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 .
DETAILED ACTION
Claims 1-3, 6-11 and 13-14 are pending. Claims 4-5 and 12 are cancelled. Claims 9-11 and 13-14 are withdrawn. Claims 1-8 are under examination.
The rejection of claims 1-3 and 6-8 under 35 U.S.C. 112(b) is withdrawn in light of the claim amendment deleting the exemplary language and correcting the indefiniteness of the claims.
The rejection of claims 1, 3 and 7-8 under 35 U.S.C. 102(a)(1) and 102(a)(2) is withdrawn in light of the claim amendment to claim 1 requiring reduction or abolishment of the enzyme corresponding to Yarrowia LIP8. Balch does not teach the enzyme corresponding to Yarrowia LIP8.
Priority
This application is a 371 of PCT/EP2020/087018 filed on 12/18/2020, which claims benefit of European Patent Office (EPO) 20187829.5 filed 7/27/2020. The effective filing date of the current application is July 27, 2020.
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.
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.
New rejection necessitated by amendment: Claims 1-3 and 6-8 are rejected under 35 U.S.C. 103 as being unpatentable over Balch et al. (WO 2019/058000 A1, published on March 28, 2019) in view of Fickers et al. (“Identification and characterisation of LIP7 and LIP8 genes encoding two extracellular triacylglycerol lipases in the yeast Yarrowia lipolytica”, Fungal Genetics and Biology, 2005, Vol. 42, pp.264-274) and Grumet et al. (“Lysosomal Acid Lipase Hydrolyzes Retinyl Ester and Affects Retinoid Turnover”, The Journal of Biological Chemistry, 2016, Vol. 291, No.34, pp.17977-17987).
Regarding claims 1 and 3, Balch teaches the biosynthesis of retinoids (title). Balch teaches a carotenoid-producing host cell, particularly fungal host cell, comprising a modification in the endogenous acyltransferase (i.e. lipase) activity, leading to reduction or abolishment of said endogenous acyltransferase activity (description p.2, lines 22-26; p.26 claims 1-2, 4 and 6-8). Balch teaches that the term “fungal host cell” includes particularly yeast as host cells, such as e.g. Yarrowia (description p.2, lines 27-28). ). Balch further teaches that preferred is expression in a fungal host cell, such as e.g. Yarrowia, more preferably expression in Yarrowia lipolytica (description p.10, lines 5-13).
Balch teaches that modification with regards to acylation activity in a process for production of retinoids using the carotenoid-producing host cell, particularly fungal host cell, means a reduction or abolishment of the endogenous genes encoding acyltransferase activity, said host cell being used for production of a retinyl acetate mix (description p.7, lines 19-24). Balch teaches when using said host cell in a vitamin A production process, the percentage of trans-isoforms, such as trans-retinyl acetate, can be increased to about 65% or more, preferably up to 100% based on the total amount of retinyl esters (description p.7, lines 27-30; p.26 claims 1-2, 4 and 6-8).
Balch does not disclose genetic modifications of lipases involved in pre-digestion of vegetable oil into glycerol and fatty acids, with a reduction or abolishment of the enzyme corresponding to Yarrowia LIP8.
However, Fickers teaches Yarrowia lipolytica contains LIP2 gene which codes for the extracellular lipase Lip2p, and additionally genes LIP7 and LIP8 genes encoding two triacylglycerol hydrolases (p.265, 1st column 1st paragraph and 2nd column top paragraph). Fickers teaches that triacylglycerol hydrolases or lipases (EC 3.1.1.3) are able to catalyse ester bond hydrolysis in acylglycerol (p.264, 1st column, 1st paragraph). Fickers further teaches developing mutants lacking one or several lipase genes (p.265, 2nd column top paragraph). Fickers further teaches that comparison of cell growth and lipase production in the deleted strain clearly demonstrates that, together with Lip2p, Lip8p is the major secreted lipase in Y. lipolytica (p.273, 2nd column, 2nd paragraph).
