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 .
Claim Status
Claims 3, 6, 8, 10, 12, 15-16, 19-20, 22, 24, 26-28 and 33 are amended (12/19/2022). Claims 4-5, 7, 11, 13-14, 21, 23, 25, and 29-32 are cancelled (12/19/2022). Thus, claims 1-3, 6, 8-10, 12, 15-20, 22, 24, 26-28 and 33 are under examination.
Priority
Claims 1-3, 6, 8-10, 12, 15-20, 22, 24, 26-28 and 33 are given a priority date of 6/12/2020, the filing date of SG10202005599R.
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 12/9/2022 and 6/11/2025 are considered.
Nucleotide and/or Amino Acid Sequence Disclosures
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 deficiencies and the required response to this Office Action are as follows:
Specific deficiency - This application fails to comply with the requirements of 37 CFR 1.821 - 1.825 because it does not contain a "Sequence Listing" as a separate part of the disclosure or a CRF of the “Sequence Listing.”.
Required response - Applicant must provide:
A "Sequence Listing" part of the disclosure; together with
An amendment specifically directing its entry into the application in accordance with 37 CFR 1.825(a)(2);
A statement that the "Sequence Listing" includes no new matter as required by 37 CFR 1.821(a)(4); and
A statement that indicates support for the amendment in the application, as filed, as required by 37 CFR 1.825(a)(3).
If the "Sequence Listing" part of the disclosure is submitted according to item 1) a) or b) above, Applicant must also provide:
A substitute specification in compliance with 37 CFR 1.52, 1.121(b)(3) and 1.125 inserting the required incorporation-by-reference paragraph, 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.
If the "Sequence Listing" part of the disclosure is submitted according to item 1) c) or d) above, applicant must also provide:
A CRF in accordance with 37 CFR 1.821(e)(1) or 1.821(e)(2) as required by 1.825(a)(5); and
A statement according to item 2) a) or b) above.
Specification
The disclosure is objected to because of the following informalities (see MPEP § 608.01):
The use of the terms, “MiSeq” (p. 5), “NCBI” (p.16, 31) and “Sigma Aldrich” (p. 20-21), “Millipore” (p. 20-21), “Invitrogen” (p. 20-21), “Beckman-Coulter” (p. 20), “Agilent” (p. 22), “Illumina” (p. 23), “Thermo Fisher” (p. 23), “Agilent Technologies” (p. 23), “Beckman” (p. 23), “10X Genomics” (p. 23) 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 27 is objected to because of the following informality:
Claim 27 at line 2: “the heterogenous population of cells comprise” should be replaced with “the heterogeneous population of cells comprises” for grammatical accuracy.
Claim 28 is objected to because of the following informality:
Claim 28 at line 2: “the heterogenous population of cells comprise” should be replaced with “the heterogeneous population of cells comprises” for grammatical accuracy.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 2-3 and 17-18 rejected under 35 U.S.C. 101 because the claimed invention is directed to an abstract idea without significantly more.
The claims recite a method for assessing transduction efficiency and/or specificity of vectors at a single-cell level, comprising classifying partitioned cells into specific cell types based on gene expression patterns and/or epigenetic features; determining transduction efficiency by calculating a percentage of cells positive for a vector and/or by comparing frequencies with which vectors are detected in cells of a specific cell type; sequencing nucleic acids to obtain sequencing reads; and matching the sequencing reads to a reference data set.
These steps amount to collecting biological information, analyzing sequencing data, and determining correlations or statistical relationships between detected vector sequences and cell types. This constitutes a judicial exception in the form of mental processes and mathematical concepts, including evaluation, comparison, and classification of information, which can be performed conceptually or using mathematical relationships.
This judicial exception is not integrated into a practical application because the claims do not include additional elements, when considered separately and in combination, that are sufficient to amount to significantly more than the judicial exception. Although the claims refer to sequencing nucleic acids and detecting vector sequences, these activities merely gather data for use in the abstract analysis and are recited at a high level of generality.
Subject Matter Eligibility Test for Products and Processes
Step 1 - Is the Claim to a Process, Machine, Manufacture or Composition of Matter? YES.
The claims provide for a method comprising:
Classifying each partitioned cell into a specific cell type based on gene expression patterns and/or epigenetic features determined from sequencing results;
Determining transduction efficiency of a specific vector against a cell type by calculating a percentage of cells of the specific cell type detected positive for the presence of the specific vector and/or by comparing frequencies with which the presence of the specific vector is detected in cells of the specific cell type relative to frequencies of detection of another vector or in other cell types; and
Matching sequencing reads obtained from nucleotide sequencing to a reference data set.
