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 communication filed 11-18-25.
Claims 1-19, 39 are pending in the instant application.
Election/Restrictions
Claims 12-14, 17-19, 39 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention or species, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on .
Applicant’s election without traverse of Group I, claims 1-11,15, 16, the Cas 7-11 polypeptide of Seq ID No. 1, the Csx29 polypeptide of SEQ ID No. 35, IL-12, steric hindrance, the linker comprising SEQ ID NO:36 in the reply filed on 11-18-25 is acknowledged.
Claim Rejections - 35 USC § 112
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 1-11,15, 16 are rejected under 35 U.S.C. 112, first paragraph, because the specification, while being enabling for the in vitro analyses of molecular interactions (e.g., Csx30-Csx31-RpoE complexes, Cas7-11-crRNA-Csx29-tgRNA), and of the in vitro inhibition of E. Coli growth does not reasonably enable methods for treating any cancer in a subject comprising administration of a Cas7-11:Csx29 complex, a guide strand specifically hybridizing a RNA target, and an apoptotic protein fused to an inhibitory peptide via a Csx30 linker.
The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims.
The following factors have been considered in determining that the specification does not enable the skilled artisan to make and/or use the invention over the broad scope claimed.
The breadth of the claims:
The claims are drawn to methods of treating cancer comprising administering an effective amount of a Cas7-11:Csx29 complex or a first nucleic acid encoding the Cas7-11:Csx29 complex, a guide RNA that specifically hybridizes to a RNA target, and an apoptotic protein fused to an inhibitory peptide via a Csx30 linker or a second nucleic acid encoding the apoptotic protein fused to the inhibitory peptide via the Csx30 linker, the apoptotic activity of the apoptotic protein is inhibited by the inhibitory peptide and the apoptotic activity of the apoptotic protein is activated upon cleavage of Csx3, which cancer cells comprise the target RNA; and Csx29 cleaves Csx30 when the Cas7-11:Csx29 complex binds to the target RNA, which Cas7-11 optionally comprises SEQ ID NO: 1, which Csx29 optionally comprises SEQ ID NO: 35, which guide RNA optionally comprises a mature pre-crRNA and is a single-strand RNA (ssRNA), and which linker optionally comprises Csx30 of SEQ D No. 36.
Teachings in the art and in the specification.
Teachings in the art:
Roberts et al (Nature Rev., Drug Discovery, Vol. 19, pages 673-694 (2020)) teaches on page 673 that “achieving efficient oligonucleotide delivery, particularly to extrahepatic tissues, remains a major translational limitation.”
Kobelt et al (Cancer Gene Therapy in Gene Therapy of Cancer: Methods and Protocols, Methods in Molecular Biology, Vol. 2521, pages 1-15 (Springer Nature 2022)) teach that limitations to cancer gene therapy relate to limitations in gene transfer efficiency (see esp. pages 3-4).
In addition, Osborn et al (Nucleic Acid Therapeutics, Vol. 28, No. 3, pages 128-136 (2018)) state the following about challenges to siRNA delivery on page 128:
…The primary challenge facing the clinical development of small interfering RNAs (siRNA) has been overcoming barriers that impede in vivo delivery. siRNAs are large, polyanionic macromolecules with intrinsically poor pharmacological properties. Unmodified siRNAs have a half-life of less than 5 min in circulation, and they do not permeate intact cellular membranes…
Damase et al (Frontiers in Bioengineering and Biotechnology, Vol. 9, Article 628137, pages 1-24 (2021)) on page 13 also address the challenges of using RNA-based drugs:
Targeted delivery is a major hurdle for effective RNA therapeutics, a hurdle that must be overcome to broaden the application of clinical translation of this type of therapeutic. …There is a need for novel delivery vehicles that will deliver the RNA drug to the site of therapeutic action facilitating the entry of the RNA drug into the cytoplasm where it may exert its effect…
Teachings in the specification:
The specification teaches the following in vitro analyses:
Figs. 1A-1D show Cryo-EM structures of the Cas7-11-erRNA-Csx29 complexes with and without the target RNA.
Fig.1A shows domain structures of Cas711 and Csx29.
Fig.1B shows nucleotide sequences of the crRNA and its target RNA. Disordered nucleotides are indicated by dashed circles PFS protospacer flanking sequence
Figure 1B discloses SEQ ID NOS 84-85 respectively, in order of appearance.
Figs. IC-1D show overall structures of Cas7-11-crRNA-Csx29
(Fig. 1C) and Cas7-11-erRNA-Csx29-tgRNA
(Fig. 1D). The bound zinc ions are shown as spheres.The disordered L1 and L2 linkers are not shown for clarity.
