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 .
Claims Status
Claims 66, 73 and 74 are amended.
Claims 68-72 and 77-81 are canceled.
Claims 84-94 are new.
Claims 1, 4, 66, 67, 73-76, and 82-94 are under examination.
Withdrawn Objections
The objection raised against the specification for containing an embedded hyperlink is withdrawn in light of the submitted amendments to the specification.
Withdrawn Rejections
Rejections under 35 U.S.C. 102
The rejection of claim 1 under 35 U.S.C. 102(a)(1) as being anticipated by Zhang et al (WO 2021/138286 A1) is withdrawn in light of Applicant argument. Applicant argue that Zhang et al’s disclosure, when taken as a whole, does not teach a person of ordinary skill in the art how to make and use the specific scAAV vector without undue experimentation. The office finds this argument to be persuasive. Thus, the rejection is withdrawn.
The rejection of claims 66,67,73, 76-77 and 81-83 under 35 U.S.C. 102(a)(1) as being anticipated by Ibraheim et al ( Nature Communication, 2021) is withdrawn in light of claim amendment. Applicant amended claim 66 to recite the limitation wherein the first and second guide RNA “ are complementary to sequences near the 5' and 3' ends of the region of the AA V vector encoding the effector protein, respectively, thereby allowing for the region of the AAV vector encoding the effector protein to be excised”. It is submitted that Ibraheim et al do not expressly teach the aforementioned limitation. Accordingly, the rejection is withdrawn.
Rejections under 35 U.S.C. 103
The rejection of claim(s) 72 and 78 under 35 U.S.C. 103 as being unpatentable over Ibraheim et al ( Nature Communication, 2021) as applied to claims 66,67,73, 76-77, and 81-83 above, and further in view of Xu et al (Biochimica et Biophysica Acta, 2003) is withdrawn in light of claims cancelation .
The rejection of claims 79-80 under 35 U.S.C. 103 as being unpatentable over Ibraheim et al ( Nature Communication, 2021) is withdrawn in light of claims cancelation .
The rejection of claim(s) 68-71 under 35 U.S.C. 103 as being unpatentable over Ibraheim et al ( Nature Communication, 2021) as applied to claims 66,67,73, 76-77, and 81-83 above, and further in view of Gersbach et al ( CA 3137248 A 1) is withdrawn in light of claims cancelation .
Edited Rejections Necessitated by Claims Amendment
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 4 and 92 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Ibraheim et al ( BioRxiv, 2020- DOI 10.1101/2020.10.09.333997- posted October 9, 2020).
Regarding claim 4 , Ibraheim et al teach a genome-editing platform comprising an adeno-associated viral (AAV) vector containing a transgene, wherein the transgene comprises:
a) a first nucleotide sequence encoding an effector protein; wherein the effector protein is Nme2Cas9;
b) a second nucleotide sequence encoding a guide nucleic acid (i.e. sgRNA ), and
c) a third nucleotide that comprises of a donor nucleic acid . (See Fig.4a and 5a).
Ibraheim et al teach utilizing the AAV comprising the aforementioned transgene in a composition to reduce the clinical manifestations in two disease models of type I hereditary
tyrosinemia (HT-I) and mucopolysaccharidosis type I (MPS-I). It should be noted that the administration of the AAV vectors via tail vein injection reads on the composition comprising the viral vector of instant claim. (See sections “In vivo validation of rAAV:HDR vectors in HT-I mice” and “In vivo validation of rAAV:HDR vectors in MPS-I mice” on pages 7-9). Accordingly, Ibraheim et al anticipate instant claims.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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.
Claims 1,84-89, and 93 are rejected under 35 U.S.C. 103 as being unpatentable over Zhang et al (WO 2021/138286 A1) in view of Ibraheim et al ( BioRxiv preprint, 2020 - posted October 9, 2020), and Karvelis et al ( Nucleic Acids Research, 2020), as evidenced by Sun et al ( Molecular Cell, 2019).
The teachings of Ibraheim et are set forth above. Ibraheim et al anticipate claim 4.
