US 12298313

Summary of US Patent 12298313B1

Title: Methods for detecting AAV

Assignee: Genzyme Corp

Inventors: Xiaoying Jin, Catherine O'Riordan, Lin Liu, Kate ZHANG

Filing Date: 2025-01-08

Issue Date: 2025-05-13

Abstract: The patent describes methods for identifying adeno-associated virus (AAV) serotypes and/or determining their heterogeneity using mass determination techniques, specifically liquid chromatography/mass spectrometry (LC/MS) or liquid chromatography/mass spectrometry-mass spectrometry (LC/MS/MS). These methods can be applied to characterize viral capsid proteins (VPs) by monitoring their protein sequence, post-translational modifications (like N-terminal acetylation), and truncations. The invention also covers recombinant AAV (rAAV) particles and AAV capsid proteins with amino acid substitutions that alter N-terminal acetylation and/or deamidation, aiming to improve stability, assembly, or transduction efficiency.

Plain-Language Overview of Independent Claims:

• Independent Claim 1: This claim describes a method to determine the specific type (serotype) of an adeno-associated virus (AAV) particle. The method involves first breaking apart (denaturing) the AAV particle. Then, the denatured particle is analyzed using liquid chromatography/mass spectrometry (LC/MS). By measuring the exact masses of the three main proteins that make up the AAV outer shell (VP1, VP2, and VP3), and comparing this unique set of masses to known values, the serotype of the AAV can be identified.

• Independent Claim 5: This claim focuses on identifying variations or inconsistencies (heterogeneity) within an AAV particle. Similar to Claim 1, it starts by denaturing the AAV particle and subjecting it to LC/MS. It then determines the masses of VP1, VP2, and VP3. The key difference is that these measured masses are compared against the expected (theoretical) masses for the AAV's known serotype. If there are any differences or "deviations" in these masses, it indicates heterogeneity within the AAV capsid, meaning there might be mixed serotypes, altered capsids, amino acid changes, shortened capsids, or other modifications.

• Independent Claim 9: This claim provides another method for determining the AAV serotype, but it involves a more detailed analysis of protein fragments. After denaturing the AAV particle, it undergoes reduction and/or alkylation (chemical treatments) and then is cut into smaller pieces (digested) to produce fragments of VP1, VP2, and/or VP3. These fragments are then analyzed using liquid chromatography/mass spectrometry-mass spectrometry (LC/MS/MS). By identifying the specific masses of these protein fragments, the AAV serotype can be determined.

• Independent Claim 13: This claim describes a method for determining the heterogeneity of an AAV particle's serotype, similar to Claim 9, but at the fragment level. After denaturation, reduction/alkylation, and digestion of the AAV particle into VP1, VP2, and/or VP3 fragments, these fragments are analyzed by LC/MS/MS. The determined masses of these fragments are then compared to the theoretical masses of fragments for the expected AAV serotype. Any deviations from these theoretical masses indicate heterogeneity in the AAV capsid.

• Independent Claim 17: This claim describes a specific recombinant AAV (rAAV) particle. This rAAV particle has an intentional change (amino acid substitution) at the second amino acid position of either VP1, VP3, or both. This substitution is designed to alter (either increase or decrease) the N-terminal acetylation of VP1 and/or VP3 compared to the original, unmodified (parental) AAV particle.

• Independent Claim 22: This claim outlines a method for improving the assembly of rAAV particles within a cell. The improvement is achieved by introducing an amino acid substitution at the second amino acid position of VP1 and/or VP3, where this substituted amino acid is N-acetylated at a higher frequency than the corresponding amino acid in the parent VP1 and/or VP3.

• Independent Claim 23: This claim describes a method to improve the ability of rAAV particles to transfer genetic material into a cell (transduction). This is done by substituting the amino acid at the second position of VP1 and/or VP3 such that this substituted amino acid has a higher frequency of N-acetylation compared to the parent VP1 and/or VP3.

• Independent Claim 24: This claim defines an AAV capsid protein that has an amino acid substitution at the second amino acid residue compared to a parent AAV capsid protein. This substitution specifically changes the N-terminal acetylation pattern of the capsid protein.

• Independent Claim 29: This claim describes a recombinant AAV (rAAV) particle that contains one or more amino acid substitutions at specific positions (A35, N57, G58 of VP1, or N382, G383, N511, G512, N715, or G716 of VP3, using AAV2 VP1 numbering). These substitutions are designed to alter the deamidation of VP1 or VP3 compared to the parent AAV particle.

• Independent Claim 30: This claim describes a method for improving the stability of an rAAV particle. This improvement is achieved by substituting one or more amino acid residues at specific positions (A35, N57, G58, N382, G383, N511, G512, N715, or G716, based on AAV2 VP1 numbering). The substitution is made to change the deamidation of VP1 and/or VP3 compared to the parent AAV particle, thereby enhancing stability.

• Independent Claim 31: This claim outlines a method for improving the assembly of rAAV particles in a cell. Similar to Claim 30, it involves substituting one or more amino acid residues at the specified positions (A35, N57, G58, N382, G383, N511, G512, N715, or G716, based on AAV2 VP1 numbering). This substitution is designed to alter the deamidation of VP1 and/or VP3, leading to improved particle assembly.

• Independent Claim 32: This claim describes a method for improving the transduction efficiency of rAAV particles in a cell. This is achieved by substituting one or more amino acid residues at specific positions (A35, N57, G58, N382, G383, N511, G512, N715, or G716, based on AAV2 VP1 numbering). The substitution alters the deamidation of VP1 and/or VP3 compared to the parent AAV particle, resulting in better transduction.

Litigation Information:

The Google Patents record for US12298313B1 indicates active litigation related to its patent family. Specifically, there is "First worldwide family litigation filed" and two PTAB cases: IPR2026-00270 (currently pending) and IPR2026-00166 (which resulted in a procedural termination). No specific dockets for US12298313B1 were found in the CAFC 2026 dockets search, although general information on accessing CAFC case records is available.