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 .
Priority
This application is a 371 of PCT/KR2021/015406 filed on 10/29/2021. PCT/KR2021/015406 claims foreign priority to 10-2020-0143486 KR filed Oct. 30, 2020.
Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a)-(d) prior to declaration of an interference, a certified English translation of the foreign application must be submitted in reply to this action. 37 CFR 41.154(b) and 41.202(e).
Failure to provide a certified translation may result in no benefit being accorded for the non-English application.
Claim Status
Claims 1-20 have been cancelled prior to examination. Claims 21-40 are pending and under examination. Claims 21, and 33 are independent claims.
Claim Interpretation
The following is a quotation of 35 U.S.C. 112(f):
(f) Element in Claim for a Combination. – An element in a claim for a combination may be expressed as a means or step for performing a specified function without the recital of structure, material, or acts in support thereof, and such claim shall be construed to cover the corresponding structure, material, or acts described in the specification and equivalents thereof.
The following is a quotation of pre-AIA 35 U.S.C. 112, sixth paragraph:
An element in a claim for a combination may be expressed as a means or step for performing a specified function without the recital of structure, material, or acts in support thereof, and such claim shall be construed to cover the corresponding structure, material, or acts described in the specification and equivalents thereof.
The claims in this application are given their broadest reasonable interpretation using the plain meaning of the claim language in light of the specification as it would be understood by one of ordinary skill in the art. The broadest reasonable interpretation of a claim element (also commonly referred to as a claim limitation) is limited by the description in the specification when 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, is invoked.
As explained in MPEP § 2181, subsection I, claim limitations that meet the following three-prong test will be interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph:
(A) the claim limitation uses the term “means” or “step” or a term used as a substitute for “means” that is a generic placeholder (also called a nonce term or a non-structural term having no specific structural meaning) for performing the claimed function;
(B) the term “means” or “step” or the generic placeholder is modified by functional language, typically, but not always linked by the transition word “for” (e.g., “means for”) or another linking word or phrase, such as “configured to” or “so that”; and
(C) the term “means” or “step” or the generic placeholder is not modified by sufficient structure, material, or acts for performing the claimed function.
Use of the word “means” (or “step”) in a claim with functional language creates a rebuttable presumption that the claim limitation is to be treated in accordance with 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph. The presumption that the claim limitation is interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, is rebutted when the claim limitation recites sufficient structure, material, or acts to entirely perform the recited function.
Absence of the word “means” (or “step”) in a claim creates a rebuttable presumption that the claim limitation is not to be treated in accordance with 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph. The presumption that the claim limitation is not interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, is rebutted when the claim limitation recites function without reciting sufficient structure, material or acts to entirely perform the recited function.
Claim limitations in this application that use the word “means” (or “step”) are being interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, except as otherwise indicated in an Office action. Conversely, claim limitations in this application that do not use the word “means” (or “step”) are not being interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, except as otherwise indicated in an Office action.
This application includes one or more claim limitations that do not use the word “means,” but are nonetheless being interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, because the claim limitation(s) uses a generic placeholder that is coupled with functional language without reciting sufficient structure to perform the recited function and the generic placeholder is not preceded by a structural modifier. Such claim limitation(s) is/are: “a gene encoding fusion partners for improving solubility and expression of recombinant proteins” in claims 21 and 35.
Because this claim limitation(s) is being interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, it is being interpreted to cover the corresponding structure described in the specification as performing the claimed function, and equivalents thereof.
If applicant does not intend to have this/these limitation(s) interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph, applicant may: (1) amend the claim limitation(s) to avoid it/them being interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph (e.g., by reciting sufficient structure to perform the claimed function); or (2) present a sufficient showing that the claim limitation(s) recite(s) sufficient structure to perform the claimed function so as to avoid it/them being interpreted under 35 U.S.C. 112(f) or pre-AIA 35 U.S.C. 112, sixth paragraph.
