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 .
The Preliminary Amendment of 27 Feb. 2024 has been entered.
Claims 1-11 are currently pending and are considered here.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
Claims 1 and 3-11 are rejected under 35 U.S.C. 112(a) as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The instant claims are drawn to a recombinant S. boulardii transformed with a gene encoding any beta-agarase. As evidenced by Martin et al., Applied microbiology and biotechnology 98.7 (2014): 2917-2935, beta-agarases are commonly expressed by marine microorganisms that colonize algae, which comprise a large, diverse and largely uncharacterized genus of organisms (p. 2917, 1st ¶ to p. 2924, 1st ¶). Martin further evidences that beta-agarases encompass at least four structurally distinct enzyme classes, GH16, GH50, GH86 and GH118 (p. 2921-2924, under Agarases). Thus, the claims encompass a large and diverse genus of potential beta-agarases.
To show possession of a claimed genus, the specification must provide sufficient distinguishing identifying characteristics of the genus, which in the case of a chemical invention requires a precise definition, such as by structure, formula, chemical name, or physical properties (MPEP 2163). The written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species sufficient to show the applicant was in possession of the claimed genus; A "representative number of species" means that the species which are adequately described are representative of the entire genus (MPEP 2163).
The instant specification discloses only the sequence of SEQ ID 1, encoding the BpGH16A agarase from B. plebeius strain DSM17135 (Published Spec. US20240294891, [0013]). The specification does not describe any additional species of beta-agarases suitable for expression in S. boulardii that could be considered a representative number of species supporting the claimed genus. The specification also fails to provide any other teachings or guidance that would allow one to identify enzymes suitable for use in the invention, such as a structure-function relationship or identifying/characteristic features. Thus, the instant specification does not evidence possession of the full scope of the claimed genus of all potential beta-agarases (see MPEP 2163 - A patentee will not be deemed to have invented species sufficient to constitute the genus by virtue of having disclosed a single species when the evidence indicates ordinary artisans could not predict the operability in the invention of any species other than the one disclosed).
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 3 and 11 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 (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 3 recites “a signal peptide that secretes a beta-agarase enzyme outside strain”. The term “strain” does not have antecedent basis in claim 1, making it unclear what is being referred to in the claim. The rejection can be overcome by amending as follows: “a signal peptide that secretes a beta-agarase enzyme outside the transformed Saccharomyces boulardii
Claim 11 recites “the product of the above step”. The term “above” is unclear. The rejection can be overcome by amending as follows: “the product of the reacting step”.
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.
Claims 1-11 are rejected under 35 U.S.C. 103 as being unpatentable over the combination of WO2019098708 to Kim et al. (Kim 2019) (citations herein are to English equivalent US20200360447) in view of Liu et al., Applied and environmental microbiology 82.8 (2016): 2280-2287 (cited in IDS of 18 Dec. 2023) and WO2020050663A to Kim et al. (Kim 2020) (citations herein are to English equivalent US20210317492).
Regarding claims 1-3, Kim 2019 teaches that beta-agarase BpGH16A (SEQ ID 1) from B. pleibeius strain DSM17135 can hydrolyze agarose (the main polysaccharide component of red algae) to produce neoagarooligosaccharides, including neoagarotetraose (DP4) and neoagarobiose (DP2), and that DP2 and DP4 can further be converted by a second B. pleibeius enzyme, a neoagarobiose hydrolase BpGH117 (SEQ ID 2), to agarotriose ([0006]-[0044]; [0085]-[0095]; Examples 1-3 and 11-14; Figs. 13-15). Kim 2019 further teaches that agarotriose can serve as a prebiotic for beneficial gut probiotic bacteria, including various species of Bifidobacterium, and can have beneficial anticancer and anti-inflammatory effects in the gut ([0014]; [0020]-[0025]; [0033]; Examples 4-9). Kim 2019 also teaches that BpGH16A (and BpGH117) can be expressed recombinantly ([0039]-[0044]; Examples 1-3, 10).
Regarding claims 7-10, Kim 2019 teaches probiotic, medicinal and/or food compositions comprising agarose and a microorganism (B. pleibeius strain DSM17135) expressing BpGH16A, wherein the compositions can produce neoagarooligosaccharides ([0008]-[0015]; [0034]-[0044]; [0078]; claims 1-6). Further regarding claims 9-10, Kim 2019 teaches that the compositions can comprise an additional probiotic microorganism other than the agarase-expressing strain, such as a Bifidobacterium ([0008]-[0015]; [0034]-[0038]; [0078]; claims 1-6).
