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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on March 10, 2026 has been entered.
DETAILED ACTION
Claims 2, 8 and 13 are cancelled. Claims 18-20 are new.
Claims 1, 3-7, 9-12 and 14-20 are pending and under examination.
Priority
This application is a CON of PCT/JP2018/036040 filed on September 27, 2018, which claims foreign benefit to JAPAN 2017-190028 filed on September 9, 2017.
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
The effective filing date of the current application is September 9, 2017.
Claim Objections
Claims 1 and 18-20 are objected to because of the following informalities:
Claim 1 is missing a colon after the phrase “selected from the group consisting of” in line 4, which should be amended to recite “selected from the group consisting of:”.
Claim 18 recites “the method for producing NMN according to claim 1, wherein contacting ribose-5- phosphate (R5P), nicotinamide (NAM), ATP and polyphosphate with (A) the transformant with enhanced expression of three enzymes: nicotinamide phosphoribosyltransferase (Nampt); phosphoribosyl pyrophosphate synthetase (Prs); and polyphosphate kinase (Ppk); or a combination of two or three transformants in which each of the two or three transformants individually enhances the expression of one or two enzymes among the three enzymes”, which is ungrammatical.
Claim 19 recites “the method for producing NMN according to claim 1, wherein contacting ribose-5- phosphate (R5P), nicotinamide (NAM), ATP and polyphosphate with (B) the cell-free protein synthesis reaction solution comprising the three enzymes”, which is ungrammatical.
Claim 20 recites “the method for producing NMN according to claim 1, wherein contacting ribose-5- phosphate (R5P), nicotinamide (NAM), ATP and polyphosphate with (C) the treated product of either the transformant, the combination of the two or three transformants, or the cell-free protein synthesis reaction solution”, which is ungrammatical.
Appropriate correction is required.
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.
Claims 18-20 are rejected under 35 U.S.C. 112(b) as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor regards as the invention.
Claim 18 recites “the method for producing NMN according to claim 1, wherein contacting ribose-5- phosphate (R5P), nicotinamide (NAM), ATP and polyphosphate with (A) the transformant with enhanced expression of three enzymes: nicotinamide phosphoribosyltransferase (Nampt); phosphoribosyl pyrophosphate synthetase (Prs); and polyphosphate kinase (Ppk); or a combination of two or three transformants in which each of the two or three transformants individually enhances the expression of one or two enzymes among the three enzymes”, which is an incomplete sentence. It is unclear what operative step is required by the wherein clause.
Claim 19 recites “the method for producing NMN according to claim 1, wherein contacting ribose-5- phosphate (R5P), nicotinamide (NAM), ATP and polyphosphate with (B) the cell-free protein synthesis reaction solution comprising the three enzymes”, which is an incomplete sentence. It is unclear what operative step is required by the wherein clause.
Claim 20 recites “the method for producing NMN according to claim 1, wherein contacting ribose-5- phosphate (R5P), nicotinamide (NAM), ATP and polyphosphate with (C) the treated product of either the transformant, the combination of the two or three transformants, or the cell-free protein synthesis reaction solution”, which is an incomplete sentence. It is unclear what operative step is required by the wherein clause.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 18-20 are rejected under 35 U.S.C. 112(d) as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 18 recites “the method for producing NMN according to claim 1, wherein contacting ribose-5- phosphate (R5P), nicotinamide (NAM), ATP and polyphosphate with (A) the transformant with enhanced expression of three enzymes: nicotinamide phosphoribosyltransferase (Nampt); phosphoribosyl pyrophosphate synthetase (Prs); and polyphosphate kinase (Ppk); or a combination of two or three transformants in which each of the two or three transformants individually enhances the expression of one or two enzymes among the three enzymes”. Claim 18 depends from claim 1, which recites contacting ribose-5- phosphate (R5P), nicotinamide (NAM), ATP and polyphosphate with at least one selected from the group consisting of (A) a transformant with enhanced expression of three enzymes: nicotinamide phosphoribosyltransferase (Nampt); phosphoribosyl pyrophosphate synthetase (Prs); and polyphosphate kinase (Ppk); or a combination of two or three transformants in which each of the two or three transformants individually enhances the expression of one or two enzymes among the three enzymes; (B) a cell-free protein synthesis reaction solution comprising the tree enzymes; and (C) a treated product of either the transformant, the combination of the two or three transformants, or the cell-free protein synthesis reaction solution, in a reaction system”. Thus, claim 18 does not further limit claim 1, and therefore is an improper dependent claim.
Claim 19 recites “the method for producing NMN according to claim 1, wherein contacting ribose-5- phosphate (R5P), nicotinamide (NAM), ATP and polyphosphate with (B) the cell-free protein synthesis reaction solution comprising the three enzymes”. Claim 19 depends from claim 1, which recites contacting ribose-5- phosphate (R5P), nicotinamide (NAM), ATP and polyphosphate with at least one selected from the group consisting of (A) a transformant with enhanced expression of three enzymes: nicotinamide phosphoribosyltransferase (Nampt); phosphoribosyl pyrophosphate synthetase (Prs); and polyphosphate kinase (Ppk); or a combination of two or three transformants in which each of the two or three transformants individually enhances the expression of one or two enzymes among the three enzymes; (B) a cell-free protein synthesis reaction solution comprising the tree enzymes; and (C) a treated product of either the transformant, the combination of the two or three transformants, or the cell-free protein synthesis reaction solution, in a reaction system”. Thus, claim 19 does not further limit claim 1, and therefore is an improper dependent claim.
Claim 20 recites “the method for producing NMN according to claim 1, wherein contacting ribose-5- phosphate (R5P), nicotinamide (NAM), ATP and polyphosphate with (C) the treated product of either the transformant, the combination of the two or three transformants, or the cell-free protein synthesis reaction solution”. Claim 20 depends from claim 1, which recites contacting ribose-5- phosphate (R5P), nicotinamide (NAM), ATP and polyphosphate with at least one selected from the group consisting of (A) a transformant with enhanced expression of three enzymes: nicotinamide phosphoribosyltransferase (Nampt); phosphoribosyl pyrophosphate synthetase (Prs); and polyphosphate kinase (Ppk); or a combination of two or three transformants in which each of the two or three transformants individually enhances the expression of one or two enzymes among the three enzymes; (B) a cell-free protein synthesis reaction solution comprising the tree enzymes; and (C) a treated product of either the transformant, the combination of the two or three transformants, or the cell-free protein synthesis reaction solution, in a reaction system”. Thus, claim 20 does not further limit claim 1, and therefore is an improper dependent claim.
Applicant may cancel the claims, amend the claims to place the claims in proper dependent form, rewrite the claims in independent form, or present a sufficient showing that the dependent claims complies with the statutory requirements.
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.
Claims 1, 5-7 and 18-19 are rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (WO 2016/198948 A1, published on December 15, 2016; previously cited) in view of Kuroda et al. (JP 2013-21967, published February 4, 2013; previously cited) and Gross et al. (“Practical Synthesis of 5-Phospho-ᴅ-ribosyl-α-1-Pyrophosphate (PRPP): Enzymatic Routes from Ribose-5-Phosphate or Ribose, Journal of the American Chemical Society, 1983, vol. 105, no. 25, pp. 7428-7435; previously cited), Whitesides et al. (US 4,411,995, issued on October 25, 1983; previously cited) and Burgos et al. (“Weak Coupling of ATP Hydrolysis to the Chemical Equilibrium of Human Nicotinamide Phosphoribosyltransferase”, Biochemistry, 2008, Vol. 47, Issue 42, pp.11086-11096; previously cited).
