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 .
DETAILED ACTION
The Amendment filed 3/3/2026 in response to Office Action of 9/17/2025, is acknowledged and has been entered. Claims 1-12, 14-15, 17-18, and 22 are now pending. Claims 1 and 17 are amended. Claims 15, 17, and 18 remain withdrawn. Claims 1-12, 14, and 22 are currently being examined.
Maintained Rejection
(Arguments Addressed)
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claim(s) 1-4, 8-11, 13, and 14 remain rejected under 35 U.S.C. 102(a)(1) as being anticipated by Wong et al (Simultaneous assembly of two target proteins using split inteins for live cell imaging. Protein Eng Des Sel. 2013 Mar;26(3):207-13; of record).
Wong teaches a method comprising delivering to eukaryotic cells (COS7, HeLa or HEK293, pg 208, Cell culture and transfection)
(a) a first vector (plasmid vectors) comprising:
(i) a nucleotide sequence encoding an N-terminal fragment of a selectable marker protein, which is upstream from a nucleotide sequence encoding an N-terminal fragment of an intein, and
(ii) a nucleotide sequence encoding a first molecule of interest and
(b) a second vector (plasmid vectors) comprising
(i) a nucleotide sequence encoding a C-terminal fragment of the intein, which is upstream from a C-terminal fragment of the selectable marker protein and
(ii) a nucleotide sequence encoding a second molecule of interest;
wherein the N-terminal fragment and the C-terminal fragment of the intein catalyze joining of the N-terminal fragment and the C-terminal fragment of the selectable marker protein to produce a full-length selectable marker protein. [Materials and methods (pg 208), see Figure 4 below] Wong teaches that the first molecule of interest and the second molecule of interest are encoded by different transgenes. [see Fig 2. Below]
Wong exemplifies this method in Figure 4: (1) a first vector comprising: (i) nucleotide sequence encoding an N-terminal fragment of a selectable marker protein (mRFP1n), which is upstream from a nucleotide sequence encoding an N-terminal fragment of an intein (NpuDnaEn), and (ii) a nucleotide sequence encoding a first molecule of interest (GCaMP2n);
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(2) a second vector comprising: (i) nucleotide sequence encoding an N-terminal fragment of a selectable marker protein (mRFP1c), which is upstream from a nucleotide sequence encoding an N-terminal fragment of an intein (NpuDnaEc), and (ii) a nucleotide sequence encoding a first molecule of interest (GCaMP2c).
Wong further demonstrates that the N-terminal fragment and the C-terminal fragment of the intein catalyze joining of the N-terminal and the C-terminal fragment of the selectable protein to produce a full-length selectable marker protein.
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Wong also demonstrates this in Fig 2 that the constructs are expressed in two different plasmids. (1) 2A (VenusN) fragment is a selectable marker upstream of the N-intein and the RFPn can be the protein of interest, and (2) the C-Intein is upstream of the VenusC fragment (selectable marker) and the protein of interest can be Mrfp1C.
Wong teaches that the selectable markers may be a fluorescent protein, such as mFRP1, Venus, GCaMP2 (as noted above in Fig. 2). Wong teaches maintaining the eukaryotic cells under conditions that permit introduction of the first and second vectors into the eukaryotic cells to produce transgenic eukaryotic cells. Wong teaches selecting the transgenic eukaryotic cells that comprise the full-length selectable marker protein. Wong teaches that the first and/or second molecule is a protein. Wong teaches that the split intein is a natural split intein, Nostoc punctiforme (NpuDnaE) intein. [pg 208, Materials and methods] Wong teaches that this method demonstrated so that a second target protein can be simultaneously formed from intein dimerization. Wong further teaches that intein proteins can accommodate large proteins on both ends without hindering protein trans-splicing. [Abstract, Introduction]
Response to Arguments
Applicant argues that the claims require two vectors to be introduced into a cell: (i) a sequence encoding a fragment of an intein; (ii) a sequence encoding a fragment of a selectable marker; and (iii) a sequence (transgene) encoding one of two functional proteins, and this leads vectors expressing three proteins: (1) a selectable marker, (2) first functional protein; and (3) a second functional protein. Applicant argues that Wong does not disclose this three-piece architecture and only includes a transgene encoding a functional protein – only a sequence encoding a fragment of a protein and Wong’s system yields two proteins total.
Applicant’s arguments have been considered but are not persuasive. Examiner provides an outline of Wong’s teachings below and points to Figure 4:
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In figure 4, Wong demonstrates:
(i) nucleotide sequence encoding an N-terminal fragment of a selectable marker protein (mRFP1n), which is upstream from a nucleotide sequence encoding an N-terminal fragment of an intein (NpuDnaEn), and (ii) a nucleotide sequence encoding a first molecule of interest (GCaMP2n);
(2) a second vector comprising: (i) nucleotide sequence encoding an N-terminal fragment of a selectable marker protein (mRFP1c), which is upstream from a nucleotide sequence encoding an N-terminal fragment of an intein (NpuDnaEc), and (ii) a nucleotide sequence encoding a second molecule of interest (GCaMP2c).
