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 I in the reply filed on March 5, 2026 is acknowledged. Claims 64, 66, 69-74 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected Group 2, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 3/5/2025. Thus, claims 1-4, 6, 8, 9, 17, 18, 22, 23, and 25 are under examination (3/5/2026).
Claim Status
Claims 4, 9, 18, 22, 23, 25, 64, 70 and 71 have been amended (3/5/2026). Claims 5, 7, 10-16, 19-21, 24, 26-63, 65, 67, 68, and 75-87 were cancelled (3/5/2026). No new matter was added. Claims 1-4, 6, 8, 9, 17, 18, 22, 23, and 25 are under examination (3/5/2026).
Priority
Claims 1-4, 6, 8, 9, 17, 18, 22, 23, and 25 receive the priority date of 12/24/2019, the filing date of U.S. Provisional Application No. 62/953,308.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Information Disclosure Statements from 6/21/2022 and 7/18/2024 are considered.
Nucleotide and/or Amino Acid Sequence Disclosures
Drawings
REQUIREMENTS FOR PATENT APPLICATIONS CONTAINING NUCLEOTIDE AND/OR AMINO ACID SEQUENCE DISCLOSURES
Items 1) and 2) provide general guidance related to requirements for sequence disclosures.
37 CFR 1.821(c) requires that patent applications which contain disclosures of nucleotide and/or amino acid sequences that fall within the definitions of 37 CFR 1.821(a) must contain a "Sequence Listing," as a separate part of the disclosure, which presents the nucleotide and/or amino acid sequences and associated information using the symbols and format in accordance with the requirements of 37 CFR 1.821 - 1.825. This "Sequence Listing" part of the disclosure may be submitted:
In accordance with 37 CFR 1.821(c)(1) via the USPTO patent electronic filing system (see Section I.1 of the Legal Framework for Patent Electronic System (https://www.uspto.gov/PatentLegalFramework), hereinafter "Legal Framework") as an ASCII text file, together with an incorporation-by-reference of the material in the ASCII text file in a separate paragraph of the specification as required by 37 CFR 1.823(b)(1) identifying:
the name of the ASCII text file;
ii) the date of creation; and
iii) the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(1) on read-only optical disc(s) as permitted by 37 CFR 1.52(e)(1)(ii), labeled according to 37 CFR 1.52(e)(5), with an incorporation-by-reference of the material in the ASCII text file according to 37 CFR 1.52(e)(8) and 37 CFR 1.823(b)(1) in a separate paragraph of the specification identifying:
the name of the ASCII text file;
the date of creation; and
the size of the ASCII text file in bytes;
In accordance with 37 CFR 1.821(c)(2) via the USPTO patent electronic filing system as a PDF file (not recommended); or
In accordance with 37 CFR 1.821(c)(3) on physical sheets of paper (not recommended).
When a “Sequence Listing” has been submitted as a PDF file as in 1(c) above (37 CFR 1.821(c)(2)) or on physical sheets of paper as in 1(d) above (37 CFR 1.821(c)(3)), 37 CFR 1.821(e)(1) requires a computer readable form (CRF) of the “Sequence Listing” in accordance with the requirements of 37 CFR 1.824.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed via the USPTO patent electronic filing system as a PDF, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the PDF copy and the CRF copy (the ASCII text file copy) are identical.
If the "Sequence Listing" required by 37 CFR 1.821(c) is filed on paper or read-only optical disc, then 37 CFR 1.821(e)(1)(ii) or 1.821(e)(2)(ii) requires submission of a statement that the "Sequence Listing" content of the paper or read-only optical disc copy and the CRF are identical.
Specific deficiency – Nucleotide and/or amino acid sequences appearing in the drawings (Figure 15) are not identified by sequence identifiers in accordance with 37 CFR 1.821(d). Sequence identifiers for nucleotide and/or amino acid sequences must appear either in the drawings or in the Brief Description of the Drawings.
