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CN-121991975-A - Capsid protein encoding nucleic acid and baculovirus vector for AAV production

CN121991975ACN 121991975 ACN121991975 ACN 121991975ACN-121991975-A

Abstract

The present disclosure relates to polynucleotides, vectors, vector systems, and insect cells for producing AAV viruses in insect cells, and methods of production.

Inventors

  • MA WENHAO
  • WU XIAOBING
  • DONG XIAOYAN

Assignees

  • 北京锦篮基因科技股份有限公司
  • 北京五加和基因科技有限公司

Dates

Publication Date
20260508
Application Date
20241031

Claims (20)

  1. 1. A variant nucleic acid encoding an AAV capsid protein, wherein the variant nucleic acid has a mutation at a nucleotide position relative to a parent nucleic acid encoding an AAV capsid protein: (i) G at position 963 is mutated to A; (ii) T mutation at position 975 to A, or (Iii) A combination of (i) and (ii), Wherein the nucleotide positions are numbered according to SEQ ID NO. 3.
  2. 2. The variant nucleic acid according to claim 1, wherein said parent nucleic acid -Comprising the nucleotide sequence of SEQ ID No. 11 at positions 955 to 966 corresponding to SEQ ID No. 3; -comprising the nucleotide sequence of SEQ ID No. 13 at positions 967 to 978 corresponding to SEQ ID No. 3; comprising the nucleotide sequence of SEQ ID NO. 15 at positions 955 to 978 corresponding to SEQ ID NO. 3, and/or -Comprising the nucleotide sequence of SEQ ID NO. 17 at positions 943 to 984 corresponding to SEQ ID NO. 3.
  3. 3. The variant nucleic acid according to claim 1 or 2, wherein the parent nucleic acid comprises the polynucleotide of SEQ ID No. 3 or a polynucleotide having at least 95%, 96%, 97% or 98% identity thereto.
  4. 4. A variant nucleic acid according to any one of claims 1-3, wherein the parent nucleic acid encodes a native or engineered AAV9 capsid protein.
  5. 5. The variant nucleic acid according to any one of claims 1-4, wherein said variant nucleic acid comprises the polynucleotide of SEQ ID No. 19 or SEQ ID No. 20, or a nucleotide sequence having at least 95%, 96% or 99% or more identity thereto, or a nucleotide sequence differing therefrom by NO more than 45 or 30 nucleotides.
  6. 6. The variant nucleic acid according to any one of claims 1-5, wherein said variant nucleic acid further comprises an insect-specific intron inserted upstream of the VP2 encoding nucleotide sequence of said parent nucleic acid (preferably between nucleotide positions 25 and 26), preferably said intron has a 5 'splice donor site GTAAGTA and a 3' splice acceptor site TATTGTTTCAG.
  7. 7. Variant nucleic acid according to claim 6, wherein said intron comprises a baculovirus gene promoter, preferably a polyhedrin gene promoter polh or a P10 protein gene promoter P10, more preferably said intron comprises or consists of the nucleotide sequence shown in SEQ ID No. 2, or a nucleotide sequence having at least 95%, 96% or 99% or more identity thereto, or a nucleotide sequence differing therefrom by NO more than 10 or 5 nucleotides.
  8. 8. The variant nucleic acid according to any one of claims 1-7, wherein said variant nucleic acid comprises or consists of the nucleotide sequence shown in SEQ ID No. 4 or 5, or a nucleotide sequence having at least 95%, 96% or 99% or more identity thereto, or a nucleotide sequence differing therefrom by NO more than 45 or 30 nucleotides.
  9. 9. The variant nucleic acid according to any one of claims 1-8, wherein expression of the variant nucleic acid in an insect cell results in an AAV capsid having an increased proportion of VP1 and/or VP2 compared to the parent nucleic acid.
  10. 10. An AAV capsid expression cassette comprising a variant nucleic acid according to any one of claims 1-9, optionally wherein the expression cassette further comprises an insect-expressible promoter operably linked to the variant nucleic acid 5', preferably the promoter is a baculovirus gene promoter, more preferably the polyhedrin gene promoter polh or the P10 protein gene promoter P10.
