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WO2024027632A1 - Nouveau squelette plasmidique pour réduire les impuretés d'adn dans la préparation de raav - Google Patents

Nouveau squelette plasmidique pour réduire les impuretés d'adn dans la préparation de raav Download PDF

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Publication number
WO2024027632A1
WO2024027632A1 PCT/CN2023/110182 CN2023110182W WO2024027632A1 WO 2024027632 A1 WO2024027632 A1 WO 2024027632A1 CN 2023110182 W CN2023110182 W CN 2023110182W WO 2024027632 A1 WO2024027632 A1 WO 2024027632A1
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Prior art keywords
polynucleotide
raav
nucleotide sequence
seq
genome
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Bo Hu
Lu Wang
Weidong Xiao
Junping Zhang
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Skyline Therapeutics Shanghai Co Ltd
Skyline Therapeutics Ltd
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Skyline Therapeutics Shanghai Co Ltd
Skyline Therapeutics Ltd
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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
    • 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
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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
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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
    • C12N2750/14143—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
    • C12N2750/14151—Methods of production or purification of viral material
    • 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
    • C12N2750/14151—Methods of production or purification of viral material
    • C12N2750/14152—Methods of production or purification of viral material relating to complementing cells and packaging systems for producing virus or viral particles

Definitions

  • the present disclosure relates to molecular biology and gene therapy.
  • the present disclosure relates to a reverse packaging inhibitory sequence (RPIS) capable of reducing DNA impurities in the genome of a recombinant adeno-associated virus (rAAV) .
  • RPIS reverse packaging inhibitory sequence
  • Adeno-associated virus is a member of Parvoviridae family. It is a simple single-stranded DNA virus, and requires a helper virus (such as adenovirus) for replication.
  • the genome of a wildtype AAV contains approximately 4.7 kilobases (kb) , comprising the cap and rep genes between two inverted terminal repeat (ITR) sequences with interrupted palindromic sequences that can fold into hairpin structures that function as primers during initiation of DNA replication.
  • ITR inverted terminal repeat
  • the cap gene encodes the viral capsid protein, and the rep gene is involved in the replication and integration of AAV.
  • AAV can infect a variety of cells.
  • rAAV Due to the advantages of high safety, low immunogenicity, the infection of a variety of tissues and non-integration, rAAV has been widely used as the vector for gene therapy.
  • hundreds of clinical trials for gene therapies using AAV as the vector have been carried out, and have shown promising prospects in a variety of diseases (Rangarajan et al., 2017, AAV5–Factor VIII Gene Transfer in Severe Hemophilia A, NEJM, 377: 2519-2530; and Pasi et al., 2020, Multiyear Follow-up of AAV5-hFVIII-SQ Gene Therapy for Hemophilia A, NEJM, 382: 29-40) .
  • the outcomes following gene therapy with rAAV are unpredictable, even in subjects without detectable pre-existing anti-AAV neutralizing antibodies.
  • the same dose and dosage regimen may show heterogeneity of therapeutic effect in different patients. This clinical heterogeneity cannot be fully attributed to adverse effects induced by the therapy and the patient’s immune response, and may also be related to DNA impurities in the genome of rAAV which may result from the design of the AAV vector (such as the transgene plasmid) .
  • AAV shuttle plasmid backbone designed to reduce the packaging of non-genomic nucleic acid impurities in rAAV products, in particular an AAV shuttle plasmid backbone comprising a reverse packaging inhibitory sequence (RPIS) capable of reducing DNA impurities in the genome of an rAAV.
  • RPIS reverse packaging inhibitory sequence
  • the present disclosure provides a polynucleotide comprising a nucleotide sequence as shown in Formula I VYHCC (I) ,
  • the polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3 and 4.
  • the present disclosure provides a vector comprising the polynucleotide of the present disclosure.
  • the vector is a transgene plasmid used for packaging a recombinant adeno-associated virus (rAAV) , or a backbone plasmid for the construction of the transgene plasmid (AAV shuttle plasmid) .
  • rAAV recombinant adeno-associated virus
  • AAV shuttle plasmid AAV shuttle plasmid
  • the vector comprises two or more copies of the polynucleotide.
  • the vector is a transgene plasmid used for packaging an rAAV, which comprises the genome of the rAAV, and the polynucleotide at 5’ and/or 3’ end of the genome.