Claim 18 is interpreted to require contacting ribose-5-phosphate (R5P), nicotinamide (NAM), ATP and polyphosphate with (A) a transformant with enhanced expression of three enzymes: nicotinamide phosphoribosyltransferase (Nampt); phosphoribosyl pyrophosphate synthetase (Prs); and polyphosphate kinase (Ppk).
Regarding claims 1 and 18, Wu teaches enzymatic systems and methods for synthesizing nicotinamide mononucleotide and nicotinic acid mononucleotide (relevant to a method for producing nicotinamide mononucleotide (NMN)) (title). Wu teaches that the system utilizes a mutated form of phosphoribosylpyrophosphate synthetase (Prs) that is rendered insensitive to its own reaction product, thus increasing its activity, and one or more other enzymes or enzyme combinations that are bound to a solid surface (relevant to (A) a transformant with enhanced expression of phosphoribosyl pyrophosphate synthetase (Prs)) (description p.2, paragraph [0007]). Wu teaches the one or more enzymes may be produced by recombinant means in one or more cells including yeast and bacteria (relevant to a transformant or a combination of transformants with enhanced expression) (description p.11, paragraph [0079]). Wu teaches that in some embodiments, the system further includes nicotinamide phosphoribosyltransferase (NAMPT) (description p.3, paragraph [0013]). Wu further teaches the system further includes adenosine triphosphate (ATP), ribose-5-phosphate, and nicotinamide (relevant to contacting ribose-5-phosphate (R5P), nicotinamide (NAM) and ATP with a transformant; relevant to wherein at least ATP is added to the reaction system) (description p.3, paragraphs [0017]-[0019]. Wu teaches the system can further include phosphoribosyl pyrophosphate (PRPP) (description p.3, paragraph [0020]).
Wu teaches that the method includes the step of contacting ribose-5-phosphate with a superactive PRS mutant in the presence of ATP, whereby PRPP is produced (relevant to a reaction with Prs to generate PRPP and AMP from R5P and ATP) (description p.3, paragraph [0022]).
Wu teaches the method further includes the steps of (a) contacting the resulting PRPP with NAMPT in the presence of nicotinamide, whereby nicotinamide mononucleotide NMN is produced (relevant to a reaction with Nampt to generate NMN from PRPP and NAM) (description p.4, paragraph [0028]).
Wu does not teach contacting polyphosphate with a transformant; a transformant with enhanced expression of polyphosphate kinase (Ppk); or an ATP regeneration reaction with Ppk to generate ATP from AMP and polyphosphate.
Kuroda teaches a method of producing ATP from AMP that includes the step of reacting polyphosphate kinase (Ppk) type 2 with polyphosphate and AMP to synthesize ATP (relevant to contacting polyphosphate; relevant to an ATP regeneration reaction with Ppk to generate ATP from AMP and polyphosphate) (Abstract; claim 1; description paragraphs [0002], [0004]). Kuroda further indicates that ATP can easily be produced from AMP (relevant to wherein at least AMP is added to the reaction system) (paragraph [0027]).
Gross teaches a method of producing phosphoribosyl pyrophosphate (PRPP) from ribose or ribose-5-phosphate (R5P) using phosphoribosyl pyrophosphate synthetase (Prs) (see abstract, scheme IV). Gross further teaches that when ribose is used as the starting material, a ribokinase (Rbk) is used to synthesize ribose-5-phosphate (R5P) (see abstract, scheme IV). Gross also teaches the use of an ATP regeneration system using pyruvate kinase (see abstract, scheme IV). Gross further teaches that PRPP is too expensive to be used in practical synthesis, which is partially due to its intrinsic instability (abstract).
Wu, Kuroda and Gross do not teach wherein the total sum of the respective moles of ATP, ADP and AMP to be added to the reaction system is 0.5 equivalents or less of the number of moles of NMN to be generated and wherein the total sum of the respective numbers of moles of ATP, ADP and AMP to be added to the reaction system is 0.5 equivalents or less of the number of moles of PRPP or NAM to be added to the reaction system at the start of the reaction.
While Whitesides does not specifically disclose a total sum of the respective numbers of moles of ATP, ADP and AMP, Whitesides does teach that the starting amounts of ATP, ADP or AMP can be varied to regenerate ATP from AMP or ADP, and coupled ATP recycling can avoid waste of adenosine moieties and reduce overall expense (specification; column 3, lines 4-14).
Burgos teaches human nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the reversible synthesis of nicotinamide mononucleotide (NMN) and inorganic pyrophosphate (PPi) from nicotinamide (NAM) and α-D-5-phosphoribosyl-1-pyrophosphate (PRPP) (abstract). Burgos teaches that with ATP hydrolysis coupled to NMN synthesis, the catalytic efficiency of the system is improved 1100-fold, substrate affinity dramatically increases and the Keq shifts -2.1 kcal/mol toward NMN formation (abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to incorporate polyphosphate kinase and polyphosphate taught by Kuroda into the method of Wu to generate PRPP, because Gross teaches that PRPP is a critical intermediate that can be generated from less complex bulk chemicals such as ribose-5-phosphate using ribokinase and ATP. One of ordinary skill in the art would have found it beneficial to do so because Kuroda teaches that integrating a method of generating ATP using polyphosphate kinase and polyphosphate is an inexpensive regeneration route for generating ATP from ADP and AMP.
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the instant application to optimize the ratio of total moles of ATP, ADP or AMP to the number of moles of NAM to be added to the reaction system at the start of the reaction to be less than or equal to 0.5 equivalents to achieve the result of the number of moles of NMN to be generated to be 2 equivalents or more of the total sum of the respective numbers of moles of the added ATP, ADP and AMP to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reasonable expectation of success that varying the amounts of ATP, ADP or AMP relative to the number of moles of NAM would result in different numbers of moles of NMN to obtain an optimal ratio of less than or equal to 0.5 with predictable results, because Whitesides teaches that small catalytic amounts of ATP can used in coupled ATP recycling, and Burgos teaches that ATP hydrolysis coupled to NMN synthesis improved the catalytic efficiency of the system 1100-fold. “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature 100°C and an acid concentration of 10%). See MPEP §2144.05.
Regarding claim 5, Wu teaches a method of synthesizing nicotinamide mononucleotide (NMN) by contacting nicotinamide riboside kinase immobilized onto a surface with nicotinamide riboside in the presence of adenosine triphosphate (ATP) whereby NMN is produced (description p. 5, paragraph [0043]).
Wu does not teach a transformant with enhanced expression of polyphosphate kinase (Ppk), wherein the Ppk belongs to the polyphosphate kinase type 2 family.
Kuroda teaches the use of polyphosphate kinase in the regeneration of ATP from ADP (abstract, description paragraphs [0002] and [0004]). Kuroda further teaches that the polyphosphate kinase belongs to the polyphosphate kinase type 2 family (relevant to wherein the Ppk belongs to the polyphosphate kinase type 2 family) (abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to incorporate polyphosphate kinase type 2 taught by Kuroda into the method of Wu, in order to add a step of regenerating ATP from ADP as taught by Kuroda in the production of NMN taught by Wu, which requires ATP. One of ordinary skill in the art would have been motivated to do so because R5P is required to generate PRPP, which is a critical intermediate for producing NMN. One of ordinary skill in the art would have found it beneficial to combine the individual reactions to produce intermediate products that could be used in subsequent reactions to create an efficient method of producing the desired end product of NMN.
