DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 10/25/24 and 4/7/25 is being considered by the examiner.
Claim Status
Claims 1-16 are pending and are examined.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1, 2, 3, 4, 5, 6, 7, 8, 14, 15, and 16 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Furukawa (“A Versatile Method for Analysis of Serine/Threonine Posttranslational Modifications by B-Elimination in the Presence of Pyrazolone Analogues.” Analytical Chemistry. 83:9060-9067. 2011).
Regarding Claim 1, Furukawa teaches a method for identifying an amino acid site in a protein to which a sugar is bonded (As an example of O-glycosylation site analysis of glycoprotein by BEP, fetuin was subjected to S-alkylation by iodoacetoamide and was subjected to BEP, followed by digestion with pronase. Pg. 9064),
the method comprising:
a first mass spectrometry step of subjecting a fragmented protein to mass spectrometry (Matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and NMR measurements were used to characterize the glycopeptides of interest. Pg. 9061);
for the protein in which the bonding of the sugar has been confirmed in the first mass spectrometry step (The model glycopeptide or glycoprotein (10 μL, 0.1 40 μg) was treated with 20 μL each of sodium hydroxide (0.3 0.6 M) and a 0.5 M methanolic solution of pyrazolone, followed by heating at various temperatures (50 95 C) for reaction times of 2 16h. pg. 9062.),
a substitution-modification step of substituting and/or modifying an amino acid at the amino acid site in the protein to which the sugar is bonded and a fragmentation step of fragmenting the protein (A mixture containing the glycopeptide (10 μL, 40μg) and 10 μL each of 0.8 M sodium hydroxide and 1 M DTT was treated with 20 μL of a 0.5 M methanolic solution of PMP, followed by heating at 75oC for 16 h. Subsequent neutralization and purification steps were performed by the same procedures as described above. Pg. 9062. 1st column, 2nd paragraph); and
a second mass spectrometry step of subjecting the substituted and/or modified, and fragmented protein obtained by the substitution-modification step and the fragmentation step, to mass spectrometry (Eluted PMP-labeled O-glycans and peptides were directly mixed with2,5-dihydroxybenzoic acid was deposited on a stainless steel target plate. MALDI-TOF data were obtainedonan Ultraflex II time-of-flight mass spectrometer. All spectra were obtained in reflectron mode with an acceleration voltage of 25 kV and a reflector voltage of 26.3 kV in the positive-ion mode. These spectra were the sum of 1000 laser shots. All peaks were selected by FlexAnalysis 3.0 and the sophisticated numerical annotation procedure (SNAP) algorithm, which fits isotopic patterns to the corresponding experimental data. The algorithm provides monoisotopic mass, intensity, area under the envelope of the isotopic cluster, and resolution of the peaks in the cluster. O-linked-type oligosaccharide structures were estimated from inputs of peak masses into the GlycoMod Tool (Swiss Institute of Bioinformatics) and into GlycoSuite (Proteome Systems Inc., Woburn, MA). See pg. 9062, 1st column, last paragraph and 2nd column, 1st paragraph).
Regarding Claim 2, Furukawa teaches the method according to claim 1, further comprising: comparing a mass spectrometry result obtained in the first mass spectrometry step with a mass spectrometry result obtained in the second mass spectrometry step (MALDI-TOF spectra acquired before and after BEP reaction (upper and lower panels, respectively) were compared. See Fig. 4 caption, pg. 9064).
Regarding Claim 3, Furukawa teaches the method according to claim 1, wherein the amino acid to which the sugar is bonded is serine and/or threonine (The aim of this study was to establish a novel analytical technique applicable to various PTMs of Ser and Thr. See pg. 9061 1st column, second paragraph. On the basis of this information, our model glycopeptide (peptide modified with T antigen at a single Ser residue. See Results. 1st paragraph under results section)).
