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 .
DETAILED ACTION
Acknowledgement is hereby made of receipt and entry of the communication filed on July 02, 2026. Claims 1, 4-11, 13-14, 16-17, 21-22, 24 and 26 are pending and are currently examined.
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.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
(Previous rejection- withdrawn) Claims 1-11, 13-14, 16-17, 21-22, 24 and 26 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
The rejection is withdrawn in view of the amendment filed on July 02, 2026.
Claim Rejections - 35 USC § 112 (Written Description)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
(Previous rejection- withdrawn) Claims 1-11, 13-14, 16-17, 21-22, 24 and 26 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The rejection is withdrawn in view of the amendment filed on July 02, 2026.
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.
(Previous rejection- withdrawn) Claims 1, 5-11, 13-14, 16-17, 21-22, 24 and 26 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Chen et al. (US 8,945,918 B2, patented on Feb. 3, 2025).
The rejection is withdrawn in view of the amendment filed on July 02, 2026.
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.
(New Rejection-necessitated by amendment) Claims 1, 5-11, 13-14, 16-17, 21-22, 24 and 26 are rejected under 35 U.S.C. 103 as being unpatentable over Chen et al. (US 8,945,918 B2, patented on Feb. 3, 2025) in view of Stoilov et al. (US20100233685 A1, published on Sep. 16, 2010).
The amended base claim 1 is an expression cassette for expressing a cap of AAV or a rep gene of AAV in an insect cell, comprising from 5' to 3' and operably linked:
a promoter capable of driving transcription in the insect cell;
an artificially constructed sequence;
an overlapping open reading frame missing a first translation start codon;
wherein the artificially constructed sequence does not comprise a translation start codon;
during a post-transcriptional processing process, through an alternative splicing function, a translation start codon AUG is formed in the artificially constructed sequence to regulate translation and expression of different protein encoding genes in the overlapping open reading frames, wherein the artificially constructed sequence comprises from 5' to 3' and operably linked: a first-5' -terminal intron donor, an adenine nucleotide, a second-5' -terminal intron donor, a 3 '-terminal intron receptor, a thymine nucleotide, and a guanine nucleotide, or the artificially constructed sequence comprises from 5' to 3' and operably linked: a first-5' -terminal intron donor, an adenine nucleotide, a thymine nucleotide, a second-5' -terminal intron donor, a 3 '-terminal intron receptor, and a guanine nucleotide.
Chen et al. teaches an expression cassette of 5' to 3' order for expressing a gene in insect cells with overlapping open reading frame (ORFs) (See Abstract) as claimed. It also teaches other elements required in the base claim as follows:
1). Teaches an insect cell-operable promoter (See e.g., Abstract).
2). Teaches a cassette expressing an AAV Cap gene encoding Cap proteins, VP1, VP2, and VP3 or a Rep gene encoding the Rep proteins, Rep78, Rep68, Rep52, and Rep40. Rep78 and (See column 5, lines 9-26).
3). Teaches an artificial intron sequence (See column 8, lines 58-67), which comprises the polh promoter and is inserted into the Rep or Cap coding sequence without containing the translation start codon (See column 9, lines 6-35).
4). Teaches that a translation start codon AUG is formed in the artificially constructed sequence to regulate translation and expression of different protein encoding genes by stating that in some aspects, the present teachings disclose using an artificial intron to express all three Cap proteins (VP1,VP2, and VP3) from a single Cap coding nucleotide without mutating the AUG translation initiation codon of VP1 protein (See column 15, lines 55-58), where the Cap gene is incorporated by an artificial intron sequence as an overlapping open-reading frame gene (See column 9, lines 19-25). The Fig. 2 of Chen below shows a post- transcriptional map of representative recombinant baculovirus expressing VP1, VP2, and VP3 within a single expression cassette. Mature VP1 mRNA is formed when the artificial intron is removed through splicing. RNA encoding VP2 and VP3 is transcribed from the promoter located inside the artificial intron (See column 7).
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5). As for the newly added 5' -terminal intron donor and 3 '-terminal intron receptor limitation, Chen teaches that in their experiments, an artificial intron comprising the polh promoter was designed using similar splicing donor and acceptor sequences. An artificial intron is inserted into the Rep78 sequence such that the Rep52 mRNA is transcribed from the polh promoter located inside the artificial intron, whereas the Rep78 pre-mRNA is transcribed from the p10 promoter located upstream from the Rep78 start codon. Upon removal by splicing of the artificial intron by the host cell, mature Rep78 mRNA is formed (See bridging column 17 and 18; Fig. 1 and below).