Figs.2A-2E show interaction between Cas7-11 and Csx29.
Fig. 2A shows structure of Csx29 in the Cas7-11-crRNA-Csx29 complex.
Fig. 2B shows interface between Cas7-11 an Csx29 in the Cas7-11-crRNA-Csx29 complex. The Cas11 and INS domains are omitted for clarity.
Fig. 2C shows location of the Csx29 active site. The catalytic residue H615 of the Csx29 protease is shown.
Figs. 2D-2E show interfaces between Cas7-1 and Csx29 in Cas7-11-crRNA-Csx29
(Fig. 2D) and Cas7-11-erRNA-Csx29 tgRNA
(Fig. 2E). Csx29 is shown as a surface representation, except for the AR, which is shown as a ribbon representation.
The AR and APD are disordered in the Cas7-11-crRNA-Csx29-tgRNA structure in (Fig. 2E).
Figs. 3A-3E show target RNA-triggered Csx30 cleavage by Csx29.
Fig. 3 shows schematic of the RNA-triggered Csx30 cleavage of the as7-11rRNA Csx29 complex TR, target RNA without a PFS CTR cognate target RNA with a non-matching PFS NTR, non-cognate target RNA with a matching PFS.
Figure3A discloses sEQ ID NO: 86.
Fig. 3B shows RNA triggered Csx30 cleavage by the Cas7-11-erRNA-Csx29 complex. The Cas7-11-crRNA-Csx29 was incubated with Csx30 at 37°C for 1 min in the presence or absence of the target RNA (CTR). The wtype (W) and catalytically inactivated
(Fig. 3D) versions of Cas7-and Csx29 were used.
Fig 3C shows effects of the complementarity between the crRNA 5' tag and tgRNAPFS on the Csx30 cleavage. The dCas7-11-crRNA-Csx29 complex was incubated withCsx30 at 37°C for 5,10,15 min in the presence of the target RNA (TR CTR or NTR).
Fig.3D shows proteolytic cleavage site in Csx30The Csx30 site cleaved by Csx2 is indicated by a triangle.The Csx30 structure was predicted using AlphaFold2, and the Ca atoms of M427and K428at the cleavage site are indicated by spheres Figure 3D discloses SEQ ID NO: 37.
Fig. 3E shows Csx29-mediated Cleavage of the Csx30 mutants. The dCas7-11-crRNA-Csx29 complex was incubated with the Csx30 mutants at 37°C for 10min in the presence or absence of the target RNA (CTR).
In (Fig. 3B), (Fig. 3C)(Fig. 3E) the proteins were analyzed by SDS-PAGE, and gel was stained with CBB.
Figs. 4A4J show effects of Csx30 and Csx31 on bacterial cell growth.
Fig. 4A shows schematic of bacterial growth assays for studying the Csx30 and Csx31 functions
Fig.4B shows Growth curves and end-point analyses
(Fig.4C) of E.coli expressing either full-length Csx30, the N-terminal fragment (residues 1 -427) of Csx30 (Csx30-1), of the C-terminal fragment (residues 428-565) of Csx30 (Csx30-2).
Figs. 4D-4E show growth curves (Fig.4D and end-point analyses
(Fig. 4E) of Ecoli expressing either Csx30-1, full-length Csx30 and Csx31, or Csx30-1 and Csx31.
In Figs 4B-4E, growth was compared between induced and uninduced expression conditions. In Figs. 4C and 4E ,,,
Fig. 4Fshows heatmap comparing the survival percentages of bacteria expressing either Csx30-1, Csx30-2, full-length Csx30 and Csx31, full-length Csx30 alone, Csx30 and Csx3, or Csx302 and Csx31, cultured at three different temperatures. …
Fig. 4H show schematic of the mammalian application of the Cas7-11-Csx29 Csx30 degron reporter system for RNA sensing in live cells.
Fig. induces cell growth inhibition as part
of anti-viral immunity. The Csx30 NTD probably binds RpoE as an anti-sigma factor, and affects cell growth and viability through unknown mechanisms.Csx31 likely functions as an antitoxin, thereby protecting the cell from the toxic effect of the Csx30 NTD
Figs. 6A-6F show Cryo-EM analysis of the Cas7-11-crRNA-Csx29 complex.
Fig. 6A shows single-particle cryo-EM image processing workflow.
Fig. 6B shows representative micrograph at a magnification of x105,000.
Fig.6C shows representative 2D averaged class images from the particles used for final reconstruction. Number of particles and resolution of reconstruction are indicated for each class.
Fig. 6D shows Fourier shell correlation (FSC) curves.
