WO2023061499A1 - Vecteurs viraux adéno-associés recombinants pour le traitement de l'amyotrophie spinale - Google Patents

Vecteurs viraux adéno-associés recombinants pour le traitement de l'amyotrophie spinale Download PDF

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WO2023061499A1
WO2023061499A1 PCT/CN2022/125518 CN2022125518W WO2023061499A1 WO 2023061499 A1 WO2023061499 A1 WO 2023061499A1 CN 2022125518 W CN2022125518 W CN 2022125518W WO 2023061499 A1 WO2023061499 A1 WO 2023061499A1
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intron
seq
expression cassette
promoter
sequence
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PCT/CN2022/125518
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Yezheng Tao
Xiaolong Zhang
Shin-Shay Tian
Xiaoping Zhao
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Shanghai Vitalgen Biopharma Co., Ltd.
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Priority to CN202280069055.0A priority Critical patent/CN118119710A/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • 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
    • 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
    • A61K48/0066—Manipulation of the nucleic acid to modify its expression pattern, e.g. enhance its duration of expression, achieved by the presence of particular introns in the delivered nucleic acid
    • 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
    • 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
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N2750/14145—Special targeting system for viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2800/00—Nucleic acids vectors
    • C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/15—Vector systems having a special element relevant for transcription chimeric enhancer/promoter combination
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/42—Vector systems having a special element relevant for transcription being an intron or intervening sequence for splicing and/or stability of RNA

Definitions

  • the present disclosure relates to gene therapy, specifically a method for treating spinal muscular atrophy (SMA) .
  • SMA spinal muscular atrophy
  • a recombinant adeno-associated viral vector comprising a nucleotide sequence encoding SMN protein.
  • viral particles comprising the rAAV vector, a pharmaceutical composition comprising the viral particles, and uses thereof.
  • SMA Spinal Muscular Atrophy
  • SMA is a neuromuscular disorder caused by loss of motor neurons in the brain stem and anterior horn of the spinal cord.
  • SMA is an inherited diseases which is often autosomal recessive.
  • SMA affects approximately 1 in 10,000 infants born in the U.S. each year. The estimated total cases in China are about 30,000-50,000, with Type I and Type II as the more frequently diagnosed types.
  • approved drugs for SMA mainly focus on replacement of the non-functional SMN1 gene by delivering recombinant adeno-associated virus (AAV) encoding a functional SMN1 gene (e.g., Zolgensma, Novartis) , or modulation of SMN2 mRNA splicing to increase the production of full-length functional SMN protein (Spinraza sold by Biogen and Evrysdi sold by Roche) .
  • AAV adeno-associated virus
  • AAV9 was known to cross the blood brain barrier (BBB) more efficiently as compared to the other AAV serotypes (Haery, L., et al., Adeno-Associated Virus Technologies and Methods for Targeted Neuronal Manipulation. Front Neuroanat, 2019. 13: p. 93. ) .
  • AAV9 is the serotype of choice in the above-mentioned approved gene therapy Zolgensma (Novartis) .
  • AAVrh. 10 and AAV PHP. B were also promising candidate AAVs which have been discussed in recent studies. However, the CNS tropism of PHP.
  • AAVrh10 has limited ability to infect spinal cord motor neurons when given intrathecally in non-human primates (Bey, K., et al., Intra-CSF AAV9 and AAVrh10 Administration in Nonhuman Primates: Promising Routes and Vectors for Which Neurological Diseases? Mol Ther Methods Clin Dev, 2020.17: p. 771-784) , while the expression of SMN protein in spinal cord is critical to achieve therapeutic benefits. Accordingly, applying AAV9 to deliver SMN1 gene would be a preferred approach to develop an effective SMA gene replacement therapy. Attempts have been made to achieve a better therapeutic effect by further optimization of the AAV9-SMN1 vector constructs.
  • WO 2018/160585 A2 discloses a recombinant AAV vector having an AAVhu68 capsid, which contains a heterogenous population of vp1, a heterogenous population of vp2 and a heterogenous population of vp3, and its efficacy in delivering a codon-optimized sequence of human SMN1 gene sequence (hSMN1) in a mouse model of SMA by intracerebroventricular administration. Results showed that survival and growth of the animals were improved.
  • WO 2019/094253 A1 discloses a recombinant AAV9 viral vector comprising a construct consisted of a chicken beta-actin (CB) promoter, a cytomegalovirus (CMV) immediate/early enhancer, hSMN protein coding sequence, a modified SV40 late 16s intron, and a bovine growth hormone (BGH) polyadenylation signal flanked by two modified AAV2 ITRs.
