DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Office Action: Notice
Any objection or rejection of record in the previous Office Action, mailed 6/4/2025,
which is not addressed in this action has been withdrawn in light of Applicants' amendments
and/or arguments. This action is FINAL
Claim Status
Applicant previously cancelled claims 2-65 (8/25/2022). Claims 1, 66-67 and 79 have been amended (7/8/2026). Claims 80-81 are new (7/8/2026). Claim 77 is cancelled (7/8/2026). No new matter was added. Thus, claims 1 and 66-76, 78-81 are under examination (3/23/2026).
Priority
Claims 1 and 66-76, 78-81 claim a priority date of 7/14/2014, the filing date of U.S. Provisional No. 62/024,373.
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 (IDS) were submitted on 7/8/2026 and are being considered by the examiner.
Rejections Maintained
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 and 66-76, 78-81 are rejected under 35 U.S.C. 101 because the claimed invention is directed to an abstract idea without significantly more.
The claims recite methods and compositions for identifying genetic elements that modulate phenotypes by providing libraries of sgRNAs, contacting cells with these libraries, selecting cells based on phenotype, and quantitating frequencies of sgRNAs to identify genetic elements that influence phenotypes. Therefore, the claims are directed to an abstract idea, including mathematical concepts and mental processes involving statistical analysis of frequency data and correlation analysis.
Specifically, the additional elements of providing sgRNA libraries, contacting cells, applying CRISPR-Cas9 systems and analyzing phenotypes are recited at a high level of generality and represent well-understood, routine, and conventional techniques in the field of genetic screening. More so, the additional limitation that at least 50% of sgRNAs comprise binding regions lacking a UUU sequence represents a constraint on sequence selection and does not alter the abstract nature of the claimed data analysis or provide a technical improvement. These steps merely perform data gathering and application of the identified relationship and do not impose a meaningful limit on the judicial exception. See MPEP 2106.05 (d).
The integration of the judicial exception into the claims does not render them patent eligible because the claims are written at a high level of generality and merely use well-known, routine, and conventional techniques in the field.
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:
identifying genetic interactions that modulate phenotypes by providing libraries of sgRNAs targeting genetic elements, contacting cells with these libraries, selecting cells based on phenotype, and quantitating frequency distributions of sgRNAs;
applying CRISPR-Cas9 systems to cells to generate test populations and analyzing the effects on phenotypes through cellular assays;
using structurally distinct sgRNAs as molecular identifiers to track genetic element targeting;
analyzing the frequency data to identify genetic elements that modulate phenotypes for scientific and therapeutic applications; and
implementing these methods through systems configured to process molecular analyses and identify genetic elements affecting phenotypes.
Thus, the claims are directed to statutory categories (i.e., processes and compositions).
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. The “mental process” of determining correlations between genetic elements and phenotypes and analyzing frequency distribution patterns corresponds to “an abstraction” (an idea having no particular concrete or tangible form).
Thus, the claimed invention describes a judicial exception, which correspond to abstractions (ideas, having no particular concrete or tangible form) and natural 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 are considered to be; (a) a mental process of evaluating/interpreting data by identifying the sub-sets of genetic elements which modulate phenotypes, identifying most frequent sgRNA species, and determining the probabilities of correspondence, (b) mathematical relations for calculating frequency distribution patterns and probability thresholds for identifying genetic elements that modulate phenotype (i.e., natural principle).
While the claims recite steps of “providing libraries of sgRNAs”, “contacting cells with libraries”, “selecting cells based on phenotype”, “quantitating frequency distributions”, these steps are recited at a high level of generality and amount to mere data fathering steps, including conventional laboratory techniques. There are no additional steps which apply either of the identified judicial exceptions into a practical application. Thus, the claims do not provide for any element/step that integrates the law of nature 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 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).
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” elements/steps of, “providing libraries of sgRNAs”, “contacting cells with these libraries”, “selecting cells based on phenotype”, “identifying interactions in test cells”, “quantitating frequency distributions” and “analyzing genetic elements that modulate phenotypes” are “physical” steps telling a practitioner to simply implement the abstract idea and are considered to be within the purview of one in the art as being routine and conventional in the art when investigating identification of genetic elements that modulate phenotypes.
For example, Kabadi et al. discloses (“Engineering synthetic TALE and CRISPR/Cas9 transcription factors for regulating gene expression”, Methods, published 7/8/2014, from IDS 2/23/2022), that there are three main classes of natural biomolecules that have been engineered to target new DNA sequences and manipulate gene expression: zinc finger proteins (ZFPs), Transcription activator-like effectors (TALEs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) system and each of these programmable DNA-binding proteins can be genetically fused to an effector domain to create custom enzymes that localize the effector function to the DNA target site (Introduction: Paragraph 1). Further, Kabadi discloses that in the naturally-occurring system of RNA-guided defense mechanisms, bacteria and archaea integrate short fragments of foreign nucleic acids (termed protospacers) into the CRISPR genomic loci and therefore functioning as molecular memory of previous invaders, allowing the CRISPR locus to be transcribed and processed into short CRISPR-derived RNAs (crRNAs) (Section 1.2 The CRSIPR/Cas system: Paragraph 1), and thus establishing these as conventional techniques.
