WO2022213745A1 - 制备重组腺相关病毒的辅助质粒及其应用 - Google Patents

制备重组腺相关病毒的辅助质粒及其应用 Download PDF

Info

Publication number
WO2022213745A1
WO2022213745A1 PCT/CN2022/078621 CN2022078621W WO2022213745A1 WO 2022213745 A1 WO2022213745 A1 WO 2022213745A1 CN 2022078621 W CN2022078621 W CN 2022078621W WO 2022213745 A1 WO2022213745 A1 WO 2022213745A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
sequences
helper plasmid
promoter
rep
Prior art date
Application number
PCT/CN2022/078621
Other languages
English (en)
French (fr)
Inventor
李华鹏
刘嘉
谢婉雯
Original Assignee
广州派真生物技术有限公司
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 广州派真生物技术有限公司 filed Critical 广州派真生物技术有限公司
Publication of WO2022213745A1 publication Critical patent/WO2022213745A1/zh
Priority to US18/377,827 priority Critical patent/US20240076319A1/en

Links

Images

Classifications

    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C07K14/01—DNA viruses
    • C07K14/015—Parvoviridae, e.g. feline panleukopenia virus, human parvovirus
    • 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
    • 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
    • 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
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing 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
    • 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
    • C12N15/861—Adenoviral 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
    • 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
    • C12N15/864—Parvoviral vectors, e.g. parvovirus, densovirus
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • 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
    • C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
    • 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
    • C12N2750/14122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • 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
    • C12N2750/14151—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
    • 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
    • C12N2800/00—Nucleic acids vectors
    • C12N2800/10—Plasmid DNA
    • C12N2800/106—Plasmid DNA for vertebrates
    • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

