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 .
Withdrawn Rejections
The rejection of claims 6-7, 12-14, and 16, under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement, is withdrawn. The amendments to the claims overcome the rejection.
The rejection of claims 4-6 and 10-17, 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or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention, is withdrawn. The amendments and arguments address the issues of indefiniteness.
The amendments necessitate modification of the rejections of record as follows:
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claim 1-3 and 11, as amended or originally presented, are rejected under 35 U.S.C. 101 because the claimed invention is directed to product of nature without significantly more.
A reminder that claim 11 is being interpreted as a being inherent to claim 2 as discussed above in the indefiniteness rejection. As such, it is in the present rejection as having the claim limitations as claim 2.
According to the 2019 Revised Patent Subject Matter Eligibility Guidelines (2019PEG), the claim is first analyzed to determine if it is directed to one of the acceptable statutory categories of invention (i.e. process, machine, manufacture, or composition of matter). Claims 1 is drawn to a composition of matter comprising nucleic acids. Thus claim 1 meets the requirements for step 1 of the analysis.
Second, the claim is assessed to determine if it is directed to a judicial exception under step 2A. Under 2019PEG, “directed to” is determined via a two-prong inquiry: (1) Does the claim recite a law of nature, a product of nature, a natural phenomenon, or an abstract idea; and (2) Does the claim recite additional element(s) that integrate the judicial exception into a practical application. The phrase, “integration of a practical application”, requires the presence of an additional claim element(s) or a combination thereof to apply, rely on or use the judicial exception in a manner that imposes a meaningful limitation on the judicial exception, such that the claim does not monopolize the judicial exception. (See MPEP § 2106.05 for examples of integration of practical application).
Regarding the first prong (1), claim 1 is directed to a “nucleic acid composition comprising” three nucleic acid encoding each nucleic acid encoding (i) a promoter comprising: one or more pairs of transcription factor (TF) binding sites operably linked to (ii) a nucleic acid coding sequence for a TF, wherein the TF binds the TF binding site. As such the claims 1 directed to three constructs encoding auto-regulation feed-back loops wherein the TF encoded by binds and regulates its own promoter. Such genetic auto-regulation, feed-back loops are found in nature in pluripotent embryonic cells of the inner cell mass cells. Tam and Lim (Tam, W.-L. and Lim, B., Genome-wide transcription factor localization and function in stem cells (September 15, 2008), Stem Book, ed. The Stem Cell Research Community, Stem Book, doi/10.3824/stembook.1.19.1, http://www.stembook.org. See printout for excerpt page 1) report, “One emergent theme arising from transcription regulation in ESCs is the revelation that a core set of factors act together in a tightly regulated framework to control similar as well as divergent downstream genes (see Table 1). Auto-regulation by top-tier TFs is a key feature. The pluripotent molecules Oct4, Sox2, Nanog and Sall4 auto-regulate through binding to their own promoters…. On its own, each factor maintains its expression that is individually required for ESC function. Collectively, all four factors regulate one another through the interconnectivity of positive feedback loops (see Table 1). The generation of these feedback circuits reinforces the transcription activity of each factor, and maintains appropriate levels of the necessary factors.” See excerpt.
PNG
media_image1.png
293
979
media_image1.png
Greyscale
As such, Tam and Lim demonstrate that three genetic auto-feedback constructs of claim 1 are natural products present in embryonic stem cells in an embryo in nature. Thus the three different recited constructs of the nucleic acid sequence independently and cumulatively are products of nature. Therefore, the claim is direct to a judicial exception and the requirements for prong 1 are meet by the claim.
Regarding the second prong (2), the only additional elements being recited by the claim are the presence of payloads nucleic acid sequence structures. Thus no recited application has been recited by the claims. Therefore, claim 1 does not integrate the judicial exception into a practical application and claim 1 meets the requirement for prong 2 and step 2A as being directed to a judicial exception.