  11. 11. A vector comprising the variant nucleic acid of any one of claims 1-10 or the expression cassette of claim 10, optionally wherein: The vector further comprises one or more polynucleotides encoding AAV Rep proteins, e.g., the nucleotide sequence of nucleotides 3852-5820 of SEQ ID NO. 7, and/or The vector comprises the nucleotide sequence of nucleotide 745 to nucleotide 5820 of SEQ ID NO. 7 or SEQ ID NO. 8 or comprises or consists of the polynucleotide of SEQ ID NO. 7 or SEQ ID NO. 8.
  12. 12. A baculovirus vector system comprising at least one vector carrying the following components: (i) Variant nucleic acid according to any one of claims 1 to 9 or AAV capsid expression cassette according to claim 10, and optionally (Ii) One or more polynucleotides encoding AAV Rep proteins, and/or (iii) a recombinant AAV genome, Preferably, components (i) and (ii) further comprise a baculovirus gene promoter, preferably a polyhedrin gene promoter polh or a P10 protein gene promoter P10, operably linked to the polynucleotide 5'.
  13. 13. The baculovirus vector system according to claim 12, wherein said one or more polynucleotides encoding AAV Rep proteins encode Rep78 and Rep52 proteins, or Rep78, rep68, rep52 and Rep40 proteins, Preferably, the component (ii) is a single Rep expression cassette capable of transcriptional expression producing a Rep78 and a Rep52 protein or a Rep78, a Rep68, a Rep52 and a Rep40 protein, wherein the Rep expression cassette comprises an insect-specific intron embedded in the insect-expressible promoter in the 5' portion.
  14. 14. The baculovirus vector system of claim 12 or 13, wherein the recombinant AAV genome comprises: a.5 'and 3' AAV Inverted Terminal Repeat (ITR) sequences, and B. A transgene of interest (GOI) expression cassette located between 5 'and 3' itrs. Preferably, wherein said GOI expression cassette comprises the following elements functionally linked to each other in the direction of transcription: -a mammalian cell-expressible promoter, -A heterologous nucleic acid encoding said GOI or an insertion site for receiving said heterologous nucleic acid; A transcription terminator, a sequence of which is defined by the sequence of the gene, Still preferably, the heterologous nucleic acid encodes a gene product of interest for gene replacement, gene suppression or gene editing, preferably the gene product of interest is a protein or RNA.
  15. 15. A baculovirus vector system according to any one of claims 12-14, wherein said system comprises: Component (i) and component (ii) on the same support or on different supports, and/or Component (iii) on the same or different support as component (i) and/or (ii).
  16. 16. A baculovirus vector system according to any one of claims 12-15, wherein said system is a single vector system, a dual vector system or a triple vector system comprising said components.
  17. 17. The vector according to claim 11 or the vector system according to any of claims 12-16, wherein the vector is a nucleic acid vector, preferably a plasmid or a bacmid, or the vector is a viral vector, preferably a recombinant baculovirus.
  18. 18. An insect cell comprising the components: (i) Variant nucleic acid according to any one of claims 1 to 9 or AAV capsid expression cassette according to claim 10, and optionally (Ii) One or more polynucleotides encoding AAV Rep proteins, and/or (iii) a recombinant AAV genome, Optionally the components (i) and/or (ii) are integrated in the genome of the insect cell.
  19. 19. An insect cell according to claim 18, comprising components (i), (ii) and (iii).
  20. 20. Use of a variant nucleic acid according to any one of claims 1-9 or an AAV capsid expression cassette according to claim 10 or a vector according to claim 11 or a vector system according to any one of claims 12-17 in the preparation of an insect cell according to claim 18 or 19.

