DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Rejections - 35 USC § 112
2. 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.
3. Claim 6 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 6 has been amended to be dependent on claim 3, however claim 3 has been canceled. For the purpose of prosecution claim 6 will be treated as though it were dependent on claim 1, as claim 1 has been amended to incorporate the limitations of claim 3.
4. The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
5. Claim 7 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 7 recites an embodiment wherein the method of wherein the cells of claim 1 are perturbed by CRISPR-Cas. Claim 1 has been amended to incorporate the limitations that the library of cells have been perturbed by CRISPR-Cas, and claim 7 does not require any particular “perturbation” that isn’t already encompassed in the amended method of claim 1. Therefore, in the embodiment of claim 7 wherein the cells are perturbed by CRISPR-Cas, the limitations of claim 7 do not further limit the claim upon which it depends.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 103
6. 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.
7. 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.
8. Claims 1, 4-7, 9-11, 19, 21 and 22 are rejected under 35 U.S.C. 103 as being unpatentable over Belhocine et al (International Patent Application No. WO 2018218226, published 29 November 2018) in view of Cao et al (Joint profiling of chromatin accessibility and gene expression in thousands of single cells, Science, 361, 6409, 1380-1385, published 28 September 2018) and Sanjana et al (Improved vectors and genome-wide libraries for CRISPR screening, Nature Methods, 11, 783-784, published 30 July 2014).
Regarding claim 1, Belhocine teaches an in vitro method for analyzing chromatin accessibility and RNA of individual cells in a library of cells ([00260] – [00261]; enhancing reads associated with accessible chromatin, applied to any library… including RNA-seq, a plurality of cells within a plurality of droplets and [00263]). Belhocine teaches that cell nuclei are isolated (i.e., obtained) from lysed cells ([00371]) and are partitioned with transposase molecules (i.e., comprise a transposome complex), and that the single nucleus comprises template DNA and template RNA molecules (i.e., DNA and RNA from the individual cells [00354]). Belhocine teaches that transposase complexes comprise a transposase, a transposon, and a first barcode (FIGs 24 and 26, [0064] and [0065]), and that the transposase causes staggered double-stranded breaks in the DNA (FIG 14a step 1b and FIG 27, additionally this is a property inherent to transposase-mediated fragmentation). Belhocine teaches that the first barcode is ligated to the dsDNA at the staggered break (FIG 27).
Belhocine teaches performing a reverse transcription comprising incubating the cell nuclei with a second barcode molecule comprising a capture sequence (i.e., reverse transcription primers) and that the second barcode molecule has the same barcode at the first barcode molecule ([00354]). Belhocine teaches that additional reagents are co-partitioned with the biological particles (e.g., cell nuclei) including dNTPs and reverse transcriptase enzymes ([00200]). Belhocine teaches the reverse transcription of RNA to cDNA ([00200]) and this process inherently requires necessary buffer components (i.e., without additional limitations from the claim, a reverse transcription buffer). Belhocine teaches that sequencing applications are performed to generate a library of DNA or cDNA fragments, and that areas of accessible chromatin are analyzed ([00264]). Belhocine teaches the processing of multiple analytes (e.g., DNA and RNA) from a single cell for simultaneous transcriptomic and genomic analysis of the cell (e.g., RNA is analyzed [00275]). Belhocine teaches that the cell nuclei are lysed (i.e., digested) in order to release (i.e., extract) the adapter-flanked template nucleic acid fragments.
Belhocine does not teach that the library of cells has been subjected to a perturbation step comprising transducing the cells with one or more vectors, each vector comprising a nucleic acid sequence encoding a Cas protein in operative association with a first promoter which controls expression of the Cas protein, and CRISPR guide RNA coding sequence in operative association with a second promoter which controls transcription thereof, and culturing the cells. Additionally, Belhocine does not teach that the transcribed RNA comprises the guide RNAs, nor do they teach performing combinatorial cellular indexing according to step (e) of amended claim 1.
However, Sanjana teaches a method of perturbing cells via transduction with a lentiviral vector (Supplemental FIG 1). Sanjana teaches that each vector comprises a nucleic acid sequence encoding a Cas protein in operative association with a first promoter, EFS. Sanjana teaches that each vector encodes a CRISPR guide RNA coding sequence in operative associated with a second promoter, U6 (FIG 1a, lentiCRISPRv2). Sanjana teaches that cells were analyzed 7 days after transduction (i.e., the cells were cultured; Supplemental FIG 1). Additionally, since CRISPR-Cas systems act by interaction with, and cleavage of, target genes the associated gRNAs are inherently present in the nuclei (i.e., the nuclei RNA of claim 1 step b inherently comprises the gRNA). Belhocine teaches that the capture sequence on the second barcoded oligonucleotide (i.e., the reverse transcription primer) is a random primer sequence (e.g., a random hexamer). Random hexamer primers inherently enable the capture and reverse transcription of all available RNA (i.e., the RNA in claim 1 step b).