Further, May et al. (US PgPub 2014/0315985 A1; filed 3/12/2014) discloses, the development of a mutation in a P-domain of the nucleic acid-targeting nucleic acid in which the P-domain starts downstream of a last paired nucleotide of a duplex between a CRISPR repeat and a tracrRNA sequence of the nucleic acid-targeting nucleic acid for the goal of inserting modifications of nucleic acid-targeting nucleic acids and site-directed polypeptides to introduce new functions to be used for genome engineering (Background of the Invention). May specifically discloses that modifications to Cas9 domains can include a site-directed polypeptide that affect the binding specificity of the enzyme for nucleic acids, where regions may share either sequence or functional homology with domains or regions as found in RNA polymerase or be bound to the tracrRNA and crRNA or a single RNA (sgRNA) (Paragraph 572, lines 1-5). These detailed specifications demonstrate that practitioners were well-versed in mutating Cas9 from the CRISPR system to generate specified DNA recognition complexes.
Further, Shalem et al. (“Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells”, Science, published 7/9/2014, from IDS 2/23/2022) discloses the simplicity of programming the CRISPR-associated nuclease Cas9 to modify specific genomic loci and therefore suggesting a new way to interrogate gene function on a genome-wide scale, including through the lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells (Abstract). Further, Shalem discloses that because the targeting specificity of Cas9 is conferred by short guide sequences, which can be easily generated at large scale by array-based oligonucleotide library synthesis it can be easily explored for the potential of Cas9 for pooled genome-scale functional screening (Paragraphs 3-4). Shalem also discloses that although RNAi is limited to transcripts, Cas9:sgRNAs can target elements across the entire genome, including promoters, enhancers, introns, and inter-genic regions and catalytically inactive mutants of Cas9 can be tethered to different functional domains to broaden the repertoire of perturbation modalities, including genome-scale gain-of-function screening using Cas9-activators and epigenetic modifiers (Paragraphs 9-10), thus establishing a high level of routine and convention.
Additionally, Wu et al. (“Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells”, Nature Biotechnology, published 4/2014) discloses a comparison of the seed regions of the four sgRNAs suggested that UUU in the seed of Nanog-sg2 might be responsible for decreased sgRNA abundance and increased specificity, consistent with a recent observation that U in PAM-proximal positions 1–4 leads to low gene-knockout efficacy14, where two mutations (U to G and U to A) in the Nanog-sg2 seed region that converted the seed (GUUUC) to the same sequence as the Phc1-sg2 seed (GGUAC), led to higher levels of sgRNA (sgRNA N2b in Fig. 4c) (Seed Sequences influence sgRNA abundance and specificity: Paragraphs 1-4). Additionally, Wu discloses considering the presence of GUUUUA adjacent to the seed and because sgRNAs are transcribed by RNA polymerase III, which is terminated by U-rich sequences40,41, and, together with the downstream U-rich region, multiple U's in the seed might induce termination of sgRNA transcription, where consistent with this, three sgRNAs with seeds UUAUU, ACUUU and UUUUU also showed very low abundance (Fig. 4c, sgRNA P3, N5 and N6) and when GUUUC was placed upstream of the seed (i.e., away from GUUUUA in the sgRNA), the sgRNA was well expressed (sgRNA C4 in Fig. 4c) (Seed Sequences influence sgRNA abundance and specificity: Paragraphs 1-4). And, thus, Wu discloses that UUU were routinely considered for sgRNA library construction.
Therefore, providing libraries of sgRNAs and determining the resultant correlation between genetic elements and phenotypes was routine and conventional before the effective filing date of the claimed invention.
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., “providing libraries of sgRNAs”, “contacting cells with these libraries”, “selecting cells based on phenotype”, “quantitating frequencies”, “identifying genetic elements that modulate phenotypes”) because those techniques were well-understood, routine, and conventional techniques that a practitioner would have thought of when instructed to analyze the natural phenomenon of how genetic elements affects phenotypes. In context with the other recited claim limitations, the language “identifying genetic elements that modulate a phenotype comprising: providing a library of sgRNAs, in which each sgRNA is represented multiple times; contacting cells with the library; selecting cells based on phenotype; and quantitating frequency distributions to identify genetic elements that influence phenotypes” indicates whether or not the relationship/correlation between providing genetic elements and phenotypes exists.
This information simply tells a practitioner about the relevant natural law (the relationship between genetic elements and phenotype outcomes), at most adding a suggestion that the medical researcher should take those laws into account. Thus, when viewed both individually and as an ordered combination, the claimed elements/steps in addition to the identified judicial exceptions (abstract idea of mathematical analysis) 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 analyzing natural phenomenon with routine, conventional activity.”
Accordingly, the claims do not qualify as patent-eligible subject matter.
Applicant’s Response: Applicant argues that claims 1 and 79 do not recite a judicial exception because they require physical CRISPR-based screening steps, including contacting cells with a defined sgRNA libraries, generating and selecting test cells based on phenotype, and quantifying sgRNA frequencies, rather than merely determining a correlation. Applicant further argues that, even if a judicial exception is recited, these physical steps and the claimed sgNRA-library architecture integrate the exception into a practical application and provide significantly more because the specific claimed features, including binding regions lacking a UUU sequence, were not well-understood, routine, and conventional.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully and fully considered and are not found persuasive, as discussed below.
Although claims 1 and 79 recite physical steps involving contacting cells with sgRNA libraries, selectin cells based on phenotype, and quantifying sgRNA frequencies, these steps primarily generate and collect the biological data used to identify the recited relationship between genetic elements and phenotype; the claims ultimately stop at identifying genetic elements associated with the phenotype. The claimed screening therefore does not meaningfully apply the identified relationship to achieve a further practical result, and the recited CRSIPR/sgRNA techniques do not, by themselves, integrate the judicial exception into a practical application.