Third, if a judicial exception is present in the claim, it is further assessed to determine if the claim recites any additional elements or steps that are sufficient to ensure that the claim as a whole amounts to significantly more than the judicial exception. As discussed above, the claim recite the sole additional structural limitation of a “payload” in teach of the three constructs of the composition. The specification does not define the term payload. As such, it must be given its common art accepted meaning which would be any genetic material intended to be delivered. As such, the payload can be additional genetic material encompasses a tag, a gene encoding a protein to be expressed, a degradation signal, a transport signal, among others. As such, the breadth of payload encompasses sequences including ones that are natural present in the nucleic acid sequence encoding the transcription factors. Thus the additional element of payload does not necessarily impart any significant or marked distinction to the judicial exception(s) of the claims. Thus, claim 1 does not meet the requirements for step 2B and thus comprises patent ineligible subject matter.
Regarding claims 2 and 11, claims 2 and 11 are drawn to a composition of matter comprising nucleic acids. Thus claim 2 meets the requirements for step 1 of the analysis.
Regarding Step 2A, Claims 2 and 11 recite the judicial exception discussed above, and further is directed to TF capable of forming homodimers. Oct4, Sox2, and Sall4 as described in Tim and Lam, all can form homodimers in autoregulation of their gene construct. As such, this recitation of homodimer formation is a function that also occurs in nature and thus is further part of the recited judicial exception(s). Thus claim 2 meets the limitations of prong 1. As discussed above not practical application is recited by the claim, as such the limitations for prong 2 is also met and the claim indeed recites a judicial exception as determined by step 2A.
Regarding Step 2B, claim 1, as amended further recites, “wherein the first DNA-binding domain, the second DNA-binding domain, and/or the third DNA-binding domain is a synthetic DNA-binding domain configured to decrease monomeric TF activity without reducing TF homodimer activity”. The recitation, “synthetic”, suggests that the DNA-binding domain was somehow synthesized. DNA-binding domains in this instant are parts of proteins and all proteins recombinant in nature or naturally occurring are synthesized. As such, the recitation “synthetic” does not impart any structural or functional distinction to the DNA-binding domains. The rest of the recitation, added by amendment, describes and limits at least one of the DNA-binding domain by its function. As such, “configured to decrease monomeric TF activity without reducing TF homodimer activity” generically encompasses decrease any TF activity by any degree of decrease without reducing any TF homodimer activity by any degree of reduction. The example from Tim and Lam demonstrates a gene circuit encompassed by the claims that comprising an Oct4 and an Oct4 TF DNA-binding domain that naturally occurs in a cell. Transcription factor Oct4 can binding its cognate TF DNA-binding domain as a monomer but it does not more weaky than an Oct4 homodimer. As such, Oct4 monomer naturally have decreased monomeric TF activity as compared to TF homodimer activity and does not reduced homodimer TF activity. As such, these new limitations do not provide any marked distinction between the claimed product and its natural counterpart. Thus the requirements for step 2B have not been met and claims 1-2 and 11 comprise patent ineligible subject matter.
Regarding claim 3, this claim further specifies that teach of the three constructs further comprise degron sequences. The requirements for step 1 of the analysis are met as discussed above. Regarding Step 2A, degron sequence are naturally present in gene species for Nanog, Sox2, Oct4, and Sall4 genes. As such, the recitation of further comprising degrons is also part of the above described judicial exception. As such, the requirement for prong 1 have been met. No additional elements that describe a practical application are present. As such, the requirements for prong 2 are met and claim 3 meets the requirements for step 2A of reciting a judicial exception. Regarding step 2B no other additional elements are recited in claim 3 that would markedly distinguish the judicial exception from its natural counterpart. As such, claim 3 does not meet the limitations of claim 3 and are thus patent ineligible.
In conclusion, claims 1-3 and 11 do meet all the requirements of the 2019PEG and therefore deemed patent ineligible.
Response to Arguments
Applicant's arguments filed have been fully considered but they are not persuasive.
Applicant traverses this rejection. However, Applicant amended claim 1 to include limitations in from claim 12. As such, these amendments overcome the rejection because claim 12 was not part of the rejection.
In response, claim 12 was subject to a species election between (i), (ii), and (iii) of claim 12. The elected species of (iii). As such, (i) and (ii) were withdrawn from consideration. As such, claim 12 was left out of the 101 rejection because only species (iii) were under consideration at that time. Species (ii) of claim 12 comprise the newly added recitation to claim 1 by amendment and was not considered. Since Applicant has now newly added it a requirement of the base claim, these limitations are under consideration and the generic nature of these limitations comprise embodiments of the claims that are products of nature and thus judicial exceptions and patent ineligible. As such, Applicant’s argument and amendment to the claims do not overcome the 101 rejection of record.
