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JP-7856218-B2 - Modified recombinant adeno-associated virus (AAV) binding protein and method for purifying AAV.

JP7856218B2JP 7856218 B2JP7856218 B2JP 7856218B2JP-7856218-B2

Inventors

  • 岩渕 圭篤
  • 栗原 健人
  • 眞鍋 友理子
  • 吉田 浩平
  • 大村 慧太
  • 田中 亨

Assignees

  • 東ソー株式会社

Dates

Publication Date
20260511
Application Date
20240522
Priority Date
20230523

Claims (12)

  1. An AAV-binding protein comprising at least the amino acid residues from the 25th serine to the 213th aspartic acid of the amino acid sequence described in Sequence ID No. 2, wherein the amino acid substitutions shown in any of (A) to ( O ) below occur in the 25th to 213th amino acid residues, and which has AAV-binding activity; (A) The 42nd asparagine in SEQ ID NO: 2 is replaced with lysine, the 114th glutamic acid in SEQ ID NO: 2 is replaced with glycine, and the 139th threonine in SEQ ID NO: 2 is replaced with alanine. (B) The 51st lysine in SEQ ID NO: 2 is replaced with arginine, the 139th threonine in SEQ ID NO: 2 is replaced with alanine, and the 174th alanine in SEQ ID NO: 2 is replaced with proline. (C) The 42nd asparagine in SEQ ID NO: 2 is replaced with lysine, the 114th glutamic acid in SEQ ID NO: 2 is replaced with glycine, the 139th threonine in SEQ ID NO: 2 is replaced with alanine, and the 174th alanine in SEQ ID NO: 2 is replaced with proline. (D) The 51st lysine in SEQ ID NO: 2 is replaced with arginine, the 114th glutamic acid in SEQ ID NO: 2 is replaced with glycine, and the 139th threonine in SEQ ID NO: 2 is replaced with proline. (E) Asparagine at position 42 of SEQ ID NO: 2 is substituted with lysine, lysine at position 51 of SEQ ID NO: 2 is substituted with arginine, glutamic acid at position 114 of SEQ ID NO: 2 is substituted with glycine, threonine at position 139 of SEQ ID NO: 2 is substituted with alanine, and alanine at position 174 of SEQ ID NO: 2 is substituted with proline (F) Asparagine at position 42 of SEQ ID NO: 2 is substituted with lysine, glutamic acid at position 114 of SEQ ID NO: 2 is substituted with glycine, threonine at position 139 of SEQ ID NO: 2 is substituted with alanine, alanine at position 174 of SEQ ID NO: 2 is substituted with proline, and glutamine at position 180 of SEQ ID NO: 2 is substituted with asparagine (G) Asparagine at position 42 of SEQ ID NO: 2 is substituted with lysine, sequence number In SEQ ID NO. 2, the 79th isoleucine is substituted with phenylalanine, the 114th glutamic acid is substituted with glycine, the 139th threonine is substituted with alanine, the 174th alanine is substituted with proline, the 180th glutamine is substituted with asparagine, and the 187th threonine is substituted with arginine (H) In SEQ ID NO. 2, the 42nd asparagine is substituted with lysine, the 107th valine is substituted with alanine, the 114th glutamic acid is substituted with glycine, the 139th threonine is substituted with alanine, the 174th alanine is substituted with proline, the 180th glutamine is substituted with asparagine, and the 187th threonine is substituted with arginine (I) Rheonine is replaced with arginine. The asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the lysine at position 168 of SEQ ID NO: 2 is replaced with arginine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine, and the threonine at position 187 of SEQ ID NO: 2 is replaced with arginine. (J) The asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the isoleucine at position 79 of SEQ ID NO: 2 is replaced with phenylalanine, the valine at position 107 of SEQ ID NO: 2 is replaced with alanine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, and the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine. , lysine at position 168 of SEQ ID NO: 2 is replaced with arginine, alanine at position 174 of SEQ ID NO: 2 is replaced with proline, and glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine (K) Asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, isoleucine at position 79 of SEQ ID NO: 2 is replaced with phenylalanine, valine at position 107 of SEQ ID NO: 2 is replaced with alanine, glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, lysine at position 168 of SEQ ID NO: 2 is replaced with arginine, alanine at position 174 of SEQ ID NO: 2 is replaced with proline, glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine, and threonine at position 187 of SEQ ID NO: 2 is replaced with arginine (L) SEQ ID NO: 27 Glycine is substituted with aspartic acid, asparagine at position 42 of SEQ ID NO: 2 is substituted with lysine, isoleucine at position 79 of SEQ ID NO: 2 is substituted with phenylalanine, valine at position 107 of SEQ ID NO: 2 is substituted with alanine, glutamic acid at