DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Status
Claims 1-15 filed on 05/24/2023 are pending and under examination.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-15 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.
Regarding claim 1, the recitation of “an insert size of about 20-40 nucleotides” in lines 8-9 of the claim is unclear. It is unclear what is encompassed by the term “insert size”. Does insert size indicate the nucleotide length of the RNA sequence that was digested and then end repaired? Does the insert size indicate the nucleotide length of the RNA sequence before the RNA library was prepared or does the insert size indicate the entire length of the nucleotide sequence prepared as an RNA library? In addition, the claim recites the limitations “the RNA” in line 3 of the claim and “the prepared RNA library” in line 8 of the claim and there is insufficient antecedent basis for these limitations in the claim. In addition, it is unclear if “the prepared library” is referring back to a construction of an RNA library or a different prepared library.
Regarding claim 7, the recitation of “UTP, CTP, GTP, TTP, dATP and dTTP” in lines 2-3 of the claim is unclear if dATP and dTTP are part of the same phosphate donor or if this is the result of a typographical error and should read “UTP, CTP, GTP, TTP, dATP, and dTTP”.
Regarding claim 11, the recitation of “a cell barcode, a sample barcode and a plate barcode” in line 2 of the claim is unclear what the differences are between a cell barcode, a sample barcode, and a plate barcode. What is limitations are required by a cell barcode versus a sample barcode versus a plate barcode. In addition, it is unclear if sample barcode and plate barcode are a part of the same type of barcode or if this is the result of a typographical error and should read “a cell barcode, a sample barcode, and a plate barcode”.
Regarding claim 12, the recitation of “for a time period of at least about 0.5, 1, 2, 4, 6, 8, 10, 12, 14 or 16 hours” in lines 3-4 of the claim is unclear if 14 and 16 hours are a part of the same time period or if this is the result of a typographical error and should read “for a time period of at least about 0.5, 1, 2, 4, 6, 8, 10, 12, 14, or 16 hours”.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claim(s) 1, 3, 4, 6-11, & 13-15 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by McGlincy (McGlincy & Ingolia; Methods, Vol. 126, pages 112-129, June 2017), as cited on the IDS dated 05/24/2023.
Regarding claim 1, McGlincy teaches a method for ribosome profiling (determining a translatome of a cell) through steps of lysing a cell, digesting the RNA of the cell lysate with a ribonuclease to generate a footprint, purifying the footprint RNA with a denaturing sample loading buffer that contains EDTA (inactivating the ribonuclease in the presence of a chelating agent of EDTA and releasing the RNA molecules from the ribosomes), treating the footprint RNA fragments (released RNA molecules) with T4 polynucleotide kinase (PNK) to prepare the fragments for ligation to a DNA linker (end repairing the released RNA molecules), constructing a library (constructing an RNA library from the end-repaired RNA molecules); analyzing and selecting for length distribution and concentration of the constructed library from a footprint size (insert size) of around 28 nt (size selecting part of the prepared RNA library for fragments having an insert size of about 20-40 nucleotides), and then sequencing the size selected library to analyze the ribosome profile (sequencing size selected RNA library and determining the translatome of the cell) (abstract lines 6-12; pg. 118 column 2 1st full paragraph lines 1-14; pg. 118 column 2 2nd full paragraph lines 1-7; pg. 118 section 3.1.2 steps 1-6; pg. 118-119 paragraph bridging pg. 118 & 119 lines 1-11; pg. 119 column 1 1st full paragraph lines 1-7; pg. 118-119 section 3.2 steps 1-4; pg. 119 column 2 2nd full paragraph lines 1-9; pg. 119 section 3.3 steps 1-3; pg. 120 column 1 1st full paragraph lines 1-4; pg. 120 section 3.4 steps 1-2; pg. 123 section 3.9 steps 1-7; pg. 124 paragraph bridging column 1 & 2 lines 1-13; pg. 124 column 2 1st full paragraph lines 1-10; pg. 124 column 2 2nd full paragraph lines 1-9; Table 3).
Regarding claims 3 & 4, McGlincy teaches purifying the footprint RNA with a denaturing sample loading buffer that contains EDTA (inactivating the ribonuclease in the presence of a chelating agent of EDTA) (pg. 119 column 2 2nd full paragraph lines 1-9; pg. 119 section 3.3 steps 1-3; Table 3).