It would have been obvious to one having ordinary skill in the art to have modified the method taught by Belhocine to have included a CRISPR-Cas based perturbation step prior to isolating the nuclei and analyzing chromatin accessibility and RNA content to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make this modification in order to compare the effects CRISPR-Cas treatment would have on gene expression between perturbed and non-perturbed cells. In addition, one having ordinary skill in the art would have recognized that the known techniques in the cited references could have been combined with predictable results because the known techniques in the cited references predictably result in methods related to the modulation and analysis of gene expression.
In addition, Cao teaches a method of combinatorial indexing to analyze both chromatin accessibility and the RNA of individual cells. Cao teaches transferring cell nuclei to a first well (i.e., compartment) prior to the tagmentation step (pg. 1, column 1, ¶ 2), Cao teaches pooling the nuclei after tagmentation and reverse transcription (i.e., after the claimed step b) and transferring the pooled nuclei to a second compartment (pg.1 column 1 ¶ 2 and column 2 ¶ 1). Cao teaches amplifying the RNA and tagmented DNA (each comprises a first index, e.g., barcode, from the first compartment) with separate primer pairs comprising a second well-specific index (i.e., a barcode unique to the second set compartment; pg. 1, column 2, ¶ 1). Cao teaches that the PCR barcode and either the reverse transcription (RT, RNA-seq) barcode or the Tn5 barcode (ATAC-seq) together comprise a cellular index (i.e., the combination of the first and second barcodes are identified as being from the same cell; FIG 1).
It would have been obvious to one having ordinary skill in the art to have simply substituted the droplet partitioning method taught by Belhocine with the combinatorial indexing method taught by Cao to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make this substitution in order to provide individual cell barcodes without having to perform the additional steps of droplet formation and optimization required by Belhocine. In addition, one having ordinary skill in the art would have recognized that the known techniques in the cited references could have been combined with predictable results because the known techniques of the cited references predictably result in the preparation of libraries for the combined analysis of chromatin accessibility and RNA.
Regarding claim 4, Cao teaches the pooling of nuclei from the first compartment and randomly distributing them into the second set of compartments (pg. 1, column 1, ¶ 1 and Supplemental pg. 3, ¶ 1).
Regarding claim 5, Belhocine teaches the transposase-mediated fragmentation of genomic DNA and the addition of a first barcode to the 5' and 3' ends of said fragment ([00256] and FIG 22). Without further language limiting the scope of this claim, the identity of the first/third/fourth barcodes are arbitrary and the first barcode taught by Belhocine comprises a sequence defined as the third barcode and a sequence defined as the fourth barcode. In that manner, when the first barcode is added to both the 5' and 3' ends of the DNA, the third barcode is added to the 5' terminal and the fourth barcode is added to the 3' terminal.
Regarding claim 6, it is reiterated that this claim is being interpreted as though it depends from claim 1 as claim 3 has been canceled. Cao teaches that DNA related to ATAC-seq (i.e., the transposase fragmented DNA) is amplified with primers specific to the barcoded Tn5 adapters (i.e., DNA fragments comprising the first barcode) and that these primers contain a second well-specific index (i.e., the claimed second barcode, pg. 1, column 2, ¶ 1). Cao additionally teaches that the forward and reverse primers comprise different barcodes, with the i5 barcode at the 5' end and the i7 barcode at the 3' end (i.e., the claimed fifth and sixth barcodes, FIG 1).
Regarding claim 7, Cao teaches an embodiment wherein cells are subjected to perturbation with dexamethasone (DEX, i.e., a chemical agent) before being lysed (pg. 1, column 2, ¶ 2 and column 3 ¶ 3).
Regarding claim 9, Sanjana does not specifically teach a method wherein cells are perturbed via transduction of vectors comprising more than one CRISPR gRNAs that are targeted to each functional unit of a cell of interest.
However, Sanjana teaches a library of vectors having the same general structure as lentiCRISPRv2, wherein each vector has a designed gene-specific gRNA, and that each gene (i.e., functional unit of interest) is targeted by 6 gRNAs in the library (Supplemental pg. 7 and 8).
It would have been obvious to one having ordinary skill in the art to have simply substituted the lentiCRISPRv2 vector targeting GFP with the library of lentiCRISPRv2 vectors taught by Sanjana to arrive at the instantly claimed method with a reasonable expectation of success. The ordinary artisan would have been motivated to make this substitution in order to screen the for the effects that knockouts of different genes have on chromatin accessibility and gene expression. In addition, the ordinary artisan would have recognized that the known techniques of the cited reference could have been combined with predictable results because the known techniques of the cited references predictably result in the transduction of cells with CRISPR-Cas systems.