Thus, the claims are directed to a statutory category (e.g., a process).
Step 2A, Prong One — Does the Claim Recite an Abstract Idea, Law of Nature, or Natural Phenomenon? YES.
Abstract ideas have been identified by the courts by way of example, including fundamental economic practices, certain methods of organizing human activities, an idea ‘of itself,’ and mathematical relationships/formulas. The claims recite a judicial exception.
Specifically, the claims recite a mental process of classifying cells based on gene expression or epigenetic information, determining transduction efficiency by calculating percentages or comparing frequencies of detected vector sequences, and matching sequencing reads to a reference data set. These limitations amount to evaluation and analysis of biological information and correlations derived from sequencing results, which corresponds to “an abstraction” (an idea having no particular concrete or tangible form). Thus, the claimed invention describes a judicial exception in the form of mental processes and mathematical relationships used to analyze biological sequencing data, which correspond to abstractions (ideas, having no particular concrete or tangible form) and natural informational principles.
Step 2A, Prong Two — Does the Claim Recite an Additional Elements that Integrate the Judicial Exception into a Practical Application? NO.
The Supreme Court has long distinguished between principles themselves, which are not patent eligible, and the integration of those principles into practical applications, which are patent eligible. However, absent are any additional elements recited in the claim beyond the judicial exceptions which integrate the exception into a practical application of the exception.
The “integration into a practical application” requires an additional element or a combination of additional elements in the claim to apply, rely on, or use the judicial exception in a manner that imposes a meaningful limit on the judicial exception, such that it is more than a drafting effort designed to monopolize the exception. The claim limitations directed to classifying cells based on sequencing-derived gene expression or epigenetic information, determining transduction efficiency by calculating percentages or comparing frequencies of detected vector sequences, and matching sequencing reads to a reference data set are not indicative of integration into a practical application.
The above claim limitations are considered simply as the recitation of: 1) a “mental” process of evaluating/interpreting data/information; and 2) mathematical relationships or correlations between detected vector sequences and specific cell types. Although the claims reference sequencing results generated in earlier steps of the method, such activities merely gather data for use in the abstract analysis and are recited at a high level of generality. The claims do not include any additional steps that improve sequencing technology, modify vector transduction techniques, or otherwise control or transform a biological system in a particular manner. There are no further additional elements that apply the identified judicial exception into a practical application. Thus, the claims do not integrate the judicial exception into a practical application.
Step 2B - Does the Claim Recite Additional Elements that Amount to Significantly More than the Judicial Exception? NO.
The Supreme Court has identified a number of considerations for determining whether ra claim with additional elements amounts to “significantly more” than the judicial exception(s) itself. The claims as a whole are analyzed to determine whether any additional element/step, or combination of additional elements/steps, in addition to the identified judicial exception(s) is sufficient to ensure that the claim amounts to “significantly more” than the exception(s).
However, the additional elements of the instant application, individually and in combination, do not amount to “significantly more.” Under the Step 2B analysis, the “physical” or additional elements/steps directed to obtaining sequencing reads from partitioned cells, detecting vector or barcode sequences, and using sequencing-derived gene expression or epigenetic information for classification and efficiency determination merely instruct a practitioner to obtain biological data and analyze the resulting information. Further, these steps (i.e., nucleotide sequencing, detection of nucleic acid sequences, and comparison or matching of sequencing reads to reference data sets) are “physical steps” that are well-understood, routine and conventional techniques in the field of single-cell sequencing and viral vector analysis.