To provide meaningful integration, the claims would need to more specifically apply the identified genetic relationship to a concrete practical use, rather than merely discover or report it-for example, by requiring administration or selection of a particular drug or treatment, modification of a specific therapeutic regimen based on the identified genetic element, or another particular treatment/action that uses the screening result to effect a practical outcome. Accordingly, Applicant’s arguments do not overcome the rejection under 35 USC 101 and the rejection is maintained.
Amended Rejections in Response to Amendments (7/8/2026)
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 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 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 and 66-76, 78-81 are rejected under 35 U.S.C. 103 as being unpatentable over May et al. (US PGPub 2014/0315985 A1; filed 3/12/2014), in view of Wu et al. (“Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells”, Nature Biotechnology, published 4/2014).
Regarding claim 1, May teaches a method for generating a library of target nucleic acids comprising: contacting a plurality of target nucleic acids with a complex comprising a site-directed polypeptide and a nucleic acid-targeting nucleic acid, cleaving the plurality of target nucleic acids, and purifying the plurality of target nucleic acids to create the library of target nucleic acids (Paragraph 59, lines 1-5). Further May teaches that the previously described method can be further expanded to allow for novel therapeutic applications, (i.e., prevention and/or treatment of: genetic diseases, cancer, fungal, protozoal, bacterial, and viral infection, ischemia, vascular disease, arthritis, immunological disorders, etc.), novel diagnostics (i.e., prediction and/or diagnosis of a condition) as well as providing for research tools (i.e., kits, functional genomics assays, and generating engineered cell lines and animal models for research and drug screening), and means for developing plants with altered phenotypes, including but not limited to, increased disease resistance, and altering fruit ripening characteristics, sugar and oil composition, yield, and color (Paragraph 503, lines 1-5). Additionally, May teaches that a site-directed polypeptide may also comprise certain regions that affect the binding specificity of the enzyme for nucleic acids, including regions that share either sequence or functional homology with domains or regions as found in RNA polymerase (i.e., tracrRNA and crRNA or a single RNA (sgRNA) (Paragraph 572, lines 1-5). Specifically, May teaches that the previously described nucleic acid guide tags include
a single guide RNA and can comprise a crRNA or a detectable label (815), and when the nucleic acid sample is ligated to the nuclei acid tag (821) (Paragraph 644, lines 1-5).
May also teaches that the previously described site-directed polypeptides within the screening method can be a type of protein, an nuclease, an endoribonuclease, a modified (i.e., shortened, mutated, lengthened) polypeptide sequence or homologue of the site-directed polypeptide and can additionally be codon optimized or enzymatically inactive, partially active, constitutively active, fully active, inducible active and/or more active, (i.e., more than the wild type homologue of the protein or polypeptide.), including Cas9 (Paragraph 198, lines 1-10).
May also teaches that the previously described site-directed polypeptides within the screening method is assembled via a recombinant DNA sequence that encodes for a modified site-directed polypeptide, and enables the expression of the modified site-directed polypeptide in a host organism and further comprises a promoter sequence, and may additionally comprise an affinity tag for purification, or an epitope tag or a sequence for the expression of the modified site-directed polypeptide (Paragraph 874, lines 1-5). Specifically, May teaches that the eluted target nucleic acids will be prepared for sequencing analysis via the generation of sequencing libraries of the eluted target nucleic acid to determine the identity and frequency of off-target binding sites of site-directed polypeptides (Paragraph 914, lines 1-5). Additionally, May teaches that the quantification of the remaining detectable labels (760) can be correlated to which sequences were represented in the nucleic acid sample (705) and which were not; including oligonucleotides that do not display a remaining detectable label (760) that correspond to sequences that were represented (or overrepresented) in the nucleic acid sample (705) and oligonucleotides that display a remaining detectable label (760) which correspond to sequences that were not represented (or underrepresented) in the nucleic acid sample (705) (Paragraph 643, lines 30-40).
May teaches that the previously described library of expression vectors comprising donor polynucleotides (sgRNAs) includes a composition comprising: a nucleic acid comprising: a spacer, wherein the spacer is between 12-30 nucleotides, inclusive, and wherein the spacer is adapted to hybridize to a sequence that is 5' to a PAM or guanosine nucleotide; a first duplex, wherein the first duplex is 3' to the spacer; a bulge, wherein the bulge comprises at least 3 unpaired nucleotides on a first strand of the first duplex and at least 1 unpaired nucleotide on a second strand of the first duplex; a linker, wherein the linker links the first strand and the second strand of the duplex and is at least 3 nucleotides in length; a P-domain; and a second duplex, wherein the second duplex is 3' of the P-domain and is adapted to bind to a site directed polypeptide. In some embodiments, the sequence that is 5' to a PAM is at least 18 nucleotides in length (19-21 nucleotides) (Paragraph 78, lines 1-5). Specifically, May teaches that the previously described sequence is 5' to a PAM is adjacent to the PAM and the PAM comprises 5'-NGG-3 (Paragraph 78, lines 1-10).
