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 .
1. Claims 1 – 15 and 16 – 20 are pending.
Election/Restrictions
2. Applicant's election with traverse of Group I (claims 1 – 8, 15, 18, 19) in the reply filed on 03/12/2026 is acknowledged. The traversal is on the ground(s) that claim 1 and its dependents are novel and non-obvious over Buerkstuemmer and the anticipation rejection is not proper where a finding cannot be made as is the case here that Buerkstuemmer discloses all features as arranged in present claim 1 in one single embodiment. This is not found persuasive because the technical feature of Groups I – VI is not a special technical feature because it does not make a contribution over the prior art in view of Buerckstuemmer as Buerckstuemmer teaches the branch point is usually located approximately between 10 and 60 nucleotides upstream of the splice acceptor as previously stated in the Restriction Requirement.
The requirement is still deemed proper and is therefore made FINAL.
3. Claims 9 – 14 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 03/12/2026.
Priority
4. This application claims foreign priority to Application EP21172761.5 filed on 05/07/2021.
Information Disclosure Statement
5. The information disclosure statement (IDS) submitted on 10/31/2023 is acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Drawings
6. The drawings are objected to because of the following informalities: there is description of color in the Specification of Figure 1 on page 16 and Figures 3 – 5 on page 17, and the various colors cannot be distinguished from each other since the figures are in black and white.
7. Color photographs and color drawings are not accepted in utility applications unless a petition filed under 37 CFR 1.84(a)(2) is granted. Any such petition must be accompanied by the appropriate fee set forth in 37 CFR 1.17(h), one set of color drawings or color photographs, as appropriate, if submitted via the USPTO patent electronic filing system or three sets of color drawings or color photographs, as appropriate, if not submitted via the via USPTO patent electronic filing system, and, unless already present, an amendment to include the following language as the first paragraph of the brief description of the drawings section of the specification:
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Color photographs will be accepted if the conditions for accepting color drawings and black and white photographs have been satisfied. See 37 CFR 1.84(b)(2).
Specification
8. The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
9. The use of the term piggybac, LongAmp, Gotaq, LSRFortessa, FlowJo, Pannoramic, which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
10. Claim 4 is objected to because of the following informalities: in line 1, “the sequence of 10” should read “a sequence of 10” because “the sequence” lacks antecedent basis. The objection does not rise to a 112(b) rejection because it is clear Applicant is referring to a sequence in the genetic element that is upstream of the splice acceptor site. Appropriate correction is required.
11. Claim 7 is objected to because of the following informalities: in line 2, “wherein two splice branch points” should read “wherein the two splice branch points” to clarify these are the same two splice branch points recited in line 1 – 2. Appropriate correction is required.
12. Claim 15 is objected to because of the following informalities: in line 2, “a genetic element” should read “the genetic element” to clarify that “genetic element” refers to the genetic element of claim 1. Appropriate correction is required.
13. Claim 19 is objected to because of the following informalities: in line 6, “the introduction” should read “the insertion” to clarify that this wherein clause refers back to the first wherein clause reciting “is inserted”. Appropriate correction is required.
14. Claim 19 is objected to because of the following informalities: in line 7, “intron the intron” should read “intron, the intron” to include punctuation. Appropriate correction is required.
15. Claim 19 is objected to because of the following informalities: in line 8, “a genetic element” should read “the genetic element” to clarify that “genetic element” refers to the genetic element of claim 1. Appropriate correction is required.
Claim Rejections - 35 USC § 112
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.
16. Claims 4, 6, 7, 8, and 19 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
17. Regarding claim 4, the phrase "preferably" renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
18. Regarding claim 6, the phrase "especially preferred" renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
19. Regarding claim 7, it is unclear if the claim is further limiting “a splice branch point” recited in claim 1 to require two or more splice branch points, or if the limitation means that the genetic element of claim 1 further comprises a second branch point, or 3 or more branch points (a branch point of claim 1 and two or more of claim 7).
20. Regarding claim 8, the phrase "preferably" renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
21. The term “stringent” in claim 19 is a relative term which renders the claim indefinite. The term “stringent” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The “splice junction consensus sequence” is rendered indefinite by the use of “stringent”.
22. The term “flexible” in claim 19 is a relative term which renders the claim indefinite. The term “flexible” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The “splice junction consensus sequence” is rendered indefinite by the use of “stringent”.