Regarding claim 6, Wu teaches the one or more enzymes may be produced by recombinant means in one or more host cells including yeast and bacteria (relevant to wherein a host of the transformant is yeast) (description p.11, paragraph [0079]).
Regarding claim 7, Wu teaches that the system utilizes a mutated form of phosphoribosylpyrophosphate synthetase (Prs) that is rendered insensitive to its own reaction product, thus increasing its activity, and one or more other enzymes or enzyme combinations that are bound to a solid surface (description p.2, paragraph [0007]). Wu teaches that in some embodiments, the system further includes nicotinamide phosphoribosyltransferase (NAMPT) (description p.3, paragraph [0013]).
Kuroda teaches the use of polyphosphate kinase (Ppk) (abstract, description paragraphs [0002] and [0004]).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the instant application to additionally transform the cell expressing Nampt and Prs from Wu with a gene expressing polyphosphate kinase (Ppk), as taught by Kuroda. One of ordinary skill in the art would have been motivated to modify the host cell which express Nampt and Prs from Wu to additionally express Ppk with a reasonable expectation of success, because each of those enzymes is part of the nicotinamide mononucleotide synthesis pathway. One of ordinary skill in the art would reasonably expect that combining one known element (Ppk) with other known elements (Nampt and Prs) would predictably result in the expression of all three enzymes.
Claim 19 is interpreted as requiring contacting of ribose-5-phosphate (R5P), nicotinamide (NAM), ATP and polyphosphate with (B) a cell-free protein synthesis reaction solution comprising the three enzymes.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to further immobilize polyphosphate kinase type 2 taught by Kuroda together with Nampt and Prs from Wu to a solid surface as taught by Wu to arrive at a cell-free protein synthesis reaction solution, because each of these enzymes is part of the nicotinamide mononucleotide synthesis pathway. One of ordinary skill in the art would reasonably expect that immobilizing one known element (Ppk) together with other known elements (Nampt and Prs) on a solid surface would predictably result in a cell-free protein synthesis reaction solution comprising the three enzymes.
Claims 3 and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (WO 2016/198948 A1, published on December 15, 2016; previously cited) in view of Kuroda et al. (JP 2013-21967, published February 4, 2013; previously cited), Gross et al. (“Practical Synthesis of 5-Phospho-ᴅ-ribosyl-α-1-Pyrophosphate (PRPP): Enzymatic Routes from Ribose-5-Phosphate or Ribose, Journal of the American Chemical Society, 1983, vol. 105, no. 25, pp. 7428-7435; previously cited), Whitesides et al. (US 4,411,995, issued on October 25, 1983; previously cited) and Burgos et al. (“Weak Coupling of ATP Hydrolysis to the Chemical Equilibrium of Human Nicotinamide Phosphoribosyltransferase”, Biochemistry, 2008, Vol. 47, Issue 42, pp.11086-11096; previously cited) as applied to claim 1 above, and further in view of Granot et al. (“Carbon source induces growth of stationary phase yeast cells, independent of carbon source metabolism”, Yeast, 1993, Vol. 9, Issue 5, pp.465-479; previously cited).
The teachings of Wu et al., Kuroda et al., Gross et al., Whitesides et al. and Burgos et al. are discussed above.
Regarding claim 3, Wu, Kuroda, Gross, Whitesides and Burgos do not teach wherein the method is performed under conditions where the transformant does not substantially proliferate.
Granot teaches that a carbon source induces growth of stationary phase yeast cells independent of carbon source metabolism (title). Granot teaches that carbon sources tested induced growth-related events in stationary phase cells, suggesting that the carbon source is the critical nutrient which stimulates growth (abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to remove a carbon source from the growth medium in the method of Wu in view of Kuroda, Gross, Whitesides and Burgos so the transformant does not substantially proliferate. One of ordinary skill in the art would have been motivated to alter the culture medium of the transformant so that it does not substantially proliferate using known methods with a reasonable expectation of success, because Granot teaches that the carbon source is critical for cell growth.
Regarding claim 20, this claim is being interpreted as requiring contacting of ribose-5-phosphate (R5P), nicotinamide (NAM), ATP and polyphosphate with C) a treated product of the transformant. As defined in the specification, examples of the treated product of the transformant include dormant cells (Specification p.40, [0072]).
Thus, claim 20 is obvious for the same reasons discussed in the rejection of claim 3 above, for cells that do not substantially proliferate (i.e. dormant).
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (WO 2016/198948 A1, published on December 15, 2016; previously cited) in view of Kuroda et al. (JP 2013-21967, published February 4, 2013; previously cited) and Gross et al. (“Practical Synthesis of 5-Phospho-ᴅ-ribosyl-α-1-Pyrophosphate (PRPP): Enzymatic Routes from Ribose-5-Phosphate or Ribose, Journal of the American Chemical Society, 1983, vol. 105, no. 25, pp. 7428-7435; previously cited), Whitesides et al. (US 4,411,995, issued on October 25, 1983; previously cited) and Burgos et al. (“Weak Coupling of ATP Hydrolysis to the Chemical Equilibrium of Human Nicotinamide Phosphoribosyltransferase”, Biochemistry, 2008, Vol. 47, Issue 42, pp.11086-11096; previously cited) as applied to claim 1 above, and further in view of Amici et al. (“Synthesis and Degradation of Adenosine 5’-Tetraphosphate by Nicotinamide and Nicotinate Phosphoribosyltransferases”, Cell Chemical Biology, May 2017, Vol. 24, Issue 5, pp. 553-564.e4; previously cited).
The teachings of Wu et al., Kuroda et al., Gross et al., Whitesides et al. and Burgos et al. are discussed above.
Regarding claim 4, Wu teaches the one or more enzymes may be produced by recombinant means in one or more cells including yeast and (description p.11, paragraph [0079]). Wu teaches the system can further comprise NAMPT, but does not disclose the source of the NAMPT (description p.3, paragraph [0013]).
Wu, Kuroda, Gross, Whitesides and Burgos do not teach wherein the Nampt is bacterium-derived.
However, Amici teaches the synthesis and degradation of adenosine 5’-tetraphosphate by nicotinamide and nicotinate phosphoribosyltransferases (title). Amici teaches evolutionary conservation in bacterial orthologs of mammalian NAMPT and NAPT. Amici further teaches recombinant A. baylyi NAMPT (NadV) (relevant to wherein the Nampt is bacterium-derived) (p.e1 – Key Resources Table).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace the Nampt enzyme taught by Wu with the recombinant A. baylyi NAMPT taught by Amici, because it would amount to a simple substitution of one known Nampt enzyme for another. One of ordinary skill in the art would reasonably expect that replacing the Nampt of Wu with the known recombinant A. baylyi NAMPT of Amici would predictably result in a transformant comprising a bacterium-derived NAMPT, because bacterium-derived NAMPT was known in the art at the time of invention.
Claims 9 and 11 are rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (WO 2016/198948 A1, published on December 15, 2016; previously cited) in view of Whitesides et al. (US 4,411,995, issued on October 25, 1983; previously cited).