Required response – Applicant must provide:
Replacement and annotated drawings in accordance with 37 CFR 1.121(d) inserting the required sequence identifiers;
AND/OR
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required sequence identifiers into the Brief Description of the Drawings, consisting of:
A copy of the previously-submitted specification, with deletions shown with strikethrough or brackets and insertions shown with underlining (marked-up version);
A copy of the amended specification without markings (clean version); and
A statement that the substitute specification contains no new matter.
Specification
The disclosure is objected to because of the following informalities (see MPEP § 608.01):
The use of the terms, “Thermo Fisher” (p. 35, 77-78), “Illumina” (p. 36, 83), “HiSeq” (p. 73), “Corning” (p. 74), “Infors Minitron” (p. 74), “Invitrogen” (p. 74-76), “Qiagen” (p. 75-82), “New England Biolabs” (p. 77-80), “Life Technologies” (p. 78), “Roche” (p. 78), “PacBio” (p. 79), “Edinburgh Genomics” (p. 79), “Lucigen” (p. 80), are trade names or marks used in commerce, has been noted in this application. The terms 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.
Claim Objections
Claim 1 is objected to because of the following informalities:
Claim 1 at step (b) at line 1: “an transcribable reporter sequence” should be replaced with “a transcribable reporter sequence.”
Claim 18 is objected to because of the following informalities:
Claim 18 at line 3: “unique molecular identifiers” should not be capitalized.
Claim 18 at line 4: spell out the entire name prior to using an acronym for “UPAS”, the first time used.
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.
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.
Claim 6 is 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.
Claim 6 is rejected. Claim 6 recites the limitation "the open reading frame of a marker gene” at line 2. There is insufficient antecedent basis for this limitation in the claim.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of pre-AIA 35 U.S.C. 103(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negated by the manner in which the invention was made.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
The factual inquiries for establishing a background for determining obviousness under pre-AIA 35 U.S.C. 103(a) are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims under pre-AIA 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of pre-AIA 35 U.S.C. 103(c) and potential pre-AIA 35 U.S.C. 102(e), (f) or (g) prior art under pre-AIA 35 U.S.C. 103(a).
Claims 1-4, 6, 8, 9, 17, 18, 22, 23, and 25 are rejected under 35 U.S.C. 103 as being unpatentable over Zheng et al. (WO 2017/139690 A1; published 8/17/2017) and Ho et al. (“A molecular barcoded yeast ORF library enables mode-of-action analysis of bioactive compounds”, Nature Biotechnology, published 2009) and in further view of Deakin et al. (“Impact of next-generation sequencing error on analysis of barcoded plasmid libraries of known complexity and sequence”, Nucleic Acids Research, published 2014).
Regarding claim 1, Zheng teaches methods, systems and compositions for single-cell analysis, including single-cell transcriptome analysis and provides a fully-integrated, droplet-based system that enables 3' mRNA digital counting of up to tens of thousands of single cells where approximately 50% of cells loaded into the system can be captured, and up to 8 samples can be processed in parallel (Paragraph 5, lines 1-5). Further, Zheng teaches that the method comprises (a) partitioning a plurality of cells of a heterogeneous cell sample into a plurality of droplets, wherein upon partitioning, a given droplet of the plurality of droplets comprises a given cell of the plurality of cells and a given bead of a plurality of beads comprising a plurality of oligonucleotide barcodes, wherein the given cell comprises a first set of polynucleotides; (b) subjecting the first set of polynucleotides to nucleic acid amplification under conditions sufficient to generate a second set of polynucleotides, wherein a given polynucleotide of the second set of polynucleotides comprises (i) a segment having a sequence of a polynucleotide of the first set or a complement thereof and (ii) a segment having a sequence of a oligonucleotide barcode or a complement thereof; (c) generating a library of polynucleotides from a pool of polynucleotides comprising a plurality of second sets of polynucleotides, including the second set of polynucleotides, from the plurality of droplets; (d) subjecting the library of polynucleotides to sequencing to yield sequencing reads, wherein barcode sequences of the plurality oligonucleotide barcodes associate sequencing reads with individual cells of the plurality of cells of the heterogeneous cell sample; (e) determining, with a sensitivity of at least about 95%), a percentage of the heterogeneous cell sample represented by the cell population using a first set of genetic aberrations and a second set of genetic aberrations obtained from processing the sequencing reads associated with individual cells of the heterogeneous cell sample, wherein the cell population represents less than about 10% of the heterogeneous cell sample (Paragraph 22, lines 1-15). Specifically, Zheng teaches that barcodes can have a variety of different formats, for example, barcodes can include: polynucleotide barcodes; random nucleic acid and/or amino acid sequences; and synthetic nucleic acid and/or amino acid sequences, where a barcode can be attached to an analyte in a reversible or irreversible manner and a barcode can be added to, for example, a fragment of a deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) sample before, during, and/or after sequencing of the sample (Paragraph 60, lines 5-10).