Regarding claims 1 and 89, Zhang et al teach compositions comprising self-complementary AAV viral vectors (scAAV) for delivery of CRISPR/Cas9 genome editing machinery. Zhang et al teach that the AAV delivery system may be used, for example, for disrupting a dystrophin splice acceptor site and inducing skipping and/or reframing of an exon of a DMD gene, thereby modifying a DMD gene in a cell or a subject. In one of the embodiments, Zhang et al expressly suggest that the effector protein (i.e. Cas9 or Cpf1) and the guide RNA may be provided on the same vector. ([0008] , [0099], and [0104]). It is submitted that Zhang et al do not provide working examples in which the scAAV contains the complete all-in-one configuration required by instant claim. Rather, in certain embodiments, Zhang et al employ an AAV vector encoding Cas9, and scAAV encoding the gRNA. Accordingly, and to the extent Applicant argue that Zhang et al working examples do not teach the same arrangement required by the claimed instant claims, Ibraheim et al supplies this teaching. As discussed above, Ibraheim et al specifically disclose single vector AAV platforms encoding Nme2Cas9 and one or more gRNAs, Ibraheim et al further recognize the limited packaging capacity of AAV vectors and employ a compact Cas9 effector to facilitate incorporation of the nucleases and guide RNA into a single AAV vector. ( See abstract). Zhang et al, on the other hand, expressly teach the desirability of packaging the effector protein and gRNA all in the same vector, and further explains in [00256], that the use of scAAV improve editing efficiency as it is less prone to DNA degradation after viral transduction. Thus, Zhang et al provide an ordinary skill in the art with a suggestion as well as a motivation for selecting scAAV configuration when implementing an AAV-mediated CRISPR system. Therefore, it would have been prima facie obvious to one with ordinary skill in the art at the time the invention was filed to modify the scAAV CRISPR system of Zhang et al according to the all-in-one strategy of Ibrahiem et al. Because Ibraheim et al, teach an all-in-one strategy for packaging an effector protein and gRNAs all in the same vector, and recognize the limited packaging capacity of AAV vectors. Therefore, in view of Ibraheim et al teachings an ordinary skill in the art would have recognized that the reduced packaging capacity of scAAV, as compared to the conventional ssAAV, presents a size constraint when attempting to package the effector nuclease and gRNAs in the same vector, and would have been motivated to select a compact CRISPR effector, as taught by Ibraheim et al, in order to reduce the size of the effector-encoding portion of the transgene and thereby facilitate packaging of the effector and guide nucleic acid within the available capacity of scAAV. An ordinary skill in the art would have had a reasonable expectation of success because Ibraheim et al demonstrate that a compact Cas9 effector and its gRNA can be functionally expressed in a single AAV vector, while Zhang et al demonstrate the use of scAAV for delivery of CRISPR components. Thus, instant claim merely amounts to combining prior art elements according to known methods to yield predictable results. See MPEP 2143 (I)(A).
Regarding claims 84- 87, and 93, following the discussion of claim 1 above. Ibraheim et al teach a gene-editing system comprising of an AAV containing a transgene encoding for dual sgRNAs and Nme2Cas9, wherein the Nme2Cas9 endonuclease comprising of 1,082 amino acids.( See abstract, and 2nd paragraph- on page 2). Ibraheim et al further teach that the first guide nucleic acid (i.e. gRNA I) and the second guide nucleic acid (gRNA II) comprise different spacer sequences targeting different sequences in the Hpd gene, wherein the spacer within the gRNA I and gRNA II are designed to precisely target the boundaries of exon 3 and 4, respectively, this reads on claim 86. ( See Fig.1e, and Material and Method-section “ Cloning of dual-guide rAAV:Nme2Cas9 plasmids” ). The gene editing systems taught by Ibraheim et al further utilized more than one promoters to drive the expression of the Nme2Cas9 and the gRNAs, including U1a and U6, this reads on claim 87. ( See Fig.1a ). Ibraheim et al further teach a transgene comprising a fourth nucleotide sequence encoding a fusion partner protein including two NLS localized at the N-terminus and C-terminus of Nme2Cas9, this reads on claim 93. (See Fig.1a). It is noted that the Nme2Cas9 endonuclease contains both the RuvC and HNH domains, as evidenced by Sun et al, and comprises of 1082 amino acids. ( See Fig.1 A provided by Sun et al). This differs from instant claims which recite an effector protein comprising of 400-800 amino acids and does not contain an HNH domain. Thus, neither Zhang nor Ibraheim teach the aforementioned limitations.