  • CB chicken beta-actin
  • CMV cytomegalovirus
  • BGH bovine growth hormone
  • WO 2019/011817 A1 discloses a rAAV vector comprising an AAV9 or AAVrh10 capsid, and a single-stranded genome comprising a CAG promoter, hSMN protein coding sequence, optionally a WPRE sequence, and the HBB2 polyadenylation sequence.
  • CN112725344A discloses a codon-optimized sequence coding for SMN protein, as well as a method for the preparation of rAAV viral particles by infecting insect cells with Baculovirus carrying an AAV genome.
  • CN112011571A discloses a recombinant AAV vector scAAV-PHP.
  • eB comprising a variant of the capsid protein of AAV9 in which two amino acids A587 and Q588 are replaced by a peptide stretch of DGTLAVPFK, rendering a higher infection efficiency as compared to AAV9.
  • CN108795946A relates to a recombinant AAV vector comprising a codon-optimized coding sequence of SMN protein under the control of a hybrid promoter and a targeting sequence complementary to human miRNA-122.
  • AAV serotypes AAV5, AAV9 and AAVrh10 are selected to build the rAAV vectors.
  • SMN protein when over-expressed in neurons, it may aggregate in neurons and leads to late-onset, dose-dependent neuronal toxicity manifested as sensorimotor deficits, synaptic loss, neurodegeneration, impaired biogenesis of small nuclear ribonucleoprotein particles (snRNPs) (van Alstyne et al., Nature Neuroscience 24, 930-940, 2021) .
  • snRNPs small nuclear ribonucleoprotein particles
  • the present inventors developed new AAV constructs including a combination of new hybrid promoter and codon-optimized hSMN protein coding sequence, which provided desirable levels of SMN protein expression comparable to the levels of the endogenous SMN protein and the inventors demonstrated expression of the SMN protein in the motor cortex and spinal cord in C57BL/6 mice treated with the invented rAAV-SMN1 vector, thus completing the invention.
  • the present application provides an isolated nucleic acid molecule, comprising a nucleotide sequence selected from a group of sequences consisting of SEQ ID NOs: 3-12, wherein the nucleotide sequence encodes human SMN polypeptide.
  • the present application provides an expression cassette comprising the isolated nucleic acid molecule of the first aspect operatively linked to a promoter.
  • the promoter is an artificially modified promoter comprising an intron sequence of the SMN1 gene.
  • the present application provides a rAAV vector, comprising the isolated nucleic acid molecule of the first aspect or the expression cassette of the second aspect.
  • the rAAV vector provides a desirable expression level of SMN protein in disease relevant tissues, including but not limited to a high expression level in e.g., brain and spinal cord, and/or an expression level similar to that from the endogenous SMN1 gene in e.g., brain and spinal cord.
  • the present application provides an AAV viral particle comprising the rAAV vector packaged into an AAV capsid, preferably a capsid with CNS tropism, more preferably AAV9 or AAV PHP. B capsid.
  • the present application provides a pharmaceutical composition
  • a pharmaceutical composition comprising the rAAV vectors of the third aspect or the viral particle of the fourth aspect, and a pharmaceutically acceptable excipient.
  • the present application provides a method for treating SMA in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the rAAV vector of the third aspect, the rAAV particle of the fourth aspect or the pharmaceutical composition of the fifth aspect.
  • the present application provides use of the rAAV vector of the third aspect, the rAAV particle of the fourth aspect or the pharmaceutical composition of the fifth aspect in treating SMA.
  • FIG. 1 shows the representative images of Western blot (WB) results from the first transfection experiment of Example 1 in SH-SY5Y, U-251 MG and U-87 MG cells.
  • FIG. 2 shows the representative images of WB results from the second transfection experiment of Example 1 in SH-SY5Y and U-251 MG cells.
  • FIG. 3 is a schematic of the construct used in codon optimization study in Example 1.
  • the gene of interest (GOI) is under the control of a CMV promoter.
  • FIG. 4 shows the schematic of the luciferase expression cassette used in the promoter screening study.
  • EF1 ⁇ promoter is used as a schematic example to illustrate the plasmid components.
  • FIGs. 5A-C show the luciferase activity driven by different promoters assessed in SH-SY5Y cells (A) , U-251 MG cells (B) and U-87 MG cells (C) . Data are presented as mean ⁇ S.E.M. from one experiment.
  • FIG. 6 shows schematics of constructs comprising EFS derived promoters and SMN1 coding sequence.
  • FIG. 7 shows representative images of WB results from transfection analysis in SH-SY5Y and U-251 MG cells of constructs shown in Fig. 6.
  • FIG. 8 shows the representative images of WB results from samples collected from SH-SY5Y and U-87 MG cells transfected with constructs harboring different combinations of promoter and codon as indicated.