position 114 of SEQ ID NO: 2 is substituted with glycine, threonine at position 139 of SEQ ID NO: 2 is substituted with alanine, lysine at position 168 of SEQ ID NO: 2 is substituted with arginine, alanine at position 174 of SEQ ID NO: 2 is substituted with proline, and glutamine at position 180 of SEQ ID NO: 2 is substituted with asparagine (M) Asparagine at position 42 of SEQ ID NO: 2 is substituted with lysine, isoleucine at position 79 of SEQ ID NO: 2 is substituted with phenylalanine, valine at position 107 of SEQ ID NO: 2 is substituted with alanine, and glutamic acid at position 114 of SEQ ID NO: 2 is substituted with glycine Substitutions occur, with the threonine at position 139 of SEQ ID NO: 2 substituted with alanine, the lysine at position 168 of SEQ ID NO: 2 substituted with arginine, the alanine at position 174 of SEQ ID NO: 2 substituted with proline, the glutamine at position 180 of SEQ ID NO: 2 substituted with asparagine, and the lysine at position 210 of SEQ ID NO: 2 substituted with glutamic acid (N) The asparagine at position 42 of SEQ ID NO: 2 substituted with lysine, the isoleucine at position 79 of SEQ ID NO: 2 substituted with phenylalanine, the valine at position 107 of SEQ ID NO: 2 substituted with alanine, the glutamic acid at position 114 of SEQ ID NO: 2 substituted with glycine, the threonine at position 139 of SEQ ID NO: 2 substituted with alanine, the lysine at position 168 of SEQ ID NO: 2 substituted with arginine, the alanine at position 174 of SEQ ID NO: 2 substituted with proline, and the glutamic acid at position 210 of SEQ ID NO: 2 The mine is replaced with asparagine, and the valine at position 212 of SEQ ID NO: 2 is replaced with isoleucine (O) The glycine at position 27 of SEQ ID NO: 2 is replaced with aspartic acid, the asparagine at position 42 of SEQ ID NO: 2 is replaced with lysine, the isoleucine at position 79 of SEQ ID NO: 2 is replaced with phenylalanine, the valine at position 107 of SEQ ID NO: 2 is replaced with alanine, the glutamic acid at position 114 of SEQ ID NO: 2 is replaced with glycine, the threonine at position 139 of SEQ ID NO: 2 is replaced with alanine, the lysine at position 168 of SEQ ID NO: 2 is replaced with arginine, the alanine at position 174 of SEQ ID NO: 2 is replaced with proline, the glutamine at position 180 of SEQ ID NO: 2 is replaced with asparagine, the lysine at position 210 of SEQ ID NO: 2 is replaced with glutamic acid, and the valine at position 212 of SEQ ID NO: 2 is replaced with isoleucine.
  2. A polynucleotide encoding an AAV-binding protein as described in claim 1.
  3. An expression vector comprising the polynucleotide described in claim 2.
  4. A transformant obtained by transforming E. coli with the expression vector described in claim 3.
  5. A method for producing an AAV-binding protein, comprising the steps of: culturing the transformant described in claim 4 to express an AAV-binding protein; and recovering the AAV-binding protein expressed from the obtained culture.
  6. An AAV adsorbent comprising an insoluble carrier and an AAV-binding protein according to claim 1, immobilized on the carrier.
  7. A column comprising the AAV adsorbent described in claim 6.
  8. A method for purifying or analyzing AAV, comprising the steps of: adding a solution containing AAV to the column described in claim 7 to adsorb the AAV onto the adsorbent; and eluting the AAV adsorbed onto the adsorbent using an eluent.
  9. The method for purifying or analyzing AAV according to claim 8, further comprising the step of washing the AAV adsorbent with an alkaline solution after the step of eluting AAV.
  10. A method for purifying AAV contained in a sample, A step of adding a sample containing AAV to an adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the insoluble carrier, and adsorbing the AAV onto the adsorbent, A step of eluting the AAV adsorbed on the adsorbent, A step of adding a fraction containing AAV eluted in the elution step to a carrier for anion exchange chromatography and adsorbing the AAV onto the carrier for anion exchange chromatography, The process includes eluting AAV adsorbed onto the carrier for anion exchange chromatography, A purification method wherein the AAV-binding protein is the AAV-binding protein described in claim 1.
  11. The purification method according to claim 10, wherein the step of eluting AAV adsorbed on a carrier for anion exchange chromatography is to elute the AAV adsorbed on the carrier using an eluent with a conductivity of 13.5 mS/cm or less, and then to elute the AAV remaining on the carrier using an eluent with a conductivity of 15.0 mS/cm or more.
  12. The purification method according to claim 11, wherein the eluate contains choline chloride.

