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 .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 November 20, 2025 has been entered.
Application Status
Applicant’s remarks, replacement drawings, Exhibit D (EP 3921423 B1), and amendments to the claims filed November 20, 2025 are acknowledged. Claims 1-2, 5-6, 8-9, 11, 13-14, 26, 32, and 40 were amended, claim 25 was canceled, and claim 41 is newly introduced. Accordingly, claims 1-6, 8-9, 11, 13-14, 21, 24, 26-27, 32, 34, 38, and 40-41 are pending.
Restriction/Election
Claim 41, which depends from claim 1, is drawn to the elected invention. Claims 32, 34, and 38 remain withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention. Claims 1-6, 8-9, 11, 13-14, 21, 24, 26-27, and 40-41 are under examination hereinafter.
Withdrawn Rejections
Applicant’s remarks and amendments have been thoroughly reviewed. The amendments to the claims overcome the § 112(b) rejections raised in the prior action. The amendments to the claims to remove the term “mutant derivatives,” and to recite specific art-recognized classes of reporter proteins or specific art-recognized reporter proteins are sufficient to overcome the § 112(a) Written Description rejections raised in the prior action.
Applicant’s remarks and amendments are not persuasive to place the claims in condition for allowance for the reasons that follow. Any objection or rejection not reiterated herein has been overcome by amendment.
Priority
Applicant’s priority claims to Application Nos. LU101118 and PCT/EP2020/052985 are acknowledged. Claims 1-6, 8-9, 11, 13-14, 21, 24, 26-27, and 40-41 find support in Application No. LU101118 and therefore, the effective filing date of the claims under examination is February 6, 2019.
Drawings
The drawings are objected to because of the following informalities:
The view numbers for partial views of Fig. 5 (sheets 18 and 23) and Fig. 6 (sheets 30-34) are followed by "(cont.)" instead of a capital letter such as FIG. 1A, FIG. 1B, etc. 37 CFR 1.84 (u)(1) states “Partial views intended to form one complete view, on one or several sheets, must be identified by the same number followed by a capital letter.”
Appropriate correction is required.
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Objections
Claim 41 is objected to because of the following informalities:
Claim 41 recites “SEQ ID NO: 33, 34, 35, 36, 36, 38, 39, or 40.” The claim recites SEQ ID NO: 36 twice, and it would be preferable to delete the extra instance of SEQ ID NO: 36.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(d)
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.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], 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 3-4 and 40 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, 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. The rejections that follow are new and necessitated by Applicant’s amendments to the claims.
Claim 3 recites “an expression product of the N-terminal splicing region of the split intein comprises at its N-terminus a cysteine or a serine.” Claim 1 already requires “an expression product of the split intein-reporter polynucleotide construct,” and that “the amino acid sequence of the N-terminal splicing region of the split intein comprises a cysteine or a serine residue at its N-terminus.” Claim 3 does not further limit the subject matter of claim 1 upon which it depends.
Claim 4 recites “an expression product of the C-terminal splicing region of the split intein comprises at its C-terminus an asparagine.” Claim 1 already requires “an expression product of the split intein-reporter polynucleotide construct,” and that “the amino acid sequence of the C-terminal splicing region of the split intein comprises an asparagine at its C-terminus.” Claim 4 does not further limit the subject matter of claim 1 upon which it depends.
Claim 40 requires a selection from a list of reporter proteins. Claim 5, from which it depends, also requires a selection from a list of reporter proteins (“wherein the reporter polynucleotide encodes a reporter protein selected from the group consisting of… wherein when the reporter protein is…). The reporter proteins recited in claim 40 are the same as those recited in claim 5, and accordingly, claim 40 does not further limit the subject matter of claim 5 upon which it depends.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 112(a) – Scope of Enablement
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 1-6, 8-9, 11, 13-14, 21, 24, 26-27, and 40-41 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph. The specification is enabling for a method of detecting a specific RNA splice event of a gene, wherein the method comprises (i) inserting a split intein-reporter polynucleotide construct into an exon in the DNA sequence encoding the gene, wherein the reporter polynucleotide encodes NanoLuc, HaloTag, scAvidin, or firefly luciferase, wherein the split intein comprises an N-terminal splicing region and a C-terminal splicing region upstream and downstream of the reporter polynucleotide, respectively, wherein the N- and C-terminal splicing regions encode the N- and C-terminal splicing regions of gp41-1 or NrdJ-1 (i.e., SEQ ID NOs: 1 and 3, or 2 and 4), and wherein the construct further encodes the complementary coiled coil domains AP6 and P5, wherein the sequence encoding AP6 is downstream of the N-terminal splicing region, such that it is positioned between the N-terminal splicing region and the reporter polynucleotide, and wherein the sequence encoding P5 is upstream of the C-terminal splicing region, such that it is positioned between the C-terminal splicing region and the reporter polynucleotide, (ii) promoting formation of a translated form of the gene comprising the split intein-reporter polynucleotide construct, wherein the translated form of the split intein-reporter polynucleotide construct excises itself from the exon in the translated form of the gene, and (iii) detecting the translated form of the split intein-reporter polynucleotide construct.