Regarding claim 10, Sanjana teaches a CRISPR-Cas vector that transcribes a single gRNA (FIG 1 lentiCRISPRv2).
Regarding claim 11, Belhocine teaches that the transposase is a Tn5 transposase ([00108]).
Regarding claim 19, Cao teaches that tagmentation is stopped by the addition of EDTA (Supplemental pg. 2, ¶ 3).
Regarding claim 21, Belhocine teaches performing both ATAC-seq and RNA-seq ([00255]).
Regarding claim 22, Belhocine teaches an in vitro method for analyzing chromatin accessibility and RNA of individual cells in a library of cells ([00260] – [00261]; enhancing reads associated with accessible chromatin, applied to any library… including RNA-seq, a plurality of cells within a plurality of droplets and [00263]). Belhocine teaches that cells are lysed to release nuclei ([00249]) and that cell nuclei are isolated (i.e., obtained) from lysed cells ([00371]) and are partitioned with transposase molecules (i.e., comprise a transposome complex), and that the single nucleus comprises template DNA and template RNA molecules (i.e., DNA and RNA from the individual cells [00354]). Belhocine teaches that transposase complexes comprise a transposase, a transposon, and a first barcode (FIGs 24 and 26, [0064] and [0065]), and that the transposase causes staggered double-stranded breaks in the DNA (FIG 14a step 1b and FIG 27, additionally this is a property inherent to transposase-mediated fragmentation). Belhocine teaches that the first barcode is ligated to the dsDNA at the staggered break (FIG 27).
9. Claims 12 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Belhocine et al (International Patent Application No. WO 2018218226, published 29 November 2018) in view of Cao et al (Joint profiling of chromatin accessibility and gene expression in thousands of single cells, Science, 361, 6409, 1380-1385, published 28 September 2018) and Sanjana et al (Improved vectors and genome-wide libraries for CRISPR screening, Nature Methods, 11, 783-784, published 30 July 2014). as applied to claim 1 above, and further in view of Corces et al (An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues, Nature Methods, 14, 959-962, published 28 August 2017).
Regarding claim 12, the method of claim 1 is discussed fully above and incorporated here.
Belhocine teaches that lysis solutions include Tween-20 and NP40 (i.e., Igepal CA630, [00278] and [00249]).
The method taught by Belhocine in view of Sanjana and Cao does not teach that cells are lysed in a resuspension buffer comprising 0.1% Tween-20 and 0.1% Igepal CA630.
However, Corces teaches the lysis of cells for ATAC-seq using a resuspension buffer comprising 0.1% NP40 (Igepal CA630) and 0.1% Tween-20 (pg. 5, column 1, ¶ 6).
It would have been obvious to one having ordinary skill in the art to have substituted the generic lysis conditions taught by Belhocine in view of Sanjana and Cao with the more exact lysis buffer taught by Corces to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make this substitution in order to have more exact reaction conditions and to reduce unneeded experimentation. In addition, the ordinary artisan would have recognized that the known techniques of the cited references could have been substituted with predictable results because the known techniques of the cited references use the same buffer components to facilitate cell lysis.
Regarding claim 18, Corces teaches that the transposition reactions (comprising the transposomes and the cell nuclei) were incubated at 37 °C for 30 min (pg. 5, column 2, ¶ 1).
10. Claims 13-14 are rejected under 35 U.S.C. 103 as being unpatentable over Belhocine et al (International Patent Application No. WO 2018218226, published 29 November 2018) in view of Cao et al (Joint profiling of chromatin accessibility and gene expression in thousands of single cells, Science, 361, 6409, 1380-1385, published 28 September 2018) and Sanjana et al (Improved vectors and genome-wide libraries for CRISPR screening, Nature Methods, 11, 783-784, published 30 July 2014). as applied to claim 1 above, and further in view of Richter et al (Glyoxal as an alternative fixative to formaldehyde in immunostaining and super-resolution microscopy, The EMBO Journal, 37, 1, 139-159, published 04 January 2018).
Regarding claim 13, the method of claim 1 is discussed fully above and incorporated here.
The method taught by Belhocine in view of Sanjana and Cao teaches that nuclei are fixed using a paraformaldehyde solution (Cao; Supplemental pg. 4 ¶ 2).
Cao does not teach that the cells are fixed prior to lysis, however MPEP 2144.04(IV)(C) states that any order or performing steps is prima facie obvious in the absence of new or unexpected results.
Cao additionally does not teach that the fixation buffer comprises about 20% (v/v) ethanol and about 3.1% glyoxal at a pH of about 5.0.
However, Richter teaches a cell fixation buffer comprising about 20% ethanol (v/v), about 3.1% glyoxal, and a pH of 4 or 5 (i.e., about 5; pg. 152, column 1, ¶ 1).