The limitation “a gene encoding fusion partners for improving solubility and expression of recombinant proteins” is interpreted under 35 U.S.C. 112(f). The term “fusion partner” fails to recite sufficiently definite structure. The specification does not use or define the term fusion partner, nor does it identify structural characteristics that would allow a person of ordinary skill in the art to understand the scope of such elements. Instead, the limitation is defined solely by the function of improving solubility and expression of recombinant proteins. Accordingly, the limitation is interpreted as a means-plus-function limitation under 35 U.S.C. 112(f), and is construed to cover the corresponding structure described in the specification and equivalents thereof. The corresponding structure is understood to include the specific fusion construct disclosed in the specification that are associated with improved solubility and/or expression, such as maltose-binding-protein (MBP), as well as structural equivalents thereof. This interpretation will be used for claims 21 and 35 and any claims that depend upon them.
Claim Rejections - 35 USC § 112
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.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
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 of carrying out his invention.
Claims 24, 38, and 39 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
The claims require that a regulatory gene causes expression of monomeric streptavidin in the periplasm of the host cell. While the specification describes regulatory genes as including elements such as ribosome bindings, promoters, and transcription factor binding sites, these elements are known to regulate transcription and translation levels, but do not direct subcellular localization of proteins. Expression of proteins in the periplasm requires signal peptides or secretion sequences that direct proteins through cellular export pathways, and such elements are not disclosed as part of the regulatory gene (see Karyolaimos et al. (Frontiers in Bioengineering and Biotechnology, 2021), pg. 3 left col. 1st para.). The specification teaches using a secretion sequence with mannose binding protein (MBP) fused monomeric streptavidin (mSA) in plasmids involved in example 4 of the specification. Though the specification does not teach how the claimed result, periplasmic expression, can be achieved using the recited regulatory gene, and a person of ordinary skill in the art would not be able to practice the full scope of the claimed invention without undue experimentation. Therefore, the claims are not enabled.
In determining whether the specification enables the full scope of the claimed invention, the factors set forth in In re Wands have been considered.
(A) The breadth of the claims: The breadth of the claims is significant, as the claims require that a regulatory gene causes expression of monomeric streptavidin in the periplasm of a host cell. The claims are not limited to any particular sequence, mechanism, or structural feature that would direct localization to the periplasm, thereby encompassing a wide range of embodiments without corresponding guidance in the specification.
(B) The nature of the invention: The nature of the invention involves recombinant protein expression and subcellular localization. Achieving expression of a protein in the periplasm requires specific biological mechanisms, including the use of signal peptides or secretion sequences that direct proteins through cellular export pathways.
(C) The state of the prior art: The art is silent towards regulatory genes that control cellular localization in gram-negative bacteria. The state of the prior art establishes that “[t]o reach the periplasm, a protein has to be equipped at its N-terminus with a cleavable signal peptide so that it can cross the cytoplasmic membrane” (see Karyolaimos et al. (Frontiers in Bioengineering and Biotechnology, 2021), pg. 3 left col. 1st para.). Periplasmic localization of proteins is achieved through known mechanisms such as signal peptides (e.g. MalE, PelB, OmpA, DsbA, PhoA) that direct proteins to the periplasm via secretion pathways. Mirzadeh et al. (Microb. Cell Fact., 2020) teaches that while different signal peptides impact periplasmic localization for recombinant proteins in an unpredictable manner (see pg. 1 Background) and that changes to the translation initiation region may help overcome this unpredictability, the signal peptides are still required (see pg. 1 Results). Such mechanisms are distinct from regulatory sequences that control transcription or translation levels. Given that the state of the art has established that signal peptide is required for periplasmic localization, a person of ordinary skill in the art would not be able to make/use the invention without undue experimentation because the claimed mechanism contradicts known periplasmic localization mechanisms and the art is silent to a regulatory gene controlling cellular localization.
(D) The level of one of ordinary skill: The level of ordinary skill in the art is relatively high, as practitioners in the field of molecular biology and recombinant protein expression are familiar with gene expression systems and protein targeting mechanisms. However, even a person of ordinary skill would require appropriate guidance to achieve the claimed results using the specific elements recited in the claims.
(E) The level of predictability in the art: Although aspects of recombinant protein expression are generally predictable, the predictability of achieving subcellular localization using the elements described in the specification is limited. In particular, regulatory sequences such as promoters and ribosome binding sites are not known to control protein localization, and therefore do not predictably result in periplasmic expression.