The specification does not reasonably provide enablement for achieving the method’s outcome (i.e., detecting a specific RNA splice event of a gene of interest) by inserting a split intein-reporter polynucleotide construct encoding any split intein with the recited amino acid residues, and any reporter polynucleotide into any exon of interest, and detecting the reporter polynucleotide and/or its expression product. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to use the invention commensurate in scope with these claims. The rejections of claims 1-6, 8-9, 11, 13-14, 21, 24, 26-27, and 40 are maintained from the prior action with modification necessitated by Applicant’s amendments and to respond to Applicant’s arguments. The rejection of claim 41 is new and necessitated by Applicant’s amendments.
The test of enablement is whether one skilled in the art could make or use the claimed invention from the disclosures in the specification coupled with information known in the art without undue experimentation (United States v. Telectronics Inc., 857 F.2d 778, 785, 8 USPQ2d 1217, 1223 (Fed. Cir. 1988)). Whether undue experimentation is needed is not based upon a single factor, but rather is a conclusion reached by weighing many factors. These factors were outlined in In re Wands, 858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988), and the most relevant factors are indicated below:
Nature of the Invention and Breadth of Claims
Claim 1 recites “A method for detecting a specific RNA splice event of a gene, wherein the specific RNA splice event creates a specific RNA splice product, which comprises an exon.” “A specific RNA splice product, which comprises an exon” is interpretated as an RNA transcribed from the gene, which is spliced in the “specific splice event,” and comprises an exon of interest, which is interpreted as a part of the RNA retained after splicing ([0031]; [0035]). The method requires (i) inserting a split intein-reporter polynucleotide construct into the exon. A “split intein” is an intein expressed in two halves – an N-terminal splicing region and a C-terminal splicing region – that catalyze splicing in trans upon association of the two domains ([0040]). The amino acid sequence of the N-terminal splicing region must comprise a cysteine or serine at its N-terminus, and the amino acid sequence the C-terminal splicing region must comprise an asparagine at its C-terminus. The genus of split inteins encompassed by the claims is limited only by two amino acids (i.e., a cysteine or serine, and asparagine). Based on the prior art, these residues are present in “virtually all inteins” and “highly conserved,” and correspond to residues involved in a conserved mechanism of trans-splicing (Shah and Muir, 2014, Chem. Sci. 2014, 5, pg. 446-461; pg. 451, left col.). The claim requires that the N-terminal and C-terminal splicing regions be “upstream of” and “downstream of” the reporter polynucleotide, respectively, in the exon. A “reporter polynucleotide” is interpreted as any nucleic acid which encodes a reporter protein recited in the claims. As described in paragraph 5 above, the skilled artisan would be familiar with reporter proteins within the classes of enzymes recited in the claim. The skilled artisan would also recognize that the reporter polynucleotides encompassed by the claims encompass many, diverse sequences.
The expression product of the split intein-reporter polynucleotide construct must excise itself from the expression product of the specific RNA splice product. The “expression product” of the specific RNA splice product is understood to be a translated polypeptide. Thus, the method requires that the translated form of the construct excise itself from the translated form of the specific RNA splice product. The amino acid C-terminal to the excision position/site must be either a cysteine, a serine, or a threonine. The method requires (ii) detecting the reporter polynucleotide and/or the expression product of the reporter polynucleotide. The detection means is not limited ([0053]), and the methods encompasses, for example, detecting the reporter polynucleotide at the DNA level, or its expression product at the RNA level, or protein level ([0037]).
