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 .
Election/Restrictions
Applicant’s election without traverse of “Group II: Claims 18-28, drawn to a method of using an rAAV particle” in the reply filed on June 17, 2026 is acknowledged.
Claims 1-17 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on June 17, 2026.
Priority/Effective Filing Date
The present application was filed on April 29, 2024. No priority claims are present in the present application as of August 2026.
Claim Status and Action Summary
This action is in response to the papers filed on June 17, 2026.
Claims 1-28 are currently pending. Claims 1-17 are withdrawn as being drawn to a nonelected invention. Claims 18-28 are under examination.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code at least on pages 9, 10, 21, 23, and 26 of the specification. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
The disclosure is objected to because of the following informalities:
In paragraph 0054, the specification references “FAM, TAMARA, and FAM fluorophores, respectively”. It is noted that “TAMARA… fluorophore” appears to be a typographical error of “TAMRA”, which is a well-known fluorophore in the molecular biology and virology arts.
Appropriate correction is required.
The use of the terms “Qiagen”, “QIAcuity”, “GenScript”, “Thermo Fisher”, “ATCC”, “DMEM”, “PENSTREP”, “Aldevron”, “Corning”, “PEIpro”, “Polyplus”, “Sartorius”, “Capture Select”, “POROS”, “AKTA PURE”, “Cytiva”, “Polysciences”, “Gibco”, “Amicon”, “Ultracel”, “Progen”, “BioTek”, “New England Biolabs”, “Teknova”, “IDT”, “Nanoplate”, “Revvity”, “GoldBio”, and “Operetta”, which are each a trade name or a mark used in commerce, has been noted in this application. Each term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claim 18 recites “AAV ITRS”. Claim 22 recites and the specification defines “AAV ITR” and “ITRs” (see specification paragraph 0033). It appears that “ITRS”, recited by claim 18, is a typographical error for the plural form of the acronym “ITR” described elsewhere in the claims and specification, and not an additional, unspecified acronym “ITRS” requiring something different from, or more than, “Inverted Terminal Repeats”.
Appropriate correction is required.
Claim 19 is objected to because of the following informalities: “The method of claim 18, wherein four or more PCR targets comprise…” appears to omit the definite article “the” between “wherein” and “four or more” to refer back to the “four or more PCR targets” recited by claim 18.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
Claims 18-25 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
As presently written, claim 18 requires only that that the “reference sample contain[s] a rAAV particle comprising… a rAAV genomic sequence comprising… four or more PCR targets”. As such, claim 18 does not appear to positively recite a step of “amplifying” or “quantifying” or “determining” or “performing quantitative PCR” on any particular one or more of the “PCR targets” (i.e. a structure) comprised by the rAAV genomic sequence. Therefore, the claim appears to encompass embodiments wherein: i) the PCR target(s) are selected from any nucleic acid sequence flanked by the two AAV ITRS, and ii) the “performing a quantitative PCR assay” requires only amplification of a single amplicon of the four or more PCR targets.
Claims 19-25 are also indefinite because they depend from, and thus include the indefinite limitations of the claims rejected as indefinite above.
Furthermore, Claim 25 recites “the rAAV genomic sequence has at least 90% sequence identity with SEQ ID NO: 1”. As presently written, it appears that the claim language broadly encompasses embodiments wherein the rAAV genomic sequence established in claims 18-24 comprises two AAV ITRs that flank four or more PCR targets, selected from “nucleic acid fragments of” [the genera recited in claim 20]. It is unclear whether the claim is intended to encompass rAAV genomic sequences comprising features that have at least 90% identity to nucleic acid fragments of the recited “targets” within the sequence of SEQ ID NO: 1, or whether the claim is intended to require that the rAAV genomic sequence is at least 90% identical to SEQ ID NO: 1 (i.e. not merely that the claimed rAAV genomic sequence comprises (i.e. “has”) fragments that are 90% or more identical to fragments of SEQ ID NO: 1).
