DETAILED ACTION
Status of the Claims
Claims 1-53 are currently pending and are the subject of this Office Action.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 07/19/2022 and 03/09/2023 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Claim Rejections – 35 U.S.C. 102
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 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 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.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Vigneault et al.
Claims 1-8, 11, 14, 17-18, 24-31, 33-39, and 45-48 are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Vigneault et al. (U.S. 9,816,088 B2, issued 11/14/2017).
Vigneault generally discloses methods of preparing a library of bar-coded immunoglobulin light and heavy chain polynucleotide sequences (e.g., as per the SUMMARY OF THE INVENTION section and esp. at col. 2, lines 8-10). Vigneault discloses (e.g., as per the SUMMARY OF THE INVENTION section and Examples 1-14) slight variations on this method, including the use of B cells or T cells, capture of mRNA onto beads using either gene specific probes or poly(T) sequences, variations on the unique barcodes, the use of fusion, palindromic, or complementary overhang tags for fusion PCR, etc. Note that Vigneault presents two sets of 14 Examples in columns 48-86.
Regarding claim 1, Vigneault discloses a method of producing two or more linked nucleic acid molecules from a single cell (e.g., as per the SUMMARY OF THE INVENTION section and/or the Examples), comprising:
(i) isolating a single cell in a first container (e.g., referred to as Emulsion Reverse Transcription in the SUMMARY and Examples, wherein emulsions of droplets are created containing a single cell and 1 or more poly(T)-coated beads, depicted in Fig. 25A), and lysing the single cell to release mRNA molecules (e.g., cells are lysed using repeated freeze-thaw cycles, depicted in Fig. 25B);
(ii) reverse transcribing the mRNA molecules to produce cDNA molecules in the first container (e.g., RT is performed in the first droplet to copy the mRNA onto the anchor sequences of the beads, for example, using MuMLV reverse transcriptase and as depicted in Fig. 25C); and
(iii) linking the cDNA molecules derived from the single cell in step (ii) in a second container, thereby producing linked nucleic acid molecules (e.g., the bead with cDNA covalently attached are recovered and washed as depicted in Fig. 25D, and subject to droplet containment, wherein one droplet has one bead as depicted in Fig. 25E, followed by fusion splicing overlap PCR to physically link the immunoglobulin chains for sequencing and/or cloning).
Regarding claim 2, Vigneault discloses the above method, wherein the first container comprises one or more solid supports attached to an oligonucleotide comprising a sequence complementary to a portion of the mRNA molecules (e.g., as per the SUMMARY section and/or Examples).
Regarding claim 3, Vigneault discloses the above method, wherein the mRNA molecules are attached to the oligonucleotide via binding to the complementary sequence (e.g., using anchor sequences which either comprise poly(T) sequences or gene specific sequences, for example, when capturing only the heavy and light chains, as per the SUMMARY section and Examples, or poly(T) as per Fig. 25A).
Regarding claim 4, Vigneault discloses the above method, wherein the reverse transcribing comprises extending the oligonucleotide with a reverse transcriptase to produce the cDNA molecules (e.g., reverse transcribing the mRNA onto the beads).
Regarding claim 5, Vigneault discloses the above method, wherein the cDNA molecules from step (ii) are covalently linked to the solid supports (e.g., as per the Examples and Fig. 25C-E).
Regarding claim 6, Vigneault discloses the above method, wherein each of the one or more solid supports is isolated in a different second container prior to step (iii) (e.g., often referred to as “Emulsion 2” as per the Examples, wherein “the beads were isolated to achieve a ratio of one bead or less per emulsion”).
Regarding claim 7, Vigneault discloses the above method, wherein the oligonucleotide is attached to the solid support by a linker (e.g., as per Fig. 25A).
Regarding claim 8, Vigneault discloses the above method, wherein the linker is located between a surface of the solid support and the sequence complementary to a portion of the mRNA molecules (e.g., a poly(T) anchor sequence as per the Examples and Fig. 25A, or gene specific anchor sequences as per the Examples).
Regarding claim 11, Vigneault discloses the above method, wherein 1 to 20 solid supports are present in the first container (e.g., “emulsion 1 may have contained more than one bead” as per several of the Examples and as detailed in the Droplet Libraries section at col. 38-40).
Regarding claim 14, Vigneault discloses the above method, wherein the solid support is a bead or particle (e.g., as per several of the Examples and as detailed in the Droplet Libraries section at col. 38-40).