Accordingly, enablement of the claims requires that one of ordinary skill in the art be able to detect an RNA splice event of any gene without undue experimentation, by I) inserting a split intein-reporter polynucleotide construct of essentially any sequence into an exon of the gene, wherein the construct must be capable of and inserted such that its translated form excises itself at a C-terminal cysteine, serine, or threonine, from the translated form of the expression product of the specific RNA splice product, and II) detecting the reporter polynucleotide and/or the expression product of the reporter polynucleotide.
Guidance in the Specification
The specification demonstrates that a gp41-1 split intein corresponding to SEQ ID NO: 16, and flanking mNeonGreen (i.e., the “reporter polynucleotide”) effectively splices from exon 2 of mouse Tubb3 ([00106]; Fig. 1A-B). The specification teaches that fast folding proteins (i.e., “expression products of the specific splice product”) present a challenge, because when the tagged protein folds faster than the split intein-reporter polynucleotide construct, “the native tertiary structure of the tagged protein might prevent the intein part from taking its final active form to excise itself out from the tagged protein and being thus trapped in its fusion form” ([00108]). The specification demonstrates this “worst case scenario” by tagging a fast folding protein (i.e., mNeonGreen) with a gp41-1 split intein corresponding to SEQ ID NO: 21 flanking NanoLuc ([00108]). The specification describes that the inclusion of “artificial anti-parallel CCs,” (i.e., coiled coils) improved construct excision efficiency, and therefore, the CCs were used in all remaining constructs downstream of the N-terminal splicing region, and upstream of the C-terminal splicing region ([00108]; “AP6” and “P5”, Fig. 2A-C).
The specification also demonstrates that a construct comprising a split intein, transmembrane domain, and CCs flanking scAvidin or HaloTag effectively spliced from mNeonGreen, and enabled detection of the splicing event in transfected cells in vitro via the membrane-anchored scAvidin and HaloTag constructs ([00113]; Fig. 2D-F). The specification teaches that “transient transfection” (such as that used in the aforementioned example) “does not always represent the behavior of stably integrated constructs in a low-copy allele-integrated physiological expression level” ([00114]). Accordingly, the specification describes an example in which the aforementioned HaloTag construct was integrated into the MAPT exon 10 ([00114]; Fig. 3A-B). The specification provides evidence that the method allowed detection of the splicing event in cells in vitro via the membrane-anchored HaloTag construct ([00115]; Fig. 3C), and that the excision of the split intein-reporter construct was “scarless” and did not impact Tau splicing ([00116]; Fig. 3D). The specification describes an adapted construct, in which HaloTag was substituted with NanoLuc, which was flanked by furin sites corresponding to SEQ ID NO: 20, and which enable release of NanoLuc into the extracellular environment, and consequently, detection of NanoLuc signal in cellular supernatant ([00118]; Fig,. 2G-H).
The specification also demonstrates a method for detecting “disease-relevant isoforms” of Tau, i.e., 3R or 4R Tau excluding or containing MAPT exon 10, respectively ([00119]-[00132]). The method comprises integrating an “optimized NLuc-based reporter (SEQ ID NO: 8) into exon 10 of MAPT” via CRISPR/Cas9 before Ser-293 ([00121]). The specification demonstrates that the splicing efficiency of Tau was unaffected by the integrated construct, and the construct (i.e., read out for the specific splicing event) could be detected with cellular resolution ([00121]-[00125]; Fig. 4-5). The specification also demonstrates that tagging a “constitutive exon” with a second, orthogonal split-intein reporter polynucleotide construct, which encodes a different split intein, CCs and reporter protein than that in the first construct, enables the skilled artisan to determine the overall levels of a protein ([00127]-[00130]; Fig. Fig. 6). The second, orthogonal construct described in the specification is firefly luciferase flanked by a N- and C- NrdJ-1 split intein corresponding to SEQ ID NO: 1 and 3 and an orthogonal coiled coil pair ([00130]). Finally, the specification describes two additional examples of using the split intein-reporter polynucleotides in different genetic contexts, i.e., FOXP1 exon 18b, and Oaz1 ([00133]-[00140]; Fig. 7).