However, Chen does not specify the first and second 5' -terminal intron donor and the locations of an adenine nucleotide (A) and a thymine nucleotide (T), and a guanine nucleotide (G), where the A and TG are specifically separated and interspersed among two distinct 5'-terminal intron donors and a 3'-terminal intron receptor.
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Stoilov et al. teaches a cassette comprising a nucleic acid with alternative splice sites (e.g., an exon flanked by two introns, an intron with two 5' splice sites and one 3' splice site, an intron with one 5' splice site and two 3' splice sites, etc.) can be inserted into a cloning site (e.g., an intronic sequence containing multiple restriction sites) which disrupts (e.g., interrupts, splits, etc.) the start (ATG) codon (See [0054]). In an example, Stoilov et al. teaches that the GFP start codon is interrupted between the "A" and "T" nucleotides by a reporter intron carrying EcoRI and BamHI restriction sites (SEQ ID NO:2) See [0138] and Fig. 1 below), which teaches that a first intron (between 110 and 140 of Fig. 1) linked to A (adenine nucleotide) and a second intron (between 140-120 of Fig. 1) linked to TG (thymine nucleotide, and a guanine nucleotide) with two 5' splice sites and one 3' splice site. Here the “splice site,” in the context of a pre-mRNA molecule, refers to the short-conserved sequence at the 5' end (donor site) or 3' end (acceptor site) of an intron to which a spliceosome binds and catalyzes the splicing of the intron from the pre-mRNA. An “alternative splice site' refers to additional donor and acceptor splice sites that can be used in to create alternative mRNAs from a single pre-mRNA. Alter native splice sites can reside in either intronic or exonic sequences. The use of alternative splice sites in pre-mRNA splicing may produce splice variants which differ in the length of an exon or in the presence or absence of an exon (See [0050]). Stoilov et al. further teaches that the present invention provides reporter constructs which advantageously detect or monitor alternative pre-mRNA splicing with increased specificity, sensitivity, and versatility over currently available splicing reporter systems. In particular, the use of two fluorescent reporter molecules that are specific for the two alternative mRNAs generated by alternative splicing enables clear separation of alternative splicing events from other cellular processes such as transcription, translation, constitutive splicing, and the like (See [0044]).
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It would have been prima facie obvious for one having ordinary skill in the art before the effective filing date of the claimed invention to introduce the alternative pre-mRNA splicing construct of Stoilov into Chen’s expression cassette for expressing a gene comprising overlapping open reading frames in an insect cell as claimed. One of ordinary skill in the art would have been motivated to do so because Stoilov teaches that their construct provides more specificity and sensitivity roles, and there would be a reasonable expectation of success to develop such an expression cassette as claimed.
Thus, the invention as a whole was clearly prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention.
Regarding claim 5, Chen et al. teaches that the overlapping open reading frames is a cap gene of AAV or a rep gene of AAV (See column 9, lines 19-23).
Regarding claims 6-8, Chen et al. teaches that the insect cell-operable promoter of a cassette can be a p10 promoter or a polh promoter (See column 5, lines 5-9) (teach claim 6). Chen et al. teaches that in other configurations, a nucleic acid cassette can include a Cap gene in which a first ORF can be a VP1 ORF and a second or additional ORF can be a VP2/VP3 ORF, as described infra. In Some configurations, both the first and the second promoters of a nucleic acid cassette can be a polh promoter (See column 5, lines 21-26) (teach claim 7). Chen et al. also teaches that the AAV2 Rep coding sequence comprising an artificial intron comprising a polyhedrin (polh) promoter (See Fig. 8, column 7, lines 55-59), where the Rep is an overlapping gene (See column 9, lines 19-22) (teach claim 8).
Regarding claims 9 and 10, Chen et al. teaches that the present teachings also include methods of expressing multiple genes in an insect cell, wherein each gene comprises overlapping ORFs. These methods comprise providing one or more insect cells harboring both a first nucleic acid cassette comprising a first gene, a first insect-operable promoter and an intron comprising a second insect-operable promoter as described herein, and a second nucleic acid cassette comprising a second gene, a third insect-operable promoter and a second intron comprising a fourth insect-operable promoter. In various aspects, the cassettes of these methods can be comprised by the same or different nucleic acids (See column 6, lines 46-59).
Regarding claim 11, Chen et al. teaches that the second cassette is in an anti-sense orientation relative to the first cassette (See column 36, claim 24).