Regarding Claim 4, Furukawa teaches the method according claim 1, wherein the substitution-modification step is a substitution reaction by a Michael addition reaction to an amino acid site from which the sugar has been eliminated by a ß-elimination reaction (Unlike most other glycan derivatization techniques, PMP derivatization proceeds under alkaline conditions. The optimal NaOH concentration is 200 400mM, which is compatible with the conditions of β-elimination employing a strong base. PMP derivatization of the released O-glycans proceeds faster than peeling degradation, thereby allowing the simultaneous release and labeling of O-glycans. In addition, since pyrazolone is a carbon carbon bond-forming Michael donor (referred to as the carbon-Michael donor),19 peptides formerly modified with phosphoric, sulfate, and various O-glycans are concomitantly labeled with the same reagent. See page 9061, 1st column, 2nd paragraph).).
Regarding Claim 5, Furukawa teaches the method according to claim 4, wherein the Michael addition reaction in the substitution-modification step is a reaction with a cyclic active methylene compound (we focused on carbohydrate derivatization chemistry with 1-phenyl-3-methyl-5-pyrazolone (PMP). See pg. 9061, 1st column, second paragraph).
Regarding Claim 6, Furukawa teaches the method according to claim 4, wherein the Michael addition reaction in the substitution-modification step is a reaction with a pyrazolone compound, a barbituric acid compound, a dimedone compound, or a hydroxycoumarin compound (Unlike most other glycan derivatization techniques, PMP derivatization proceeds under alkaline conditions. The optimal NaOH concentration is 200 400mM, which is compatible with the conditions of β-elimination employing a strong base. PMP derivatization of the released O-glycans proceeds faster than peeling degradation, thereby allowing the simultaneous release and labeling of O-glycans. In addition, since pyrazolone is a carbon carbon bond-forming Michael donor (referred to as the carbon-Michael donor),19 peptides formerly modified with phosphoric, sulfate, and various O-glycans are concomitantly labeled with the same reagent. See page 9061, 1st column, 2nd paragraph).
Regarding Claim 7, Furukawa teaches the method according to claim 4, wherein the Michael addition reaction in the substitution-modification step is a reaction with a pyrazolone compound (Unlike most other glycan derivatization techniques, PMP derivatization proceeds under alkaline conditions. The optimal NaOH concentration is 200 400mM, which is compatible with the conditions of β-elimination employing a strong base. PMP derivatization of the released O-glycans proceeds faster than peeling degradation, thereby allowing the simultaneous release and labeling of O-glycans. In addition, since pyrazolone is a carbon carbon bond-forming Michael donor (referred to as the carbon-Michael donor),19 peptides formerly modified with phosphoric, sulfate, and various O-glycans are concomitantly labeled with the same reagent. See page 9061, 1st column, 2nd paragraph).
Regarding Claim 8, Furukawa teaches the method according to claim 7, wherein the pyrazolone compound is 3-methyl-1-phenyl-pyrazolone (we focused on carbohydrate derivatization chemistry with 1-phenyl-3-methyl-5-pyrazolone (PMP). See pg. 9061, 1st column, second paragraph).
Regarding Claim 14, Furukawa teaches a kit for performing the method according to claim 1, the kit comprising: a cyclic active methylene compound (we focused on carbohydrate derivatization chemistry with 1-phenyl-3-methyl-5-pyrazolone (PMP). See pg. 9061, 1st column, second paragraph).
Regarding Claim 15, Furukawa teaches the kit according to claim 14, further comprising: a unit for purifying a fragmented protein (where cleavages of glycan moieties are dominant and structural information of peptide moieties are scarce, that of PMP-labeled peptide (m/z812.4) provided many fragments due to the cleavage of peptide bonds (Figure S-4c, Supporting Information), thus advantageous to identify the peptide identification and the O-glycan bonding site. These results demonstrate the feasibility of BEP for the analysis of. See pg. 9064, 1st column, last paragraph and 2nd column, 1st paragraph).
Regarding Claim 16, Furukawa teaches the method according to claim 1, wherein a labeled amino acid residue is identified in the range of the amino acid residue which has been found to glycosylated in the first mass spectrometry step by the subsequent substitution-modification step, the fragmentation step, and the second mass spectrometry step (.The simultaneous cleavage and labeling of O-glycan and the concomitant labeling of peptide moiety by BEP was also observed for a glycopeptide in which the O-glycan was attached to a single Thr residue, indicating that the described approach is applicable to O-GalNAc type modifications occurring on Ser or Thr (data not shown). See pg. 9063, last paragraph of 2nd column, pg. 9064, 1st column, 1st paragraph).
