WO2023219394A1 - Variant de protéine smn1 humaine et son utilisation - Google Patents

Variant de protéine smn1 humaine et son utilisation Download PDF

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WO2023219394A1
WO2023219394A1 PCT/KR2023/006293 KR2023006293W WO2023219394A1 WO 2023219394 A1 WO2023219394 A1 WO 2023219394A1 KR 2023006293 W KR2023006293 W KR 2023006293W WO 2023219394 A1 WO2023219394 A1 WO 2023219394A1
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vector
hsmn1
cmv
seq
amino acid
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Korean (ko)
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공영윤
이준우
강종설
김지훈
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서울대학교산학협력단
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • 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.
  • SMA Spinal Muscular Atrophy
  • SMA1 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.
  • siRNA such as Spinraza
  • the cost of one administration is very high.
  • treatments that treat SMA with a single dose such as Zolgensma
  • 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.
  • 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.
  • 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.
  • SMA spinal muscular atrophy
  • 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.
  • 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.
  • motor neuron 1 Provides protein variants.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • it may be SMN1 isoform d containing SEQ ID NO: 1 or a variant thereof.
  • variant 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.
  • 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.
  • 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).
  • the substituted amino acid may be a non-polar amino acid.
  • 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.
  • 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.
  • the lysine (K) may be replaced with alanine (A), arginine (R), serine (S), glutamine (Q), aspartic acid (D), or tyrosine (Y).
  • 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.
  • 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.
  • the lysine (K) may be replaced with alanine (A) or arginine (R).
  • the human SMN1 variant may include one amino acid sequence selected from the group consisting of SEQ ID NOs: 13 to 18.
  • 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.
  • the lysine (K) may be replaced with alanine (A) or arginine (R).
  • the human SMN1 variant may include one amino acid sequence selected from the group consisting of SEQ ID NOs: 19 to 24.
  • 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.
  • the lysine (K) may be replaced with alanine (A) or arginine (R).
  • the human SMN1 variant may include one amino acid sequence selected from the group consisting of SEQ ID NOs: 25 to 30 and 41.
  • 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 human SMN1 protein variants.
  • the polynucleotide may be DNA or RNA.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the signal sequence is cleaved within the lumen of the ER by cellular enzymes commonly known as signal peptidases.
  • 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.
  • tPa tissue Plasminogen Activation
  • HSV gDs Synignal sequence of Herpes simplex virus glycoprotein D
  • growth hormone a secretion signal sequence used in higher eukaryotic cells, including mammals.
  • 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.
  • 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.
  • 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.
  • 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, ⁇ gt10, ⁇ gt11, ⁇ ZAP, etc.
  • the viral vector may be a retrovirus, adenovirus vector, adeno-virus associated vector (AAV), lentivirus vector, insect virus vector (Baculovirus, etc.), or vaccinia vector.
  • AAV adeno-virus associated vector
  • lentivirus vector lentivirus vector
  • insect virus vector Bacillus, etc.
  • vaccinia vector vaccinia vector
  • the viral vector may be an adeno-virus associated vector (AAV).
  • AAV adeno-virus associated vector
  • 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.
  • 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.
  • the vector can additionally load a polynucleotide encoding the human ⁇ -glucuronidase promoter, chicken ⁇ -actin promoter, or CMV promoter.
  • the polynucleotide encoding the SMN1 gene variant may be operably linked to the promoter.
  • the promoter is capable of expressing a polynucleotide encoding a human SMN 1 variant in neurons of the spinal cord.
  • the promoter can express SMN1 in motor neurons of the spinal cord.
  • a polynucleotide encoding a CMV promoter can be additionally loaded.
  • the CMV promoter may include the amino acid sequence of SEQ ID NO: 52.
  • 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.
  • the vector may contain one or more polynucleotides selected from the group consisting of SEQ ID NOs: 33, 34, and 50 to 57.
  • 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.
  • SMA spinal muscular atrophy
  • 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.
  • SMA Spinal Muscular Atrophy
  • the spinal muscular atrophy may be SMA type 1, SMA type 2, or SMA type 3.
  • 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.
  • the subject may be a primate, and may specifically be a pediatric or adult subject.
  • 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.
  • the pharmaceutical composition 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.
  • 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.
  • “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.
  • 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.
  • 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.
  • the pharmaceutical composition 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.
  • a suitable carrier 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 are mammals and humans, and humans are particularly preferred.
  • the pharmaceutical composition of the present application may further include any compounds or natural extracts known to have a SMA treatment effect.
  • Table 1 below shows the amino acid sequences of hSMN1 and hSMN1 K186R used in the experiment.
  • hSMN1 Amino acid sequence hSMN1 (SEQ ID NO: 1) MAMSSGGSGGGVPEQEDSVLFRRGTGQSDDSDIWDDTALIKAYDKAVASFKHALKNG DICETSGKPKTTPKRKPAKKNKSQKKNTAASLQQWKVGDKCSAIWSEDGCIYPATIAS IDFKRETCVVVYTGYGNREEQNLSDLLSPICEVANNIEQNAQENENESQVSTDESENS RSPGNKSDNIKP K SAPWNSFLPPPPPMPGPRLGPGKPGLKFNGPPPPPPPPPPPPHLLSC WLPPFPSGPPIIPPPPPICPDSLDDADALGSMLISWYMSGYHTGYYMGFRQNQKEGRC SHSLN hSMN1 K186R (SEQ ID NO: 7) MAMSSGGSGGGVPEQEDSVLFRRGTGQSDDSDIWDDTALIKAYDKAVASFKHALKNG DICETSGKPKTTPKRKPAKKNKSQ
  • Table 2 shows the nucleic acid sequence of hSMN1 used in the experiment.
  • 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.
  • PEI polyethylenimine
  • Opti-MEM TM Opti-MEM TM
  • Example 1.2 AAV production and purification
  • 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.
  • lysis buffer 5 M NaCl, 1 M Tris HCl, ultrapure water
  • 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 ⁇ L 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 ⁇ L of phenol red
  • 1xPBS-MK 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.
  • qPCR quantitative PCR
  • 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)/ ⁇ l, and was finally diluted 1/10 times from 1x10 7 to 1x10 1 for the standard curve.
  • VG vector genomes
  • DNase1 1 U/ ⁇ l
  • step condition Step1 95°C, 5 min, 1 cycle
  • Step2 95°C, 10 min, 1 cycle
  • Step3 95°C, 10 s / 60°C 40 s / 72°C, 1 s, 40 cycles
  • Step4 40°C, 10 s, 1 cycle
  • 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.
  • 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.
  • SMA type 1.5 Smn-/-; SMN ⁇ 7+/-; SMN2+/+ mice 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.
  • mice injected with AAV were maintained under the care of their mothers in their birth cages, and their survival and weight were recorded daily.
  • righting reflex negative geotaxis
  • tail suspension Hindlimb clasping
  • 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.
  • 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.
  • 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.
  • 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.
  • mice injected with AAV9-CMV-hSMN1 K186R kicked their legs without crossing them.
  • the legs were crossed for more than 20 seconds out of the 30 seconds measured, and kicking the legs upward was rarely observed.
  • 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.
  • 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.
  • mice injected with the SMA gene as in Example 2 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.
  • 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.
  • 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).
  • GA gastrocnemius muscle
  • 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.
  • qRT-PCR was performed as follows.
  • RNA 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.
  • 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 ⁇ m 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.
  • EDTA antigen retrieval buffer (1mM EDTA, 0.05% Tween 20, pH 8.0
  • 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.
  • 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.

