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 .
This action is in response to the papers filed on 05/07/2026. Claims 1, 3-16, 18-20, 31 and 53 are currently pending as per claims filed on 05/07/2026
Applicant’s election without traverse of Group I, claims 1, 3-16, 18-20 in the reply filed on 05/07/2026 is acknowledged.
In addition, Applicant further elects the species in Group I as follows:
For Claim 3, the species of human wildtype intron of intron 1 of MTERFD3.
For Claim 4, the species of intron 1 of MTERFD3 comprising a sequence set forth in SEQ ID NO:2.
For Claim 8, the species of SEQ ID NO:5.
For Claim 9, the species of 5' splice site is GTGAG.
For Claim 10, the species of canonical 3' splice site is CAG.
For Claim 11, the species of at least one cryptic 3' splice site is AAG.
For Claim 12, the species wherein each of the plurality of cryptic 3' splice sites is AAG.
For Claim 13, the species of all of the characteristics.
For Claim 18, the species of recurrent change-of-function mutation in SF3B1 results in an amino acid substitution of K700E.
Claims 31 and 53 are withdrawn from further consideration by Applicants pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim.
The requirement is still deemed proper and is therefore made FINAL.
Therefore, claims 1, 3-16, 18-20 are examined on their merits to which the
following grounds of rejection are applicable. Claims 1 is an independent claim.
Priority
The instant application is a 371 of PCT/US2021/056273 filed on 10/22/2021.
The instant application claims domestic benefit to US provisional patent
application number 63/160,405 filed on 3/12/2021 and US provisional patent application
number 63/105,143 filed on 10/23/2020.
Thus, the earliest possible priority for the instant application is 10/23/2020.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 04/20/2023, 09/17/2024, and 01/27/2025, were filed after the mailing date of the current office action. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Claim Objection
Claim 12 is in improper Markush form; a Markush group should be in the form “selected from the group consisting of A, B, and C” and not “selected from”. Appropriate correction is requested
Claim Rejections - 35 USC § 112 (b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 5 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 5 is indefinite in its use of the term "optionally" at line 8. As written, it is unclear exactly what that applicant intends to claim. Therefore, the metes and bounds are unclear and the claim is rendered indefinite. Applicant must plainly recite what they intend to claim.
Claim 5 is indefinite in it’s use of the phrase “and the like” in line 6.
A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) is considered indefinite, since the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). Note the explanation given by the Board of Patent Appeals and Interferences in Ex parte Wu, 10 USPQ2d 2031, 2033 (Bd. Pat. App. & Inter. 1989), as to where broad language is followed by "such as" and then narrow language. The Board stated that this can render a claim indefinite by raising a question or doubt as to whether the feature introduced by such language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims. Note also, for example, the decisions of Ex parte Steigewald, 131 USPQ 74 (Bd. App. 1961); Ex parte Hall, 83 USPQ 38 (Bd. App. 1948); and Ex parte Hasche, 86 USPQ 481 (Bd. App. 1949).
In the present instance, claim 5 recites the broad recitation “and the like”, and the claim also recites specific types of nucleotides in the consensus 5' splice site which is the narrower statement of the range/limitation.
Therefore, the metes and bounds are unclear and the claim is rendered indefinite.
Claim 5 is indefinite in its use of the phrase “MaxEntScan” in line 14 . It is unclear what is “MaxEntScan”. The term " MaxEntScan " is not defined by the claim, the specification does not provide sufficient detail as to how one would use MaxEntScan. Therefore, the metes and bounds are unclear and the claim is rendered indefinite.
It is noted that the term “canonical” is defined in the specification at para 0078 as “a splice site whose usage results in preservation of the open reading frame if the intron is inserted into a coding DNA sequence and subsequently spliced, such that no in-frame termination codons are introduced into the coding sequence if the canonical 3′ splice site is used during the splicing process”
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.