Regarding claim 17, Vigneault discloses the above method, wherein linking the cDNA molecules in step (iii) comprises amplifying and linking the cDNA molecules by overlap extension PCR (e.g., overlap PCR as per the SUMMARY section and several of the Examples).
Regarding claim 18, Vigneault discloses the above method, wherein the cDNA molecules are released from the solid support in the second container prior to step (iii) (e.g., as depicted in Fig. 25F).
Regarding claim 24, Vigneault discloses the above method, wherein the single cell is an immune system cell (e.g., as per the Samples section on col. 24-25).
Regarding claim 25, Vigneault discloses the above method, wherein the single cell is a B cell (e.g., as per the Samples section on col. 24-25).
Regarding claim 26, Vigneault discloses the above method, wherein the mRNA molecules encode a heavy chain variable region and a light chain variable region (e.g., as per the Samples section on col. 24-25).
Regarding claim 27, Vigneault discloses the above method, wherein the cDNA molecules encode a cognate pair of heavy and light chain variable regions (e.g., as per the Samples section on col. 24-25).
Regarding claim 28, Vigneault discloses the above method, wherein the single cell is a T cell (e.g., as per the Samples section on col. 24-25).
Regarding claim 29, Vigneault discloses the above method, wherein the single cell is a natural killer T (NKT) cell (e.g., as per the Samples section on col. 24-25).
Regarding claim 30, Vigneault discloses the above method, wherein the cDNA molecules encode a cognate pair of T cell receptor alpha and beta chains (e.g., as per the Samples section on col. 24-25).
Regarding claim 31, Vigneault discloses the above method, wherein the first or second container comprises an aqueous droplet in an emulsion (e.g., as per several of the Examples and as detailed in the Droplet Libraries section at col. 38-40).
Regarding claim 33, Vigneault discloses the above method, further comprising digesting the mRNA following step (ii) (e.g., “1 μl of RNAse H (Enzymatics) was added and the reaction was incubated for an additional 15 mins at 37°C” as per Examples 11-12).
Regarding claim 34, Vigneault discloses the above method, wherein the mRNA is digested in the first container, or between steps (ii) and (iii) (e.g., “1 μl of RNAse H (Enzymatics) was added and the reaction was incubated for an additional 15 mins at 37°C” as per Examples 11-12).
Regarding claim 35, Vigneault discloses a method for producing a library of linked nucleic acid molecules (e.g., as per the SUMMARY OF THE INVENTION section and/or the Examples), comprising:
a) isolating a plurality of single cells in a plurality of first containers, where the first containers comprise a single cell (e.g., referred to as Emulsion Reverse Transcription in the SUMMARY and Examples, wherein emulsions of droplets are created containing a single cell and 1 or more poly(T)-coated beads, depicted in Fig. 25A);
b) lysing the single cells to release mRNA molecules in the first container (e.g., cells are lysed using repeated freeze-thaw cycles, depicted in Fig. 25B);
c) reverse transcribing the mRNA molecules to produce cDNA molecules derived from single cells in the first container (e.g., RT is performed in the first droplet to copy the mRNA onto the anchor sequences of the beads, for example, using MuMLV reverse transcriptase and as depicted in Fig. 25C);
d) linking the cDNA molecules from step (c) in a second container (e.g., the bead with cDNA covalently attached are recovered and washed as depicted in Fig. 25D, and subject to droplet containment, wherein one droplet has one bead as depicted in Fig. 25E, followed by fusion splicing overlap PCR to physically link the immunoglobulin chains for sequencing and/or cloning);
e) combining the linked cDNA molecules from step (d) to produce a library of linked nucleic acid molecules (e.g., combining for Illumina sequencing as per the Examples).
Regarding claims 36-38, Vigneault discloses the above method, wherein the single cells are B cells, T cells, or NK cells (e.g., as per the Samples section on col. 24-25), but does not specifically comment on the percentage of heavy or alpha chain variable regions that are correctly paired with the cognate light or beta chain variable regions in the library being increased compared to a method where steps (c) and (d) are performed in the same container. It is noted that In re Best (195 USPQ 430) and In re Fitzgerald (205 USPQ 594) discuss the support of rejections wherein the prior art discloses subject matter which there is reason to believe inherently includes functions that are newly cited or is identical to a product instantly claimed. In such a situation the burden is shifted to the applicants to "prove that subject matter shown to be in the prior art does not possess characteristic relied on" (205 USPQ 594, second column, first full paragraph). In the instant case, Vigneault is performing all of the same steps as presently claimed, so there is reason to believe that the reference will achieve the same or similar results as the present claims, absent evidence to the contrary.