Zheng also teaches that in some cases, these reporter sequences can be greater than about 5 nucleotides in length, greater than about 10 nucleotides in length, greater than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 or even 200 nucleotides in length and in some cases, these reporter nucleotides may be less than about 250 nucleotides in length, less than about 200, 180, 150, 120 100, 90, 80, 70, 60, 50, 40, or even 30 nucleotides in length (Paragraph 219, lines 1-10). Specifically, Zheng teaches the application of methods and systems disclosed herein for analyzing cell lines and External RNA Controls Consortium (ERCC) (Paragraph 52, lines 1-10), and Figures 22H and 221 show the distribution of normalized UMI counts vs. GC content and gene length in 293T cells, respectively. UMI counts were normalized by RNA content (Paragraph 52, lines 10-15).
Regarding claims 2-3, Zheng teaches that one particularly useful application, the methods and systems described may be used to characterize cell features, such as cell surface features (proteins, receptors), and in a particular example a library of potential cell binding ligands, e.g., antibodies, antibody fragments, cell surface receptor binding molecules, or the like, maybe provided associated with a first set of nucleic acid reporter molecules, e.g., where a different reporter oligonucleotide sequence is associated with a specific ligand, and therefore capable of binding to a specific cell surface feature (Paragraph 162, lines 1-5). Zheng also teaches that in some aspects, different members of the library may be characterized by the presence of a different oligonucleotide sequence label, e.g., an antibody to a first type of cell surface protein or receptor would have associated with it a first known reporter oligonucleotide sequence, while an antibody to a second receptor protein would have a different known reporter oligonucleotide sequence associated with it and prior to co-partitioning, the cells would be incubated with the library of ligands, that may represent antibodies to a broad panel of different cell surface features, e.g., receptors, proteins, etc., and which include their associated reporter oligonucleotides (Paragraph 162, lines 5-10).
Regarding claims 4 and 6, Zheng teaches that without the need for lysing the cells within the partitions, one could then subject the reporter oligonucleotides to the barcoding operations described above for cellular nucleic acids, to produce barcoded, reporter oligonucleotides, where the presence of the reporter oligonucleotides can be indicative of the presence of the particular cell surface feature, and the barcode sequence will allow the attribution of the range of different cell surface features to a given individual cell or population of cells based upon the barcode sequence that was co- partitioned with that cell or population of cells and as a result, one may generate a cell-by-cell profile of the cell surface features within a broader population of cells (Paragraph 163, lines 1-5).
