result, as shown in Figure 15, when AAV9-CMV-hSMN1 was injected, excessive accumulation of SMN RNA was observed. This trend was consistent with the Western blot results shown in Figures 13 and 14. In addition, when AAV-CMV-hSMN1 ^K186R was injected, higher expression of IGF1 and IGFALS in the liver was observed than when AAV-CMV-hSMN1 was injected.

Experimental Example 5.4. Immunohistochemical analysis

To analyze the phenotype of the liver tissue of the experimental group rats, the following immunohistochemical (IHC) analysis was performed.

First, the tissue was fixed using 4% PFA. Afterwards, the tissue was moved to 15% and 30% sucrose solution and left until the tissue settled. Then, the settled tissue was placed in a tube containing OCT compound and rapidly frozen in liquid nitrogen. IHC was performed by cutting the frozen sample into 7 ㎛ sections using a cryostat. The IHC process is as follows. The OCT was removed by washing with 1X PBS for 15 minutes, and in the case of spinal cord sections, additional fixing was performed by placing them in cold methanol for 30 minutes. Afterwards, the slide was placed in EDTA antigen retrieval buffer (1mM EDTA, 0.05% Tween 20, pH 8.0), and antigen retrieval was performed at 105°C for 10 minutes. Afterwards, the 1X PBS washed section was blocked and then treated with primary antibody. The primary antibody was rabbit anti-Ki67 (1:500 dilution), and was incubated at 4°C overnight. After washing with 1x PBS, the secondary antibody (Alexa Fluor 594 goat anti-rabbit IgG (1:400)) matching each primary antibody was treated at room temperature for 1 hour, and then a mounting solution containing DAPI was added. The process was completed by covering it with a coverslip.

As a result, as shown in Figures 16 and 17, no difference in Ki67 positive cells was observed at PND12, where no difference in body weight was observed, but at PND25, where a significant difference in body weight and exercise capacity was observed, AAV9-CMV-hSMN1 ^K186R was injected. It was confirmed that the rat liver tissue had a higher density of Ki67-positive hepatocytes than the liver tissue of rats injected with AAV9-CMV-hSMN1. In addition, as shown in Figure 18, when Ki67 positive cells were quantified, there was no significant difference in PND12 in the liver tissue of mice injected with AAV9-CMV-hSMN1 ^K186R compared to the liver tissue of mice injected with AAV9-CMV-hSMN1. There were more at PND25, and this trend was consistent with Figures 16 and 17. This showed that the hepatocytes of mice injected with AAV9-CMV-hSMN1 ^K186R at PND25 had a higher proliferation rate than the hepatocytes of mice injected with AAV9-CMV-hSMN1, and also showed a tendency to be more similar to the liver of a normal subject.

Claims (16)

1. A human SMN1 (Survival of motor neuron 1) protein in which lysine at positions 41, 51, 184, and/or 186 in an amino acid sequence selected from the group consisting of SEQ ID NOs. 1 to 6 is replaced with another amino acid. variant.
2. According to paragraph 1,

   The variant is a human SMN1 protein variant in which ubiquitination is inhibited compared to the human SMN1 protein containing one amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6.
3. According to paragraph 1,

   The substituted amino acids include alanine (A), arginine (R), asparagine (N), aspartic acid (D), cysteine (C), glutamine (Q), glutamic acid (E), glycine (G), and histidine ( H), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), proline (P), serine (S), threonine (T), tryptophan (W), tyrosine (Y) and valine ( V), any one selected from the group consisting of human SMN1 protein variants.
4. According to paragraph 1,

   The variant is a human SMN1 protein variant comprising one amino acid sequence selected from the group consisting of SEQ ID NOs: 7 to 30 and 36 to 41.
5. A polynucleotide encoding the human SMN1 protein variant of any one of claims 1 to 4.
6. According to clause 5,

   A polynucleotide encoding a human SMN1 protein variant, wherein the polynucleotide is DNA or RNA.
7. According to clause 6,

   A polynucleotide encoding a human SMN1 protein variant, comprising one or more base sequences selected from the group consisting of SEQ ID NOs: 35 and 42 to 49.
8. A vector loaded with the polynucleotide of any one of claims 5 to 7.
9. According to clause 8,

   The vector is a viral vector or a plasmid vector.
10. According to clause 9,

    The viral vector is an adenovirus vector, an adeno-virus associated vector (AAV), a lentivirus vector, or a vaccinia vector.
11. According to clause 8,

    A vector additionally loaded with the CMV promoter.
12. According to clause 8,

    The vector is SEQ ID NO: 33, 34, and 50 to 57.
13. A pharmaceutical composition for treating or preventing spinal muscular atrophy (SMA), comprising the vector of any one of claims 8 to 12 as an active ingredient.
14. According to clause 13,

    A pharmaceutical composition, wherein the spinal muscular atrophy is SMA type 1, SMA type 2, or SMA type 3.
15. A method of treating or preventing spinal muscular atrophy comprising administering the pharmaceutical composition of any one of claims 13 to 14 to a subject.
16. Use of the human SMN1 protein variant of any one of claims 1 to 4 for treating or preventing spinal muscular atrophy (SMA).

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