For example, Brock (WO 2018/031864 A1, published 2/15/2018) discloses methods and platforms related to modulating expression of a gene of interest within a select population of cells comprising: providing a population of cells; providing a vehicle, plasmid, vector or recombinant virus, or equivalent thereof, capable of stably expressing a guide nucleic acid comprising randomized barcodes, thereby producing a population of barcoded cells; allowing said barcoded cell to divide, thereby forming a barcoded progeny of cells; saving an aliquot of cells; identifying the barcode in a lineage of interest from the barcoded progeny of cells; reconstituting the aliquot of saved cells, and transforming the reconstituted aliquot of cells with a transcriptional element comprising a transcriptional effector, the barcode of the lineage of interest, and a gene of interest; utilizing the transcriptional effector to modify expression of the gene of interest within the lineage of interest (Abstract). Specifically, Brock discloses that the demonstration that expressed gRNA barcodes can be used to efficiently perform lineage-specific manipulation of gene expression opens up the possibility for a broad range of studies investigating the potential of lineage-specific perturbations within the context of a heterogeneous, evolving cell population, where the ability to concurrently track clonal fitness dynamics and generate lineage-specific genomic and transcriptomic data over longitudinal studies will provide unprecedented insight into cancer adaptation and other diseases with an evolutionary basis (p. 34, Paragraph 1). Brock also discloses that cells in the lineage of interest can be selected in a variety of ways, known to those of skill in the art; where for example, cells can be selected on the basis of phenotype, wherein the phenotype can be created from the gene of interest and selecting the cells on the basis of phenotype can comprise selecting the cells on the basis of protein expression, RNA expression, or protein activity and in some cases selecting the cells on the basis of the phenotype comprises fluorescence activated cell sorting, affinity purification of cells, or selection based on cell motility (cell sorting can be done using single cell sorting, fluorescent activated cell sorting (FACS), physical cell manipulation, laser capture, or magnetic cell sorting (p. 28, Paragraphs 1-2).
In addition, Zincarelli et al. (“Analysis of AAV Serotypes 1-9 Mediated Gene Expression and Tropism in Mice After Systematic Injection”; The American Society of Gene Therapy, published February 11, 2016, and cited in the Information Disclosure Statement filed 12/9/2022) disclose that viral vectors have been identified as a promising means for gene delivery because they have important advantages over other vectors because they do not exhibit pathogenicity in humans and they provide significantly longer transgene expression (Introduction: Paragraph 1). Further, Zincarelli discloses that in order to further analyze the transduction efficiency of different AAV serotypes, luciferase enzyme activity in various tissues (Figure 4) was examined via imaging to monitor expression levels (Luciferase protein expression in selected tissues: Paragraph 1). Zincarelli also discloses that robust cardiac localization for several serotypes was evaluated via transthoracic echocardiography to evaluate whether cardiac function is affected by AAV transduction, therefore showcasing the investigation of gene therapy for a potential of diseases (i.e., heart failure).
Wang et al. (“Advances and Applications of Single Cell Sequencing Technologies; Mol. Cell. , published 5/21/2015 and cited in the Information Disclosure Statement 12/9/2022) disclose that single cell sequencing has emerged as a powerful new set of technologies for studying rare cells and delineating complex populations, including in translational applications in the clinic (Abstract). Further, Wang discloses that in order to sequence a single cell, it must first be captured and while the methods for isolating single cells from abundant populations have been well-established, the isolation of rare single cells (<1%) remains a formidable technical challenge; therefore in order to isolate a single cell randomly from an abundant population, several approaches can be employed: mouth pipetting, serial dilution, robotic micromanipulation, flow-assisted cell sorting (FACS) and microfluidic platforms (Table 1) (Single Cell Isolation Methods: Paragraph 1). Specifically, Wang discloses that many of these approaches require cells or nuclei in suspension, and therefore cannot preserve their spatial context in tissues; however, this limitation can be overcome using Laser-capture-microdissection (LCM), which can also be used to isolate rare cells (Single Cell Isolation Methods: Paragraph 1). Wang also discloses that the development of DNA SCS methods has proven to be more challenging than RNA, since a single cell contains only 2 copies of each DNA molecule, but thousands of copies of most RNA molecules, thus resulting in technical errors; however both the Hi-C approach and bisulfite sequencing have been used to identify physical chromatin interactions in single cells, as well as the measurement of cytosine methylation modifications at 1.5 million CpG sites in a single cell (Single Cell Epigenomic Sequencing Methods: Paragraph 1), showcasing the simplicity of RNA single cell sequencing over the technical complications of DNA single cell sequencing.
Therefore, when considered as an ordered combination, the claims merely collect biological information and perform statistical or comparative analysis to determine transduction efficiency or specificity metrics, which does not provide an inventive concept sufficient to transform the abstract idea into patent eligible subject matter.
Simply appending routine and conventional activities previously known to the industry specified at a high level of generality to the judicial exception and/or generally linking the use of the judicial exception(s) to a particular technological environment or field of use, are not found to be enough to qualify as “significantly more.” Nothing is added by identifying the techniques to be used (i.e., obtaining sequencing reads from partitioned cells, detecting vector or barcode sequences, classifying cells based on sequencing-derived gene expression or epigenetic information, determining transduction efficiency by calculating percentages or comparing detection frequencies, and matching sequencing reads to a reference data set) because those techniques were well-understood, routine, and conventional techniques that a practitioner would have employed when analyzing sequencing data in the context of vector transduction studies. In context with the other recited claim limitations, the language directed to determining whether a relationship or correlation exists between detected vector sequences and specific cell types based on sequencing results merely instructs one to observe and evaluate the relevant biological information.