  • the present invention relates to genetic engineering and AAV production in human cells, in particular to a new type of AAV packaging auxiliary plasmid and its application in AAV production.
  • rAAV recombinant adeno-associated virus
  • the three-plasmid transfection method is simple, rapid, and widely used for AAV production.
  • the method involves co-transfection of HEK293 cells with three plasmids: a helper plasmid, pADhelper, which provides the four adenoviral elements; and a helper plasmid, pRC, which provides the coding sequences for the Rep and Cap proteins of AAV, where the Rep protein is responsible for the replication and assistance of the AAV genome.
  • the Cap protein constitutes the AAV shell; a plasmid including the target sequence, which can be referred to as pGOI for short, has a 5'ITR and 3'ITR sequence in the natural AAV genome upstream and downstream of the target sequence.
  • the coding sequence in the pRC plasmid of an AAV serotype X consists of Rep2 from AAV2 and the coat protein CapX encoding AAVX.
  • the P5 promoter is the key promoter for the expression of Rep and Cap.
  • the P5 promoter is placed in RepCap.
  • RepCap Before encoding, as shown in "P5+RC" in Figure 2.
  • X. Xiao et al. have reported that placing a P5 promoter in the upstream and downstream of RC can increase the yield of AAV [1].
  • the ITR placed upstream and downstream of the target gene (or target sequence), whose single sequence is 145 bp in full length, is considered to be related to the replication and guide packaging of the AAV genome. Because there is a reverse self-complementary sequence in the ITR, it will self-complement to form a T-shaped double-stranded structure in the case of single-stranded DNA.
  • T.R.Flotte et al. have found that the complete ITR has promoter activity and drives the expression of the CFTR gene [2]. However, in terms of driving gene expression, ITRs have very low transcriptional activity compared to traditional promoters [3].
  • helper plasmid for preparing a recombinant adeno-associated virus comprising: 1) Rep and Cap protein coding sequences of AAV; 2) at least one promoter sequence; and 3) at least one DA' sequence or The reverse complement of the DA' sequence AD' sequence.
  • the at least one DA' or AD' sequence is located upstream of the Rep and Cap protein coding sequences.
  • the at least one DA' or AD' sequence is located downstream of the Rep and Cap protein coding sequences.
  • helper plasmids in order from 5' to 3', include:
  • the promoter is a P5 promoter.
  • the P5 promoter sequence comprises the sequence set forth in SEQ ID NO:2 or a functional variant having at least 90% sequence identity with the sequence set forth in SEQ ID NO:2.
  • the DA' sequence comprises the sequence set forth in SEQ ID NO:4 or is a functional variant having at least 90% sequence identity to the sequence set forth in SEQ ID NO:4.
  • the AD' sequence comprises the sequence set forth in SEQ ID NO:3 or is a functional variant having at least 90% sequence identity to the sequence set forth in SEQ ID NO:3.
  • the Rep and Cap proteins are from adeno-associated virus type 2.
  • provided herein is a method for increasing the production capacity of recombinant adeno-associated virus, comprising transforming a host cell with the helper plasmid described above.
  • the present invention proposes a class of auxiliary packaging plasmids encoding Rep/Cap (including all its serotypes), which can significantly increase the AAV yield of single cells when AD' or DA' sequences are added.
  • Figure 1 is a schematic diagram of the ITR structure of the AAV genome.
  • Figure 2 is a schematic diagram of the experimental design for studying the functions of DA' and AD' in the Examples.
  • Figure 3 shows the comparison results of the viral titers in the lysates of each group of plasmids tested in the Examples and the control vector.
  • Adeno-associated virus was first discovered in adenovirus (AdV) preparations and later found in human tissues. It belongs to the genus Dependoparvovirus of the family Parvoviridae, is a defective virus with a simple structure, and requires the participation of a helper virus (eg, adenovirus) for replication. Adeno-associated viruses are currently believed to not cause any human disease, which is the basis for their adaptation for gene therapy.
  • the genome of adeno-associated virus is about 4.7kb, which is a single-stranded DNA molecule, including two reading frames (rep gene and cap gene) and two inverted terminal repeats (inverted terminal repeat, ITR) located at the end of the genome.
  • the rep gene encodes multiple proteins (Rep78, Rep68, Rep52, and Rep40) for viral replication
  • the cap gene encodes three subunits of the capsid protein (VP1, VP2, and VP3).
  • ITRs play a key role in adeno-associated virus replication and packaging, and are involved in the integration and escape of viral genomes into the host genome.
  • the ITR sequence (usually 145 bp) can form a T-shaped structure. As shown in Figure 1, based on the position on the T-shaped structure, the ITR sequence can be divided into multiple segments such as A, B, B', C', C, A', D and D. A and A' are complementary in sequence, B and B' are complementary, C and C' are complementary, and D and D' are complementary.
  • the ITR sequence includes two arm palindromes (B-B' and C-C') and one long stem palindrome (A-A').
  • the D' region is present at the 5' end of the AAV genome, and the D segment is present at the 3' end.
  • Recombinant adeno-associated virus refers to an adeno-associated virus obtained by improving the genome of wild-type adeno-associated virus. It usually has the capsid protein and structure of wild-type AAV, but the protein-coding sequences (rep and cap genes) in the genome are replaced by the gene of interest (eg, a therapeutic gene), leaving only the ITR sequences at both ends.