Regarding claim 39, Vigneault discloses the above method, wherein step (d) comprises amplifying and linking the cDNA molecules by overlap extension PCR (e.g., fusion splicing overlap PCR to physically link the immunoglobulin chains for sequencing and/or cloning as per the Examples).
Regarding claim 45, Vigneault discloses a method for producing two or more linked nucleic acid molecules from a single cell (e.g., as per the SUMMARY OF THE INVENTION section and/or the Examples), comprising:
(i) isolating a single cell in a first container (e.g., referred to as Emulsion Reverse Transcription in the SUMMARY and Examples, wherein emulsions of droplets are created containing a single cell and 1 or more poly(T)-coated beads, depicted in Fig. 25A), and lysing the single cell to release mRNA molecules (e.g., cells are lysed using repeated freeze-thaw cycles, depicted in Fig. 25B);
(ii) hybridizing the mRNA molecules to a capture oligonucleotide attached to a solid support, wherein the capture oligonucleotide comprises a sequence complementary to a portion of the mRNA sequence (e.g., gene-specific or poly(T)-coated beads, depicted in Fig. 25);
(iii) reverse transcribing the mRNA molecules to produce cDNA molecules attached to the solid support in the first container (e.g., RT is performed in the first droplet to copy the mRNA onto the anchor sequences of the beads, for example, using MuMLV reverse transcriptase and as depicted in Fig. 25C);
(iv) linking the cDNA molecules derived from step (iii) in a second container, thereby producing linked nucleic acid molecules (e.g., the bead with cDNA covalently attached are recovered and washed as depicted in Fig. 25D, and subject to droplet containment, wherein one droplet has one bead as depicted in Fig. 25E, followed by fusion splicing overlap PCR to physically link the immunoglobulin chains for sequencing and/or cloning).
Regarding claim 46, Vigneault discloses the above method, wherein the capture oligonucleotide further comprises a linker positioned between the solid support and the sequence complementary to a portion of the mRNA sequence (e.g., a poly(T) anchor sequence as per the Examples and Fig. 25A, or gene specific anchor sequences as per the Examples).
Regarding claim 47, Vigneault discloses the above method, wherein the linker is cleaved, releasing the cDNA molecules from the solid support prior to step (iv) (e.g., as depicted in Fig. 25F).
Regarding claim 48, Vigneault discloses the above method, wherein step (iv) comprises amplifying and linking the cDNA molecules by overlap extension PCR (e.g., fusion splicing overlap PCR to physically link the immunoglobulin chains for sequencing and/or cloning as per the Examples).
Claim Rejections – 35 U.S.C. 103(a)
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 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.
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.
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 set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied 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.
Vigneault et al.
Claims 1-8, 11-18, 24-39, and 45-48 are rejected under 35 U.S.C. 103 as being unpatentable over Vigneault et al. (U.S. 9,816,088 B2, issued 11/14/2017).
Vigneault is relied on as above.
Regarding claims 12-13, Vigneault discloses the method as detailed herein, wherein the first container comprises one or more beads following a Poisson distribution
Regarding claims 15-16 and 32, Vigneault discloses the above method, wherein “[t]he droplets range in size from roughly 0.5 micron to 500 micron in diameter, which corresponds to about 1 pico liter to 1 nano liter. However, droplets can be as small as 5 microns and as large as 500 microns. Preferably, the droplets are at less than 100 microns, about 1 micron to about 100 microns in diameter. The most preferred size is about 20 to 40 microns in diameter (10 to 100 picoliters).” This encompasses the ranges as claimed in claims 15-16 and 32. As per MPEP § 2144.05(I), in the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists, citing In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976) and In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990)
Vigneault et al. and Mikkelsen et al.
Claims 1-18, 24-39, and 45-48 are rejected under 35 U.S.C. 103 as being unpatentable over Vigneault et al. (U.S. 9,816,088 B2, issued 11/14/2017) in view of Mikkelsen et al. (ACS Comb. Sci., 2018, 20, 377−399).
Vigneault is relied on as above, however, the reference is silent regarding the limitations of the linker being a photocleavable linker, as set forth in claim 9, and wherein the cDNA molecules are released from the solid support by exposing the photocleavable linker to light in the second container, as set forth in claim 10.
Mikkelsen discloses cleavage of DNA from solid supports using photocleavable linkers and cleaving by exposure to the appropriate wavelength of light (e.g., as per the Abstract and Table 1).