Specifically Zheng teaches that this example is schematically illustrated in FIG. 5 and as shown, a population of cells, represented by cells 502 and 504 are incubated with a library of cell surface associated reagents, e.g., antibodies, cell surface binding proteins, ligands or the like, where each different type of binding group includes an associated nucleic acid reporter molecule associated with it, shown as ligands and associated reporter molecules 506, 508, 510 and 512 (with the reporter molecules being indicated by the differently shaded circles) and where the cell expresses the surface features that are bound by the library, the ligands and their associated reporter molecules can become associated or coupled with the cell surface (Paragraph 164, lines 1-5). Further Zheng teaches that individual cells are then partitioned into separate partitions, e.g., droplets 514 and 516, along with their associated ligand/reporter molecules, as well as an individual barcode oligonucleotide bead as described elsewhere herein, e.g., beads 522 and 524, respectively and as with other examples described herein, the barcoded oligonucleotides are released from the beads and used to attach the barcode sequence the reporter molecules present within each partition with a barcode that is common to a given partition, but which varies widely among different partitions (Paragraph 164, lines 5-10).
Also, Zheng teaches that for example, as shown in FIG. 5, the reporter molecules that associate with cell 502 in partition 514 are barcoded with barcode sequence 518, while the reporter molecules associated with cell 504 in partition 516 are barcoded with barcode 520 and as a result, one is provided with a library of oligonucleotides that reflects the surface ligands of the cell, as reflected by the reporter molecule, but which is substantially attributable to an individual cell by virtue of a common barcode sequence, allowing a single cell level profiling of the surface characteristics of the cell, and as will be appreciated, this process is not limited to cell surface receptors but may be used to identify the presence of a wide variety of specific cell structures, chemistries or other characteristics (Paragraph 164, lines 10-15).
Regarding claim 8, Zheng teaches that in an example method of cellular RNA analysis and in reference to FIG. 10, a cell is co-partitioned along with a barcode bearing bead, and other reagents such as reverse transcriptase, reducing agent and dNTPs into a partition (e.g., a droplet in an emulsion), where in operation 1050, the cell is lysed while the barcoded oligonucleotides 1002 are released (e.g., via the action of the reducing agent) from the bead, and the poly-T segment 1014 of the released barcode oligonucleotide then hybridizes to the poly-A tail of mRNA 1020 and next at operation 1052, the poly-T segment is then extended in a reverse transcription reaction using the mRNA as template to produce a cDNA transcript 1022 of the mRNA and also includes each of the sequence segments 1020, 1008, 1012, 1010, 1016, and 1014 of the barcode oligonucleotide (Paragraph 203, lines 1-5). Also, Zheng teaches that within any given partition, all of the cDNA transcripts of the individual mRNA molecules will include a common barcode sequence segment 1012, however, by including the unique random N-mer sequence, the transcripts made from different mRNA molecules within a given partition will vary at this unique sequence and as described elsewhere herein, this provides a quantitation feature that can be identifiable even following any subsequent amplification of the contents of a given partition, e.g., the number of unique segments associated with a common barcode can be indicative of the quantity of mRNA originating from a single partition, and thus, a single cell (Paragraph 203, lines 5-10). Further, Zheng teaches that at operation 1054 a second strand is synthesized and at operation 1056 the T7 promoter sequence can be used by T7 polymerase to produce RNA transcripts in in vitro transcription. At operation 1058 the transcripts are fragmented (e.g., sheared), ligated to additional functional sequences, and reverse transcribed and the functional sequences may include a sequencer specific flow cell attachment sequence 1030, e.g., a P5 sequence, as well as functional sequence 1028, which may include sequencing primers, e.g., a Rl primer binding sequence, as well as functional sequence 1032, which may include a sample index, e.g., an i5 sample index sequence and at operation 1060 the RNA transcripts can be reverse transcribed to DNA, the DNA amplified (e.g., via PCR), and sequenced to identify the sequence of the cDNA transcript of the mRNA, as well as to sequence the barcode segment and the unique sequence segment (Paragraph 203, lines 10-15). Specifically, Zheng teaches that in some cases, operations 1050 and 1052 can occur in the partition, while operations 1054, 1056, 1058 and 1060 can occur in bulk (e.g., outside the partition) and in the case where a partition is a droplet in an emulsion, the emulsion can be broken and the contents of the droplet pooled in order to complete operations 1054, 1056, 1058 and 1060 (Paragraph 203, lines 15-20).