Grumet teaches lysosomal acid lipase (LAL) hydrolyzes retinyl ester and affects retinoid turnover (title). Grumet teaches that murine LAL (mLAL) hydrolyzes retinyl esters (REs), and pharmacological inhibition of LAL in the human hepatocyte cell line HepG2 led to an accumulation of REs in endosomal/lysosomal-enriched fractions, suggesting that LAL is limiting for the clearance of endocytosed REs (p.17977, 2nd column last paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to delete the LIP8 gene and LIP2 gene for lipase, as taught by Fickers, in the Yarrowia lipolytica host cell taught by Balch, because Fickers teaches that Yarrowia lipolytica LIP8 gene encodes the major secreted lipase in Y. lipolytica. One of ordinary skill in the art would have been motivated to abolish the function of the LIP2 or LIP8 lipase genes because Grumet teaches that lysosomal acid lipase is known to hydrolyze retinyl esters and affect retinoid turnover. One of ordinary skill in the art would have found it beneficial to abolish the function of LIP8 and inhibit lipase activity in the Yarrowia lipolytica host to prevent the hydrolysis of retinyl esters.
Regarding claims 2, 3 and 8, Balch teaches a carotenoid-producing host cell further comprising a modification in the endogenous acyltransferase activity, wherein the endogenous acyltransferase activity has been reduced or abolished (description p.26, claim 4).
Balch does not teach the host cell further comprising reduction or abolishment of one or more genes expressing lipase activities selected from Yarrowia LIP2.
However, Fickers teaches Yarrowia lipolytica contains LIP2 gene which codes for the extracellular lipase Lip2p, and additionally genes LIP7 and LIP8 genes encoding two triacylglycerol hydrolases (p.265, 1st column 1st paragraph and 2nd column top paragraph). Fickers teaches that triacylglycerol hydrolases or lipases (EC 3.1.1.3) are able to catalyse ester bond hydrolysis in acylglycerol (p.264, 1st column, 1st paragraph). Fickers teaches developing mutants lacking one or several lipase genes (p.265, 2nd column top paragraph). Fickers teaches a lack of measurable lipase activity in the culture supernatants of the triple mutant strain, which lacks Lip2p, Lip7p and Lip8p lipases (i.e. reduction/abolishment/inactivation accomplished by deletion) (p.273, 1st column, last paragraph). Fickers further teaches that comparison of cell growth and lipase production in the deleted strain clearly demonstrates that, together with Lip2p, Lip8p is the major secreted lipase in Y. lipolytica (p.273, 2nd column, 2nd paragraph).
Grumet teaches lysosomal acid lipase (LAL) hydrolyzes retinyl ester and affects retinoid turnover (title). Grumet teaches that murine LAL (mLAL) hydrolyzes retinyl esters (REs), and pharmacological inhibition of LAL in the human hepatocyte cell line HepG2 led to an accumulation of REs in endosomal/lysosomal-enriched fractions, suggesting that LAL is limiting for the clearance of endocytosed REs (p.17977, 2nd column last paragraph).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to delete both LIP8 and LIP2 genes for lipase, as taught by Fickers, in the Yarrowia lipolytica host cell taught by Balch, because Grumet teaches that lysosomal acid lipase is known to hydrolyze retinyl esters and affect retinoid turnover. One of ordinary skill in the art would have found it beneficial to inhibit lipase activity and further abolish the function of LIP2 gene in the Yarrowia lipolytica host to prevent the hydrolysis of retinyl esters.
Regarding claim 6, Balch teaches a carotenoid-producing host cell, particularly fungal host cell, as above and defined herein, wherein the acetyl transferase [EC 2.3.1.84], preferably an enzyme with acetyl transferase 1 activity, catalyzes the conversion of retinal to a retinyl acetate mix, wherein the mix comprises up to 100% retinyl acetate in trans-isoform (description p.16, lines 15-19). Balch further teaches said host cell being capable or used for production of a retinyl acetate mix comprising at least about 65% in trans-isoform compared to a host cell expressing the respective endogenous acyltransferases prior to the modification of the host cell, i.e. wherein the endogenous acyltransferases are still active, and the percentage of trans-isoforms can be increased to 70, 75, 80, 85, 90, 95, 98 or up to 100% based on the total amount of retinyl esters (description p.7, lines 23-27; p.26 claims 1-2, 4, and 6-8).