  • rAAV Recombinant adeno-associated virus
  • Helper plasmid a helper plasmid for the preparation of rAAV.
  • transfection of host cells typically requires co-transfection of three plasmids: the helper plasmid pADhelper, which provides the four adenovirus elements; and the helper plasmid pRC, which provides the coding sequences for the Rep and Cap proteins of the AAV.
  • the Rep protein is responsible for the replication of the AAV genome and assists in the assembly of the AAV genome particles
  • the Cap protein forms the AAV coat; and the plasmid including the target sequence (eg, a therapeutic gene) can be abbreviated as pGOI.
  • AD' sequence refers to the sequence formed by the combination of the A segment and the D' segment described above.
  • the AD' sequence has the sequence shown in SEQ ID NO:3.
  • DA' sequence refers to the sequence formed by the combination of the above-mentioned D segment and A' segment.
  • the DA' sequence has the sequence shown in SEQ ID NO:4.
  • AD' sequences and DA' sequences are used interchangeably, eg when reference to an AD' sequence is to be understood as a reference to a DA' sequence as well.
  • Sequence Identity When referring to a nucleotide sequence, the term “Sequence identity” (also referred to as “sequence identity”) refers to the identity between two nucleotide sequences (eg, a query sequence and a reference sequence) The amount of degree, usually expressed as a percentage. Typically, prior to calculating the percent identity between two nucleotide sequences, the sequences are aligned and gaps (if any) are introduced. If at a certain alignment position, the bases in the two sequences are the same, the two sequences are considered to be identical or matched at that position; if the bases in the two sequences are different, they are considered to be inconsistent or mismatched at that position.
  • the number of matched positions is divided by the total number of positions in the alignment window to obtain sequence identity. In other algorithms, the number of gaps and/or the gap length are also taken into account.
  • the published alignment software BLAST available at ncbi.nlm.nih.gov
  • BLAST can be used to obtain optimal sequence alignments and calculate the difference between two nucleotide sequences by using default settings. sequence identity between.
  • the pRC helper plasmids provided herein may include the P5 promoter sequence that includes the sequence set forth in SEQ ID NO:2.
  • the P5 promoter sequence may be a functional variant having at least 90% (eg, at least 95% or at least 98%) sequence identity to the sequence set forth in SEQ ID NO:2.
  • the DA' sequence included in the pRC helper plasmids provided herein may include the sequence set forth in SEQ ID NO:4. In other embodiments, the DA' sequence is a functional variant having at least 90% (eg, at least 92%, at least 95%, or at least 98%) sequence identity to the sequence set forth in SEQ ID NO:4. In some embodiments, the AD' sequence included in the pRC helper plasmids provided herein may include the sequence set forth in SEQ ID NO:3. In other embodiments, the AD' sequence is a functional variant having at least 90% (eg, at least 92%, at least 95%, or at least 98%) sequence identity to the sequence set forth in SEQ ID NO: 3
  • All pRC series vectors involved in the present invention are obtained by conventional molecular cloning methods.
  • the production of rAAV can be significantly improved by the combined use of the P5 promoter and the DA' or AD' sequence.
  • Rep2Cap2 candidate helper vectors were constructed by conventional molecular cloning methods based on the backbone vector (SEQ ID NO: 1) by designing a combination of the number and placement of multiple DA' sequences or AD' and P5 promoters ( Figure 2). ), and then parallelly compared the toxigenic titers of each helper vector in the general three-plasmid transfection system.
  • the pRC and pAdHleper auxiliary vector (vector source: UPENN vector core PL-F-PVADF6 of the University of Pennsylvania Vector Department) and the AAV plasmid pAAV.CAG.EGFP vector of the fluorescent protein gene driven by the CAG promoter are 0.5 ⁇ g: 0.5 ⁇ g : 0.5 ⁇ g was added to 0.5 mL of DMEM, then 3 ⁇ L of PEI (1 ⁇ g/ ⁇ L) was added, mixed immediately, placed at room temperature for 10 minutes, the medium in the 24-well plate was aspirated, added to the transfection mixed medium and put back into 37 °C cell incubator (5% CO 2 concentration) culture;
  • the primers FWD ITR (5'-GGAACCCCTAGTGATGGAGTT) (SEQ ID NO: 5), REV ITR (5'-CGGCCTCAGTGAGCGA) (SEQ ID NO: 6) were used to specifically detect the sequence of all types of AAV vector ITRs.
  • reaction system components are listed in the table below.
  • Reagent X1 Nuclease-free Water 15.8 ⁇ L 10x DNase Buffer 2 ⁇ L DNase I, RNase free l (70U/ ⁇ L) (purchased from Roch) 0.2 ⁇ L Sample 2 ⁇ L
  • Standard preparation Take the plasmid standard containing 2E+8AAV copies/ ⁇ L and make 6 serial dilutions with 8 ⁇ L+72 ⁇ L water. Then the first gradient concentration is 2E+8GC/ ⁇ L, and its concentration in the software is set to 8E+14GC/mL to reflect the dilution gradient of the sample.
  • the gradients of the latter series were 8E+13GC/mL, 8E+12GC/mL, 8E+11GC/mL, 8E+10GC/mL, 8E+9GC/mL.
  • reaction system components are listed in the table below.
  • Cycle 40cycle: 95°C for 15sec; 60°C for 1min
  • helper plasmid of a specific serotype (Rep2Cap2) is used.
  • Rep2Cap2 specific serotype
  • the present invention is not limited to this specific serotype, but can utilize other serotypes currently known and may continue to be discovered in the future. helper plasmids to practice the present invention.
  • nucleic acid sequences mentioned herein are as follows.
  • Plasmid backbone sequence (SEQ ID NO:1)

