DETAILED ACTION
Status
Claims 33- 45 are pending.
Claims 33- 36 are considered on the merits.
Claims 37- 45 are withdrawn.
Claims 33- 36 are rejected.
No claims are allowed.
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election without traverse of Group I, claims 33-36 in the reply filed on 2026 April 16 is acknowledged.
Claims 37- 45 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to nonelected inventions, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 2026 April 16.
Claim Objections
Claim 33 is objected to because of the following informalities: Claim 33 has two periods. Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 33- 36 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 33, recites “a lentiviral vector comprising human U6 (hU6) promoter, mouse U6 (mU6) promoter and human H1 (hH1) promoter expressing an array of three or more barcoded guide RNAs ("gRNAs") oligo pairs,” Figure 6, among other locations in the specifications, describes “oligo pairs” as “annealed” oligos. A lentiviral vector with unidirectional promoters as described in the claim and specifications does not express an oligo pair. Therefore, claim 33 is indefinite.
Claim 34 contains the trademark/trade name “CombiGEM-CRISPR v2.0.” Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe a method of assembling a “combinatorial gRNA library” and, accordingly, the identification/description is indefinite.
Furthermore regarding claim 34, the recitation “a combinatorial gRNA library is assembled by CombiGEM-CRISPR v2.0” is indefinite because it is unclear what steps and/or structures are defined by the term “CombiGEM-CRISPR v2.0.” The specification provides an “overview” of “CombiGEM-CRISPR v2.0.” in Figure 2. However, it is not clear which details of the steps and/or structures of “CombiGEM-CRISPR v2.0” as illustrated in Figure 2 are required for this term. Therefore, claim 34 is indefinite.
Those claims included in the statement of rejection but not otherwise discussed are rejected for depending from a rejected claim but failing to remedy the indefiniteness therein.
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.
Claim(s) 33- 36 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wong (Wong et al. Proc Natl Acad Sci U S A. 2016 Mar 1;113(9):2544-9.) as evidenced by Addgene (Addgene. 2026. https://www.addgene.org/browse/sequence/138725/), and further in view of Najm (Najm et al. Nat Biotechnol. 2018 Feb;36(2):179-189.), and Nageshwaran (Nageshwaran et al. J Vis Exp. 2018 Oct 2;(140):57998.).
Regarding claim 33, Wong teaches “This technology leverages the simplicity of the CRISPR-Cas9 system for multiplexed targeting of specific genomic loci and the versatility of combinatorial genetics en masse (Combi-GEM)” (Abstract). Wong further teaches “These libraries can be delivered into human cells by lentiviruses” (page 2544, 2nd column, top); “a library of barcoded gRNA target sequences was first synthesized, annealed, and pooled in equal ratios for cloning downstream of a U6 promoter in the [pAWp28] storage vector” (page 2544, 2nd column; supplement page 2); and provides a diagram in Fig. 1 (shown below). Addgene’s sequence analyzer confirms the U6 promoter in the pAWp28 vector used by Wong is human U6. Wong continues to describe the expression vector “pooled inserts of the barcoded sgRNA expression units were prepared by restriction digestion of the storage vectors” (page 2545, 1st column). Wong further describes the vector comprising three or more barcoded gRNAs “This process can be iteratively repeated to generate higher-order barcoded combinatorial gRNA libraries”(page 2545, 1st column). Wong provides a workflow and diagram of the vector in Fig. 1 (shown below).
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Additionally, Wong teaches modification of the promoters’ 3’ end to facilitate paired annealing of the barcoded gRNA oligo pairs (Fig. S1 shown below).
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Therefore, Wong teaches all of the elements of the claim, except a mouse U6 promoter (mU6) or human H1 promoter (hH1).
Najm describes their field of endeavor “Combinatorial genetic screening using CRISPR–Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks.” (Abstract). As such, Najm teaches “We designed a lentiviral construct, pPapi, to express SaCas9 and two sgRNAs from the U6 and H1 promoters” (page 179, 2nd column, bottom). Najm teaches embodiments of both human and “the mouse U6 promoter” in Fig. 3d (page 183, 1st column). Najm goes further to provide a motivation for a combination of promoters “repetitive elements in lentiviral vectors, including the U6 promoter, lead to high levels of recombination and decrease combinatorial screen efficiency” (page 179, 1st column).
Therefore, Najm teaches the elements a mouse U6 promoter (mU6) and human H1 promoter (hH1).
Nageshwaran teaches lentiviral construction comprising a U6 promoter with a modified 3’ end for paired annealing of the gRNA “Append CACCG to the 5’ end of the oligo [gRNA]. Note: Upon ligation to the pSB700 backbone, this sequence will comprise the 3’ end of the U6 promoter driving the transcription of the gRNA. The “CACC” sequence ensures that the oligo is compatible with the overhangs of the BsmBI-digested pSB700 vector.” (page 2, section 2.1.).
Therefore, Nageshwaran teaches the element “the promoters having a 3' end comprising modified U6 promoter sequences for paired annealing of the barcoded gRNAs oligo pairs.”