Description

This disclosure relates to proteins that bind to adeno-associated viruses (AAVs). In one embodiment, this disclosure relates to improved recombinant AAV-binding proteins, such as those with enhanced alkali resistance. In one embodiment, this disclosure relates to a method for purifying adeno-associated virus (AAV). Furthermore, in one embodiment, this disclosure relates to a method for conveniently purifying, for example, an AAV vector containing genes. Adeno-associated viruses (AAVs) are non-enveloped viruses classified in the Parvoviridae family and Dependovirus genus. The outer shell of an AAV particle is composed of three types of proteins (VP1, VP2, and VP3), with approximately 60 protein molecules mixed and assembled in a ratio of roughly VP1:VP2:VP3 = 1:1:10, forming an icosahedron shape with a diameter of 20 to 30 nm. In nature, AAV lacks the ability to replicate independently; replication relies on helper viruses such as adenoviruses and herpesviruses. When these helper viruses are present, the AAV genome replicates within the host cell, forming complete AAV particles containing the AAV genome, which are then released from the host cell. Conversely, when the helper viruses are absent, the AAV genome remains maintained in the episome or is integrated into the host chromosome (latent state). AAV is attracting attention as a potential gene transfer vector for the treatment of congenital genetic disorders because it can infect cells of a wide range of species, including humans, and can infect non-dividing cells that have completed differentiation, such as blood cells, muscle cells, and nerve cells; it is not pathogenic to humans, thus reducing concerns about side effects; and the viral particles are physicochemically stable. The production of recombinant AAV vectors (hereinafter also simply referred to as AAV vectors) is typically carried out by introducing nucleic acids encoding elements essential for AAV particle formation into cells to create cells capable of producing AAV (hereinafter also referred to as AAV-producing cells), and then culturing these cells to express the elements essential for AAV particle formation. The produced AAV vectors are recovered and purified from the AAV-producing cells to obtain therapeutic AAV vector preparations. One method for recovering and purifying AAV vectors from AAV-producing cells involves affinity chromatography based on the binding affinity to AAV, using an adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the carrier. This method allows for the recovery and purification of the vector from a solution containing the AAV vector even if it is contaminating other substances. As a specific example, Patent Document 1 describes a method for achieving high-purity purification of AAV vectors by using a polypeptide containing extracellular domain 1 (PKD1) and domain 2 (PKD2) of KIAA0319L (UniProt No. Q8IZA0), but with improved stability against heat, acid, and alkali by substituting amino acid residues at specific positions in these domains with other amino acid residues, as an AAV-binding protein (hereinafter also simply referred to as "ligand protein") immobilized on an insoluble carrier. On the other hand, when the above-mentioned adsorbent is used for purification purposes, after AAV purification, the remaining AAV and contaminants are usually washed with a high-concentration (e.g., 0.1 M to 0.5 M) aqueous sodium hydroxide solution. Therefore, the creation of ligand proteins that can withstand this alkaline washing has been desired. The manufactured AAV vectors include those containing genes (Full AAV vectors) and those not containing genes (Empty AAV vectors). Of these, Empty AAV vectors may reduce the efficacy of the drug as a therapeutic agent or induce side effects due to overdose (Non-Patent Literature 2). Therefore, in order to use them as therapeutic AAV vector preparations, it is necessary to purify the Full AAV vectors from the manufactured AAV vectors to a high degree of purity. A method for purifying AAV vectors is known, which involves affinity chromatography using an AAV adsorbent containing an insoluble carrier and an AAV-binding protein immobilized on the carrier (Patent Documents 1 and 2, and Non-Patent Document 2). However, even with this affinity chromatography method, it has been difficult to remove empty AAV vectors. One known method for obtaining full AAV vectors involves ultracentrifugation of a solution containing the manufactured AAV vectors to obtain a fraction rich in full AAV vectors. However, ultracentrifugation requires complex steps such as preparing a density gradient solution, ultracentrifugation, and fraction acquisition, and generally takes several days to obtain full AAV vectors. Furthermore, because it is difficult to distinguish the fraction rich in full AAV vectors, there is a risk of acquiring layers before or after the full AAV vector during fraction recovery, and there is a high risk of cont