Regarding claims 13-14, Chen et al. teaches a transgene/exogenous gene and AAV ITRS at in some configurations, an insect cell can further include an additional nucleic acid comprising a transgene of interest to be expressed by the host insect cell. Such a nucleic acid can comprise, in some aspects, an additional cassette comprising, in 5' to 3' order, a first inverted terminal repeat (ITR) of an AAV, a mammalian cell-operable promoter, a transgene, a polyadenylation signal, and a second ITR of an AAV (See column 5, lines 60-67), where the transgene can be a reporter gene. Such as a chloramphenicol acetyltransferase, a B-galactosidase, a B-glucoronidase, a renilla luciferase, a firefly luciferase, a green fluorescent protein (GFP), or a red fluorescent protein (RFP) (See column 6, lines 2-12).
Regarding claims 16-17, Chen et al. teaches a mammalian cell-operable promoter; a transgene, and a second ITR of an AAV. In various configurations, one or more of the cassettes and the additional nucleic acid can be comprised in one or more vectors (See column 7, lines 5-9) (claim 16), and the results, presented in Table 1, show that high titers of rAAV vectors can be produced in Sf9 cells using the recombinant baculoviruses that carry the Rep and Cap coding sequences comprising the artificial intron, respectively (See column 19, lines 48-52), where the engineered baculovirus can be the insect cytocompatible vector (teach claim 17).
Regarding claims 21-22, Chen et al. discloses nucleic acid cassettes for expressing in an insect cell (See Abstract) (claim 21), and a nucleic acid comprising a transgene of interest or a cassette comprising a gene having multiple ORFs such as a Rep gene and/or a Cap gene of an AAV can be integrated into the genome of a host insect cell (See column 6, paragraph 2) (claim 22).
Regarding claims 24 and 26, Chen et al. teaches that they demonstrate that VP1, VP2, and VP3 proteins are properly packaged in virions by using AAV Cap coding sequences comprising the artificial intron (See Example 6, column 22) (claim 26), and these methods further comprise culturing the insect cells in a culture medium (See claim 24).
(New Rejection-necessitated by amendment) Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Chen et al. (US 8,945,918 B2, patented on Feb. 3, 2025) and in view of Stoilov et al. (US20100233685 A1, published on Sep. 16, 2010) as applied to claims 1, 5-11, 13-14, 16-17, 21-22, 24 and 26 above and further in view of S. Clancy (Nature Education 1(1):31, 2008) as evidenced by RNA splicing (Wikipedia, https://en.wikipedia.org/wiki/RNA_splicing).
Regarding claim 4, Chen et al. teaches that the 5'-terminus nucleotides of the intron sequences of SEQ ID NO. 1 (See Table 1) has 5'-terminus nucleotide GTNN and 3'-terminus nucleotides NNAG. Chen et al. also teaches that 12 oligos are used to form the artificial intron (See table 2 below and column 18, lines 20-65), where SEQ ID NO: 5 has the GTNN, and SEQ ID NOs: 2 and 8 have the NNAG.
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Nevertheless, Clancy teaches that most commonly, the RNA sequence that is removed begins with the dinucleotide GU at its 5′ end, and ends with AG at its 3′ end. These consensus sequences are known to be critical, because changing one of the conserved nucleotides results in inhibition of splicing. Another important sequence occurs at what is called the branch point, located anywhere from 18 to 40 nucleotides upstream from the 3′ end of an intron (See page 1, paragraph 4). This teaching can be evidenced by Wikipedia ‘s RNA splicing. Wikipedia teaches that within introns, a donor site (5' end of the intron), a branch site (near the 3' end of the intron) and an acceptor site (3' end of the intron) are required for splicing. The splice donor site includes an almost invariant sequence GU at the 5' end of the intron, within a larger, less highly conserved region. The splice acceptor site at the 3' end of the intron terminates the intron
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with an almost invariant AG sequence (See page 1, paragraph 4 and below).
It would have been prima facie obvious for one having ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Chen and Clancy to arrive at the invention as claimed. Because Clancy teaches that the consensus sequences are known to be critical for RNA splicing, one of skill in the art would have been motivated to introduce the GTNN at 5’-teminus and NNAG at 3’-teminus based on Clancy’s teachings, and there would be a reasonable expectation of success to develop an expression cassette as claimed.
Responses to Applicant’s Remarks
Applicant’s arguments filed on July 02, 2026 has been received and fully considered.
1). Applicant’s amendments regarding the rejection under 35 U.S.C. 112(b) is considered and the rejection is withdrawn.
2). Applicant’s amendment regarding the rejection under 35 U.S.C. § 112 (a) is considered and the rejection is withdrawn.
3). Applicant’s amendment regarding the rejection under 35 U.S.C. 102 is considered and the rejection is withdrawn.