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Immunology (AREA)
  • Cell Biology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Saccharide Compounds (AREA)

Abstract

提供了一种用于制备重组腺相关病毒的辅助质粒，其包括：1)AAV的Rep和Cap蛋白编码序列；2)至少一个启动子序列；以及3)至少一个DA'序列或DA'序列的反向互补序列AD'序列。还提供了该辅助质粒在提高重组腺相关病毒生产能力方面的应用。在加入AD'或DA'序列的情况下，提供的辅助质粒可显著提升细胞的AAV产量。

Description

制备重组腺相关病毒的辅助质粒及其应用
相关申请
本申请要求于2021年4月8日提交至中国专利局，申请号为202110377907.X的中国专利申请的优先权，在此通过引用将其全文并入本文。
技术领域
本发明涉及基因工程和在人源细胞中的AAV生产，具体涉及一类新型的AAV包装辅助质粒，及其在AAV生产中的应用。
背景技术
目前，基因治疗从四十年前的科学家的梦想正逐步变成了现实。多种病毒、非病毒载体经过多年的演变、淘汰，目前重组腺相关病毒(rAAV)载体是公认为符合安全性、有效性、特异性三个标准的基因治疗载体。但是，由于业界大规模生产AAV的能力有限，因而临床应用的普及程度也受此制约。
因此，AAV的产量瓶颈亟待打破。在生产上游改进、创新，提高AAV产率是目前降低目前基因治疗生产成本的关键点之一。目前，三质粒转染法简单、快速，是被广泛采用的AAV生产方法。该方法涉及使用三个质粒共转染HEK293细胞：一个辅助质粒pADhelper，提供四个腺病毒元件；一个辅助质粒pRC，提供AAV的Rep和Cap蛋白编码序列，其中Rep蛋白负责AAV基因组的复制与协助AAV基因组颗粒的组装，Cap蛋白组成AAV外壳；一个包括目的序列的质粒，可简称为pGOI，目的序列的上下游各有一个天然AAV基因组中的5’ITR和3’ITR序列。
通常情况下，一个AAV血清型X的pRC质粒中编码序列由来自AAV2的Rep2和编码AAVX的外壳蛋白CapX组成，P5启动子是启动Rep、Cap表达的关键启动子，通常P5启动子置于RepCap编码前，如图2中“P5+RC”所示。X.Xiao等人曾报道在RC的上下游各放置一个P5启动子，可以提高AAV的产率[1]。在目的质粒pGOI中，置于目的基因(或者目的序列)的上下游的ITR，其单个序列全长145bp，被认为与AAV基因组的复制与引导包装相关。因为ITR中存在反向自互补序列，因此在单链DNA的状况下会自互补形成T形的双链结构。T.R.Flotte等人曾发现完整的ITR具有启动子活性，驱动CFTR基因的表达[2]。但是，就驱动基因表达而言，ITR与传统启动子相比，仅具有非常低的转录活性[3]。
发明内容
一方面，本文提供了一种用于制备重组腺相关病毒的辅助质粒，其包括：1)AAV的Rep和Cap蛋白编码序列；2)至少一个启动子序列；以及3)至少一个DA’序列或DA’序列的反向互补序列AD’序列。
在一些实施方案中，所述至少一个DA’或AD’序列位于Rep和Cap蛋白编码序列的上游。
在一些实施方案中，所述至少一个DA’或AD’序列位于所述Rep和Cap蛋白编码序列的下游。
在一些实施方案中，所述辅助质粒从5’至3’方向依次包括：
i)DA’或AD’序列、启动子序列以及Rep和Cap蛋白编码序列；
ii)DA’或AD’序列、启动子序列、Rep和Cap蛋白编码序列以及DA’或AD’序列；
iii)DA’或AD’序列、启动子序列、Rep和Cap蛋白编码序列、DA’或AD’以及启动子序列；或
iv)Rep和Cap蛋白编码序列、DA’或AD’序列以及启动子序列。
在一些实施方案中，所述启动子为P5启动子。
在一些实施方案中，所述P5启动子序列包括SEQ ID NO：2所示序列或与SEQ ID NO：2所示序列具有至少90％序列一致性的功能性变体。
在一些实施方案中，所述DA’序列包括SEQ ID NO：4所示序列或为与SEQ ID NO：4所示序列具有至少90％序列一致性的功能性变体。
在一些实施方案中，所述AD’序列包括SEQ ID NO：3所示序列或为与SEQ ID NO：3所示序列具有至少90％序列一致性的功能性变体。
在一些实施方案中，所述Rep和Cap蛋白来自2型腺相关病毒。
另一方面，本文提供了一种提高重组腺相关病毒生产能力的方法，其包括使用上述辅助质粒转化宿主细胞。
本发明提出一类编码Rep/Cap(包括其所有血清型)辅助包装质粒，在加入AD’或DA’序列的情况下，可显著提升单细胞的AAV产量。
附图说明
图1为AAV基因组ITR结构示意图。
图2为实施例中研究DA’和AD’功能的实验设计示意图。
图3显示了实施例中检测的各组质粒的裂解液病毒滴度与对照载体的比较结果。
具体实施方式
除非另有说明，本文使用的所有技术和科学术语具有本领域普通技术人员所通常理解的含义。