  • FIG. 9 shows ablation of the SMN protein expression in the puromycin-resistant SH-SY5Y cells infected with a lentivirus expressing SMN1 specific shRNA (shSMN1) .
  • shNT refers to a short hairpin RNA targeting a non-coding sequence used as a negative control.
  • FIG. 10 shows the WB results of SMN protein expressions after the candidate AAV vectors were used to infect a SMN1 knock-down SH-SY5Y cell line.
  • FIG. 11A-B show the rescue efficiency of different transgene constructs in normalization of the mRNA levels of Small nuclear ribonucleoprotein-associated protein B (SmB) , and Small nuclear ribonucleoprotein D1 (SmD1) in the SMN1 knock-down SH-SY5Y cell line.
  • SmB Small nuclear ribonucleoprotein-associated protein B
  • SmD1 Small nuclear ribonucleoprotein D1
  • FIG. 12 shows the study protocol of the primary human motor neuron differentiation and treatment schedule.
  • Motor neuron differentiation medium II (MNP medium II) was applied for 14 days.
  • FIG. 13 shows the representative images of human motor neurons after being treated with different rAAVs packaged with different SMN1 transgene constructs.
  • FIG. 14 shows the effects of rAAVs packaged with key representative transgene constructs in inducing SMN protein expression in the SMN1 knocked-down primary human motor neurons.
  • FIG. 15 and FIG. 16 show the SMN protein levels in motor cortex of the wild type C57BL/6 mice which received i.c.v. injection of rAAVs packaged with the indicated transgene constructs.
  • FIG. 17 and FIG. 18 show the SMN protein levels in the spinal cord of the wild-type C57BL/6 mice which received i.c.v. injection of rAAVs packaged with the indicated transgene constructs.
  • the wording “comprise” and variations thereof such as “comprises” and “comprising” will be understood to imply the inclusion of a stated element, e.g. an amino acid sequence, a nucleotide sequence, a property, a step or a group thereof, but not the exclusion of any other elements, e.g. amino acid sequences, nucleotide sequences, properties and steps.
  • the term “comprise” or any variation thereof can be substituted with the term “contain” , “include” or sometimes “have” or equivalent variation thereof.
  • the wording “comprise” also include the scenario of “consisting of” .
  • expression cassette herein refers to a DNA component included in a vector (e.g., rAAV vector) and consisted of a gene (e.g., SMN1 gene) to be expressed in a host cell transfected by the vector and regulatory sequence (s) .
  • the expression cassette of SMN1 inserted in an AAV vector can achieve higher and more consistent SMN protein expression in motor neurons.
  • the present disclosure first provides a group of codon-optimized nucleotide sequences encoding for SMN protein, specifically the human SMN protein having an amino acid sequence as shown in SEQ ID NO: 32, and an isolated nucleic acid sequence comprising any of these nucleotide sequences.
  • isolated nucleic acid means a DNA or RNA which is removed from all or a portion of a polynucleotide in which the isolated polynucleotide is found in nature, or is linked to a polynucleotide to which it is not linked in nature.
  • An isolated nucleic acid molecule "comprising" a specific nucleotide sequence may include, in addition to the specified sequence, operably linked regulatory sequences that control expression of the coding region of the recited nucleic acid sequences. Due to the codon degeneracy, one skilled in the art understands that any specific amino acid sequence can be coded by several different nucleotide sequences.
  • Codon-optimized coding sequence herein refers to a nucleotide sequence coding for SMN1 protein modified from the wild-type SMN1 coding sequence accommodating codon bias. Optimization may be achieved by reducing sequence complexity, adjusting GC content, adjusting codon usage and/or avoiding rare codons.
  • the coding sequence which has been codon optimized usually shows an increased translational efficiency of the gene of interest (GOI) , leading to a higher protein expression.
  • Tools e.g., JCat
  • the codon of the SMN1 coding sequence of the present application has a Codon Adaptation Index (CAI) greater than 0.8.
  • CAI is a measure of codon bias.
  • the codon-optimized coding sequence for human SMN protein comprises or consists of a nucleotide sequence selected from a group consisting of the nucleotide sequences of SEQ ID Nos: 3-12. More preferably, the codon-optimized coding sequence for human SMN protein comprises or consists of a nucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 9.
  • an expression cassette can comprise one or more regulatory sequences in addition to coding sequence.
  • Regulatory sequence can be selected from one or more of promoter, enhancer, polyadenylation sequence, and translation termination signal.
  • a certain combination of regulatory sequences of the present disclosure can achieve unexpected effect in improving the expression efficiency of the coding sequence.