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Abstract

La présente invention concerne une protéine SMN1 humaine mutée, un polynucléotide codant pour celle-ci, et une composition pharmaceutique pour le traitement de l'atrophie musculaire spinale, comprenant un vecteur la portant. La composition réduit la toxicité hépatique observée dans la thérapie génique SMN1 existante par mutation de hSMN1, qui peut être utilisée directement chez l'homme, tout en augmentant la stabilité, ce qui permet de traiter de manière efficace l'atrophie musculaire spinale due à un effet remarquable par rapport à la même dose et à un effet durable.
PCT/KR2023/006293 2022-05-10 2023-05-09 Variant de protéine smn1 humaine et son utilisation WO2023219394A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20160003792A (ko) * 2013-05-01 2016-01-11 젠자임 코포레이션 척수성 근위축증을 치료하기 위한 조성물 및 방법
KR101835490B1 (ko) * 2009-05-02 2018-03-08 젠자임 코포레이션 신경변성 질환에 대한 유전자 요법
KR20180086266A (ko) * 2015-12-14 2018-07-30 더 트러스티스 오브 더 유니버시티 오브 펜실바니아 척수성 근위축증의 치료에 유용한 아데노-관련 바이러스 벡터
KR20210099025A (ko) * 2018-11-30 2021-08-11 노파르티스 아게 Aav 바이러스 벡터 및 이의 용도
WO2022028472A1 (fr) * 2020-08-05 2022-02-10 Hangzhou Jiayin Biotech Ltd. Constructions d'acides nucléiques et leurs utilisations pour le traitement de l'amyotrophie spinale

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101835490B1 (ko) * 2009-05-02 2018-03-08 젠자임 코포레이션 신경변성 질환에 대한 유전자 요법
KR20160003792A (ko) * 2013-05-01 2016-01-11 젠자임 코포레이션 척수성 근위축증을 치료하기 위한 조성물 및 방법
KR20180086266A (ko) * 2015-12-14 2018-07-30 더 트러스티스 오브 더 유니버시티 오브 펜실바니아 척수성 근위축증의 치료에 유용한 아데노-관련 바이러스 벡터
KR20210099025A (ko) * 2018-11-30 2021-08-11 노파르티스 아게 Aav 바이러스 벡터 및 이의 용도
WO2022028472A1 (fr) * 2020-08-05 2022-02-10 Hangzhou Jiayin Biotech Ltd. Constructions d'acides nucléiques et leurs utilisations pour le traitement de l'amyotrophie spinale

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