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 .
Office Action: Notice
Any objection or rejection of record in the previous Office Action, mailed 3/3/2026,
which is not addressed in this action has been withdrawn in light of Applicants' amendments
and/or arguments. This action is a second NON-FINAL.
Election/Restrictions
Applicant’s election with traverse of Group I, claims 16-21, in the reply filed on November 26, 2025 is acknowledged. Claims 22-30 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected Groups 2 through 4, there being no allowable generic or linking claim. Election was made with traverse in the reply filed on 11/26/2025.
Applicant’s traversal is not persuasive. The present application is being examined as a national stage application under 35 USC 372 guidelines, and therefore the determination of unity by the International Searching Authority is not binding to the USPTO (see MPEP 1893.03 (d)). The claims continue to be directed to distinct inventions including (i) a method of isolating a replication origin, (ii) nucleic acid compositions and vectors comprising the origin, (iii) a method of gene expression, and (iv) a computer program product. Accordingly, the restriction (10/7/2025) requirement is maintained.
Thus, claims 16-21 are under examination (2/16/2023).
Claim Status
Claims 16-21 are under examination (5/27/2026). Claims 16 and 19-21 have been amended (5/27/2026). No new matter was added.
Priority
Claims 16-21 receive a priority date of 9/6/2021, the effective filing date of EP20305987.8.
Objections Withdrawn
Specification:
The objections to the specification due to the use of a trademark and browser code are withdrawn in view of Applicant’s amendments.
The objection to the specification due to the lack of Sequence Listing has been withdrawn in view of Applicant’s incorporation of a new Sequence Listing, to comply with 37 CFR 1.831(b).
Claims:
The objection to claims 16 and 19-21 in request to correct minor informalities, is withdrawn due to Applicant’s amendments.
Rejections Withdrawn
Claim Rejections - 35 USC § 112(b)
The rejections of claims 16-21 under 35 U.S.C. 112(b) or pre-AIA 35 U.S.C. 112, 2nd paragraph, are withdrawn in view of Applicant’s amendments of claim 16 to correct lack of antecedent basis.
Claim Rejections - 35 USC § 102 (a)(1), 102 (a)(2)
The rejections of claims 16-21 under 35 U.S.C. 102(a)(1) and 102 (a)(2) as being anticipated by Nadimpalli et al. (US PGPub 2003/0005483 A1; published 1/2/2003), are withdrawn in view of Applicant’s arguments (5/27/2026). Specifically, the Applicant’s arguments are persuasive because Nadimpalli’s disclosure of nucleic-acid probes and general sequence/GC-content parameters does not disclose the specific claimed process of dividing genomic DNA into 500-b- windows at 100-bp intervals and identifying windows according to the recited nucleotide-content and G-content criteria. Accordingly, Nadimpalli does not disclose each and every limitation of claim 16 as arranged in the claim, and the rejection under 35 USC 102 is withdrawn.
Rejections Maintained
Claim Rejections—35 USC § 103
Claims 16-21 are rejected under 35 U.S.C. 103 as being unpatentable over Nadimpalli et al. (US PGPub 2003/0005483 A1; published 1/2/2003), in view of Bartholdy et al. (“Allele-specific analysis of DNA replication origins in mammalian cells”, Nature Communications, published 5/19/2015) and in further view of Rhodes et al., (“G-quadruplexes and their regulatory roles in biology”, Nucleic Acids Research, 2015).
Regarding claims 16-17, Nadimpalli teaches isolated prolifera nucleic acids and their encoded proteins, including methods and compositions relating to altering prolifera levels in plants (i.e., invention further provides recombinant expression cassettes, host cells, transgenic plants, and antibody compositions) (Abstract). Nadimpalli incorporates in these teachings, an isolated nucleic acid comprising a member selected from the group consisting of (a) a polynucleotide having a specified sequence identity to a polynucleotide encoding a polypeptide of the present invention; (b) a polynucleotide which is complementary to the polynucleotide of (a); and, (c) a polynucleotide comprising a specified number of contiguous nucleotides from a polynucleotide of (a) or (b) where the isolated nucleic acid can be DNA (Paragraph 9, lines 1-5). Specifically, Nadimpalli teaches that replication control can be modified on a more local level, thereby allowing specific replication origins in some parts of the genome to be activated repeatedly providing a selective advantage for gene amplification and in Drosophila, chondrion genes have been shown to be selectively amplified by this process, where selectively expressing a prolifera polynucleotide of the present invention with the Origin Recognition Complex (ORC), at a set of replication origins of specific genes, copy number in cells can be controlled (Paragraph 86, lines 1-5).
