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 .
Status of the Claims
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 10/03/2025 was previously entered.
Applicant’s response of 02/27/2026 has been received and entered into the application file.
Claim 74 was amended in the claim set filed 02/27/2026.
Claims 1, 2, 4, 13, 23-26, 28, and 71-77 are pending, of which claim 28 was previously withdrawn from consideration.
Accordingly, claims 1, 2, 4, 13, 23-26, and 71-77 are pending and under consideration.
Election/Restrictions
Applicants previously elected Group I, drawn to a complex comprising a Cas polypeptide and a capped-sgRNA (comprising an m7G cap or analog thereof, a spacer capable of sequence-specific hybridization, and a direct repeat capable of binding to the Cas polypeptide) (claims 1-4, 13, 18, and 23-26), without traverse.
Claims 1-4, 13, 18, and 23-26 were previously examined on the merits. Claims 28, 30, 31, 55, 56, 61-63, 65, and 66 were previously withdrawn from consideration, pursuant to 37 CFR 1.142(b), as being directed to non-elected inventions, there being no allowable generic or linking claim at this time. Claims 29-70 have since been cancelled. Accordingly, claim 28 is withdrawn from consideration, pursuant to 37 CFR 1.142(b), as being directed to a non-elected invention, there being no allowable generic or linking claim at this time.
Claims 1, 2, 4, 13, 23-26, and 71-77 are under consideration.
Status of Prior Rejections
RE: Claim Objections
Claim 74 was previously objected to for minor informalities.
The amendments to instant claim 74 have obviated the basis of the objection. The objection of record is hereby withdrawn.
RE: Claim Rejections - 35 USC § 103
►Claims 1, 2, 4, 13, 18, 23, 26, and 71-74 were previously rejected under 35 U.S.C. 103 as being unpatentable over US 2017/0306335 A1 (hereinafter Zhang; of record) in view of Mu et al., 2019 (of record), Ramanathan et al., 2016 (of record), He et al., 2016 (of record), and Hinnebusch, 2011 (as cited in the IDS filed 02/23/2022), as evidenced by Cowling et al., 2010 (of record), and Jiang and Doudna, 2017 (of record).
►Claim 24 was previously rejected under 35 U.S.C. 103 as being unpatentable over US 2017/0306335 A1 (hereinafter Zhang; of record) in view of Mu et al., 2019 (of record), Ramanathan et al., 2016 (of record), He et al., 2016 (of record), and Hinnebusch, 2011 (as cited in the IDS filed 02/23/2022), as evidenced by Cowling et al., 2010 (of record) and Jiang and Doudna, 2017 (of record), as applied to claim 23, and further in view of Wolter and Puchta, 2018 (of record).
►Claim 25 was previously rejected under 35 U.S.C. 103 as being unpatentable over US 2017/0306335 A1 (hereinafter Zhang; of record) in view of Mu et al., 2019 (of record), Ramanathan et al., 2016, (of record) He et al., 2016 (of record), Hinnebusch, 2011 (as cited in the IDS filed 02/23/2022), and Wolter and Puchta, 2018 (of record), as evidenced by Cowling et al., 2010 (of record) and Jiang and Doudna, 2017 (of record), as applied to claim 24, and further in view of Faure et al., 2018 (of record).
►Claims 75-77 were previously rejected under 35 U.S.C. 103 as being unpatentable over US 2017/0306335 A1 (hereinafter Zhang; of record) in view of Mu et al., 2019 (of record), Ramanathan et al., 2016 (of record), He et al., 2016 (of record), and Hinnebusch, 2011 (as cited in the IDS filed 02/23/2022), as evidenced by Cowling et al., 2010 (of record) and Jiang and Doudna, 2017 (of record), as applied to claim 1, and further in view of Martin et al., 2011.
Applicant has traversed the rejections of record, asserting that Hinnebusch does not teach or suggest targeting an exogenous m7G cap proximal to a start codon of an RNA, as recited at claim 1.
In response, while Applicant’s arguments have been fully considered, they are not found persuasive.
While the Examiner acknowledges that Hinnebusch does not teach or suggest targeting an exogenous m7G cap proximal to a start codon of an RNA, as recited at claim 1, this deficiency is cured by the cited art. As previously set forth, Zhang and Mu et al., 2019 collectively disclose the exogenous m7G cap of the instantly claimed sgRNA, while Ramanathan discloses the claimed m7G cap species. Hinnebusch was applied to motivate the chosen target site proximal to a target start codon of the RNA molecule relative to an endogenous 5’ m7G cap of the targeted RNA molecule.