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) 1-3, 6-7, 9-11, 12, and 16, as amended or originally presented, is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Tam and Lim (Tam, W.-L. and Lim, B., Genome-wide transcription factor localization and function in stem cells (September 15, 2008), Stem Book, ed. The Stem Cell Research Community, Stem Book, doi/10.3824/stembook.1.19.1, http://www.stembook.org. See printout for excerpt page 1).
As discussed in more detail above in the 101 rejection, claim 1-3 encompass naturally occurring system present in the ES cells.
Regarding claim 1, Tam and Lim disclose ES cell that comprising a first nucleic acid encoding the Oct4 promoter, which comprises an Oct4 transcription binding site, and a Oct4 coding sequences and inherently comprises degron and signaling sequences (i.e. first payload). Tam and Lim further disclose that ES cell comprising a second nucleic acid encoding the Sox2 promoter, which comprises an Sox2 transcription binding site, and a Sox 2 coding sequences and inherently degron and signaling sequences (i.e. second payload). Tam and Lim further disclose that ES cell comprising a third nucleic acid encoding the Nanog promoter, which comprises an Nanog transcription binding site, and a Nanog coding sequences and inherently degron and signaling sequences (i.e. third payload). Alternatively, Tam and Lim further disclose that ES cell comprising a third nucleic acid encoding the Sall4 promoter, which comprises an Sall4 transcription binding site, and a Sall4 coding sequences and inherently degron and signaling sequences (i.e. third payload). Thus Tam and Lim expressly or inherently disclose all the limitations of the claims.
Regarding the amendments to claim 1, Oct4 DNA-binding domain is configured to bind monomers more weakly than Oct4 homodimers. As such, inherently the newly added wherein clause are disclosed by Tam and Lim.
Regarding claims 2-11, the oct4, Nanog, Sox2,and Sall4 promoters are all four inherently capable of forming homodimers binding these TFs as the claims require. As such, Tam and Lim expressly or inherently discloses the limitations of claims 2 and 11.
Regarding claim 3, Oct4, Sox2, Sall4 and Nanog all have degron sequences inherently. As such, Tam and Lim expressly or inherently discloses the limitations of claim 3.
Regarding claim 6, this claim recites multiple possible dimerization domains that are derived from multiple proteins. Option (iii) recites a list of proteins having dimerization domains and further recites, “portions thereof” or “variants thereof”. The breadth of portions or variant encompasses dimerization domains that have at least one or two amino acid residues in common with the listed proteins. The dimerization domains of Oct4, Sox2, Nanog and Sall4 all have at least one or two amino acids in common with the portions or variants there of as claimed. As such, the disclosures by Tam and Lim meet the limitations of claim 6.
Regarding claim 7, the claim recites dimerization ligands derivatives, which are interpreted as discussed above. Oct4, Sox2, Nanog, and Sall4 have at least one or two residue in common with the claimed derivatives. As such, Tam and Lim meet the limitations of the claims.
Regarding claims 9-10, Oct4, Sox2, and Sall4 are all able to form heterodimers as claimed.
Regarding claim 12, option (i) recites a list of protein species, or “portions thereof”. The DNA binding domain of Oct4, Sox2, Nanog, and Sall4 have portions of the DNA binding domain in common with the claimed protein species. As such, Tam and Lim meet the limitations of claim.
Regarding claim 16, this claim recite “derivatives” of degron species. Derivatives, given its broadest reasonable interpretation encompass any sequences with at least one or two amino acids in common with the recited degron species. Oct4, Sox2, Nanog and Sall4 as disclosed by Tam and Lim all comprise degron sequences that have at least one or two amino acids in common which encompasses “derivatives” as claimed.
Response to Arguments
Applicant's arguments filed 3/16/2025 have been fully considered but they are not persuasive.
Applicant traverses the 102 rejection on the same grounds as the 101. In response, Applicant’s arguments are not found persuasive for reasons discussed above in addressing the 101 rejection and the modified 102 rejection.
