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 .
Status of Application, Amendments, And/Or Claims
The Applicants amendments/remarks received 1/5/2026 are acknowledged. Claims 1-2, 4-7, 12-14, 16, 21, 26 and 30 are amended; claims 3, 8-9, 15, 17-18, 20, 22, 24-25 and 27-29 are canceled; no claims are withdrawn; claims 1-2, 4-7, 10-14, 16, 19, 21, 23, 26 and 30 are pending and have been examined on the merits.
Drawings
Replacement sheets for the drawings have been received 1/5/2026. Applicant has correctly amended “FIGURE” to “Fig.”; however, inexplicably, the sheet numbers have been removed.
The drawings are objected to for the following reason:
37 CFR 1.84 (t) states " Numbering of sheets of drawings. The sheets of drawings should be numbered in consecutive Arabic numerals, starting with 1, within the sight as defined in paragraph (g) of this section. These numbers, if present, must be placed in the middle of the top of the sheet, but not in the margin. The numbers can be placed on the right-hand side if the drawing extends too close to the middle of the top edge of the usable surface. The drawing sheet numbering must be clear and larger than the numbers used as reference characters to avoid confusion. The number of each sheet should be shown by two Arabic numerals placed on either side of an oblique line, with the first being the sheet number and the second being the total number of sheets of drawings, with no other marking.”
The sheets were correctly numbered with “1/8” through “8/8” at the middle top of the sheet on the original drawings received 12/8/2022. Please restore the sheet numberings on the replacement sheets. If limited space was the reason for removal of the sheet numbers, one could put the figure indications under the figures instead of above the figures as they are currently.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Rejections - 35 USC § 112
The rejection of claims 26 and 30 under 35 U.S.C. § 112(b), as set forth at p. 5 of the previous Office Action, is withdrawn in view of the amendment of the claims.
Claim Rejections - 35 USC § 102
The rejection of claims 1-7, 10-14, 16, 19, 21, 23 and 26 under 35 U.S.C. § 102(a)(1)/102(a)(2) over Knight et al., WO 2020/030799 (cite N, PTO-892, 9/5/2025; herein “Knight”) as set forth at pp. 5-8 of the previous Office Action, is moot regarding claim 3 due to cancelation of the claim and is withdrawn regarding the remaining claims in view of the amendment of the claims.
Claim Rejections - 35 USC § 103
The rejection of claims 1-7, 10-14, 16, 19, 21, 23, 26 and 30 under 35 U.S.C. § 103 over Knight in view of Tsuji et al., 1980 (NPL cite 30, IDS, 12/20/2023; herein “Tsuji”), Jiang et al., CN112375117 (Foreign patent cite 24, IDS, 12/20/2023; herein “Jiang”) and Sawada et al., US 4579819 (US Patent cite 3, IDS, 12/20/2023; herein “Sawada”) as set forth at pp. 8-10 of the previous Office Action, is moot regarding claim 3 due to cancelation of the claim and is withdrawn regarding the remaining claims in view of the amendment of the claims.
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, 4-7, 10-14, 16, 19, 21, 23 and 26 are rejected under 35 U.S.C. 103 as being unpatentable over Knight et al., WO 2020/030799 (cite N, PTO-892, 9/5/2025; herein “Knight” in view of Ruprecht et al., 2015 (cite U, attached PTO-892; herein “Ruprecht”) in light of “Alignment of Knight’s fadE34#2 (Query) to Ruprecht’s R. jostii RHA1 CasC (Sbjct)” (cite V, attached PTO-892) and “Alignment of Knight’s fadE34#2 (Query) to Knight’s fadE34 (Sbjct)” (cite W, attached PTO-892).