Regarding claim 66, May teaches a library composition of small guide RNAs (sgRNAs) or microarrays that comprise a plurality of polynucleotide probes or genetic elements, including about 1, 10, 100, 1000, 5000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 110000, 120000 or more probes (Paragraph 655, lines 1-3). Further, May teaches that a library of expression vectors comprising donor polynucleotides (sgRNAs) includes expression vectors comprising polynucleotide sequences encoding for differing genetic elements of interest but the same reporter elements or the library can comprise expression vectors comprising polynucleotide sequences encoding for differing genetic elements of interest and differing reporter elements (Paragraph 785, lines 1-5). Specifically, May teaches that the second method can use a sequence-specific RNA or DNA-based probe to quantify only the nucleic acid containing the probe sequence (Paragraph 650, lines 20-30). Also, May teaches that the previously described library of expression vectors or sgRNAs include an affinity tag comprising a sequence that can bind to a conditionally enzymatically inactive site-directed polypeptide via the affinity tag comprising a sequence that can bind to a conditionally enzymatically inactive endoribonuclease and can contain a sequence that can bind to a conditionally enzymatically inactive Csy4 protein (Paragraph 946, lines 1-5). Specifically, May teaches that the conditionally enzymatically inactive site-directed polypeptide will bind, but not cleave, the affinity tag and that the affinity tag or binding region comprises the nucleotide sequence 5'-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3' or therefore lacking a UUU sequence (Paragraph 946, lines 1-10).
May teaches that the previously described library of expression vectors comprising donor polynucleotides (sgRNAs) includes a composition comprising: a nucleic acid comprising: a spacer, wherein the spacer is between 12-30 nucleotides, inclusive, and wherein the spacer is adapted to hybridize to a sequence that is 5' to a PAM or guanosine nucleotide; a first duplex, wherein the first duplex is 3' to the spacer; a bulge, wherein the bulge comprises at least 3 unpaired nucleotides on a first strand of the first duplex and at least 1 unpaired nucleotide on a second strand of the first duplex; a linker, wherein the linker links the first strand and the second strand of the duplex and is at least 3 nucleotides in length; a P-domain; and a second duplex, wherein the second duplex is 3' of the P-domain and is adapted to bind to a site directed polypeptide. In some embodiments, the sequence that is 5' to a PAM is at least 18 nucleotides in length (19-21 nucleotides) (Paragraph 78, lines 1-5). Specifically, May teaches that the previously described sequence is 5' to a PAM is adjacent to the PAM and the PAM comprises 5'-NGG-3 (Paragraph 78, lines 1-10).
Regarding claim 67, May teaches that the previously described library of expression vectors comprising donor polynucleotides (sgRNAs) includes a composition comprising: a nucleic acid comprising: a spacer, wherein the spacer is between 12-30 nucleotides, inclusive, and wherein the spacer is adapted to hybridize to a sequence that is 5' to a PAM or guanosine nucleotide; a first duplex, wherein the first duplex is 3' to the spacer; a bulge, wherein the bulge comprises at least 3 unpaired nucleotides on a first strand of the first duplex and at least 1 unpaired nucleotide on a second strand of the first duplex; a linker, wherein the linker links the first strand and the second strand of the duplex and is at least 3 nucleotides in length; a P-domain; and a second duplex, wherein the second duplex is 3' of the P-domain and is adapted to bind to a site directed polypeptide. In some embodiments, the sequence that is 5' to a PAM is at least 18 nucleotides in length (19-21 nucleotides) (Paragraph 78, lines 1-5). Specifically, May teaches that the previously described sequence is 5' to a PAM is adjacent to the PAM and the PAM comprises 5'-NGG-3 (Paragraph 78, lines 1-10).
Regarding claims 68-69, May teaches that the previously described library of expression vectors comprising donor polynucleotides (sgRNAs) includes at least one of the plurality of nucleic acid molecules encodes for a nucleic acid-targeting nucleic acid and one of the plurality of nucleic acid molecules encodes for a site-directed polypeptide; and a fusion polypeptide, wherein the fusion polypeptide comprises a plurality of the nucleic acid-binding proteins, wherein the plurality of nucleic acid-binding proteins are adapted to bind to their cognate nucleic acid-binding protein binding sites delivering the composition to the subcellular location; an expression vector comprising a polynucleotide sequence encoding for a genetic element of interest; and a reporter element, wherein the reporter element comprises a polynucleotide sequence encoding a site-directed polypeptide, and one or more a nucleic acids, wherein the one or more nucleic acids comprises a sequence comprising at least 50% sequence identity to a crRNA over 6 contiguous nucleotides and a sequence comprising at least 50% sequence identity to a tracrRNA or sgRNA over 6 contiguous nucleotides; and a vector comprising a polynucleotide sequence encoding a nucleic acid (Paragraph 87, lines 30-40).
Regarding claim 70, May teaches that the previously described library of expression vectors comprising donor polynucleotides (sgRNAs) includes specified binding regions, consisting of a polynucleotide sequence, or fragment (i.e., nucleotides, exogenous or endogenous to a cell, a cell-free environment, a gene or fragment thereof, DNA, RNA, one or more analogs (i.e., altered backbone, sugar, or nucleobase)) (Paragraph 173, lines 1-5). Specifically, May teaches that some non-limiting examples of analogs include those with a GC percentage of about 40-60% including: 5-bromouracil, peptide nucleic acid, xeno nucleic acid, morpholinos, locked nucleic acids, glycol nucleic acids, threose nucleic acids, dideoxynucleotides, cordycepin, 7-deaza-GTP, florophores (i.e., rhodamine or flurescein linked to the sugar), thiol containing nucleotides, biotin linked nucleotides, fluorescent base analogs, CpG islands, methyl-7-guanosine, methylated nucleotides, inosine, thiouridine, pseudourdine, dihydrouridine, queuosine, and wyosine (Paragraph 173, lines 1-10). Further, May teaches that the previously described targeted gene or engineered nucleic acid-targeting nucleic acid can comprise at least two hairpins; the first hairpin or binding region comprising a duplex between the minimum CRISPR repeat and the minimum tracrRNA or sgRNA sequence and the second hairpin can be downstream of the first hairpin and the second hairpin can start at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides or basepairs downstream of the last paired nucleotide of the first duplex, while the second hairpin can start at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more nucleotides or basepairs downstream of the last paired nucleotide of the first duplex (Paragraph 799, lines 1-8).