(F) The amount of direction provided by the inventor: The specification provides minimal direction or guidance regarding how to achieve periplasmic expression using the claimed regulatory gene. While regulatory elements such as promoters and ribosome binding sites are described, the specification does not teach the sue of signal peptides or other localization sequences required to direct proteins to the periplasm.
(G) The existence of working examples: The specification does not provide working examples demonstrating expression of monomeric streptavidin in the periplasm of a host cell using the claimed regulatory gene. In example 4 of the specification, the applicants utilized a plasmid containing a secretion sequence with the MBP-mSA fusion, which is separate from the claimed regulatory gene. The absence of examples using the regulatory gene to drive periplasmic expression weighs against enablement, particularly given the specific localization required by the claims.
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure: The quantity of experimentation required to practice the full scope of the invention would be substantial. A person of ordinary skill in the art would need to identify and incorporate appropriate signal peptides or secretion mechanisms not disclosed in the specification, and further determine how to achieve the claimed periplasmic expression, thereby requiring undue experimentation.
Considering the above factors together, undue experimentation would be required for a person of ordinary sill in the art to practice the full scope of the claimed invention. Accordingly, claims 24, 38, and 39 are not enabled.
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.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 22, 24-27, 30, 33-40 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, 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.
Where applicant acts as his or her own lexicographer to specifically define a term of a claim contrary to its ordinary meaning, the written description must clearly redefine the claim term and set forth the uncommon definition so as to put one reasonably skilled in the art on notice that the applicant intended to so redefine that claim term. Process Control Corp. v. HydReclaim Corp., 190 F.3d 1350, 1357, 52 USPQ2d 1029, 1033 (Fed. Cir. 1999). The term “regulatory gene” in claims 21-30, 33-34 is used by the claim to mean “non-coding regulatory sequences,” while the accepted meaning is “a segment of DNA that contains instructions for building one or more molecules.(see NIH, “Gene” The term is indefinite because the specification does not clearly redefine the term.
Furthermore, Claims 22 and 30 recite the limitation "the gene encoding monomeric streptavidin" in line 2. There is insufficient antecedent basis for this limitation in the claims.
Claim 24 recites the limitation “the monomeric streptavidin” spanning lines 1 and 2. There is insufficient antecedent basis for this limitation in the claim.
The term “total Gibbs free energy change (ΔGtotal)” in claims 25 and 34 is indefinite. The claim recite the “regulatory gene has a total Gibbs free energy change of (ΔGtotal) of 0 or less.” However, a gene, as a sequence of nucleotides, does not inherently possess a Gibbs free energy change absent a defined process or interaction. Gibbs free energy change is a thermodynamic parameter that depends on specific conditions and interactions, such as RNA folding, ribosome binding, or hybridization, none of which are specified in the claim. It is therefore unclear what physical or biochemical process the recited ΔGtotal refers to, and a person of ordinary skill in the art would not be able to determine the scope of the claim with reasonable certainty. Accordingly, the claim is indefinite.
The term “translation initiation rate controlled within a predetermined range” in claim 26 is indefinite. The claim recites that “the regulatory gene has a translation initiation rate (TIR) controlled within a predetermined range.” However, translation initiation rate is not a property of a gene itself, but rather a property of the process of translation of an mRNA molecule, which depends on factors such as ribosome binding, mRNA secondary structure, and cellular conditions. It is therefore unclear how a gene, as a nucleotide sequence, can be said to “have” a translation initiation rate. Additionally, the phrase “controlled within a predetermined range” lacks clarity, as the claim does not specify the range, the method of control, or how the translation initiation rate is measured. Accordingly, a person of ordinary skill in the art would not be able to determine the scope of the claim with reasonable certainty, and the claim is indefinite.
In regards to claim 27 and 34, the claim recites “the translation initiation rate of the regulatory gene is 50 to 45,000 AU.” However, as discussed with respect to claim 26, translation initiation rate is not a property of a gene itself. Furthermore, the recitation of arbitrary units (AU) renders the scope of the claim unclear, as the claim does not define the basis for these units, the method of measurement, or the model used to determine the translation initiation rate. Accordingly, a person of ordinary skill in the art would not be able to determine the scope of the claim with reasonable certainty, and the claim is indefinite.