  • the transgene plasmid comprises the genome of the rAAV, and the polynucleotides at 5’ and 3’ ends of the genome.
  • the polynucleotides at 5’ and 3’ ends of the genome comprise the same or different nucleotide sequence (s) .
  • the present disclosure provides a host cell comprising the polynucleotide or the vector of the present disclosure.
  • the present disclosure provides a method of producing rAAV with reduced DNA impurities in the genome, comprising transfecting a host cell with the transgene plasmid of the present disclosure, a packaging plasmid, and a helper plasmid.
  • the present disclosure provides an rAAV with reduced DNA impurities in the genome produced by the method of the present disclosure.
  • the present disclosure provides a pharmaceutical composition comprising the rAAV of the present disclosure.
  • Fig. 1 shows the structures of the genome of AAV2 at the 3’ end (A) and an exemplified transgene plasmid for packaging rAAV (B) , the rAAV comprises the RPIS-1 and RPIS-2 of the present disclosure, in which GOI is the gene of interest including the whole expression cassette comprising the coding sequence and regulatory elements.
  • Fig. 2 shows the designed constructs for testing the effect of the RPIS.
  • Fig. 3 shows the maps for the transgene plasmid (A) , the packaging plasmid (B and C) and the helper plasmid (D) .
  • Fig. 4 shows the effect of the RPIS on reducing DNA impurities by qPCR analysis of the rAAVs GCB204, GCB206, GCB207 and GCB208, left panel: the percentage of DNA impurities from the packaging and helper plasmids (Kana vs. transgene) , and right panel: the percentage of DNA impurities from the backbone of the transgene plasmid (ORI vs transgene) .
  • Fig. 5 shows the comparison of the effects achieved by various RPISs by qPCR analysis of the DNA impurities in the rAAVs GCB204 (lack of RPISs) , GCB208 (comprising RPIS-1 and RPIS-2) , and GCB607 (comprising RPIS-3 and RPIS-4) , left panel: the percentage of DNA impurities from the packaging and helper plasmids (Kana vs. transgene) , and right panel: the percentage of DNA impurities from the backbone of the transgene plasmid (ORI vs transgene) .
  • Fig. 6 shows the effect of the RPIS on reducing DNA impurities by qPCR analysis of the rAAVs SKG401, and SKG403 left panel: the percentage of DNA impurities from the packaging and helper plasmids, and right panel: the percentage of DNA impurities from the backbone of the transgene plasmid.
  • Fig. 7 shows the effect of the RPIS on reducing DNA impurities by sequencing the genome of the rAAVs GCB204, GCB206, GCB207 and GCB208.
  • Fig. 8 shows the FIX activity in HemB mice injected with the rAAVs GCB204, GCB206, GCB207 and GCB208.
  • Adeno-associated virus is a member of Parvoviridae family. It is a simple single-stranded DNA virus, and requires a helper virus (such as adenovirus) for replication.
  • the genome of a wildtype AAV contains approximately 4.7 kilobases (kb) , comprising the cap and rep genes between two inverted terminal repeat (ITR) sequences, approximately 145 nucleotides in length, with interrupted palindromic sequences that can fold into hairpin structures that function as primers during initiation of DNA replication.
  • ITR inverted terminal repeat
  • the cap gene encodes the viral capsid protein
  • the rep gene is involved in the replication and integration of AAV.
  • AAV can infect a variety of cells, and the viral DNA can be integrated into human chromosome 19 in the presence of the rep product.
  • ITRs inverted terminal repeats
  • AAV viral cis-elements named due to their symmetry. These elements are essential for efficient multiplication of an AAV genome.
  • ITR refers to ITRs of known natural AAV serotypes, to chimeric ITRs formed by the fusion of ITR elements derived from different serotypes, and to functional variant thereof.
  • Examples for the serotypes of AAV infecting human include, but are not limited to, human serotype 1 AAV (hAAV1) , hAAV2, hAAV3, hAAV4, hAAV5, hAAV6, hAAV7, hAAV8, hAAV9, hAAV10, and hAAV11.
  • the production of a recombinant AAV particle may involve three plasmids, a transgene plasmid comprising an expression construct for expressing an exogenous polynucleotide, a packaging plasmid encoding the REP and/or CAP proteins, and a helper plasmid.