  • Promoter refers to a DNA sequence enables initiation of transcription of a downstream gene under the control of said promoter. Promoters include but not limited to constitutive promoters, cell type-specific promoters, tissue-specific promoters, development stage-specific promoters. Promoter can be a naturally occurring promoter of a gene, a modified version of a naturally occurring promoter or a synthetic promoter.
  • the promoter of the present disclosure can be a constitutive promoter.
  • the promoter can be chicken ⁇ -actin promoter or EFS (a short version of EF1 ⁇ ) promoter, or a promoter derived therefrom.
  • Enhancer is a regulatory DNA sequence which can enhance the transcription of the GOI in AAV together with the promoter.
  • the expression cassette of the present application comprises of an enhancer. More preferably, the enhancer can be a CMV enhancer, e.g., in a CBh promoter.
  • intron sequences functioning as enhancers can be included.
  • an intron sequence originated from the intron of GOI can be included in the expression cassette.
  • promoter together with an enhancer and/or an intron sequence are collectively referred as “promoter” or “promoter element” .
  • hybrid with reference to a nucleotide sequence, for example a promoter or an intron sequence, means that said nucleotide sequence is obtained by combining two or more sequences which do not exist consecutively in nature.
  • a hybrid promoter can be obtained by combining a complete or partial sequence of a naturally occurring promoter or a variant thereof with an intron sequence from a different gene of the promoter, or with an intron sequence from the same gene of the promoter in an order different from the natural state.
  • the promoter is the CBh promoter. In another preferred embodiment, the promoter is consisted of the EFS promoter and an intron sequence.
  • the intron sequence has a total length of about or less than 200bp, about or less than 250bp, about or less than 300bp, about or less than 350bp, about or less than 400 bp.
  • the intron sequence is a hybrid intron sequence obtained by combining at least two intron fragments derived from the same gene or from different genes.
  • the hybrid intron comprises or consists of intron fragments derived from one or more of the following introns: intron of chicken ⁇ -actin (CBA) gene, minute virus of mice (MMV) intron, intron of human ⁇ -globin gene, intron of immunoglobulin heavy chain gene, and intron of adenovirus.
  • the hybrid intron comprises or consists of fragments derived from (1) an intron of chicken ⁇ -actin (CBA) gene and a minute virus of mice (MMV) intron; (2) an intron of human ⁇ -globin gene and an intron of immunoglobulin heavy chain gene; or (3) an intron of adenovirus and an intron of immunoglobulin heavy chain gene.
  • the intron sequence comprises or consists of the nucleotide sequence of any one of SEQ ID NOs: 17-19, preferably SEQ ID NO: 17.
  • the intron sequence of the present disclosure is derived from the gene of interest.
  • the intron sequence is consisted of one or more fragments derived from one or more intronic regions of the gene of interest, in particular one or more intronic regions of human SMN1 gene, e.g. intron 1, intron 2, intron 3, intron 4, intron 5, intron 6, or intron 7.
  • the intron sequence is derived from an intronic region which locates at a position closer to the promoter region, e.g. intron 1 or intron 2 of human SMN1 gene.
  • the intron sequence can be a truncated version of intron 1 or intron 2 of human SMN1 gene, e.g. with one or more portions deleted, e.g. with middle portion deleted.
  • the intron sequence has a nucleotide sequence as shown by nucleotide positions 215-614 of either SEQ ID NOs: 27 or 28.
  • the intron sequence is derived from an intronic region of a gene other than the gene of interest.
  • the intron sequence is SV40 small t antigen intron, e.g. having a sequence of SEQ ID NO: 20.
  • the intron has a nucleotide sequence of SEQ ID NO: 17, or a nucleotide sequence at nucleotide positions 215-614 of either SEQ ID NOs: 27 or 28.
  • the promoter comprises or consists of a nucleotide sequence selected from a group consisting of the nucleotide sequences of SEQ ID Nos: 22-25 (EFShI1, EFShI2, EFShI3, EFSI4) and SEQ ID Nos: 28-29 (EFSdI1 and EFSdI2) .
  • the promoter comprises or consists of a nucleotide sequence of SEQ ID NO: 22 (EFShI1) .
  • the promoter or promoter/intron element has a length of no more than 1000 bp, no more than 900 bp, no more than 850 bp, no more than 800 bp, no more than 700 bp, no more than 600 bp, no more than 500 bp, or no more than 400 bp, due to the limited packaging capacity of AAV.
  • the intron sequence when the intron sequence is derived from an intronic region of the gene of interest, it can be inserted into the coding sequence (e.g., codon-optimized coding sequence) at a position corresponding to the position where it locates in the gene in nature, e.g., between two exons, instead of locating at a position 5’-upstream of the coding sequence and constituting a promoter/intron element.