As previously set forth and as depicted in Figure 1 of Hinnebusch, 2011, the endogenous 5’ m7G cap is known to be located upstream of the AUG start codon and facilitates scanning for said AUG start codon (in a suitable sequence context) by the 43S preinitiation complex (PIC). After the start codon is identified, scanning stops and a cascade of chemical and conformational changes occur to initiate the elongation phase of protein synthesis (page 435, column 1, paragraphs 2 and 3). This disclosure establishes that the endogenous 5’ m7G cap must necessarily be located upstream of the endogenous start codon in order to facilitate activation of the elongation phase of protein synthesis, in part by directing 43S PIC scanning. Therefore, a capped sgRNA (as in Mu et al., 2019) targeted to an mRNA transcript (as in Zhang, paragraph [0100]) to activate translation of said mRNA transcript must facilitate the same 43S PIC scanning to locate the endogenous start codon of the targeted mRNA transcript. Furthermore, in order to effectively activate translation, this capped sgRNA (taught by Zhang and Mu et al., 2019) must direct its m7GpppG cap (Cap 0; as disclosed in Ramanathan et al., 2016) downstream of the endogenous 5’ m7G cap so that the endogenous 43S PIC scanning does not physically block or otherwise compete with the 43S PIC scanning initiated by the m7GpppG-capped sgRNA. A person of ordinary skill in the art would have therefore arrived at the instantly claimed subject matter prior to the effective filing date of the instant application, as all the principles of this system were known in the art prior to the effective filing date of the instant application, as set forth above.
While Applicant has asserted that arriving at the instantly claimed subject matter requires the use of hindsight reasoning in view of the cited art, this is not found persuasive, as all of the principles of the instantly claimed system, including capped guide RNAs and the mechanisms by which capped transcripts are translated, were known in the art prior to the effective filing date of the instant application.
Applicant has further traversed the rejections of record regarding claims 75-77, asserting that while Martin discloses an element downstream of the start codon that governs translation, this element is an RNA structure which interacts with the endogenous m7G cap. Applicant further asserts that Martin does not teach or suggest targeting a complex comprising an additional exogenous m7G cap to a sequence proximal to a start codon of an RNA molecule relative to an endogenous 5’ m7G cap of the RNA molecule and downstream of the target start codon, as required by claim 75.
In response, while Applicant’s arguments have been fully considered, they are not found persuasive.
While the Examiner acknowledges that Hinnebusch does not teach or suggest targeting an exogenous m7G cap proximal to a start codon of an RNA, this deficiency is cured by the cited art. As previously set forth, Zhang and Mu et al., 2019 collectively disclose the exogenous m7G cap of the instantly claimed sgRNA, while Ramanathan discloses the claimed m7G cap species. Martin was applied to motivate the chosen target site proximal to a start codon of an RNA molecule relative to an endogenous 5’ m7G cap of the RNA molecule and downstream of the target start codon.
As previously set forth, while the canonical, endogenous pathway by which capped mRNA initiates translation involves scanning of the 43S PIC in the 5’ to 3’ direction (reviewed in Hinnebusch, 2011), Martin et al., 2011 discloses that translation can also be initiated by factors downstream of the start codon of mRNAs that tether ribosomal particles and sequester the m7G cap to facilitate direct positioning of the ribosome on the cognate start codon (abstract). Accordingly, Martin et al., 2011 discloses that translation initiation may be governed both by scanning that initiates upstream of the endogenous start codon (as in Hinnebusch, 2011) and by factors downstream of the endogenous start codon. Accordingly, one of ordinary skill in the art would be motivated to target a region downstream of the target start codon of the mRNA in order to ensure that user-directed translation initiates properly, efficiently, and robustly.
Although the instant claim set does not recite the RNA structures of Martin, Martin nonetheless clearly establishes that a person of ordinary skill in the art would be aware, prior to the effective filing date of the claimed invention, that translation may be initiated by upstream scanning (as in Hinnebusch) as well as by downstream factors (as in Martin). Therefore, a person of ordinary skill in the art would have been motivated to target sites either upstream and/or downstream of the endogenous start codon in order to ensure that user-directed translation initiates properly, efficiently, and robustly.
Accordingly, the rejections of record are hereby maintained, as set forth below.
New/Maintained Grounds of Rejection
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.