In regards to claim 33, the recites “a method of screening a regulatory gene…” comprising introducing genes and measuring an expression level. However, the claim does not specify how the measured expression level is use to perform the recited “screening,” nor does it provide a comparison, threshold, or decision criterion by which a candidate regulatory gene is identified as regulating expression. Additionally, the claim doesn’t specify whether expression level refers to transcription (e.g. mRNA production) or translation (e.g. protein production). These represent distinct biological measurements that may yield different results depending on the system used, which is also not specified in the claim. The existence of claim 36, which requires the vector to be transformed into a host cell, would imply that this limitation does not already exist in claim 33 and that cell free systems for production may be evaluated. As such, it is unclear how the recited steps constitute a screening method, and the scope of the claim is not reasonably certain.
Neither claims 34 nor 35 rectify the deficiencies noted above with respect to claim 33 for which they both depend and are likewise indefinite.
Claim 36 depends from claim 33 and recites that the expression level is measured from a host cell transformed with the vector. While this limitation provides some context for where expression occurs, it does not remedy the deficiencies noted above. In particular, claim 36 still fails to define how the measured expression level is used to perform the recited screening, including the absence of any comparison or decision criterion for identifying candidate regulatory genes. Claim 40 depends from claim 36, but does not remedy the deficiencies noted above.
Claim 37 depends on claim 36 and recites determining that a candidate regulatory gene increases expression when “the expression level of monomeric streptavidin expressed from the host cell is higher than that before the candidate regulatory gene is introduced.” However, the claim does not specify how the “before” expression level is to be established or measure. It is unclear whether this baseline refers to expression in the same host cell prior to introduction, a separate control cell lacking the candidate regulatory gene, or another reference condition. The claim further fails to define whether the measurements are performed under identical condition or using the same assay. Because multiple reasonable interpretations exist for establishing the comparison, it is unclear how the determination is to be made and whether the limitation is satisfied. Accordingly, the metes and bounds of the claim are not reasonably certain.
Claim 38 depends on claim 36, which recites a “host cell” without limitation to a particular type of cell. The scope of the claim therefore encompasses a wide range of host cells including eukaryotic cells such as mammalian, plant, and insect cells. Claim 38, further requires measuring expression of mSA “in a periplasm of the transformed host cell.” However, many host cells within the scope of claim 36 do not possess a periplasmic compartment. As a result, it is unclear how the recited measurement steps is to be performed for such embodiments, or whether the limitation is satisfied. Accordingly, the scope of the claim is not reasonably certain.
In regards to claim 39, the claim depends on claim 38 and further recites a determination based on expression of mSA “in the periplasm of the transformed host cell.” For the reasons discussed above with respect to claim 38,the scope of the claim encompasses embodiments in which the host cell lacks a periplasmic compartment, rendering it unclear how the recited condition is satisfied. Additionally, the claim recites that “when the monomeric streptavidin is expressed in the periplasm of the transformed host cell, it is determined that the candidate regulatory gene is a gene that increases expression.” This limitation introduces a conditional statement without specifying a comparison or baseline for determining an increase in expression. The claim does not define how the determination is made or what constitutes an “increase,” and appears to equate the localization with an increase in expression, further contributing to the lack of clarity. Accordingly, the metes and bounds of the claim are not reasonably certain.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 21-23, 29 are rejected under 35 U.S.C. 103 as being unpatentable over Demonte et al. (“Expression and purification of soluble monomeric streptavidin in Escherichia coli”, Applied Microbiology and Biotechnology, 2014, on IDS, as evidenced by Addgene plasmid #52319).
Demonte teaches expression and purification of soluble monomeric streptavidin, including a gene construct comprising: a gene encoding biotin-binding protein (monomeric streptavidin); a gene encoding fusion partners for improving solubility (maltose binding protein; MBP) and expression of recombinant proteins (using an expression plasmid in E. coli).