Nadimpalli teaches that the previously shown method conditions are sequence-dependent and will be different in different circumstances and by controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing) and can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing) where generally, a probe is less than about 1000 nucleotides in length, optionally approximately 500 nucleotides in length (Paragraph 61, lines 1-5).Nadimpalli also teaches that the previously explained method includes polynucleotides that can encode a polypeptide having a subsequence having at least 10, 15, 20, 25, 30, 35, 40, 45, or 50, contiguous amino acids from the prototype polypeptide and where further, the number of such subsequences encoded by a polynucleotide of the instant embodiment can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4, or 5 and the subsequences can be separated by any integer of nucleotides from 1 to the number of nucleotides in the sequence such as at least 5, 10, 15, 25, 50, 100, or 200 nucleotides (Paragraph 118, lines 5-10). Nadimpalli also teaches that complementary bases associate through hydrogen bonding in double stranded nucleic acids where for example, the following base pairs are complementary: guanine and cytosine; adenine and thymine; and adenine and uracil (Paragraph 124, lines 1-5). Further, Nadimpalli teaches that as indicated in (g), above, the present invention provides isolated nucleic acids comprising polynucleotides which comprise at least 15 contiguous bases from the polynucleotides of sections (A) through (F) as discussed above where the length of the polynucleotide is given as an integer selected from the group consisting of from at least 15 to the length of the nucleic acid sequence from which the polynucleotide is a subsequence of, for example, polynucleotides of the present invention are inclusive of polynucleotides comprising at least 15, 20, 25, 30, 40, 50, 60, 75, or 100 contiguous nucleotides in length from the polynucleotides of interest and optionally, the number of such subsequences encoded by a polynucleotide of the instant embodiment can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4, or 5, where the subsequences can be separated by any integer of nucleotides from 1 to the number of nucleotides in the sequence such as at least 5, 10, 15, 25, 50, 100, or 200 nucleotides (Paragraph 126, lines 1-10).
Nadimpalli teaches that for DNA-DNA hybrids, the content includes 41-61% of guanosine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs.
Nadimpalli teaches that in optional embodiments, the stringency allows for selective hybridization of sequences having at least 70%, 75%, 80%, 85%, 90%, 95%, or 98% sequence identity over the length of the hybridized region where full-length enriched cDNA libraries can be normalized to increase the representation of rare sequences (Paragraph 130, lines 10-15). Further, Nadimpalli teaches the primers are complementary to a subsequence of the target nucleic acid which they amplify but may have a sequence identity ranging from about 85% to 99% relative to the polynucleotide sequence which they are designed to anneal to and those skilled in the art will appreciate, the sites to which the primer pairs will selectively hybridize are chosen such that a single contiguous nucleic acid can be formed under the desired nucleic acid amplification conditions where the primer length in nucleotides is selected from the group of integers consisting of from at least 15 to 50 and thus, the primers can be at least 15, 18, 20, 25, 30, 40, or 50 nucleotides in length where those of skill will recognize that a lengthened primer sequence can be employed to increase specificity of binding (i.e., annealing) to a target sequence (Paragraph 109, lines 1-10). Further, Nadimpalli teaches that a non-annealing sequence at the 5'end of a primer (a "tail") can be added, for example, to introduce a cloning site at the terminal ends of the amplicon (Paragraph 109, lines 1-10).
Nadimpalli teaches that the expression of isolated nucleic acids encoding a protein of the present invention will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter (which is either constitutive or regulatable), followed by incorporation into an expression vector and the vectors can be suitable for replication and integration in either prokaryotes or eukaryotes where typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding a protein of the present invention (Paragraph 178, lines 1-10). Further, Nadimpalli teaches that to obtain high level expression of a cloned gene, it is desirable to construct expression vectors which contain, at the minimum, a strong promoter to direct transcription, a ribosome binding site for translational initiation, and a transcription/translation terminator and one of skill would recognize that modifications can be made to a protein of the present invention without diminishing its biological activity and some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located purification sequences (restriction sites or termination codons can also be introduced) (Paragraph 178, lines 10-20).
Nadimpalli also teaches that a variety of eukaryotic expression systems such as yeast, insect cell lines, plant and mammalian cells, are known to those of skill in the art (Paragraph 183, lines 1-5).