Claim Interpretation
Claim 1 recites the claim term “reference standard”. It is noted that the specification provides the following special definition: “The terms “positive control” and “standard” mean, in the context of quantitative PCR, a control template for the polymerization reaction that provides a positive reaction demonstrating that the reaction conditions and assay are correct (e.g., lack of inhibition)” (Specification, paragraph 0035).
Claim 1 recites the claim term “ITRS”. The specification defines: ““ITR” means, within the AAV genome, inverted terminal repeat sequences.” (Specification, paragraph 0033).
Claim 20 recites the claim terms, “CMV”, “SV40”, “bGH”, “hGH”, “WPRE”, and “eGFP”. The specification defines: “bGH” means… bovine growth hormone… “WPRE” means a woodchuck hepatitis virus posttranscriptional regulatory element. “CMV” means a cytomegalovirus. “eGFP” means an enhanced green fluorescent protein… “SV40” means simian virus 40.” (Specification, paragraph 0034).
Claim 25 recites “the rAAV genomic sequence has at least 90% sequence identity with SEQ ID NO: 1”. Under the broadest reasonable interpretation of the plain meaning of the claim as presently written, it appears that the claim language broadly encompasses embodiments wherein the rAAV genomic sequence established in claims 18-24 comprises two AAV ITRs that flank four or more PCR targets, selected from “nucleic acid fragments of” [the genera recited in claim 20] wherein the fragments have at least 90% identity to nucleic acid fragments of the recited “targets” within the sequence of SEQ ID NO: 1.
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.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 18-24 and 26 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Wagner et al., “A Novel Method for the Quantification of Adeno-Associated Virus Vectors for RNA Interference Applications Using Quantitative Polymerase Chain Reaction and Purified Genomic Adeno-Associated Virus DNA as a Standard” Human Gene Therapy Methods 24:355-363 (December 2013) as evidenced by FPBase “Emerald” https://web.archive.org/web/20190818200247/https://www.fpbase.org/protein/emerald/ (published 2019, accessed Aug. 22, 2026).
Regarding claim 18, Wagner et al. teach methods for quantitation of recombinant adenovirus associated virus (rAAV) vectors using virus genomic DNA extracted from purified rAAV particles as a standard (Wagner et al., Abstract). Wagner et al. teach the rAAV genome (i.e. flanked by inverted terminal repeats (ITRs)) comprises four PCR targets (amplified with four primer sets targeting different regions within the AAV vector genome (VG)) (Wagner et al., Abstract). Wagner et al. teach determining the genome copy titer of rAAV-containing samples by qPCR comprising comparison to a standard curve constructed from serial 10-fold dilutions of the standardized AAV reference sample (Wagner et al., page 357, column 1-2 bridging paragraph).
Regarding claim 19, Wagner et al. teach four or more PCR targets comprise fragments of PU6 promoter, PCMV promoter, emGFP (i.e. a gene sequence), and/or a shRNA (i.e. a gene sequence).
Regarding claim 20, Wagner et al. teach the rAAV reference comprises sequences (i.e. PCR targets) comprising any number of nucleic acid fragments from a: CMV promoter and an emerald GFP (emGFP). FPBase teaches that emGFP is a green fluorescent protein that was derived from eGFP with four amino acid substitutions (i.e. emGFP is an eGFP).
Regarding claim 21, Wagner et al. teach the AAV rep and cap proteins are AAV serotype 2 (Wagner et al., page 357, column 1, paragraph 2).
Regarding claims 22-24, Wagner et al. teach the AAV ITRs flank a transgene encoding a functional protein that is a reporter protein that is emGFP (i.e. an eGFP) (Wagner et al., Figure 5).
Regarding claim 26, Wagner et al. teach using rAAV particles as a reference standard for an analytical assay demonstrating the expression of emGFP (i.e. a reporter protein encoded by the rAAV genome), wherein host cells are infected with rAAV particles comprising AAV-2 rep and cap proteins, and a rAAV genome comprising two AAV ITRs that flank four or more PCR targets including fragments of a CMV promoter and an emGFP (i.e. an eGFP) gene that encodes a functional reporter protein (emGFP) (Wagner et al., Figures 1 and 5).