腺相关病毒(adeno-associated virus,AAV)：腺相关病毒最早在腺病毒(AdV)制剂中被发现，之后也在人体组织中找到。其属于微小病毒科(Parvoviridae)依赖病毒属(Dependoparvovirus)，为结构简单的缺陷型病毒，复制需要辅助病毒(例如腺病毒)的参与。目前认为腺相关病毒不会导致任何人类疾病，这也是将其改造而用于基因治疗的基础。腺相关病毒的基因组大概为4.7kb，为单链DNA分子，包括两个阅读框(rep基因和cap基因)和位于基因组末端的两个反向末端重复(inverted terminal repeat,ITR)。rep基因编码用于病毒复制的多个蛋白(Rep78、Rep68、Rep52和Rep40)，cap基因则编码衣壳蛋白的三个亚基(VP1、VP2和VP3)。ITR在腺相关病毒的复制和包装过程中起着关键作用，并且参与了病毒基因组在宿主基因组上的整合和逃逸过程。ITR序列(通常145bp)可形成T型结构。如图1所示，基于在T型结构上的位置，可将ITR序列划分出A、B、B’、C’、C、A’、D和D等多个区段。在序列上A和A’互补，B和B’互补，C和C’互补，D和D’互补。从结构上看，ITR序列包括两个臂回文(B-B’和C-C’)和一个长茎回文(A-A’)。D’区存在AAV基因组5’端，D区段存在于在3’端。
重组腺相关病毒(rAAV)：指对野生型腺相关病毒的基因组进行了改进而获得的腺相关病毒。其通常具有野生型AAV的衣壳蛋白和结构，但基因组中的蛋白编码序列(rep和cap基因)被目的基因(例如治疗性基因)所替换，仅保留了两端的ITR序列。rAAV的一个重要用途是作为载体用于基因治疗。
辅助质粒：为用于制备rAAV的辅助质粒。为了产生rAAV，转染宿主细胞(如哺乳动物或昆虫细胞)时通常需要共转染三个质粒：辅助质粒pADhelper，提供四个腺病毒元件；辅助质粒pRC，提供AAV的Rep和Cap蛋白编码序列，其中Rep蛋白负责AAV基因组的复制与协助AAV基因组颗粒的组装，Cap蛋白形成AAV外壳；以及包括目的序列(例如治疗基因)的质粒，可简称为pGOI。
AD’序列：指上文所述A区段和D’区段组合形成的序列。在一个具体实例中，AD’序列具有SEQ ID NO:3所示序列。
DA’序列：指上文所述D区段和A’区段组合形成的序列。在一个具体实例中，DA’序列具有SEQ ID NO:4所示序列。
在本文描述的实施方案中，除非另有说明，AD’序列和DA’序列可互换使用，例如当提及AD’序列时，应理解为也同时提及DA’序列。
序列一致性：提及核苷酸序列时，术语“序列一致性(Sequence identity)”(也称为”序列同一性”)指两核苷酸序列(例如查询序列和参照序列)之间一致性程度的量，一般以百分比表示。通常，在计算两核苷酸序列之间的一致性百分比之前，先进行序列比对(alignment)并引入缺口(gap)(如果有的话)。如果在某个比对位置，两序列中的碱基相同，则认为两序列在该位置一致或匹配；两序列中的碱基不同，则认为在该位置不一致或错配。在一些算法中，用匹配位置数除以比对窗口中的位置总数以获得序列一致性。在另一些算法中，还将缺口数量和/或缺口长度考虑在内。出于本发明的目的，可以采用公 开的比对软件BLAST(可在网页ncbi.nlm.nih.gov找到)，通过使用缺省设置来获得最佳序列比对并计算出两核苷酸序列之间的序列一致性。
功能性变体：指与作为比较基础的核酸分子相比，在序列上具有至少一个核苷酸变化(包括替换、增加或删除)，但这种变化后的核酸分子基本上保留了其原来的生物学功能。在给出示例性核酸分子序列后，本领域技术人员能够通过常规实验方法寻求获得这些功能性变体。因此，一些实施方案中，本文提供的pRC辅助质粒包括的P5启动子序列可包括SEQ ID NO：2所示序列。在另一些实施方案中，该P5启动子序列可为与SEQ ID NO：2所示序列具有至少90％(例如至少95％或至少98％)序列一致性的功能性变体。在一些实施方案中，本文提供的pRC辅助质粒包括的DA’序列可包括SEQ ID NO：4所示序列。在另一些实施方案中，该DA’序列为与SEQ ID NO：4所示序列具有至少90％(例如至少92％、至少95％或至少98％)序列一致性的功能性变体。在一些实施方案中，本文提供的pRC辅助质粒包括的AD’序列可包括SEQ ID NO：3所示序列。在另一些实施方案中，该AD’序列为与SEQ ID NO：3所示序列具有至少90％(例如至少92％、至少95％或至少98％)序列一致性的功能性变体
本发明中涉及的全部pRC系列载体均通过常规的分子克隆方法获得。
在本文提供的pRC辅助质粒中，通过P5启动子和DA’或AD’序列的联合使用，可显著提供rAAV的产量。
以下通过具体实施例来进一步阐述本发明。
实施例
通过设计含有多种DA’序列或AD’和P5启动子的数量和放置位置的组合，通过常规的分子克隆方法基于骨架载体(SEQ ID NO：1)构建出不同的Rep2Cap2候选辅助载体(图2)，再平行比较各辅助载体在通用的三质粒转染体系中的产毒滴度。
三质粒转染生产AAV方法简要步骤：
1)铺293T细胞(293T，来源于
Figure PCTCN2022078621-appb-000001
CRL-3216 TM)约3x10 5至24孔板中，使用高糖DMEM培养基，含10％胎牛血清、2mM L-glutamine(ATCC 30-2214)、1％Penicillin/Streptomycin，在37℃，5％CO 2条件下培养约16小时，转染时细胞密度约为60～70％；