23. The metes and bounds of claim 19 is unclear because it recites a method of introducing an intron sequence into an exon or between two exons but recites a wherein clause of “the intron is inserted into an intron insertion site”. Further, the claim recites “the steps of” but only recites one active step of “selecting”.
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.
24. Claim 19 is rejected under 35 U.S.C. 101 because the claimed invention is directed to “selecting” which is an abstract ideal that is a mental process without significantly more. The claim(s) recite(s) “selecting” which is a mental process.
The Office published Office's new guidance document entitled 2019 Revised Patent Subject Matter Eligibility Guidance, published January 7, 2019. Applicant is directed to the Federal Register, Volume 4, No. 4, pages 50-57 at page 74621.
Claim 19 recites a method of introducing an intron sequence into an exon or between two exons of a gene, comprising the steps of selecting the exon or one of the two exons, respectively, which is positioned within the first 50% base pairs (bp) of a protein coding-sequence of the gene; and wherein the intron is inserted into an intron insertion site containing either a stringent splice junction consensus sequence or a flexible splice junction consensus sequence; and wherein after the introduction of the intron the intron is separating exons on the intron' s 5' and 3' sides with the exons being each at least 60 bp in length, wherein the intron sequence is a genetic element of claim 1.
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Step 1 of the USPTO' s eligibility analysis entails considering whether the claimed subject matter falls within the four statutory categories of patentable subject matter identified by 35 U.S.C. 101: Process, machine, manufacture, or composition of matter. The claims are directed to a process (step 1, Yes).
Step 2A of the 2019 Revised Patent Subject Matter Eligibility Guidance is a two-prong inquiry. In Step 2A Prong One, examiners evaluate whether the claim recites a judicial exception. The process is directed to an abstract idea of “selecting” (Step 2A, prong 1, Yes). The markedly different characteristics analysis is used to determine if the abstract idea (“selecting”) is a mental process. Nothing in the claim precludes the step of selecting from being performed in the human mind. Therefore, the claimed step of “selecting” is a mental process.
In Step 2A Prong Two, examiners evaluate whether the claim recites additional elements that integrate the exception into a practical application of that exception. This evaluation is performed by (a) identifying whether there are any additional elements recited in the claim beyond the judicial exception, and (b) evaluating those additional elements individually and in combination to determine whether the claim as a whole integrates the exception into a practical application. Besides the mental process of “selecting”, the claim recites “a method of introducing an intron sequence into an exon or between two exons of a gene” and “the intron is inserted into an intron insertion site” and a preferred outcome of the distance separating exons. An evaluation of whether this limitation is insignificant extra-solution activity is then performed. Note that because Step 2A Prong Two analysis excludes consideration of whether a limitation is well-understood, routine, conventional activity, this evaluation does not take into account whether or not the limitation is well-known. When so evaluated, this additional element is insignificant extra-solution activity because “exon” and “gene” are recited so generally and no limitations are recited regarding how the exon or gene is selected. The claim does not impose any limitations on selecting the exon or the gene or how the selection is performed. Therefore, the claim covers any possible exon and selection of the exon being performed in the human mind and fails to meaningfully limit the claim because it is at best the equivalent of merely adding the words “apply it” to the judicial exception. Accordingly, the mental process of selecting is not integrated into a practical application and the claim is therefore directed to a mental process that is a judicial exception(Step 2A, prong 2, No).
In Step 2B, the eligibility analysis evaluates whether the claim as a whole amounts to significantly more than the recited exception, i.e., whether any additional element, or combination of additional elements adds an inventive concept into the claim. As discussed with respect to Step 2A Prong Two, the claim recites the claim recites “a method of introducing an intron sequence into an exon or between two exons of a gene” and “the intron is inserted into an intron insertion site”, which is at best the equivalent of merely adding the words “apply it” to the judicial exception. Mere instructions to apply an exception cannot provide an inventive concept. At Step 2B, the evaluation of the insignificant extra-solution activity consideration takes into account whether or not the extra-solution activity is well-known. Here, recitation of “exon” and “gene” is recited at a high level of generality. Thus, the preamble of the claim does not amount to significantly more and does not provide an inventive concept (Step 2B: No).
Limitations that were found not to be enough to qualify as ‘‘significantly more” when recited in a claim with a judicial exception include: Adding the words ‘‘apply it” (or an equivalent) with the judicial exception, or mere instructions to implement an abstract idea on a computer; simply appending well-understood, routine and conventional activities previously known to the industry, specified at a high level of generality, to the judicial exception, e.g., a claim to an abstract idea requiring no more than a generic computer to perform generic computer functions that are well-understood, routine and conventional activities previously known to the industry; adding insignificant extrasolution activity to the judicial exception, e.g., mere data gathering in conjunction with a law of nature or abstract idea; or generally linking the use of the judicial exception to a particular technological environment or field of use.
