US 11698377

US Patent 11698377, titled "Methods for detecting AAV," was granted to Genzyme Corp. The inventors listed are Xiaoying Jin, Catherine O'Riordan, Lin Liu, and Kate Zhang. The application was filed on August 14, 2017, and the patent was issued on July 11, 2023.

Abstract:
The patent describes methods for serotyping and/or determining the heterogeneity of a viral particle, specifically an adeno-associated virus (AAV) particle, using mass determination techniques such as liquid chromatography/mass spectrometry (LC/MS) or liquid chromatography/mass spectrometry-mass spectrometry (LC/MS/MS). These methods can be used to monitor protein sequences and post-translational modifications at the intact protein level, identify N-terminal acetylations, and characterize viral capsid protein heterogeneity in gene therapy development. They can also confirm VP sequences in capsid engineering and study the impact of post-translational modifications on transfection potency and intracellular protein trafficking. Furthermore, the methods can be used to design AAV particles with altered stability and/or transduction efficiency by modifying N-terminal acetylation or deamidation.

Plain-Language Overview of Independent Claims:

• Independent Claim 1: This claim describes a method to identify the serotype of an adeno-associated virus (AAV) particle. It involves three main steps:

  1. Denaturing the AAV particle (breaking it down).
  2. Subjecting the denatured particle to liquid chromatography/mass spectrometry (LC/MS).
  3. Determining the masses of the three main capsid proteins (VP1, VP2, and VP3) of the AAV particle.
     The specific combination of these measured protein masses is then used to indicate the AAV serotype.

• Independent Claim 9: This claim focuses on determining the heterogeneity of an AAV particle. It includes:

  1. Denaturing the AAV particle.
  2. Subjecting the denatured particle to liquid chromatography/mass spectrometry (LC/MS).
  3. Determining the masses of VP1, VP2, and VP3.
  4. Comparing these measured masses to the known theoretical masses for the specific AAV serotype.
     Any deviation in one or more of these measured masses from the theoretical masses indicates heterogeneity within the AAV capsid. Heterogeneity can include mixed serotypes, variant capsids, amino acid changes, truncated capsids, or modified capsids.

• Independent Claim 17: This claim also describes a method for determining the serotype of an AAV particle, but it involves additional sample preparation steps:

  1. Denaturing the AAV particle.
  2. Subjecting the denatured particle to reduction and/or alkylation (chemical treatments to break disulfide bonds).
  3. Digesting the treated particle to create smaller fragments of VP1, VP2, and/or VP3.
  4. Subjecting these protein fragments to liquid chromatography/mass spectrometry-mass spectrometry (LC/MS/MS).
  5. Determining the masses of these fragments.
     The specific combination of the masses of these fragments is indicative of the AAV serotype.

• Independent Claim 25: Similar to Claim 17, this claim focuses on determining the heterogeneity of an AAV particle's serotype using fragmented proteins:

  1. Denaturing the AAV particle.
  2. Subjecting the denatured particle to reduction and/or alkylation.
  3. Digesting the treated particle to generate fragments of VP1, VP2, and/or VP3.
  4. Subjecting these fragments to liquid chromatography/mass spectrometry-mass spectrometry (LC/MS/MS).
  5. Determining the masses of these fragments.
  6. Comparing these measured fragment masses to the theoretical masses of fragments for that AAV serotype.
     A deviation in one or more of these fragment masses indicates heterogeneity of the AAV capsid. Heterogeneity can include mixed serotypes, variant capsids, amino acid changes, truncated capsids, or modified capsids.

• Independent Claim 33: This claim introduces a modified recombinant AAV (rAAV) particle. It claims an rAAV particle with a specific amino acid change at residue 2 of VP1 and/or VP3, where this change alters the N-terminal acetylation compared to the original, unmodified AAV particle.

• Independent Claim 37: This claim is directed to an AAV capsid protein (e.g., VP1 or VP3) that has an amino acid substitution at residue 2 compared to a parent AAV capsid protein. This substitution is designed to alter the N-terminal acetylation of the capsid protein.

• Independent Claim 40: This claim describes a method to improve the stability of an rAAV particle. It involves substituting amino acid residue 2 of VP1 and/or VP3 in a way that alters the N-terminal acetylation of these proteins compared to the parent VP1 and/or VP3.

• Independent Claim 41: This claim describes a method to improve the assembly of rAAV particles within a cell. It involves substituting amino acid residue 2 of VP1 and/or VP3 such that the N-terminal acetylation is altered compared to the parent proteins.

• Independent Claim 42: This claim describes a method to improve the transduction (delivery of genetic material) of rAAV particles into a cell. This improvement is achieved by substituting amino acid residue 2 of VP1 and/or VP3 to alter N-terminal acetylation compared to the parent proteins.

• Independent Claim 43: This claim describes a method to reduce the transduction of rAAV particles in a cell. Similar to Claim 42, it involves substituting amino acid residue 2 of VP1 and/or VP3 to alter N-terminal acetylation compared to the parent proteins.

• Independent Claim 45: This claim introduces a recombinant AAV (rAAV) particle comprising one or more amino acid substitutions at specific residues (A35, N57, G58, N382, G383, N511, G512, N715, or G716) of VP1 or VP3 (residue numbering based on AAV2 VP1). The purpose of these substitutions is to alter deamidation compared to the parent AAV particle.

• Independent Claim 48: This claim describes an AAV capsid protein with an amino acid substitution that alters its deamidation compared to the parent AAV capsid protein.

• Independent Claim 49: This claim details a method to improve the stability of an rAAV particle by substituting one or more amino acid residues (A35, N57, G58, N382, G383, N511, G512, N715, or G716, based on AAV2 VP1 numbering). These substitutions are designed to alter deamidation compared to the parent AAV particle.

• Independent Claim 50: This claim describes a method to improve the assembly of rAAV particles in a cell through the substitution of specific amino acid residues (A35, N57, G58, N382, G383, N511, G512, N715, or G716, based on AAV2 VP1 numbering). These substitutions alter deamidation compared to the parent AAV particle.

• Independent Claim 51: This claim describes a method to improve the transduction of rAAV particles in a cell, similar to Claim 50, by substituting specific amino acid residues (A35, N57, G58, N382, G383, N511, G512, N715, or G716, based on AAV2 VP1 numbering) to alter deamidation compared to the parent AAV particle.

Litigation Status:
US Patent 11698377 is currently active and is involved in litigation. A PTAB case, IPR2026-00167, has been filed and is pending.