Claims 1, 2, 4, 13, 23, 26, and 71-74 are rejected under 35 U.S.C. 103 as being unpatentable over US 2017/0306335 A1 (hereinafter Zhang; of record) in view of Mu et al., 2019 (of record), Ramanathan et al., 2016 (of record), He et al., 2016 (of record), and Hinnebusch, 2011 (as cited in the IDS filed 02/23/2022; of record), as evidenced by Cowling et al., 2010 (of record), and Jiang and Doudna, 2017 (of record).
PNG
media_image1.png
202
353
media_image1.png
Greyscale
With regard to amended claim 1, which recites “a complex comprising a Cas polypeptide; and a capped-sgRNA,” in which the sgRNA itself comprises “(i) an m7G cap or analog thereof; (ii) a spacer capable of specifically hybridizing with a target sequence in an RNA molecule, wherein the target sequence is proximal to a target start codon of the RNA molecule relative to an endogenous 5’ m7G cap of the RNA molecule; and (iii) a direct repeat capable of binding to the Cas polypeptide; wherein the capped-sgRNA has the following structure [(shown to the right)]: where a’ is a guanosine or adenine, b’ is a linker, and c’ is the spacer,” Zhang discloses an "engineered, non-naturally occurring CRISPR-Cas RNA-targeting system" that comprises a Cas protein and an RNA-targeting guide RNA. The guide RNA of Zhang may comprise both a guide sequence and a tracr sequence (paragraph [0020]). Per Jiang and Doudna, 2017, a guide RNA is defined as a native crRNA-tracrRNA (or an sgRNA), with the crRNA sequence conferring targeting specificity (page 512, paragraph 2) and the tracrRNA encoding noncoding RNA with homology to repeat sequences for recruitment of Cas endonucleases such as Cas9 (page 506, Figure 1 caption; page 512, paragraph 2). Thus, the RNA-targeting guide RNA of Zhang, which comprises both a guide sequence and a tracr sequence, is considered to read on the instantly claimed “spacer capable of specifically hybridizing with a target sequence in an RNA molecule” as well as the instantly claimed “direct repeat capable of binding to the Cas polypeptide.” Thus, Zhang discloses all the limitations of instant claim 1, with the exception of the capped guide RNA taught therein and that the target sequence is proximal to a target start codon of the RNA molecule relative to an endogenous 5’ m7G cap of the RNA molecule.
However, Mu et al., 2019 discloses capping of sgRNA with m7G (Figure 1A). Per the disclosure of Mu et al., 2019, capping sgRNA with m7G upstream of the sequence-specific-targeting guide sequence (Figures 1 and 2) resulted in enhanced sgRNA stability, facilitated a higher level of genome disruption, and mediated efficient activation of endogenous gene expression (page 225, column 2, paragraph 3). While Mu et al., 2019 does not disclose the capped-sgRNA structure of amended claim 1, specifically wherein “a’ is a guanosine or adenine, b’ is a linker, and c’ is the spacer,” these elements are all disclosed in the prior art.
As taught in Ramanathan et al., 2016 and shown below, a dinucleotide 5’ m7G cap in which both nucleotides of the dinucleotides are guanosine (i.e. an m7GpppG cap) is known in the art. The cap structure of amended claim 1 (pictured above) comprises the established m7GpppG (Cap 0) structure taught in Ramanathan et al., 2016 in which the variable RNA species is attached to the cap structure at the same position as the instantly claimed linker connecting the instantly claimed sgRNA. Ramanathan et al., 2016 further discloses that this cap 0 structure is known to be required for efficient translation (page 7515, column 2, paragraph 2; page 7519, column 2, paragraph 3). Thus, Ramanathan et al., 2016 discloses the cap structure of amended claim 1, with the exception of elements b’ (a linker) and c’ (a spacer), while Mu et al., 2019 discloses an sgRNA attached to a cap placed upstream of the sequence-specific-targeting guide sequence (which reads on the instantly claimed spacer (c’)). He et al., 2016
PNG
media_image2.png
349
883
media_image2.png
Greyscale
remedies this deficiency by disclosing the role(s) of linkers in gRNA design.
He et al., 2016 discloses generation of functional, synthetic conjugated sgRNAs with linkers “not necessarily…limited to the triazole linker” disclosed within, which may include amide, disulfide, thioester, hydrozone, or other linkers (page 1811, column 1, paragraph 2). As set forth above, Jiang and Doudna, 2017 disclose that sgRNAs combine the crRNA and tracrRNA sequences into a single RNA transcript (page 509, paragraph 2) with the crRNA sequence conferring targeting specificity (page 512, paragraph 2), which is considered to read on the instantly claimed spacer sequence. He et al., 2016 further discloses that a benefit of using linkers to conjugate sgRNAs is “the possibility of incorporating chemical modifications” (page 1809, column 2, paragraph 1), such as the instantly claimed cap. While He et al., 2016 does not disclose linking an sgRNA to the instantly claimed cap, their disclosure does establish that sgRNAs are known to function with attached linkers and that these linkers facilitate the incorporation of chemical modifications (i.e. to modulate the function of the sgRNA).