In summary, the specification provides predictability for achieving the method’s outcome (i.e., detecting a specific splice event) by utilizing a split intein-reporter polynucleotide construct comprising one of two different split inteins, i.e., the gp41-1 pair corresponding to SEQ ID NOs: 2 and 4 and the NrdJ-1 pair corresponding to SEQ ID NOs: 1 and 3, flanking specific reporter polynucleotides, i.e., NanoLuc, HaloTag, scAvidin, and firefly luciferase, wherein the construct further comprises the antiparallel coiled coil pair described in the specification, i.e., AP6 and P5, in the positions described in the specification. The specification fails to provide predictability for achieving the method’s outcome with a split intein-reporter polynucleotide construct comprising a split intein limited by amino acids which are present in “virtually all inteins” or “highly conserved” e.g., those that splice slower than gp41-1 or NrdJ-1, and/or those requiring specific extein sequences for splicing. Without inclusion of the antiparallel coiled coils described in the specification, the specification also fails to provide predictability for achieving the method’s outcome with the many, diverse genes encompassed by the claims, e.g., genes which do not have the requisite sequences to mediate effective split intein splicing, and/or genes which fold at a rate that prohibits the split intein from excising from the gene’s expression product’s exon.
State of the Prior Art
The prior art was thoroughly searched for examples of the instantly claimed method, and guidance in performing the instantly claimed method. None of the prior art reviewed during the search provides a working example of detecting a specific RNA splice event of a gene using the instantly claimed method. The closest prior art method is described by Muller (Ramsden et al., 2011, BMC Biotechnology, 11:71, pg. 1-11; of record). However, as described below, Muller’s method detects a specific protein splicing event of a gene. Neither Muller, or the remaining prior art reviewed in the search suggest the use of Muller’s method for detecting a specific RNA splice event of a gene, or provide additional predictability for achieving the instantly claimed method’s outcome across its full scope.
Carvajal-Vallejos (Carvajal-Vallejos et al., 2012, Journal of Biological Chemistry, 287(34), pg. 28686-2696; of record) teaches that there are many natural and engineered split inteins (pg. 28686, right col. to pg. 28687, left col.). Carvajal-Vallejos teaches the mechanism through which split inteins trans-splice, which requires association of the intein fragments, formation of a reactive intermediate at the N-terminal cysteine or serine residue of the split intein (“In general, X can be sulfur or oxygen”), formation of a branched intermediate at a cysteine, serine, or threonine at the first C-terminal position in the C-extein (“first residue in the C-extein, which can be Cys, Ser, or Thr”), and cyclization of the conserved C-terminal asparagine residue of the split intein (“cyclization of the conserved Asn residue at the C terminus of the intein”) (Fig. 1A and description, pg. 28687; pg. 28689, left col.). Carvajal-Vallejos teaches that many artificial split inteins require refolding or show low solubility in fusion constructs, which limits their application in various biotechnology applications (pg. 28687, left col.). Carvajal-Vallejos also teaches that split intein’s splicing efficiency is influenced by native extein residues directly flanking the intein sequences (pg. 28694, left col.). Carvajal-Vallejos teaches that these additional, native extein residues are undesirable for certain applications, because they can add additional residues to a target protein, for example (pg. 28687, left col.). Carvajal-Vallejos teaches that split inteins have different splicing rates and yields, and requirements for these native extein sequences (pg. 28692-28694; Figs. 2-4, Tables 4-5). Carvajal-Vallejos teaches four split inteins which splice “exceptionally fast and efficiently” (pg. 28689, left col.; Fig. 2-3) and exhibit no promiscuity, thereby allowing for use of different split inteins in the same host cell (pg. 28692, right col.). Three of the split inteins – gp41-1, gp41-8, and NrdJ-1 – still splice without native extein sequences (pg. 28692, left col.; Table 4). Carvajal-Vallejos teaches that “the four inteins characterized in th[eir] study show the highest reaction rates and efficiencies in the trans-splicing reaction” (pg. 28695, left col.).
In summary, Carvajal-Vallejos teaches that split inteins share a conserved trans-splicing mechanism involving a cysteine or serine residue at the N-terminus of an N-terminal splicing region, and an asparagine at the C-terminus of a C-terminal splicing region (Fig. 1). Carvajal-Vallejos’ teachings strongly suggest that split inteins are not universally applicable to methods which require their insertion into, and excision from an exon of interest due to their differing splice rates and efficiencies, and reliance on specific extein sequences. Carvajal-Vallejos does teach three split inteins, i.e., gp41-1, gp41-8, and NrdJ-1, which splice quickly and effectively, and retain splicing capability without native extein sequences. However, Carvajal-Vallejos does not characterize the splicing efficiency of these split inteins in the context of a split intein-reporter polynucleotide construct.
