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 .
Claims 1-20 are currently pending and under examination.
Information Disclosure Statement
The Information Disclosure Statements filed January 93, 2024; and March 07, 2024 have been 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/patents-application- process/filing-online/legal-framework-efs-web), 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 - The incorporation by reference paragraph required by 37 CFR 1.834(c)(1),
1.835(a)(2), or 1.835(b)(2) is missing, defective or incomplete.
Required response – Applicant must:
Amend the Sequence Listing Incorporation by Reference paragraph at page 1 of the
specification. It is noted the Sequence Listing Incorporation by Reference paragraph is included however fails to list the size of the ASCII text file. The ASCII text file itself lists the size as 20,889 bytes.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code (see Page 50, Lines 18, 21, 23, and 27, Page 51, Lines 1-2, Page 52, Lines 7, 9, 17 and 26, Page 56, Line 24, Page 57, Lines 2-3). Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
The use of the terms Triton® X-100, Phusion® (see Page 41, Line 9, Page 43, Line 8, Page 46, Line 3, Page 47, Lines 13, 15 and 25 and Page 49, Line 17), which are a trade names or a marks used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim 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 1-20 are rejected under 35 U.S.C. 101 because the claimed invention is directed to one or more judicial exceptions (i.e., product of nature, a law of nature, a natural phenomenon, or an abstract idea) without significantly more.
Every claimed invention must be examined to determine whether the claimed invention complies with 35 U.S.C. 101, particularly whether the claimed invention falls within a 35 U.S.C. 101 judicial exception of non-patentable subject matter (e.g., an abstract idea, law of nature, natural phenomenon, natural product etc.). Phenomena of nature, though just discovered, natural products, mental processes, and abstract intellectual concepts are not patentable, as they are the basic tools of scientific and technological work. See MPEP 2106. As per the “2019 Revised Subject Matter Eligibility Guidance” (Federal Register Vol. 84, No. 4, available 01-07-2019), claims drawn to a process, machine, manufacture or composition of matter are further analyzed according to a two-part process to determine if A) the claim(s) is/are “directed to” a judicial exception because the claims(s) recite(s) a judicial exception (i.e. prong one) that is not integrated into a practical application (i.e. prong two) and, if so, if B) the claim(s) provide(s) an inventive concept, i.e. recite(s) additional elements that amount to significantly more than the judicial exception.
Subject Matter Eligibility Test for Products and Processes
Step 1 - Is the Claim to a Process, Machine, Manufacture or Composition of Matter? YES
Claims 1-20 are directed to one of the statutory classes. Claims 1-20 are directed a method for analyzing an ability of target nucleic acid sequences to impact gene expression (process).
Step 2A, Prong One — Does the Claim Recite an Abstract Idea, Law of Nature, or Natural Phenomenon? YES
Claims 1-20 recite the abstract idea of using mathematical relationships, receiving, comparing and processing data using mental steps. Claims directed to nothing more than abstract ideas, natural phenomena, and laws of nature are not eligible for patent protection (see MPEP 2106.04). Abstract ideas include mathematical concepts, (mathematical formulas or equations, mathematical relationships and mathematical calculations), certain methods of organizing human activity, and mental processes (including procedures for collecting, observing, determining, comparing, evaluating, and organizing information (See MPEP 2106.04(a)(2)). In particular, these abstract ideas include:
• Analyzing an ability of target nucleic acid sequences to impact gene expression (mental process, human mind is capable of receiving/ collecting data, observing/evaluating, organizing information).
• Associating the target nucleic acid sequence with the associated barcode nucleic acid sequence based on the long-read sequencing information (mental process, human mind is capable of receiving/collecting data, observing/evaluating, organizing information).
• Analyzing the target nucleic acid sequences by comparing the long-read sequencing information and the short-read sequencing information (mental process, human mind is capable of receiving/collecting data, observing/evaluating, organizing information).
• Associating barcodes detected in the short-read sequencing information from extracted DNA, total mRNA, and polysome-bound mRNA with the target nucleic acid sequences from the long-read sequencing information. (mental process, human mind is capable of receiving/ collecting data, observing/evaluating, organizing information).
• Determining a number of target nucleic sequences, a number of RNA molecules translated from the target nucleic acid sequences, and a number of polysome-bound mRNA molecules from the long-read nucleic acid sequencing information and the short-read sequencing information. (mental process, human mind is capable of receiving/ collecting data, observing/evaluating, organizing information, mathematical relationships and mathematical calculations).