Further Applicant refers to the working examples that describe an engineered ZF TF binding domain that weakly binds monomers but more strongly bind homodimers. In response, the working example referred to by Applicant is not commensurate in scope with the claims. Further, similar to the working example, the breadth of the claims reads on the naturally occurring Oct4 DNA-binding domain that like the working example binds the monomer weakly and the homodimer strongly. As such, the disclosure of the Oct4 DNA-binding domain by Tam and Lim meet the limitations of the amended claims.
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.
(1) Claim(s) 1, 4, 17, and 18 is/are rejected under 35 U.S.C. 103 as being unpatentable over Tam and Lim (Tam, W.-L. and Lim, B., Genome-wide transcription factor localization and function in stem cells (September 15, 2008), Stem Book, ed. The Stem Cell Research Community, Stem Book, doi/10.3824/stembook.1.19.1, http://www.stembook.org. See printout for excerpt page 1), as applied to claims 1-3, 6-7, 9, 12, and 16 above, and further in view of Mack (US 2010/0041054 A1).
Tam and Lim teach the nucleic acid composition of claim 1, 4, and 17 as discussed above. Tam and Lim does not does not teach that the payload can be a diagnostic fluorescent protein (species elections for claim 17). Tam and Lim does not teach that the promoter operably linked to TF gene are in a polycistronic transcript with the payload (claim 4)
However, Mack teaches a method of reprogramming a cell that introduces a multiple nucleotide sequences encoding a reprogramming factor genes, IRES sequence, and a reporter genes. Figure 1 for example, [0009]. The use of a reporter sequence linked to a reprogramming factor via and IRES allows for simultaneous expression of reporter genes and the reprogramming factor which aids in selection reprogrammed cells. See [0010]. Mack teaches that the reprogramming factor can be any reprogramming factor, such as Oct4, Sox2, Nanog. See [0014] for example. Mack teaches that the reporter can be a fluorescent protein reporter gene such as GFP, RFP, BFP, YFP, among others [0013].
As such, it would have been obvious to an artisan of ordinary skill before the time of filing to include the reprogramming factor transcription factor gene taught in Tam and Lim with an polycistronic transcript with a reporter via an IRES sequence as taught by Mac to predictably arrive at the limitations of claims 1, 4, and 17. The artisan would have a reasonable expectation of success because Mack demonstrates that compositions comprising reprogramming factor genes linked to a fluorescence reporter gene via an IRES sequence were successfully being used in the prior art long before effective filing. Further the artisan would have been motivated to place the reprogramming genes in linkage with different fluorescent protein reporter genes because Mack teaches the use of a reporter sequence linked to a reprogramming factor via and IRES allows for simultaneous expression of reporter genes and the reprogramming factor which aids in selection reprogrammed cells. As such, Tam and Lim in view of Mack render claims 1, 4, and 17 obvious.
(2) Claim(s) 1, 4-5, 14 and 15 is/are rejected under 35 U.S.C. 103 as being unpatentable over Tam and Lim (Tam, W.-L. and Lim, B., Genome-wide transcription factor localization and function in stem cells (September 15, 2008), Stem Book, ed. The Stem Cell Research Community, Stem Book, doi/10.3824/stembook.1.19.1, http://www.stembook.org. See printout for excerpt page 1), as applied to claims 1-3, 6-7, 9, 12, and 16 above, and further in view of Nagy (WO 2010/012077).
Tam and Lim teach the nucleic acid composition as described above. Tam and Lim do not teach that the first, second, and third promoter further comprise a transactivator recognition sequence that a transactivator is capable of binding. Or a basal expression motif.
However, Nagy discloses multiple nucleic acid vectors or transposon sequences, each one comprising a reprogramming transcription factors selected from Oct4, Sox2, Klf4, c-Myc, Lin28, and Nanog each vector or transposon sequence comprising a promoter, more specifically an inducible promoter. In an embodiment, the vector or transposon sequence comprising TetO2 tetracycline/doxycycline (dox) inducible promoter sequence and cell comprising these elements are cultured in the presence of dox and inducible control of the reprogramming transcription factor expression (p. 4, line 19 to p. 5, line 31).