Topilina (Topilina and Mills, 2014, Mobile DNA, 2014, 5:5, pg. 1-14; of record) teaches that inteins have been used for a wide variety of applications, e.g., genetic markers (Fig. 3H; “Inteins as genetic markers”, pg. 4-5), various labelling applications (Fig. 4, “In-cell protein labeling,” pg. 5-6), and as biosensors (Fig. 6, pg. 8-10). Topilina, referencing the work of Muller, teaches that a split intein (“interrupted inteins”) flanking selectable markers was able to splice and serve as a selectable marker for expression of a gene of interest (pg. 4, right col.). Muller teaches the method uses the Pch PRP8 intein, which is artificially split with a genetic marker, i.e., hygromycin B phosphotransferase, His5, or aminoglycoside phosphotransferase (“G148R”), and inserted into the “central sequence of the CMD1 [calmodulin] gene” in yeast (Fig. 4-5). Muller demonstrates that the artificially split intein-genetic marker constructs produced various levels of spliced and un-spliced products (Fig. 5B). Muller does not provide splicing data from the hygromycin B phosphotransferase construct. Although some spliced products were formed from the G148R constructs as evidenced by Fig. 5B, Muller concluded that both the hygromycin B phosphotransferase and G148R intein constructs “were not suitable for tagging” CMD1 (Fig. 5B; pg. 5, right col.). Muller teaches that they are “activity investigating ways to boost the level of stable protein and the efficiency of splicing through changes in codon usage and the composition of the linkers between the various domains” (pg. 7, right col.).
Taken together, Muller provides a similar method to that of the instant claims; however, Muller’s method does not detect a specific RNA splicing event, but rather, detects a specific protein splicing event (i.e., that of the artificially split Pch PRP8 intein flanking His5 or G148 R from the calmodulin gene in yeast). Indeed, Davis (Davis et al., 7 November 1986, Cell, Vol. 47, Issue 3, pg. 423-431; of record) teaches that the yeast CMD1 gene does not actually undergo RNA splicing (“The gene (designated CMD1) is a unique, single-copy locus, contains no introns”, Summary, pg. 423). While Muller demonstrates that a split intein-reporter polynucleotide construct may be used as a genetic marker, neither Muller, Topilina, nor the remaining art reviewed during the search provides any working examples of a method of detecting an RNA splice event with a split intein-reporter polynucleotide construct. The prior art also does not provide predictability for achieving the instantly claimed method’s outcome with:
any split intein which comprises the specific N-terminal splicing region’s N-terminal cysteine or serine, or C-terminal splicing region’s C-terminal asparagine. As evidenced by the prior art, these specific amino acids relate to a conserved trans-splicing mechanism of split inteins. However, there is no evidence in the art to support to support that the mere presence of these two amino acids which are present in “virtually all inteins” or “highly conserved” are sufficient to promote excision from the many, diverse reporter polynucleotide and gene contexts encompassed by the claim. Indeed, Carvajal-Vallejos teaches split inteins which despite having this conserved trans-splicing mechanism may not work in the method due to extein dependence or slow splice kinetics. Beyond the guidance related to gp41-1, gp41-8, and NrdJ-1, there is insufficient guidance to determine which split inteins with the recited amino acids would function in the method. Indeed, Shah teaches that only a small fraction of known inteins have been experimentally characterized in any way, which represents a “major deficiency in the intein field” (pg. 459, left col.).
any reporter polynucleotide or gene encompassed by the claim. Indeed, Muller and others strongly suggest that some reporter polynucleotides and/or genes of interest may not be permissive to mediate excision of the split intein-reporter polynucleotide construct (e.g., hygromycin B phosphotransferase in calmodulin), and provide no guidance to predict which reporter polynucleotides and/or genes of interest would be permissive for excision. Indeed, Friedel (Friedel et al., 3 October 2018, Chem. Sci. 2019, 10, pg. 239-251) teaches that “the origin of intein dependence on certain flanking amino acids is not well understood and the engineering of inteins with greater or unlimited tolerance is challenging” (pg. 247, left col.). Friedel teaches that “there is little precedent for non-native extein residues to promote, instead of to impair, the protein splicing pathway,” and concludes that “it is at present unpredictable” which inteins would be favorably amenable to unnatural flanking moieties (pg. 247, right col.; pg. 248, right col.).