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 18-24 and 26-28 are rejected under 35 U.S.C. 103 as being unpatentable over Wagner et al., “A Novel Method for the Quantification of Adeno-Associated Virus Vectors for RNA Interference Applications Using Quantitative Polymerase Chain Reaction and Purified Genomic Adeno-Associated Virus DNA as a Standard” Human Gene Therapy Methods 24:355-363 (December 2013) in view of: D’Costa et al., “Practical utilization of recombinant AAV vector reference standards: focus on vector genomes titration by free ITR qPCR” Molecular Therapy-Methods & Clinical Development (2016), 16019, Girard et al., WO 2019/195729 A1, and Zhu et al., “A Novel Adeno-Associated Virus-Based Genetic Vaccine Encoding the Hepatitis C Virus NS3/4 Protein Exhibits Immunogenic Properties in Mice Superior to Those of an NS3-Protein-Based Vaccine” PLoS ONE 10(11):e0142349 (November 2015).
Regarding claim 18, Wagner et al. teach methods for quantitation of recombinant adenovirus associated virus (rAAV) vectors using virus genomic DNA extracted from purified rAAV particles as a standard (Wagner et al., Abstract). Wagner et al. teach the rAAV genome (i.e. flanked by inverted terminal repeats (ITRs)) comprises four PCR targets (amplified with four primer sets targeting different regions within the AAV vector genome (VG)) (Wagner et al., Abstract). Wagner et al. teach determining the genome copy titer of rAAV-containing samples by qPCR comprising comparison to a standard curve constructed from serial 10-fold dilutions of the standardized AAV reference sample (Wagner et al., page 357, column 1-2 bridging paragraph)
Regarding claims 19-20, Wagner et al. teach four or more PCR targets comprise fragments of PU6 promoter, PCMV promoter, emGFP (i.e. a gene sequence), and/or a shRNA (i.e. a gene sequence). Wagner et al. teach the rAAV reference comprises sequences (i.e. PCR targets) comprising any number of nucleic acid fragments from a: CMV promoter and an emerald GFP (emGFP).
Wagner et al. do not explicitly teach that all four of the PCR targets are flanked by the AAV ITRs (Wagner et al. utilize a set of primers that are specific to the 5’ AAV ITR).
However, D’Costa et al. teach quantitation of AAV ITRs is negatively influenced by the secondary structure of said ITRs without nucleolytic cleavage of the ITRs (D’Costa et al., Table 1 and abstract). D’Costa et al. teach nucleolytic cleavage of the secondary structures and additional qPCR amplicons (transgene SV40 polyadenylation sequence, GFP transgene, and digested ITR5’ and ITR3’) confirm the apparent discrepancy inherent to measures of native-structured ITR2 relative to qPCR measures of AAV2 transgene sequences (D’Costa et al., Abstract and page 8, column 1-2 bridging paragraph).
Furthermore, Girard et al. teach that each of the following nucleic acid constituents are conventional components of AAV2 vectors with known advantages for controlling expression of transgenes encoded on the AAV2 genome (i.e. flanked by AAV2 ITRs): Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), which “is important for high-level expression of native mRNA transcripts” (Girard et al., paragraph 0142), bovine growth hormone (bGH) polyadenylation sequence, which “is used to enhance gene expression… higher than other polyA sequences” (Girard et al., paragraph 0143), Cytomegalovirus (CMV) enhancer (Girard et al., paragraph 0141), and an eGFP transgene for measuring transduction efficiency (Girard et al., paragraph 0323).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have selected any one of these known advantageous rAAV constituent sequences as an additional, or alternative qPCR target in the methods taught by Wagner et al. and D’Costa et al. The ordinary artisan would have been particularly motivated to select an additional non-ITR qPCR target for the four amplicon methods taught by Wagner et al. by the teachings of D’Costa et al. that accurate quantitation of the rAAV2 ITRs requires an additional step of nucleolytic cleavage of the ITR secondary structures, as the secondary structure was shown to contribute to inconsistent quantitation in qPCR relative to non-ITR structured amplicons in the rAAV2 genome.