Regarding the target sequence, which is recited to be proximal to a target start codon of the RNA molecule relative to an endogenous 5’ m7G cap of the RNA molecule, neither Zhang, Mu et al., 2019, Ramanathan et al., 2016, nor He et al., 2016 disclose targeting the instantly claimed capped-sgRNA complex to a site proximal to a target start codon of the targeted RNA molecule relative to its endogenous 5’ m7G cap. However, one of ordinary skill in the art would have been motivated to design the sgRNA (as reviewed in Jiang and Doudna, 2017-see Figure 2 and its associated caption) to target a site proximal to a start codon of the targeted RNA molecule based on the disclosure of Hinnebusch, 2011. As depicted in Figure 1 of Hinnebusch, 2011, the endogenous 5’ m7G cap is known to be located upstream of the AUG start codon and facilitates scanning for said AUG start codon (in a suitable sequence context) by the 43S preinitiation complex (PIC). After the start codon is identified, scanning stops and a cascade of chemical and conformational changes occur to initiate the elongation phase of protein synthesis (page 435, column 1, paragraphs 2 and 3). This disclosure establishes that the 5’ m7G cap must necessarily be located upstream of the endogenous start codon in order to facilitate activation of the elongation phase of protein synthesis, in part by directing 43S PIC scanning. Therefore, a capped sgRNA (as in Mu et al., 2019) targeted to an mRNA transcript (as in Zhang, paragraph [0100]) to activate translation of said mRNA transcript must facilitate the same 43S PIC scanning to locate the endogenous start codon of the targeted mRNA transcript. Furthermore, in order to effectively activate translation, this capped sgRNA must direct its m7GpppG cap (Cap 0; as disclosed in Ramanathan et al., 2016) downstream of the endogenous 5’ m7G cap so that the endogenous 43S PIC scanning does not physically block or otherwise compete with the 43S PIC scanning initiated by the m7GpppG-capped sgRNA.
With regard to claim 2, which recites “the RNA molecule [targeted by the complex of instant claim 1] is a messenger RNA,” Zhang discloses that “the target sequence may be a…messenger RNA” (paragraph [0100]). Thus, Zhang discloses the targeted RNA molecule of instant claim 2.
With regard to claim 4, which recites “the target sequence is downstream of the endogenous 5’ m7G cap,” this limitation is considered to be an inherent, endogenous property of cellular mRNA per the teachings of Cowling et al., 2010. Cowling et al., 2010 teaches that the m7G cap added to the 5’ end of mRNA is “essential for efficient gene expression” and “necessary for the translation of most cellular mRNAs” (abstract), meaning mRNAs are inherently assumed to have endogenous m7G caps, as they are essential and necessary for the majority of cellular mRNAs. Additionally, Cowling et al., 2010 teaches that mRNA cap methylation occurs co-transcriptionally (figure 3), with mRNA transcription elongation proceeding after capping (section “mRNA Cap Methylation-Dependent Transcription,” pages 288-289), meaning the mRNA target sequence would necessarily be downstream of the endogenous m7G cap, which marks the 5’ end of the mRNA transcript itself.
With regard to claim 13, which recites “the spacer [of instant claim 1] is at least 80% complementary to the target sequence,” Zhang discloses that the RNA-targeting guide sequence, which is considered to read on the instantly claimed spacer as set forth above, is “about or more than about…80%, 85%, 90%, 95%, 97.5%, 99%, or more” (paragraph [0100]) complementary to its corresponding target RNA sequence. Thus, Zhang discloses the recited percent complementarity of instant claim 13
With regard to claims 23 and 26, which recite “the Cas polypeptide [of instant claim 1] is a nuclease-deficient Cas (dCas) polypeptide, wherein the dCas comprises an inactivated target cleavage domain and a retained guide cleavage domain” and further that “the nuclease deficient Cas polypeptide is a nuclease-deficient Cas9 (dCas9)” and are not disclosed in Zhang, Mu et al., 2019 discloses the use of dCas9 (Figure 2) with m7G-capped-sgRNAs (Figures 1 and 2), which are set forth above. Mu et al., 2019 discloses that dCas9 (which reads on the dCas polypeptide of instant claim 23 as well as the dCas9 of instant claim 26) lacks endonuclease activity (which reads on the instantly claimed “inactivated target cleavage domain”) while maintaining DNA binding capability (which reads on the instantly claimed “retained guide cleavage domain”) (page 223, column 1, paragraph 1). Mu et al., 2019 further discloses that dCas9 can enable regulation of gene transcription (page 223, column 1, paragraph 1), which is consistent with the teachings of Jiang and Doudna, 2017. Jiang and Doudna, 2017 teach that dCas9 effectively uncouples cleavage activity from targeting activity, facilitating additional Cas functions such as transcriptional regulation, epigenetic modification, live-cell imaging, and nucleotide editing (Figure 2). Thus, Mu et al., 2019 discloses the nuclease deficient Cas (dCas) species of instant claims 23 and 26.