• Quantitating mRNA transcript levels by determining a ratio of the number of RNA molecules translated from the target nucleic acid sequences to the number of target nucleic sequences. (Comparing/receiving/organizing data using mathematical concepts such as mathematical formulas/equations, mathematical relationships and mathematical calculations).
• Comparing mRNA transcript levels of a wild-type target nucleic acid sequence to mRNA transcript levels of a mutant target nucleic acid sequence. (mental process, human mind is capable of receiving/collecting data, comparing date, observing/evaluating, organizing information).
• Quantitating mRNA translation levels by determining a ratio of the number of polysome-bound mRNA molecules to the number of RNA molecules translated from the target nucleic acid sequences. (Comparing/receiving/organizing data using mathematical concepts such as mathematical formulas/equations, mathematical relationships and mathematical calculations).
• Comparing mRNA translation levels of a mutant target nucleic acid sequence to mRNA translation levels of a wild-type target nucleic acid sequence. (mental process, human mind is capable of receiving/collecting data, comparing date, observing/evaluating, organizing information).
Therefore, the claims recite elements that constitute one or more judicial exceptions
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 analysis continues with identifying additional elements beyond the judicial exceptions that might evidence integration of the judicial exceptions into a practical application. The steps or elements in addition to the judicial exceptions are: “cloning the target nucleic acid sequences and associated barcode nucleic acid sequences into a plurality of plasmids”, “transducing the plurality of plasmids into a plurality of cells”, “extracting DNA, total mRNA, and polysome-bound mRNA from the plurality of cells” and “sequencing the barcode nucleic acid”, which is not indicative of integration into practical application. These steps, recited at a high level of generality, comprise routine data gathering, which is considered an insignificant extra-solution activity. This data gathering is required for using the judicial exceptions. (See MPEP 2106.05(g)). There are no further/additional steps which applies either the identified judicial exception into practical application. Thus, a careful evaluation of the claim as a whole fails to reveal the practical application of the judicial exception to, e.g., effect an improvement to the functioning of a computer or other technology/technical field, effect a particular treatment or prophylaxis for a disease or medical treatment, implement a particular machine that is integral to the claim, or effect a transformation or reduction of a particular article to a different state or thing, or to apply the judicial exception in another meaningful way beyond generally linking its use to a particular technological environment. Accordingly, the claims do not integrate the judicial exception(s) into a practical application and is therefore directed to a judicial exception.
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 a 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).
The eligibility analysis proceeds with identifying any additional elements or limitations, separate from the judicial exceptions, that might potentially render the claims directed to a judicial exception patent eligible. To render the claims patent- eligible, these elements must comprise meaningful limitations that add to or transform the judicial exception to the effect that it amounts to significantly more than the natural correlation or abstract idea itself - i.e. provide an “inventive concept’. The elements that are in addition to the judicial exception comprise: cloning the target nucleic acid sequences and associated barcode nucleic acid sequences into a plurality of plasmids, transducing the plurality of plasmids into a plurality of cells and extracting nucleic acids from the plurality of cells and sequencing the barcode nucleic acid. When considered separately and in combination, these elements do not add significantly more to the judicial exception. These steps are well-understood, routine and conventional activities in the field. For example, Cottrell et al. “PTRE-seq reveals mechanism and interactions of RNA binding proteins and miRNAs”, Nat Commun 9, 301, published January 19, 2018, cited on the IDS filed January 03, 2024, and Zhao et al. (“Massively parallel functional annotation of 3′ untranslated regions”. Nat Biotechnol 32, 387–393, published March 16, 2014), cited on the IDS filed January 03, 2024, as well as Aiden et al. (U.S. Patent Application Publication US 2020/0255828 A1, published August 13, 2020), disclose cloning the target nucleic acid sequences and associated barcode nucleic acid sequences into a plurality of plasmids, transducing the plurality of plasmids into a plurality of cells and extracting nucleic acids from the plurality of cells and sequencing the barcode nucleic acid. The claims recite an abstract idea with additional elements. Because these elements are not inventive concepts, the claims do not integrate the abstract idea into a practical application. The judicial exception alone cannot provide that inventive concept or practical application (MPEP 2106.05). The claims therefore do not include additional elements that are sufficient to amount to significantly more than the judicial exception.
Accordingly, the claims do not qualify as patent-eligible subject matter.