Regarding claim 9, Wu teaches enzymatic systems and methods for synthesizing nicotinamide mononucleotide and nicotinic acid mononucleotide (relevant to a method for producing nicotinamide mononucleotide (NMN)) (title). Wu teaches that the system utilizes a mutated form of phosphoribosylpyrophosphate synthetase (Prs) that is rendered insensitive to its own reaction product, thus increasing its activity, and one or more other enzymes or enzyme combinations that are bound to a solid surface (description p.2, paragraph [0007]). Wu teaches that in some embodiments, the system further includes nicotinamide phosphoribosyltransferase (NAMPT) (relevant to a cell-free protein synthesis reaction solution with nicotinamide phosphoribosyltransferase (Nampt)) (description p.3, paragraph [0013]). Wu further teaches the system further includes adenosine triphosphate (ATP) and nicotinamide (relevant to contacting nicotinamide (NAM) and ATP; relevant to wherein at least ATP is added to the reaction system) (description p.3, paragraphs [0017]-[0019]. Wu teaches the system can further include phosphoribosyl pyrophosphate (PRPP) (relevant to contacting phosphoribosyl pyrophosphate (PRPP)) (description p.3, paragraph [0020]).
Wu teaches the method further includes the steps of (a) contacting the resulting PRPP with NAMPT in the presence of nicotinamide, whereby nicotinamide mononucleotide NMN is produced (relevant to a reaction with Nampt to generate NMN from PRPP and NAM) (description p.4, paragraph [0028]).
Wu does not teach coupling with a reaction to hydrolyze pyrophosphate into two molecules of phosphate.
Whitesides teaches the use of pyrophosphorylase (PPase) to hydrolyze pyrophosphate in the enzymatic coupling reaction of NMN (relevant to coupling with a reaction to hydrolyze pyrophosphate into two molecules of phosphate) (reaction scheme on page 3; specification column 2, lines 60-62).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the instant application to add pyrophosphorylase (PPase) taught by Whitesides in the reaction mix with Nampt and PRPP, as taught by Wu, because each of these enzymes are part of the nicotinamide mononucleotide synthesis pathway. One of ordinary skill in the art would reasonably expect that combining one known element (PPase) with other known elements (Nampt and PRPP) would predictably result in a reaction that produces NMN, because it was known in the art at the time of invention that each of these enzymes are part of the nicotinamide mononucleotide synthesis pathway.
Regarding claim 11, Wu teaches that a variety of cell-free translation methods are known in the art, for example the use of reticulocyte lysate to facilitate enzyme production (relevant to wherein the treated product is a purified enzyme) (description p.11-12, paragraph [0079]). Wu teaches embodiments where the superactive PRS mutant is purified from cells (description p.2, paragraph [0011]). Wu further teaches embodiments where NAMPT is purified from cells (description p.3, paragraph [0014]).
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (WO 2016/198948 A1, published on December 15, 2016; previously cited) in view of Whitesides et al. (US 4,411,995, issued on October 25, 1983; previously cited) as applied to claim 9 above, and further in view of Granot et al. (“Carbon source induces growth of stationary phase yeast cells, independent of carbon source metabolism”, Yeast, 1993, Vol. 9, Issue 5, pp.465-479; previously cited).
The teachings of Wu et al. and Whitesides et al. are discussed above.
Regarding claim 10, Wu and Whitesides do not teach wherein the method is performed under conditions where the transformant does not substantially proliferate.
Granot teaches that carbon source induces growth of stationary phase yeast cells independent of carbon source metabolism (title). Granot teaches that carbon sources tested induced growth-related events in stationary phase cells, suggesting that the carbon source is the critical nutrient which stimulates growth (abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to remove a carbon source from the growth medium in the method of Wu and Whitesides to modify the culture medium of the transformant so it does not substantially proliferate. One of ordinary skill in the art would have been motivated to alter the culture medium of the transformant using known methods with a reasonable expectation of success, so that the transformant does not substantially proliferate, because Granot teaches that the carbon source is critical for cell growth.
Claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (WO 2016/198948 A1, published on December 15, 2016; previously cited) in view of Whitesides et al. (US 4,411,995, issued on October 25, 1983; previously cited) as applied to claim 9 above, and further in view of Burgos et al. (“Weak Coupling of ATP Hydrolysis to the Chemical Equilibrium of Human Nicotinamide Phosphoribosyltransferase”, Biochemistry, 2008, Vol. 47, Issue 42, pp.11086-11096; previously cited).
Regarding claim 16, Wu does not teach wherein the total sum of the respective moles of ATP, ADP and AMP to be added to the reaction system is 0.5 equivalents or less of the number of moles of NMN to be generated and wherein the total sum of the respective numbers of moles of ATP, ADP and AMP to be added to the reaction system is 0.5 equivalents or less of the number of moles of PRPP or NAM to be added to the reaction system at the start of the reaction.
While Whitesides does not specifically disclose a total sum of the respective numbers of moles of ATP, ADP and AMP, Whitesides does teach that the starting amounts of ATP, ADP or AMP can be varied to regenerate ATP from AMP or ADP and coupled ATP recycling can avoid waste of adenosine moieties and reduce overall expense (specification column 3, lines 4-14).
Burgos teaches human nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the reversible synthesis of nicotinamide mononucleotide (NMN) and inorganic pyrophosphate (PPi) from nicotinamide (NAM) and α-D-5-phosphoribosyl-1-pyrophosphate (PRPP) (abstract). Burgos teaches that with ATP hydrolysis coupled to NMN synthesis, the catalytic efficiency of the system is improved 1100-fold, substrate affinity dramatically increases and the Keq shifts -2.1 kcal/mol toward NMN formation (abstract).
Thus, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the instant application to optimize the ratio of total moles of ATP, ADP or AMP to the number of moles of NMN to be generated and the number of moles of PRPP or NAM to be added to the reaction system at the start of the reaction to be less than or equal to 0.5 to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reasonable expectation of success that varying the amounts of ATP, ADP or AMP would result in different numbers of moles of NMN to obtain an optimal ratio of less than or equal to 0.5 with predictable results, because Whitesides teaches that small catalytic amounts of ATP can used in coupled ATP recycling, and Burgos teaches that ATP hydrolysis coupled to NMN synthesis improved the catalytic efficiency of the system 1100-fold. “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature 100°C and an acid concentration of 10%). See MPEP 2144.05.
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (WO 2016/198948 A1, published on December 15, 2016; previously cited) in view of Sinclair et al. (WO 2015/069860, published on May 14, 2015; previously cited), Kinney et al. (“Pyridine Nucleotide Cycle of Salmonella typhimurium: In Vitro Demonstration of Nicotinamide Mononucleotide Deamidase and Characterization of pnuA Mutants Defective in Nicotinamide Mononucleotide Transport”, Journal of Bacteriology, Nov. 1979, vol. 140, no. 3, pp. 607-611; previously cited) and Koepke et al. (US 20130316364, published on November 28, 2013; previously cited).
