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 .
Application Status and Election
Claims 79-92 are pending.
Applicant’s election of a meganuclease targeting a GTAT center sequence and having K48A, Q50K, G71, S72R, V73A and S74 relative to SEQ ID NO 1 in the reply filed April 22, 2026 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)). After an extensive search of I-CreI variants having each of the recited substitutions (K48A, Q50K, S72R and V73A) relative to SEQ ID NO 1 and wild-type I-CreI, Examiner concludes that the elected species is free of the prior art. Although engineering I-CreI recombinases is routine in the art and amino acid substitutions at each of residue positions have been described in the prior art, there is no teaching or suggestion to combine them. There is also no teaching as to the effect of the substitutions at each of those positions on the ability to bind and/or cleave a specific central sequence. The search was widened to include any set of substitutions recited in claims 86. In view of the sheer breadth of different combinations for meganucleases claimed in claims 79-92, the claims that are directed to meganucleases that can comprise different first and second subunits and can bind and cleave central sequences recited in claims 79 and 80, claims 79-85 are withdrawn.
Claims 86-92 are under examination.
Specification
The use of the terms Lipofectamine®, 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 Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 88-90 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claims 88-90 recite “a polynucleotide”, “A recombinant DNA construct comprising a polynucleotide” and “a recombinant virus comprising a polynucleotide” comprising a nucleic acid encoding said engineered meganuclease of claim 86. Thus claims 88-90 are directed to only nucleic acids, whereas claim 86 only recites “an engineered meganuclease” which is a protein. 35 CFR 1.75 states “One or more claims may be presented in dependent form, referring back to and further limiting another claim or claims in the same application.” Because claims 88-90 refer back claim 86, they are dependent claims of claim 86. However, polynucleotides and polypeptides are distinct and mutually exclusive molecules. Polynucleotides are composed of nucleotides and not amino acids. Thus claims 88-90 do not include all the limitations of the claim from which it depends.
To overcome this rejection, it is suggested that claim 86 be amended to recite “An engineered meganuclease or a nucleic acid encoding the meganuclease, wherein the meganuclease binds and cleaves…”
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 112(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.
Claims 86 and 88-92 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.
Claim 86 recites “an engineered meganuclease” that has a function of binding and cleaving a center sequence and has “an amino acid substitution at one or more positions corresponding to position 48, 50, 71, 72, 73 and 74 of SEQ ID NO: 1” and then recites three options (A), (B) and (C), with additional residue options for each position. In Options (A) and (B), there is a conjunction “and” between the last and second to last – between (e) and (f) – such that it is clear that each recited position must have one of the recited amino acids. However, there is no conjunction between the last (e) and second to last (d) options of embodiment (C). As such it is not clear if each of the substitutions (a)-(e) are needed, a subset, or just 1 substitution.
Claims 88-92 are rejected for depending from claim 86 and not remedying the indefiniteness. It is noted that claim 87 is not included in this rejection since it recites specific options for each of the positions.
Claim Rejections - 35 USC § 112(a)
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 86-92 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.
MPEP 2163.II.A3.(a).(i) states, “whether the specification shows that applicant was in possession of the claimed invention is not a single, simple determination, but rather is a factual determination reached by considering a number of factors. Factors to be considered in determining whether there is sufficient evidence of possession include the level of skill and knowledge in the art, partial structure, physical and/or chemical properties, functional characteristics alone or coupled with a known or disclosed correlation between structure and function, and the method of making the claimed invention.”
For claims drawn to a genus, MPEP 2163.II.A3.(a).(ii) states, “written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species” where “representative number of species' means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus.”
Claim 86 recites a meganuclease comprising a first and second subunit and then limits only the structure of the first subunit to being 85% identical to SEQ ID NO 1 and having a set of substitutions at one or more of position 48, 50, 71, 72, 73 and 74. Claim 86 also requires the function that the engineered nuclease bind to and cleave a 22-base pair recognition sequence comprising a center sequence of GTAG, GTAT, GTGA, GTCA, GTGG or GTGT. Claim 86 does not limit or recite any structural features of the “second subunit”, so the structure of half of the meganuclease is completely undefined. For the first subunit, SEQ ID NO 1 is 163 amino acids in length. A subunit having a minimum of 85% identity to SEQ ID NO allows a maximum of 24 varied amino acids, 4-6 of which need to be in the recited positions and are limited by a subset of amino acids. The genus of such first subunits of the meganuclease comprises over 1029 different subunits. Such subunits can have any amino acid at up to 18 other positions in the protein, and thus the genus is structurally diverse. Additionally, since there are no structural limitations for the second subunit in terms of size or sequence, the genus of meganucleases is actually infinite. For the reasons provided below, Applicant has not described sufficient number of meganucleases in the genus that have the function of recognizing a specific central sequence.
For the purposes of brevity, the analysis below focuses on the genus meganucleases that recognize the elected central sequence: GTAT. However, the claims are not limited to only meganucleases that recognize GTAT. If Applicant did not sufficiently describe the subset of meganucleases that recognize GTAT, then it can be concluded that they also did not sufficiently describe the genus of meganucleases that can bind and cleave GTAG, GTGA, GTGC, GTGG, and GTGT.
