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 .
Applicants previously canceled claims 3-4 and 16. Claims 7-15 remain withdrawn from consideration as being directed to non-elected inventions. Applicants amend claims 1 and withdrawn claim 7. Claims 1-2, 5-6, and 17-18 are under examination.
Any objection or rejection of record in the previous Office Action, mailed March 14, 2025, which is not addressed in this action has been withdrawn in light of Applicants’ amendments and/or arguments. This action is FINAL.
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.
Claims 1-2, 5-6, and 17-18 are rejected under 35 U.S.C. 103 as being unpatentable over Liu et al. (US Patent Application Publication No. 2015/0166980 A1, cited in the Information Disclosure Statement filed January 18, 2015; see the entire reference) in view of Rogozin et al. (8(6) Nature Immunology 647-656 and 1/88-88/88 of Supplementary Materials (2007); see the entire reference), Mateus et al. (16 Endangered Species Research 183-198 (February 29, 2012); see the entire reference), Zhao et al. (PCT Patent Application Publication No. WO 2016/069283 (published May 6, 2016; see the entire reference), Doyon et al. (U.S. Patent No. 8,772,008, issued July 8, 2014); see the entire reference), Rieder et al. (1(3) Cell Cycle 169-175 (2002)); see the entire reference) and Zhang et al. (PCT Patent Application Publication No. 2014/018423, published January 30, 2014; see the entire reference). This rejection is maintained.
Regarding claims 1, 2, 5, 6, 17 and 18, Liu discloses methods of targeted nucleic acid editing comprising contacting a DNA molecule with (a) a fusion protein comprising a nuclease-inactive Cas9 domain (i.e., deficient in two DNA cleavage abilities), (GGGS), linker, and a deaminase domain; and (b) an sgRNA targeting the fusion protein of (a) to a target nucleotide sequence of the DNA strand, wherein the DNA molecule is contacted with the fusion protein and the sgRNA in an amount effective and under conditions suitable for the deamination of a nucleotide base (e.g., paragraphs [0008]-[00011], [0037], [0040] and [0057]). Liu et al teach the method where the deaminase is a cytidine deaminase, such as an APOBEC family deaminase, such as activation-induced cytidine deaminase (AID) (e.g., paragraphs [0025], [0037], [0040] and [0051]). Liu discloses the application of the method to editing DNA in cells (e.g., paragraphs [0037] and [0093]). Liu discloses that a number of different deaminases are suitable for use in the method (e.g., paragraphs [0058] and [0083]). Liu et al teach that a human cell is contacted with an expression construct encoding a Cas9 deaminase protein and an appropriately designed sgRNA targeting the fusion protein to a mutation site in a PI3KCA gene, for example (paragraphs [0088] and [0093]; Examples 2-6). Liu discloses that the article such as “a,” “an,” and “the” may mean more than one (paragraphs [0017] and [0193]). The instant claims require “modifying a plurality of targeted sites.” The claims reasonable encompass the method where the target sites are in the same position in a population of cells. Liu discloses contacting cells with an expression construct encoding the Cas9 deaminase fusion protein and an appropriately designed sgRNA targeting the fusion protein to a mutation site in the genome (paragraphs [0010]-[0011] and [0088]).
Liu does not discloses that the cytidine deaminase is Petromyzon marinus cytosine deaminase 1 (PmCDA1) with the amino acid sequence set forth in SEQ ID NO: 2. Liu does not disclose the method where the human cell is cultured at a temperature of 20°C to 25°C at least temporarily, or overnight. Liu does not disclose that a plurality of targeted sites are in different positions or that the expression construct is an expression vector which is not capable of autonomous replication in the host cell.
Rogozin discloses lamprey cytosine deaminases that are members of the AID-APOBEC cytosine deaminase family (pages 650-651). Rogozin discloses PmCDA1 is a polypeptide of 208 residues encoded by a single exon (page 650, paragraph bridging columns; supplementary Fig. 6). Supplementary Fig. 6 teaches the amino acid sequence of PMCDA1 (EF094823), which is identical to instant SEQ ID NO: 2 (Appendix I). Rogozin discloses that expression of human AID in Escherichia coli generates cytosine deamination sites that cause transition mutations of C:G nucleotide pairs to A:T pairs (page 651, paragraph bridging columns). Rogozin discloses that expression of PmCDAI in E. coli also produced dC-to-dT and dG-to-dA transition mutations (page 651, paragraph bridging columns; Supplementary Tables | and 2). Further, Rogozin discloses that PMCDA1 was highly mutagenic when expressed in ung- yeast (paragraph bridging pages 651-652; Supplementary Table 3).