  • the term “D sequence” refers to a segment of nucleotide sequence next to the terminal resolution site (trs) .
  • the D sequence is about 20 nucleotides in length, e.g., with a nucleotide sequence of SEQ ID NO: 5.
  • the D sequence is conserved between serotypes of AAVs (see Grimm et al., 2006, Liver Transduction with Recombinant adeno-associated virus is primarily restricted by capsid serotype not vector genotype, JOURNAL OFVIROLOGY, Vol. 80, No. 1, p. 426-439) .
  • expression construct refers to a single-stranded or double-stranded polynucleotide, which is isolated from a naturally occurring gene or modified to contain a nucleic acid segment that does not naturally occur.
  • the expression construct may contain the control sequences required to express the coding sequence of the present invention.
  • polynucleotide usually refers to generally a nucleic acid molecule (e.g., 100 bases and up to 30 kilobases in length) and a sequence that is either complementary (antisense) or identical (sense) to the sequence of a messenger RNA (mRNA) or miRNA fragment or molecule.
  • mRNA messenger RNA
  • miRNA fragment or molecule usually refers to DNA or RNA molecules that are either transcribed or non-transcribed.
  • exogenous polynucleotide refers to a nucleotide sequence that does not originate from the host in which it is placed. It may be identical to the host’s DNA or heterologous. An example is a sequence of interest inserted into a vector. Such exogenous DNA sequences may be derived from a variety of sources including DNA, cDNA, synthetic DNA, and RNA. Exogenous polynucleotides also encompass DNA sequences that encode antisense oligonucleotides.
  • expression includes any step involved in the production of a polypeptide, including but not limited to transcription, post-transcriptional modification, translation, post-translational modification, and secretion.
  • control sequence includes all elements necessary or beneficial for the expression of the polynucleotide encoding the polypeptide of the present invention.
  • Each control sequence may be natural or foreign to the nucleotide sequence encoding the polypeptide, or natural or foreign to each other.
  • control sequences include, but are not limited to, leader sequence, polyadenylation sequence, pro-peptide sequence, promoter, enhancer, signal peptide sequence, and transcription terminator. At a minimum, control sequences include a promoter and signals for the termination of transcription and translation.
  • control sequence may be a suitable promoter sequence, a nucleotide sequence recognized by the host cell to express the polynucleotide encoding the polypeptide of the present invention.
  • the promoter sequence contains a transcription control sequence that mediates the expression of the polypeptide.
  • the promoter may be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, for example, lac operon of E. coli.
  • the promoters also include mutant, modified and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides, which are homologous or heterologous to the host cell.
  • operably linked refers to a configuration in which a control sequence is placed at an appropriate position relative to the coding sequence of the polynucleotide sequence, whereby the control sequence directs the expression of the polypeptide coding sequence.
  • GLA and “ ⁇ -Gal A” are exchangeable when referring to a protein, and mean alpha galactosidase A.
  • the deficiency of GLA in a subject results in the Fabry disease, and thus, an rAAV expressing GLA can be used to treat the Fabry disease.
  • factor IX and “FIX” are exchangeable, and refer to the blood coagulation factor that can cleave inactive factor X in the presence of factor VIII, resulting in active factor X (FXa) .
  • FXa active factor X
  • the deficiency of FIX in a subject tresults in hemophilia B (HemB) , and thus, an rAAV expressing FIX can be used to treat HemB.
  • the polynucleotide encoding the protein to be expressed can be subjected to various manipulations to improve the expression of the polypeptide. Before the insertion thereof into a vector, manipulation of the polynucleotide according to the expression vector or the host, such as codon optimization, is desirable or necessary.
  • recombinant refers to nucleic acids, vectors, polypeptides, or proteins that have been generated using DNA recombination (cloning) methods and are distinguishable from native or wild-type nucleic acids, vectors, polypeptides, or proteins.
  • polypeptide and “protein” are used interchangeably herein and refer to a polymer of amino acids and includes full-length proteins and fragments thereof.
  • a “motif” means a set of nucleotides or amino acids that are essential for the structure, the stability, or the function of a polynucleotide or protein while nucleotides or amino acids at other positions can vary between variants of the polynucleotide or protein.
  • the term “host cell” refers to, for example microorganisms, yeast cells, insect cells, and mammalian cells, that can be, or have been, used as recipients of rAAV vectors.