It would have been prima facie obvious to a person of ordinary skill in the art prior to the effective filing date of the application to substitute the uracil linker of Vigneault with the photocleavable linker of Mikkelsen. One of ordinary skill in the art would have been motivated to do so since such linkers are able to be cleaved quickly, under mild conditions, and without the expense of uracil cleaving enzymes (e.g., as per the Abstract).
One of ordinary skill in the art would have had a reasonable expectation of success as of the application’s effective filing date in combining the teachings of the prior art references to arrive at the invention as presently claimed since Mikkelsen discloses several examples of performing synthesis or attachment of DNA to solid supports via a photocleavable linker and noting that such beads were also commercially available as kits.
Vigneault et al. and Wetmur et al.
Claims 1-8, 11-20, 24-41, and 45-50 are rejected under 35 U.S.C. 103 as being unpatentable over Vigneault et al. (U.S. 9,816,088 B2, issued 11/14/2017) in view of Wetmur et al. (Nucleic Acids Research, 2005, 33(8):2615-2619).
Vigneault is relied on as above, however, the reference is silent regarding the limitations of the overlap extension PCR comprises amplifying the cDNA molecules using one or more internal primers comprising a biotin tag, as set forth in claims 19, 40, and 49, wherein cDNA molecules comprising the biotin tag are removed after step (iii), as set forth in claims 20, 41, and 50.
Vigneault discloses embodiments using excess P5 and P7 primers (e.g., as per the Examples) as a means to increase the amount of final, full-length product over the intermediate PCR products. As an alternative, Wetmur discloses similar overlap extension PCR, but with the use of biotinylated internal primers such that “unlinked biotinylated amplicons were removed on streptavidin-coated magnetic beads” (e.g., as per p. 2617, right column) as a means to only leave full-length products in the end.
It would have been prima facie obvious to a person of ordinary skill in the art prior to the effective filing date of the application to use biotinylated internal primers as per Wetmur in the nucleic acid linking methods of Vigneault. One of ordinary skill in the art would have been motivated to do so since such a method allows for the virtual elimination of unlinked products, rather than simply a reduction. Further, as per MPEP 2143(I)(A), the rationale to support a conclusion that the claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination yielded nothing more than predictable results to one of ordinary skill in the art.
One of ordinary skill in the art would have had a reasonable expectation of success as of the application’s effective filing date in combining the teachings of the prior art references to arrive at the invention as presently claimed since the use of biotinylated primers and streptavidin capture techniques were well known in the art at the time, including the availability of commercial kits for it.
Vigneault et al. and Nikiforov et al.
Claims 1-8, 11-18, 21-39, 42-48, and 51-53 are rejected under 35 U.S.C. 103 as being unpatentable over Vigneault et al. (U.S. 9,816,088 B2, issued 11/14/2017) in view of Nikiforov et al. (PCR Methods and Applications, 1994, 3:285-291).
Vigneault is relied on as above, however, the reference is silent regarding the limitations of the overlap extension PCR comprises amplifying the cDNA molecules using one or more external primers chemically modified to resist nuclease degradation, as set forth in claims 21, 42, and 51, wherein the one or more external primers are chemically modified to include phosphorothioate bonds, as set forth in claims 22, 43, and 52, and wherein the cDNA molecules are contacted with a 5'-exonuclease after step (iii), as set forth in claims 23, 44, and 53.
Nikiforov discloses methods of using PCR primers comprising nuclease-resistant phosophorothioate nucleotides at the 5’-termini followed by 5’-exonuclease PCR clean up (e.g., as per the MATERIALS AND METHODS section on pp 285-286).
It would have been prima facie obvious to a person of ordinary skill in the art prior to the effective filing date of the application to use PCR primers comprising nuclease-resistant phosophorothioate nucleotides at the 5’-termini followed by 5’-exonuclease PCR clean up as per Nikiforov in the nucleic acid linking methods of Vigneault. One of ordinary skill in the art would have been motivated to do so since Nikiforov discloses it as an effective and efficient method of PCR clean up to isolate the final PCR product from any intermediate, non-productive amplificons (e.g., as per the DISCUSSION section on pp. 288-289).
One of ordinary skill in the art would have had a reasonable expectation of success as of the application’s effective filing date in combining the teachings of the prior art references to arrive at the invention as presently claimed since PCR primers with phosphorothioate nucleotides at the 5’-termini are compatible with most PCR protocols and are commercially available to order from custom oligonucleotide suppliers.
Conclusion
No claims are allowed.
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/JEREMY C FLINDERS/
Primary Examiner, Art Unit 1684