Regarding claim 9, Zheng teaches that FIG. 8 shows images of individual Jurkat cells co-partitioned along with barcode oligonucleotide containing beads in aqueous droplets in an aqueous in oil emulsion and as illustrated, individual cells may be readily co-partitioned with individual beads and as will be appreciated, optimization of individual cell loading may be carried out by a number of methods, including by providing dilutions of cell populations into the microfluidic system in order to achieve the desired cell loading per partition as described elsewhere herein via amplification and barcoding, as well as attachment of other functional sequences (Paragraph 147, lines 1-5). Zheng also teaches that as noted above, fragmentation may be accomplished through the co-partitioning of shearing enzymes, such as endonucleases, in order to fragment the nucleic acids into smaller fragments and these endonucleases may include restriction endonucleases, including type II and type lis restriction endonucleases as well as other nucleic acid cleaving enzymes, such as nicking endonucleases, and the like and in some cases, fragmentation may not be desired, and full length nucleic acids may be retained within the partitions, or in the case of encapsulated cells or cell contents (Paragraph 147, lines 5-10).
Zheng also teaches that in some cases, these reporter sequences can be greater than about 5 nucleotides in length, greater than about 10 nucleotides in length, greater than about 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 150 or even 200 nucleotides in length and in some cases, these reporter nucleotides may be less than about 250 nucleotides in length, less than about 200, 180, 150, 120 100, 90, 80, 70, 60, 50, 40, or even 30 nucleotides in length (Paragraph 219, lines 1-10). Specifically, Zheng teaches the application of methods and systems disclosed herein for analyzing cell lines and External RNA Controls Consortium (ERCC) (Paragraph 52, lines 1-10), and Figures 22H and 221 show the distribution of normalized UMI counts vs. GC content and gene length in 293T cells, respectively. UMI counts were normalized by RNA content (Paragraph 52, lines 10-15).
Regarding claim 17, Zheng teaches that the nucleic acid barcode sequences can include from 6 to about 20 or more nucleotides within the sequence of the oligonucleotides and in some cases, the length of a barcode sequence may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 nucleotides or longer (Paragraph 136, lines 1-5).
Regarding claim 18, Zheng teaches that the barcode sequencing libraries were quantified by quantitative PCR (qPCR) (KAPA Biosystems Library Quantification Kit for Illumina® platforms P/N KK4824) and sequencing libraries were loaded at 2. lpM on an Illumina® NextSeq500 with 2 x 75 paired-end kits using the following read length: 98bp Readl, 14bp 17 Index, 8bp 15 Index and lObp Read2 (Paragraph 305, lines 15-20).
Regarding claims 22, 23 and 25, Zheng teaches that although operations with various barcode designs have been discussed individually, individual beads can include barcode oligonucleotides of various designs for simultaneous use and in addition to characterizing individual cells or cell sub-populations from larger populations, the processes and systems described herein may also be used to characterize individual cells as a way to provide an overall profile of a cellular, or other organismal population and a variety of applications require the evaluation of the presence and quantification of different cell or organism types within a population of cells, including, for example, microbiome analysis and characterization, environmental testing, food safety testing, epidemiological analysis, e.g., in tracing contamination or the like (Paragraphs 210-217). Zheng teaches that in particular, the analysis processes described above may be used to individually characterize, sequence and/or identify large numbers of individual cells within a population and that this characterization may then be used to assemble an overall profile of the originating population, which can provide important prognostic and diagnostic information via oligonucleotide sequences (Paragraphs 210-217).