Demonte additionally teaches expression control elements within the construct, including T7 promoter which regulates expression of the gene encoding the biotin-binding protein. Such promoters and associated regulatory sequences are well known in the art to control transcription and expression of recombinant genes in host cells. Demonte further teaches that the construct comprises an ribosome binding site (RBS), that helps regulate ribosome binding and subsequent protein production. These components read on the limitation requiring “a regulatory gene that regulates expression of the gene encoding biotin-binding protein“ in claim 21. Accordingly, Demonte teach all of the limitations of claim 21.
In regards to claim 22, Demonte teaches that the T7 and RBS are located upstream of the gene encoding monomeric streptavidin.
In regards to claim 23, Demonte teaches the use of a ribosome binding site.
In regards to claims 28 and 29, Demonte teaches the use of a ribosome binding site with a sequence length of 15-39 bp (23 bp), which comprises SEQ ID 5 (AGG). Furthermore, Demonte also teaches a T7 promoter sequence which is 19 bp in length and includes SEQ IDS. 5-7 (ATAGG).
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In regards to claim 30, as shown above in regards to claims 28 and 29, the spacing between SEQ ID: 5 and the start of the gene encoding monomeric streptavidin is between 6 to 13 bp.
In regards to claim 31, Demonte teaches a recombinant vector comprising the gene construct of claim 21 (see Addgene plasmid #52319).
In regards to claim 32, Demonte teaches transforming E. coli to express the recombinant vector (see Demonte pg. 6286 right col. last para.) .
Claims 21-24 and 28-32 are rejected under 35 U.S.C. 103 as being unpatentable over Demonte et al. (Applied Microbiology and Biotechnology, 2014, as evidenced by Addgene plasmid #52319) as applied to claims 21-23, and 28-32 above, and included here for reasons supra, in view of Dammeyer et al. (“Broad host range vectors for expression of proteins with (Twin-) Strep-tag, His-tag and engineered, export optimized yellow fluorescent protein”, Microbial Cell Factories, 2013, as evidence by Addgene plasmid #45944).
Demonte teaches a gene construct comprising a gene encoding a biotin-binding protein, namely monomeric streptavidin, fused toa fusion partner (MBP) for improving solubility and expression, and further comprising regulatory sequences including a ribosome binding site that regulate expression of the encoded protein. Demonte additionally teaches that the construct is engineered to produce soluble protein and reduce formation of inclusion bodies during expression in Escherichia coli. Demonte does not teach expression in the periplasm of a host cell.
Dammeyer teaches that recombinant proteins expressing in E. coli may be directed to the periplasmic space through the use of signal peptides, such as PelB leader sequence, and that periplasmic expression provides advantages including enhanced protein folding and disulfide bond formation (see Dammeyer pg. 3 left col. 2nd para.). Dammeyer further teach that periplasmic targeting via such signal sequences results in localization of expressed proteins in the periplasm of the host cell (see Dammeyer Fig. 4).
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to modify the construct of Demonte to include a signal peptide, such as the PelB leader sequence taught by Dammeyer in order to direct expression of the monomeric streptavidin to the periplasm of the host cell. Such as modification would have been motivated by the well-known advantages of periplasmic expression, including improved protein folding and formation of disulfide bonds, particularly for proteins prone to misfolding or aggregation.
Although Demonte utilizes cytoplasmic expression conditions, including engineered host strains, to promote proper folding and reduce inclusion body formation, this does not teach away from periplasmic expression. Rather, it reflects one of multiple known strategies in the art for improving protein folding and solubility. Dammeyer, explicitly teaches an alternative approach, periplasmic targeting via signal peptides, to achieve a similar goal. A person of ordinary skill in the art would have recognized these approaches as interchangeable and would have selected between them as a matter of routine optimization depending on the desired expression outcome.
Claims 21-40 are rejected under 35 U.S.C. 103 as being unpatentable over Demonte et al. (Applied Microbiology and Biotechnology, 2014, as evidenced by Addgene plasmid #52319) in view of Dammeyer et al. (Microbial Cell Factories, 2013, as evidence by Addgene plasmid #45944) as applied to claims 21-24, and 28-32 above, and included here for reasons supra, in view of Salis, H. (“Ribosome Binding Site Calculator”, Methods in Enzymology, 2011).