  • the intron sequence is derived from intron 1 of human SMN1 gene, e.g., a truncated version of intron 1 of human SMN1 gene, it can be inserted at a position between exon 1 and exon 2 of the coding sequence.
  • the polyadenylation sequence of the present disclosure can be bGH polyA, SPA or SV40 polyA, preferably SV40 polyA. Any one of the polyA can be combined with a Woodchuck Hepatitis Virus posttranscriptional regulatory element (WPRE) , which is a DNA sequence, when transcribed, creates a tertiary structure to enhance protein expression, and configured as WPRE-bGH polyA, WPRE-SPA or WPRE-SV40 poly A, respectively.
  • WPRE Woodchuck Hepatitis Virus posttranscriptional regulatory element
  • a modified version of WPRE can also be used, which contains five-point mutations in the putative promoter region and 1 nucleotide change in the start codon of woodchuck hepatitis virus X protein (WHx) open reading frame.
  • Kozak consensus sequence (Kozak sequence) , named after the scientist who discovered it, is a nucleic acid sequence motif present in most eukaryotic mRNA transcripts that functions as the protein translation initiation site. Kozak sequence ensures the protein is accurately and efficiently translated.
  • the expression cassette comprises an EFS promoter, a hybrid intron, a Kozak sequence, an optimized SMN1 codon sequence, WPRE, and SV40 polyA.
  • the expression cassette of the present disclosure comprises or consists of a nucleotide sequence as shown in any one of SEQ ID NO: 33, SEQ ID NO: 34, or SEQ ID NO: 35.
  • the nucleic acid molecule or the expression cassette of the present disclosure can be constructed into a recombinant AAV (rAAV) vector, to obtain rAAV particles for delivery into a subject in need thereof.
  • rAAV recombinant AAV
  • the rAAV vectors are in single stranded form.
  • the rAAV vector is comprised of two inverted terminal repeat (ITR) sequences at both ends of the inserted nucleotide sequence.
  • ITR inverted terminal repeat
  • the ITR of the present disclosure can be ITR derived from any AAV serotypes.
  • serotype of AAV ITR the phrase “derived from” means that the ITR can be the ITR of a certain serotype or a variant derived therefrom with modification (s) .
  • the rAAV vector comprises two ITRs derived from AAV2.
  • the rAAV vector comprises two AAV2 ITRs, or comprises a wild-type AAV2 ITR and a truncated version of AAV2 ITR lacking the region C or region C’.
  • the wild-type AAV2 ITR locates at the 5’ of the inserted nucleotide sequence, while the AAV2 ITR variant locates at the 3’ of the inserted nucleotide sequence; or vice versa.
  • the rAAV genome was packaged into an AAV capsid.
  • the capsid can be derived from any AAV serotype known in the art or characterized in the future.
  • the capsid and ITRs can be derived from the same serotype of AAV or from different serotypes of AAV.
  • the capsid is suitable for nervous system delivery, e.g., intrathecal, intracisterna magna, intracerebroventricular delivery or intravenous.
  • the AAV vector comprises a capsid of AAV1, AAV2, AAV4, AAV5, AAV7, AAV8, AAV9, AAVrh10, AAV PHP. B, AAV2.7m8, or AAVAnc80L65 serotype, or a variant thereof.
  • the capsid is the AAV9 capsid.
  • the term “pharmaceutical composition” refers to a composition suitable for delivering to a subject.
  • the pharmaceutical composition of the present disclosure comprises the isolated nucleic acid, the rAAV vector or the viral particle of the present disclosure and a pharmaceutically acceptable excipient.
  • Conventional pharmaceutically acceptable excipients are known in the art and can be solid or liquid excipients.
  • the pharmaceutical composition can be a liquid for injection.
  • administration when applied to a subject, e.g., an animal, including human, or to cell, tissue, organ, or biological fluid, means contact of an exogenous pharmaceutical, therapeutic, diagnostic agent, or composition with the subject, cell, tissue, organ, or biological fluid.
  • Treatment of a cell encompasses contact of a reagent with the cell, as well as contact of a reagent with a fluid, where the fluid is in contact with the cell.
  • administration and treatment also include in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding compound, or by another cell.
  • the rAAV vector of the present application can be delivered via intravenous or intra-CSF administration.
  • the rAAV vector is delivered via intra-CSF route.
  • the rAAV vector can be administered via a single dose or multiple doses. In a specific embodiment, the rAAV vector is administered via a single injection.
  • the dosage of the rAAV vector can be varied based on the administration route. For example, intravenous injection, as a systemic route, usually needs a delivery of a higher dose of rAAV to achieve sufficient therapeutic effects, given that SMA is a CNS disease.
  • the dosage can also be varied based on the body weight of the subject. Therefore, the dose range can be within a broad scope which covers 1 ⁇ 10 13 -5 ⁇ 10 14 vg.