Finally, Selgrade (Selgrade et al., 2014, J Am Chem Soc, 8;135(20), pg. 7713-7719; of record) teaches that complementary coiled coil domains (“EE” and “RR”; “LZB” and “LZA”) can “drive the specific association of [] intein/extein fusion proteins” and mediate VMA split intein trans-splicing (pg. 7713-7714; Fig. 1-2). Selgrade demonstrates that the coiled coil domains must be complementary, or inefficient trans-splicing results (Fig. 1D). Selgrade teaches that the system may be used with “two separate pairs of inteins with little cross-reactivity” (pg. 7717, right col.). Selgrade’s method differs from the instant method in that I) the construct is not inserted into a gene of interest in an exon of interest, II) the reporter polynucleotide, while flanked by N- and C- terminal splicing regions of the split intein, is itself split into N- and C- terminal domains. Selgrade appears to provide predictability for the use of two pairs of complementary coiled coil domains in the construct of the instant method (“EE” and “RR”; “LZB” and “LZA”), because as evidenced by Selgrade, the coiled coil domains drive split intein trans-splicing of at least one split intein. Selgrade does not provide guidance as to the location of the coiled coil domains within the construct, or any guidance as to complementary coiled coil domains that may not successfully drive trans-splicing.
In summary, the prior art fails to teach any working examples of the claimed method. The prior art provides predictability for achieving the method’s outcome (i.e., detecting a specific splice event) with one additional split intein, i.e., gp41-8 taught by Carvajal-Vallejos. The prior art also provides predictability for two additional complementary coiled coil pairs (“EE” and “RR”; “LZB” and “LZA”). The prior art does not provide predictability for achieving the method’s outcomes using any combination of split intein, reporter polynucleotide, and gene of interest. Indeed, the prior art strongly suggests that the efficiency of split intein splicing (and thereby, the outcome of the method) is highly dependent on the split intein’s splicing kinetics, and its interaction with surrounding sequences which mediate its splicing. Like the specification, Selgrade provides predictability for using complementary coiled coil domains within the construct, but none of the prior art reviewed during the search provides evidence which would suggest that the incorporation of complementary coiled coil domains are a universal “cure” that transforms slow splicing, inefficient, or extein sequence-constrained split inteins into split inteins suitable for the claimed method. Neither Selgrade, or the remaining art reviewed during the search provide guidance as the optimal locations of the coiled coil domains in the construct, or which, if any, of the known complementary coiled coil domain pairs are unsuitable for driving trans-splicing.
Experimentation Required and Level of Skill in the Art
In order to practice the invention, a large amount of highly unpredictable experimentation would be required. The skilled artisan would need to prepare split intein-reporter polynucleotides commensurate with the claims, i.e., constructs including sufficient representatives of natural and artificial split inteins, and sufficient representatives of reporter polynucleotides encoding the many, diverse proteins encompassed by the classes recited in the claim. The constructs would then need to be inserted into exons in genes of interest commensurate with the claims, i.e., those with diverse sequences flanking the insertion site. The skilled artisan would then need to detect the reporter polynucleotide and/or expression product of the reporter polynucleotide using any means, e.g., PCR, western blot, etc. The skilled artisan would need to confirm whether the expression product of the construct excised from the expression product of the specific splice product at the recited position, and whether the method actually enabled detection of a specific splice event of the gene.
The level of skill in the art is high, and the aforementioned experimentation would be well within the purview of the skilled artisan. However, such experimentation is undue because the claims at present still encompass virtually any split intein, any reporter polynucleotide (i.e., any nucleic acid encoding virtually any protein encompassed by the broad classes recited in the claims), and any gene of interest containing an exon with any sequence. The specification and prior art teach that the ability of the split intein-reporter polynucleotide construct to splice effectively from its integrated location is highly dependent on the folding kinetics of the host protein, the kinetics of the split intein’s splicing, and the interactions of the split intein with surrounding extein sequences, and to some undetermined degree based on Muller, the reporter polynucleotide sequence. Neither the specification nor prior art provide working examples commensurate with the broad scope of the claims. The prior art also provides examples on constructs which are minimally or not effective at excision (see Muller’s example with HygBR). Neither the specification nor prior art provide sufficient guidance for the skilled artisan to predict which of the many, incredibly diverse split intein-reporter polynucleotide constructs, would effectively splice from the many, incredibly diverse exons encompassed by the claim.