Mateus discloses that spawning of the sea lamprey, Petromyzon marinus L.., starts at 15°C, and eggs hatch and survive at temperatures between 15 and 23°C (page 189, column 2, first full paragraph).
Zhao discloses that zinc finger nuclease (ZFN) and TALE nuclease (TALEN) mediated gene disruption had been reported in the art to be more efficient when cells were transiently exposed to mild hypothermia (Example 1). Zhao discloses that the same phenomenon is observed with a CRISPR-Cas9 system comprising Cas9 and guide RNA (e.g., Example 1). Zhao discloses the electro-transfer of in vitro transcribed RNA encoding Cas9 and guide RNA into human T cells, which were cultured for 1 day at 32°C after electro-transfer, which resulted in 2.5-fold better gene disruption than when cells were cultured at 37°C, which is interpreted as including overnight culturing (Example 1; Figs. 2C and 2D). Zhao discloses that the CRISPR/Cas system can simultaneously target multiple genomic loci by co-expressing a single Cas9 protein with two or more gRNAs, making the system suited for multiplex gene editing (e.g., Example 5, at the top of the second page of the example; Example 11).
Doyon discloses subjecting cells expressing a zinc finger nuclease to hypothermic conditions (cold shock) to increase activity of the nuclease for genomic modifications (Abstract; paragraph bridging columns 1-2). Doyon discloses cold shock of mammalian cells at 33°C, 29°C, 28°C, 27°C and even lower (column 7, lines 19-25; column 15, lines 26-39). Doyon discloses that the temperature can vary during the period of cold-shocking, so long as it remains low enough so that the cells are not dividing or are dividing at a reduced rate (column 15, lines 26-39). Doyon discloses culturing at the cold shock temperature for between 1 and 4 days (e.g., column 2, lines 14-21), which includes overnight. Doyon discloses that cold shock of mammalian cells improves the efficiency of editing by a zinc finger nuclease to 2- to 10-fold or more (Examples 2-3; Figs. 1-3).
Rieder discloses that mammalian cells have optimum growth at temperatures of 35-37°C in culture but may continue to grow and divide at 25-33°C at a rate substantially slower than optimal growth temperatures (page 169, Introduction, first paragraph). Rieder discloses culturing the cells at 20-21°C or 24-25°C for 15-20 hours, and up to 5 days at 24-25°C (page 565, last paragraph; page 566, 3" and 4" paragraphs), which includes overnight. Rieder discloses that rat kangaroo kidney (Prtk1) and human pancreatic ductal adenocarcinoma (CFPAC-1) cells take 8-10 hours for spindle formation at 19-20°C as compared to about 50 minutes at 35-37°C ( page 169, Introduction, second paragraph). Rieder discloses that the mitotic index of most growing mammalian cell cultures falls to near-zero in 24-48 hours, which includes overnight, when culture temperatures are shifted from 37°C to <20°C (page 169, Introduction, third paragraph). Rieder discloses that the cells are capable of diving once the culture temperature is returned to 37°C (page 169, Introduction, 3" paragraph).