Regarding claim 21, Wagner et al. teach the AAV rep and cap proteins are AAV serotype 2 (Wagner et al., page 357, column 1, paragraph 2).
Regarding claims 22-24, Wagner et al. teach the AAV ITRs flank a transgene encoding a functional protein that is a reporter protein that is emGFP (i.e. an eGFP) (Wagner et al., Figure 5). Additionally, both D’Costa et al. (D’Costa et al., table 3) and Girard et al. (Girard et al., paragraph 0323) teach that eGFP is a useful marker for determining ITR2 titration by qPCR and for determining infectivity by observing GFP expression in cells following infection with the rAAV2 standard.
Regarding claim 26, Wagner et al. teach using rAAV particles as a reference standard for an analytical assay demonstrating the expression of emGFP (i.e. a reporter protein encoded by the rAAV genome), wherein host cells are infected with rAAV particles comprising AAV-2 rep and cap proteins, and a rAAV genome comprising two AAV ITRs that flank four or more PCR targets including fragments of a CMV promoter and an emGFP (i.e. an eGFP) gene that encodes a functional reporter protein (emGFP) (Wagner et al., Figures 1 and 5).
Regarding claims 27-28, Wagner et al. do not teach that the host cell is HepG2.
However, Zhu et al. teach HepG2 cells are a useful study system to confirm infectivity of AAV2 vectors encoding an eGFP reporter gene prior to animal trials of a HCV vaccine formulation comprising AAV2-expression of HCV NS3/4 protein (antigens) in host cells (Zhu et al., page 3, paragraph 3).
Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have utilized the rAAV-2 standards for analytical assays for reporter gene expression (i.e. confirming/quantifying rAAV particle infectivity) as taught by Wagner et al. in the specific host cells (HepG2) recited by claim 27 and 28 because of the teaching of Zhu et al. that rAAV-2 encoding HCV NS3/4 and eGFP (as a reporter gene) was effectively evaluated for in vitro infectivity on HepG2 cells prior to transduction into live mice as a trial of immunization against HCV infection.
Claim 25 is rejected under 35 U.S.C. 103 as being unpatentable over Wagner et al. in view of D’Costa et al., Girard et al., and Zhu et al. as applied to claims 18-24 and 26-28 above, and further in view of Choi et al., “Optimization of AAV expression cassettes to improve packaging capacity and transgene expression in neurons” Molecular Brain 7, 17 (2014), and GenBank KJ411917.1, KJ411915.1, and KJ411912.1.
Under the broadest reasonable interpretation of the plain meaning of the claim as presently written, it appears that the claim language broadly encompasses embodiments wherein the rAAV genomic sequence established in claims 18-24 comprises two AAV ITRs that flank four or more PCR targets, selected from “nucleic acid fragments of” [the genera recited in claim 20] wherein the fragments have at least 90% identity to nucleic acid fragments of the recited “targets” within the sequence of SEQ ID NO: 1.
Several fragments of the claimed SEQ ID NO: 1 have greater than 90% identity to known sequences (see NCBI BLAST results below and attached).
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584
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It is noted that the following Subject sequences identified by the BLAST described above include the following elements within the matching sequence regions (see Choi et al., figure 2, reproduced below for clarity:
KJ411917.1 “pAAV-CW3A-EGFP”: two flanking rAAV ITRs, eGFP, WPRE
KJ411915.1 “pAAV-CW3L-EGFP”: two flanking rAAV ITRs, eGFP, WPRE
KJ411912.1 “pAAV-CW3B-EGFP”: two flanking rAAV ITRs, eGFP, WPRE, bGHpA
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364
332
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While the constructs taught by Choi et al. utilize a cell-type specific CaMKII promoter to drive eGFP expression, Choi et al. explicitly state “In this study, we used the CaMKIIα promoter to drive transgene expression in neurons, but it would also be possible to substitute this promoter with other ubiquitous promoters, cell-type–specific promoters, or even conditional promoters for different applications.”