Given that Zhang discloses an engineered, non-naturally occurring CRISPR-Cas RNA-targeting system comprising an RNA-targeting guide, itself comprising a spacer sequence with at least 80% complementarity to its target sequence and a direct repeat sequence, that Mu et al., 2019 discloses that m7G capping of sgRNAs enhances their stability and facilitates efficient activation of endogenous gene expression, that Ramanathan et al., 2016 discloses that the 5’ m7GpppG cap (in which the variable RNA species is attached to the cap structure at the same position as the instantly claimed linker) is required for efficient translation, that He et al., 2016 discloses that synthetic linkers do not interfere with sgRNA function and may be used to incorporate chemical modifications, and that Hinnebusch, 2011 discloses that the 5’ m7G cap functions in part by directing 43S PIC scanning to identify the downstream start codon, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to cap the gRNA of Zhang (as in Mu et al., 2019) with an m7GpppG cap (as in Ramanathan et al., 2016) using a linker to connect the sgRNA (as in He et al., 2016) to the m7GpppG cap structure depicted in Ramanathan et al., 2016. Furthermore, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to design the capped sgRNA such that it targets a region proximal to a target start codon of the RNA molecule (such as an mRNA, as disclosed in Zhang) relative to an endogenous 5’ m7G cap of the RNA molecule so that the endogenous 43S PIC scanning does not physically block or otherwise compete with the 43S PIC scanning initiated by the Cap0-capped sgRNA based on the disclosure of Hinnebusch, 2011. Collectively, these designs and modifications thereof would predictably direct a user-provided cap to a user-specified mRNA transcript, thereby activating translation of the targeted mRNA transcript. One would have been motivated to make such a modification to the construction of the capped sgRNA of Zhang in order to receive the expected benefit of generating m7GpppG-capped sgRNA with the added functionality of influencing translation of the targeted mRNA.
Additionally, with regard to claim 71, which recites “the target sequence [of the complex of claim 1] comprises the target start codon of the RNA molecule,” as set forth above, Hinnebusch, 2011 discloses that the endogenous mRNA 5’ m7G cap must necessarily be located upstream of the endogenous start codon in order to facilitate activation of the elongation phase of protein synthesis, in part by directing 43S PIC scanning (page 435, column 1, paragraphs 2 and 3). Therefore, a capped sgRNA (as in Mu et al., 2019) targeted to an mRNA transcript (as in Zhang, paragraph [0100]) to activate translation of said mRNA transcript must facilitate the same 43S PIC scanning to locate the endogenous start codon of the targeted mRNA transcript. Furthermore, in order to effectively activate translation, this capped sgRNA must direct its m7GpppG cap (Cap 0; as disclosed in Ramanathan et al., 2016) downstream of the endogenous 5’ m7G cap so that the endogenous 43S PIC scanning does not physically block or otherwise compete with the 43S PIC scanning initiated by the m7GpppG-capped sgRNA. Additionally, Jiang and Doudna, 2017 teach that sgRNA spacer sequences have a set length (i.e. 20 nucleotides for Cas9 sgRNA spacer sequences) and confer target specificity (page 512, paragraph 3). Thus, given that the instantly claimed capped sgRNA must direct its m7GpppG cap downstream of the endogenous 5’ m7G cap to facilitate 43S PIC scanning for the endogenous start codon of the mRNA transcript (based on the disclosure of Hinnebusch, 2011), one of ordinary skill in the art would have been motivated to ensure that the claimed capped sgRNA would effectively facilitate 43S PIC scanning and start codon recognition by designing the sgRNA spacer sequence to target a sequence comprising the endogenous start codon so that the 43S PIC (once recruited) can quickly scan for and recognize the start codon due to its close proximity to the user-provided m7GpppG cap, as defined by the spacer sequence of the sgRNA (reviewed in Jiang and Doudna, 2017).