Description

Capsid protein encoding nucleic acid and baculovirus vector for AAV production Technical Field The invention relates to the technical field of recombinant adeno-associated virus (AAV) production. In particular, the invention relates to polynucleotides, vectors, vector systems and insect cells for producing AAV viruses in insect cells and methods of production. Background Adeno-associated virus (AAV) vectors have been identified as a safe and reliable gene transfer vector for efficient transgene delivery in vivo and in vitro. AAV vectors have been widely used in gene therapy drug development as a promising gene and cell therapy delivery vehicle. Glybera, luxturna, zolgenesma, hemgenix, roctavian, upstaza and Elevidys(Model-Informed Approaches and Innovative Clinical Trial Design for Adeno-Associated Viral Vector-Based Gene Therapy Product Development:A White Paper.Clin.Pharmacol.Ther.2023;114:515–529.doi:10.1002/cpt.2972.)., which are currently approved by the FDA/EMA, require the development of AAV production systems based on mammalian and insect cells as the demand for high quality adeno-associated virus (AAV) products increases with gene and cell therapy. Mammalian HEK-293 production systems are one of the most widely used techniques for generating recombinant AAV (rAAV) on a laboratory scale. Typically, the system includes plasmids encoding AAV Rep and Cap proteins, the vector genome of interest, and adenovirus helper genes, and has been optimized for transfection of HEK-293 cells. Larger scale clinical trials and commercialization of rAAV products require expanded production, resulting in the development of baculovirus-insect (Sf 9) cell systems for rAAV production. Although the baculovirus-Sf 9 cell system has been successfully used for AAV production on a variety of scales, it was observed that rAAV produced in insect cells reduced VP1 content (VP 1: VP2: VP3 ratio of about 1:1:30 to 1:1:60, whereas wild-type AAV is 1:1:10) and was less potent than rAAV produced in mammalian cells. See Yoko Marwidi et al ,A robust and flexible baculovirus-insect cell system for AAV vector production with improved yield,capsid ratios and potency,Molecular Therapy:Methods&Clinical Development Vol.32June2024. thus, there is a need to increase the proportion of VP1 in AAV to increase AAV transduction. Patent application CN101522903a proposes that Cap proteins are expressed using alternative start codons or integrated insect-specific introns to optimize VP ratio (VP 1: VP2: VP 3), thereby producing rAAV vectors with enhanced infectivity. Patent CN101405033a proposes to increase VP1/VP3 ratio and improve infectivity of the resulting AAV vector by delivering additional VP1 genes in addition to VP1, VP2 and VP3 to insect cells. Although the proposed methods improve the production of recombinant AAV to some extent, there is a continuing need in the art for improved Baculovirus Expression Vector (BEV) systems for clinical use of recombinant AAV to meet the need for large-scale production of high quality recombinant AAV vectors. Summary of The Invention In eukaryotic cells, mRNA splicing is a mechanism that regulates gene expression at the post-transcriptional level. During this splicing process, primary transcripts produced by transcription from the DNA template strand are typically spliced at splice recognition site sites under the action of a biologically unique splicing mechanism to produce mature mRNA encoding the protein. However, for exogenous transgenes expressed in a particular host cell, the presence of a potentially missplice site recognized by the host cell is often disadvantageous in that it will trigger a missplice event, create unwanted byproducts or reduce the desired product of interest. In intensive studies, the inventors have surprisingly found that in the nucleic acid encoding an AAV capsid protein, there are some unintended splice recognition sites that may lead to erroneous splicing resulting in reduced abundance of intact VP1 and VP2 mRNA, and thus lower VP1, 2 ratios. For this reason, the present inventors have designed and explored modifications to capsid expression genes to eliminate mis-splicing without changing amino acids and to increase the ratio of VP1, VP 2. More specifically, the inventors found that the ratio of VP1 and VP2 in AAV capsids can be significantly increased by introducing a G-to-A (herein, abbreviated as c.963G > A) single-site mutation at the nucleotide 963 position corresponding to SEQ ID NO:3, or introducing G-to-A and T-to-A (herein, abbreviated as c.963G > A+c975T > A) double-site mutation at the nucleotide 963 and 975 positions corresponding to SEQ ID NO:3, respectively, in an AAV capsid protein encoding nucleic acid comprising an intron. Further studies have demonstrated that the use of recombinant AAV viruses produced by packaging nucleic acids having such mutations in insect cells has increased transduction capacity in vitro assays for a variety of mammalian cell lines and i