Kravelis et al supplement Zhang and Ibraheim et al by describing miniature effector proteins such as CRISPR-Cas12f nucleases and demonstrate that such nucleases are capable of targeted double-stranded DNA cleavage in a PAM-dependent manner. In particular, Kravelis et al teach that CRISPR-Cas12f nucleases are miniature class 2 type V-F CRISPR-Cas comprising of 400-603 amino acids, wherein Cas12f contain RuvC domain but lacks HNH domain. ( See abstract, and Fig.1a). Kravelis et al further teach that these miniature nucleases have the potential utility in genome-editing tools. ( See abstract). Kravelis et al , for example, state that “our results significantly improve our understanding of novel CRISPR-Cas systems and pave the way for the adoption of programmable miniature nucleases for genome editing ap plications” .( See the last pargraph of the Discussion section). As such, one with ordinary skill in the art at the time the invention was filed would have been motivated to modify the scAAV delivery system of Zhang et al in view of Ibraheim and Karvelis by selecting a miniature CRISPR-cas12f nuclease as the CRISPR effector. Because Ibraheim et al teach an all-in-one AAV configuration comprising a CRISPR nuclease and one or more guide RNAs in a single vector and recognize the packaging limitations associated with AAV vector. Karvelis et al teach a miniature Casf12 nucleases having sizes of about 400-600 amino acids and demonstrate that such nucleases are capable of programmable, RNA-guided double stranded DNA cleavage, and expressly suggest utilizing these effector proteins in genome-editing systems. Thus, one with ordinary skill in the art would have been motivated to select a miniature effector protein, as taught by Karvelis, for the all-in-one AAV configuration of Ibraheim et, and implement such confirguration in the scAAV system of Zhang et al, because doing so would allow for all of the components of the CRISPR system to be packaged in one viral vector including the nuclease and any additional elements such as multiple gRNAs or a donor polynucleotide for HDR. Thus, instant claims are merely amounts to combining prior art elements according to known methods to yield predictable results. See MPEP 2143 (I)(A).
Regarding claim 88, following the discussion of claim 1 above, Ibraheim et al further teach incorporating nuclease target sites into the vector to provide self-inactivation of the vector, following accumulation of the Nme2Cas9/sgRNA complex, thereby limiting the continued Cas9 expression. ( See abstract, Fig.3 , and Discussion). Thus, it would have been obvious to one with ordinary skill in the art to employ the self-inactivating feature taught by Ibraheim et al in the scAAV system of Zhang et al. An ordinary skill in the art would be motivated to do so to limit the duration of nuclease expression following genome editing, thus reducing continued nucleases activity and potential undesired editing.
Claims 4, 90-91 are rejected under 35 U.S.C. 103 as being unpatentable over Ibraheim et al ( BioRxiv preprint, 2020 - posted October 9, 2020), in view of Karvelis et al ( Nucleic Acids Research, 2020).
The teachings of Ibraheim et al and Kravelis are set forth above.
Regarding claims 90-91, following the discussion of claim 4 above. Ibraheim et al anticipate claim 4. It is submitted that the genome editing platform of Ibraheim et al comprises of nucleic acid encoding Nme2Cas9 endonuclease which is approximately 1082 amino acids in length and comprises both RuvC and HNH domains. This differs from instant claims which recite an effector protein comprising of 400-800 amino acids and does not contain an HNH domain.