Species described in the Specification
Applicant describes screening for I-CreI variants for the ability to bind a cleave a GTAT central sequences in an already engineered I-CreI variant (that recognizes a specific half site: CAAAACGTA (Fig 1; Example 19). For the GTAT screening, Applicant mutated the first subunit, but used the second subunit of the engineered I-CreI, which appears to have the sequence in SEQ ID NO 6 (page 178). Applicant discovered 32 variants with various residues at 9 positions that were able to bind and cleave the LOX3.4-GTAT central sequence (Tables 161 and 162). Thus, Applicant described 32 species of meganucleases, each of which has the exact same “second subunit” and each having at least 94% identity to SEQ ID NO 1. Applicant does not attempt to rationally combine all the various combinations at the 9 sites, so any structure-function relationship between each of the substitutions and the ability to recognize bases in the central sequence is unknown. Applicant also does not attempt to structurally model the substitutions to determine how they may interact with the DNA bases in the central sequence. The 32 species described by Applicant do not represent the diversity of the genus since there isn’t a defined or known structure-function relationship between the varied residues and their role in central sequence recognition. Thus, in view of the Specification, it was not predictable which additional 18 residues could be changed in the I-CreI and still allow binding and cleaving, and 2) what combinations for substitutions in the 6 claimed sites can maintain binding/cleaving of the GTAT central sequence.
State of the Art
Attempts to engineer I-CreI recombinases have been made since a crystal structure was first solved in 2001. For instance, Smith reports semi-rational mutagenesis approach coupled with high-throughput screening to derive endonuclease variants to alter substrate specificity (Smith et al., Nucleic Acids Research (2006), 34:22, pages 1-12; page 1, last ¶). Smith found two domains in I-CreI that are responsible for contacting two regions, 10NNN and 5NNN, of the inverted repeat, but these regions did not interact with the central sequence (Fig 3). Arnould tested the ability of one I-Cre variant, D75N to recognize different central sequences (Arnould et al., Journal of Molecular Biology (2007), 371: 49-65). Whereas wild type I-CreI could tolerate changes to in the central sequence, the D75N I-CreI variant could no longer bind and cleave Cs at the +2 position or any other nucleotide except T at the +1 position (Fig 5). Another variant, M3, with amino acid substitutions at positions 19, 32, 69, 85, 87 and 109) (Fig 4) completely lost the ability to bind to a cleave sites other than “GT” at the +2 and +1 position (Fig 5). From this, it can be concluded that amino acid substitutions at other positions than those in the claims can alter the ability of I-CreI variants from binding and cleaving certain central sequences. Gouble teaches I-CreI variants with the claimed Q50R or G71R substitutions for targeting an IL3RG3 allele (US 20110091441 A1). However, Gouble is silent as to the effect of binding and cleaving different central sequences. Molina further studied I-CreI – DNA interactions, specifically focusing on the central 4 bp of the I-CreI recognition sequence (Molina et al., Nucleic Acids Research (2012), 40: 6939-6945). Molina teaches that only one residue in I-CreI, K139, contacts the backbone of the central sequence and no residues contact the bases (page 6937, ¶2). Nevertheless, Molina teaches that modifications in the K139 region have a strong effect on DNA binding and cleaving (page 6937, ¶2). Molina varied the central sequence and determined DNA bases that affected DNA binding and cleaving by one I-CreI variant (Figure 1). Molina found that specific central sequences can affect binding/cleaving even through the 10NNN and 5NNN sequences would have promoted site binding (page 6943, ¶3). This lends support to the conclusion that there does not appear to be a strong structure-function relationship between the central sequence and the I-CreI recombinase structure such that the skilled artisan could predict 1) which additional 18 residues could be changed in the I-CreI and still allow binding and cleaving, and 2) what combinations for substitutions in the 6 claimed sites can maintain binding/cleaving of the central sequence.
Taken together based on 1) the discovery by Applicant of only 32 I-CreI variants that are capable of binding/cleaving a GTAT central sequence out of the extremely large genus of claimed structural variants, and 2) the lack of disclosure in the Specification or in the prior art about a structure-function relationship between I-CreI structure and recognition of the 2NN central sequence bases, the skilled artisan would reasonably conclude that Applicant did not possess the extremely large genus of meganuclease variants as claimed.
Dependent claims
Claims 88-92 do not limit the genus size of the claimed meganucleases, and thus lack sufficient written description for the reasons described for claim 86.
Claim 87 recites options for the residues 48, 50, 71, 72, 73 and 74 in reference to SEQ ID NOs. However, the recitation does not require the entire protein to have the recited SEQ ID NOs. The SEQ ID NOs are only used to provide options for the recited residue positions, which appear to be mostly the same options of claim 86. Claim 87 also does not limit the structure of the second subunit. Thus, claim 87 lacks sufficient written description for the reasons described for claim 86.
Conclusion
No claims are allowable.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CATHERINE KONOPKA whose telephone number is (571)272-0330. The examiner can normally be reached Mon - Fri 7- 4.
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If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Ram Shukla can be reached at (571)272-0735. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/CATHERINE KONOPKA/Primary Examiner, Art Unit 1635
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