Zhang discloses that vectors are tools that allow or facilitate the transfer of an entity from one environment to another, such as the transfer of a heterologous DNA segment into a target cell, where such vector can be an expression vector (paragraphs [00117]-[00118]). Zhang discloses viral vector gene delivery systems are commonly used in gene transfer and gene therapy applications (paragraph [00219]). Zhang discloses that adenovirus is not associated with severe human pathology and is extremely efficient in introducing its DNA into the host cell (paragraph [0220]). Further, Zhang discloses that adenovirus may infect a wide variety of cells and can be rendered replication defective and/or non-replicating by deletions in the early region 1 (E1) of the viral genome (paragraph [00220]). Moreover, Zhang discloses that more than El can be deleted such that vector is capable of providing more carrying capacity while remaining replication defective and/or non-replicating (paragraphs [00226]-[00228]).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of targeted editing of Liu et al to include the Petromyzon marinus cytidine deaminase (PmCDA1) of instant SEQ ID NO: 2 as taught by Rogozin as the cytidine deaminase of the fusion protein, because Liu discloses that it is within the ordinary skill in the art to use a variety of different cytidine deaminases for targeted editing, such as an APOBEC family deaminase, such as activation-induced cytidine deaminase (AID), and Rogozin discloses that PMCDA1 is a member of the AID-APOBEC cytosine deaminase family and has the same activity as human AID with regard to generating transition mutations. Because Liu discloses the use of AID cytidine deaminases for targeted editing, and Rogozin discloses that PmCDAI is an AID capable of gene editing through transition mutations, one would have had a reasonable expectation of success in substituting one AID for another in order to achieve the predictable result of gene editing. Furthermore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of targeted editing of Liu to include the transient, mild hypothermia at 25°C, including overnight, because Liu et al teach it is within the skill of the art to use human cells for editing with the nuclease-deficient Cas9-cytidine deaminase fusion protein, and Zhao discloses the treatment of human cells with transient, mild hypothermia for a culture period that includes overnight after the introduction of a Cas9 protein for gene editing as had been taught in the art for editing with ZFN and TALEN. Doyon et al specifically teach transient mild hypothermia for editing with ZFN at temperatures 33°C, 29°C, 28°C, 27°C and even lower, which encompasses the claimed range of 20-25°C and the specific temperature of 25°C, for periods of time that includes overnight. Furthermore, Doyon discloses that the period of reduced temperature or cold-shock is used to keep the cells from dividing or to allow them to divide at a reduced rate, and Rieder discloses that at 25°C mammalian cells are dividing at a reduced rate, and at < 20°C, mammalian cells are not dividing when the cells are cultured for a period that includes overnight. Thus, one would have selected a temperature of 20-25°C or 25°C for a culture period including overnight in order to reduce the rate of cell division and improve the editing process. One would have had an expectation of success in using the PmCDA1 of Rogozin at this temperature, because PmCDA1 is from the sea lamprey whose native habitat is at or near the range of 20-25°C, as taught by Mateus. Furthermore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of targeted editing of Liu et al to include targeting a plurality of sites at different positions, because Liu discloses targeting a site, and Zhao discloses that the CRISPR Cas9/guide RNA system can be used to target multiple sites simultaneously by providing different guide RNAs. One would have made such a modification in order to be able to target multiple genes in a single cell according to Zhao.
One of ordinary skill in the art would have been motivated to temporarily culture the cells, for a period including overnight, at 20-25°C or 25°C in order to achieve the benefit of improving the efficiency of editing as compared to standard culture conditions at 37°C as disclosed by Zhao and Doyon at a temperature and time known to reduce cell division as taught by Rieder. Furthermore, one would have been motivated to include multiple guide RNAs to target different genes in a single cell in order to carry out multiplex editing to create new combinations of alterations.
It would also have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the teachings of Liu, Rogozin, Mateus, Zhao, Doyon, and Rieder to include substitute the expression construct of Liu with the replication defective and/or non-replicating adenoviral expression vector disclosed by Zhang because Liu discloses it is within the ordinary skill in the art to use an expression construct to express Cas9 and guide RNA, and Zhang discloses the use of a replication defective and/or non-replicating adenoviral expression vector for the introduction into and expression of a nucleic acid in cells. One would have made such a substitution in order to receive the expected benefit of using a specific type of expression construct known to function as a way to introduce nucleic acid into a cell and to express the nucleic acid in the cell.
One would have been motivated to make such a modification in order to receive the expected benefit of using a vector that has a large carrying capacity as a result of the gene deletions to render the vector incapable of autonomous replication in a cell at high efficiency in many cell types as taught by Zhang.
Response to Amendments and Arguments
Regarding the rejection under 35 U.S.C. § 103, based upon Liu, Rogozin, Mateus, Zhao, Doyon, and Rieder, Applicants’ amendments and arguments, as well as the Declaration under 37 C.F.R. § 1.132, have been fully considered but are not deemed to be persuasive.