For further information, please see the latest revision of MPEP 2104-2106 {Patent Subject Matter Eligibility Under 35 U.S.C. 101}, including MPEP 2106.04 {Eligibility Step 2A: Whether a Claim is Directed to a Judicial Exception} and 2106.05 {Eligibility Step 2B: Whether a Claim Amounts to Significantly More}, as well as the guidance on Subject Matter Eligibility, including the 2019 Guidance issued Jan. 7, 2019, and the October 2019 Update, provided on the USPTO website at https:/Awww.uspto.gov/patent/laws-and-regulations/examination-policy/subject-matter- eligibility.
Claim Rejections - 35 USC § 112
Claims 4-5 and 19 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 4 is vague and indefinite for the following reasons:
In claim 4, lines 3-4, the terms “quantitating mRNA transcript levels by determining a ratio of the number of RNA molecules translated from the target nucleic acid sequences to the number of target nucleic sequences” are unclear and confusing. It is unclear how one is to determine transcript levels by determining the number of translated molecules? For the purposes of examination the claim is interpreted as meaning “quantitating mRNA transcript levels by determining a ratio of a number of RNA molecules transcribed from the target nucleic acid sequences to the number of target nucleic sequences”.
Claim 5 depends from claim 4 and is therefore included in this rejection.
Claim 19 is vague and indefinite for the following reasons:
In claim 19, lines 20-21, the terms “quantitating mRNA transcript levels by determining a ratio of the number of RNA molecules translated from the target nucleic acid sequences to the number of target nucleic sequences” are unclear and confusing. It is unclear how one is to determine transcript levels by determining the number of translated molecules? For the purposes of examination the claim is interpreted as meaning “quantitating mRNA transcript levels by determining a ratio of the number of RNA molecules transcribed from the target nucleic acid sequences to the number of target nucleic sequences”.
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 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 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 the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-20 are rejected under 35 U.S.C. 103 as being unpatentable over Cottrell et al. (“PTRE-seq reveals mechanism and interactions of RNA binding proteins and miRNAs”, Nat Commun 9, 301, published January 19, 2018), cited on the IDS filed January 03, 2024, and Zhao et al. (“Massively parallel functional annotation of 3′ untranslated regions”. Nat Biotechnol 32, 387–393, published March 16, 2014), cited on the IDS filed January 03, 2024, in view of Aiden et al. (U.S. Patent Application Publication US 2020/0255828 A1, published August 13, 2020).
Regarding claim 1, Cottrell teaches a method for analyzing an ability of target nucleic acid sequences to impact gene expression (Abstract and Page 2, Left Column, Last Paragraph). Cottrell teaches cloning the target nucleic acid sequences and associated barcode nucleic acid sequences into a plurality of plasmids (Page 3, Left Column, First—Second Paragraph, Page 10, Right Column, Fourth—Last Paragraph and Fig. 1). Cottrell teaches sequencing the plurality of plasmids to provide sequencing information based on a target nucleic acid sequence of the target nucleic acid sequences and an associated barcode nucleic acid sequence within a plasmid of the plurality of plasmids (Page 2, Right Column, Last Paragraph—Page 3, Left Column, Second Paragraph, Page 10, Right Column, Fourth—Last Paragraph and Fig. 1). Cottrell teaches associating the target nucleic acid sequence with the associated barcode nucleic acid sequence based on the sequencing information (Page 2, Right Column, Last Paragraph—Page 3, Left Column, Second Paragraph, Page 10, Right Column, Fourth—Last Paragraph and Page 11, Sixth-Seventh Paragraph). Cottrell teaches transducing the plurality of plasmids into a plurality of cells (Page 3, Left Column, Second Paragraph). Cottrell teaches extracting total mRNA, and polysome-bound mRNA from the plurality of cells (Page 3, Right Column, Second Paragraph). Cottrell teaches sequencing the barcode nucleic acid sequences in the extracted DNA, total mRNA, and polysome-bound mRNA to provide short-read sequencing information (Page 3, Right Column, Second Paragraph and Page 11, Left column, Sixth-Seventh Paragraph). Cottrell teaches analyzing the target nucleic acid sequences by comparing the plasmids and the short-read sequencing information (Page 3, Left Column, Last Paragraph—Right Column First Paragraph).
Regarding claim 2, Cottrell teaches associating barcodes detected in the short-read sequencing information from extracted DNA, total mRNA, and polysome-bound mRNA (Page 3, Right Column, Second Paragraph and Page 11, Left column, Sixth-Seventh Paragraph).