Conclusion
Taking into consideration the factors outlined above, including the nature of the invention, the breadth of the claims, the lack of working examples to meet the scope of the claims in either the specification or prior art, and the guidance in the specification and prior art which strongly suggests to the skilled artisan that the method’s outcome would not be achieved over the full scope of the claimed invention, it is the conclusion that undue experimentation would be required to make and use the invention as claimed.
Dependent Claims
Claims 2-6, 8-9, 11, 13-14, 21, 24, 26-27, and 40-41 do not further limit the method so as to resolve the scope of enablement issues above, and are therefore, not sufficiently enabled for the reasons above.
Response to Remarks - 35 USC § 112(a) – Scope of Enablement
Applicant’s remarks regarding the § 112(a) rejections raised in the prior action have been thoroughly reviewed. Applicant argues that the amendments to claim 1 to require “specific types of reporter polynucleotides and split inteins comprising specific residues, a skilled person will be able to make or use the invention” without undue experimentation based on the guidance in the specification and prior art. This argument is not convincing. While the claims have been amended to encompass reporter polynucleotides encoding specific classes of reporter proteins, the genus of reporter polynucleotides is still extremely broad, and encompasses many, diverse sequences. The guidance in the specification is limited to only a very small percentage of reporter polynucleotides encompassed by the claims. The prior art does not offer additional guidance to determine which of the many different reporter polynucleotides encompassed by the claims would be conducive for excision of the many split inteins encompassed by the claims. Although Applicant argues that the split inteins have been sufficiently limited by the requirement for two amino acid residues at particular locations in the N- and C-terminal splicing regions, based on the prior art these residues correspond to residues involved in a conserved mechanism of trans-splicing and are present in “virtually all inteins” or “highly conserved” (Shah, pg. 451, left col.). Based on the prior art, there are known inteins which exhibit extein sequence dependence, and/or have slow splicing kinetics which may make them unsuitable for the claimed method. There are also many known inteins that have not been experimentally characterized in any way (Shah, pg. 459, left col.). The guidance in the specification is limited to two split inteins (gp41-1 and NrdJ-1) in very narrow reporter polynucleotide and gene contexts compared to the instant claims. The prior art provides support for using one additional split intein, i.e., gp41-8 taught by Carvajal-Vallejos, but does not provide sufficient guidance to determine which of the “virtually all inteins” that have the recited residues would function in the method as claimed, due to their diverse, and in many cases completely uncharacterized, degrees of extein dependence and splicing kinetics.
Applicant, referencing MPEP 2164.01(b), states “as long as the Specification discloses at least one method of making and using the claimed invention that bears a reasonable correlation to the entire scope of the claim, then the enablement requirement… is satisfied.” Applicant refers to the “successful use of the claimed method in Fig 1A and 1B wherein a reporter protein encoded by a reporter polynucleotide, i.e., mNeonGreen, coupled to gp41-1, i.e., a split intein comprising a cysteine or a serine at its N-terminus and an asparagine residue at its C-terminus, effectively splices from an exon of a gene.” This argument and Applicant’s working example are not convincing evidence of enablement across the method’s full scope. The claims are examined for compliance with the enablement requirement based on an analysis of the Wands factors. MPEP 2164.01 states that “The specification may require a reasonable amount of experimentation to make and use the invention and what is reasonable will depend on the nature of the invention and the underlying art.” The nature of the invention and the underlying art are highly unpredictability, such that practicing the method across its claimed scope would require an unreasonable amount of experimentation.
For example, Carvajal-Vallejos teaches that many artificial split inteins require refolding or show low solubility in fusion constructs, which limits their application in various biotechnology applications (pg. 28687, left col.). With respect to naturally occurring inteins, Shah teaches that only a small fraction have been experimentally characterized in any way, which represents a “major deficiency in the intein field” (pg. 459, left col.). Carvajal-Vallejos teaches that a split-intein’s splicing efficiency is influenced by native extein residues directly flanking the intein sequences (pg. 28694, left col.). Friedel teaches that “the origin of intein dependence on certain flanking amino acids is not well understood and the engineering of inteins with greater or unlimited tolerance is challenging” (pg. 247, left col.). Friedel teaches that “there is little precedent for non-native extein residues to promote, instead of to impair, the protein splicing pathway,” and concludes that “it is at present unpredictable” which inteins would be favorably amenable to unnatural flanking moieties (pg. 247, right col.; pg. 248, right col.). These teachings are highly relevant to the claimed method, which requires that virtually any split intein, flanking any one reporter polynucleotide selected from an extremely broad and diverse group of sequences, be capable of trans-splicing when inserted into any exon of a gene with virtually any sequence.