Regarding claim 12, Wu teaches enzymatic systems and methods for synthesizing nicotinamide mononucleotide and nicotinic acid mononucleotide (relevant to a method for producing nicotinamide mononucleotide (NMN)) (title). Wu teaches that the system utilizes a mutated form of phosphoribosylpyrophosphate synthetase (Prs) that is rendered insensitive to its own reaction product, thus increasing its activity, and one or more other enzymes or enzyme combinations that are bound to a solid surface (description p.2, paragraph [0007]). Wu teaches that in some embodiments, the system further includes nicotinamide phosphoribosyltransferase (NAMPT) (description p.3, paragraph [0013]). Wu further teaches the system further includes adenosine triphosphate (ATP) and nicotinamide (which suggests contacting nicotinamide (NAM) and ATP with a transformant; relevant to wherein at least ATP is added to the reaction system) (description p.3, paragraphs [0017]-[0019]. Wu teaches the system can further include phosphoribosyl pyrophosphate (PRPP) (relevant to contacting phosphoribosyl pyrophosphate (PRPP)) (description p.3, paragraph [0020]).
Wu teaches the method further includes the steps of (a) contacting the resulting PRPP with NAMPT in the presence of nicotinamide, whereby nicotinamide mononucleotide NMN is produced (relevant to a reaction with Nampt to generate NMN from PRPP and NAM) (description p.4, paragraph [0028]).
Wu does not teach a transformant with enhanced expression of Nampt, wherein the transformant has a disruption or deletion of a gene encoding (d) nicotinamide mononucleotide deamidase and a gene encoding one or more of the enzymes listed above.
Sinclair teaches a method for producing nicotinamide mononucleotide (NMN) comprising providing an isolated cell that overexpresses nicotinamide phosphoribosyltransferase (Nampt) (relevant to a transformant with enhanced expression of nicotinamide phosphoribosyltransferase (Nampt)) (Figure 1; specification page 1, lines 26-28) and phosphoribosyl pyrophosphate synthetase (Prs) (specification page 1, lines 30-31 and page 7, lines 28-30) and culturing the cell in the presence of nicotinamide (NAM) (Figure 1). Sinclair further teaches that PRS1 and Nampt combined with PRPP is involved in generating NMN (Figure 1). Sinclair teaches modified nucleic acid molecules including deletion of genes. The modified nucleic acid molecules can encode modified polypeptides (specification page 23, lines 31-34 through page 24, line 1). Sinclair further teaches creating variants of polypeptides with one or more modifications to the primary amino acid sequence of the polypeptide. These modifications which create a variant can be made to reduce or eliminate an activity of a polypeptide. Sinclair further teaches that these modifications can include deletions, point mutations, truncations, and so on (specification page 24, lines 24-33).
Sinclair does not teach modifications of 5’-nucleotidase, purine-nucleoside phosphorylase, nicotinamide mononucleotide deamidase, purine nucleosidase, uridine nucleosidase, or NMN nucleosidase.
Kinney teaches that nicotinamide mononucleotide deamidase is used to degrade NMN and recycle it into nicotinamide adenine dinucleotide (NAD) (abstract, figure 1). Kinney further teaches that there are many similarities in the genetic and biochemical pyridine nucleotide cycle of Salmonella typhimurium and Escherichia coli (p. 607, column 1). Kinney further teaches that mutations in the nicotinamide mononucleotide deamidase enzyme cause strains to be defective in the transport of NMN across the cell membrane (p. 607, column 2).
Koepke teaches that one or more genes in a biosynthesis pathway for a vitamin or other essential nutrient can be used as an effective selective marker to identify cells transformed with an exogenous nucleic acid (abstract). Koepke additionally teaches that 5’-nucleotidase (EC 3.1.3.5), purine-nucleoside phosphorylase (EC 2.4.2.1), and purine nucleosidase (EC 3.2.2.1) are part of the nicotinic acid biosynthesis pathway (specification page 8, table 3).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the instant application to delete the gene encoding nicotinamide mononucleotide deamidase EC 3.5.1.42 taught by Kinney and delete the gene encoding 5’-nucleotidase (EC 3.1.3.5), purine-nucleoside phosphorylase (EC 2.4.2.1), or purine nucleosidase (EC 3.2.2.1) taught by Koepke, in the transformant taught by Wu, using known methods of gene deletion or gene disruption taught by Sinclair. One of ordinary skill in the art would reasonably expect that deleting these genes would predictably result in a variant without nicotinamide mononucleotide deamidase activity and without 5’-nucleotidase, purine-nucleoside phosphorylase, or purine nucleosidase activity, because Sinclair teaches that modifications which create a variant can be made to reduce or eliminate an activity of a polypeptide, such as an enzyme. One of ordinary skill in the art would have been motivated to apply the teachings of Sinclair to specifically disrupt or delete a gene encoding nicotinamide mononucleotide and 5’-nucleotidase, purine-nucleoside phosphorylase, or purine nucleosidase using known methods (gene deletion or disruption) to obtain predictable results (alteration of gene expression to reduce activity of an enzyme) in the transformant of Wu to arrive at the instantly claimed invention. It was known in the art at the time of invention that 5’-nucleotidase (EC 3.1.3.5), purine-nucleoside phosphorylase (EC 2.4.2.1), or purine nucleosidase (EC 3.2.2.1) were all part of the nicotinic acid biosynthesis pathway; and it was known in the art at the time of invention that disrupting the activity of nicotinamide mononucleotide deamidase prevented NMN transport across the cell membrane, thereby increasing the amount of NMN produced.
Claim 17 is rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (WO 2016/198948 A1, published on December 15, 2016; previously cited) in view of Sinclair et al. (WO 2015/069860, published on May 14, 2015; previously cited), Kinney et al. (“Pyridine Nucleotide Cycle of Salmonella typhimurium: In Vitro Demonstration of Nicotinamide Mononucleotide Deamidase and Characterization of pnuA Mutants Defective in Nicotinamide Mononucleotide Transport”, Journal of Bacteriology, Nov. 1979, vol. 140, no. 3, pp. 607-611; previously cited) and Koepke et al. (US 20130316364, published on November 28, 2013; previously cited) as applied to claim 12 above, and further in view of Whitesides et al. (US 4,411,995, issued on October 25, 1983; previously cited) and Burgos et al. (“Weak Coupling of ATP Hydrolysis to the Chemical Equilibrium of Human Nicotinamide Phosphoribosyltransferase”, Biochemistry, 2008, Vol. 47, Issue 42, pp.11086-11096; previously cited).
The teachings of Wu et al., Sinclair et al., Kinney et al. and Koepke et al. are discussed above.
Regarding claim 17, Wu, Sinclair, Kinney and Koepke do not teach wherein the total sum of the respective moles of ATP, ADP and AMP to be added to the reaction system is 0.5 equivalents or less of the number of moles of NMN to be generated and wherein the total sum of the respective numbers of moles of ATP, ADP and AMP to be added to the reaction system is 0.5 equivalents or less of the number of moles of PRPP or NAM to be added to the reaction system at the start of the reaction.
While Whitesides does not specifically disclose a total sum of the respective numbers of moles of ATP, ADP and AMP, Whitesides does teach that the starting amounts of ATP, ADP or AMP can be varied to regenerate ATP from AMP or ADP and coupled ATP recycling can avoid waste of adenosine moieties and reduce overall expense (specification column 3, lines 4-14).