As discussed above, Kravelis et al supplement Ibraheim et al by teaching a miniature Casf12 nucleases having sizes of about 400-600 amino acids and demonstrate that such nucleases are capable of programmable, RNA-guided double stranded DNA cleavage, and expressly suggest utilizing these effector proteins for genome-editing system application. Thus, one with ordinary skill in the art would have been motivated to select a miniature effector protein, as taught by Karvelis, and implement that in the all-in-one system of Ibraheim et al, instead of Nme2Cas9, because doing so would allow for all of the components of the CRISPR system to be packaged in one viral vector including the nuclease and any additional elements such as multiple gRNAs or a donor polynucleotide for HDR. Thus, instant claims are merely amounts to combining prior art known elements according to known methods to yield predictable results. See MPEP 2143 (I)(A).
Claims 66-67, 73-76,82-83, and 94 are rejected under 35 U.S.C. 102(a)(1) as being unpatentable over Ibraheim et al ( BioRxiv preprint, 2020 - posted October 9, 2020), in view of Police et al ( WO 2019/092505 A1), and Karvelis et al ( Nucleic Acids Research, 2020).
The teachings of Ibraheim et al and Kravelis are set forth above.
Regarding claims 66 and 82- 83, following the discussion of claim 4 above, Ibraheim et al teach a genome-editing platform comprising an adeno-associated viral (AAV) vector comprising a transgene, wherein the transgene comprises:
a) a first nucleotide sequence encoding an effector protein; wherein the effector protein is Nme2Cas9;
b) a second nucleotide sequence encoding a first guide nucleic acid (i.e. sgRNA I); and
c) a third nucleotide sequence encoding a second guide nucleic acid (i.e. sgRNA II).
Ibraheim et al refer to the construct as dual-sgRNA AAV:Nme2Cas9 vector. (See Fig.1a). Ibraheim et al demonstrate that the co-expression of Nme2Cas9 and the two sgRNAs (i.e. sgRNA I and II) can efficiently be utilized to induce segmental deletions in targeted nucleic acid in cultured cells and in vivo. Specifically, Ibraheim et al utilized the dual-sgRNA AAV:Nme2Cas9 vector to excise a 606-bp fragment spanning exons 3-4 of the Hpd gene in cultured cells and then in HT-I mouse model, this reads on claim 66 and 82-83. It should be noted that the first guide nucleic acid (i.e. gRNA I) and the second guide nucleic acid (gRNA II), taught by Ibraheim et al, comprise different spacer sequences targeting different sequences in the Hpd gene, wherein the spacer within the gRNA I and gRNA II are designed to precisely target the boundaries of exon 3 and 4, respectively. ( See Fig.1e, and Material and Method-section “ Cloning of dual-guide rAAV:Nme2Cas9 plasmids” ). Ibraheim et al further recognize the incorporation of the self-inactivating feature in the all-in-one platform as desirable feature. ( See abstract). However, Ibraheim et al do not expressly teach positioning the two gRNAs at the 5’ and 3’ position flanking the effector protein, such that the effector itself is excised.
Police et al supplement Ibraheim et al by teaching a self-inactivating genome editing system comprising of CRISPR-Cas system, in which the nucleic acid encoding Cas9 contains one or more self-inactivating (SIN) sites, and a guide RNA contains a DNA-targeting sequences complementary to the SIN site. As such, the guide RNA directs Cas9 to cleave itself (i.e. results in self-excision). Importantly, Police et al expressly teach that the SIN sites can be positioned at the 5’ end and/or 3’ end of the segment encoding the effector protein. ([00571-00574]). Police et al further teach a second SIN site and expressly provide embodiments in which the first and second SIN sites have different nucleotide sequences. ( [ 00578]). Police et al further describe the use of two gRNAs to target the two SIN sites. In example 3, Police et al teach a first gRNA1 comprising a first spacer and targets SIN site 1, while sgRNA2 comprises a different spacer and targets different SIN site2. [00680-00681].