Regarding claim 3, Cottrell teaches wherein analyzing the target nucleic acid sequences further comprises determining a number of target nucleic sequences, a number of RNA molecules translated from the target nucleic acid sequences, and a number of polysome-bound mRNA molecules from the short-read sequencing information (Page 2, Right Column, First—Fourth Paragraph, Page 3, Left Column, First Paragraph—Right Column, First Paragraph, Page 5, Right Column, Last Paragraph, Page 6, Left Column Last Paragraph—Right Column, Last Paragraph, Page 9, Left Column, First Paragraph—Right Column, First Paragraph, Page 10, Left Column, First Paragraph, Page 11, Left Column, Fifth-Sixth Paragraph).
Regarding claim 4, Cottrell teaches analyzing the target nucleic acid sequences further comprises quantitating mRNA transcript levels by determining a ratio of the number of RNA molecules translated from the target nucleic acid sequences to the number of target nucleic sequences (Page 2, Right Column, Last Paragraph—Page 3, Left Column, Second Paragraph).
Regarding claim 5, Cottrell teaches comparing mRNA transcript levels of a wild-type target nucleic acid sequence to mRNA transcript levels of a mutant target nucleic acid sequence. (Page 3, Left Column, Last Paragraph—Right Column, First Paragraph, Page 2, Right Column, Last Paragraph and Fig. 3).
Regarding claim 6, Cottrell teaches analyzing the target nucleic acid sequences further comprises quantitating mRNA translation levels by determining a ratio of the number of polysome-bound mRNA molecules to the number of RNA molecules translated from the target nucleic acid sequences (Page 3, Left Column, Last Paragraph—Right Column, First Paragraph).
Regarding claim 7, Cottrell teaches comparing mRNA translation levels of a mutant target nucleic acid sequence to mRNA translation levels of a wild-type target nucleic acid sequence (Page 3, Left Column, Last Paragraph—Right Column, First Paragraph, Page 2, Right Column, Last Paragraph and Fig. 3).
Regarding claim 8, Cottrell teaches the target nucleic acid sequences include one or more untranslated regions (UTRs) (Page 2, Right Column, Last Paragraph).
Regarding claim 9, Cottrell teaches the one or more UTRs are selected from a 5' UTR, a 3' UTR, and combinations thereof (Page 2, Right Column, Last Paragraph).
Regarding claim 10, Cottrell teaches a target nucleic acid sequence of the target nucleic acid sequences has a length in a range of about 40 base pairs to about 3,000 base pairs (Page 2, Right Column, Last Paragraph).
Regarding claim 11, Cottrell teaches the plasmid further comprises a promoter sequence (Page 3, Left Column, First-Second Paragraph and Fig. 1).
Regarding claim 12, Cottrell teaches the promoter nucleic acid sequence is disposed at a 5' end of the target nucleic acid sequence (Page 3, Left Column, First-Second Paragraph and Fig. 1).
Regarding claim 13, Cottrell teaches the plasmid further comprises a reporter nucleic acid sequence (Page 3, Left Column, First Paragraph—Right Column First Paragraph and Fig. 1).
Regarding claim 14, Cottrell teaches the reporter nucleic acid sequence is disposed at a 3' end of the target nucleic acid sequence (Page 3, Left Column, First Paragraph—Right Column First Paragraph and Fig. 1).
Regarding claim 15, Cottrell teaches the reporter nucleic acid sequence is disposed at a 5' end of the barcode nucleic acid sequence (Page 3, Left Column, First Paragraph—Right Column First Paragraph and Fig. 1).
Regarding claim 16, Cottrell teaches the barcode nucleic acid sequences include nucleic acid sequences consisting of a random nucleic acid sequence (Page 11, Left Column, Sixth Paragraph and Right Column Fifth Paragraph).
Regarding claim 17, Cottrell teaches introducing a plurality of mutations into a plasmid of the plurality of plasmids (Page 3, Left Column, Last Paragraph—Right Column, First Paragraph, Page 2, Right Column, Last Paragraph and Fig. 3).
Regarding claim 18, Cottrell teaches the analyzed target nucleic acid sequence as discussed above.