With regard to claim 72, which recites “the 5’ end of the target sequence [of the complex of claim 1] is upstream of the target start codon of the mRNA,” as set forth above, Hinnebusch, 2011 discloses that the endogenous mRNA 5’ m7G cap must necessarily be located upstream of the endogenous start codon in order to facilitate activation of the elongation phase of protein synthesis, in part by directing 43S PIC scanning (page 435, column 1, paragraphs 2 and 3). Therefore, a capped sgRNA (as in Mu et al., 2019) targeted to an mRNA transcript (as in Zhang, paragraph [0100]) to activate translation of said mRNA transcript must facilitate the same 43S PIC scanning to locate the endogenous start codon of the targeted mRNA transcript. Accordingly, one of ordinary skill in the art would have been motivated by the disclosure of Hinnebusch, 2011 to design the claimed capped sgRNA such that the spacer sequence therein targets a region upstream of the target start codon of the mRNA in order to facilitate 43S PIC scanning in the endogenous 5’ to 3’ direction (as depicted in Figure 1 of Hinnebusch, 2011).
With regard to claims 73 and 74, which respectively recite “the 5’ end of the target sequence [of the complex of claim 72 or 73] is between 1 and 50 nucleotides [or 1 to 15 nucleotides] upstream of the first nucleotide of the target start codon of the mRNA,” as set forth above, one of ordinary skill in the art would have been motivated by the disclosure of Hinnebusch, 2011 to design the claimed capped sgRNA such that the spacer sequence therein targets a region upstream of the target start codon of the mRNA in order to facilitate 43S PIC scanning in the endogenous 5’ to 3’ direction (as depicted in Figure 1 of Hinnebusch, 2011). Hinnebusch, 2011 further discloses that endogenous mRNA entry (i.e. upstream) and exit (i.e. downstream) channels (which form as the mRNA passes through the 43S PIC) each comprise ~12 nucleotides (page 435, column 1, paragraph 4-page 435, column 2, paragraph 1; Figure 2). Accordingly, one of ordinary skill in the art would have been motivated by the disclosure of Hinnebusch, 2011 to target a region within ~12 nucleotides upstream of the start codon in order to position the scanning complex such that the requisite machinery is optimally positioned to recognize the start codon and initiate the elongation phase of protein synthesis as quickly and efficiently as possible, as depicted in Figure 2B of Hinnebush, 2011 and set forth above.
Given that Zhang, Mu et al., 2019, Ramanathan et al., 2016, He et al., 2016, and Hinnebush, 2011 collectively disclose the complex of instant claim 1 (as set forth above), and that Hinnebusch, 2011 further discloses that endogenous mRNA entry and exit channels each comprise ~12 nucleotides as the mRNA is scanned and elongation is initiated, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to target a region within ~12 nucleotides upstream of the start codon to predictably position the scanning complex such that the requisite machinery is optimally positioned to recognize the start codon and initiate the elongation phase of protein synthesis as quickly and efficiently as possible. One would have been motivated to make such a modification in order to receive the expected benefit of positioning the scanning complex such that the requisite machinery is optimally positioned to recognize the start codon and initiate the elongation phase of protein synthesis as quickly and efficiently as possible.
Claim 24 is rejected under 35 U.S.C. 103 as being unpatentable over US 2017/0306335 A1 (hereinafter Zhang; of record) in view of Mu et al., 2019 (of record), Ramanathan et al., 2016 (of record), He et al., 2016 (of record), and Hinnebusch, 2011 (as cited in the IDS filed 02/23/2022; of record), as evidenced by Cowling et al., 2010 (of record) and Jiang and Doudna, 2017 (of record), as applied to claim 23 above, and further in view of Wolter and Puchta, 2018 (of record).
The combined disclosures of Zhang, Mu et al., 2019, Ramanathan et al., 2016, He et al., 2016, Cowling et al., 2010, Hinnebusch, 2011, and Jiang and Doudna, 2017 are described above and applied as before. However, these disclosures do not teach the nuclease-deficient Cas13 species of instant claim 24.