This information simply tells a practitioner about the relevant natural relationships reflected in sequencing data, at most adding a suggestion that such relationships be taken into account when evaluation transduction efficiency or specificity. Thus, when viewed both individually and as an ordered combination, the claimed elements/steps in addition to the identified judicial exception are found insufficient to supply an inventive concept because the elements/steps are considered conventional and specified at a high level of generality. The claim limitations do not transform the abstract idea that they recite into patent-eligible subject matter because “the claims simply instruct the practitioner to implement the abstract idea with routine, conventional activity.”
Accordingly, the claims do not qualify as patent-eligible subject matter.
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.
Claims 1-3, 6, 8-10, 12, 15-20, 22, 24, 26-28 and 33 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.
Claim 1 is rejected. Claim 1 recites the limitation "the transduction efficiency” in line 1. There is insufficient antecedent basis for this limitation in the claim.
Claim 1 is further rejected. Claim 1 recites the limitation "the presence” at step (e) at line 1. There is insufficient antecedent basis for this limitation in the claim.
Claims 2-3, 6, 8-10, 12, 15-20, 22, 24, 26-28 and 33 are included in this rejection due to their dependency on claim 1.
Claim 3 is further rejected. Claim 3 recites the limitation "the specific cell type” in line 3. There is insufficient antecedent basis for this limitation in the claim.
Claim 12 is further rejected. Claim 12 recites the limitation "the production” in line 9. There is insufficient antecedent basis for this limitation in the claim.
Claim 15 is included in this rejection due to its dependency on claim 12.
Claim 18 is further rejected. Claim 18 recites the limitation "the genomes and/or the transcriptomes” in line 2. There is insufficient antecedent basis for this limitation in the claim.
Claim Rejections - 35 USC § 102
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 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 1-3, 6, 8-10, 12, 15-20, 22, 24, 26-28 and 33 are rejected under 35 U.S.C. 102 (a)(1) and (a)(2) as being anticipated by Brock et al. (WO 2018/031864 A1, published 2/15/2018).
Regarding claims 1-2, Brock teaches methods and platforms related to modulating expression of a gene of interest within a select population of cells comprising: providing a population of cells; providing a vehicle, plasmid, vector or recombinant virus, or equivalent thereof, capable of stably expressing a guide nucleic acid comprising randomized barcodes, thereby producing a population of barcoded cells; allowing said barcoded cell to divide, thereby forming a barcoded progeny of cells; saving an aliquot of cells; identifying the barcode in a lineage of interest from the barcoded progeny of cells; reconstituting the aliquot of saved cells, and transforming the reconstituted aliquot of cells with a transcriptional element comprising a transcriptional effector, the barcode of the lineage of interest, and a gene of interest; utilizing the transcriptional effector to modify expression of the gene of interest within the lineage of interest (Abstract). Brock further teaches methods for delivering vectors or other nucleic acid (such as RNA) into mammalian cells in culture (termed transfection) are routine, and a number of transfection methods find use with the invention and these include but are not limited to calcium phosphate precipitation, electroporation, lipid-based methods (liposomes or lipoplexes) such as Transfectamine® (Life Technologies™) and TransFectin™ (Bio-Rad Laboratories), cationic polymer transfections, for example using DEAE-dextran, direct nucleic acid injection, biolistic particle injection, and viral transduction using engineered viral carriers (termed transduction, using e.g., engineered herpes simplex virus, adenovirus, adeno-associated virus, vaccinia virus, Sindbis virus), and sonoporation (p. 12, Paragraph 1). Specifically, Brock teaches that the demonstration that expressed gRNA barcodes can be used to efficiently perform lineage-specific manipulation of gene expression opens up the possibility for a broad range of studies investigating the potential of lineage-specific perturbations within the context of a heterogeneous, evolving cell population, where the ability to concurrently track clonal fitness dynamics and generate lineage-specific genomic and transcriptomic data over longitudinal studies will provide unprecedented insight into cancer adaptation and other diseases with an evolutionary basis (p. 34, Paragraph 1). Brock also teaches that cells in the lineage of interest can be selected in a variety of ways, known to those of skill in the art; where for example, cells can be selected on the basis of phenotype, wherein the phenotype can be created from the gene of interest and selecting the cells on the basis of phenotype can comprise selecting the cells on the basis of protein expression, RNA expression, or protein activity and in some cases selecting the cells on the basis of the phenotype comprises fluorescence activated cell sorting, affinity purification of cells, or selection based on cell motility (cell sorting can be done using single cell sorting, fluorescent activated cell sorting (FACS), physical cell manipulation, laser capture, or magnetic cell sorting (p. 28, Paragraphs 1-2).