Regarding claim 19, Cottrell teaches a method for analyzing an ability of target nucleic acid sequences to impact gene expression (Abstract and Page 2, Left Column, Last Paragraph). Cottrell teaches cloning the target nucleic acid sequences and associated barcode nucleic acid sequences into a plurality of plasmids (Page 3, Left Column, First—Second Paragraph, Page 10, Right Column, Fourth—Last Paragraph and Fig. 1). Cottrell teaches sequencing the plurality of plasmids to provide sequencing information based on a target nucleic acid sequence of the target nucleic acid sequences and an associated barcode nucleic acid sequence within a plasmid of the plurality of plasmids (Page 2, Right Column, Last Paragraph—Page 3, Left Column, Second Paragraph, Page 10, Right Column, Fourth—Last Paragraph and Fig. 1). Cottrell teaches associating the target nucleic acid sequence with the associated barcode nucleic acid sequence based on the sequencing information (Page 2, Right Column, Last Paragraph—Page 3, Left Column, Second Paragraph, Page 10, Right Column, Fourth—Last Paragraph and Page 11, Sixth-Seventh Paragraph). Cottrell teaches transducing the plurality of plasmids into a plurality of cells (Page 3, Left Column, Second Paragraph). Cottrell teaches extracting total mRNA, and polysome-bound mRNA from the plurality of cells (Page 3, Right Column, Second Paragraph). Cottrell teaches sequencing the barcode nucleic acid sequences in the extracted DNA, total mRNA, and polysome-bound mRNA to provide short-read sequencing information (Page 3, Right Column, Second Paragraph and Page 11, Left column, Sixth-Seventh Paragraph). Cottrell teaches analyzing the target nucleic acid sequences by comparing the plasmids and the short-read sequencing information (Page 3, Left Column, Last Paragraph—Right Column First Paragraph). Cottrell teaches wherein analyzing the target nucleic acid sequences further comprises determining a number of target nucleic sequences, a number of RNA molecules translated from the target nucleic acid sequences, and a number of polysome-bound mRNA molecules from the short-read sequencing information (Page 2, Right Column, First—Fourth Paragraph, Page 3, Left Column, First Paragraph—Right Column, First Paragraph, Page 5, Right Column, Last Paragraph, Page 6, Left Column Last Paragraph—Right Column, Last Paragraph, Page 9, Left Column, First Paragraph—Right Column, First Paragraph, Page 10, Left Column, First Paragraph, Page 11, Left Column, Fifth-Sixth Paragraph). Cottrell teaches analyzing the target nucleic acid sequences further comprises quantitating mRNA transcript levels by determining a ratio of the number of RNA molecules translated from the target nucleic acid sequences to the number of target nucleic sequences (Page 2, Right Column, Last Paragraph—Page 3, Left Column, Second Paragraph). Cottrell teaches comparing mRNA transcript levels of a wild-type target nucleic acid sequence to mRNA transcript levels of a mutant target nucleic acid sequence. (Page 3, Left Column, Last Paragraph—Right Column, First Paragraph, Page 2, Right Column, Last Paragraph and Fig. 3).
Regarding claim 20, Cottrell teaches a method for analyzing an ability of target nucleic acid sequences to impact gene expression (Abstract and Page 2, Left Column, Last Paragraph). Cottrell teaches cloning the target nucleic acid sequences and associated barcode nucleic acid sequences into a plurality of plasmids (Page 3, Left Column, First—Second Paragraph, Page 10, Right Column, Fourth—Last Paragraph and Fig. 1). Cottrell teaches sequencing the plurality of plasmids to provide sequencing information based on a target nucleic acid sequence of the target nucleic acid sequences and an associated barcode nucleic acid sequence within a plasmid of the plurality of plasmids (Page 2, Right Column, Last Paragraph—Page 3, Left Column, Second Paragraph, Page 10, Right Column, Fourth—Last Paragraph and Fig. 1). Cottrell teaches associating the target nucleic acid sequence with the associated barcode nucleic acid sequence based on the sequencing information (Page 2, Right Column, Last Paragraph—Page 3, Left Column, Second Paragraph, Page 10, Right Column, Fourth—Last Paragraph and Page 11, Sixth-Seventh Paragraph). Cottrell teaches transducing the plurality of plasmids into a plurality of cells (Page 3, Left Column, Second Paragraph). Cottrell teaches extracting total mRNA, and polysome-bound mRNA from the plurality of cells (Page 3, Right Column, Second Paragraph). Cottrell teaches sequencing the barcode nucleic acid sequences in the extracted DNA, total mRNA, and polysome-bound mRNA to provide short-read sequencing