Claim 1, 3-16, 18-20 rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for:
Sixteen different artificial nucleic acid intron constructs comprising the introns of, MTERFD3 identified by SEQ ID NOS: 56-71 each with a 5’ splice site, a canonical 3’ splice site, a cryptic 3’ splice site, and pyrimidine rich domain, and a branchpoint (table 1, page 34 of published application) which comprise two exon domains for herpes simplex virus thymidine kinase (HSV-TK) system,
does not reasonably provide enablement for any artificial nucleic acid intron constructs, comprising any intron comprising: a 5' splice site; a canonical 3' splice site; at least one cryptic 3' splice site, that is within about 100 nucleotides upstream of the canonical 3' splice site or within about 50 nucleotides downstream of the canonical 3' splice site; a pyrimidine-rich domain comprising at least 6 consecutive nucleotides, wherein the sequence of the pyrimidine-rich domain is at least 60% pyrimidine nucleotides, and wherein the pyrimidine-rich domain is within at least 50 nucleotides of a cryptic 3' splice site; and at least one branchpoint at least 15 nucleotides upstream of the canonical 3' splice site and can comprise any two exons for encoding any protein of interest with the intended use, for example, of being spliced differently in a cancer cell comprising a change-of function or loss-of-function mutation in a recurrently mutated SF3Bl RNA splicing factor gene relative to the splicing pattern of the intron in a cell lacking a change-of-function or loss-of function mutation in a recurrently mutated RNA splicing factor gene as required in claim 16
The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make or use the invention commensurate in scope with these claims.
The factors to be considered in determining whether undue experimentation is
required are summarized in In re Wands, 858 F.2d 731, 737, 8 U.S.P.Q.2d 1400, 1404
(Fed. Cir. 1988) (a) the breadth of the claims; (b) the nature of the invention; (c) the state of the prior art; (d) the level of one of ordinary skill; (e) the level of predictability in the art; (f) the amount of direction provided by the inventor; (g) the existence of working examples; and (h) the quantity of experimentation needed to make or use the invention based on the content of the disclosure.
While all of these factors are considered, a sufficient number are discussed below so as to create a prima facie case.
the breadth of the claims; the nature of the invention:
Claim 1 is directed to a vast genus of artificial nucleic acid intron construct, comprising a vast genus of introns comprising: a 5' splice site; a canonical 3' splice site; at least one cryptic 3' splice site, that is within about 100 nucleotides upstream of the canonical 3' splice site or within about 50 nucleotides downstream of the canonical 3' splice site (i.e. a wide range of positions for the at least one cryptic 3’ splice stie); a pyrimidine-rich domain comprising at least 6 consecutive nucleotides, wherein the sequence of the pyrimidine-rich domain is at least 60% pyrimidine nucleotides, and wherein the pyrimidine-rich domain is within at least 50 nucleotides of a cryptic 3' splice site (i.e. a wide range of positions for the at least one cryptic 3’ splice stie); and at least one branchpoint at least 15 nucleotides upstream of the canonical 3' splice site such that when the artificial nucleic acid intron construct is placed inside a coding sequence, the construct can produce a functional protein mainly in mutant cells while remaining inactive or altered in normal cells. Claim 3 further limits claim 1 to be from intron 1 of MTERFD3. Claim 4 further limits claim 3 to be for a sequence set forth in SEQ ID NO: 2. Claim 5 further limits claim 3 wherein the intron further comprises several features. Claim 6-8 further limits claim 4 to the intron having specific location ranges for the 3’ and 5’ splice site and identity to SEQ ID NO: 5. Claim 9-12 further limit claim 1 to specific sequences to the splice sites. Claim 13 further limits claim 1 to characteristics of the pyrimidine-rich domain. Claim 14 and 15 further limits claim 1 to features of the branchpoint. Claim 16 and 18 further limits claim 1 to the function of the intron. Claim 19 and 20 further limits claim 1 to exon domains flanking the intron.