Burgos teaches human nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the reversible synthesis of nicotinamide mononucleotide (NMN) and inorganic pyrophosphate (PPi) from nicotinamide (NAM) and α-D-5-phosphoribosyl-1-pyrophosphate (PRPP) (abstract). Burgos teaches that with ATP hydrolysis coupled to NMN synthesis, the catalytic efficiency of the system is improved 1100-fold, substrate affinity dramatically increases and the Keq shifts -2.1 kcal/mol toward NMN formation (abstract).
Thus, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the instant application to optimize the ratio of total moles of ATP, ADP or AMP to the number of moles of NMN to be generated and the number of moles of PRPP or NAM to be added to the reaction system at the start of the reaction to be less than or equal to 0.5 and arrive at the instantly claimed invention. One of ordinary skill in the art would have a reasonable expectation of success that varying the amounts of ATP, ADP or AMP relative to the number of moles of PRPP or NAM would result in different numbers of moles of NMN to obtain an optimal ratio of less than or equal to 0.5 with predictable results, because Whitesides teaches that small catalytic amounts of ATP can used in coupled ATP recycling, and Burgos teaches that ATP hydrolysis coupled to NMN synthesis improved the catalytic efficiency of the system 1100-fold. “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature 100°C and an acid concentration of 10%). See MPEP 2144.05.
Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over are rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (WO 2016/198948 A1, published on December 15, 2016; previously cited) in view of Sinclair et al. (WO 2015/069860, published on May 14, 2015; previously cited), Whitesides et al. (US 4,411,995, issued on October 25, 1983; previously cited) and Burgos et al. (“Weak Coupling of ATP Hydrolysis to the Chemical Equilibrium of Human Nicotinamide Phosphoribosyltransferase”, Biochemistry, 2008, Vol. 47, Issue 42, pp.11086-11096; previously cited).
Regarding claim 14, Wu teaches enzymatic systems and methods for synthesizing nicotinamide mononucleotide and nicotinic acid mononucleotide (relevant to a method for producing nicotinamide mononucleotide (NMN)) (title). Wu teaches that the system utilizes a mutated form of phosphoribosylpyrophosphate synthetase (Prs) that is rendered insensitive to its own reaction product, thus increasing its activity, and one or more other enzymes or enzyme combinations that are bound to a solid surface (description p.2, paragraph [0007]). Wu teaches that in some embodiments, the system further includes nicotinamide phosphoribosyltransferase (NAMPT) (relevant to a transformant with enhanced expression of nicotinamide phosphoribosyltransferase (Nampt) and phosphoribosyl pyrophosphate synthetase (Prs)) (description p.3, paragraph [0013]). Wu further teaches the system further includes adenosine triphosphate (ATP) and nicotinamide (relevant to contacting nicotinamide (NAM) and ATP with a transformant; relevant to wherein at least ATP is added to the reaction system) (description p.3, paragraphs [0017]-[0019]. Wu teaches the system can further include phosphoribosyl pyrophosphate (PRPP) (description p.3, paragraph [0020]).
Wu teaches that the method includes the step of contacting ribose-5-phosphate with a superactive PRS mutant in the presence of ATP, whereby PRPP is produced (relevant to contacting ribose-5-phosphate (R5P); relevant to a reaction with Prs to generate PRPP and AMP from R5P and ATP) (description p.3, paragraph [0022]).
Wu teaches the method further includes the steps of (a) contacting the resulting PRPP with NAMPT in the presence of nicotinamide, whereby nicotinamide mononucleotide NMN is produced (relevant to a reaction with Nampt to generate NMN from PRPP and NAM) (description p.4, paragraph [0028]).
Wu does not teach a transformant with enhanced expression, or wherein the total sum of the respective numbers of moles of the added ATP, ADP and AMP is 0.5 equivalents or less of the number of moles of the added NAM, and wherein the number of moles of the generated NMN is 2 equivalents or more of the total sum of the respective numbers of moles of the added ATP, ADP and AMP. .
Sinclair teaches a method for producing nicotinamide mononucleotide (NMN) comprising providing an isolated cell that overexpresses nicotinamide phosphoribosyltransferase (Nampt) (Figure 1; specification page 1, lines 26-28) and phosphoribosyl pyrophosphate synthetase (Prs) (specification page 1, lines 30-31 and page 7, lines 28-30) (relevant to a transformant with enhanced expression of two enzymes, Nampt and Prs) and culturing the cell in the presence of nicotinamide (NAM) (Figure 1). Sinclair further teaches that PRS1 and Nampt combined with PRPP is involved in generating NMN (Figure 1).
Burgos teaches human nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the reversible synthesis of nicotinamide mononucleotide (NMN) and inorganic pyrophosphate (PPi) from nicotinamide (NAM) and α-D-5-phosphoribosyl-1-pyrophosphate (PRPP) (abstract). Burgos teaches that with ATP hydrolysis coupled to NMN synthesis, the catalytic efficiency of the system is improved 1100-fold, substrate affinity dramatically increases and the Keq shifts -2.1 kcal/mol toward NMN formation (abstract).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace the cell expressing Prs and Nampt taught by Wu with a cell that overexpresses Nampt and Prs taught by Sinclair in the method of Wu. Both Wu and Sinclair teach a cell expressing Prs and Nampt to generate NMN. One of ordinary skill in the art would reasonably expect that replacing a cell that expresses Prs and Nampt with a cell that overexpresses Prs and Nampt would predictably result in a higher production of the desired product NMN, because it was known in the art at the time of invention that a cell could overexpress two enzymes of Prs and Nampt.
While Whitesides does not specifically disclose a total sum of the respective numbers of moles of ATP, ADP and AMP, Whitesides does teach that the starting amounts of ATP, ADP or AMP can be varied to regenerate ATP from AMP or ADP and coupled ATP recycling can avoid waste of adenosine moieties and reduce overall expense (specification column 3, lines 4-14).
Burgos teaches human nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the reversible synthesis of nicotinamide mononucleotide (NMN) and inorganic pyrophosphate (PPi) from nicotinamide (NAM) and α-D-5-phosphoribosyl-1-pyrophosphate (PRPP) (abstract). Burgos teaches that with ATP hydrolysis coupled to NMN synthesis, the catalytic efficiency of the system is improved 1100-fold, substrate affinity dramatically increases and the Keq shifts -2.1 kcal/mol toward NMN formation (abstract).
Thus, it would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the instant application to optimize the ratio of total moles of ATP, ADP or AMP to the number of moles of NMN to be generated and the number of moles of PRPP or NAM to be added to the reaction system at the start of the reaction to be less than or equal to 0.5 in the method of Wu in view of Sinclair to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reasonable expectation of success that varying the amounts of ATP, ADP or AMP relative to the number of moles of NAM would result in different numbers of moles of NMN to obtain an optimal ratio of less than or equal to 0.5 with predictable results, because Whitesides teaches that small catalytic amounts of ATP can used in coupled ATP recycling, and Burgos teaches that ATP hydrolysis coupled to NMN synthesis improved the catalytic efficiency of the system 1100-fold. “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation.” In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955) (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature 100°C and an acid concentration of 10%). See MPEP 2144.05.