Mapmap FSC curve was calculated between the two independently refined half-maps after masking (blue line), and the overall resolution was determined by gold standard FSC = 0.143 criterion. Map-to Model FSC was calculated between the refined atomic models and maps (red line).
Fig. 6E shows directional FSC plots calculated in the 3DFSC server.
Fig. 6Fshows Euler angle distribution of particles in the final reconstruction.
Figs. 7A-7F show Cryo-EM analysis of the Cas7-11-crRNA-Csx29-tgRNA complex.
Fig. 7A shows single-particle cryo-EM image processing workflow.
Fig. 7B shows representative micrograph at a magnification of x105,000.
Fig. 7C shows representative 2D averaged class images from the particles used for final reconstruction. Number of particles and resolution of reconstruction are indicated for each class.
Fig. 7D shows FSC curves. Map-to-map FSC curve was calculated between the two independently refined halfmaps after masking, and the overall resolution was determined by gold standard FSC = 0.143 criterion. MaptoModel FSC was calculated between the refined atomic models and maps.
Fig. 7E shows directional FSC plots calculated in the 3DFSC server.
Fig. 7F shows Euler angle distribution of particles in the final reconstruction.
Figs. 8A-8D show Cryo-EM density maps.
Figs. 8A-8B show Cryo-EM density maps for Cas7-11-crRNA-Csx29 (Fig. 8A) and Cas7-11-crRNA-Csx29-tgRNA (Fig. 8B).
Figs. 8C-8D show Cryo-EM density maps for Cas7-11-erRNA-Csx29 (Fig. 8C) and Cas7-11- crRNA-Csx29-tgRNA (Fig. 8D).
Figs. 9A-9C show structural comparison of the Cas7 11 complexes in different states.
Figs. 9A-9C show structures of Cas7-11-crRNA-tgRNA (PDB ID: 7WAH) (Fig. 9A), Cas7-11- crRNA-Csx29
(Fig. 9B), and Cas7-11-crRNA-Csx29-tgRNA (Fig. 9C). The bound zinc ions are shown. The disordered L1 and L2 linkers are not shown for clarity. The disordered regions (residues 10431126) in the INS domain are indicated by dashed circles in
(Fig. 9B) and (Fig.9C). The bound RNA molecules are shown on the right of the complexes.
Figs. 10A10C show RNA recognition by Cas711.
Fig. 10A shows recognition of the crRNA 5' end by the Cas7.1 domain. The density map is shown as a gray mesh. The possible location of U(-16) and the pre-crRNA processing site are indicated by a dashed circle and a triangle,respectively.
Figs.10B-10C show recognition of the guide-target duplex by Cas7-11
(Fig.10B) and Csm (PDB ID: 6IFY)
(Fig. 10C). The catalytic residues (D429A/D654A of Cas7-11 and D33N of Csm) are depicted as space-filling models. The target RNA cleavage sites are indicated by triangles. The thumb-like ß-hairpins are indicated by circles in the schematics.
Fig. 11 shows structural comparison between Csx29 and human separase. Overall structures of Csx29 and huma separase (PDB ID: 7NJ1). The catalytic residues are depicted as space-filling models. Securin (separase inhibitor) is colored gray.The close-up views of the protease active sites are shown in insets.
Figs.12A12D show interaction between Cas711 and Csx29.
Fig. 12A shows interface between Cas7-11 and Csx29 in the Cas7-11-crRNA-Csx29 complex.Cas711 and Csx29 are shown as ribbon and surface representations, respectively. The INS and CTE domains of Cas7-11 are omitted for clarity.
Fig. 12B-12D show structures of the Cas7.1-Cas7.4 domains in Cas7-11- crRNA-tgRNA (PDB ID: 7WAH)
(Fig. 12B) Cas7-11-crRNA-Csx29 (Fig. 12C) and Cas7-11- crRNA-Csx29-tgRNA
(Fig. 12D) The bound zinc ions are shown as spheres.The α-helical insertion in the Cas7.4 ZF motif is highlighted.
Figs.13A-13D show interface between Cas7-11 and Csx29.
Fig.13A shows interface between Cas7-11 Cas7.4 and Csx29 NTD.
Fig.13B shows interface between Cas7-11 Cas7.3/L2 and Csx29 NTD.
Fig.13C shows interface between Cas7-11 L2 and the Csx29 NTD/TPR.
Fig.13D shows interface between Cas7-11 Cas7.3 and Csx29 TPR1/2.
Figs. 14A14D show target RNA induced conformational change in the Cas7-11-Csx29 complex.
Figs 14A-14B show interfaces between Cas7-11 and Csx29 in Cas7-11-erRNA--Csx29
(Fig. 14A) and Cas7-11-crRNA-Csx29-tgRNA (Fig.14B).