Regarding claim 7, Balch teaches using carbon source 5% corn oil in mineral oil (relevant to vegetable oil as carbon source) (description p.19, line9-10).Balch teaches said when using said host cell in a vitamin A production process, the percentage of trans-isoforms, such as trans-retinyl acetate, can be increased to about 65% or more, preferably up to 100% based on the total amount of retinyl esters (description p.7, lines 27-30; p.26 claims 1-2, 4 and 6-8 ).
Response to Arguments
Applicant argues that Balch’s disclosure is silent on modification of certain lipase activities in the host cell in order to increase the percentage of retinyl acetate and decrease the conversion of retinol into unwanted long-chain retinyl esters (See Remarks dated 11/20/2025, p.6 last paragraph). Applicant argues that as shown in Examples of the present specification, deletion of Lip8 in Yarrowia resulted in 70% retinyl acetate as compared to “not detectable” retinyl acetate levels in a strain wherein the lipase genes, particularly Lip8, is still active (See Remarks dated 11/20/2025, p.6 last paragraph). Applicant argues that this level of retinyl acetate could be increased via introduction of additional deletions (e.g. deletions of Lip2, Lip3 and Lip4) (See remarks dated 11/20/2025, p.6 last paragraph). Applicant argues these modifications are not taught or suggested in Balch, nor are they rendered obvious by combining Balch with Fickers and/or Grumet; and neither Fickers nor Grumet specifically refer to deletion of Lip8 in order to boost retinyl acetate production (See Remarks dated 11/20/2025, p.7, top paragraph).
Applicant's arguments filed November 20, 2025 have been fully considered but they are not persuasive. As discussed in the rejection above, Balch teaches a Yarrowia fungal host cell, and teaches a modification in the endogenous acyltransferase (i.e. lipase) activity, leading to reduction or abolishment of said endogenous acyltransferase activity. Fickers a mutant where Lip8p and Lip2 genes have been deleted, resulting in decreased lipase activity. Grumet teaches that lysosomal acid lipase is known to hydrolyze retinyl esters and affect retinoid turnover, and pharmacological inhibition of LAL (lipase) in the human hepatocyte cell line HepG2 led to an accumulation of REs (retinyl esters) in endosomal/lysosomal-enriched fractions, suggesting that LAL is limiting for the clearance of endocytosed REs. Thus, one of ordinary skill in the art would have been motivated to modify the host cell taught by Balch by deleting Lip2 and Lip8p genes taught by Fickers because Grumet teaches that lipase affects retinoid turnover. One of ordinary skill in the art would have found it beneficial to abolish Lip8p activity to decrease retinoid turnover, because Fickers teaches that Lip8p is the main source of lipase activity in Yarrowia and abolishing Lip8p would thereby increase retinoid accumulation in the cell.
Regarding applicant’s argument that references do not teach the deletion of Lip8 in order to boost retinyl acetate production: Balch teaches a reduction or abolishment of the endogenous genes encoding acyltransferase activity. Balch further teaches when using said host cell in a vitamin A production process, the percentage of trans-isoforms, such as trans-retinyl acetate, can be increased to about 65% or more, preferably up to 100% based on the total amount of retinyl esters. Fickers teaches deleting Lip8p. The boosting of production of retinyl acetate is a direct result of deleting Lip8, which is taught by Fickers. Thus, Balch in view of Fickers would produce a genetically modified host where Lip8p is deleted, resulting in the desired effect of increased retinyl acetate production.
Conclusion
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.
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/DEEPA MISHRA/Examiner, Art Unit 1657
/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657