information (Page 3, Right Column, Second Paragraph and Page 11, Left column, Sixth-Seventh Paragraph). Cottrell teaches analyzing the target nucleic acid sequences by comparing the plasmids and the short-read sequencing information (Page 3, Left Column, Last Paragraph—Right Column First Paragraph). Cottrell teaches wherein analyzing the target nucleic acid sequences further comprises determining a number of target nucleic sequences, a number of RNA molecules translated from the target nucleic acid sequences, and a number of polysome-bound mRNA molecules from the short-read sequencing information (Page 2, Right Column, First—Fourth Paragraph, Page 3, Left Column, First Paragraph—Right Column, First Paragraph, Page 5, Right Column, Last Paragraph, Page 6, Left Column Last Paragraph—Right Column, Last Paragraph, Page 9, Left Column, First Paragraph—Right Column, First Paragraph, Page 10, Left Column, First Paragraph, Page 11, Left Column, Fifth-Sixth Paragraph). Cottrell teaches analyzing the target nucleic acid sequences further comprises quantitating mRNA translation levels by determining a ratio of the number of polysome-bound mRNA molecules to the number of RNA molecules translated from the target nucleic acid sequences (Page 3, Left Column, Last Paragraph—Right Column, First Paragraph). Cottrell teaches comparing mRNA translation levels of a mutant target nucleic acid sequence to mRNA translation levels of a wild-type target nucleic acid sequence (Page 3, Left Column, Last Paragraph—Right Column, First Paragraph, Page 2, Right Column, Last Paragraph and Fig. 3).
Cottrell does not teach or suggest sequencing the plurality of plasmids to provide long-read sequencing information based on a target nucleic acid sequence of the target nucleic acid sequences and an associated barcode nucleic acid sequence within a plasmid of the plurality of plasmids. Cottrell does not teach or suggest associating the target nucleic acid sequence with the associated barcode nucleic acid sequence based on the long-read sequencing information. Cottrell does not teach or suggest comparing the long-read sequencing information and the short-read sequencing information. Cottrell does not teach or suggest associating barcodes detected in the short-read sequencing information from extracted DNA, total mRNA, and polysome-bound mRNA with the target nucleic acid sequences from the long-read sequencing information. Cottrell does not teach or suggest determining a number of target nucleic sequences, a number of RNA molecules translated from the target nucleic acid sequences, and a number of polysome-bound mRNA molecules explicitly from the long-read nucleic acid sequencing information. Cottrell does not teach or suggest confirming the analyzed target nucleic acid sequences with a process selected from clustered regularly interspaced short palindromic repeats (CRISPR)- mediated base editing.
Zhao teaches analyzing an ability of ability of target nucleic acid sequences to impact gene expression (Abstract, Page 387, Left Column, Last Paragraph—Right Column, First Paragraph and Page 390, Right Column, Last Paragraph). Zhao teaches the target includes a 3’UTR (Fig. 1). Zhao teaches extracting, analyzing and sequencing DNA and total mRNA (Abstract, Page 392, Left Column, Third Paragraph and Fig. 1). Zhao teaches extract DNA and mRNA that can be used as a template for sequencing to analyze an ability of target nucleic acid sequence to impact gene expression (Abstract). Zhao teaches cloning the target nucleic acid sequences and associated barcode nucleic acid sequences into a plurality of plasmids and associating barcodes detected in the short-read sequencing information from extracted DNA, total mRNA (Page 387, Left Column, Last Paragraph—Right Column, First Paragraph and Fig. 1). Zhao teaches quantitating mRNA transcript levels by determining a ratio of the number of RNA molecules transcribed from the target nucleic acid sequences to the number of target nucleic sequences (Page 393, Left Column, Second—Third Paragraph). Zhao teaches comparing mRNA transcript levels of a wild-type target nucleic acid sequence to mRNA transcript levels of a mutant target nucleic acid sequence (Fig. 1). Zhao teaches introducing a plurality of mutations into a plasmid of the plurality of plasmids (Fig. 1). Zhao teaches using these methods allows for directly quantifying the effects of regulatory elements (3’UTR sequence variation on gene expression as well as steady-state mRNA abundance, mRNA stability and protein production (Page 387, Left Column, First Paragraph—Right Column, First Paragraph and Page 390, Left Column, First Paragraph).