Regarding claims 6 & 7, McGlincy teaches treating the footprint RNA fragments (released RNA molecules) with T4 polynucleotide kinase (PNK) to prepare the fragments for ligation to a DNA linker (end repairing the released RNA molecules) in which the T4 PNK transfers phosphates to itself (a phosphate donor that is not ATP) (pg. 120 column 1 1st full paragraph lines 1-4; pg. 120 section 3.4 steps 1-2).
Regarding claim 8, McGlincy teaches the method may be used to treat HEK293 cells (translatome of two or more cells is determined) (pg. 118 column 2 1st full paragraph lines 1-8).
Regarding claim 9, McGlincy teaches that after library construction (step v) the constructed library is treated with a DNA binding buffer to purify the products, loading the products on a gel and excising the full-length libraries corresponding to insert products (pooling the constructed RNA libraries after step v) and before step vi)) (pg. 123 section 3.9 steps 3-7).
Regarding claim 10, McGlincy teaches preparation of a library of end-repaired footprint RNA fragments (library preparation step v)) comprising ligation of linker to the 5’ end and McGlincy teaches dual-ligation strategies that employ sequential ligation of 3’ & 5’ adapters to the footprint during library preparation (ligating and first adapter and a second adapter to the end-repaired RNA molecules), reverse transcribing the linker-ligated sample (reverse-transcribing the adapter-ligated RNA molecules to obtain cDNA), and amplifying the sample with reverse and forward primers (amplifying the cDNA with a first and second primer) wherein barcodes can be added during linker ligation and at the PCR stage using indexed reverse library PCR primers (preferably at least one adapter comprises a barcode and preferably at least one of the first and second primer comprises a barcode) (pg. 116 column 2 5th full paragraph lines 1-6; pg. 117 column 2 1st full paragraph lines 1-9; pg. 120 section 3.4 steps 2 & 3; pg. 121 section 3.6 steps 1-7; pg. 123 section 3.9 steps 1-8; pg. 128 column 1 2nd full paragraph lines 1-8; Table 16).
Regarding claim 11, it is noted, as discussed above, the recitation of “a cell barcode, a sample barcode and a plate barcode” in line 2 of the claim is unclear what the differences are between a cell barcode, a sample barcode, and a plate barcode. What is limitations are required by a cell barcode versus a sample barcode versus a plate barcode. Therefore, for the purposes of this rejection, a cell barcode is given its broadest reasonable interpretation to comprise a barcode used in a sample and a cell barcode is given its broadest reasonable interpretation to comprise a barcode used in a cell.
McGlincy teaches barcodes can be added to the sample derived from the cell during linker ligation and at the PCR stage using indexed reverse library PCR primers (barcode in step a) and/or c) is a cell barcode or a sample barcode) (pg. 117 column 2 1st full paragraph lines 1-9).
Regarding claim 13, McGlincy teaches end repairing the released RNA molecules comprises ligation of a linker (adapter) performed in a buffer that comprises PEG-800 wherein a highest ligation efficiency may be achieved by increasing the concentration of PEG-800 to 25% w/v (ligation of first and/or second adapter is performed in a buffer comprising PEG in a concentration of preferably about 15%-25%) (pg. 120 column 1 1st full paragraph lines 6-11).
Regarding claim 14, McGlincy teaches performing a gel and excising full-length libraires compared to no-insert products of library amplification products (a complexity reduction step comprising resulting in the enrichment of full-length libraries (a specific target sequence) within the complex starting material) (pg. 123 section 3.9 step 7).
Regarding claim 15, McGlincy teaches the method may be used to treat HEK293 cells (the cell is preferably a human cell) (pg. 118 column 2 1st full paragraph lines 1-8).
Claim Rejections - 35 USC § 103
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.
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) 2 & 5 is/are rejected under 35 U.S.C. 103 as being unpatentable over McGlincy (McGlincy & Ingolia; Methods, Vol. 126, pages 112-129, June 2017), as cited on the IDS dated 05/24/2023, in view of Reid (Reid, Shenolikar, & Nicchitta; Methods, Vol. 91, pages 69-74, July 2015), as cited on the IDS dated 05/24/2023.
The teachings of McGlincy with respect to claim 1 is discussed above and incorporated herein.