As such, it would have been obvious to an artisan of ordinary skill before the time of effectively filing to further add an inducible promoter element, such as TetO2 tetracycline/doxycycline (dox) inducible promoter sequence, taught by Nagy to the three different nucleic acid constructs of Tam and Lim to predictable arrive at the limitations of claims 1, 4, and 5. Further the artisan would have a reasonable expectation of success because Nagy teaches that TetO2 tetracycline/doxycycline (dox) inducible promoter sequence have been used with reprogramming transcription factor vector constructs since at least 2010. Further, the artisan would be motivated to further include TetO2 tetracycline/doxycycline (dox) inducible promoter sequence because Nagy teaches that it allows for control of reprogramming factor expression in cells. As such, Tam and Lim in view of Nagy render claims 1, 4, and 5 obvious.
Regarding claim 14, this claim recites “portions thereof having transactivation activity”. The tetracycline/doxycycline (dox) inducible promoter sequence has at least one or two amino acids in common with the TFs taught by Tam and Lim in view of Nagy and thus teaches the requisite limitations of the claim.
Regarding claim 15, Tam and Lim in view of Nagy teach an inducible Tet responsive promoter transactivator recognition sequence as discussed above.
Regarding claim 17, Tam and Lim in view of Nagy teaches and IRES sequence as discussed above.
Response to Arguments
Applicant's arguments filed 3/16/2026 have been fully considered but they are not persuasive.
In remarks addressing each of the 103 rejections of record, Applicant submits that the secondary references do not cure the deficiencies of Tam and Lim. In response, Tam and Lim are not found to be deficient for reasons discussed above. Applicant has not address specifically how the secondary reference further would be supplement the teachings of Tam and Lim. As such, the secondary references cited do cure the deficiencies in Tam and Lim as reiterated above in the 103 rejections. Therefore the rejections are maintained.
The following new rejection is necessitated by the amendments to the claims:
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 1-18 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 written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
When determining if a recited genus has adequate written description for a genus:
(1) the broadest reasonable interpretation of the genus is determined;
(2) the disclosure is examined to determine if the specification has provided a representative number of species to describe the complete structure of the genus;
(3) the disclosure is examined to determine whether a representative number of species have been sufficiently described by other relevant characteristics, specified features and functional attributes that would distinguish different members of the claimed genus; and
(4) the state of the art is examined to the determine if it supports/supplement the genus description in the specification in a manner that would demonstrate the application was in possession of the claimed genus at the time of effectively filing.
Amended claim 1 newly recites the genus, “a synthetic DNA-binding domain configured to decrease monomeric TF activity without reducing TF homodimer activity”
Breadth of the Genus:
The breadth of the genus encompasses any DNA-binding domain that has the functional property of imparting decreased monomeric TF activity without reducing TF homodimer activity. The breadth of TF activity is any activity associated with a TF, including but not limited to binding to its cognate domain, regulating promoter activity, inducing transcription, among others. As such, the breadth of the recitation is a genus of DNA binding-domain that in any way has differentiation physical or functional interactions or effects with a TF monomer as claimed to a TF homodimer. This encompasses a wide range of possible DNA binding domains sole described by a capability of that DNA-binding domain. As such, the genus is structurally and functionally very diverse.
Specification Description:
The specification describes the following (citations from the published application):
[0017] In some embodiments, the DNA-binding domain is a synthetic DNA-binding domain configured to decrease monomeric TF activity without reducing TF homodimer activity. In some embodiments, a DNA-binding domain comprises or is derived from a zinc finger DNA-binding domain. In some embodiments, the zinc finger (ZF) DNA-binding domain comprises or is derived from ErbB2 ZF, BCRZF, HIV1ZF, HIV2ZF, 37ZF (37-12 array), 42ZF (42-10 array), 43ZF (43-8 array), 92ZF (92-1 array), and/or 97ZF (97-4 array). In some embodiments, the ZF DNA-binding domain comprises one or more arginine-to-alanine mutations. In some embodiments, the ZF DNA-binding domain comprises three fingers that bind weakly as monomers to 9 bp target sites and bind at least about 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or 100-fold, more strongly as homodimers to 18 bp tandem binding site pairs. In some embodiments, a DNA-binding domain comprises an amino acid sequence at least 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, or 99 percent identical to ErbB2ZFWT (SEQ ID NO: 6), ErbB2ZFR39A (SEQ ID NO: 7), ErbB2ZFR2AR39A (SEQ ID NO: 8), ErbB2ZFR2AR39AR67A (SEQ ID NO: 9), 37ZFWT (SEQ ID NO: 10), 37ZFR39A (SEQ ID NO: 11), 37ZFR2AR39A (SEQ ID NO: 12), 37ZFR2AR39AR67A (SEQ ID NO: 13), 42ZFR2AR39AR67A (SEQ ID NO: 14), 92ZFWT (SEQ ID NO: 15), 92ZFR39A (SEQ ID NO: 16), 92ZFR2AR39A (SEQ ID NO: 17), 92ZFR2AR39AR67A (SEQ ID NO: 18), 97ZFWT (SEQ ID NO: 19), 97ZFR39A (SEQ ID NO: 20), 97ZFR2AR39A (SEQ ID NO: 21), BCRZF (SEQ ID NO: 22), BCRZFR39A (SEQ ID NO: 23), HIV1ZFWT (SEQ ID NO: 24), HIV1ZFR39A (SEQ ID NO: 25), HIV1ZFR2AR39A (SEQ ID NO: 26), HIV1ZFR2AR39AR67A (SEQ ID NO: 27), HIV2ZFWT (SEQ ID NO: 28), HIV2ZFR39A (SEQ ID NO: 29), HIV2ZFR2AR39A (SEQ ID NO: 30), and/or HIV2ZFR2AR39AR67A (SEQ ID NO: 31).