In the instant case, the limitations of the claims do not impose limits on the claim scope such that they are not markedly different from a mental process being performed in the human mind.
Claim Interpretation
25. For the purpose of applying prior art, “preferably” in claim 4 is interpreted as optional limitations that are not required by the claim.
26. For the purpose of applying prior art, the nucleic acid sequences recited in claim 6 are interpreted to be the same sequence in the SEQ ID NOs recited in parentheses.
27. For the purpose of applying prior art, “especially preferred” in claim 6 is interpreted as optional limitations that are not required by the claim.
28. For the purpose of applying prior art, claim 7 is interpreted as the genetic element of claim 1 further comprising a second branch point.
29. For the purpose of applying prior art, “preferably” in claim 8 is interpreted as optional limitations that are not required by the claim. Therefore, claim 8 is interpreted as a genetic vector comprising the genetic element of claim 1.
30. For the purpose of applying prior art, claim 19 is interpreted as a method comprising the step of selecting the exon because this is the only active step recited in the claim.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
31. Claim(s) 1 – 8, 15, 18, 19, and 20 is/are rejected under 35 U.S.C. 103 as being unpatentable over Guzzardo (Guzzardo, Paloma M., et al. Scientific reports 7.1 (2017): 16770.), hereinafter Guzzardo which is cited on the IDS filed 10/31/2023 in view of Skarnes (WO-2017203275-A1; Filed 05/27/2016; Published 11/30/2017), hereinafter Skarnes which is cited on the IDS filed 10/31/2023.
Regarding claim 1, Guzzardo teaches a cassette (“genetic element”) comprising a splice donor site, a first recombinase recognition site, a splice branch point, a second recombination site and a splice acceptor site (Table 1; page 2, para. 4; Figure 1C). Guzzardo does not teach “wherein the splice branch point is at a distance of 10 to 56 nucleotides in length from the splice acceptor site, or a reverse complementary sequence”.
Regarding claim 2, Guzzardo teaches Variants 1 and 2 cassettes in which the branch point is 6 nucleotides from the second loxP site in Table 1.
Regarding claim 3, Guzzardo teaches the splice donor site (green in Figure 1C) is at a distance of 80 to 5000 nucleotides in length from the splice acceptor site (blue in Figure 1C) in Figure 1C.
Regarding claim 4, Guzzardo teaches a 10 nucleotide polypyrimidine tract (underlined in Figure 1C) that is 5’ adjacent to the splice acceptor site (blue in Figure 1C) that contains 9 pyrimidine nucleotides where 4 are C and 5 are T in Figure 1C.
Regarding claim 5 and 20, Guzzardo teaches the nucleic acid sequence of the splice donor site (green in Figure 1C) is GTAAG (“GTPuAG” of claim 5), the splice branch point (yellow in Figure 1C) comprises the sequence CTGAT (“CTPuAPy” of claims 5 and 20), and the splice acceptor sequence (blue in Figure 1C) comprises CAG (“AG” of claims 5 and 20) in Figure 1C.
Regarding claim 8, Guzzardo teaches the cassette is present on a plasmid (page 3, para. 1).
Regarding claim 15, Guzzardo teaches a plasmid encoding the cassette and a Cas9 expression plasmid (page 3, para. 1 – 3).
Regarding claim 18, Guzzardo teaches a plasmid encoding the cassette and a Cas9 expression plasmid and a plasmid encoding gRNA targeting an exon in a target gene (page 3, para. 1 – 3; Table 2; Figure 1; page 5, para. 2).
Regarding claim 19, Guzzardo teaches a method of introducing an artificial intron sequence into an exon comprising targeting (“selecting”) the exons of human genes CD46 and METTL16 because CD46 is cell-surface expressed and can be easily detected by FACS and METTL16 is an essential gene and cassette activation should trigger cell death (page 7, para. 2 – 8; page 3; Figure 4). Guzzardo does not teach “which is positioned within the first 50% base pairs (bp) of a protein coding-sequence of the gene”.