the amount of direction provided by the inventor; the existence of working
examples:
As recited above, claim 1 is broadly but reasonable interpreted as comprising a vast genus of artificial nucleic acid intron construct, comprising a vast genus of introns comprising: a 5' splice site; a canonical 3' splice site; at least one cryptic 3' splice site with a wide range of positions for the at least one cryptic 3’ splice stie; a pyrimidine-rich domain comprising at least 6 consecutive nucleotides, wherein the sequence of the pyrimidine-rich domain is at least 60% pyrimidine nucleotides, and wherein the pyrimidine-rich domain has a wide range of positions; and at least one branchpoint at least 15 nucleotides upstream of the canonical 3' splice site with the intended use, for example, of be spliced differently in a cancer cell comprising a change-of function or loss-of-function mutation in a recurrently mutated SF3Bl RNA splicing factor gene relative to the splicing pattern of the intron in a cell lacking a change-of-function or loss-of function mutation in a recurrently mutated RNA splicing factor gene as required in claim 16. The specification does not provide support for the genus of constructs with a wide range of splicing locations in the intron of the construct such that it would provide the claimed function of being spliced differently in a cancer cell with a mutation compared to cell lacking a mutation when introducing “ to the cell an expression cassette comprising a coding sequence (CDS) interrupted by at least one artificial nucleic acid intron as described herein, wherein the expression cassette further comprises a promoter operatively linked to the CDS” (para [0034] of the published application) .
The specification describes use of 6 endogenous introns to develop synthetic introns (para 0242 of published application). The specification describes that, ultimately, the intron 1 of MTERFD3 was used in the artificial intron constructs due to being more efficiently excised in the SF3B1-mutant cells (para 0245 of published application), and the MTERFD3 constructs were used for further optimization of key sequence features such as splicing locations (para 0246-0247 of published application; Fig 2E-G). Furthermore, the specification discloses that herpes simplex virus thymidine kinase (HSV-TK) system was used as the cancer treatment agent in the vector. More specifically, two exons of HSV-TK flanked the 3’ and 5’ end of the intron and when the intron is excised by the mutated SF3B1 which is only found in cancer cells the two exons come together to then allow translation of the full HSV-TK thus eliciting its effects on the cancer cell; para 0186 of published application. While the specification describes massive parallel assaying to determine key features such as 3’ and 5’ splicing regions required for function of the construct (para 0237), 8 synthetic constructs for the MTERFD3 intron were used for single construct studies and revealed the essentiality of the immediate sequence context of the cryptic 3’ss at -11 nt and far distal consensus branchpoints are important (para 0257 of published application).
Therefore, the specification only provides details to synthetic constructs comprising a MTERFD3 intron with specific key features for splicing as described in table 1 on page 86 and the exons that are interrupted by inclusion of the MTERFD3 intron correspond to HSV-TK such that when the intron is excised by SF3B1 mutant cells, HSV-TK protein is expressed (i.e. expression of protein of interest after intron excision; page 68, line 27-30).
the state of the prior art; the level of predictability in the art:
Use of synthetic constructs for targeting expression of genes of interest in biomedical applications has been known in the art, however the use of intron modifications to achieve that are spliced differently depending on the cell’s RNA-splicing defect of cancer cells in relation to normal cells remains an area of research as these technologies are still in their infancy stage of development. Haddad-Mashadrizeh teaches that while, intron-based gene therapies show potential, “each regulator of spliceosome catalytic elements (SCEs), exon junction complexes (EJCs), intron-defined regulatory elements (IDREs), and intron splicing control sequences (ISCSs) has a specific function that modulates gene expression at different levels. Therefore, harnessing this potential relies on having precise knowledge about introns and their regulatory elements, as well as understanding their functions” (page 40, left col, para 1). Nissim teaches that there is potential off-target activity of circuit components (i.e. artificial intron constructs) and affect specificity of the constructs (page 1145, right col, Discussion) and that future work will be needed to optimize and characterize
specific immunomodulatory combinations in the constructs (page 1147, left col, para 1).