  • treat includes to cure or at least to alleviate the symptoms of SMA, or conditions of diseases related to SMA.
  • the viral vector encoding for SMN protein of the present application can be used to treat a subject having SMA.
  • the subject can be an infant, a child, an adolescent or an adult.
  • the subject can be an infant or a child within two years old.
  • a subject having SMA can be diagnosed by one or more of the following measures: checking the blood creatine kinase levels, performing genetic tests for the SMN1 gene, performing nerve conduction tests to check the electrical activity of muscles and nerves, and performing muscle tissue biopsy to identify muscle loss and atrophy.
  • This example describes the method for optimizing the SMN protein coding sequence.
  • Codon optimized sequences were designed by codon optimization tools such as JCat. Ten sequences which obtained a high CAI value containing higher frequency codons were selected for evaluation of the protein levels expressed in biologically relevant cell lines transfected with plasmids carrying the candidate sequences.
  • the wild-type SMN1 sequence and a previously reported codon-optimized SMN1 sequence, SMNopi (referred as “SMN-2011” hereinafter, Dominguez, E., et al., Intravenous scAAV9 delivery of a codon-optimized SMN1 sequence rescues SMA mice. Hum Mol Genet, 2011. 20 (4) : p. 681-93. ) were used as references.
  • Ten candidate coding sequences (SEQ ID Nos: 3-12) as shown below and the two reference sequences (SMN-WT and SMNopt-2011, as shown in SEQ ID Nos: 1 and 2) were inserted into an scAAV (self-complementary AAV) vector plasmid under the control of a CMV promoter, respectively.
  • the schematic of the plasmid map is shown in FIG. 3, using the wild-type sequence as an example. As shown in FIG.
  • the construct comprises from 5’ to 3’: 5’ AAV2 ITR (a truncated version of 3’ ITR; SEQ ID NO: 13) , a CMV enhancer/promoter (SEQ ID NO: 15) , the SMN1 coding sequence, an SV40 poly (A) signal (SEQ ID NO: 16) , and a 3’ AAV2 ITR (SEQ ID NO: 14) .
  • All of the 12 recombinant vectors including SMN-WT (wild-type) , SMN-BS (Biosune) , SMN-GS (Genescript) , SMN-JCat, SMN-SAgs (Sangon, modified) , SMN-GW (Genewiz) , SMN-NP (NovoPro) , SMN-TK (Tsingke) , SMN-TY (Tianyihuiyuan) , SMN-GScg (Genscript modified) , SMN-GWcg (Genewiz modified) , SMN-2011, and a negative control which only expresses GFP were transfected into three cell lines, SH-SY5Y (Procell, CL-0208) , U-251 MG (China National collection of authenticated cell cultures, Cat.
  • the protein expression levels of SMN and the housekeeping gene ⁇ -tubulin were detected by an antibody against the human SMN (Sigma, #HPA045271) and ⁇ -tubulin (Proteintech, #66240-1-Ig) , respectively.
  • the results of Western blots were shown in FIG. 1. Among all the candidates evaluated here, SAgs and TY performed better in terms of SMN protein expression.
  • This example describes the developing of an optimized expression cassette of SMN coding sequence.
  • the strength of promoter in an AAV vector is known to be critical for the expression of the exogenous gene.
  • the strength of 11 different promoters and promoter/intron elements, including constitutive promoters and in-house designed EFS/intron chimeric promoters (Table 1) was evaluated in a luciferase assay as described below to identify the most desirable promoter element for our use.
  • CBh promoter is a previously reported hybrid promoter (Gray, S. J., et al., Optimizing promoters for recombinant adeno-associated virus-mediated gene expression in the peripheral and central nervous system using self-complementary vectors. Hum Gene Ther, 2011. 22 (9) : p. 1143-53) which contains a hybrid intron 1.
  • Hybrid intron 1 (hI1; SEQ ID NO: 17) is derived from the chicken ⁇ -actin (CBA) and minute virus of mice (MMV) introns.
  • Hybrid intron 2 (hI2; SEQ ID NO: 18) is chimeric intron of the human ⁇ -globin and immunoglobulin heavy chain genes.
  • Hybrid intron 3 (hI3; SEQ ID NO: 19) is from the adenovirus and immunoglobulin heavy chain genes.
  • Intron 4 (I4; SEQ ID NO: 20) is the SV40 small t antigen intron.
  • promoter Nos. 8-11 (SEQ ID NOs: 22-25) were designed and constructed by the present inventors.
  • FIG. 4 is a schematic drawing exemplifying the structure of a representative construct by using EF1 ⁇ as an example. After transfection, the cells were collected and homogenized in 1 ⁇ Passive Lysis Buffer (Promega, E1910) and stored in -80°C.