Regarding claim 18, Nadimpalli teaches that replication control can be modified on a more local level, thereby allowing specific replication origins in some parts of the genome to be activated repeatedly providing a selective advantage for gene amplification and in Drosophila, chondrion genes have been shown to be selectively amplified by this process, where selectively expressing a prolifera polynucleotide of the present invention with the Origin Recognition Complex (ORC), at a set of replication origins of specific genes, copy number in cells can be controlled (Paragraph 86, lines 1-5). Nadimpalli also teaches that the expression of isolated nucleic acids encoding a protein of the present invention will typically be achieved by operably linking, for example, the DNA or cDNA to a promoter (which is either constitutive or regulatable), followed by incorporation into an expression vector and the vectors can be suitable for replication and integration in either prokaryotes or eukaryotes where typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding a protein of the present invention (Paragraph 178, lines 1-10).
Regarding claims 19-21, Nadimpalli teaches that the previously shown method conditions are sequence-dependent and will be different in different circumstances and by controlling the stringency of the hybridization and/or washing conditions, target sequences can be identified which are 100% complementary to the probe (homologous probing) and can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing) where generally, a probe is less than about 1000 nucleotides in length, optionally approximately 500 nucleotides in length (Paragraph 61, lines 1-5). Nadimpalli also teaches that the previously explained method includes polynucleotides that can encode a polypeptide having a subsequence having at least 10, 15, 20, 25, 30, 35, 40, 45, or 50, contiguous amino acids from the prototype polypeptide and where further, the number of such subsequences encoded by a polynucleotide of the instant embodiment can be any integer selected from the group consisting of from 1 to 20, such as 2, 3, 4, or 5 and the subsequences can be separated by any integer of nucleotides from 1 to the number of nucleotides in the sequence such as at least 5, 10, 15, 25, 50, 100, or 200 nucleotides (Paragraph 118, lines 5-10).
Nadimpalli teaches that typical expression vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of the expression of the DNA encoding a protein of the present invention (Paragraph 178, lines 5-10). Further, Nadimpalli teaches that Further, Nadimpalli teaches the primers are complementary to a subsequence of the target nucleic acid which they amplify but may have a sequence identity ranging from about 85% to 99% relative to the polynucleotide sequence which they are designed to anneal to and those skilled in the art will appreciate, the sites to which the primer pairs will selectively hybridize are chosen such that a single contiguous nucleic acid can be formed under the desired nucleic acid amplification conditions (Paragraph 109, lines 1-10).
Nadimpalli does not teach or suggest identifying mammalian genomic DNA replication origins using the claimed 500-bp sliding-window analysis and the specific G/A/T-content thresholds and variations between adjacent windows, followed by functional selection of the identified fragment as a replication origin. Further, Nadimpalli does not teach or suggest that the 500 bp window sequence containing tandemly G4 structures (12x) or the resultant fragment containing histone acetylation marks.
Bartholdy teaches that mammalian DNA replication origins are characterized by asymmetric nucleotide composition, particularly pronounced G/C and A/T skew, and that strong replication origins contain large G-rich regions (Abstract). Specifically, Bartholdy teaches via analysis of G-content of 100-bp windows centered on replication-origin subpeaks and identifies approximately 200-500-bp G-rich regions associated with highly efficient origins (Origins are profoundly G/C and A/T skewed). Also, Bartholdy teaches that G-density and G/C skew are determined using 50-bp sliding windows with a one-basepair step, demonstrating the use of moving window nucleotide content analysis to characterize mammalian replication origins (Figure 6; Methods: GC-content and G-content analysis, Analysis of DNA skew).
Rhodes teaches that DNA and RNA G-quadruplexes are in various cellular pathways including DNA replication, gene expression and telomere maintenance (Abstract). Rhodes also teaches that G-quadruplex structures are topologically very polymorphic and can arise from the intra- or inter- molecular folding of G-rich strands and intra-molecular folding requires the presence of four or more G-tracts in one strand, whereas inter-molecular folding can arise from two or four strands giving rise to parallel or antiparallel structures depending on the orientation of the strands in a G-quadruplex (Introduction: Paragraphs 1-2). Specifically, Rhodes teaches that a recent investigation on the role of the RNA helicase eIF4A, using ribosome footprinting to provide snapshots of translation across the transcriptome, has revealed that the hallmark of eIF4A dependent transcripts is a 12-nucleotide pG4 signature (CGG)4 that can form RNA G-quadruplex structures (Figure 5; G-quadruplexes in transcription and translation: Paragraphs 2-3).