Therefore, the prior art highlights the unpredictability of the combination of genomic elements such as exon junction and intron splicing sequences such that expression of genes of interest can be altered leading to undesirable results including off-target activity.
the quantity of experimentation needed to make or use the invention based on the
content of the disclosure:
The skilled artisan would be required to perform under levels of experimentation
in order to practice the claimed invention. The instant specification does not reduce to
practice the claimed invention; the instant specification does not provide guidance on making any artificial nucleic acid construct with any intron other than: Sixteen different artificial nucleic acid intron constructs comprising the introns of, MTERFD3 identified by SEQ ID NOS 56-71 each with a 5’ splice site, a canonical 3’ splice site, a cryptic 3’ splice site, and pyrimidine rich domain, and a branchpoint and comprises herpes simplex virus thymidine kinase (HSV-TK) system as the exon domains for expressing HSV-TK. Thus, the skilled artisan would be forced to 1) determine which introns can be inserted in the construct, 2) where the 3’ and 5’ and cryptic splice sites should be in the construct, and 3) determine is the introns would be able to be spliced differently in a cancer cell comprising a mutation in SF3B1 compared to a cell without the mutation.
the level of one of ordinary skill:
The level of one of ordinary skill is a PhD holder.
Conclusion:
When all of the Wands factors are considered together, they establish a prima
facie case that the specification is not enabling for the claims. The Specification only
provides details for artificial nucleic acid constructs with the introns of MTERFD3 identified by SEQ ID NOS 56-71 each with a 5’ splice site, a canonical 3’ splice site, a cryptic 3’ splice site, and pyrimidine rich domain, and a branchpoint and comprises herpes simplex virus thymidine kinase (HSV-TK) system. Use of the HSV-TK system in the artificial intron construct is described in the specification as the coding sequence (i.e. exons) being divided by the intron such that, upon excision of the intron by mutant SF3B1 cells, the HSK-TK is ultimately translated to protein (para [0186]).
Claim interpretation
Claim 3 recites the phrase “wherein the intron is derived from a human wildtype intron”. This phrase is interpreted to mean that the intron can be 100% sequence matched to the wildtype intron as the 100%-sequence-matching intron would be completely “derived” from the wildtype intron.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claim(s) 1, 3-16, and 18-20 are rejected under 35 U.S.C. 103 as being unpatentable over Enenkel et al (US 20160348143 A1; as cited in IDS), and further in view of Maguire et al (Journal of Pathology, 2015, pages 571–580), NCBI Reference Sequence NC_000012.12 (Homo sapiens chromosome 12, GRCh38.p14 Primary Assembly MTERFD3, first published Aug 29, 2002, pages 1-8), Nissim et al (Cell, 2017, pages 1138–1150), and University of Maryland (RNA Info: Splice Site Consensus, first published online 07/16/2019), as evidenced by Wikipedia (Polypyrimidine tract, downloaded 5/21/2026, pages 1-2)
Regarding claim 1, Enenkel teaches novel artificial introns that can be derived from a natural intron from a gene of interest (para 0046) and compositions comprising artificial introns where in artificial introns contain at least three highly conserved sequence elements essential for splicing: a 5′ splice site, a branch point and a 3′ splice site containing a run of pyrimidines called a polypyrimidine tract and the branch point or branch site is usually located approximately between 10 and 60 nucleotides upstream of the 3′ splice site (para 0039), and that splice sites include cryptic splice sites (para 0041), rendering obvious an artificial nucleic acid intron construct, comprising an intron comprising: a 5' splice site; a canonical 3' splice site; at least one cryptic 3' splice site.