Regarding claim 11, Kim 2019 teaches a method of producing neoagarooligosaccharides comprising reacting a culture extract of a strain expressing BpGH16A (B. pleibeius strain DSM17135) with agarose, and separating and purifying the neoagarooligosaccharides ([0011]-[0013]; [0085]-[0095]; Examples 1-3 and 11-14; Figs. 13-15; claims 11-15).
Claims 1-11 differ from Kim 2019 in that: the strain expressing the agarase is a recombinant Saccharomyces boulardii transformed with a gene encoding the beta-agarase (claim 1); the gene has a sequence of SEQ ID 1 (claim 2); the Saccharomyces boulardii is transformed with a gene encoding a signal peptide for secretion of the beta-agarase (claim 3); the signal peptide is chicken lysozyme signal peptide (claim 4) of SEQ ID 2 (claim 5); the transformation is via CRISPR-Cas9 or a vector (claim 6).
Regarding claims 1 and 6, Liu teaches methods for transforming S. boulardii to express one or more heterologous genes of interest using CRISPR-Cas9, allowing S. boulardii to be used as a probiotic host for delivery of therapeutic proteins (p. 2280, last ¶ to p. 2281, 1st ¶; p. 2284-2285, under Metabolic pathway introduction into S. boulardii; and Secretion of human lysozyme by S. boulardii; p. 2286, last ¶). Liu exemplifies introduction of a new metabolic pathway comprising three genes that allows for fermentation of the carbohydrate xylose, as well as introduction of a secreted human enzyme (lysozyme) that allows for hydrolysis of cell wall polysaccharides (p. 2284-2285, under Metabolic pathway introduction into S. boulardii; and Secretion of human lysozyme by S. boulardii). Liu teaches that S. boulardii is a generally recognized as safe (GRAS) probiotic capable of surviving in the intestinal tract, allowing for in-gut production of therapeutic proteins beneficial for gut health (p. 2280, ¶1-2; p. 2885, under Secretion of human lysozyme by S. boulardii).
Regarding claim 2, Kim 2020 teaches a gene sequence encoding beta-agarase BpGH16A from B. pleibeius strain DSM17135 (SEQ ID 4), which comprises instantly claimed SEQ ID 1 with 100% identity (see Sequence Search Result #2, under us-18-571-437-1.rnpbm).
Regarding claims 3-5, the lysozyme was associated with a chicken lysozyme signal peptide having 100% identity to instant SEQ ID 2 (see attached alignment) (p. 2285, under Secretion of human lysozyme by S. boulardii; Supp. Materials, p. 5 (showing signal peptide sequence)).
Regarding claim 6, Liu teaches that the CRISPR-Cas9 system used to transform the S. boulardii (see above) has numerous advantages, including rapid and efficient targeting without off-target effects, and an absence of unnecessary scars and/or residual genetic elements (p. 2285, last ¶ to p. 2286, 1st ¶).
It would have been obvious to one of ordinary skill in the art at the time the invention was made to prepare a composition for converting agarose to neoagarooligosaccharides comprising a strain expressing beta-agarase BpGH16A (and optionally BpGH117) as taught by Kim 2019 wherein the strain is an S. boulardii strain engineered via CRISPR-Cas9 as taught by Liu with the BpGH16A gene taught by Kim 2020 because it would have been obvious to combine prior art elements according to known methods to yield predictable results. One of ordinary skill would have been motivated to use an S. boulardii strain engineered via CRISPR-Cas9 as taught by Liu as the agarase-expressing strain because Liu teaches that S. boulardii is GRAS and can survive in the intestinal tract allowing for continuous delivery of therapeutic proteins in the gut (e.g., to allow for conversion of agarose in red alae and other foods into neoagarooligosaccharides having beneficial prebiotic and therapeutic effects). Using an S. boulardii strain engineered via CRISPR-Cas9 as taught by Liu as the agarase-expressing strain in the composition of Kim 2019 would have led to predictable results with a reasonable expectation of success because Liu teaches a general method for engineering S. boulardii with a gene of interest (see e.g., Liu, p. 2286, last ¶) and exemplifies other carbohydrate-active enzymes, and BpGH16A is known and demonstrated to be expressible and a recombinant protein by Kim 2019.
Conclusion
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/ROBERT J YAMASAKI/Primary Examiner, Art Unit 1657