Regarding claim 71, May teaches that the previously described library of expression vectors comprising donor polynucleotides (sgRNAs) includes homologous recombination (HR) that can be used to introduce barcode sequences into a cell and/or a cell population (i.e., a human cell, a mammalian cell, a yeast, a fungi, a protozoa, an archaea) where a library of donor plasmids (i.e., comprising the donor polynucleotide) can be prepared with randomized sequences in the donor cassette (Paragraph 830, lines 1-5). Further, May teaches that the donor polynucleotide (i.e., the additional sequence of a donor polynucleotide between two homologous ends) can comprise a marker where a marker can comprise a visualization marker (i.e., a fluorescent marker such as GFP) or a random polynucleotide sequence (i.e., such as a random hexamer sequence) or a barcode (Paragraph 828, lines 1-5).
Regarding claim 72, May teaches that the previously described library of expression vectors comprising donor polynucleotides (sgRNAs) includes binding regions or a first duplex that is at least 6 nucleotides in length where the 3 unpaired nucleotides of the bulge comprise 5'-AAG-3' and adjacent to the 3 unpaired nucleotides is a nucleotide that forms a wobble pair with a nucleotide on the second strand of the first duplex and the polypeptide binds to a region of the nucleic acid selected from the group consisting of: the first duplex, the second duplex, and the P-domain, or any combination thereof for structurally distinct regions (Paragraph 78, lines 20-30).
Regarding claim 73, May teaches that the previously described library of expression vectors comprising donor polynucleotides (sgRNAs) includes a composition comprising: an engineered nucleic acid-targeting nucleic acid comprising a 3' hybridizing extension or non-overlapping region, and a donor polynucleotide, wherein the donor polynucleotide is hybridized to the 3' hybridizing extension or non-overlap; where the 3' hybridizing extension or non-overlap is adapted to hybridize to at least 5 nucleotides from the 3' of the donor polynucleotide or to at least 5 nucleotides from the 5' of the donor polynucleotide or to at least 5 adjacent nucleotides in the donor polynucleotide or to all of the donor polynucleotide (Paragraph 15, lines 1-5). Further, May teaches that the 3' hybridizing extension or non-overlapping region is RNA where the engineered nucleic acid-targeting nucleic acid is an isolated engineered nucleic acid-targeting nucleic acid (Paragraph 15, lines 1-10). May also teaches that the previously described method includes a method for introducing a donor polynucleotide into a target nucleic acid comprising: contacting the target nucleic acid with a composition comprising: an engineered nucleic acid-targeting nucleic acid comprising a 3' hybridizing extension or non-overlapping region, and a donor polynucleotide, wherein the donor polynucleotide is hybridized to the 3' hybridizing extension incorporating cleaving the target nucleic acid to produce a cleaved target nucleic acid via a site-directed polypeptide (Paragraph 16, lines 1-5).
Regarding claim 74, May teaches that the previously described library of expression vectors comprising donor polynucleotides (sgRNAs) includes a target nucleic acid can refer to a chromosomal sequence or an extrachromosomal sequence, (i.e., an episomal sequence, a minicircle sequence, a mitochondrial sequence, a chloroplast sequence, etc.), including DNA, RNA, polynucleotide, a nucleic acid sequence that may not be related to any other sequence in a nucleic acid sample by a single nucleotide substitution or a nucleic acid sequence that may not be related to any other sequence in a nucleic acid sample by a 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide substitutions (Paragraph 202, lines 1-8).
Regarding claims 75-76, May teaches that the previously described library of expression vectors comprising donor polynucleotides (sgRNAs) includes an engineered nucleic acid-targeting nucleic acid comprising: a mutation in a P-domain of the nucleic acid-targeting nucleic acid where the P-domain or transcription start site starts downstream of a last paired nucleotide of a duplex between a CRISPR repeat and a tracrRNA sequence of the nucleic acid-targeting nucleic acid, including a linker sequence to link the CRISPR repeat and the tracrRNA sequence (Paragraph 4, lines 1-5). Further, May teaches that a tracrRNA or sgRNA extension sequence can provide stability and/or provide a location for modifications of a nucleic acid-targeting nucleic acid and can have a length of from about 1 nucleotide to about 400 nucleotides (Paragraph 335, lines 1-5). May also teaches that a tracrRNA or sgRNA extension sequence can have a length between 25 and 100 basepairs or nucleotides (Paragraph 335, lines 1-5).
May also teaches that the previously described method includes non-limiting examples of suitable moieties can include: 5' cap (i.e., a 7-methylguanylate cap (m7 G)), a riboswitch sequence (i.e., to allow for regulated stability and/or regulated accessibility by proteins and protein complexes), a sequence that forms a dsRNA duplex (i.e., a hairpin), a sequence that targets the RNA to a subcellular location (i.e., nucleus, mitochondria, chloroplasts, and the like), a modification or sequence that provides for tracking (i.e., direct conjugation to a fluorescent molecule, conjugation to a moiety that facilitates fluorescent detection, a sequence that allows for fluorescent detection, etc.), a modification or sequence that provides a binding site for proteins (i.e., proteins that act on DNA, including transcriptional activators, transcriptional repressors, DNA methyltransferases, DNA demethylases, histone acetyltransferases, histone deacetylases, and the like) a modification or sequence that provides for increased, decreased, and/or controllable stability, or any combination thereof where a spacer extension sequence can comprise a primer binding site, a molecular index (i.e., barcode sequence, affinity tag) (Paragraph 294, lines 1-10).