Knight is drawn to producing genetically modified bacteria for bioconversion of steroidal substrates to a steroidal product of interest (Abst.), i.e., non-naturally occurring steroid degrading microbial organisms, wherein the non-naturally-occurring steroid-degrading microbial organism can be a Rhodococcus sp. or a Mycobacterium sp. (p. 1, ll. 1-3), more specifically, the non-naturally occurring steroid degrading microbial organism can be derived from Rhodococcus rhodochrous, Rhodococcus jostii, Mycobacterium smegmatis or Mycobacterium neoaurum (p. 14, ll. 13-19). Knight discloses that Rhodococcus sp. and Mycobacterium sp. degrade sterol compounds (p. 1, ll. 30-34) and that genetic modification to block the degradation pathway prior to the breakdown of the polycyclic ring system and at various points in the side-chain oxidation of the steroid allows the accumulation of the steroid product of interest (p. 2, ll. 3-7). Knight teaches that the genetic modifications to allow the accumulation of the steroid product of interest can comprise inactivation of one or more of fadE34 (amino acid sequence is Knight SEQ ID NO: 12), fadE34#2 (amino acid sequence is Knight SEQ ID NO: 22), or homologs thereof (p. 16, ll. 1-3), wherein the homolog comprises at least 50% amino acid sequence identity (p. 16, ll. 23-31) and inactivation of kshA1 (Knight SEQ ID NO: 1), kshA2 (Knight SEQ ID NO: 2), kshA3 (Knight SEQ ID NO: 3), and kshA4 (Knight SEQ ID NO: 4) (p. 15, ll. 29-31). Inactivation of kshA1, kshA2, kshA3 and kshA4 disrupts aerobic 9,10-seco degradation activity and steroid 9-alpha-hydroxylation activity because kshA1-5 are 3-ketosteroid-9-alpha-hydroxylases. Inactivation of fadE34, fadE34#2 and/or homologs thereof disrupts 4-(steroid-17-yl)-pentanoyl side chain degradation activity because fadE34 and fadE34#2 are acyl CoA dehydrogenases (ACADs) with pentanoyl side chain cleavage activity.
Disruption of FadE34 in Rhodococcus jostii would comprise disrupting the expression sequence at RHA1_RS21845 because FadE34 is at RHA1_RS21845 in Rhodococcus jostii (specification, p. 20). Disruption of FadE34 in Mycobacterium smegmatis would comprise disrupting the expression sequence at MSMEG_6041 because FadE34 is at MSMEG_6041 in Mycobacterium smegmatis (specification, p. 31). Disruption of FadE34 in Mycobacterium neoaurum would comprise disrupting the expression sequence at MyAD_RS24250 because FadE34 is at MyAD_RS24250 in Mycobacterium neoaurum (specification, p. 34).
Knight does not specifically disclose that fadE34#2 comprises CasC activity; however, it would appear that fadE34#2 is a CasC homolog rather than an FadE34 homolog because Knight’s fadE34#2 is more closely related to Rhodococcus jostii RHA1 CasC (amino acid sequence in Fig. S3 of Ruprecht; R. jostii RHA1 CasC is 99% identical to instant SEQ ID NO: 76), wherein the amino acid sequence identity between fadE34#2 and Rhodococcus jostii RHA1 CasC is 74% (see “Alignment of Knight’s fadE34#2 (Query) to Ruprecht’s R. jostii RHA1 CasC (Sbjct)”) while the amino acid sequence identity between Knight’s fadE34 and Knight’s fadE34#2 is only 47% (see “Alignment of Knight’s fadE34#2 (Query) to Knight’s fadE34 (Sbjct)”).
However, a person of ordinary skill in the art at the time of filing would have found it obvious to practice the production of non-naturally occurring steroid degrading microbial organisms taught by Knight wherein the disruption of 4-(steroid-17-yl)-pentanoyl side chain degradation activity comprises the disruption of CasC activity in view of the disclosure of Ruprecht.
Ruprecht discloses that Rhodococcus (R. jostii RHA1 specifically) FadE34 and CasC are both pentanoyl side chain cleavage enzymes with a specificity for 5-carbon (i.e., pentanoyl) side chains and are inactive toward 3-carbon side chains (Abst.; p. 1362, ¶1). Ruprecht discloses that CasC has a broader substrate specificity than FadE34 wherein CasC has a similar specificity for cholyl-CoA, deoxycholyl-CoA, and 3β-hydroxy-5-cholen-24-oyl-CoA, while FadE34 has specificity constants at least 2 orders of magnitude higher for 3β-hydroxy-5-cholen-24-oyl-CoA than for cholyl-CoA or deoxycholyl-CoA (p. 1363, “Specificities of FadE34, CasC, and FadE28-FadE29.”) Thus, a person of ordinary skill in the art at the time of filing would have found it obvious that CasC should also be inactivated to further disrupt 4-(steroid-17-yl)-pentanoyl side chain degradation activity.