Rhodes also teaches that, based on observations from replication studies in avian DT40 cells, it was concluded that transcriptional silencing occurred via G-quadruplex impeded DNA replication (Figure 5) affecting transcription through the inappropriate inheritance of epigenetic histone marks where, epigenetic instability can arise from a G-quadruplex located at a distance of up to 3500 base pairs from the transcription start-site and although these observations provide compelling evidence for the involvement of pG4s in the regulation of gene expression in vivo, whether the mechanism for transcriptional regulation is directly through G-quadruplex formation remains to be proven (G-quadruplexes in transcription and translation: Paragraphs 2-3).
It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Nadimpalli, in view of Rhodes and Bartholdy. Nadimpalli teaches methods of identifying and isolating nucleic acid fragments associated with replication control, including selecting fragments based on sequence-dependent characteristics and defined fragment lengths influenced by sequence composition and specific genomic regions identification. Rhodes, however, teaches that G4 structures arise in G-rich nucleic acid regions are likewise implicated in DNA replication control within localized genomic regions, transcriptional regulation and chromatin-associated processes. Bartholdy further teaches that mammalian DNA replication origins exhibit pronounced G/C and A/T nucleotide-distribution skew and analyzes G-content in windows surrounding replication-origin peaks, identifying approximately 200-500 bp G-rich regions associated with strong replication origins.
Because Nadimpalli recognizes that replication control is sequence-dependent and Rhodes teaches that G-rich tandem G4 motifs affect replication and chromatin regulation, it would have been obvious to select or identify replication origin fragments containing tandem G-quadruplex-forming sequences or G-rich repeated elements as part of a method for isolating functional replication origins. Bartholdy further provides evidence that localized nucleotide composition, including G-content and G/C or A/T skew, is characteristic of mammalian replication origins. Thus, it would have been obvious to analyze localized windows of mammalian genomic DNA according to nucleotide composition to identify candidate replication-origin regions. The modification merely applies known sequence-structure relationships to the selection of replication-associated DNA fragments. Rhodes also teaches that G4 structures are associated with chromatin regulation and epigenetic effects, including interactions affecting histone modification and transcriptional silencing. Because replication origin activity and chromatin state are functionally linked, it would have been obvious to select or identify replication origin fragments containing polycomb binding sites or histone acetylation marks in order to further influence replication initiation and gene regulation, which reflects the predictable application of established principles in molecular biology.
Nadimpalli further teaches selection of nucleic acid fragments of defined lengths association with replication-related activity, including fragments less than about 1000 nucleotides. Selecting a specific core initiation origin sequence length within such a disclosed or suggested range constitutes routine optimization (see MPEP 2144.05) of a known result-effective variable, namely a fragment length sufficient to support replication initiation (i.e., 716 bp). In the absence of evidence demonstrating criticality of a particular length or unexpected results, choosing a specific value within a known range would have been obvious to one of ordinary skill in the art.
Therefore, a person of ordinary skill in the art would have had a reasonable expectation of success in combining the teachings of Nadimpalli, Bartholdy and Rhodes because the references collectively address sequence-dependent characteristics association with replication and chromatin processes, and Bartholdy specifically demonstrates that localized nucleotide-composition patterns are associated with mammalian replication origins. The relationship between G-rich motifs, nucleotide-composition patterns, chromatin-associated marks, and replication activity was well understood at the time of the invention. The proposed combination involves the application of known techniques and known biological relationships and would have yielded predictable results without undue experimentation.
Applicant’s Response: The Applicant argues that Nadimpalli does not teach the claimed method of identifying mammalian genomic DNA replication origins using the recited sliding-window nucleotide-content criteria and therefore does not anticipate the claims. Applicant further argues that the cited art does not teach or suggest the claimed combination of specific window sizes, nucleotide-content threshold, and sequence characteristics for identifying and isolating a functional mammalian replication origin.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully considered and were found persuasive, as discussed below.
Specifically, with respect to the 102 rejections, Nadimpalli does not expressly or inherently disclose the claimed combination of sliding-window nucleotide content criteria for identifying mammalian genomic DNA replication origins; accordingly, the 102 rejection is withdrawn. However, the arguments do not overcome the newly presented 103 rejections because Bartholdy teaches localized window-based nucleotide composition analysis associated with mammalian replication origins, while Nadimpalli and Rhodes provide the additional teachings regarding sequence-dependent replication control and G-rich replication-associated regions; the combined teachings provide an articulated basis for the claimed methodology with a reasonable expectation of success. See MPEP 2141 and 2143.
Conclusion
No claim is allowed.
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/ELIZABETH ROSE LAFAVE/Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684
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