Claim 15 is rejected under 35 U.S.C. 103 as being unpatentable over are rejected under 35 U.S.C. 103 as being unpatentable over Wu et al. (WO 2016/198948 A1, published on December 15, 2016; previously cited) in view of Sinclair et al. (WO 2015/069860, published on May 14, 2015; previously cited), Whitesides et al. (US 4,411,995, issued on October 25, 1983; previously cited) and Burgos et al. (“Weak Coupling of ATP Hydrolysis to the Chemical Equilibrium of Human Nicotinamide Phosphoribosyltransferase”, Biochemistry, 2008, Vol. 47, Issue 42, pp.11086-11096; previously cited) as applied to claim 14 above, and further in view of Gross et al. (“Practical Synthesis of 5-Phospho-ᴅ-ribosyl-α-1-Pyrophosphate (PRPP): Enzymatic Routes from Ribose-5-Phosphate or Ribose, Journal of the American Chemical Society, 1983, vol. 105, no. 25, pp. 7428-7435; previously cited).
The teachings of Wu et al., Sinclair et al., Whitesides et al. and Burgos et al. are discussed above.
Regarding claim 15, Wu, Sinclair, Whitesides and Burgos do not teach contacting ribose and ATP with a transformant with enhanced expression of ribokinase in a reaction system to perform a reaction with Rbk to generate R5P and ADP from ribose and ATP.
Gross further teaches the use of ribokinase and ribose to generate R5P and the use of polyphosphate to regenerate ATP from AMP (abstract, scheme IV).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the instant application to additionally transform cells expressing Nampt and Prs, as taught by Wu in view of Whitesides, with genes expressing ribokinase, as taught by Gross. One of ordinary skill in the art would have been motivated to modify the cell to additionally express ribokinase with a reasonable expectation of success because conventional techniques to yield cells expressing specific molecules of interest were known in the art at the time of invention. It is obvious to combine the expression of one known element (ribokinase) with another (Nampt and Prs) to obtain predictable results.
Response to Arguments
Regarding claims 1 and 5-7, Applicant argues that the cited references are silent as to the ratio of the total number of moles of added ATP, ADP and AMP to the added NAM, or the generated NMN to the total number of moles of the added ATP, ADP and AMP as set forth in the claims (See Remarks dated 3/10/2026, p.7, 3rd paragraph). Applicant argues that the present disclosure entails the feature that a small amount of ATP can produce a large amount of NMN by combining a first reaction of R5P with Prs and ATP to produce PRPP, and a second reaction of PRPP with Nampt and NAM to produce NMN when coupled with ATP regeneration by Ppk from polyphosphate (See Remarks dated 3/10/2026, p.7 5th paragraph). Applicant argues that there is no reasonable expectation of success to arrive at the claimed ratios; as Whitesides merely teaches the use of small catalytic amounts of ATP in recycling and Burgos discusses improved catalytic efficiency without addressing the specific ratios claimed, while the present invention uniquely combines reactions to achieve these ratios which are not disclosed in the prior art (See Remarks dated 3/10/2026, p.8 1st paragraph). Applicant argues that the claimed embodiment achieves unexpected results by producing large amounts of NMN with a small amount of ATP, which is accomplished through a novel combination of reactions involving R5P, Prs, ATP, PRPP, Nampt, and NAM coupled with ATP regeneration by Ppk from polyphosphate and these results are not obvious from the prior art (See Remarks dated 3/10/2026, p.8 2nd paragraph). Applicant argues that Burgos discloses an NMN/ATP ratio of 1 equivalent or less, which contradicts the claimed ratios, indicating the claimed ratios are not achievable by Burgos alone and there is no reasonable expectation of success in arriving at the claimed invention, and the specific ratios claimed are critical to the invention’s success as they enable the efficient production of NMN and lead to improved results that are not obvious (See Remarks dated 3/10/2026, p.8 paragraphs 2-3).
Applicant’s remarks dated March 10, 2026 have been fully considered by they are not persuasive. The result of generating a number of moles of NMN that is 2 equivalents or more is directed to a result obtained by practicing the claimed method, as the amount of NMN produced is unknown at the beginning of the reaction. One of ordinary skill in the art would be unable to predict whether the total number of moles of ATP, ADP and AMP added is 0.5 equivalents or less relative to the generated number of moles of NMN being 2 equivalents or more until the reaction has completed. As discussed in the rejection above, Wu teaches the combination of more than one reaction, and that NAM is a starting material along with ATP in the production of NMN. Whitesides teaches that starting amounts of ATP, ADP or AMP can be varied to regenerate ATP from AMP and ADP, and coupled ATP recycling can avoid waste of adenosine moieties and reduce overall expense. Burgos teaches that when ATP hydrolysis is coupled to NMN synthesis, the catalytic efficiency of the system is improved 1100-fold, and the Keq shifts towards NMN formation. Therefore, one of ordinary skill in the art would have found it obvious to vary the amount of ATP, ADP and AMP, and reasonably expect that doing so would predictably result in achieving the desired result of 2 equivalents or more of NMN produced.
It is noted that the prior art teaches all the required components of the claimed method. The claimed method is directed towards producing nicotinamide mononucleotide. Applicant has not demonstrated the criticality of the ratio of the total sum of the moles of ATP, ADP and AMP of being 0.5 equivalents or less of the moles of NAM added, nor demonstrated an unexpected effect resulting from adding 0.5 equivalents or less. It is known in the art that adding ATP, ADP or AMP to a reaction will allow regeneration of ATP and the addition will increase the production efficiency of NMN. Furthermore, Applicant has not demonstrated that 2 equivalents or more NMN can be produced using less than 0.5 equivalents of ATP, ADP and AMP. The examples in the specification demonstrate the addition of ATP only. See MPEP §2163.05 III: “With respect to changing numerical range limitations, the analysis must take into account which ranges one skilled in the art would consider inherently supported by the discussion in the original disclosure. In the decision in In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976), the ranges described in the original specification included a range of "25%- 60%" and specific examples of "36%" and "50%." A corresponding new claim limitation to "at least 35%" did not meet the description requirement because the phrase "at least" had no upper limit and caused the claim to read literally on embodiments outside the "25% to 60%" range, however a limitation to "between 35% and 60%" did meet the description requirement”. See also MPEP § 716.02(d) II: “To establish unexpected results over a claimed range, applicants should compare a sufficient number of tests both inside and outside the claimed range to show the criticality of the claimed range. In re Hill, 284 F.2d 955, 128 USPQ 197 (CCPA 1960)”.
Regarding claims 3 and 4, Applicant argues that claims 3 and 4 depend from claim 1, and are not rendered obvious over Wu, Kuroda and Gross for the same reasons mentioned above, and Granot and Amici do not cure the deficiencies of Wu, Kuroda and Gross (See Remarks dated 3/10/2026, p.8 section II).
Applicant’s arguments are not found persuasive for the same reasons discussed above.
Regarding claims 9 and 11, Applicant argues that the office action overlooks the specific role of PPase in hydrolyzing pyrophosphate (PPi), which is crucial in the synthesis process, and the prior art does not teach or suggest the specific combination and conditions claimed (See Remarks dated 3/10/2026, p.9, 3rd paragraph). Applicant argues that Burgos describes that hydrolysis of the phospho-enzyme is required for each catalytic turnover in NMN synthesis in the presence of ATP and the ability of exogenous PPi to enhance ATPase indicates that hydrolysis might be triggered by this product signaling that the reaction is complete. Based on the disclosure of Burgos, a person with ordinary skill in the art would understand that the hydrolysis of PPi inhibits production of NMN and a person of ordinary skill in the art would not have reason to combine Whitesides with Wu (See Remarks dated 3/10/2026, p.9, 4th paragraph). Applicant argues that the claimed invention involves a specific and non-obvious combination of elements that are not suggested by the prior art, and the unique interaction between ATP regeneration and PRPP generation is not disclosed or suggested by Wu, Whitesides or Burgos (See Remarks dated 3/10/2026, p.9 5th paragraph).