[0113] There is provided herein a new circuit architecture termed MultiFate that advantageously allows the engineering of multistable circuits. The system is based, in some embodiments, on a few key properties. First, in some embodiments, it uses transcription factors that activate when dimerized, with much weaker activity as monomers.
[0152] A TF monomer can have at least about 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 400-fold, 600-fold, 800-fold, or a number or a range between any of these values, less binding affinity for a pair of TF binding sites as compared to a TF homodimer. In some embodiments, a first promoter, second promoter, third promoter, and/or nth supplemental promoter induces transcription at least about 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 400-fold, 600-fold, 800-fold, or a number or a range between any of these values, less in the presence of a TF monomer can compared to a TF homodimer. In some embodiments, TF homodimerization and heterodimerization occur with a substantially equal dissociation constant (K.sub.d).
[0156] The DNA-binding domain can be a synthetic DNA-binding domain configured to decrease monomeric TF activity without reducing TF homodimer activity. A DNA-binding domain can comprise or can be derived from a zinc finger DNA-binding domain. The zinc finger (ZF) DNA-binding domain can comprise or can be derived from ErbB2 ZF, BCRZF, HIV1ZF, HIV2ZF, 37ZF (37-12 array), 42ZF (42-10 array), 43ZF (43-8 array), 92ZF (92-1 array), and/or 97ZF (97-4 array). The ZF DNA-binding domain can comprise one or more arginine-to-alanine mutations. The ZF DNA-binding domain can comprise three fingers that bind weakly as monomer to 9 bp target sites and bind at least about 1.1-fold, 1.3-fold, 1.5-fold, 1.7-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 200-fold, 400-fold, 600-fold, 800-fold, or a number or a range between any of these values, more strongly as homodimers to 18 bp tandem binding site pairs.
Working Examples:
Engineered Zinc Finger Transcription Factors Enable Homodimer-Dependent Self-Activation and Heterodimer-Dependent Inhibition.
[0257] Synthetic zinc finger (ZF) transcription factors provide, in some embodiments, a platform to implement the MultiFate circuit. They can recognize and activate a promoter containing target DNA binding sites with high specificity. Further, engineered ZF DNA-binding domains containing three fingers bind weakly as monomers to 9 bp target sites, but can bind much more strongly as homodimers to 18 bp tandem binding site pairs. Without being bound by any particular theory, this property enables homodimer-dependent transcriptional activity and inhibition through heterodimerization.