Hence, a person of ordinary skill in the art at the time of filing would have found it obvious to produce non-naturally occurring steroid degrading microbial organisms as taught by Knight, which have disrupted aerobic 9,10-seco degradation activity through the inactivation of kshA1-4 and disrupted 4-(steroid-17-yl)-pentanoyl side chain degradation activity through the inactivation of fadE34 and fadE34#2, wherein the Rhodococcus CasC gene, as disclosed by Ruprecht, is also inactivated because Knight teaches the inactivation of fadE34#2 or homologs with at least 50% amino acid identity to fadE34#2, Rhodococcus CasC is 74% identical to fadE34#2 and Ruprecht discloses that Rhodococcus CasC has the pentanoyl side chain cleavage activity of FadE34 as well as activity toward cholyl-CoA and deoxycholyl-CoA; therefore, claims 1-2, 5-7, 10, 12, 14, 19, 21 and 23 are prima facie obvious.
Knight discloses that the genetic modifications to allow the accumulation of the steroid product of interest can comprise inactivation of kstD1, kstD2 and kstD3, which are 3-ketosteroid delta-1 dehydrogenases (p. 15, ll. 33-35; p. 37, ll. 9-10) and Knight discloses that the microorganism can be Rhodococcus rhodochrous, Rhodococcus jostii, Mycobacterium smegmatis or Mycobacterium neoaurum (p. 14, ll. 13-19); hence, a person of ordinary skill in the art at the time of filing would have found it obvious for the non-naturally occurring microbial organism to be derived from the genus Mycobacterium further comprising disrupted KstD enzymatic activity; therefore, claims 5 and 13 are prima facie obvious. Disruption of KstD in Rhodococcus jostii would comprise disrupting the expression sequence at RHA1_RS22090 because KstD is at RHA1_RS22090 in Rhodococcus jostii (specification, p. 23); therefore, claim 11 is prima facie obvious.
Knight discloses a method of making a steroid comprising growing Rhodococcus rhodochrous strain LM9 (LM9 is ΔFadE34 ΔFadE34#2 ΔKshA1 ΔKshA2 ΔKshA3 ΔKshA4 ΔKshA5 ΔKstD1 ΔKstD2 ΔKstD3 wherein “Δ” means the gene is deleted/inactivated; pp. 37-40; p. 69) on β-sitosterol (i.e., one or more phytosterols) to produce KCEA (p. 40; Fig. 8B); hence, a person of ordinary skill in the art at the time of filing would have found it obvious to practice a method of making a steroid comprising growing the non-naturally occurring microorganism made obvious by Knight in view of Ruprecht which comprise disrupted aerobic 9,10-seco degradation activity and disrupted CasC activity on one or more phytosterols to produce 3-ketochol-4-enoic acid (KCEA); therefore, claim 26 is prima facie obvious.
Regarding claim 4, Knight teaches adding back kshA5, a 3-ketosteroid-9-alpha-hydroxylase, into a R. rhodochrous strain (LM19 derived from RG41 derived from RG32; pp. 40-41, Example 4; pp. 37-38, Example 1) wherein kshA1-5 have been inactivated, in order to produce variant steroid products comprising a 9-hydroxyl group (pp. 40-41, Example 4); hence, a person of ordinary skill in the art at the time of filing would have found it obvious to produce the non-naturally occurring steroid degrading microbial organism made obvious by Knight in view of Ruprecht comprising disrupted aerobic 9, 10-seco degradation activity and disrupted CasC and FadE34 activity further comprising one or more 3-ketosteroid-9-alpha-hydroxylase isoenzymes because Knight demonstrates that adding 3-ketosteroid-9-alpha-hydroxylase activity allows the non-naturally occurring steroid degrading microbial organism to produce wanted variant steroid products; therefore, claim 4 is prima facie obvious.
Regarding claim 16, Knight does not specifically teach inactivating ChsE1, i.e., FadE28, and ChsE2, i.e., FadE29; however, a person of ordinary skill in the art at the time of filing would have found it obvious for the non-naturally occurring steroid degrading microbial organism produced by Knight to further comprise inactivation of FadE28 and FadE29 in view of the disclosure of Ruprecht.
Ruprecht, as disclosed above, is drawn to characterizing ACADs involved in bacterial steroid degradation. Ruprecht teaches that FadE34 and CasC are both unusual ACADs in that they contain two fused acyl-CoA dehydrogenase domains in a single polypeptide wherein one ACAD domain (C-terminal) has the catalytic glutamate base required for enzyme activity while the other ACAD domain (N-terminal) contains an arginine required for ionic interactions with the pyrophosphate of the flavin adenine dinucleotide (FAD) cofactor (Abst.) Ruprecht discloses that the N-terminal domains of FadE34 and CasC are similar to FadE28 while the C-terminal domains of FadE34 and CasC are similar to FadE29 (pp. 1365-1366, “Discussion”, ¶1-2). Ruprecht discloses that FadE28/29 is active toward cholesterol metabolites containing a 3-carbon side chain (pp. 1365-1366, “Discussion”, ¶1-2).