With regard to claim 24, which recites “the nuclease-deficient Cas polypeptide [of instant claim 23] is a nuclease-deficient Cas13 (dCas13) polypeptide, wherein the dCas13 is dCas13b or dCas13d,” Wolter and Puchta, 2018 disclose that catalytic residues of Cas13 can be deactivated through mutation, as with Cas9, to generate catalytically dead Cas13 (dCas13) (page 772, column 2, paragraph 2). Per Jiang and Doudna, 2017, deactivating catalytic Cas domains effectively uncouples cleavage activity from targeting activity, facilitating additional Cas functions such as transcriptional regulation, epigenetic modification, live-cell imaging, and nucleotide editing (Figure 2). Wolter and Puchta, 2018 additionally disclose that Cas13 species such as Cas13b can be used to edit RNA in a similar manner as Cas9 can be used to edit DNA (page 772, column 2, paragraph 3), and that such species of Cas13 offer practical advantages, including its modular composition, which enables simple and fast design, large scalability, and induction of subtle RNA manipulations (using mutant Cas13 species such as dCas13) (page 773, column 1, paragraph 4-page 773, column 2, paragraph 1). Given that Cas13 can be transformed into dCas13 by mutating its catalytic residues alone, one of ordinary skill in the art would not expect these mutations to impact the practical advantages listed above, which do not depend on the activity of its catalytic residues.
Given that the disclosure of Wolter and Puchta, 2018 teaches the mutability of Cas13 to form dCas13 as well as the practical advantages associated with using Cas13 species such as Cas13b, it would have been obvious to one of ordinary skill in the art before the filing date of the claimed invention to utilize dCas13 species such as dCas13b, as in instant claim 24, as the nuclease-deficient Cas polypeptide species of instant claim 23 to predictably generate a system benefitting from the modular nature of Cas13, allowing simpler and faster design, larger scalability, and greater ability to manipulate RNA using mutant Cas13 species such as dCas13. One would have been motivated to make such a modification to the Cas polypeptide of the complex in order to receive the expected benefit of a simpler and more powerful Cas system.
Claim 25 is rejected under 35 U.S.C. 103 as being unpatentable over US 2017/0306335 A1 (hereinafter Zhang; of record) in view of Mu et al., 2019 (of record), Ramanathan et al., 2016, (of record) He et al., 2016 (of record), Hinnebusch, 2011 (as cited in the IDS filed 02/23/2022; of record), and Wolter and Puchta, 2018 (of record), as evidenced by Cowling et al., 2010 (of record) and Jiang and Doudna, 2017 (of record), as applied to claim 24 above, and further in view of Faure et al., 2018 (of record).
The combined disclosures of Zhang, Mu et al., 2019, Ramanathan et al., 2016, He et al., 2016, Cowling et al., 2010, Hinnebusch, 2011, Wolter and Puchta, 2018, and Jiang and Doudna, 2017 are described above and applied as before. However, these disclosures do not teach the direct repeat capable of binding to a dCas13 polypeptide of instant claim 25.
With regard to claim 25, which recites “the direct repeat is capable of binding to a nuclease-deficient Cas13 (dCas13) polypeptide, wherein the dCas13 is dCas13b or dCas13d,” Faure et al., 2018 discloses that class 2 CRISPR-Cas systems comprise three distinct types with multiple subtypes, including type II (Cas9) and type VI (Cas13) (page 435, column 2, paragraph 1). Additionally, Faure et al., 2018 discloses that while all subtypes of type II CRISPR-Cas systems require tracrRNAs (which bind to the Cas endonuclease, as previously set forth per Jiang and Doudna, 2017), type VI CRISPR-Cas systems do not require these tracrRNAs (page 442, column 2, paragraph 3). In fact, Faure et al., 2018 discloses that “the structures of the single gRNA and the effector protein” are wholly different in type VI CRISPR-Cas systems as compared to type II CRISPR-Cas systems (page 443, column 1, paragraph 1). Thus, the disclosure of Faure et al., 2018 clearly indicates that direct repeats capable of binding to Cas polypeptides widely vary between types of CRISPR-Cas systems and would not be interchangeable. One of ordinary skill in the art would thus be clearly motivated to utilize Cas binding sequences appropriate for Cas13 (type VI) in order to effectively use Cas13, as instantly claimed.
Given that the disclosure of Faure et al., 2018 sets forth that binding sequences capable of interacting with Cas polypeptides vary between subtypes of class 2 CRISPR-Cas systems and that the complex from which instant claim 25 depends comprises type VI (Cas13) species, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to tailor the direct repeat sequence of the capped sgRNA to bind to type VI (Cas13) species to predictably generate a complex capable of functioning properly. One would have been motivated to make such a modification to the direct repeat sequence of the capped sgRNA in order to receive the expected benefit of a CRISPR complex capable of functioning.