Guzzardo does not teach “wherein the splice branch point is at a distance of 10 to 56 nucleotides in length from the splice acceptor site, or a reverse complementary sequence” of claim 1 or “FRT site” of claim 6 or “two or more splice branch points” of claim 7 or “which is positioned within the first 50% base pairs (bp) of a protein coding-sequence of the gene” of claim 19. However, Guzzardo teaches there is a large unmet need for tools to facilitate the study of essential genes (page 1, para. 3). Guzzardo teaches conditional inactivation of essential genes remains a challenge (Abstract). Guzzardo teaches the method using the small cassette results in clones bearing the conditional knockout at frequencies above 5% and loss of gene expression as judged by flow cytometry, Western blot or immunofluorescence (Abstract). Guzzardo teaches the method using the cassette highlights the broad utility of the approach for conditional gene inactivation and suggests that this tool could be used to study the loss-of-function phenotypes of essential genes (Abstract; page 2, para. 3). Guzzardo teaches the cassette is a small intron cassette and is used in a method to generate conditional gene knockouts where the cassette is inserted into the coding sequence of an exon (page 5, para. 2). Guzzardo teaches to overcome limitations of previous similar approaches, the size of the intron was minimized (page 5, para. 2). Guzzardo teaches the method causes the removal of the branch point by Cre/loxP to cripple the intron, thus triggering the translation of truncated intronic sequences (page 5, para. 2; Figure 1). Guzzardo teaches addition of three stop codons would then lead to translational termination of the gene and would thus abrogate gene expression (page 5, para. 2). Guzzardo teaches the cassette readily allows the tagging of endogenous genes, poising them for conditional gene activation and the system is robust, triggering a profound decrease in gene expression and may be the first approach that can be truly applied at a large scale (page 10, para. 1).
Guzzardo teaches that since the intron was very small, the precise architecture of the cassette was not straightforward and the two major risks included (i) cassette modification could disturb intron splicing, hence leaving the cassette in a constant “on-state”; (ii) as the branch point sequence is quite degenerate, nearby sequences may take over branch point function upon its removal (page 5, para. 3). Guzzardo teaches to overcome these difficulties, an assay based on NanoLuc reporter expression was established to test a variety of cassette configurations (page 5, last para.). Guzzardo teaches an ideal cassette leaves NanoLuc expression unaffected (“off-state”) but triggers a profound decrease upon Cre/loxP recombination (“on-state”) (page 6, para. 1). Guzzardo teaches testing variations of the cassette where one variant showed decreased NanoLuc expression in the “off-state”, and two variants did not result in reduction of NanoLuc expression upon Cre recombination (suggesting that alternative branch points had taken over) (page 6, para. 2; Figure 2). Guzzardo teaches variant 4 shown in Figure 1C was well tolerated in the “off-state” and triggered a profound decrease in NanoLuc expression upon cassette activation (Figure 2; page 6, para. 2). Guzzardo teaches the main difference between the variants is the location of the loxP sites (Table 1). Guzzardo teaches splicing is strongly dependent on the sequence context and requires certain specific sequences at the intron/exon boundary and different introns have different preferences for the sequences upstream of the splice donor and downstream of the splice acceptor sites (page 6, last para.). Thus, Guzzardo teaches a method for routine optimization of the sequences of the cassette containing a splice donor site, two recombinase recognition sites, a splice branch point, and a splice acceptor site.
Regarding “wherein the splice branch point is at a distance of 10 to 56 nucleotides in length from the splice acceptor site, or a reverse complementary sequence” of claim 1, Skarnes teaches a cassette for conditional gene knockout comprising a splice donor, a branch point, and a splice acceptor (page 11, lines 11 – 20). Skarnes teaches in Figure 1b the cassette comprises two pairs of loxP sites for Cre mediated recombination, splicing donor and acceptor sites including two branching points for spliceosome recognition and intron excision (page 4, lines 7 – 18). Skarnes teaches embodiments where the branch point is 46 and 56 nucleotides upstream from the splice acceptor (page 11, lines 11 – 20). Skarnes teaches the sequence of the splice donor site is GTAAG; the sequence of the splice acceptor site is TTTCCCTCCCTTAG; and the sequence of the branch point is CTGAT or CTGAC (page 11, lines 21 – 30). Skarnes teaches the nucleic acid encoding the cassette is present on a vector (page 14, lines 17 – 23).
Regarding claim 6, Skarnes teaches the recombinase sites include loxP and FRT sites and FLP-recombinase is a tyrosine family site-specific recombinase which recognizes FRT sites (page 16, lines 7 – 18 and 30 – 33).