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Furukawa (“A Versatile Method for Analysis of Serine/Threonine Posttranslational Modifications by B-Elimination in the Presence of Pyrazolone Analogues.” Analytical Chemistry. 83:9060-9067. 2011), in view of Shinohara (WO 2012111775).
Regarding Claim 9, Furukawa teaches the method according to claim 1.
Furukawa is silent to the sugar is N-acetylglucosamine.
Shinohara teaches in the related art of post-translational modification analysis of serine and threonine. See Abstract. In particular, a glycan bonded through an O atom on the amino acid side (hereinafter, a glycan bonded through an O atom on the amino acid side is sometimes referred to as an O-glycan. Similarly, it is bonded through an O atom on the amino acid side. Phosphoric acid group and sulfuric acid group may be referred to as O-phosphoric acid group and O-sulfuric acid group, respectively.) As sugars bonded to O atom, N-acetylgalactosamine, mannose, fucose, N-acetylglucosamine. Those starting from either xylose or glucose, or sugars binding to the O atom, starting from any of N-acetylgalactosamine, mannose, fucose, xylose and glucose, and further extended by binding of sugars may be used.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have used N-acetylglucosamine, as taught by Shinohara, as an alternative sugar with an O-glycan, as taught by Shinohara.
Claims 10, 11, and 12 are rejected under 35 U.S.C. 103 as being unpatentable over Furukawa (“A Versatile Method for Analysis of Serine/Threonine Posttranslational Modifications by B-Elimination in the Presence of Pyrazolone Analogues.” Analytical Chemistry. 83:9060-9067. 2011), in view of Jin (WO 2018/035059).
Regarding Claims 10, 11, and 12, Furukawa teaches the method according to claim 1.
Furukawa is silent to wherein the protein is a capsid of a virus, the virus is an adeno-associated virus, the virus is an adeno-associated virus 5 referred to as AAV5.
Jin teaches in the related art of virus particles. [0076] encapsulated in a viral particle, particularly an AAV particle. A rAAV vector can be packaged into an AAV capsid to generate a "recombinant adeno-associated viral particle (rAAV particle)". [0077] An "rAAV virus" or "rAAV viral particle" refers to a viral particle composed of at least one AAV capsid protein and an encapsidated rAAV vector genome. [0204] To assay glycosylation, AAV particles with capsid proteins having a mutated 2.sup.nd position (e.g., iMet X) are used to infect cells (e.g., HeLa cells). AAV particles from infected cells are subjected to one or more assays including without limitation chemical detection of glycosylation (e.g., applying a commercially available digoxigenin (DIG) glycan detection and/or fluorescent glycoprotein detection kit on denatured and electrophoretically separated capsid proteins) and mass spectrometry (e.g., FT-ICR MS), as compared to wild-type AAV particles used to infect the same cell line (see, e.g., Murray, S. et al. (2006) . Virol. 80:6171- 6176 for a description of such assays).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have used a protein such as a capsid of a virus, the virus is an adeno-associated virus, the virus is an adeno-associated virus 5 referred to as AAV5, as taught by Jin, in the method of Furukawa, to allow for determining the serotype of a virus particle and/or or determining the heterogeneity of a virus particle (e.g., an AAV particle), as taught by Jin, See Abstract.
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Furukawa (“A Versatile Method for Analysis of Serine/Threonine Posttranslational Modifications by B-Elimination in the Presence of Pyrazolone Analogues.” Analytical Chemistry. 83:9060-9067. 2011).
Regarding Claim 13, Furukawa teaches the method according to claim 1 and The model glycopeptide or glycoprotein (10 μL, 0.1- 40 μg) (See pg. 9062, 1st column, 1st paragraph).
Furukawa is silent to an amount of the protein to be subjected to the spectrometry is 100 fmol or less.
A smaller amount of protein can be subjected to mass spectrometry. Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have used 100 fmol or less of protein subjected to the spectrometry to allow for a small amount of sample to be used and to save additional sample for other testing methods.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to JACQUELINE BRAZIN whose telephone number is (571)270-1457. The examiner can normally be reached M-F 8-5.
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/JB/
/CHARLES CAPOZZI/Supervisory Patent Examiner, Art Unit 1798