  • the term includes the progeny of the original cell which has been transduced.
  • a “host cell” as used herein generally refers to a cell which has been transduced with an exogenous DNA sequence. It is understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to natural, accidental, or deliberate mutation.
  • pharmaceutically acceptable refers to molecular entities and compositions that are physiologically tolerable and do not typically produce toxicity or an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human.
  • subject includes, but is not limited to, humans, nonhuman primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
  • farm animals such as cattle, sheep, pigs, goats and horses
  • domestic mammals such as dogs and cats
  • laboratory animals including rodents such as mice, rats and guinea pigs, and the like.
  • rodents such as mice, rats and guinea pigs, and the like.
  • the term does not denote a particular age or sex. Thus, adult and newborn subjects, as well as fetuses, whether male or female, are intended to be covered.
  • the present disclosure provides a polynucleotide comprising a reverse packaging inhibitory sequence (RPIS) which is capable of reducing the packaging of the non-genomic nucleic acid impurities into an rAAV when it is present in the transgene plasmid, e.g., at one or both ends of the rAAV genome.
  • RPIS reverse packaging inhibitory sequence
  • D sequence is essential for the packaging of rAAV, and the introduction of alteration into the D sequence may result in erroneous packaging of plasmid backbone in rAAV (Zhang et al., ASGCT 2020) .
  • the RPIS of the present disclosure is designed according to the D sequence.
  • the RPIS is designed to be different from the D sequence in at least 5 positions, preferably at least 5 contiguous positions, more preferably the 5 positions at the 3’ end.
  • the RPIS of the present disclosure comprises a motif which is designed corresponding to at least the 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 positions from the 3’ end of the D sequence.
  • the motif is designed corresponding to positions 16-20, 15-20, 14-20, 13-20, 12-20, 11-20, 10-20, 9-20, 8-20, 7-20, or 6-20 of SEQ ID NO: 5.
  • the RPIS is designed according to the entire D sequence. The preferability of the RPIS increases with the difference between the RPIS and the D sequence.
  • the polynucleotide of the present disclosure comprises a nucleotide sequence as shown in Formula I VYHCC (I)
  • the polynucleotide of the present disclosure comprises a nucleotide sequence as shown in Formula VI AYWCC (VI)
  • the polynucleotide of the present disclosure comprises a nucleotide sequence as shown in Formula II CCCCMVYHCC (II)
  • the polynucleotide of the present disclosure comprises a nucleotide sequence as shown in Formula VII CCCCAAYWCC (VII)
  • the polynucleotide of the present disclosure comprises a nucleotide sequence as shown in Formula III AAAAMCCCCMVYHCC (III)
  • the polynucleotide of the present disclosure comprises a nucleotide sequence as shown in Formula VIII AAAAMCCCCAAYWCC (VIII)
  • the polynucleotide of the present disclosure comprises a nucleotide sequence as shown in Formula IV
  • N any nucleotide
  • Y C or T
  • W A or T
  • S G or C
  • M A or C
  • H A or C or T
  • V A or G or C.
  • the polynucleotide of the present disclosure comprises a nucleotide sequence as shown in Formula V
  • the polynucleotide of the present disclosure comprises the at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 positions from the 3’ end of a nucleotide sequence selected from SEQ ID NOs: 1, 2, 3 and 4, i.e., at least the nucleotides at positions 16-20, 15-20, 14-20, 13-20, 12-20, 11-20, 10-20, 9-20, 8-20, 7-20, or 6-20 of any of SEQ ID NOs: 1, 2, 3 and 4.
  • the polynucleotide of the present disclosure comprises the nucleotides at positions 16-20 of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3 and 4. In some embodiments, the polynucleotide of the present disclosure comprises the nucleotides at positions 11-20 of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3 and 4. In some embodiments, the polynucleotide of the present disclosure comprises the nucleotides at positions 6-20 of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3 and 4.
  • the polynucleotide of the present disclosure comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3 and 4.
  • the present disclosure provides a vector comprising a polynucleotide comprising the RPIS of the present disclosure.