Brock further teaches that specifically, disclosed herein is a method of generating a population of cells that display a desired characteristic when exposed to a candidate agent, the method comprising: providing a population of cells; providing a vehicle, plasmid, vector or recombinant virus, or equivalent thereof, capable of stably expressing a guide nucleic acid comprising randomized barcodes, thereby producing a population of barcoded cells; saving an aliquot of cells; exposing the barcoded cells to one or more candidate agents; identifying a desired characteristic in a barcoded cell exposed to a candidate agent; reconstituting the aliquot of cells and exposing the reconstituted aliquot of cells to a nucleic acid comprising a transcriptional activator, a barcode, and a gene of interest, wherein the barcode is the same as that of the barcoded cell with the desired characteristic; utilizing the transcriptional activator to drive expression of the gene of interest; identifying and selecting barcoded cells with the desired characteristic; and allowing the selected barcoded cell to divide, thereby forming generating a population of cells that display a desired characteristic when exposed to a candidate agent (p. 29, Paragraph 1).
Regarding claim 3, Brock teaches that the previously described method includes application to polypeptides of two or more sequences or subsequences that are the same ("identical") or have a specified percentage of amino acid residues or nucleotides that are
identical ("percent identity") when compared and aligned for maximum correspondence with a
second molecule, as measured using a sequence comparison algorithm (e.g., by a BLAST alignment, or any other algorithm known to persons of skill), or alternatively, by visual inspection (p. 20, Paragraph 2).
Regarding claims 6 and 8, Brock teaches methods and compositions wherein each cell in a population is uniquely tagged with a stably integrated barcode-gRNA under control of a constitutive promoter and following barcode instantiation, cells are permitted to proliferate and at intervals the genomically encoded barcode region is sequenced for quantitation of clonal barcodes; a parallel sample portion is archived for retroactive analysis, where RNA sequencing of barcode gRNA can be performed directly in one example and lineage dynamics may inform the identification of specific lineages of interest for subsequent gene activation in archival samples (p. 22, Paragraph 1).
Regarding claims 9-10, Brock teaches that methods for delivering vectors or other nucleic acid (such as RNA) into mammalian cells in culture (termed transfection) are routine, and a number of transfection methods find use with the invention; including but are not limited to calcium phosphate precipitation, electroporation, lipid-based methods (liposomes or lipoplexes) such as Transfectamine® (Life Technologies™) and TransFectin™ (Bio-Rad Laboratories), cationic polymer transfections, for example using DEAE-dextran, direct nucleic acid injection, biolistic particle injection, and viral transduction using engineered viral carriers (termed transduction, using e.g., engineered herpes simplex virus, adenovirus, adeno-associated virus, vaccinia virus, Sindbis virus), and sonoporation (p.11, Paragraph 1).
Also, Brock teaches that Portions of polynucleotides can be any length, for example, at least 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300 or 500 or more nucleotides in length (p. 21, Paragraph 3).
Regarding claim 12, Brock teaches that there are many uses for this versatile tool, including driving lineage specific expression of a reporter, allowing lineage isolation via cell sorting and other uses include driving lineage specific expression of a lethal protein, thereby allowing for targeted cell death of a specific lineage; use of an auxotrophic marker; use of a drug resistance gene/protein to allow for the targeted selection of a specific lineage of interest; or a differentiation marker to allow for lineage specific differentiation, where barcoded guide nucleotide can also be co-expressed with libraries of small non-coding RNA (microRNA) for functional assessment of microRNA (p. 22, Paragraph 1).
