DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
This Office action is in response to the communications filed 5-6-26.
Claims 106-125 are pending in the instant application.
Election/Restrictions
Claims 113, 116-125 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention or species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 5-6-26.
Applicant’s election without traverse of Group I, claims 106-112, 114, 115, SEQ ID Nos. 1676 and 584, in the reply filed on 5-6-26 is acknowledged.
Claim Rejections - 35 USC § 112
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 106-112, 114, 115 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.
The breadth of the claims
The claims are broadly drawn to engineered nuclease systems comprising:(a) an endonuclease configured to have selectivity for a protospacer adjacent motif (PAM) sequence comprising nnnnCC, wherein said endonuclease is a class 2, type II Cas endonuclease, and said endonuclease comprises a PAM-Interacting (PI) domain having at least 80% amino acid sequence identity to SEQ ID NO: 1633; and an engineered guide nucleic acid structure configured to form a complex with said endonuclease, which endonuclease optionally comprises an amino acid sequence comprising at least 80% sequence identity to SEQ ID NO: 484, which engineered guide nucleic acid structure comprises:(i) a targeting nucleic acid sequence configured to hybridize to a target deoxyribonucleic acid sequence; and (ii) any tracr ribonucleic acid sequence configured to bind to said endonuclease, which endonuclease optionally further comprises one or more of a RuvC domain optionally comprising an amino acid sequence having at least 80% sequence identity to a RuvC domain of SEQ ID NO: 484 or an HNH domain of SEQ ID NO: 484, which tracr ribonucleic acid sequence optionally comprises a sequence having at least 80% sequence identity to SEQ ID NO: 1661, which engineered guide nucleic acid structure comprises a sequence having at least 80% sequence identity to SEQ ID NO: 584, which targeting nucleic acid sequence comprises a spacer sequence configured to hybridize to a region of a TRAC locus or a region of an AAVS 1 locus, or which said target deoxyribonucleic acid sequence optionally comprises a sequence having at least 80% sequence identity to at least 18 consecutive nucleotides of SEQ ID No. 1676, or a reverse complement thereof.
Teachings in the specification
Example 2 - Discovery of MG44, MG46, MG71, MG72, MG73, MG74, MG87, MG88 and MG89 Families of CRISPR systems
[00150] Analysis of the data from the metagenomic analysis of Example 1 revealed new clusters of previously undescribed putative CRISPR systems comprising 9 families (MG44, MG46, MG71, MG72, MG73, MG74, MG87, MG88 and MG89). The corresponding protein and nucleic acid sequences for these new enzymes and their exemplary subdomains are presented as SEQ ID NOs: 1-549 or 602-1276.
Example 3 - Determination of Protospacer-Adjacent Motifs
[00151] PAM sequences were determined by sequencing plasmids containing randomly- generated PAM sequences that could be cleaved by putative endonucleases expressed in an E. coli lysate-based expression system (myTXTL, Arbor Biosciences). In this system, an E. coli codon optimized nucleotide sequence was transcribed and translated from a PCR fragment under control of a T7 promoter. A second PCR fragment with a tracr sequence under a T7 promoter and a minimal CRISPR array composed of a T7 promoter followed by a repeat-spacer-repeat sequence was transcribed in the same reaction. Successful expression of the endonuclease and tracr sequence in the TXTL system followed by CRISPR array processing provided active in vitro CRISPR nuclease complexes.
[00152] A library of target plasmids containing a spacer sequence matching that in the minimal array followed by 8N mixed bases (putative PAM sequences) was incubated with the output of the TXTL reaction. After 1-3 hr, the reaction was stopped and the DNA was recovered ... Adapter sequences were blunt-end ligated to DNA with active PAM sequences that had been cleaved by the endonuclease, whereas DNA that had not been cleaved was inaccessible for ligation. DNA segments comprising active PAM sequences were then amplified by PCR with primers specific to the library and the adapter sequence. The PCR amplification products were resolved on a gel to identify amplicons that corresponded to cleavage events. The amplified segments of the cleavage reaction were also used as template for preparation of an NGS library. Sequencing this resulting library, which was a subset of the starting 8N library, revealed the sequences which contain the correct PAM for the active CRISPR complex. For PAM testing with a single RNA construct, the same procedure was repeated except that an in vitro transcribed RNA was added along with the plasmid library and the tracr/minimal CRISPR array template was omitted.
For endonucleases where NGS libraries were prepared, seqLogo … representations were constructed and are presented in FIG The seqLogo module used to construct these representations takes the position weight matrix of a DNA sequence motif (e.g. a PAM sequence) and plots the corresponding sequence logo as introduced by Schneider and Stephens (see e.g. Schneider et al. Nucleic Acids Res. 1990 Oct 25;18(20):6097-100. The characters representing the sequence in the seqLogo representations have been stacked on top of each other for each position in the aligned sequences (e.g. PAM sequences). The height of each letter is proportional to its frequency, and the letters have been sorted so the most common one is on top.
Example 7 - Gene editing outcomes at the DNA level for TRAC and AAVS1 in K562 cells
[00160] Nucleofection of MG71-2, MG73-1, and MG89-2 mRNA along with the matching guide RNA from Table 4 below (500 ng mRNA/150 pmol guide) was performed into K562 cells (200,000) using the Lonza 4D electroporator. Cells were harvested and genomic DNA prepared three days post-transfection. PCR primers appropriate for use in NGS-based DNA sequencing were generated, optimized, and used to amplify the individual target sequences for each guide RNA. The amplicons were sequenced on an Illumina MiSeq machine and analyzed with a proprietary Python script to measure gene editing (FIG. 4).
Table 4: sgRNAs and Associated Sequences Targeted within AAVS1 and TRAC Sites Used in Example 7
[Emphases added] [Citations omitted]
The teachings in the specification and summarized in Table 4 and Example 7 are not representative of the various combinations instantly claimed. The specification fails to provide the requisite guidance for making and using the large genus of components of engineered nuclease systems instantly claimed. Since the disclosure fails to describe the common attributes and characteristics concisely identifying members of the proposed genus of engineered nuclease systems and the combined components, and because the claimed genus is highly variant, the description provided is insufficient.
One of skill in the art would reasonably conclude that the disclosure fails to provide a representative number of species to describe the vast genus of combinations of components instantly claimed, whereby endonuclease activity is predictably obtained.
Thus, Applicant was not in possession of the broadly claimed genus.
Allowable Subject Matter
SEQ ID No. 1633 appears free of the prior art searched and of record.
Conclusion
Certain papers related to this application may be submitted to Art Unit 1637 by facsimile transmission. The faxing of such papers must conform with the notices published in the Official Gazette, 1156 OG 61 (November 16, 1993) and 1157 OG 94 (December 28, 1993) (see 37 C.F.R. ' 1.6(d)). The official fax telephone number for the Group is 571-273-8300. NOTE: If Applicant does submit a paper by fax, the original signed copy should be retained by applicant or applicant's representative. NO DUPLICATE COPIES SHOULD BE SUBMITTED so as to avoid the processing of duplicate papers in the Office.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Jane Zara whose telephone number is (571) 272-0765. The examiner’s office hours are generally Monday-Friday, 10:30am - 7pm. If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Jennifer Dunston, can be reached on (571)-272-2916. Any inquiry of a general nature or relating to the status of this application should be directed to the Group receptionist whose telephone number is (703) 308-0196.
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Jane Zara
6-8-26
/JANE J ZARA/Primary Examiner, Art Unit 1637
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