Regarding claims 77-78, May teaches a library composition of small guide RNAs (sgRNAs) or microarrays that comprise a plurality of polynucleotide probes or genetic elements, including about 1, 10, 100, 1000, 5000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000, 90000, 100000, 110000, 120000 or more probes (Paragraph 655, lines 1-3). Further, May teaches that a library of expression vectors comprising donor polynucleotides (sgRNAs) includes expression vectors comprising polynucleotide sequences encoding for differing genetic elements of interest but the same reporter elements or the library can comprise expression vectors comprising polynucleotide sequences encoding for differing genetic elements of interest and differing reporter elements (Paragraph 785, lines 1-5). Specifically, May teaches that the second method can use a sequence-specific RNA or DNA-based probe to quantify only the nucleic acid containing the probe sequence (Paragraph 650, lines 20-30). Also, May teaches that the previously described library of expression vectors or sgRNAs include an affinity tag comprising a sequence that can bind to a conditionally enzymatically inactive site-directed polypeptide via the affinity tag comprising a sequence that can bind to a conditionally enzymatically inactive endoribonuclease and can contain a sequence that can bind to a conditionally enzymatically inactive Csy4 protein (Paragraph 946, lines 1-5). Specifically, May teaches that the conditionally enzymatically inactive site-directed polypeptide will bind, but not cleave, the affinity tag and that the affinity tag or binding region comprises the nucleotide sequence 5'-GUUCACUGCCGUAUAGGCAGCUAAGAAA-3' or therefore lacking a UUU sequence (Paragraph 946, lines 1-10).
Regarding claim 79, May teaches a method for generating a library of target nucleic acids comprising: contacting a plurality of target nucleic acids with a complex comprising a site-directed polypeptide and a nucleic acid-targeting nucleic acid, cleaving the plurality of target nucleic acids, and purifying the plurality of target nucleic acids to create the library of target nucleic acids (Paragraph 59, lines 1-5). Further May teaches that the previously described method can be further expanded to allow for novel therapeutic applications, (i.e., prevention and/or treatment of: genetic diseases, cancer, fungal, protozoal, bacterial, and viral infection, ischemia, vascular disease, arthritis, immunological disorders, etc.), novel diagnostics (i.e., prediction and/or diagnosis of a condition) as well as providing for research tools (i.e., kits, functional genomics assays, and generating engineered cell lines and animal models for research and drug screening), and means for developing plants with altered phenotypes, including but not limited to, increased disease resistance, and altering fruit ripening characteristics, sugar and oil composition, yield, and color (Paragraph 503, lines 1-5). Additionally, May teaches that a site-directed polypeptide may also comprise certain regions that affect the binding specificity of the enzyme for nucleic acids, including regions that share either sequence or functional homology with domains or regions as found in RNA polymerase (i.e., tracrRNA and crRNA or a single RNA (sgRNA) (Paragraph 572, lines 1-5). Specifically, May teaches that the previously described nucleic acid guide tags include
a single guide RNA and can comprise a crRNA or a detectable label (815), and when the nucleic acid sample is ligated to the nuclei acid tag (821) (Paragraph 644, lines 1-5).
May also teaches that the previously described site-directed polypeptides within the screening method can be a type of protein, an nuclease, an endoribonuclease, a modified (i.e., shortened, mutated, lengthened) polypeptide sequence or homologue of the site-directed polypeptide and can additionally be codon optimized or enzymatically inactive, partially active, constitutively active, fully active, inducible active and/or more active, (i.e., more than the wild type homologue of the protein or polypeptide.), including Cas9 (Paragraph 198, lines 1-10).
May also teaches that the previously described site-directed polypeptides within the screening method is assembled via a recombinant DNA sequence that encodes for a modified site-directed polypeptide, and enables the expression of the modified site-directed polypeptide in a host organism and further comprises a promoter sequence, and may additionally comprise an affinity tag for purification, or an epitope tag or a sequence for the expression of the modified site-directed polypeptide (Paragraph 874, lines 1-5). Specifically, May teaches that the eluted target nucleic acids will be prepared for sequencing analysis via the generation of sequencing libraries of the eluted target nucleic acid to determine the identity and frequency of off-target binding sites of site-directed polypeptides (Paragraph 914, lines 1-5). Additionally, May teaches that the quantification of the remaining detectable labels (760) can be correlated to which sequences were represented in the nucleic acid sample (705) and which were not; including oligonucleotides that do not display a remaining detectable label (760) that correspond to sequences that were represented (or overrepresented) in the nucleic acid sample (705) and oligonucleotides that display a remaining detectable label (760) which correspond to sequences that were not represented (or underrepresented) in the nucleic acid sample (705) (Paragraph 643, lines 30-40).
May also teaches that the location of where to modify a site-directed polypeptide (i.e., a Cas9 variant) can be determined using sequence and/or structural alignment where sequence alignment can identify regions of a polypeptide that similar and/or dissimilar (i.e., conserved, not conserved, hydrophobic, hydrophilic, etc.) and in some instances, a region in the sequence of interest that is similar to other sequences is suitable for modification and in some instances, a region in the sequence of interest that is dissimilar from other sequences is suitable for modification (Paragraph 507, lines 1-5).