Wong further demonstrates that the N-terminal fragment and the C-terminal fragment of the intein catalyze joining of the N-terminal and the C-terminal fragment of the selectable protein to produce a full-length selectable marker protein. Wong also demonstrates this in Fig 2 that the constructs are expressed in two different plasmids. (1) 2A (VenusN) fragment is a selectable marker upstream of the N-intein and the RFPn can be the protein of interest, and (2) the C-Intein is upstream of the VenusC fragment (selectable marker) and the protein of interest can be Mrfp1C.
Contrary to Applicant’s Argument, Wong teaches the incorporation of two molecules of interest, each encoded by a different transgene, in addition to a selectable marker. Wong demonstrates that the constructs are expressed on two different plasmids, with a single selectable marker (VenusN), and two molecules of interest (RFPn) and (MRFP1c). Thus, he teaches two separate functional proteins. Wong teaches the instantly claimed method and also also states that two intein construct halves can be placed under the control of different promoters, and can be used in this manner to improve tissue-specific targeting; and that the expression of the two intein construct halves were driven under the same promoter, the two intein constructs can conveniently be redesigned to act under two orthogonal promoters to allow for greater tissue specificity. [pgs 211-212] Although the use of Wong’s method may be different than what Applicant argues above, the method of claim 1 is anticipated by Wong’s teachings.
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.
Claim(s1-12, 14, and 22 remain rejected under 35 U.S.C. 103 as being unpatentable over Wong et al (Simultaneous assembly of two target proteins using split inteins for live cell imaging. Protein Eng Des Sel. 2013 Mar;26(3):207-13, of record), in view of Bergwerf et al (US20150232507, of record A1, Published 8/20/215) and Freitas et al (Modified blasticidin S resistance gene (bsrm) as a selectable marker for construction of retroviral vectors, Journal of Biotechnology, Vol 95, Issue 1, pgs 57-62, April 25, 2002, of record).
The teachings of Wong are recited above. However, Wong does not teach that (1) selectable marker protein is an antibiotic resistance protein and (2) the split intein is an engineered intein, as required by claims 5-7, 12, 22 and 23.
Bergwerf teaches methods of using split inteins, comprising the use of fusion proteins that have two components, the first component comprises an intein domain and a heterologous polypeptide C-terminal to the intein domain, and a second component that has an intein domain and a heterologous polypeptide that is N-terminal to the intein domain. [0143, 0177-0180] Bergwerf teaches a vector that is useful for preparing this fusion protein comprising N-terminal and C-terminal regions of polypeptides that is connected by trans-splicing reactions between both of the intein domains. [0201] Bergwerf teaches that the vector may further comprise a polynucleotide encoding a marker protein, that confers antibiotic resistance, including blasticidin. [0244] Bergwerf also teaches that split inteins may be derived from prokaryotic organisms or are engineered. [0003, 0011]
Freitas teaches that blasticidin S is a microbial antibiotic that inhibits protein synthesis in eukaryotes. Freitas teaches that the inhibitory action of blasticidin S can be controlled by bsr. Freitas teaches that bsr (blasticidin S deaminase gne) makes it an attractive use for vectors due to the small size. [Abstract and Introduction]
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to use a selectable marker that is an antibiotic resistance protein that confers resistance to blasticidin in the method of Wong. One would have been motivated to, and have a reasonable expectation of success, because: (1) Wong teaches a method of delivering two vectors to eukaryotic cells, where each vector contains a selectable protein, an intein, and a molecule of interest, (2) Bergwerf teaches a method of using vectors to encode fusion proteins that comprise split intein domains, and that the vectors may encode a marker protein that confer antibiotic resistance to blasticidin, and (3) Freitas demonstrates that bsr is an attractive use for vectors due to the small size, and teaches that it encodes for the resistance of blasticidin. Given the known method of utilizing split intein and selectable markers of the instant claims, and given the known need to confer resistance to antibiotics, one of skill in the art could have pursued using the antibiotic resistance protein that confers resistance to blasticidin in the method of Wong, with a reasonable expectation of success.
It would have been prima facie obvious to one of ordinary skill in the art at the time the invention was filed to use an engineered intein in the method of Wong. One would have been motivated to, and have a reasonable expectation of success, because: (1) Wong teaches a method of delivering two vectors to eukaryotic cells, where each vector contains a selectable protein, an intein, and a molecule of interest, and (2) Bergwerf teaches a method of using vectors to encode fusion proteins that comprise split intein domains, and teaches that spit intein domains may be natural occurring or engineered. Given the known methods of utilizing split inteins and given the known types of split inteins, natural occurring or engineered, one of skill in the art could have pursued using an engineered split intein in the method of Wong, with a reasonable expectation of success.
Response to Arguments
The applicant traverses it on the same grounds as asserted with respect to the anticipation rejection (that Wong does not teach certain limitations, and that the secondary references fail to make up for that deficiency). The arguments are not found persuasive for the reasons asserted above.
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/SARAH A ALSOMAIRY/Examiner, Art Unit 1646
/Zachariah Lucas/Supervisory Patent Examiner, Art Unit 1600
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