As outlined above, Demonte teaches the limitations of claim 21 for which claims 25 depends. Demonte teaches a gene construct comprising a gene encoding a biotin-binding protein (monomeric streptavidin), a fusion partner (MBP) for improving solubility and expression, and regulatory sequences including ribosome binding site that regulates expression of the encoded protein. Demonte does not teach the physical parameters of the regulatory gene, specifically related to the Gibbs free energy.
Salis, however, teaches that expression of recombinant proteins can be controlled by designing regulatory sequences, including ribosome binding sites and adjacent untranslated regions, using thermodynamic models, including calculations of the total Gibbs free energy change (ΔGTotal) associated with ribosome binding and mRNA folding (see Salis Section 4, pgs. 27-38). Salis further teaches that optimizing ΔGTotal, including values of 0 or less, improves translation initiation and protein expression.
It would have been prima facie obvious to one of ordinary skill in the art at the time of filing to optimize the regulatory sequence of the construct of Demonte, including ribosome binding site and adjacent untranslated region, using known thermodynamic design principles as taught by Salis, in order to improve expression of the recombinant protein. Such optimization of ΔGTotal represents routine tuning of known expression elements to achieve predictable improvements in protein production, which is a stated objective of Demonte.
In regards to claims 26 and 27, Demonte teaches a gene construct comprising a gene encoding a biotin-binding protein and a regulatory sequence including a ribosome binding site that regulates expression of the encoded protein. Demonte does not teach controlling the regulatory gene translation initiation rate (TIR).
However, Salis teaches that translation initiation rate can be controlled by designing regulatory sequences, including ribosome binding sites and adjacent untranslated region, and that such rates can be tuned within the desired ranges by modifying sequence features affecting ribosome binding and mRNA structure (see section 2.5, pg. 24). Salis further teaches that translation initiation rate can be tuned across a wide range by modifying sequence features and that such rates are commonly expressed in arbitrary units based on predictive models, such that “[a] protein CDS translated at 1000 au will produce 10 times more protein than one translated at 100 au, assuming that all other conditions are equal…” (see section 1.1, pg. 20).
It would have been obvious to a person of ordinary skill in the art to modify the regulatory sequence of the construct of Demonte to control translation initiation rate within the desired range as taught by Salis, in order to achieve predictable levels of protein expression. Such optimization of expression parameters, including tuning thermodynamic properties and translation initiation rates, represents routine experimentation to achieve predictable improvements in protein expression.
In regards to claim 33, Demonte teaches introducing a gene encoding a biotin-binding protein, namely monomeric streptavidin, into an expression vector comprising regulatory sequences, including a ribosome binding site, and expressing the protein in a host cell. Demonte further teaches evaluating expression of the recombinant protein, including obtaining soluble protein, and measuring expression levels (see Demonte Figs. 2-6).
Salis teaches that regulatory sequences, including ribosome binding sites and untranslated regions, can be varied and designed to control expression of a gene, and that expression levels of recombinant proteins are measured to evaluate the effect of such regulatory sequences.
It would have been obvious to a person of ordinary skill in the art to introduce candidate regulatory sequences into an expression vector comprising a gene encoding monomeric streptavidin and to measure expression levels in order to identify regulatory sequences that modulate expression, as this represents routine screening and optimization of gene expression systems.
In regards to claim 34, Demonte teaches introducing a gene encoding a biotin-binding protein, namely monomeric streptavidin, into an expression vector comprising regulatory sequences, including a ribosome binding site, and expressing the protein in a host cell. Demonte further teaches evaluating expression of the recombinant protein, including obtaining soluble protein, and measuring expression levels (see Demonte Figs. 2-6).