  • EFShI1 (SEQ ID NO: 22) , which is consisted of a EFS promoter plus a hybrid intron 1 (hI1; SEQ ID NO: 17) also showed good expression and was much better than EFS combined with other introns (hI2, hI3 and I4; SEQ ID NOs 18, 19 and 20, respectively) .
  • the EF1 ⁇ promoter composed of the 212 bp EFS core promoter and a 939 bp long intron is much longer than EFS combined with the 229 bp hybrid intron 1, and may exceed the packaging capacity of scAAV when combines with other required expression elements. Therefore, CBh and EFShI1 promoters were selected for further analysis.
  • Example 3 Further improving the SMN protein expression by including an SMN1 intron
  • This example continued promoter optimization by evaluating potential expression-improving introns derived from the SMN1 gene.
  • ⁇ intron1 SEQ. NO. 27
  • ⁇ intron2 dI2, SEQ. NO. 28
  • SMN1 NM _000344.4
  • Each of the newly formed 400 bp intron was inserted into its original position in the SMN1 gene (e.g., dI1 was inserted into where intron 1 resides, between exon 1 and exon 2 sequences) or a position 3’ of the promoter in the vector.
  • EFS promoter was used in this study due to its smaller size in combination with the wild-type SMN1 (SMN1-WT. SEQ ID No: 1) as the GOI.
  • Seven vectors including four constructs (EFS-SMN1dI1, EFS-SMN1dI2, EFSdI1-SMN1, EFSdI2-SMN1) comprising either dI1 or dI2 inserted inside or 5’ of the WT SMN1, and three reference constructs CMV-SMN1, EFS-SMN1, and EFShI1-SMN1 were constructed as shown in FIG. 6, with WPRE and SV40 poly (A) signal as elements downstream of the GOI.
  • the vectors were used to transfect two cell lines, SH-SY5Y and U-251 MG. The transfection experiments were conducted same as described above.
  • the SMN protein levels were evaluated by WB to determine the additive effects of the intron addition and the results are shown in FIG. 7.
  • dI2 enhanced SMN1 expression when placed either in its natural location inside the gene or between the EFS promoter and the GOI, while dI1 only enhanced the expression when located 3’ of EFS promoter in the U-251 MG cells.
  • the EFShI1 promoter showed relatively high strength in SH-SY5Y and U-251 MG cells.
  • the top two candidate coding sequences (SMN-SAgs and SMN-TY) and the top four promoters (CBh, EFShI1, EFSdI1, and EFSdI2) were selected for further evaluation.
  • CBh, EFShI1, EFSdI1, and EFSdI2 the top four promoters
  • EFShI1 and EFSdI2 performed better than EFSdI1 when directing the same coding sequence.
  • SAgs showed slightly better performance than TY in SH-SY5Y and U-87 MG cells.
  • SAgs codon in combination with promoters CBh, EFShI1, and EFSdI2 were selected for further evaluation.
  • This example shows the performance of viral particles derived from candidate vectors in restoring SMN function in vitro.
  • an SMN1 knock-down cellular model was established by lentivirus mediated gene knockdown in the SH-SY5Y cell line.
  • a pLenti plasmid which harbors shRNA (SEQ ID NO: 30) targeting SMN1 was packaged as lentivirus and then added onto SH-SY5Y cells.
  • SEQ ID NO: 30 shRNA targeting SMN1
  • the puromycin-resistant cells were collected and analyzed to confirm the knockdown of the SMN protein (FIG. 9) .
  • the SMN1 KD cell line was named SMN1-KD-SH.
  • a negative control shNT targeting non-coding gene (SEQ ID NO: 31) was used.
  • SMN protein results in impaired cellular function, including defective assembly of U-rich small nuclear ribonucleoprotein particles (UsnRNPs) and down-regulation of Sm transcripts, like SmB and SmD. Re-introduction of SMN protein should rescue these cellular defects (Prusty, A.B., et al., Impaired spliceosomal UsnRNP assembly leads to Sm mRNA down-regulation and Sm protein degradation. J Cell Biol, 2017. 216 (8) : p. 2391-2407. ) . Afore-mentioned 3 candidates and 2 benchmarks were tested in the presence of 1 ⁇ M of MG-132 in the SMN1-KD-SH cells.
  • a human motor neuron in vitro assay was established to further evaluate the candidate AAV vectors.
  • iPSC Human induced pluripotent stem cell
  • MNP motor neural progenitor
  • HopStem Cat. No. HopCell-MPC
  • the MNPs were cultured in a differentiation medium (HopStem, motor neuron differentiation medium with supplemental factor II) for 7 days, then the lentivirus expressing SMN1 shRNA was applied to the cells at MOI 20 (FIG. 12) .