  • the vector of the present disclosure comprises a polynucleotide comprising a nucleotide sequence as shown in Formula I VYHCC (I)
  • the polynucleotide comprises a nucleotide sequence as shown in Formula VI AYWCC (VI)
  • the polynucleotide comprises a nucleotide sequence as shown in Formula II CCCCMVYHCC (II)
  • the polynucleotide comprises a nucleotide sequence as shown in Formula VII CCCCAAYWCC (VII)
  • the polynucleotide comprises a nucleotide sequence as shown in Formula III AAAAMCCCCMVYHCC (III)
  • the polynucleotide comprises a nucleotide sequence as shown in Formula VIII AAAAMCCCCAAYWCC (VIII)
  • the polynucleotide comprises a nucleotide sequence as shown in Formula IV NMCCSAAAAMCCCCMVYHCC (IV)
  • N any nucleotide
  • Y C or T
  • W A or T
  • S G or C
  • M A or C
  • H A or C or T
  • V A or G or C.
  • the polynucleotide comprises a nucleotide sequence as shown in Formula V KACCGAAAAMCCCCAAYWCC (V)
  • the polynucleotide comprises the at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 positions from the 3’ end of a nucleotide sequence selected from SEQ ID NOs: 1, 2, 3 and 4, i.e., at least the nucleotides at positions 16-20, 15-20, 14-20, 13-20, 12-20, 11-20, 10-20, 9-20, 8-20, 7-20, or 6-20 of any of SEQ ID NOs: 1, 2, 3 and 4.
  • the polynucleotide comprises the nucleotides at positions 16-20 of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3 and 4. In some embodiments, the polynucleotide comprises the nucleotides at positions 11-20 of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3 and 4. In some embodiments, the polynucleotide comprises the nucleotides at positions 6-20 of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3 and 4.
  • the polynucleotide comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3 and 4.
  • the vector is the transgene plasmid for packaging an rAAV.
  • the vector comprises the polynucleotide of the present disclosure and a genome of rAAV.
  • the polynucleotide can be at the 5’ and/or 3’ ends of the genome.
  • the vector comprises, in the order of 5’ to 3’, a first polynucleotide, a genome of rAAV, and a second polynucleotide.
  • the first and second polynucleotides comprise the same or different nucleotide sequence (s) .
  • the vector is a vector providing the insert for the transgene plasmid, wherein the insert is a recombinant construct comprising the polynucleotide of the present disclosure and a genome of rAAV.
  • the polynucleotide can be at the 5’ and/or 3’ ends of the genome.
  • the recombinant construct comprises, in the order of 5’ to 3’, a first polynucleotide, a genome of rAAV, and a second polynucleotide.
  • the first and second polynucleotides comprise the same or different nucleotide sequence (s) .
  • the vector is a vector providing the backbone of the transgene plasmid.
  • the vector comprises a first polynucleotide, a second polynucleotide, and optionally a cloning site between the first and second polynucleotide.
  • the cloning site comprises one or more restriction sites.
  • the first and second polynucleotides comprise the same or different nucleotide sequence (s) .
  • the first and second polynucleotides comprise a nucleotide sequence as shown in Formula I VYHCC (I)
  • the first and second polynucleotides comprise a nucleotide sequence as shown in Formula VI AYWCC (VI)
  • the first and second polynucleotides comprise a nucleotide sequence as shown in Formula II CCCCMVYHCC (II)
  • the first and second polynucleotides comprise a nucleotide sequence as shown in Formula VII CCCCAAYWCC (VII)
  • the first and second polynucleotides comprise a nucleotide sequence as shown in Formula III AAAAMCCCCMVYHCC (III)
  • the first and second polynucleotides comprise a nucleotide sequence as shown in Formula VIII AAAAMCCCCAAYWCC (VIII)
  • the first and second polynucleotides comprise a nucleotide sequence as shown in Formula IV NMCCSAAAAMCCCCMVYHCC (IV)
  • N any nucleotide
  • Y C or T
  • W A or T
  • S G or C
  • M A or C
  • H A or C or T
  • V A or G or C.
  • the first and second polynucleotides comprise a nucleotide sequence as shown in Formula V KACCGAAAAMCCCCAAYWCC (V)
  • the first and second polynucleotides comprise the at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 positions from the 3’ end of a nucleotide sequence selected from SEQ ID NOs: 1, 2, 3 and 4, i.e., at least the nucleotides at positions 16-20, 15-20, 14-20, 13-20, 12-20, 11-20, 10-20, 9-20, 8-20, 7-20, or 6-20 of any of SEQ ID NOs: 1, 2, 3 and 4.