Further, May teaches that the previously described method of identifying genetic elements includes a method for detecting if two complexes are in proximity to one another comprising: contacting a first target nucleic acid with a first complex, wherein the first complex comprises a first site-directed polypeptide, a first modified nucleic acid-targeting nucleic acid, and a first effector protein, wherein the effector protein is adapted to bind to the modified nucleic acid-targeting nucleic acid, and wherein the first effector protein comprises a non-native sequence that comprises a first portion of a split system, and contacting a second target nucleic acid with a second complex, wherein the second complex comprises a second site-directed polypeptide, a second modified nucleic acid-targeting nucleic acid, and a second effector protein, wherein the effector protein is adapted to bind to the modified nucleic acid-targeting nucleic acid, and wherein the second effector protein comprises a non-native sequence that comprises a second portion of a split system including the ability for the first target nucleic acid and the second target nucleic acid to be on the same polynucleotide polymer or two or more protein fragments that individually are not active, but, when formed into a complex, result in an active protein complex or an interaction between the first portion and the second portion. In some embodiments, the detecting indicates the first and second complex are in proximity to one another or where the site-directed polypeptide is adapted to be unable to cleave the target nucleic acid (Paragraph 20, lines 1-15).
May teaches that the previously described library of expression vectors comprising donor polynucleotides (sgRNAs) includes a composition comprising: a nucleic acid comprising: a spacer, wherein the spacer is between 12-30 nucleotides, inclusive, and wherein the spacer is adapted to hybridize to a sequence that is 5' to a PAM or guanosine nucleotide; a first duplex, wherein the first duplex is 3' to the spacer; a bulge, wherein the bulge comprises at least 3 unpaired nucleotides on a first strand of the first duplex and at least 1 unpaired nucleotide on a second strand of the first duplex; a linker, wherein the linker links the first strand and the second strand of the duplex and is at least 3 nucleotides in length; a P-domain; and a second duplex, wherein the second duplex is 3' of the P-domain and is adapted to bind to a site directed polypeptide. In some embodiments, the sequence that is 5' to a PAM is at least 18 nucleotides in length (19-21 nucleotides) (Paragraph 78, lines 1-5). Specifically, May teaches that the previously described sequence is 5' to a PAM is adjacent to the PAM and the PAM comprises 5'-NGG-3 (Paragraph 78, lines 1-10).
Regarding claims 80-81, May teaches that the previously described library of expression vectors comprising donor polynucleotides (sgRNAs) includes a composition comprising: a nucleic acid comprising: a spacer, wherein the spacer is between 12-30 nucleotides, inclusive, and wherein the spacer is adapted to hybridize to a sequence that is 5' to a PAM or guanosine nucleotide; a first duplex, wherein the first duplex is 3' to the spacer; a bulge, wherein the bulge comprises at least 3 unpaired nucleotides on a first strand of the first duplex and at least 1 unpaired nucleotide on a second strand of the first duplex; a linker, wherein the linker links the first strand and the second strand of the duplex and is at least 3 nucleotides in length; a P-domain; and a second duplex, wherein the second duplex is 3' of the P-domain and is adapted to bind to a site directed polypeptide. In some embodiments, the sequence that is 5' to a PAM is at least 18 nucleotides in length (19-21 nucleotides) (Paragraph 78, lines 1-5). Specifically, May teaches that the previously described sequence is 5' to a PAM is adjacent to the PAM and the PAM comprises 5'-NGG-3 (Paragraph 78, lines 1-10).
May does not teach or suggest designing sgRNA binding regions to exclude U-rich motifs, such as UUU sequences, nor does it address sequence constraints within such binding regions.
Wu teaches that bacterial type II CRISPR-Cas9 systems have been widely adapted for RNA-guided genome editing and transcription regulation in eukaryotic cells, yet their in vivo target specificity is poorly understood, via mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs) (Abstract). Specifically, Wu teaches that each of the four sgRNAs we tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM) (Abstract). Further, Wu teaches a comparison of the seed regions of the four sgRNAs suggested that UUU in the seed of Nanog-sg2 might be responsible for decreased sgRNA abundance and increased specificity, consistent with a recent observation that U in PAM-proximal positions 1–4 leads to low gene-knockout efficacy14, where two mutations (U to G and U to A) in the Nanog-sg2 seed region that converted the seed (GUUUC) to the same sequence as the Phc1-sg2 seed (GGUAC), led to higher levels of sgRNA (sgRNA N2b in Fig. 4c) (Seed Sequences influence sgRNA abundance and specificity: Paragraphs 1-4). Additionally, Wu teaches considering the presence of GUUUUA adjacent to the seed and because sgRNAs are transcribed by RNA polymerase III, which is terminated by U-rich sequences40,41, and, together with the downstream U-rich region, multiple U's in the seed might induce termination of sgRNA transcription, where consistent with this, three sgRNAs with seeds UUAUU, ACUUU and UUUUU also showed very low abundance (Fig. 4c, sgRNA P3, N5 and N6) and when GUUUC was placed upstream of the seed (i.e., away from GUUUUA in the sgRNA), the sgRNA was well expressed (sgRNA C4 in Fig. 4c) (Seed Sequences influence sgRNA abundance and specificity: Paragraphs 1-4).