It would have been obvious to one having ordinary skill in the art to have modified the method taught by Belhocine to include the fixation step taught by Cao, and to further have substituted the paraformaldehyde fixation solution taught by Cao with the glyoxal fixation solution taught by Richter to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to fix the cells in order to preserve chromatin arrangement during handling or make the cells more stable for storage, and the ordinary artisan would have been further motivated to replace the paraformaldehyde fixation solution with glyoxal because Richter specifically teaches that glyoxal is less toxic that paraformaldehyde (abstract). In addition, the ordinary artisan would have recognized that the known techniques in the cited references could have been combined with predictable results because the known techniques of the cited references predictably result in the fixation of cells.
Regarding claim 14, Richter does not specifically teach that the cells are fixed for 7 minutes.
However, Richter teaches that cells fixed with glyoxal at pH 5 become permeable to a cell impermeable dye within 10 minutes of fixation, and specifically show permeabilization has occurred at 6 minutes and 10 minutes (FIG 1B), at room temperature (pg. 141, column 2, ¶ 1).
It would have been obvious to one having ordinary skill in the art to have tested fixation time points between 6 and 10 minutes. The ordinary artisan would have been motivated to test these time points during routine experimental optimization.
11. Claims 15 and 16 are rejected under 35 U.S.C. 103 as being unpatentable over Belhocine et al (International Patent Application No. WO 2018218226, published 29 November 2018) in view of Cao et al (Joint profiling of chromatin accessibility and gene expression in thousands of single cells, Science, 361, 6409, 1380-1385, published 28 September 2018) and Sanjana et al (Improved vectors and genome-wide libraries for CRISPR screening, Nature Methods, 11, 783-784, published 30 July 2014). as applied to claim 1 above, and further in view of Picelli et al (Tn5 transposase and tagmentation procedures for massively scaled sequencing projects, Genome Research, 24, 2033-2040, published 30 July 2014).
Regarding claim 15, the method of claim 1 is discussed fully above and incorporated here. Belhocine in view of Sanjana and Cao teaches that tagmentation occurs in an appropriate buffer containing magnesium.
Belhocine in view of Sanjana and Cao does not teach that the tagmentation buffer comprises H2O, 5 mM Mg2+, a hydrophilic solvent in a zwitterionic buffer at a pH of about 8.5.
However, Picelli teaches a 5X tagmentation buffer that is 50 mM TAPS-NaOH (pH 8.5, TAPS is a zwitterionic buffer), 25 mM MgCl2, and 50% DMF (a hydrophilic solvent, Table 1).
It would have been obvious to one having ordinary skill in the art to have simply substituted the generic tagmentation buffer taught by Belhocine in view of Sanjana and Cao with the specific tagmentation buffer taught by Picelli to arrive at the instantly claimed invention with a reasonable expectation of success. The ordinary artisan would have been motivated to make this substitution in order to have a more specific experimental procedure and reduce undue experimentation. In addition, the ordinary artisan would have recognized that the known techniques in the cited references could have been combined with predictable results because the known techniques in the cited references predictably result in the buffer for the tagmentation of DNA.
Regarding claim 16, Picelli teaches a tagmentation buffer that is 50 mM TAPS-NaOH (pH 8.5), 25 mM MgCl2, and 50% DMF (Table 1).
Picelli does not specifically teach that the tagmentation buffer comprises an RNAse inhibitor.
However, Picelli teaches using an RNAse inhibitor when lysing cells for RNA-seq experiments (pg. 2039, column 2, ¶ 2), such as those performed by the methods of Belhocine discussed in the rejection of claim 1 above.
It would have been obvious to one having ordinary skill in the art to have modified the tagmentation buffer taught by Picelli to include an RNAse inhibitor. The ordinary artisan would have been motivated to make this modification because Picelli specifically teaches the use of an RNAse inhibitor when lysing cells for RNA-seq applications (pg. 2039, column 2, ¶ 2). In addition, the ordinary artisan would have recognized that the known techniques in the cited reference could have been combined with predictable results because the known techniques of the cited reference predictably result in the stabilization of RNA for RNA-seq library preparation.
Response to Arguments
12. Applicant’s arguments with respect to claims 1, 4-7, 9-16, 18, 19, 21 and 22 have been considered but are moot because the new ground of rejection does not rely on any reference applied in the prior rejection of record for any teaching or matter specifically challenged in the argument.
13. 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.
Conclusion
14. No claims are allowed.
15. Any inquiry concerning this communication or earlier communications from the examiner should be directed to BRIAN ELLIS YOUNG whose telephone number is (703)756-5397. The examiner can normally be reached M-F 0730 - 1700.
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/BRIAN ELLIS YOUNG/Examiner, Art Unit 1684
/JULIET C SWITZER/Primary Examiner, Art Unit 1682
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