In view of Enenkel’s polypyrimidine tract, it would have been obvious to design a a pyrimidine-rich domain comprising at least 6 consecutive nucleotides, wherein the sequence of the pyrimidine-rich domain is at least 60% pyrimidine nucleotides, based on influential considerations in the design of the assay such that the polypyrimidine tract promotes the assembly of the spliceosome and is rich with pyrimidine nucleotides and is usually 15–20 base pairs long, located about 5–40 base pairs before the 3' end of the intron to be spliced, as evidenced by Wikipedia (page 1, para 1, downloaded 5/21/2026).
Enenkel does not teach the that cryptic splice site is within about 100 nucleotides upstream of the canonical 3' splice site or within about 50 nucleotides downstream of the canonical 3' splice sites; the pyrimidine-rich domain is within at least 50 nucleotides of a cryptic 3' splice site; and the at least one branchpoint at least 15 nucleotides upstream of the canonical 3' splice site, however it would have been prima facie obvious to one of ordinary skill in the art at the time of the effective filing date, to modify the teachings of Enenkel to optimize for the aforementioned nucleotide positions for the cryptic splice site, the pyrimidine- rich domain, and the branchpoint for the artificial intron through routine experimentation to generate the artificial intron of interest based on gene structure and based on influential considerations that are implemented in splice site optimization tools.
Regarding claim 3, 16, and 18 the teachings of Enenkel render obvious claim 1.
Enenkel does not teach wherein the intron is derived from a human wildtype intron selected from intron 1 of MTERFD3, wherein the intron is configured to be spliced differently in a cancer cell comprising a change-of- function or loss-of-function mutation in a recurrently mutated SF3B1 RNA splicing factor gene relative to the splicing pattern of the intron in a cell lacking a change-of-function or loss-of- function mutation in a recurrently mutated RNA splicing factor gene, and wherein the recurrent change-of-function mutation in SF3B1 results in an amino acid substitution K700E.
However, one of ordinary skill in the art would have considered the teachings of Maguire, NCBI, and Nissim, as these references are analogous prior art pertaining to the use of intron-based gene therapies for cancer treatment and the relevance of SF3B1 and MTERFD3 as a downstream target for cancer treatment.
Nissim teaches synthetic gene circuits (i.e. intron construct) that are designed to achieve a tumor-specific production of immunomodulators (page 1139, left col, para 2) wherein the synthetic gene circuit uses a microRNA intron inserted within a gene of interest, resulting in two flanking exons from the gene of interest and the microRNA is spliced out by canonical splicing mechanisms and permits translation of the gene of interest from the two exons (see Figure 2, page 1141; page 1140, left col, RNA-Based AND Gate Circuit). Nissim teaches that the tumor-specificity of the construct is driven by cancer-specific promoters of the construct (page 1140, right col, Synthetic Cancer-Specific Promoters).
Maguire teaches that MTERFD3 is a gene that shows differential splicing in breast cancer cells due to SF3B1 K700E mutations (page 576, left col, para 1) and that targets for SF3B1 (and, by default, spliced gene targets of SF3B1 such as MTERFD3) would be novel therapeutic targets in breast cancer (abstract).
NCBI teaches the gene sequence of MTERFD3, including the location of introns.
It would have been prima facie obvious to one of ordinary skill, in the art at the time of the effective filing date, to modify the teachings of an artificial intron construct that can be derived from a natural intron from a gene of interest from Enenkel with the teachings of Nissim for an intron construct that can be tumor-specific wherein the tumor-specific artificial intron construct comprises the intron of MTERFD3, instead of a microRNA, as MTERFD3 is differentially spliced in breast cancer cells that have a mutated SF3B1 gene as taught by Maguire and the nucleotide sequence for the MTERFD3 gene can be found from NCBI. Furthermore, while Nissim teaches microRNA is spliced out by canonical splicing mechanisms and permits translation of the gene of interest from the two exons, one could instead use mutated splicing mechanisms by a cancer cell to drive specificity of the construct to only cancer cells as taught by Maguire. One would be motivated to do this to generate an intron construct that could be specifically spliced by mutated SF3B1 in a cancer cell and lead to specific expression of a gene of interest that can therapeutically target the cancer and not require designing cancer-specific promoters.