Claims 75-77 are rejected under 35 U.S.C. 103 as being unpatentable over US 2017/0306335 A1 (hereinafter Zhang; of record) in view of Mu et al., 2019 (of record), Ramanathan et al., 2016 (of record), He et al., 2016 (of record), and Hinnebusch, 2011 (as cited in the IDS filed 02/23/2022; of record), as evidenced by Cowling et al., 2010 (of record) and Jiang and Doudna, 2017 (of record), as applied to claim 1 above, and further in view of Martin et al., 2011 (of record).
The combined disclosures of Zhang, Mu et al., 2019, Ramanathan et al., 2016, He et al., 2016, Cowling et al., 2010, Hinnebusch, 2011, and Jiang and Doudna, 2017 are described above and applied as before. However, these disclosures do not teach the target sequence downstream of the target start codon of instant claims 75-77.
With regard to claim 75, which recites “the 5’ end of the target sequence [of the complex of claim 1] is downstream of the target start codon of the mRNA,” as set forth above, the canonical, endogenous pathway by which capped mRNA initiates translation involves scanning of the 43S PIC in the 5’ to 3’ direction (reviewed in Hinnebusch, 2011). However, Martin et al., 2011 discloses that translation can also be initiated by factors downstream of the start codon of mRNAs that tether ribosomal particles and sequester the m7G cap to facilitate direct positioning of the ribosome on the cognate start codon (abstract). Accordingly, Martin et al., 2011 discloses that translation initiation may be governed both by scanning that initiates upstream of the endogenous start codon (as in Hinnebusch, 2011) and by factors downstream of the endogenous start codon. Accordingly, one of ordinary skill in the art would be motivated to target a region downstream of the target start codon of the mRNA in order to ensure that user-directed translation initiates properly and robustly.
With regard to claims 76 and 77, which respectively recite “the 5’ end of the target sequence [of the complex of claim 72 or 73] is between 1 and 50 nucleotides [or 1 to 15 nucleotides] downstream of the last nucleotide of the target start codon of the mRNA,” as set forth above, one of ordinary skill in the art would have been motivated by the disclosure of Martin et al., 2011 to target a region downstream of the target start codon of the mRNA in order to ensure that user-directed translation initiates properly and robustly. Additionally, as set forth above regarding instant claims 73 and 74, Hinnebusch, 2011 further that endogenous mRNA entry (i.e. upstream) and exit (i.e. downstream) channels (which form as the mRNA passes through the 43S PIC) each comprise ~12 nucleotides (page 435, column 1, paragraph 4-page 435, column 2, paragraph 1; Figure 2). Accordingly, one of ordinary skill in the art would have been motivated by the disclosure of Hinnebusch, 2011 and Martin et al., 2011 to target a region within ~12 nucleotides downstream of the start codon such that the requisite machinery recruited for recognizing the start codon and initiating the elongation phase of protein synthesis is firmly positioned within the region spanning the mRNA exit channel, thereby initiating the elongation phase of protein synthesis as quickly and efficiently as possible.
Given that Zhang, Mu et al., 2019, Ramanathan et al., 2016, He et al., 2016, and Hinnebush, 2011 collectively disclose the complex of instant claim 1 (as set forth above), that Hinnebusch, 2011 further discloses that endogenous mRNA entry and exit channels each comprise ~12 nucleotides, and that Martin et al., 2011 discloses that cap-dependent translation initiation may also be initiated by factors downstream of the start codon, it would have been obvious to someone of ordinary skill in the art before the effective filing date of the claimed invention to target a region within ~12 nucleotides downstream of the start codon to predictably position the requisite machinery for recognizing the start codon and initiating the elongation phase of protein synthesis within the region spanning the mRNA exit channel, thereby initiating the elongation phase of protein synthesis as quickly and efficiently as possible. One would have been motivated to make such a modification in order to receive the expected benefit of positioning the scanning complex such that the requisite machinery is optimally positioned to recognize the start codon and initiate the elongation phase of protein synthesis as quickly and efficiently as possible.
Conclusion
No claims are allowed.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Sarah E Allen whose telephone number is (571)272-0408. The examiner can normally be reached M-Th 8-5, F 8-12.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Jennifer Dunston can be reached at 571-272-2916. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/SARAH E ALLEN/ Examiner, Art Unit 1637
/J. E. ANGELL/ Primary Examiner, Art Unit 1637