Zheng teaches that oligonucleotide-based reporter molecules provide advantages of being able to generate significant diversity in terms of sequence, while also being readily attachable to most biomolecules, e.g., antibodies, etc., as well as being readily detected, e.g., using sequencing or array technologies and in the example process, the binding groups include oligonucleotides attached to them and thus, a first binding group type, e.g., antibodies to a first type of cell surface feature, will have associated with it a reporter oligonucleotide that has a first nucleotide sequence where different binding group types, e.g., antibodies having binding affinity for other, different cell surface features, will have associated therewith reporter oligonucleotides that comprise different nucleotide sequences, e.g., having a partially or completely different nucleotide sequence (Paragraphs 210-217). Zheng teaches that in some cases, for each type of cell surface feature binding group, e.g., antibody or antibody fragment, the reporter oligonucleotide sequence may be known and readily identifiable as being associated with the known cell surface feature binding group and these oligonucleotides may be directly coupled to the binding group, or they may be attached to a bead, molecular lattice, e.g., a linear, globular, cross-slinked, or other polymer, or other framework that is attached or otherwise associated with the binding group, which allows attachment of multiple reporter oligonucleotides to a single binding group.
Specifically, Zheng teaches that in the case of multiple reporter molecules coupled to a single binding group, such reporter molecules can comprise the same sequence, or a particular binding group will include a known set of reporter oligonucleotide sequences and as between different binding groups, e.g., specific for different cell surface features, the reporter molecules can be different and attributable to the particular binding group (Paragraphs 210-217).
However, Zheng does not teach or suggest that the barcode associated with the plurality of synthetic nucleic acids is characterized by a specified complexity with a range of 10E7 – 10E12. Further, Zheng does not teach or suggest that the transcribable reporter sequence associated with these synthetic nucleic acids is an operably linked open reading frame of a marker gene.
Ho teaches a yeast chemical-genomics approach designed to identify genes that when mutated confer drug resistance, thereby providing insight about the modes of action of compounds, and specifically developed a molecular barcoded yeast open reading frame (MoBY-ORF) library in which each gene, controlled by its native promoter and terminator, is cloned into a centromere-based vector along with two unique oligonucleotide barcodes where the MoBY-ORF resource has numerous genetic and chemical-genetic applications, but focused on cloning wild-type versions of mutant drug-resistance genes using a complementation strategy and on simultaneously assaying the fitness of all transformants with barcode microarrays (Abstract). Further, Ho teaches that the MoBY-ORF library consists of plasmids that each carry a pair of oligonucleotide barcodes and a single yeast ORF that is flanked by its native upstream and downstream genomic sequences and the plasmid vector p5472 carries a URA3 selectable marker and a yeast centromere, which maintains one to three copies of the plasmid per cell (Fig. 1). Specifically, Ho teaches that the vector was designed to be compatible with an in vivo bacterial cloning method, mating-assisted genetically integrated cloning (MAGIC), which facilitates the rapid construction of recombinant DNA molecules, enabling the barcoded clones to be transferred efficiently to other vector backbones, such as a high-copy vector and the barcodes were obtained from the yeast deletion mutant collection and comprise two unique 20-nucleotide DNA sequences (labeled the UPTAG and DNTAG) flanking a dominant selectable marker (KanMX) that confers resistance to the drug G418/kanamycin where the barcodes can be amplified with universal primers, enabling cells carrying a specific ORF to be quantitatively detected with a microarray having probes that hybridize to the barcode sequences (Results: Paragraph 1).