  • the first and second polynucleotides comprise the nucleotides at positions 16-20 of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3 and 4. In some embodiments, the first and second polynucleotides comprise the nucleotides at positions 11-20 of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3 and 4. In some embodiments, the first and second polynucleotides comprise the nucleotides at positions 6-20 of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3 and 4.
  • the first and second polynucleotides comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3 and 4.
  • An rAAV can be packaged by a system comprising a transgene plasmid comprising the genome of the rAAV, a packaging plasmid encoding the REP and/or CAP proteins, and a helper plasmid (see Crosson SM, et al., Helper-free Production of Laboratory Grade AAV and Purification by Iodixanol Density Gradient Centrifugation. Mol Ther Methods Clin Dev. 2018; 10: 1-7) .
  • the present disclosure provides a method of producing rAAV with reduced DNA impurities in the genome, comprising a step of transfecting a host cell with a vector of the present disclosure, together with a packaging plasmid encoding the REP and/or CAP proteins, and a helper plasmid.
  • the present disclosure further provides the host cell comprising or transfected with a vector of the present disclosure.
  • the vector comprises, in the order of 5’ to 3’, a first polynucleotide, a genome of rAAV, and a second polynucleotide.
  • the first and second polynucleotides comprise the same or different nucleotide sequence (s) .
  • the first and second polynucleotides comprise a nucleotide sequence as shown in Formula I VYHCC (I)
  • the first and second polynucleotides comprise a nucleotide sequence as shown in Formula VI AYWCC (VI)
  • the first and second polynucleotides comprise a nucleotide sequence as shown in Formula II CCCCMVYHCC (II)
  • the first and second polynucleotides comprise a nucleotide sequence as shown in Formula VII CCCCAAYWCC (VII)
  • the first and second polynucleotides comprise a nucleotide sequence as shown in Formula III AAAAMCCCCMVYHCC (III)
  • the first and second polynucleotides comprise a nucleotide sequence as shown in Formula VIII AAAAMCCCCAAYWCC (VIII)
  • the first and second polynucleotides comprise a nucleotide sequence as shown in Formula IV NMCCSAAAAMCCCCMVYHCC (IV)
  • N any nucleotide
  • Y C or T
  • W A or T
  • S G or C
  • M A or C
  • H A or C or T
  • V A or G or C.
  • the first and second polynucleotides comprise a nucleotide sequence as shown in Formula V KACCGAAAAMCCCCAAYWCC (V)
  • the first and second polynucleotides comprise the at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 positions from the 3’ end of a nucleotide sequence selected from SEQ ID NOs: 1, 2, 3 and 4, i.e., at least the nucleotides at positions 16-20, 15-20, 14-20, 13-20, 12-20, 11-20, 10-20, 9-20, 8-20, 7-20, or 6-20 of any of SEQ ID NOs: 1, 2, 3 and 4.
  • the first and second polynucleotides comprise the nucleotides at positions 16-20 of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3 and 4. In some embodiments, the first and second polynucleotides comprise the nucleotides at positions 11-20 of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3 and 4. In some embodiments, the first and second polynucleotides comprise the nucleotides at positions 6-20 of the nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3 and 4.
  • the first and second polynucleotides comprise a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3 and 4.
  • the method of the present disclosure can achieve a reduction of DNA impurities in the rAAV genome by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%as compared to the method with the transgene plasmid not comprising the RPIS of the present disclosure.
  • the DNA impurities are derived from the backbone of the transgene plasmid and/or from the packaging and/or helper plasmid.
  • the present disclosure provides an rAAV produced by the method as described above, wherein the rAAV comprises reduced DNA impurities in the genome.
  • the rAAV is of a serotype selected from the group consisting of hAAV1, hAAV2, hAAV3, hAAV4, hAAV5, hAAV6, hAAV7, hAAV8, hAAV9, hAAV10, and hAAV11.
  • the rAAV of the present disclosure comprises a reduced DNA impurities in the rAAV genome by 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%or 100%as compared to the rAAV produced with the transgene plasmid not comprising the RPIS of the present disclosure.
  • the DNA impurities are derived from the backbone of the transgene plasmid and/or from the packaging and/or helper plasmid.
  • compositions containing the AAV of the invention can be formulated in any conventional manner by mixing a selected amount of the rAAV with one or more pharmaceutically acceptable carriers or excipients.