Salis teaches that regulatory sequences, including ribosome binding sites and adjacent untranslated regions, can be systematically designed and varied to control gene expression, including by optimizing thermodynamic parameters such as total Gibbs free energy change of ribosomal binding (see Salis Section 4, pgs. 27-38) and translation initiation rate (see Salis section 2.5, pg. 24), and that such parameters may be tuned across a wide range, including values expressed in arbitrary units (see Salis see section 1.1, pg. 20). Salis further teaches that regulatory sequences typically comprise short nucleotide sequences and that expression can be optimized by adjusting sequence length and spacing between the ribosome binding site and the initiation codon, including spacing within defined ranges. It would have been obvious to one of ordinary skill in the art to select candidate regulatory sequences satisfying one or more of the recited conditions, including thermodynamic parameters, translation initiation rates, sequence lengths, and spacing relative to the initiation codon, as such parameters are known to affect gene expression and are routinely optimized. With respect to the recitation that “the candidate regulatory gene comprises a gene sequence represented by any one of SEQ ID NOs: 5 to 7,” Demonte teaches regulatory sequences, including ribosome binding sites and promoter region, that inherently include short nucleotide motifs corresponding to the recited sequences. For example, Shine-Dalgarno sequences commonly include motifs such as “AGG,” and promoter regions similarly contain short nucleotide sequences overlapping with the recited sequences of “AGG”, “TAGG”, or “ATAGG” of SEQ ID NOs: 5 to 7, respectively. Given the extremely short length of these sequences (3-5 nucleotides), such motifs are ubiquitous in regulatory regions, and their presence in the prior art construct, would have been recognized by a person of ordinary skill in the art. Accordingly, the claimed subject matter would have been obvious.
In regards to claim 35, Demonte teaches method comprising introducing a gene encoding a biotin-binding protein, namely monomeric streptavidin, into an expression vector comprising regulatory sequences and expressing the protein in a host cell (see Demonte pg. 6286 right col. last para.). Demonte further teaches that the gene encoding the biotin-binding protein is fused to a gene encoding a fusion partner, namely maltose binding protein (MBP), which improves solubility and expression of the recombinant protein, and that this fusion construct is introduced into the vector for expression (see Demonte pg. 6286 left col. 2nd para.).
In regards to claims 36 and 37, Demonte teaches transforming a host cell, such as Escherichia coli, with a vector to express recombinant monomeric streptavidin. Demonte further teaches producing and isolating soluble monomeric streptavidin from the host cell, and measuring the expression levels (see Demonte Figs. 2-6. Pg. 6288 right col. 3rd para.). Salis teaches varying regulatory sequences to modulate expression and measuring expression levels to evaluate the effect of such variations. It would have been obvious to a person of ordinary skill in the art to compare expression levels obtained using different regulatory sequences to determine that a given regulatory sequence increases expression when it produces a higher expression level relative to a baseline, as such comparison and interpretation represent routine evaluation of experimental results. Therefore, the claimed subject matter would have been obvious.
In regards to claims 38 and 39, Demonte teaches a method comprising introducing a gene encoding monomeric streptavidin into an expression vector, transforming a host cell such as E. coli with the vector, and expressing the recombinant protein, thereby enabling measurement of expression levels of monomeric streptavidin produced by the host cell (see Demonte Fig. 2-6). Dammeyer teaches that recombinant protein expressing in E. coli may be directed to the periplasmic space through the use of signal peptides, such as the PelB leader sequence, and that such periplasmic expression enables production and measurement of proteins localized in the periplasm (see Dammeyer Fig. 2). It would have been obvious to one of ordinary skill in the art to modify the method of Demonte to direct expression of monomeric streptavidin to the periplasm of the host cell using known signal peptides, as taught by Dammeyer in order to improve protein folding and stability.
Furthermore, it would have been obvious to define criteria for determining whether a regulatory sequence increases expression based on observed experimental outcomes, including protein localization, as such criteria represent routine interpretation of experimental data, regardless of whether such criteria directly correlate with actual expression levels.
In regards to 40, Demonte, Dammeyer, and Salis each teach methods in which an expression vector is introduced into host cells and measuring expression levels. In order to evaluate the expression levels, the cell must be cultured after transformation. As such, the combination of Demonte, Dammeyer, and Salis teach or at least render obvious, culturing the transformed host cells.
Conclusion
No claim is allowed
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/MATTHEW HAROLD RAYMONDA/Examiner, Art Unit 1684
/AARON A PRIEST/ Primary Examiner, Art Unit 1681
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