Aiden teaches a method for analyzing an ability of target nucleic acid sequences to impact gene expression (Page 1, [0007]-[0008] and Page 2, [0021]-[0023]). Aiden teaches cloning the target nucleic acid sequences and associated barcode nucleic acid sequences into a plurality of plasmids (Page 8, [0140]-[0141], Page 28, [0283], Page 96, [0810]). Aiden teaches sequencing the plurality of plasmids to provide long-read sequencing information based on a target nucleic acid sequence of the target nucleic acid sequences and an associated barcode nucleic acid sequence within a plasmid of the plurality of plasmids (Page 96, [0810], Page 22, [0240] and Page 25, [0261]-[0262]). Aiden teaches associating the target nucleic acid sequence with the associated barcode nucleic acid sequence based on the long-read sequencing information (Page 96, [0810], Page 22, [0240] and Page 25, [0261]-[0263]). Aiden teaches transducing the plurality of plasmids into a plurality of cells (Page 28, [0283], Page 50, [0487] and Pages 95-96, [0809]-[0810]). Aiden teaches extracting DNA and RNA from the plurality of cells (Page 24, [0252], Page 54, [0511] and Page 97, [0822]). Aiden teaches sequencing the barcode nucleic acid sequences in the extracted DNA and RNA to provide short-read sequencing information (Page 16, [0201], Page 22, [0240], Page 25, [0262], Page 85, [0740], Page 95, [0806] and Page 96, [0810]). Aiden teaches analyzing the target nucleic acid sequences by comparing the long-read sequencing information and the short-read sequencing information including associated barcodes (i.e., targets sequenced using short-read sequencing technologies or long read sequencing technologies, determining two or more junctions or a set of target junctions in which one may be short-read sequenced and one may be long-read sequenced as well as the identity of RNA and DNA may be determined by Sanger, SBS, Nanopore and SMRT sequencing and analysis of (comparing the two) the results of those sequencing is performed to analyze gene expression; Page 22, [0240]-[0241], Page 254, [0261]-[0263] and Pages 95-96, [0809]-[0810]). Aiden teaches quantifying the transcript level (Page 15, [0192], Page 16, [0198]-[0199], Pages 88-89, [0773] and Page 98, [0835]) Aiden teaches determining transcription levels of wild type target nucleic acids and mutant targets (Pages 28-29, [0284], Page 36, [0348]-[0349], Page 38, [0363]-[0366] and Page 64, [0599]). Aiden teaches analyzing the level of translation for gene expression (Page 28, [0280], Pages 28-29, [0284], Page 33, [0313] and Page 64, [0599]). Aiden teaches a 3’ UTR (Page 51, [0492]). Aiden teaches the target length is in the range of 40 to 3,000 base pairs (Page 21, [0238]). Aiden teaches the plasmid comprises a promoter sequence (Page 49, [0476]). Aiden teaches the plasmid comprises a reporter nucleic acid sequence (Page 96, [0810]). Aiden teaches introducing a plurality of mutations into a plasmid of the plurality of plasmids (Page 96, [0810]). Aiden teaches confirming the analyzed target nucleic acid sequences with clustered regularly interspaced short palindromic repeats (CRISPR)- mediated base editing (Page 5, [0060]-[0061], Page 40, [0401]-[0409]), Page 82, [0713], Page 21, [0233] and Page 17, [0206]). Aiden using these methods allows for modulating and analyzing gene expression (Page 1, [0004], Page 34, [0318] and Page 38, [0360]).
It would have been obvious to one having ordinary skill in the art before the effective filing date of the invention to modify the teachings of Cottrell with the teaching of Zhao to use extracted DNA and total mRNA that can be used as a template for sequencing. Using these methods would allow for directly quantifying the effects of regulatory elements (3’UTR), sequence variation on gene expression, as well as steady-state mRNA abundance, mRNA stability and protein production as taught by Zhao (Page 387, Left Column, First Paragraph—Right Column, First Paragraph and Page 390, Left Column, First Paragraph).
It would have been obvious to one having ordinary skill in the art before the effective filing date of the invention to modify the teachings of Cottrell and Zhao with the teachings of Aiden, to use long-read sequencing and compare the long-read with the short read sequencing as well as confirming the analyzed target nucleic acid sequences with a process selected from clustered regularly interspaced short palindromic repeats (CRISPR)- mediated base editing. Using these methods would allow for modulating and analyzing gene expression as taught by Aiden (Page 1, [0004], Page 34, [0318] and Page 38, [0360]).
Conclusion
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/JESSICA D PARISI/Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684
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