  • a differentiation medium HopStem, motor neuron differentiation medium with supplemental factor II
  • the lentivirus expressing SMN1 shRNA was applied to the cells at MOI 20 (FIG. 12) .
  • Two days post lentivirus addition the candidate AAVs together with the two benchmarks were added.
  • the morphology of the AAV vector transduced cells was normal without any obvious defects in neurite formation, with neurite outgrow observed 5 days post AAV administration (14 days post differentiation) (FIG. 13) .
  • the treated motor neuron samples were collected by N-PER TM neuronal protein extraction reagent (Thermo fisher, 87792) and the SMN protein levels were determined by WB.
  • the results showed that CBh-SAgs gave a better expression than the other candidate vectors (FIG. 14) in the lentivirus treated cells, but only EFShI1-SAgs expressed the same level of SMN as that in the PBS+GFP group which can be considered as the endogenous level of SMN in human motor neurons.
  • Example 6 Expression of SMN protein in brain and spinal cord in the wild-type C57BL/6 mice by administration of rAAV vector via i.c.v
  • the candidate AAV vectors comprsing expression cassette CBh-SAgs, EFShI1-SAgs, and EFSdI2-SAgs (SEQ ID NO: 33, SEQ ID NO: 34 and SEQ ID NO: 35, respectively) together with the two benchmarks CBs-WT and CBh-2011 were injected into the lateral ventricle of the wild-type C57BL/6 mice (i.c.v) on postnatal day 1. After 21 days, the mice were euthanized and tissue samples were collected in N-PER TM neuronal protein extraction reagent (Thermo fisher, 87792) and proteins were extracted after tissue homogenization.
  • N-PER TM neuronal protein extraction reagent Thermo fisher, 87792
  • SMN protein was elevated in both the motor cortex and spinal cord samples collected from the five treatment groups as compared to the CMV-GFP control group (FIGs. 15-18) , and among the three candidates, EFShI1-SAgs expressed the most SMN protein.

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Abstract

L'invention concerne des séquences à codon optimisé codant pour le polypeptide SMN et des vecteurs de virus adéno-associés recombinants (rAAV) comprenant l'une desdites séquences sous le contrôle d'un promoteur hybride. L'invention concerne également des particules virales comprenant le vecteur rAAV, une composition pharmaceutique comprenant le vecteur rAAV ou les particules virales, et leurs utilisations dans le traitement de l'amyotrophie spinale (SMA).
PCT/CN2022/125518 2021-10-15 2022-10-14 Vecteurs viraux adéno-associés recombinants pour le traitement de l'amyotrophie spinale WO2023061499A1 (fr)

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Citations (6)

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WO2014178863A1 (fr) * 2013-05-01 2014-11-06 Genzyme Corporation Compositions et procédés pour traiter l'amyotrophie spinale
WO2018160585A2 (fr) * 2017-02-28 2018-09-07 The Trustees Of The University Of Pennsylvania Compositions utiles dans le traitement de l'amyotrophie spinale
WO2019011817A1 (fr) * 2017-07-08 2019-01-17 Genethon Traitement de l'amyotrophie spinale
WO2019094253A1 (fr) * 2017-11-08 2019-05-16 Avexis Inc. Moyens et procédé de préparation de vecteurs viraux et leurs utilisations
WO2020127813A1 (fr) * 2018-12-21 2020-06-25 Genethon Cassettes d'expression pour vecteurs de thérapie génique
CN112725344A (zh) * 2020-12-28 2021-04-30 中吉智药(南京)生物技术有限公司 密码子优化的smn1基因、腺相关病毒表达质粒及基因药物

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WO2014178863A1 (fr) * 2013-05-01 2014-11-06 Genzyme Corporation Compositions et procédés pour traiter l'amyotrophie spinale
WO2018160585A2 (fr) * 2017-02-28 2018-09-07 The Trustees Of The University Of Pennsylvania Compositions utiles dans le traitement de l'amyotrophie spinale
WO2019011817A1 (fr) * 2017-07-08 2019-01-17 Genethon Traitement de l'amyotrophie spinale
WO2019094253A1 (fr) * 2017-11-08 2019-05-16 Avexis Inc. Moyens et procédé de préparation de vecteurs viraux et leurs utilisations
WO2020127813A1 (fr) * 2018-12-21 2020-06-25 Genethon Cassettes d'expression pour vecteurs de thérapie génique
CN112725344A (zh) * 2020-12-28 2021-04-30 中吉智药(南京)生物技术有限公司 密码子优化的smn1基因、腺相关病毒表达质粒及基因药物

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