Taken together, instant claims would have been obvious to a person of ordinary skill in the art at the time the invention was filed, because Ibraheim et al teach all-in-one genome editing platform and recognize the desirability to self-inactivate the system, while Police et al provide an ordinary skill in the art with the experimental basis to accomplish such objective by incorporating CRISPR-targetable SIN sites at the 5’ and 3’ end of Cas9-encoding nucleic acid and providing gRNAs complementary to those sites. An ordinary skill in the art would have been motivated to modify the system of Ibraheim et al by incorporating the self-inactivating system taught by Police et al because doing so would have predictably resulted in cleaving the effector protein and self-inactivating the system. Such modification represents a combination of prior art known elements according to known methods to yield predictable results. See MPEP 2143 (I)(A).
Regarding claims 67, 73 ,76, and 94 , Ibraheim et al teach different configurations for the construction of the dual-sgRNA AAV:Nme2Cas9 vector with designs 1 and 4 being the most efficient configuration enabling packaging of the full-length vector genomes. It should be noted that design 4 comprises of a first U6 promoter driving the expression of gRNA I, a U1a promoter driving the expression of the effector protein (i.e. Nme2Cas9), and a second U6 promoter driving the expression of gRNA II, this reads on claim 67. Design 4 also comprises of two NLS localized at the N-terminus and C-terminus of Nme2Cas9), this reads on claims 76 and 94. (See Fig.1a).
Regarding claims74-75, following the discussion of claims 66 and 84-85 , the genome editing platform of Ibraheim et al comprises of Nme2Cas9 endonuclease which is about 1082 amino acids and contains both the RuvC and HNH domains. This differs from instant claims which recite an effector protein comprising of 400-600 amino acids and does not contain an HNH domain. The teachings of Kravelis are set forth above.
As discussed above, Kravelis et al, supplement Ibraheim et al by teaching a miniature Casf12 nucleases having sizes of about 400-600 amino acids and demonstrate that such nucleases are capable of programmable, RNA-guided double stranded DNA cleavage, and expressly suggest utilizing these effector proteins for genome-editing system application. Thus, one with ordinary skill in the art would have been motivated to modify the all-in-one AAV configuration of Ibraheim et al by incorporating a miniature effector protein, as taught by Karvelis because doing so would allow for all of the components of the CRISPR system to be packaged in one viral vector including the nuclease and any additional elements such as multiple gRNAs or a donor polynucleotide for HDR. Thus, instant claims are merely amounts to combining prior art known elements according to known methods to yield predictable results. See MPEP 2143 (I)(A).
Response to Arguments
Applicant's arguments filed 07/15/2026 have been fully considered but they are not persuasive.
Applicants argue that the specification of priority application U.S. Serial No.
63/225,210 provide an adequate support for the limitation of "a third nucleotide sequence encoding a second nucleic acid” recited in step (c) of instant claim 66 evident by the recitation that states “In some instances, the AAV vector comprises one or more guide nucleic acids that comprise a region that is complementary to sequences near the 5' and 3' ends of the region of the AAV vector encoding the effector protein, thereby allowing for the region of the AAV vector encoding the effector protein to be excised.” [0044].
The office finds this argument to be persuasive, therefore, the priority date for claim 66 and its dependent claims is 07/23/2021.
Applicants also argue that lbraheim et al relied upon in the previous office
action was published on Nov 1st, 2021. Thus, lbraheim is not valid as prior art under 35 U.S.C. 102 or 103 , as it was published after the July 23,2021 effective filing date of the instant application.
The office agrees with Applicant that the November 1, 2021, Nature Communications publication was published after the effective filing date. However, the office intended to rely upon the corresponding preprint, which was publicly available prior to the Applicants claimed priority date. Specifically, the corresponding preprint posted on bioRxiv on October 9,2020, and therefore was publicly available prior to July 23,2021. In other words, the reference to the Nature Communications in the previous rejection was a typographical error. Therefore, Applicant arguments directed solely to the November 1,2021 of the Nature Communications article does not overcome the rejections based on the October 9, 2020, bioRxiv publication.
Conclusion
No claim is allowed.
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/FATIMAH KHALAF MATALKAH/Examiner, Art Unit 1638
/Tracy Vivlemore/Supervisory Primary Examiner, Art Unit 1638
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