As described in Examiner’s response in the prior action, the references cited by Applicant in an attempt to support enablement of the claims also support unpredictability for the claimed method. Exhibit A, teaches that despite substantial effort, “at the moment no reliable a priori prediction can be made about whether an intein will be active in a particular non-native extein context or estimations about the splicing efficiency” (pg. 1, right col.). Exhibit C teaches that “the full scope of [intein] application is limited by two common properties of most inteins: (1) slow splicing and cleavage reaction and (2) dependence on local extein sequences composition (“Caveats of protein splicing,” pg. 11-12). Exhibit C teaches that no “universal” intein exists, and that such an intein may never exist (pg. 13).
The prior art supports that it would not be possible for the skilled artisan to determine which of the many split intein-reporter polynucleotides encompassed by the claims would effectively splice from the many exons encompassed by the claims. The specification discloses only a few working examples which represent a very small fraction of the methods encompassed by the claims. The specification does not provide sufficient guidance to overcome the lack of predictability based on the nature of the invention and underlying art, e.g., by providing means to overcome extein dependence, and/or improve splicing kinetics, such that virtually any intein could be used with the many, diverse reporter polynucleotides and genes encompassed by the claims. After weighing the factual considerations of the Wands factors, it is concluded that the amount of experimentation necessary to practice the claimed invention across its scope is unreasonable.
Applicant argues that “because the prior art [cited in the enablement rejection] is directed to different, albeit related technology, the lack of enablement rejection is not reasonably supported by the prior art cited.” For example, Applicant argues that “the teaching of Ramsden [Muller]… cannot be applied to support the allegation that the claimed subject matter lacks enablement, because Ramsden et al., is applied to a gene known not to be spliced.” Applicant also argues that “Carvajal-Vallejos… is distinct from the claimed subject matter because “it does not characterize the splicing efficient of split inteins in the context of a split intein-heterologous polynucleotide construct.” Applicant does, however, seek to rely on Carvajal-Vallejos’ teaching regarding the amino acid residues recited in claim 1 to “support that the claimed subject matter is enabled.” Applicant makes similar arguments for Selgrade (“a skilled artisan will appreciate that the model system of Selgrade et al., is distinct enough from the claimed subject matter that its findings and shortcomings cannot be applied.”). Applicant’s remarks regarding the alleged inapplicability of the cited art are not convincing. First, Examiner notes that the rejections in each of the prior actions and above clearly outline the differences between the instantly claimed use of split inteins, and the uses of split inteins described in the prior art. Applicant’s remarks directed to differences between the prior art methods and instant claims are acknowledged, but it is noted that these remarks do not add anything more than what was pointed out by Examiner in the rejection.
The cited prior art is highly relevant to the instantly claimed use of split inteins, and can, indeed, be used to reasonably support lack of enablement for the claimed subject matter. As Applicant’s concede, the cited prior art is directed to “related technology.” Applicant appears to be arguing that the only evidence which can be used to support an enablement rejection is evidence which actually teaches the claimed invention. This argument is not supported by any evidence, and is not consistent with examination practice to determine compliance with the enablement requirement as described in MPEP 2164. MPEP 2164 is replete with guidance which requires an Examiner to evaluate the technology most closely related to the invention.
In the instant case, the prior art does not teach a working example of the instantly claimed method. However, the skilled artisan would recognize that the instantly claimed method closely relates to the methods described by Ramsden (i.e., Muller), Carvajal-Vallejos, and Selgrade. Specifically, the claims and cited prior art describe uses of split inteins which require the split inteins to effectively trans-splice from an expression product (i.e., a polypeptide sequence). The teachings of the prior art related to split intein splicing kinetics, extein dependence, the use of coiled-coil domains to improve splicing efficacy, etc., are imperative to understanding the nature of the invention and the underlying art, which are Wands factors which must be weighed to determine enablement.
Finally, Applicant indicates that Exhibit D provides subject matter which was granted under European practice. Examiner thanks Applicant for bringing European Patent No. EP 3921423 to their attention. However, the determination of patentability in the instant application is independent from patentability determinations in other applications. Applicant’s arguments related to Exhibit D are not convincing.
Conclusion
No claims are allowed.
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/JENNA L PERSONS/Examiner, Art Unit 1637
/Soren Harward/Primary Examiner, TC 1600