Deakin teaches Barcoded vectors are promising tools for investigating clonal diversity and dynamics in hematopoietic gene therapy and analysis of clones marked with barcoded vectors requires accurate identification of potentially large numbers of individually rare barcodes, when the exact number, sequence identity and abundance are unknown and to explore this potential application empirically, without prior assumptions, barcode libraries were sequenced of known complexity containing 1, 10 and 100 Sanger-sequenced barcodes via an Illumina platform, where libraries containing 1 and 10 barcodes were distinguished from false barcodes generated by sequencing error by a several log-fold difference in abundance (Abstract). Further, Deakin teaches that analyses of vector integration sites (ISs), which uniquely tag individual gene-marked HPC clones, are yielding important insights into clonal complexity, clonal dynamics and genotoxicity following gene therapy (Introduction: Paragraph 1) and individual HPCs would be uniquely tagged provided the barcoded vector stock has sufficiently high complexity (Introduction: Paragraph 2). Deakin also teaches that since the sequence identities of barcodes in a barcode library of the complexity required for clinical use would be unknown, the veracity of barcode variants identified using NGS must be assumed and unless there is a clear distinction between true barcodes and erroneously generated sequences and in this study, the feasibility of vector barcoding coupled with NGS was investigated for potential use in analyzing clonal diversity, using mixtures of defined complexity comprising known barcode sequences (Discussion: Paragraphs 1-3; Figure 4). Further, Deakin teaches that they describe an empirical approach to evaluate the size of a barcode library that can be resolved by NGS, using over 100 000-fold coverage in two independent sequencing runs to analyze the same library of known bar code sequences and in doing so, similar limitations applicable to independent NGS technologies were identified, pertaining to the analyzable degree of complexity, sensitivity and
specificity of analysis and assessable clone size and in the context of our experimental configuration, sequencing error was found to impose an upper limit on the degree of complexity that could be resolved using NGS versus barcode libraries of low complexity, containing just one
or 10 unique barcodes, could readily be distinguished from background produced by error (Discussion: Paragraphs 1-3). Overall, Deakin teaches that in the experimental configuration used here, the upper limit on the analyzable degree of complexity thus lies between 10 and
100 known barcodes (Discussion: Paragraphs 1-3).
It would have been obvious to a person of ordinary skill in the art at or before the time of the invention to modify the system of Zheng to include the barcoded open reading frame library constructs taught by Ho, as further informed by Deakin, in order to achieve improved identification, tracking, and quantification of nucleic acid molecules with complex populations. Specifically, a person of ordinary skill in the art would have been motivated to incorporate the ORF-based constructs of Ho into the system of Zheng because Zheng already relies on nucleic acid reporter sequences for identifying and quantifying biological features, and Ho provides a known and well-characterized framework for linking nucleic acid sequence identity to expressed gene products under regulatory control. Substituting or incorporating ORF-based reporter constructs into Zheng’s system would have predictably enhanced the ability to measure gene expression or cellular responses using known reporter or marker genes, which were widely used in the art for such purposes. Further, Deakin teaches that barcode libraries used in sequencing applications inherently require sufficient complexity to uniquely identify individual molecules or clones, and that barcode diversity and library complexity directly impact the accuracy and resolution of sequencing-based analyses. Accordingly, it would have been obvious to select or optimize barcode complexity values within the claimed ranges as a matter of routine design choice to ensure adequate diversity and minimize barcode collision, particularly in systems such as Zheng and Ho that rely on unique barcode-to-entity correspondence.
A person of ordinary skill in the art would have had a reasonable expectation of success in making these modifications because all cited references operate within the same technological field of high-throughput sequencing, barcoding, and nucleic acid library construction, and each reference demonstrates compatible components (barcodes, regulatory elements, ORFs, and sequencing workflows) that are routinely combined in the art. The substitution of known ORF-based reporter constructs into a known barcoding and sequencing framework represents the predictable use of prior art elements according to their established functions.
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ELIZABETH ROSE LAFAVE whose telephone number is (703)756-4747. The examiner can normally be reached Compressed Bi-Week: M-F 7:30-4:30.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Heather Calamita can be reached on 571-272-2876. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/ELIZABETH ROSE LAFAVE/ Examiner, Art Unit 1684
/HEATHER CALAMITA/ Supervisory Patent Examiner, Art Unit 1684
Prosecution Insights