Applicant’s arguments are not found persuasive. As taught by Whitesides, the function of pyrophosphorylase (PPase) is to hydrolyze pyrophosphate in the enzymatic coupling reaction of NMN. One of ordinary skill in the art would recognize that PPase is used to hydrolyze pyrophosphate, and would have a reasonable expectation that combining known parts of the nicotinamide mononucleotide synthesis pathway together would predictably result in the production of nicotinamide mononucleotide, because it would amount to combining known elements together in a predictable way. One of ordinary skill in the art would have been motivated to combine known elements of ATP regeneration together with PRPP generation to produce NMN based on the teachings of Wu and Whitesides for the reasons stated above.
Regarding claim 12, Applicant argues that the Office suggests it would be obvious to modify the transformant of Wu using the teachings of Kinney and Koepke with Sinclair’s methods, but this overlooks the specific and unique combination of elements in the claimed embodiment that are not suggested by the prior art (See remarks dated 3/10/2026, p.10, 3rd paragraph). Applicant argues that the claimed embodiment involves a specific and non-obvious combination of genetic modifications that are not disclosed or suggested by the prior art, and the unique approach to altering gene expression to achieve the claimed results is not predictable from the teachings of Wu, Sinclair, Kinney and Koepke (See Remarks dated 3/10/2026, p.10, 4th paragraph).
Claim 12 requires that a disruption or deletion of a gene encoding one or more of the following (a), (c), (g) (h) and (i). Sinclair teaches modifying nucleic acid molecules including deletion of genes, with modified nucleic acid molecules encoding modified polypeptides, and further creating variants of polypeptides with one or more modifications to create a variant that can reduce or eliminate activity of a polypeptide. Kinney teaches mutations in the nicotinamide mononucleotide deamidase enzyme cause strains to be defective in the transport of NMN across the cell membrane. Koepke teaches that 5’-nucleotidase (EC 3.1.3.5), purine-nucleoside phosphorylase (EC 2.4.2.1), and purine nucleosidase (EC 3.2.2.1) are part of the nicotinic acid biosynthesis pathway. Thus, it would have been obvious to one of ordinary skill in the art to modify the transformant taught by Wu using the teachings of Kinney and Koepke using the methods of Sinclair, because Sinclair teaches modifications can create a variant can be made to reduce or eliminate an activity of a polypeptide such as an enzyme. One of ordinary skill in the art would have been motivated to apply the teachings of Sinclair to specifically disrupt or delete a gene encoding nicotinamide mononucleotide and 5’-nucleotidase, purine-nucleoside phosphorylase, or purine nucleosidase using known methods (gene deletion or disruption) to obtain predictable results (alteration of gene expression to reduce activity of an enzyme) in the transformant of Wu to arrive at the instantly claimed invention. It was known in the art at the time of invention that 5’-nucleotidase (EC 3.1.3.5), purine-nucleoside phosphorylase (EC 2.4.2.1), or purine nucleosidase (EC 3.2.2.1) were all part of the nicotinic acid biosynthesis pathway; and it was known in the art at the time of invention that disrupting the activity of nicotinamide mononucleotide deamidase prevented NMN transport across the cell membrane, thereby increasing the amount of NMN produced. Further, if NMN is not transported out of the cell, it would accumulate in the cell, and be recoverable from the cell later.
Applicant’s arguments are not found persuasive. Applicant’s arguments regarding the specific and non-obvious combination of genetic modifications and the uniqueness of the approach cannot take the place of evidence in the record. See MPEP 716.01(c).
Regarding claim 17, Applicant argues that claim 17 depends from claim 12 and is not rendered obvious over Wu, Sinclair, Kinney and Koepke and Whitesides and Burgos do not cure the deficiencies of Wu, Sinclair, Kinney and Koepke (See Remarks dated 3/10/2026, p.10 section V).
Applicant’s arguments are not found persuasive for the same reasons discussed above.
Regarding claim 14, Applicant argues that the Office action suggests that it would be obvious to replace a cell expressing Prs and Nampt with one that overexpresses these enzymes, however this overlooks the specific conditions and interactions required to achieve the claimed embodiment, which are not disclosed or suggested by the prior art (See Remarks dated 3/10/2026, p.11, section VI. 3rd paragraph). Applicant argues that while Wu and Sinclair teach cells expressing Prs and Nampt, there is no motivation to combine these teachings with Whitesides, Burgos and Gross in the manner claimed (See Remarks dated 3/10/2026, p.11, section VI 4th paragraph). Applicant argues that the specific combination and overexpression strategy are not suggested by the prior art; the claimed embodiment involves a unique approach to overexpressing Prs and Nampt that is not disclosed or suggested by the cited references, and the specific method and conditions for achieving higher NMN production are not predictable from the teachings of Wu, Sinclair, Whitesides, Burgos and Gross (See Remarks dated 3/10/2026, p.11 section VI 4th paragraph).
Applicant’s arguments are not persuasive. Wu teaches that the method includes the step of contacting ribose-5-phosphate with a superactive PRS mutant in the presence of ATP, whereby PRPP is produced (description p.3, paragraph [0022]). Wu teaches the method further includes the steps of (a) contacting the resulting PRPP with NAMPT in the presence of nicotinamide, whereby nicotinamide mononucleotide NMN is produced (description p.4, paragraph [0028]). Sinclair teaches a method for producing nicotinamide mononucleotide (NMN) comprising providing an isolated cell that overexpresses nicotinamide phosphoribosyltransferase (Nampt) (Figure 1; specification page 1, lines 26-28) and phosphoribosyl pyrophosphate synthetase (Prs) (specification page 1, lines 30-31 and page 7, lines 28-30) and culturing the cell in the presence of nicotinamide (NAM) (Figure 1). Sinclair further teaches that PRS1 and Nampt combined with PRPP is involved in generating NMN (Figure 1). Burgos teaches human nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the reversible synthesis of nicotinamide mononucleotide (NMN) and inorganic pyrophosphate (PPi) from nicotinamide (NAM) and α-D-5-phosphoribosyl-1-pyrophosphate (PRPP) (abstract). Burgos teaches that with ATP hydrolysis coupled to NMN synthesis, the catalytic efficiency of the system is improved 1100-fold, substrate affinity dramatically increases and the Keq shifts -2.1 kcal/mol toward NMN formation (abstract). It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to replace the cell expressing Prs and Nampt taught by Wu with a cell that overexpresses Nampt and Prs taught by Sinclair in the method of Wu to arrive at the claimed invention. Both Wu and Sinclair teach a cell expressing Prs and Nampt to generate NMN. One of ordinary skill in the art would reasonably expect that replacing a cell that expresses Prs and Nampt with a cell that overexpresses Prs and Nampt would predictably result in a higher production of the desired product NMN, because it was known in the art at the time of invention that a cell could overexpress two enzymes of Prs and Nampt.
Conclusion
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/LOUISE W HUMPHREY/Supervisory Patent Examiner, Art Unit 1657
/DEEPA MISHRA/Examiner, Art Unit 1657
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