  • the carrier or excipient is within the skill of the administering professional and can depend upon a number of parameters. These include, for example, the mode of administration (i.e., systemic, oral, local, topical or any other mode) and the disease to be treated.
  • Pharmaceutical carriers or vehicles suitable for administration of the rAAV provided herein include any such carriers known to those skilled in the art to be suitable for the particular mode of administration.
  • Transgene plasmids comprising a rAAV genome encoding FIX and hGLA, respectively, with or without RPISs were constructed by Gibson Assembly with a pGCB108 plasmid as the backbone.
  • the resulted transgene plasmids were shown in Table 1.
  • the structures of pGCB204-pGCB208 were shown in Fig. 2.
  • the maps of pGCB208, pSKG401 and pSKG403 were shown in Figs. 3A, 3B and 3C as examples.
  • 293 cells were cultured in DMEM medium (Thermo Fisher) containing 10%fetal bovine serum (FBS, Corning) .
  • HEK-293T cells were plated in Hyperflask culture flasks (Corning) two days before the transfection, so that the cells grew to 70%-90%confluence on the day of transfection.
  • helper plasmid pGCB011, see Fig. 3F
  • packaging plasmid pGCB005 for AAV5 (see Fig. 3D or pGCB009 for AAV9, see Fig.
  • the supernatant was collected in a tube, and mixed with 1/4 volume of 40%PEG8000 solution was added (final concentration of 8%PEG8000, 0.5M NaCl) , and the mixture was placed at 4°C overnight.
  • the rAAV in the supernatant was concentrated (by centrifugation, 3700g, 30min, 4°C) .
  • the adherent cells were trypsinized, and collected by centrifugation (3700g, 30min, 4°C) .
  • the pellet was resuspended in lysis buffer (50 mM Tris, 0.15 M NaCl, 2 mM MgCl2, pH 8) , and the suspension was placed in a -80°C freezer for freezing. Then, the rAAV in the cells was released by 3 cycles of freezing at -80°C and thawing at 37°C.
  • the concentrated virus suspension from the supernatant and the virus released from the cell lysis were mixed. Then, the nuclease (Benzonase, Sigma aldrich) were added, followed by the incubation at 37°C for 40 min, then 25%sodium deoxycholate solution was added, followed by the incubation at 37°C for 30 minutes. After centrifugation at 3, 700g for 15 minutes at 4°C, the obtained supernatant was subjected to subsequent iodixanol density gradient (15%iodixanol 5.5 mL, 25%iodixanol 5.5 mL, 40%iodixanol 4 mL, 60%iodixanol 5mL) centrifugation for purification.
  • iodixanol density gradient (15%iodixanol 5.5 mL, 25%iodixanol 5.5 mL, 40%iodixanol 4 mL, 60%iodixanol 5mL
  • the 40%iodixanol layer was carefully collected with 18G needle, followed by ultrafiltration and concentration using 50k Da Amicon Ultra-15 ultrafiltration tubes (Sigma aldrich) , the resulted rAAV was suspended in DPBS buffer containing 0.001%Pluronic TM F68 (PF68) .
  • the qPCR mixture (10 ⁇ L) comprised 5 ⁇ L of 2x TaqMan Universal PCR Master Mix (Thermo) , primers (0.2 ⁇ M of each) and probe (0.1 ⁇ M) and 4 ⁇ L template (see Table 3) .
  • qPCR was performed in a QuantStudio 5 PCR system with cycles of: 50°C for 2 minutes, 95°C for 10 minutes, 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.
  • Table 4 The titers of the rAAVs as measured by qPCR.
  • This Example was carried out to verify the effect of RPIS on reducing DNA impurities from the plasmids used in the packaging.
  • RPIS reduced the impurities from the packaging and helper plasmids (Fig. 4, left panel) and the backbone of the transgene plasmid (Fig. 4, right panel) in the genome of rAAVs (GCB206 and GCB208, which were AAV5) as compared to the rAAV GCB204 and GCB207, respectively, which did not comprise RPIS.
  • NGS next-generation sequencing
  • the genomic DNA of the rAAVs GCB204, GCB206, GCB207 and GCB208 was extracted using Qiagen DNeasy Blood &Tissue Kits according to the instructions. 10 ⁇ g of genomic DNA from each of the rAAVs was provided for sequencing by GENEWIZ, Inc. (Suzhou, China) and the data were also analyzed by GENEWIZ, Inc.