Regarding claim 7, Skarnes teaches the cassette comprises two splice branch points (Figure 1b; page 4, lines 7 – 18). Skarnes does not teach the distance between the two splice branch points is 1 to 10 nucleotides from each other. However, it would be obvious to adjust the location of the branch points to arrive at branch points that are 1 to 10 nucleotides in distance from each other as it is a result-effective variable dependent on the splicing machinery of a given cell/organism because Guzzardo teaches that a major risks of small cassettes for conditional knockout included (i) cassette modification could disturb intron splicing, hence leaving the cassette in a constant “on-state”; (ii) as the branch point sequence is quite degenerate, nearby sequences may take over branch point function upon its removal (page 5, para. 3) and Guzzardo teaches in the optimization of the cassette sequence two variants did not result in reduction of NanoLuc expression upon Cre recombination (suggesting that alternative branch points had taken over) (page 6, para. 2; Figure 2).
Regarding claim 19, Skarnes teaches the one-step method of producing a conditional knockout using the cassette combines invertible intronic cassette (FLIP) with high efficiency Cas9-assisted gene editing (page 17, lines 10 – 24; page 20 lines 17 – 21). Skarnes teaches to maximize the potential for a null mutation, the target exon must lie within the first 50% of the protein-coding sequence (“which is positioned within the first 50% base pairs of a protein-coding sequence of the gene” of claim 19) (page 25, lines 20 – 35). Skarnes teaches based on the minimum size of mammalian exons (50 bp) the size of the split exons was set to be at least 60 bp and insertion points that match the consensus sequence for mammalian splice junctions were chosen for optimal splicing (page 25, lines 20 – 35). Skarnes teaches using a dsRed cassette to insert dsRed in the eGFP cDNA in Example 1 where Cre recombinase mediated cassette inversion resulting in loss of eGFP expression and maintaining red fluorescence (page 24, lines 5 – 25; Figure 1c and d). Skarnes teaches using a cassette conditional modification into mouse beta catenin and several human genes (page 24, lines 25 – 37; page 25, lines 1 – 20). Skarnes teaches a bioinformatics analysis that revealed over 1 million insertion sites and corresponding gRNA binding sites covering over 15,000 genes in the mouse and human genomes (page 25, lines 20 – 35). Skarnes teaches the cassette and method of using the cassette can be applied to any gene, including non-coding RNA genes (page 26, lines 1 – 7). Skarnes teaches while simple constitutive knockouts are useful and informative, in many cases it is desirable to engineer conditional loss-of-function models, particularly for essential genes required for cell viability or embryonic development (page 1, lines 30 – 35).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Guzzardo regarding a cassette comprising a splice donor site, a first recombinase recognition site, a splice branch point, a second recombination site and a splice acceptor site for use in producing conditional knockouts with the teachings of Skarnes regarding a cassette for conditional gene knockout comprising a splice donor, a branch point, and a splice acceptor where the branch point is 46 and 56 nucleotides upstream from the splice acceptor to arrive at the claimed genetic element comprising: a splice donor site, a first recombinase recognition site, a splice branch point, a second recombinase recognition site, a splice acceptor site, wherein the splice branch point is at a distance of 10 to 56 nucleotides in length from the splice acceptor site, or a reverse complementary sequence thereto. One would have been motivated to combine the teachings of Guzzardo and Skarnes in a cassette for creating conditional knockouts as Guzzardo teaches there is a large unmet need for tools to facilitate the study of essential genes and Guzzardo teaches conditional inactivation of essential genes remains a challenge and Guzzardo teaches the method using the cassette highlights the broad utility of the approach for conditional gene inactivation and suggests that this tool could be used to study the loss-of-function phenotypes of essential genes and Skarnes teaches while simple constitutive knockouts are useful and informative, in many cases it is desirable to engineer conditional loss-of-function models, particularly for essential genes required for cell viability or embryonic development. One would have a reasonable expectation of success in combining the teachings as Guzzardo and Skarnes teach successful use of their cassettes for conditional knockout of several genes in human cells and Guzzardo teaches the cassette readily allows the tagging of endogenous genes, poising them for conditional gene activation and the system is robust, triggering a profound decrease in gene expression and may be the first approach that can be truly applied at a large scale and Skarnes teaches the cassette and method of using the cassette can be applied to any gene, including non-coding RNA genes.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ZANNA M BEHARRY whose telephone number is (571)270-0411. The examiner can normally be reached Monday - Friday 8:45 am - 5:45 pm.
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/ZANNA MARIA BEHARRY/Examiner, Art Unit 1632
Prosecution Insights