5). Applicant’s amendments and arguments regarding the rejection under 35 U.S.C. 103 are not persuasive as follows:
(i). Applicant argued that Chen completely fails to teach or suggest that the individual nucleotides of the translation start codon (A, T, and G) are specifically separated and interspersed among two distinct 5'-terminal intron donors and a 3'-terminal intron receptor (See Remarks, page 10).
Applicant’s argument is not persuasive.
First, based on applicant’s amendment, Chen was withdrawn as a reference for the rejection under 35 U.S.C. 102. Chen is used as a prior art reference under 35 U.S.C. 103 rejection.
Second, Chen teaches using an artificial intron comprising the polh promoter was designed using similar splicing donor and acceptor sequences as reported by Chisholm and Henner J Virol. 62(9):3193-3200 (1988) (See bridging columns 17 and 18), which indicates a similar structure as claimed is used in Chen’s experiment. In addition, the description also indicates that the donor and acceptor technique is a common technology in the art. One of skilled in the art can develop a comparable 5'-terminal intron donors and a 3'-terminal intron receptor system based on the routine experimental optimization.
Third, a support reference of Stoilov is cited for addressing the detailed 5'-terminal intron donors and a 3'-terminal intron receptor system as claimed under 35 U.S.C. 103 rejection. For example, the Fig. 1 of Stoilov teaches the “A and “TG” are separated by two introns among the marked numbers of 110, 140 and 120.
(ii). Applicant argued that Stoilov is entirely devoid of any teaching or suggestion of the complex, dual-donor architecture defined in the amended claim 1. Stoilov utilizes a single 5' splice site and a single 3' splice site to simply interrupt the start codon, acting merely as a binary switch (the AUG is either assembled or not assembled) (See Remarks, page 10).
Applicant’s argument is not persuasive.
Stoilov teaches that “An ‘alternative splice site' refers to additional donor and acceptor splice sites that can be used in to create alternative mRNAs from a single pre-mRNA” (See 0050]), which can include a second donor sequence. For example, the Fig. 1 of Stoilov teaches the “A and “TG” are separated by two introns among the marked numbers of 110, 140 and 120.
(iii). Applicant argued that a person having ordinary skill in the art would have no motivation to modify Chen's expression cassette by incorporating Stoilov's simple A-intron-TG reporter construct to arrive at the Applicant's highly complex, multi-donor design. Such a combination would completely fail to achieve the precise regulation of the stoichiometric ratio of multiple overlapping proteins (e.g., AAV VP1:VP2:VP3) (See remarks, page 11).
Applicant’s argument is not persuasive.
First, Chen teaches an artificial intron comprising the polh promoter was designed using splicing donor and acceptor sequences, where artificial intron is inserted into the Rep78 sequence such that the Rep52 mRNA is transcribed from the polh promoter located inside the artificial intron, whereas the Rep78 pre-mRNA is transcribed from the p10 promoter located upstream from the Rep78 start codon. Upon removal by splicing of the artificial intron by the host cell, mature Rep78 mRNA is formed (See bridging columns 17 and 18). Here the description teaches the intricate spatial layout employing the 5'-terminal intron donors and the 3'-terminal intron receptor. Based on the teaching of Chen, one of ordinary skill art can insert additional 5'-terminal intron donors to control the gene splicing to express the four splicing proteins of Rep78 or 68 and Rep52 or 40. Here the 3'-terminal intron receptor obviously needs to be shared for these 5'-terminal intron donors because they are the overlapping ORFs (See column 7, Fig. 1; column 5).
Second, Stoilov, as a combination reference to Chen, teaches the details for how the “donor” and “receptor” works (See Fig. 1 below) and discloses that an “alternative splice site' refers to additional donor and acceptor splice sites that can be used in to create alternative mRNAs from a single pre-mRNA (See [0050]). Stoilov teaches that their invention provides reporter constructs which advantageously detect or monitor alternative pre mRNA splicing with increased specificity, sensitivity, and versatility over currently available splicing reporter systems. In particular, the use of two fluorescent reporter molecules that are specific for the two alternative mRNAs generated by alternative splicing enables clear separation of alternative splicing events from other cellular processes such as transcription, translation, constitutive splicing, and the like (See 0044).
Accordingly, a person having ordinary skill in the art would have motivation to modify Chen's expression cassette by incorporating Stoilov's simple A-intron-TG reporter construct to arrive at the Applicant's highly complex, multi-donor design.
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Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any extension fee pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to RUIXUE WANG whose telephone number is (571)272-7960. The examiner can normally be reached Monday-Friday 8:00 am-5:00 pm, EST.
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/RUIXUE WANG/Examiner, Art Unit 1672
/NICOLE KINSEY WHITE/ Primary Examiner, Art Unit 1672
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