Regarding claim 4, the teachings of Enekel, Nissim, Maguire, and NCBI render obvious claims 1 and 3. Moreover, NCBI teaches the intron 1 of MTERFD3 with 100% alignment to a sequence set forth in SEQ ID NO:2 of the instant application See alignment on next page. Asterisks indicate matching nucleotides.
Reference = NCBI MTERFD3 sequence, SEQ_ID_2 = instant SEQ ID NO
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875
718
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:2
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873
736
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863
725
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97
732
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Regarding claim 5, Enekel, Nissim, Maguire, and NCBI render obvious claims 1 and 3. Moreover, Enekel teaches “The branch point or branch site is usually located approximately between 10 and 60 nucleotides upstream of the 3′ splice site (para 0039), rendering obvious the artificial nucleic acid intron construct of claim 3, wherein the intron is derived from a human wildtype intron 1 of MTERFD3, and wherein the intron further comprises at least one branchpoint at least 20 nucleotides upstream of the canonical 3' splice site.
Regarding claim 6 and 7, Enekel, Nissim, Maguire, and NCBI render obvious claims 1, 3, and 4. Furthermore, as NCBI teaches the intron recited in claims 3 and 4 with 100% sequence alignment, the intron would, by default, have a 5' end domain with about 10 to about 150 nucleotides having at least 50% sequence identity to a sequence of the 5'-most 10 to about 150 nucleotides of the wildtype intron and a 3' end domain with about 50 to about 350 nucleotides having at least 50% sequence identity to a sequence of the 3'-most 50 to about 350 nucleotides of the wildtype intron.
Regarding claim 8, Enekel, Nissim, Maguire, and NCBI render obvious claims 1, 3, and 4. Furthermore, NCBI teaches an intron sequence that has 83% query sequence identity to SEQ ID NO: 5 of the instant application. See alignment below.
Qy = instant SEQ ID NO:2, Db = NCBI MTERFD3 sequence
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367
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Regarding claim 9-12, the teachings of Enenkel render obvious claim 1.
Enenkel does not teach wherein the 5’ splice site comprises a sequence GTGAG (claim 9), wherein the 3’ canonical splice site comprises a sequence CAG (claim 10), wherein at least one cryptic 3’ splice site comprises a sequence AAG (claim 11), and wherein the intron comprises a plurality of cryptic 3' splice sites within about 100 nucleotides upstream of the canonical 3' splice site or within about 100 nucleotides downstream of the canonical 3' splice site, and wherein each of the plurality of the cryptic 3' splice sites comprises a sequence AAG (claim 12).
University of Maryland (UMD) teaches that “It is well-established that nearly all splice sites conform to consensus sequences” (page 1, para1) and that “Splice site consensus sequences for U2 (major class) introns in pre-mRNA generally conform to the following consensus sequences: 5' splice sites: MAG|GTRAGT where M is A or C and R is A or G (page 1, para 2) and 3' splice sites: CAG|G (page 1, para 2). Hence, UMD teaches the sequence GTGAG as recited in claim 9 and recited in claim 10. UMD teaches that “These consensus sequences include nearly invariant dinucleotides at each end of the intron, GT at the 5' end of the intron, and AG at the 3' end of the intron” (page 1, para 1), hence rendering obvious the AAG sequence for the 3’ cryptic splice site recited in claim 11 and 12.
Regarding the recitation “the intron comprises a plurality of cryptic 3' splice sites within about 100 nucleotides upstream of the canonical 3' splice site or within about 100 nucleotides downstream of the canonical 3' splice site” as recited in claim 12, it would have been prima facie obvious to one of ordinary skill in the art at the time of the effective filing date, to modify the teachings of Enenkel to optimize for the for the intron to comprise a plurality of cryptic 3’ splice sites and the aforementioned locations upstream of or downstream of the canonical 3’ splice site through routine experimentation to generate the artificial intron of interest based on gene structure.