Wagner et al. teach various recombinant AAV standards comprising AAV ITRs flanking an eGFP driven by a strong CMV promoter (Wagner et al., figure 1).
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356
564
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Therefore, it would have been prima facie obvious prior to the effective filing date of the claimed invention for one of ordinary skill in the art to have combined the known rAAV vector sequences taught by Wagner et al. and Choi et al. each comprising fragments having >90% identity to fragments of the recited SEQ ID NO: 1 because of the teachings of Choi et al. that the WPRE and bGH polyadenylation sequences are optimized for expression of eGFP from a cell-type specific promoter and that said construct can readily be adapted to expression in other cell types by exchanging the “CaMKII” promoter with, for example, a “ubiquitous promoter” (e.g. the strong CMV promoter taught by Wagner et al.
Conclusion
No claim is allowed.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Aurnhammer et al., “Universal Real-Time PCR for the Detection and Quantification of Adeno-Associated Virus Serotype 2-Derived Inverted Terminal Repeat Sequences” Human Gene Therapy Methods” Part B 23:18-28 (February 20-12).
Ayuso et al., “Manufacturing and Characterization of a Recombinant Adeno-Associated Virus Type 8 Reference Standard Material” Human Gene Therapy 25:977-987 (November 2014).
Addgene “AAV Titration by qPCR Using SYBR Green Technology” March 24, 2023 Wayback Machine Archive
Thomas et al., “A Real-Time PCR Method to Rapidly Titer Adenovirus Stocks” Methods in Molecular Medicine, Vol. 130: Adenovirus Methods and Protocols, Second Edition, vol. 1: Adenoviruses Ad Vectors, Quantitation and Animal Models, pp 185-192 (2007)
Noyszewski et al., “A Novel Reference Standard for Assessing AAV Titer and Genome Integrity” ASGCT 28th Annual Meeting Abstracts Molecular Therapy, 33, pg. 1, 750-751 (2025)
Lock et al. (b), “Characterization of a Recombinant Adeno-Associated Virus Type 2 Reference Standard Material” Human Gene Therapy 21:1273-1285 (October 2010).
Penaud-Budloo et al., “Stability of the adeno-associated virus 8 reference standard material” Gene Therapy (2019) 26:211-215.
Rohr et al., “Fast and reliable titration of recombinant adeno-associated virus type-2 using quantitative real-time PCR” Journal of Virological Methods 106 (2002) 81-88.
Grieger et al., “Production of Recombinant Adeno-associated Virus Vectors Using Suspension HEK293 Cells and Continuous Harvest of Vector from the Culture Media for GMP FIX and FLT1 Clinical Vector” Molecular Therapy vol. 24, no. 2, 287-297 (February 2016)
Grimm et al., “Titration of AAV-2 particles via a novel capsid ELISA: packaging of genomes can limit production of recombinant AAV-2” Gene Therapy 6, 1322-1330 (1999)
Lim et al., “Measuring rAAV Genomic Titer, Viral Particle Titer and Full/Empty Capsid Ratio on the Applied BioSystems QuantStudio Absolute QTM dPCR System” Molecular Therapy Vol 31 no4s1, pp 1, 384-385 (April 2023)
Lock et al., (a) “Rapid, Simple, and Versatile Manufacturing of Recombinant Adeno-Associated Viral Vectors at Scale” Human Gene Therapy 21:1259-1271 (October 2010).
Rohr et al., “Quantitative real-time PCR for titration of infectious recombinant AAV-2 particles” Journal of Virological Methods 127 (2005) 40-45
US 2021/0062161 A1 (Vink)
US 2014/0287938 A1 (Zhang)
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/Z.M.T./Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682
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