  • the single-stranded DNA was complemented to form double-stranded DNA by 3’ ITR extension.
  • the double-stranded DNA was then subjected to fragmentation into 200-300bp, and the adapter was added to the ends of the fragments to generate the library, followed by sequencing on the Novaseq 2x150bp platform (Illumina) . 10.0 Gb of data were detected for each sample.
  • the proportion of cis-packaging fragments, trans-packaging fragments, helper plasmid fragments in each rAAV genome were analyzed upon alignment of the sequencing data.
  • the rAAVs GCB206 and GCB208 comprising RPIS showed lower percentage of reads aligned with the backbone of the transgene plasmid, the packaging plasmid and the helper plasmid, and higher percentage of reads aligned with ITR-ITR, than rAAVs GCB204 and GCB207 which did not comprise RPIS.
  • the percentage of the reads from various source are shown in Table 6.
  • FIX activity in the plasma was detected using Coatest SP FIX kit (Dipharma, Bedford, USA) according to the instructions.
  • the standard was diluted to 1.2-100%with the sample diluting solution (mixed with 0.5%plasma from HemB model) for generating the standard curve.
  • the tested samples were diluted at a ratio of 1: 200.25 ⁇ l of sample or standard diluent was incubated at 37°C for 5min, followed by the addition of 25 ⁇ l of CaCl 2 (pre-heated at 37°C) . The mixture was incubated at 37°C for 5 min.
  • FIX activity was calculated based on the absorbance at 405 nm and 490 nm. A standard curve was generated based on the FIX activities shown by the standard diluents, and the FIX activities in the plasma were according to the standard curve.
  • mice injected with the rAAVs comprising RPIS showed higher FIX activities in the plasma than the mice injected with rAAVs without RPIS (GCB204 and GCB207) .

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Abstract

La présente invention concerne un polynucléotide capable de réduire les impuretés d'ADN dans le génome d'un rAAV pendant l'emballage et un vecteur comprenant le polynucléotide. La présente invention concerne également un procédé de production de rAAV à impuretés d'ADN réduites dans le génome.
PCT/CN2023/110182 2022-08-01 2023-07-31 Nouveau squelette plasmidique pour réduire les impuretés d'adn dans la préparation de raav Ceased WO2024027632A1 (fr)

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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011133890A1 (fr) * 2010-04-23 2011-10-27 University Of Massachusetts Vecteurs aav ciblant le système nerveux central et leurs procédés d'utilisation
US20190071671A1 (en) * 2016-03-18 2019-03-07 The Children's Hospital Of Philadelphia Therapeutic for treatment of diseases including the central nervous system
US20210317474A1 (en) * 2017-11-08 2021-10-14 Novartis Ag Means and method for producing and purifying viral vectors

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011133890A1 (fr) * 2010-04-23 2011-10-27 University Of Massachusetts Vecteurs aav ciblant le système nerveux central et leurs procédés d'utilisation
US20190071671A1 (en) * 2016-03-18 2019-03-07 The Children's Hospital Of Philadelphia Therapeutic for treatment of diseases including the central nervous system
US20210317474A1 (en) * 2017-11-08 2021-10-14 Novartis Ag Means and method for producing and purifying viral vectors

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
CROSSON SM ET AL.: "Helper-free Production of Laboratory Grade AAV and Purification by Iodixanol Density Gradient Centrifugation", MOL THER METHODS CLIN DEV, vol. 10, 2018, pages 1 - 7
PASI ET AL.: "Multiyear Follow-up of AAV5-hFVIII-SQ Gene Therapy for Hemophilia A", NEJM, vol. 382, 2020, pages 29 - 40
RANGARAJAN ET AL.: "AAVS-Factor VIII Gene Transfer in Severe Hemophilia A", NEJM, vol. 377, 2017, pages 2519 - 2530
SEE GRIMM ET AL.: "Liver Transduction with Recombinant adeno-associated virus is primarily restricted by capsid serotype not vector genotype", JOURNAL OFVIROLOGY, vol. 80, no. 1, 2006, pages 426 - 439, XP002453976, DOI: 10.1128/JVI.80.1.426-439.2006

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