Regarding claim 2, McGlincy teaches digesting the RNA of the cell lysate with a ribonuclease of RNase 1 to generate a footprint and that micrococcal nuclease (MNase) has been utilized in ribosomal profiling (pg. 127 column 2 2nd full paragraph lines 1-14).
McGlincy does not teach that the ribonuclease in step ii) is MNase.
Reid teaches a method of ribosomal profiling that uses MNase to generate ribosome footprints in cellular extracts (abstract lines 5-7; pg. 70 column 1 2nd full paragraph lines 1-8; pg. 70 section 2.2 steps 1-3). Reid also teaches that this method is simplified enabling the removal of expensive purification ribosomes such as RNase I with the addition of simpler and more cost effective ribonuclease of MNase and should ease the barrier to employing ribosomal profiling (abstract lines 5-11; pg. 69 column 2 1st full paragraph lines 1-9).
McGlincy and Reid are considered to be analogous to the claimed invention because they are all in the same field of ribosomal profiling (determining a translatome) of a cell. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method digesting the RNA of the cell lysate with RNase I in McGlincy to incorporate the use of the MNase as taught in Reid because Reid teaches that doing so would provide a simpler and more cost effective method for conducting ribosomal profiling of a cell.
Regarding claim 5, McGlincy does not teach that step iii) further comprises a chaotropic agent.
Reid teaches adding guanidinium thiocyanate to the completed digestion reaction (step iii) further comprising the presence of a chaotropic agent which is preferably guanidium thiocyanate) (pg. 70 section 2.3 step (8)). Reid also teaches that this method is simplified enabling the removal of expensive purification ribosomes such as RNase I with the addition of simpler and more cost effective ribonuclease of MNase and should ease the barrier to employing ribosomal profiling (abstract lines 5-11; pg. 69 column 2 1st full paragraph lines 1-9).
McGlincy and Reid are considered to be analogous to the claimed invention because they are all in the same field of ribosomal profiling (determining a translatome) of a cell. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method purifying the footprint RNA with a denaturing sample loading buffer that contains EDTA in McGlincy to incorporate the addition of a chaotropic agent in the buffer as taught in Reid because Reid teaches that doing so would provide a simpler and more cost effective method for conducting ribosomal profiling of a cell.
Claim(s) 12 is/are rejected under 35 U.S.C. 103 as being unpatentable over McGlincy (McGlincy & Ingolia; Methods, Vol. 126, pages 112-129, June 2017), as cited on the IDS dated 05/24/2023, in view of Heyer (Heyer et al.; Nucleic Acids Research, Vol. 43, pages 1-14, December 2014).
The teachings of McGlincy with respect to claims 1 & 10 is discussed above and incorporated herein.
Regarding claim 12, McGlincy does not teach ligating linker (adapter) at a temperature below about 10°C.
Heyer teaches obtaining RNA fragments from ribonucleoprotein particle footprinting (determining a translatome) in which the ligation of adapters to the RNA are optimized through ligation at different temperatures and time periods including ligation of adapters to RNA at 4 °C for a time period of 0 to 24 hours (ligation of first and/or second adapter is performed at a temperature below about 10°C, preferably about 4°C, preferably for a time period of at least about 0.5, 1, 2, 4, 6, 8, 10, 12, 14, or 16 hours) (abstract lines 7-11; pg. 4-5 paragraph bridging pg. 4 & 5 lines 1-21; Figure 2F). Heyer also teaches that the use of T4 RNA ligases for library preparation employ a wide range of reaction times and temperatures but colder temperature should stabilize secondary structures of the RNA (pg. 4-5 paragraph bridging pg. 4-5 lines 1-8).
McGlincy and Heyer are considered to be analogous to the claimed invention because they are all in the same field of preparing libraries for ribosomal profiling (determining a translatome) of a cell. Therefore, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of end repairing the released RNA molecules comprising ligation of a linker (adapter) in McGlincy to incorporate performing ligation of adapters at a temperature of below about 10°C as taught in Heyer because Heyer teaches that doing so would provide a method to stabilize the RNA when ligating an adapter for preparation of a library for analysis of ribonucleoprotein footprinting.
Conclusion
Claims 1-15 are rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to BAILEY C BUCHANAN whose telephone number is (703)756-1315. The examiner can normally be reached Monday-Friday 8:00am-5:00pm ET.
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/BAILEY BUCHANAN/Examiner, Art Unit 1682
/JEHANNE S SITTON/Primary Examiner, Art Unit 1682