2)把pRC与pAdHleper辅助载体(载体来源：美国宾夕法尼亚大学载体部UPENN vector core PL-F-PVADF6)及CAG启动子驱动的荧光蛋白基因的AAV质粒pAAV.CAG.EGFP载体按0.5μg：0.5μg：0.5μg加入0.5mL DMEM中，再加入PEI 3μL(1μg/μL)，马上混匀，常温下放置10分钟后，吸去24孔板中培养基，加入转染混合培养基中再放回37℃细胞培养箱(5％CO 2浓度)培养；
3)培养72小时后收集细胞和上清，在37℃水浴和干冰-乙醇浴中反复冻融三次(解冻与冻结每次各2分钟)，10000g离心10分钟后上清为AAV粗提液。
AAV滴度测定方法：
使用引物FWD ITR(5'-GGAACCCCTAGTGATGGAGTT)(SEQ ID NO：5)，REV ITR(5'-CGGCCTCAGTGAGCGA)(SEQ ID NO：6)特异性检测所有类型AAV载体ITR的序列。
(1)Dnase I消化样品
反应体系成分列在下表中。
Reagent X1
Nuclease-free Water 15.8μL
10x DNase Buffer 2μL
DNase I,RNase free l(70U/μL)(购买自Roch) 0.2μL
Sample 2μL
取5μL样品，稀释20倍。分相应数目的PCR管，每管18μL消化液，分别加入2μL稀释后样品、和2μL用于制作标准曲线的质粒标准品(含4E+8AAV拷贝(Genome Copies,GC)，作为DNase消化对照)，相当于稀释10倍，37℃孵育30min。消化后，取5μL样品加入95μL水中，连续稀释2次，共计稀释80000倍，RefAAV(质粒标准品)共计稀释4000倍。
(2)SYBR GreenqPCR
标准品准备：取含2E+8AAV拷贝数/μL的质粒标准品，按8μL+72μL水做6个连续稀释。则第一个梯度浓度为2E+8GC/μL，将其在软件中浓度设置为8E+14GC/mL，以反映样品的稀释梯度。后面系列的梯度依次为8E+13GC/mL，8E+12GC/mL，8E+11GC/mL，8E+10GC/mL，8E+9GC/mL。
反应体系成分列在下表中。
Reagent Vol.per reaction
SYBR PCR试剂(2X) 10μL
ROX(50X) 0.4
FWD ITR(50μM) 0.1μL
REV ITR(50μM) 0.1μL
Nuclease-free water 4.4μL
样品DNA 5μL
按每个样品做3个重复孔计算，配制相应体积的混合物，每孔分18μL，再各自加入2μL样品。
(3)SYBR Green qPCR条件
预变性:95℃10min
循环:40cycle：95℃15sec；60℃1min
全部滴度检测结果转换为与对照载体P5+RC的倍数关系后如图3所示。结果表明，与单独的P5启动子驱动的Rep2Cap2载体“P5+RC”相比，含有DA’或AD’序列的辅助质粒具有增加rAAV病毒产量的作用。
实施例中采用了具体血清型(Rep2Cap2)的辅助质粒，本领域技术人员应理解的是，本发明并不限于该具体血清型，而是可利用目前已知和未来可能继续发现的其他血清型辅助质粒来实施本发明。
本文提及的一些核酸序列如下。
质粒骨架序列(SEQ ID NO:1)
Figure PCTCN2022078621-appb-000002
Figure PCTCN2022078621-appb-000003
P5启动子序列(SEQ ID NO:2)
Figure PCTCN2022078621-appb-000004
AD’序列(SEQ ID NO:3)
Figure PCTCN2022078621-appb-000005
DA’序列(SEQ ID NO:4)
Figure PCTCN2022078621-appb-000006
参考文献：
1.Xiao X,Li J,Samulski RJ.Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.J Virol.1998 Mar；72(3):2224-32.doi:10.1128/JVI.72.3.2224-2232.1998.PMID:9499080；PMCID:PMC109519.
2.Flotte TR,Afione SA,Solow R,Drumm ML,Markakis D,Guggino WB,Zeitlin PL,Carter BJ.Expression of the cystic fibrosis transmembrane conductance regulator from a novel adeno-associated virus promoter.J Biol Chem.1993 Feb 15；268(5):3781-90.PMID:7679117.
3.Zhang L,Wang DH,Fischer H,Fan PD,Widdicombe JH,Kan YW,Dong JY.Efficient expression of CFTR function with adeno-associated virus vectors that carry shortened CFTR genes.PNAS.1998 August 18；95(17)10158-10163.