Fig. 14C shows recognition of the tgRNA non-matching PFS by Cas711. The density map for the RNA molecules is shown as a gray mesh.
Fig. 14D shows superimposition of Cas7-11-crRNA-Csx29 and Cas7-11-crRNA- Csx29tgRNA. A potential steric clash between the tgRNA non-matching PFS and Csx29 (TPR1 and AR2) is indicated by a dashed circle.
Figs. 15A-15B show target RNA and Csx30 cleavage by theCas7-11-Csx29 complex.
Fig. 15A shows the Cas7-11-crRNACsx29 complex was incubated with a 5'-Cy5-labeled ssRNA FII11725504.1 target at 37°C for 10 min, and then analyzed by 5% TBEurea PAGE. The gels were visualized, using either Cy5 or SYBR Gold fluorescence. The wild-type (W) and catalytically inactivated (D)versions of Cas711 and Csx29 were used.
Fig. 15B shows RNA-triggered Csx30 cleavage by the Cas7-11-crRNA-Csx29 complex. The Cas7-11-crRNA Csx29 complex was incubated with Csx30 at 37°C for 5 min in the presence of the target RNA (CTR). The wild-type (W) and catalytically inactivated (D) versions of Cas7-11 and Csx29 were used.
Fig. 16 shows N-terminal analysis of Csx30. Elution profiles for N-terminal seven residues in the -15 kDa Csx30 fragment (Csx30-2) were shown.
Figs. 17A17D show effects of Csx30 and Csx31 on bacterial cell growth.
Fig. 17A shows growth curves of E. coli expressing the non-induced full-length Csx30, the N-terminal fragment (residues 1-427) of Csx30 (Csx30-1), or the C-terminal fragment (residues 428-565) of Csx30(Csx302). These curves serve as noninduced controls for ihe curves in Figure 4B.
Fig17B shows effects of Csx30 growth at a range concentrations End-point analysis of E. coli expressing arabinose-inducible full-length Csx30, the N-terminal fragment (residues 1-427) of Csx30 (Csx30-1), the C-terminal fragment (residues 428-565) of Csx30 (Csx30-2), or full-length and N- or C terminal Csx30 fragments conjugated to Csx31. OD600 values are shown for bacteria at concentrations ranging from 0 to 2% arabinose in the growth media, including the 1% value used for other experiments in the study.
Fig. 17C shows electrostatic surface potential of the Csx30 and Csx31 structures predicted using AlphaFold2. The predicted structures suggested that Csx30 and Csx31have negatively and positively charged surfaces, respectively.
Fig. 17D shows growth curves of E. coli expressing non-induced Csx30-1, full-length Csx30 and Csx31, or Csx30-1 and Csx31. These curves serve as non-induced controls for the curves in Figure 4D.
Figs.18A18E show interaction between Csx30, Csx31, and RpoE.
Figs.18A-18B show elution profiles of the Csx30-Csx31-RpoE complex from a gelfiltration column. Csx30, His6-tagged Csx31 ("His6" disclosed as SEQ ID NO: 83), and His6-tagged RpoE ("His6" disclosed as SEQ ID NO: 83) were co-expressed in E. coli, and purified by Ni-NTA and HiLoad16/600 Superdex 200 columns.
In (Fig.18A) the Csx30-Csx31RpoEcomplex was loaded onto a Superdex 200 Increase column
(Fig.18B) the Csx30-Csx31-RpoE complex was incubated with the Cas7-11-crRNA-Csx29-tgRNA complex, and then loaded onto a Superdex 200 Increase column. The fractions indicated by orange lines were analyzed by SDS-PAGE…
[Emphases added][Citations omitted].
The examples provided in the instant specification, of the in vitro analyses of molecular interactions (e.g., Csx30-Csx31-RpoE complexes, Cas7-11-crRNA-Csx29-tgRNA), and of the in vitro inhibition of E. Coli growth, are not representative or correlative of the ability to treat cancer in a subject as instantly claimed.
In light of the teachings in the art and the specification, one skilled in the art would not accept on its face the examples provided in the instant disclosure as being correlative or representative of the ability to provide treatment effects in a subject. Since the specification fails to provide the requisite guidance for the treatment in any subject, and since determination of the factors required for accomplishing this in any subject is highly unpredictable, it would require undue experimentation to practice the invention over the broad scope claimed.
For these reasons, the instant rejection for lacking enablement over the full scope claimed is proper.
Allowable Subject Matter
SEQ ID No. 36 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
1-31-26
/JANE J ZARA/Primary Examiner, Art Unit 1637