Applicants and the 1.132 Declaration assert that the use of a temporary culture of mammalian cells at 20 °C to 25 °C unexpectedly exhibit beneficial effects, such as improvement of the function of pmCDA1-dCas9 complexes, which lead to stable and efficient editing of multiple target sites. Applicants and the 1.132 Declaration assert that neither Liu nor Zhang teach or suggest culturing cells at the lowered temperature. Applicants assert and the 1.132 Declaration that the prior art cultures the cells at a much longer period than overnight.
To begin, it appears that Applicants are attacking each prior art reference individually. However, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., Inc., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
Here, Liu and Zhang are cited, not for the temperature of culture, but for the a) use of a dCas9-deaminase fusion protein, where the domains are connected with a linker, as well as an sgRNA that targets the fusion protein to a target sequence (Liu) and b) the use of a replication defective and/or non-replicating adenoviral expression vector for the introduction into and expression of a nucleic acid in cells (Zhang). Thus, in turn, Zhang provides the expected benefit of using a specific type of expression construct known to function as a way to introduce nucleic acid into a cell and to express the nucleic acid in the cell. One would have been motivated to make such a modification in order to receive the expected benefit of using a vector that has a large carrying capacity as a result of the gene, and to do so would also provide for the expected benefit of a more compact vector, a higher alteration efficiency, and lower off-target effects. Applicants and the Declaration point only to these two references and note that they do not provide for culturing temporarily at the lowered temperatures of 20 °C to 25 °C.
However, as noted in the Office Action, neither Liu nor Zhang (nor Rogozin, which discloses PmCDA1 and its sequence nor Mateus, which discloses sea lampreys (Petrormyzon marinus) spawn and hatch eggs at low temperatures) discloses culturing the cells at the lowered temperatures of 20 °C to 25 °C. Rather, Doyon discloses cold shock of cells at temperatures of 27 C and lower for a period ranging from 1 day to 4 days. A one day cold-shock is deemed to be a temporary lowering of temperature and a 1 day cold shock is deemed to encompass an overnight cold shock. Doyon discloses that editing efficiency by zinc finger nucleases is increased using such a low temperature period of culture.
Zhao also discloses zinc finger nucleases and TALE nuclease mediated gene disruption is more efficient at low (hypothermic) temperatures, and further discloses that the same holds for CRISPR-Cas9 gene disruption.
Rieder also discloses that mammalian cells can be cultured at temperatures of 20 °C-21 °C or 24 °C to 25 °C for 15-20 hours, and up to 5 days, which timer periods encompass a temporary and/or overnight culture at such temperatures. Rieder discloses that lowering temperature accordingly increases the time for spindle formation in mammalian cells, and that the cells are capable of dividing again upon increasing the temperature.
Thus, the disclosures of Doyon and Rieder are not discussed in either Applicants’ arguments of the Declaration. And Applicants assertion of unexpected benefits/results would actually have been expected in light of Doyon and Rieder. Zhao and Doyon specifically even demonstrates increased gene disruption and editing efficiency at temporarily low temperatures. Neither Applicants’ assertions not the 1.132 Declaration provide any evidence, in light of the combination of Liu, Zhang, Mateus with the low temperature disclosures of Doyon, Zhao, and Reider, that the cited prior art references, taken together and as a whole, would not have resulted in the same beneficial editing efficiency as claimed by the instant application. Thus, the increased editing efficiency, while beneficial, is not deemed to be unexpected in light of the cited prior art.
For all these reasons, and those listed above, the combination of upon Liu, Rogozin, Mateus, Zhao, Doyon, Rieder, and Zhang are deemed to render claims 1-2, 5-6, and 17-19 obvious and the rejection is maintained.
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Chen et al. (24(3) Molecular Therapy 447-457 (September 4, 2015)) disclose replication-defective viruses have a role as delivery vehicles for CRISPR Cas9 nucleases (Abstract). Chen et al. disclose that replication-defective viral vectors have been extensively used in academia and industry to deliver foreign genetic payloads into virtually any cell type of interest (page 447, paragraph bridging columns).
THIS ACTION IS MADE FINAL. 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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to NANCY J LEITH whose telephone number is (313)446-4874. The examiner can normally be reached Monday - Thursday 8:00 AM - 6:30 PM.
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NANCY J. LEITH
Primary Examiner
Art Unit 1636
/NANCY J LEITH/Primary Examiner, Art Unit 1636