Regarding claims 15-18, Brock teaches that methods for delivering vectors or other nucleic acid (such as RNA) into mammalian cells in culture (termed transfection) are routine, and a number of transfection methods find use with the invention; including but are not limited to calcium phosphate precipitation, electroporation, lipid-based methods (liposomes or lipoplexes) such as Transfectamine® (Life Technologies™) and TransFectin™ (Bio-Rad Laboratories), cationic polymer transfections, for example using DEAE-dextran, direct nucleic acid injection, biolistic particle injection, and viral transduction using engineered viral carriers (termed transduction, using e.g., engineered herpes simplex virus, adenovirus, adeno-associated virus, vaccinia virus, Sindbis virus), and sonoporation (p.11, Paragraph 1). Brock also teaches that currently, cell populations carrying unique heritable barcode identifiers are bulk processed for quantitation of barcode frequency by sequencing (p. 2, Paragraph 1). Further Brock teaches methods and compositions wherein each cell in a population is uniquely tagged with a stably integrated barcode-gRNA under control of a constitutive promoter, and following barcode instantiation, cells are permitted to proliferate and at intervals the genomically encoded barcode region is sequenced for quantitation of clonal barcodes; a parallel sample portion is archived for retroactive analysis (p. 21, Paragraphs 4-5).
Specifically, Brock teaches that an existing workflow is established for the generation of high diversity barcode-tagged cell populations in patient-derived cultures, including reference standards, the efficiency of cellular lineage tracking and retrieval is tested using a) a reference set of low diversity barcodes and then b) a reference barcode in the background of a high diversity library of -106 barcodes (p. 38, Paragraph 1).
Regarding claim 19, Brock teaches that for developmental studies, screening in human and mouse pluripotent cells may pinpoint genes required for pluripotency or for differentiation into distinct cell types and to distinguish cell types, fluorescent or cell surface marker reporters of gene expression may be used and cells may be sorted into groups or compartments based on expression level; where any phenotype that is compatible with rapid sorting or separation may be harnessed for pooled screening (p. 29, Paragraph 3).
Regarding claim 20, Brock teaches methods and compositions wherein each cell in a population is uniquely tagged with a stably integrated barcode-gRNA under control of a constitutive promoter and following barcode instantiation, cells are permitted to proliferate and at intervals the genomically encoded barcode region is sequenced for quantitation of clonal barcodes; a parallel sample portion is archived for retroactive analysis, where RNA sequencing of barcode gRNA can be performed directly in one example and lineage dynamics may inform the identification of specific lineages of interest for subsequent gene activation in archival samples (p. 22, Paragraph 1).
Regarding claim 22, Brock teaches that to confirm the specificity and efficiency of lineage-specific expression, recall was tested in the presence of a large diverse barcoded population; and the high-diversity barcode gRNA library was constructed with the template: GNSNWNSNWNSNWNSNWNSN (SEQ ID NO: 1), having a diversity potential greater than 500,000,000 unique sequences (Fig. 2) and this gRNA library was ligated into a gRNA expression lentiviral transfer vector and assembled into a pooled gRNA barcoded lentivirus (p. 33, Paragraph 3).
Regarding claims 24 and 26-28, Brock further teaches methods for delivering vectors or other nucleic acid (such as RNA) into mammalian cells in culture (termed transfection) are routine, and a number of transfection methods find use with the invention and these include but are not limited to calcium phosphate precipitation, electroporation, lipid-based methods (liposomes or lipoplexes) such as Transfectamine® (Life Technologies™) and TransFectin™ (Bio-Rad Laboratories), cationic polymer transfections, for example using DEAE-dextran, direct nucleic acid injection, biolistic particle injection, and viral transduction using engineered viral carriers (termed transduction, using e.g., engineered herpes simplex virus, adenovirus, adeno-associated virus, vaccinia virus, Sindbis virus), and sonoporation (p. 12, Paragraph 1).
Regarding claim 33, Brock teaches that the previously described method can be applied to, for example, an ex vivo patient-specific tool, tumor cells are labeled with a library (more than 106 unique tags) of novel expressed barcodes, cultured as patient-derived organoids (i.e., breast, lung, ovarian) and treated with the same first-line treatment as patients where in multiple parallel samples, one can monitor the growth dynamics of the post-treatment population and determine which clones survive the treatment or may even have a growth advantage and using BAAR, these resistant clones of interest can be purified from an untreated population and evaluated to identify appropriate second and third line treatments that target the resistant survivor cell population (p. 37, Paragraphs 3-4).
Brock teaches each and every limitation of claims 1-3, 6, 8-10, 12, 15-20, 22, 24, 26-28 and 33, and therefore Brock anticipates claims 1-3, 6, 8-10, 12, 15-20, 22, 24, 26-28 and 33.
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.
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/ELIZABETH ROSE LAFAVE/Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684