It would have been obvious to one of ordinary skill in the art at the time of the invention to modify the sgRNA libraries of May in view of Wu so that the sgRNAs, particularly the binding regions of those sgRNAs, were selected to avoid UUU motifs. A person of ordinary skill in the art, starting from May’s disclosure of pooled sgRNA libraries for targeting genetic elements, would have understood that effectiveness of such a library depends on adequate transcription, abundance, and functionality of the individual sgRNAs. Wu expressly teaches that U-rich motifs within the sgRNA seed or targeting region can reduce sgRNA abundance through premature transcription termination (Figure 4). Accordingly, Wu would have provided a clear reason to alter the sgRNA sequence design of May so as to avoid such motifs in the binding regions, because doing so would have predictably improved expression of the sgRNAs used in May’s library-based screening methods.
Moreover, May’s disclosed spacer length of 12-30 nucleotides encompasses the presently claimed binding region ranges of 10-40 nucleotides and 19-21 nuckeotides; accordingly, the claimed ranges do not distinguish over May because an overlapping prior art range establishes a prima facie case of obviousness. See MPEP 2144.05. In particular, the newly recited range of 19-21 nucleotides is fully encompassed by May’s disclosed range of 12-30 nucleotides and therefore does not overcome the rejection absent evidence that the claimed narrower range is critical or produces unexpected results. See MPEP 2144.05.
Further, one of ordinary skill in the art would have had a reasonable expectation of success in making this modification because Wu provides both a mechanistic explanation and experimental evidence showing that U-rich motifs in sgRNA binding regions can impair transcription and that removal of such motifs improves sgRNA abundance and performance. Sequence optimization of guide RNAs was a predictable and routine aspect of CRISPR design, and applying Wu’s teaching to the sgRNA libraries of May would merely have involved selecting guides with preferred sequence characteristics. For the same reason, selecting a library in which at least 50% of the sgRNA binding regions lack a UUU sequence would have been the product of routine optimization of a result-effective variable, namely the sequence composition of the guide region to improve transcriptional efficiency and functional utility across the library.
Applicant’s Response: The Applicant argues that May and Wu fail to teach or suggest the claimed sgRNA library, particularly the requirement that at least 50% of the sgRNA binding regions lack a UUU sequence, because Wu’s teachings concerning a U-rich sequence are directed to particular seed-region effects rather than the claimed library-wide limitation. Applicant further contends that the cited art provides no sufficient motivation or reasonable expectation of success for modifying May’s library to arrive at the claimed composition and arrangement.
Examiner’s Response to Traversal: Applicant’s arguments have been carefully and fully considered and are not found persuasive, in view of the newly combined teachings of May and Wu.
Applicant argues that May does not expressly disclose a library wherein at least 50% of the sgRNA binding regions lack a UUU sequence and that Wu’s discussion of U-rich sequences does not provide sufficient motivation to arrive at the claimed library. However, the rejection does not rely upon May alone for this limitation. May teaches the underlying pooled sgRNA library and CRISPR screening system, while Wu teaches that U-rich sequences, including UUU-containing sequences within the target-recognition portion of an sgRNA, can adversely affect sgRNA transcription and abundance through premature RNA polymerase III termination and further demonstrates improved sgRNA abundance following alteration of such sequences.
Accorindingly, one of ordinary skill in the art, when constructing the sgRNA library taught by May, would have had reason to apply Wu’s known sequence-design principle by preferentially selecting sgRNAs lacking the identified detrimental UUU sequence in their binding regions, with a reasonable expectation of obtaining improved sgRNA transcript, abundance, and functional representation within the library. The proposed modiciation therefore represents application of a known technique to May’s known sgRNA library to obtain the predictable improvement expressly identified by Wu. See MPEP 2143, particularly the rationale of applying known technique to a known product or method ready for improvement to yield predictable results.
Applicant’s argument concerning the claimed “at least 50%” does not overcome the rejection because the claimed percentage does not require a different type of sgRNA or mechanism; rather, it specifies the proportion of the library possessing the sequence characteristic that Wu teaches is advantategous. In view of Wu’s teaching to avoid the determinantal U-rich sequence characteristic, a skilled artisan constructing May’s pooled library would have had reason to increase the representation of guides lacking that characteristic. However, the Exqaminer does not rely solely on the proposition that the precise 50% threshold is inherently a result-effective variable; rather, the rejection is based on the predictable application of Wu’s sequene-selection teaching across May’s library. See MPEP 2143 and 2143.02; obviousness requires a reasonable, rather than absolute, expectation of success.
Applicant’s amendments therefore do not overcome the rejection because they further define the proportion of sgRNAs having the sequence characteristic without intodroducing a structural or functional limitation that distinguishes the claimed library from the predictable application of Wu’s teaching to May. Moreover, merely specifying a numerical proportion does not establish patentatbility where the prior art supplies a reason to preferentially select the underlying characteristic, absent persuasive evidence that the claimed proportion produces an unexpected result or otherwise represents more than the predictable implementation of the prior art teaching. See MPEP 2144.05 regarding claimed amounts and proportions; notably, a result-effective variable rationale requires evidence that the relevant parameter was recognized as affecting the result.
To overcome the rejection, Applicant may amend the claims to recite a specific structural or functional limitation, such as one establishing a specific relationship between the UUU-free-binding region composition and the claimed screening method rather than merely specifying the percentage of such guides. Alternatively, Applicant may provide persuasive evidence that the claimed 50% composition produces an unexpected technical result relative to the sequence optimization suggested by Wu.
Accordingly, the combination of May and Wu teaches or renders obvious the claimed sgRNAs comprising binding regions with the recited sequence characteristics, under the new rejection under 35 USC 103.
Conclusions
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/ELIZABETH ROSE LAFAVE/Examiner, Art Unit 1684
/HEATHER CALAMITA/Supervisory Patent Examiner, Art Unit 1684