[0258] To engineer ZF transcription factors, the ErbB2 ZF DNA-binding domain was fused to a GCN4 homodimerization domain and a VP48 transcriptional activation domain to create the synthetic transcription factor, termed ZF-GCN4-AD (FIG. 2A). A transcription factor (ZF-AD) lacking GCN4 was used as a monomeric control. To assay their transcriptional activity, a reporter was constructed containing 18 bp homodimer binding sites driving the expression of Citrine. Each transcription factor was then co-transfected together with the reporter and an mTagBFP2 co-transfection marker into Chinese hamster ovary K1 (CHO-K1) cells, and analyzed for Citrine expression by flow cytometry 36 hours later (FIG. 2A and FIG. 9A) (See, “Additional Methods” below). The wild-type (WT) ZF-GCN4-AD factors strongly activated the reporter whereas ZF-AD exhibited weaker basal activity (FIG. 2A and FIG. 9B). Arginine-to-alanine mutations were introduced at key positions in the ZF known to weaken DNA binding, which decreased monomeric activity without reducing homodimer activity (FIG. 2A, red square). Replacing the GCN4 with the FKBP12F36V (FKBP) homodimerization domain allowed for dose-dependent control of dimerization with the small molecule AP1903 (FIG. 2B). Finally, this general design was repeated to engineer a set of additional homodimer-dependent ZF transcription factors with orthogonal DNA-binding specificities (FIG. 9B and FIG. 9C).
As such, the specification while generically contemplating the genus, “a synthetic DNA-binding domain configured to decrease monomeric TF activity without reducing TF homodimer activity”, the specification solely provides description of a handful of ZF with engineered mutations that bind weakly as a monomer and stronger as a homodimer. However, the genus is much more broadly encompassing all DNA-binding domains that have mutation or that naturally exist with the claimed configuration that decreases monomer TF activity without reducing TF homodimer activity. As such, the small number of described species does not serve as a representative number of species that would be descriptive of the complete genus.
State of the Art:
A review of the art before the time of effective filing and continuing post-filing teaches that any changes to the amino acid sequence or nucleic acid sequences encoding the generic proteins as the breadth of the genus requires is unpredictable. For example, Sotomayor-Vivas et al ((2022) Linking protein structural and functional change to mutation using amino acid networks. PLoS ONE 17(1): e0261829. https://doi.org/10.1371/journal.pone.0261829. Pages 1-23) report, “Proteins are complex biomolecules that have been subject to mutational dynamics for billions
of years and whose tasks are essential for the maintenance, development, and survival of well-functioning cells. They start from a sequence of amino acids that folds into a three-dimensional (3D) structure that determines their function…. Understanding the underlying relations between sequence, structure, and function of a protein has been an active research topic in molecular biology for decades….Structure and function prediction from the amino acid sequence has been an open problem even prior to Anfinsen’s discovery of the thermodynamic hypothesis, which states that, under normal conditions, the protein sequence is responsible for the native configuration of a protein… The replacement of an amino acid in the sequence—a mutation—can have structural consequences on the resulting protein and thus has a potential effect on its function.” Pp. 1-2. Sotomayor-Vivas further states “In general mutations occur natural and have no effect on protein function” the means of predicting which mutations alter function and which do not have long been under investigation (See Introduction). As such, while most mutations that occur in a primary amino acid sequence do not alter function, only at must as one amino acid residue change in a critical part of the primary structure can have significant impact on the protein function and means of distinguishing these critical function altering mutations are unpredictable and still in the process of being developed.
The prior art discusses mutations resulting unpredictable functional effects. Further, the genus is even larger than mutated forms of the DNA-binding domain. As such, the art before the time of the invention does not supplement the shortcomings of the specification.
Thus the genus, a synthetic DNA-binding domain configured to decrease monomeric TF activity without reducing TF homodimer activity”, lack adequate written description because the description of a small number of engineered ZF DNA-binding domains does not provide sufficient description of a representative number of species to describe the complete structure of the claimed genus. Further, the art teaches while modified forms of DNA-binding domains can be designed, arriving at the functional property of “configured to decrease monomeric TF activity without reducing TF homodimer activity” is not predictable. As such, an artisan of ordinary skill at the before the time of effective filing and after would not be able to envision the complete structure of “a synthetic DNA-binding domain configured to decrease monomeric TF activity without reducing TF homodimer activity”. Therefore, the ordinary artisan would not come to the conclusion that the application was in possession of the genus before the time of effective filing.
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to MARCIA STEPHENS NOBLE whose telephone number is (571)272-5545. The examiner can normally be reached M-F 9-5:30.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Peter Paras can be reached at 571-272-4517. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
MARCIA S. NOBLE
Primary Examiner
Art Unit 1632
/MARCIA S NOBLE/Primary Examiner, Art Unit 1632
Prosecution Insights