Regarding claim 13, the teachings of Enenkel render obvious claim 1.
Moreover, Enenkel teaches a 3′ splice site (splice acceptor site) containing a run of pyrimidines called a polypyrimidine tract (para 0039) and “the consensus sequence for a splice acceptor site consists of the sequence set forth in SEQ ID NO: 21.” (para 0043). SEQ ID NO: 21 of Enenkel consists of the sequence:
yyyyyyyyyy yyyyyyyyyy yyyyyyyyyy yyyyyyyyyy nyagr;
wherein “y” indicates a pyrimidine C or T.
Hence, Enenkel renders obvious the characteristics recited in claim 13: the pyrimidine-rich domain comprises at least 15 consecutive nucleotides, the pyrimidine-rich domain has a sequence with at least 60% pyrimidine nucleotides and is at least 40% thymine nucleotides, the pyrimidine-rich domain is within at least 30 nucleotides of a cryptic 3' splice site, the pyrimidine-rich domain has a sequence with at least 50% sequence identity to any 20 nucleotides selected from the sequence set forth as SEQ ID NO:49. See sequence alignment below showing 80% identity in any 20 nucleotides of Enenkel SEQ ID NO: 21 and instant application SEQ ID NO: 49 with at least 60% pyrimidine nucleotides and is at least 40% thymine nucleotides.
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Qy = instant application SEQ ID NO: 49, Db = example of Enenkel SEQ ID NO: 21
Regarding claim 14 and 15, the teachings of Enenkel render obvious claim 1. Moreover, Enenkel teaches “The branch point or branch site is usually located approximately between 10 and 60 nucleotides upstream of the 3′ splice site and forms during the splicing process via its conserved adenosine residue” (para 0039) and “the preferred branch point sequence is TACTAAC” (para 0039), rendering obvious wherein the at least one branchpoint is at least 20 nucleotides upstream of the canonical 3' splice site, and wherein the branchpoint nucleotide is an adenine and wherein the branchpoint and surrounding sequence context has sequence identity of at least 60% to the sequence tactaAca, where the uppercase A is the branchpoint nucleotide..
Regarding claim 19 and 20, the teachings of Enenkel render obvious claim 1.
Enenkel does not teach the artificial nucleic acid intron construct of claim 1, further comprising a first exon domain and a second exon domain, wherein the intron is disposed between the first exon domain and the second exon domain.
Nissim teaches synthetic gene circuits (i.e. intron construct) that are designed to achieve a tumor-specific production of immunomodulators (page 1139, left col, para 2) wherein the synthetic gene circuit uses a microRNA intron inserted between two flanking exons (i.e. gene of interest) and the microRNA is spliced out permitting protein translation of the gene of interest from the two exons (see Figure 2, page 1141).
It would have been prima facie obvious to one of ordinary skill, in the art at the time of the effective filing date, modify the teachings of an artificial intron construct that can be derived from a natural intron from a gene of interest from Enenkel with the teachings of Nissim for an intron construct that can be tumor-specific wherein the tumor-specific artificial intron construct is placed between two exons that, when the intron is excised, come together to be translated into a protein of interest. One would be motivated to do this to have a construct that could selectively express a protein of interest in a tumor cell due to excision of the intron and the protein of interest could be used as a cancer treatment. As use of artificial intron constructs has been shown in the art, one would have a reasonable expectation of success.
Conclusion
Claims 1, 3-16, and 18-20 are rejected.
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/JULIANA IRENE CANDELARIA/ Examiner, Art Unit 1634
/MARIA G LEAVITT/ Supervisory Patent Examiner, Art Unit 1634
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