Claims (10)

  1. 用于制备重组腺相关病毒的辅助质粒，包括：
    1)AAV的Rep和Cap蛋白编码序列；
    2)至少一个启动子序列；以及
    3)至少一个DA’序列或DA’序列的反向互补序列AD’序列。
  2. 如权利要求1所述的辅助质粒，其中至少一个DA’或AD’序列位于Rep和Cap蛋白编码序列的上游。
  3. 如权利要求1或2所述的辅助质粒，其中至少一个DA’或AD’序列位于所述Rep和Cap蛋白编码序列的下游。
  4. 如权利要求1-3任一项所述的辅助质粒，其中，所述辅助质粒从5’至3’方向依次包括：
    i)DA’或AD’序列、启动子序列以及Rep和Cap蛋白编码序列；
    ii)DA’或AD’序列、启动子序列、Rep和Cap蛋白编码序列以及DA’或AD’序列；
    iii)DA’或AD’序列、启动子序列、Rep和Cap蛋白编码序列、DA’或AD’序列以及启动子序列；或
    iv)Rep和Cap蛋白编码序列、DA’或AD’序列以及启动子序列。
  5. 如权利要求1-4任一项所述的辅助质粒，其中所述启动子为P5启动子。
  6. 如权利要求1-5任一项所述的辅助质粒，其中所述P5启动子序列包括SEQ ID NO：2所示序列或与SEQ ID NO：2所示序列具有至少90％序列一致性的功能性变体。
  7. 如权利要求1-6任一项所述的辅助质粒，其中所述DA’序列包括SEQ ID NO：4所示序列或为与SEQ ID NO：4所示序列具有至少90％序列一致性的功能性变体。
  8. 如权利要求1-7任一项所述的辅助质粒，其中所述AD’序列包括SEQ ID NO：3所示序列或为与SEQ ID NO：3所示序列具有至少90％序列一致性的功能性变体。
  9. 如权利要求1-8任一项所述的辅助质粒，其中所述Rep和Cap蛋白来自2型腺相关病毒。
  10. 提高重组腺相关病毒生产能力的方法，包括使用权利要求1-9任一项所述的辅助质粒转化宿主细胞。
PCT/CN2022/078621 2021-04-08 2022-03-01 制备重组腺相关病毒的辅助质粒及其应用 WO2022213745A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US18/377,827 US20240076319A1 (en) 2021-04-08 2023-10-08 Helper plasmid and method for preparing recombinant adeno-associated virus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110377907 2021-04-08
CN202110377907.X 2021-04-08

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US18/377,827 Continuation US20240076319A1 (en) 2021-04-08 2023-10-08 Helper plasmid and method for preparing recombinant adeno-associated virus

Publications (1)

Publication Number Publication Date
WO2022213745A1 true WO2022213745A1 (zh) 2022-10-13

Family

ID=83545133

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/078621 WO2022213745A1 (zh) 2021-04-08 2022-03-01 制备重组腺相关病毒的辅助质粒及其应用

Country Status (3)

Country Link
US (1) US20240076319A1 (zh)
CN (1) CN115197967B (zh)
WO (1) WO2022213745A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115851837A (zh) * 2022-01-25 2023-03-28 广州派真生物技术有限公司 一种提高杆状病毒系统生产腺相关病毒的方法及应用
CN117660532A (zh) * 2023-12-13 2024-03-08 广州派真生物技术有限公司 一种降低重组腺相关病毒中rcAAV残留的辅助质粒及应用

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002097056A2 (en) * 2001-05-31 2002-12-05 The Rockefeller University Method for generating replication defective viral vectors that are helper free
WO2016183422A1 (en) * 2015-05-14 2016-11-17 St. Jude Children's Research Hospital, Inc. Nucleic acid molecules containing spacers and methods of use thereof
US20190231901A1 (en) * 2018-02-01 2019-08-01 Homology Medicines, Inc. Adeno-associated virus compositions for pah gene transfer and methods of use thereof
WO2020081415A1 (en) * 2018-10-15 2020-04-23 Regenxbio Inc. Method for measuring the infectivity of replication defective viral vectors and viruses
WO2020146899A1 (en) * 2019-01-11 2020-07-16 Chan Zuckerberg Biohub, Inc. Targeted in vivo genome modification