It would have been obvious to a person having ordinary skill in the art (PHOSITA) at the time of filing to have made Wong’s lentiviral construct with Najm’s mU6 and hH1 promoters, and further modified the promoters according to Nageshwaran to arrive at the multiplexed genome editing system of the instant claim because it amounts to combining prior art elements according to known methods to yield predictable results. A PHOSITA would have known of Wong’s, Najm’s and Nageshwaran’s lentiviral vectors. Similarly, a PHOSITA would have recognized the need to include a promoter to control downstream gene expression in their construct. The promoters hU6, mU6, hH1 each share this functionality. Similarly, a PHOSITA would have recognized that each of these promoters were used by Wong and Najm in a similar manner. Therefore, they would have been able to predict the results of incorporating these promoters and had a reasonable expectation of success making such substitutions.
Furthermore, Nageshwaran demonstrates modifying the 3’ end of a U6 promoter for paired annealing and because the structures of the mU6 and hH1 promoters were known, then a PHOSITA would have had a reasonable expectation of success in applying the method of Nageshwaran to the mU6 and hH1 promoters. To emphasize this point, Nageshwaran’s 3’ modification is identical to the modifications shown in the instant application’s specification page 16.
Therefore, Wong (as evidenced by Addgene), Najm, and Nageshwaran teach all of the elements of claim 33.
Regarding claim 34, Wong, Najm, and Nageshwaran teach all of the elements of claim 33. Wong further teaches claim element (a), “Oligos with the gRNA scaffold sequence were inserted into the pooled storage vector library”(Fig. 1 legend). Wong further teaches claim element (c), the effectiveness of their lentiviral construct “CombiGEM-CRISPR can be used to efficiently assemble and deliver barcoded combinatorial gRNA libraries into human cells” (page 2545, 1st column, 1st paragraph). Wong continues to teach claim elements (d) and (e), high-throughput analytical methods post transfection, “Illumina HiSeq was used to quantify the representation of individual barcoded combinations in … the infected human cell pools”(page 2546, 1st column, 1st paragraph).
Wong (in view of Najm’s promoter modifications described above) further teaches claim element (b), “a combinatorial gRNA library is assembled by CombiGEM-CRISPR v2.0.”
The specification of the instant application section titled “figure 2. Overview of CombiGEM-CRISPR v2.0” provides some guidance, “Barcoded combinatorial gRNA library is assembled multiplicatively using one-pot reactions as described in Figure 6.”
This examiner is currently interpreting Fig. 6 of the instant specification, gRNA library assembly, to be 4 steps:
Oligo pairs (Oligo-F and Oligo-R, respectively) were synthesized and annealed to create double-stranded inserts with 20 bp gRNA target sequences, two Bbsl sites, 8 bp barcodes, and 5' overhangs at their ends.
The inserts were mixed in equal molar ratio and cloned into three Bbsl and Mfe digested storage vectors, which contain the hH1, hU6, or mU6 promoters, with one-pot ligation reactions via their compatible ends to create the pooled storage vector libraries.
The 3' end of the promoter sequences of hH1 and mU6 were modified such that the same pool of gRNA oligos can be used for building the libraries.
Subsequent single pot ligation reactions were performed with the Bbsl-digested pooled storage vector libraries and an insert containing the gRNA scaffold sequence (WT, v1, or v2), BamHI and EcoRI sites, and 5' overhangs at their ends to assemble the barcoded gRNA library pools.
Wong teaches each of these steps as shown in the workflow shown in Fig. S1 above and described in Wong’s Fig. S1 legend:
“Forward and reverse oligo pairs (Oligo-F and Oligo-R, respectively) were synthesized and annealed to create double stranded inserts with 20 bp gRNA target sequences, two BbsI sites, 8 bp barcodes, and 5’ overhangs at their ends.”
“To create the pooled storage vector library, the annealed inserts were mixed in 1:1 ratio and cloned in the storage vector (digested with BbsI and MfeI) with a one-pot ligation reaction via their compatible ends.”
Notice, in the Fig. S1, the promoter’s sticky end matches the gRNA sticky end. Furthermore, recall Najm teaches modification of hH1, hU6, or mU6 promoters’ 3’ ends.
“To create the barcoded gRNA library pool, another single-pot ligation reaction was performed with the pooled storage vector library digested with BbsI, and an insert containing the gRNA scaffold sequence, BamHI and EcoRI sites, and 5’ overhangs at their ends”
Therefore, Wong teaches all of the elements of claim 34.
Regarding claims 35 and 36, as shown in Fig. S1 above, Wong teaches the ligation of the gRNA scaffold into the vector, align such that the promoter’s 5’ overhang at the 3’ end overlaps the 5’ overhang at the 5’ end of the RNA scaffold “We then applied the CombiGEM method for assembly of combinatorial gRNA libraries” (page 2545, 1st column, top); and “To create the barcoded gRNA library pool, another single-pot ligation reaction was performed with the pooled storage vector library digested with BbsI, and an insert containing the gRNA scaffold sequence” (Fig. S1 legend).
As described above, the combination of Wong-Najm-Nageshwaran’s teaches modified hU6, mU6 and hH1 promoters of the gRNA libraries, identical to the instant application’s promoter modifications.
Therefore, Wong (as evidenced by Addgene), Najm, and Nageshwaran teach all of the elements of claims 35 and 36.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to AARON DUREL WARD whose telephone number is (571)272-8495. The examiner can normally be reached Monday to Thursday 8:00AM 6:00PM.
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/AARON DUREL WARD/Examiner, Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636
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