Hence, a person of ordinary skill in the art at the time of filing would have found it obvious to produce the non-naturally occurring steroid degrading microbial organism comprising disrupted aerobic 9, 10-seco degradation activity and disrupted CasC and FadE34 activity made obvious by Knight in view of Ruprecht further comprising inactivated FadE28 and FadE29 in order to prevent degradation of cholesterol metabolites containing a 3-carbon side chain; therefore, claim 16 is prima facie obvious.
Response to Arguments
Applicant's arguments filed 1/5/2026 have been fully considered but they are not persuasive. Arguments of the Applicant’s Response on pp. 7-8 regarding the rejections under 35 U.S.C. §§ 112(b), 102(a)(1)/102(a)(2) and 103 are moot as the rejections have been withdrawn. The rejection set forth above fully addresses all limitations of amended claims 1-2, 4-7, 10-14, 16, 19, 21, 23 and 26.
Claims 1-2, 4-7, 10-14, 16, 19, 21, 23, 26 and 30 are rejected under 35 U.S.C. 103 as being unpatentable over Knight in view of Ruprecht, Tsuji et al., 1980 (NPL cite 30, IDS, 12/20/2023; herein “Tsuji”), Jiang et al., CN112375117 (Foreign patent cite 24, IDS, 12/20/2023; herein “Jiang”) and Sawada et al., US 4579819 (US Patent cite 3, IDS, 12/20/2023; herein “Sawada”).
The discussion of Knight and Ruprecht regarding claims 1-2, 4-7, 10-14, 16, 19, 21, 23 and 26 set forth in the rejection above is incorporated herein.
Knight does not teach that the method of producing KCEA and/or KCDA further comprises converting the KCEA or KCDA to ursodeoxycholic acid (UCDA). However, a person of ordinary skill in the art at the time of filing would have found it obvious for the method to comprise converting the KCEA or KCDA to UCDA in view of the disclosures of Tsuji, Jiang and Sawada.
Sawada teaches that UCDA is a clinically valuable reagent because it is effective at solubilizing cholesterol gallstones when orally administered to a patient (col. 1, ll. 35-40). Hence, a person of ordinary skill in the art at the time of filing would have found it obvious to produce UCDA because it is an effective drug.
Tsuji teaches the catalytic hydrogenation of 3-oxo-4-ene and -1,4-diene steroids to 5β steroids (Title; p. 2729, ¶1); hence, a person of ordinary skill in the art at the time of filing would have found it obvious to convert KCEA or KCDA to 3-keto-5β-cholanic acid with the method of Tsuji.
Jiang teaches methods for hydrogenating (i.e., reducing) 3-ketosteroids to lithocholic acid (p. 2 of English translation, “Summary of the invention”); hence, a person of ordinary skill in the art at the time of filing would have found it obvious to hydrogenate the 3-keto-5β-cholanic acid produced from KCEA or KCDA in the method of Tsuji to produce lithocholic acid with the method of Jiang.
Sawada teaches producing UCDA from lithocholic acid (Abst.); hence, a person of ordinary skill in the art at the time of filing would have found it obvious to produce UCDA from the lithocholic acid produced by the method of Jiang.
Hence, a person of ordinary skill in the art at the time of filing would have found it obvious to use the combined methods of Tsuji, Jiang and Sawada to produce UDCA from the KCEA and KCDA because UCDA is a clinically valuable drug; therefore, claim 30 is prima facie obvious.
Response to Arguments
Regarding the rejection of claims 1-7, 10-14, 16, 19, 21, 23, 26 and 30 under 35 U.S.C. 103 over Knight in view of Tsuji, Jiang and Sawada, Applicant argues that none of the cited art teaches or suggests knocking out CasC activity (p. 8). The rejection set forth above fully addresses all limitations of amended claims 1-2, 4-7, 10-14, 16, 19, 21, 23, 26 and 30.
Conclusion
No claims are allowed.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Trent R Clarke whose telephone number is (571)272-2904. The examiner can normally be reached M-F 10-7 MST.
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/TRENT R CLARKE/Examiner, Art Unit 1651
/DAVID W BERKE-SCHLESSEL/Primary Examiner, Art Unit 1651
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