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10137283A1 (de) * 2001-08-01 2003-02-27 Deutsches Krebsforsch AAV-Vektor-Verpackungsplasmide zur Helfervirus-freien Herstellung pseudotypisierter AAV-Partikel über Einzeltransfektion
CN1286980C (zh) * 2004-11-30 2006-11-29 华中科技大学同济医学院附属同济医院 表达人类反义受磷蛋白基因的重组腺相关病毒及其制备方法
ES2856090T3 (es) * 2014-09-24 2021-09-27 Hope City Variantes de vectores de virus adenoasociados para la edición genómica de alta eficacia y sus métodos
CN106884014B (zh) * 2015-12-16 2020-11-13 北京五加和基因科技有限公司 腺相关病毒反向末端重复序列突变体及其应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002097056A2 (en) * 2001-05-31 2002-12-05 The Rockefeller University Method for generating replication defective viral vectors that are helper free
WO2016183422A1 (en) * 2015-05-14 2016-11-17 St. Jude Children's Research Hospital, Inc. Nucleic acid molecules containing spacers and methods of use thereof
US20190231901A1 (en) * 2018-02-01 2019-08-01 Homology Medicines, Inc. Adeno-associated virus compositions for pah gene transfer and methods of use thereof
WO2020081415A1 (en) * 2018-10-15 2020-04-23 Regenxbio Inc. Method for measuring the infectivity of replication defective viral vectors and viruses
WO2020146899A1 (en) * 2019-01-11 2020-07-16 Chan Zuckerberg Biohub, Inc. Targeted in vivo genome modification

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
MUSATOV, S. ET AL.: "A cis-Acting Element That Directs Circular Adeno-Associated Virus", JOURNAL OF VIROLOGY, vol. 76, no. 24, 31 December 2002 (2002-12-31), XP002325443, DOI: 10.1128/JVI.76.24.12792-12802.2002 *
QINGZHANG ZHOU, WENHONG TIAN, CHUNGUO LIU, ZHONGHUI LIAN, XIAOYAN DONG, XIAOBING WU: "Deletion of the B-B’ and C-C’ regions of inverted terminal repeats reduces rAAV productivity but increases transgene expression", SCIENTIFIC REPORTS, vol. 7, no. 1, 1 December 2017 (2017-12-01), XP055707318, DOI: 10.1038/s41598-017-04054-4 *

Also Published As

Publication number Publication date
US20240076319A1 (en) 2024-03-07
CN115197967A (zh) 2022-10-18
CN115197967B (zh) 2023-09-15

Similar Documents

Publication Publication Date Title
WO2022213745A1 (zh) 制备重组腺相关病毒的辅助质粒及其应用
AU2017248840B2 (en) Methods of enhancing biological potency of baculovirus system-produced recombinant adeno-associated virus
US8409842B2 (en) Recombinant adeno-associated virus production
US7943379B2 (en) Production of rAAV in vero cells using particular adenovirus helpers
CN117304256B (zh) 腺相关病毒突变体及其应用
US20230399658A1 (en) Aav chimeras
CN115960177B (zh) 腺相关病毒突变体及其应用
CN115925819B (zh) 腺相关病毒突变体及其应用
CN114150021B (zh) 一种包含重叠开放阅读框的基因的表达盒及其在昆虫细胞中的应用
Wagner et al. A novel method for the quantification of adeno-associated virus vectors for RNA interference applications using quantitative polymerase chain reaction and purified genomic adeno-associated virus DNA as a standard
CN116004921A (zh) 重组腺相关病毒载体制品中rcAAV含量测定方法及检测试剂盒
CN116063404A (zh) 可提高AAV包装能力的衣壳蛋白MutD及其应用
CN117285608A (zh) 腺相关病毒突变体及其应用
WO2023124487A1 (zh) 重组腺相关病毒基因组及由其包装的单极rAAV载体
CN116813719B (zh) 腺相关病毒突变体及其应用
CN117660534A (zh) 一种降低重组腺相关病毒中宿主细胞dna残留的辅助质粒及应用
CN116284262A (zh) 高效感染ht-22细胞的腺相关病毒突变体
AU2021327855A1 (en) Method of making recombinant aavs
CN116063405A (zh) 可提高AAV病毒包装能力的衣壳蛋白突变体MutC及其应用
CN116023513A (zh) 一种适用于大鼠肝细胞特异性感染腺相关病毒突变体

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22783815

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE