DETAILED ACTION
Receipt of Arguments/Remarks filed on November 17 2025 is acknowledged. Claims 1-37 and 57 were/stand cancelled. Claims 38-39, 41, 47-50, 52, 55, 59-60, 65 and 68 were amended. Claims 38-56 and 58-70 are pending.
This action is second action non-final due to new rejections which are not the result of claim amendments.
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 .
Withdrawn Objections/Rejections
The amendments filed November 17 2025 are sufficient to overcome the objection of claims 38, 48 and 49 .
The amendments filed November 17 2025 are sufficient to overcome the rejection of claim 39, 41, 48, 49 and 55 under 35 USC 112(b). The reorganization of the claims has clarified the scope. The amendments have corrected many of the identified issues.
Claim Objections
Claim 39 is objected to because of the following informalities: the various species (X, X1, X2, etc. see bottom of page 4) need to be separated by semi-colons (;) instead of commas so that the separation between species is clear. Appropriate correction is required.
Claim 39 is objected to because of the following informalities: a semi-colon (;) instead of a comma should be between “heteroaryl” and “R’” to more clearly separate the various groups/species. Appropriate correction is required.
Claim 48 is objected to because of the following informalities: a colon “:” is required after NO in line 4. Appropriate correction is required.
Claim Rejections - 35 USC § 112-Indefinitness
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 39, 41-50, 54-55, 59 and 66-67 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 39 as currently written is vague and indefinite. Claim 39 recites “the non-nucleotide linker” in line 2. This claim depends from claim 38. Claim 38 recites two different non-nucleotide linkers, specifically one part of lgRNA and one that covalently links the one or more molecules to the lgRNA. It isn’t clear if the non-nucleotide linker in claim 39 is limited the non-nucleotide linker of the lgRNA, the non-nucleotide linker connecting the one or more molecules to the lgRNA or both.
Claim 41 as currently written is vague and indefinite. Firstly, the structures are very difficult to read. For example, B44 and B45 recite R groups. It isn’t clear if the claim recites R11 or R14 (B-44) and R11 or R15 (B-45). New structures are needed in which all substituents on the structures are clear.
Claim 41 as currently written is vague and indefinite. Claim B-39 recites Y5, however, there is no definition of Y5 provided in the description. This makes the scope of the claim unclear.
Claim 41 as currently written is vague and indefinite. The claim recites a definition for R’. But then the claim recites or NR’2 is… While the definition of NR’2 is clear, it isn’t clear of the R’ recited earlier can also apply to NR’2. Since R’ is included in other structures besides NR’2. To make it clear the claim should recite something to the effect of “or when R’ is part of NR’2 then NR’2 is a heterocyclic group linked at its N”.
Claims 42-46 all recites the complex of claim 38 comprises a spacer. However claim 38 never recites a spacer. Therefore, it isn’t clear if this is a further element (which if the case the claim should recite further comprising) or if this spacer is supposed to further limit a particular structure in claim 38. Furthermore, the claims never indicate where the spacer is located. Is it in the Cas protein, gRNA or somewhere else.
Claim 47 as currently written is vague and indefinite. Claim 47 depends from claim 38 which recites a single strand nucleic acid template. Claim 47 recites said nucleic acid template comprises two sequences. The structure of the two sequences. Are these two separate sequences which are connected into one? Since these limitations are limiting the single strand nucleic acid template the presence of the word two sequences creates confusion as to the scope.
Claim 47 as currently written is vague and indefinite. The claim recites “wherein said covalent linker is a non-nucleotide linker”. What covalent linker? The covalent linker in claim 38 is already claimed to be a non-nucleotide linker. It is unclear if this is just a repeat limitation from claim 38 or if the covalent linker is supposed to be referring some sort of connection between the two sequences and gene editing sequence.
Claim 48 as currently written is vague and indefinite. The claim recites “wherein n is any nucleotide” but never indicate where n is present. The examiner suggests stating “wherein n in SEQ ID No: 84-110 is any nucleotide” to make it clear that the n is present in the claimed sequences.
Claim 50 as currently written is vague an indefinite. While the recitation “comprising a mixture of lgRNA conjugates” is not indefinite. The claim states comprising a mixture of lgRNA conjugates of various spacers targeting at different loci. The spacers referred to are not clear. No spacer is recited in claim 38. Clearly the claim is attempting to be directed to more than a mixture of lgRNA conjugates of claim 38 but the metes and bounds of the claim are unclear.
Claim 54 as currently written is vague and indefinite. The claim recites “covalently linked molecules for targeted cellular delivery”. Are these additional elements? If so, then the claim needs to state "further comprising". Is this attempting to limit the one or more molecules in claim 38, then the claim needs to clarify that the “molecules for targeted cellular delivery” are the one or more molecules. If it is the latter then the response must also indicate how it is further limiting. Claim 38 recites the one or more molecules are an antibody, an aptamer or a single strand nucleic acid template. It isn’t clear how “molecules for targeted cellular delivery” limit those structures.
Claim 55 as currently written is vague and indefinite. Step (a) recites cleaving target gene DNA to be edited. But the claim never specifies what cleaves the target gene DNA. Specifically the claim never requires actually administration of the complex or contacting cells with the complex. Nor the claim never species that it is the CRISPR-Cas protein which cleaves the target.
Claim 55 as currently written is vague and indefinite. The claim is directed to “target gene editing” but the last two lines of the claim states using the template to edit the target gene by introducing insertions, deletions or point mutations included in said template. The way the claim reads, the scope encompasses introducing insertions in the template. But I think the scope is that the target gene is edited by introducing insertions from said template? The claim language creates ambiguity in the scope of how the insertions, deletions or point mutations are introduced.
Claim 59 as currently written recites “said nucleic acid template is a DNA”. However, this claim depends from claim 51 which depends from claim 38 which recites a “single strand nucleic acid template”. Claim 59 does not make it clear that the DNA is single-strand which is required by claim 38. Applicants could amend the claim to recite said single strand nucleic acid template is a DNA. The examiner notes that claim 60 also just refers to a DNA template but since it does not depend from claim 38, the indefiniteness is not addressed.
Claim 66 as currently written is vague and indefinite. The species here are confusing. Is the claims scope that the mRNA, plasmid or viral vector encodes a Cas protein? The separation of species needs to be clarified. Also does the "to form conjugates(s) of CRISPR-Cas protein-guide RNA complex in targeted cells" modify all or just the (viral vector)? The claim scope is unclear.
Claim 67 as currently written is vague and indefinite. Where does the Markush grouping end? The different species of the Markush are not clear. Is it after "and" in line 5 or "or" in line 6? The claim scope is unclear.
Claim 49 is included in the rejection as they depend on a rejected base claim and they do not clarify the issues.
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 of this title, 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 factual inquiries set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied 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 38-47, 50-51, 54-56, 61-62, 64-66 and 69-70 are rejected under 35 U.S.C. 103 as being unpatentable over Zhong (USPGPUB No. 20160215275) in view of Lee et al. (eLife, 2017, cited in the Office action mailed on July 17 2025).
Applicant Claims
The instant application claims a conjugate of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas protein-guide RNA(s) complex, comprising (a) an IgRNA-Cas protein (RNP) complex and (b) one or more molecules selected from the group consisting of, antibodies, aptamers, and a single strand nucleic acid template, wherein said one or more molecules are covalently linked to said IgRNA by one or more non-nucleotide linkers and said IgRNA comprises a crRNA, a tracrRNA and at least one non-nucleotide linker located after its first nucleotide and before its last nucleotide.
Determination of the Scope and Content of the Prior Art
(MPEP §2141.01)
Zhong is directed to chemically ligated RNAs for CRISPR/CAS-9-LGRNA complexes as antiviral therapeutic agents. The chemically ligated guide RNA oligonucleotides (lgRNA) comprises two functional RNA modules (crgRNA and tracrgRNA) joined by chemical nNt-linkers (i.e. non-nucleotide linker), their complexes with CRISPR-Cas9 (paragraph 0014; claim 1; 0024). In some embodiments the crRNA and tracrRNA are truncated at 3’-end and 5-end (paragraph 0079).
Ascertainment of the Difference Between Scope the Prior Art and the Claims
(MPEP §2141.02)
While Zhong et al. teach a complex between Cas9 and IgRNA, Zhong does not teach one or molecules covalently linked to said IgRNA. However, this deficiency is cured by Lee et al.
Lee et al. is directed to synthetically modified guide RNA and donor DNA are versatile platform for CRISPR-Cas9 engineering The CRISPR/Cas9 system uses a gRNA to target and cleave DNA sequences with specificality and can result in precise genome editing via homology directed repair if a donor DNA template is simultaneously delivered along with the CRISPR/Cas9 system (page 1). It is taught that chemical modification of the gRNA and donor DNA has great potential for improving the gene editing efficiency of Cas9 and Cpf1. It was demonstrated that the gRNA and donor DNA can be directly conjugated together into one molecule and that this conjugate is 3 times better at transfection cells and inducing HDR with cationic polymers than unconjugated gRNA and donor DNA (abstract; Fig. 1; page 8). A gRNA-donor DNA conjugate (gDonor) was synthesized by conjugating an azide terminated donor DNA with an alkyne modified crRNA (page 8). The donor DNA includes a short single stranded DNA (Figure 2a; page 4). As shown in Figure 2a, the DNA can be attached at the 3’-end of the crRNA via a non-nucleotide linker:
PNG
media_image1.png
326
370
media_image1.png
Greyscale
Finding of Prima Facie Obviousness Rationale and Motivation
(MPEP §2142-2143)
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Zhong et al. and Lee et al. and conjugate (i.e. covalently link) the donor DNA to the crRNA of Zhong et al. One skilled in the art would have been motivated to form this conjugate as it has been demonstrated that this allows for better transfection than unconjugated as taught by Lee et al.
Regarding claims 38 and 65-66, as shown in claim 1 of Zhong there is an nNt-linker in the lgRNA located after its first nucleotide and before its last nucleotide (paragraph 0014; claim 1; 0024). Conjugation of the donor DNA reads on the instantly claimed one or more molecules (specifically a single strand nucleic acid template) which is connected by a non-nucleotide linker. Zhong teaches a complex between the CRISPR-Cas protein and the guide RNA.
Regarding claim 39, as shown in claim 1 of Zhong, examples of nNt-linkers include:
PNG
media_image2.png
222
574
media_image2.png
Greyscale
which contains an M-1 core, two L wherein one L is L1 with n=6, the other L comprises L4 wherein n is 3 and m is 4, Nuc-2, Nuc-3. Regarding the non-nucleotide linker connecting the template to the crRNA, the structure shown in Figure 2a of Lee et al. comprises M-3, L1 wherein n is 0, Nuc-2, Nuc-3. Furthermore, Zhong teaches that the nNt-linker comprises a 1,4-disubstituted 1,2-3-triazole formed between an alkyne and an azide (paragraph 0035). This is the same reaction to connect the DNA in Lee with the crRNA. Therefore, one skilled in the art would have been motivated to utilize any corresponding ligand which can be used in the azide-alkyne reaction as both Lee et al. and Zhong teach it is useful in forming non-nucleotide linkers.
Regarding claim 40, Zhong teaches cell-penetrating peptide (CPP) is conjugated to a purified recombinant Cas9 protein (with appended Cys (i.e. cysteine) residue at the C terminus) (paragraph 0127). This reads on a non-PEG polymer or a peptide being covalently bound to the Cas protein.
Regarding claim 41, as shown in claim 1 of Zhong, the crgRNA comprises nucleotides modified at sugar moieties which includes at least II-6, II-4, II-5, II-7, II-8 and II-3.
Regarding claim 42, as shown in claim 1 and 5 of Zhong, the IgRNA comprises a spacer in crgRNA which is 12 to 20 nt in HIV genomes, of which each thymine is replaced with uracil.
Regarding claim 43, as shown in claim 1 and 6 of Zhong, the IgRNA comprises a spacer in crgRNA which is 12 to 20 nt in HBV genomes in which each thymine is replaced with uracil.
Regarding claim 44, as shown in claim 1 and 7 of Zhong, the IgRNA comprises a spacer in crgRNA and the spacer is 12 to 20 nt in HSV genomes of which each thymine is replaced with uracil.
Regarding claim 45, as shown in claim 1 and 8 of Zhong, the IgRNA comprises a spacer in crgRNA and the spacer is 12 to 20 nt in EBV genomes, of which each thymine is replaced with uracil.
Regarding claim 46, claim 12 of Zhong indicates that there can be mixtures of IgRNAs with various spacers at different loci of viral genomes. The spacer RNA oligomers have the same sequences as the sense strands of the genomes (see paragraphs 0043-0046).
Regarding claims 47 and 50, Lee et al. teaches that donor DNA can be conjugated wherein this donor DNA contains the desired edit. Lee et al. also teaches that extending the guide RNA by adding aptamer sequences can enable researchers to specifically activate the genes that have been edited. Lee et al. also teaches that extension of the gRNA with aptamer sequences can be used to recruit RNA binding counterparts within the cell that enable either the visualization or transcription of specific DNA sequences in the genome. Incorporation of modified RNA bases into the gRNA results in reduced immunogenicity, increased stability and enhanced gene editing efficiency. Finally chemical modification of the donor DNA with phosphorothioates dramatically increases HDR efficiency by increasing donor DNA stability (introduction).
Therefore the use of aptamers and donor DNA which reads on “two sequences” would allow for editing as well as activation of the genes in combination with the gRNA.
Regarding claim 51, Zhong teaches a recombinant Cas9 protein (example 7; claim 10) which is a recombinant engineered class 2 endonuclease (paragraph 0061).
Regarding claims 54 and 70, Zhong teaches cell-penetrating peptide (CPP) is conjugated to a purified recombinant Cas9 protein (with appended Cys (i.e. cysteine) residue at the C terminus) (paragraph 0127). This results in the complex comprising covalently linked molecules for targeted cellular delivery.
Regarding claim 55 and 61-62, Zhong teaches that Cas9 cleaves leaving a blunt end DNA double strand break (paragraph 0061). Taught is the use of the CRISPR-Cas9-IGRNA systems for gene editing (paragraph 0107). As taught by Lee et al., the gRNA was designed to cut the BFP gene and donor DNA was designed to covert the BFP gene into the GFP gene (see Figure 4; page 8). Therefore, the combination of Zhong and Lee et al. suggest the claimed method of target gene editing.
Regarding claim 56, this claim is directed to the conjugate. The wherein clause is interpreted as what happens to the complex in cells when the complex is encoded by a viral plasmid. Zhong teaches that the Cas9 protein and RNA have been introduced by plasmid transfection (paragraph 0010). Since the same complex is suggested by the cited prior art, the same self-assembly would be present.
Regarding claim 64, Lee et al. teaches the donor DNA includes a short single stranded DNA (Figure 2a; page 4).
Regarding claim 69, Zhong teaches IgRNA incubated with purified Cas9protein and then complexed with cationic lipids (paragraph 0125).
Claims 52-53, 58-60, 63 and 67-68 are rejected under 35 U.S.C. 103 as being unpatentable over Zhong in view of Lee et al. as applied to claims 38-47, 50-51, 54-56, 61-62, 64-66 and 69-70 above and in further view of Donohoue et al. (USPGPUB No. 20160362667, cited in the Office action mailed on July 17 2025) and Lim et al. (Nature Communications, 2019, cited in the Office action mailed on July 17 2025).
Applicant Claims
The instant application claims said Cas protein is a recombinant engineered endonuclease comprising at least two cysteines, and at least one of the said cysteines are introduced by site directed mutations of internal amino acids, and the said cysteines are conjugated with molecules for epitope masking and/or targeted delivery, and wherein the wild type cysteines are optionally mutated to avoid deactivation of said Cas protein due to covalent conjugations.
The instant application claims said complex is PEGylated.
The instant application claims said CRISPR-Cas protein is a fusion protein of nickase or catalytically inactive Cas further comprising one or more functional protein domains selected from the group consisting of FokI, DNA polymerase, reverse transcriptase, DNA methyltransferase, nucleic acid deaminases, transcription activator(s), transcription repressor(s), and histone acetyltransferase and deacetylase.
The instant application claims said Cas-effector fusion protein is a Cas protein fused with a DNA polymerase, and wherein said DNA polymerase uses a DNA template for gene editing.
The instant application claims a complex comprising a fusion protein of a Cas protein and a DNA polymerase, wherein said DNA polymerase uses a DNA template for templated DNA synthesis, said Cas protein nicks a target gene by cutting one of its double strands or cleaves a target gene to form a double strand break by cutting its both strands, and wherein said complex does not comprise a cytosine/adenine deaminase.
Determination of the Scope and Content of the Prior Art
(MPEP §2141.01)
The teachings of Zhong and Lee et al. are set forth above. Zhong teaches cell-penetrating peptide (CPP) is conjugated to a purified recombinant Cas9 protein (with appended Cys (i.e. cysteine) residue at the C terminus) (paragraph 0127).
Ascertainment of the Difference Between Scope the Prior Art and the Claims
(MPEP §2141.02)
While Zhong teaches cysteine can be appended to allow for covalent bonding of CPP, Zhong does not teach the Cas protein is mutated to include two cys residues. Zhong does not teach the complex is pegylated. Zhong does not teach a nickase or a functional protein or DNA polymerase. However, these deficiencies are cured by Lim et al. and Donohue et al.
Lim et al. is directed to a conjugation platform for CRISPR-Cas9 that allows efficient β-cell engineering. CRISPR-Cas9 is a DNA endonuclease that can be targeted to a genomic site using a guide RNA (gRNA) bearing sequence complementarity to the target site. Genetic fusion of Cas9 with effector domains (e.g., a transcription activator) has yielded transformative technologies; however, this approach is limited to fusions that are generally linear, polypeptidic, and located on the termini of Cas9. A conjugation platform that allows the creation of fusions that are non-polypeptidic (e.g., nucleic acids, small molecules, polyethylene glycol [PEG] chains), unnatural peptides/proteins, internally located on Cas9, and branched with a multivalent display, would provide a greater diversity of technologies and applications. Appending PEG chains to Cas9 may reduce the immunogenicity of the protein (page 1, main). Taught is thiol-maleimide chemistry and DNA-base pairing which is amendable to a wide range of substrates (page 1, main). Cas9 was scanned to choose residence replaceable with engineered cysteines to which molecules could be efficiency appended without loss of cas9 activity (page Internal residues on Cas9 that can be modified using thiol-maleimide chemistry without compromising Cas9 activity were identified (discussion and results). Figure S2 shows that PEG-maleimide was reacted with cysteine on Cas9 (pages 1-2, Main). Figure S11 shows two cysteine residues which are labeled with conjugates (see also multivalent display of ssODN on Cas9; page 5).
Donohoue et al. is directed to CRISPR-Cas compositions and methods. It is taught that mutated forms of Cas9 protein can be used. These include Cas9 proteins that contain a single inactive catalytic domain. Such modifications cleave only one strand of a target DNA thus creating a single-strand break (aka nickase). With these modified Cas protein NHEJ (non-homologous end joining) is less likely to occur (paragraph 0217). Taught are fusion proteins which are created by joining two or more proteins. The modifications results in fusion proteins which possess additional activity. These activities include polymerase activity (paragraph 0088). Taught is Cas proteins comprising a cys substation of a non-cys amino acid residue (claim 12; paragraph 0018).
Finding of Prima Facie Obviousness Rationale and Motivation
(MPEP §2142-2143)
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Zhong, Lee et al., Lim et al. and Donohoue et al. and utilize a mutated Cas9 where at least two cysteine residues are introduced. One skilled in the art would have been motivated to add cysteine residues in order to provide conjugation sites as taught by Lim et al. Since this type of mutation, i.e. residues which can be mutated which retain activity, is well known as taught by Lim et al. and Donohoue et al. and Zhong teaches the use of cysteine for conjugation there is a reasonable expectation of success. Rendering claims 52 obvious.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Zhong, Lee et al., Lim et al. and Donohoue et al. and utilize known conjugation groups such as PEG and effector domains (e.g., a transcription activator). One skilled in the art would have been motivated to utilize these types of conjugation groups as Lim et al. teaches that effector domains have yielded transformative technologies and PEG chain may reduce the immunogenicity of the protein. Rendering claim 53 obvious.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Zhong, Lee et al., Lim et al. and Donohoue et al. and utilize a fusion protein. One skilled in the art would have been motivated to utilize a fusion protein as both Lim et al. and Donohoue et al. teach the use of fusion proteins to provide additional activities. One skilled in the art would have been motivated to utilize a nickase in order to reduce NHEJ (non-homologous end joining) as taught by Donohoue et al. One skilled in the art would have been motivated to utilize a DNA polymerase in order to provide for polymerase activity as suggested by Donohoue et al. Rending claims 58-60, 63, 67-69 obvious.
Claims 48-49 are rejected under 35 U.S.C. 103 as being unpatentable over Zhong in view of Lee et al. as applied to claims 38-47, 50-51, 54-56, 61-62, 64-66 and 69-70 above and in further view of Feng et al. (USPGPUB No. 20170198302)
Applicant Claims
The instant application claims said gene editing sequence comprises an oligonucleotide sequence to introduce one or more stop codons selected from the group consisting of 5'-(tga)-3', 5'- (taa)-3', 5'-(tag)-3' and SEQ ID NO 84-110.
The instant application claims gene editing sequence comprises an oligonucleotide sequence to introduce one or more transcription cis-regulatory elements.
Determination of the Scope and Content of the Prior Art
(MPEP §2141.01)
The teachings of Zhong and Lee et al. are set forth above. Both suggest the use of a CRISPR-Cas complex for editing genes.
Ascertainment of the Difference Between Scope the Prior Art and the Claims
(MPEP §2141.02)
Zhong does not teach the inclusion of a stop codon or cis-regulatory elements. However, this deficiency is cured by Feng et al.
Feng et al. is directed to methods and systems for targeted gene manipulation. Taught are methods for inserting a reporter into an active gene locus or silence genome locus via CRISPR-induced homology directed repair (claim 22-23). The donor construct can comprise multiple stop codons (claim 15). It is taught that three stop codons are included in different reading frames to prevent the restoration of copGFP expression by NHEJ-mediated repair (paragraph 0205). Sequences taught as including the stop codon include XJ-5 which contain multiple TGA segments (paragraph 0254; table 3). The donor constructs can include promoters. Promoters include cis- and trans- acting transcription control elements and regulatory sequences that are involved in regulating or modulating the timing and/or rate of transcription of a gene. The promoter can be a cis-acting transcriptional control element. These sequences typically interact with proteins or other biomolecules to carry out (turn on/off, regulate, modulate, etc.) gene transcription (paragraph 0113).
Finding of Prima Facie Obviousness Rationale and Motivation
(MPEP §2142-2143)
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Zhong, Lee et al. and Feng et al. and utilize a stop codon and cis-acting transcription control elements in the donor nucleotide sequence. One skilled in the art would have been motivated to utilize cis-acting transcription control elements in order to regulate transcription as taught by Feng et al. One skilled in the art would have been motivated to utilize stop codons in order to prevent the restoration of expression by NHEJ-mediated repair as taught by Feng et al.
Response to Arguments
Applicants’ arguments filed November 17 2025 have been fully considered but they are not persuasive.
Applicant argues that (1; page 18-19) Lee teaches conjugation of donor DNA to crRNA via an alkyne-azide click chemistry linker at the 5’-end of he crRNA. However, Lee does not teach or suggest an lgRNA with an internal non-nucleotide linkers nor conjugates of such an lgRNA. In Lee, the crRNA is a standard RNA without internal non-nucleotide linkers. Furthermore, Lee’s tracrRNA is not covalently linked to form a single-guide structure and thus does not teach a crRNA-tracr-RNA single molecule with a covalently attached ssDNA. With these distinct gaps, Zhong and Lee cannot be combined to arrive at the claimed invention.
Regarding Applicant’s first argument, while neither reference individually teaches the claimed invention, this is not requirement for an obviousness rejection. One cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). The test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981). Therefore, while Lee et al. does not tech the lgRNA as claimed, this limitation is taught by Zhong. The question for obviousness is would it have been obvious to one of ordinary skill in the art to conjugate the donor DNA to the sgRNA. Since Zhong teaches the crRNA is conjugated to the tracrRNA via a non-nucleotide linker, this portion is taught by Zhong. Lee et al. teaches conjugation of the donor DNA to the 5’-end of the crRNA. Since the 5’-end of the crRNA of Zhong is of a similar scope as Lee et al. one skilled in the art would have a reasonable expectation of success in conjugating the donor to the 5’-end of the crRNA of Zhong with the same/similar click chemistry which is a non-nucleotide linker.
Applicant argues that (2, page 19) that modifying Zhong’s lgRNA with Lee’s terminal crRNA conjugation would require substantial redesign including chemical modifications to incorporate internal linkers while maintaining functionality of guide RNA. Such modifications were not trivial or predictable before the effective filing as evidenced by Taemaitree et al. and Nat. Biotechnol.
Regarding Applicant’s argument, the examiner notes that the references Applicant point to have not been submitted. If Applicant wishes the examiner to consider these references, then they must be submitted and made of record. None the less Zhong expressly discloses how to make a lgRNA with a crRNA and a tracrRNA with a nNt linker. Therefore, while other references might suggest the difficulties, Zhong clearly shows that they can be made. Additionally, as set forth above Lee et al. expressly shows how to chemically attach the donor DNA to the crRNA. Furthermore, if making the lgRNA with the various nNt linkers were so difficult and challenging, then Applicants would only be enabled for the specific examples shown in the instant specification. However, the examiner is of the position, that one skilled in the art would recognize the chemistry needed to make the lgRNA. While Applicants are correct that modifications could affect the gRNA, Lee et al. makes the same comment (little is known about how chemical modifications that are tolerated by the gRNA of the CRISPR/Cas9 system, page 1, first paragraph). However, in conclusion Lee et al. demonstrate that the gRNAs and donor DAN can be chemically modified at their terminal positions without losing activity (conclusions). Therefore, the state of the are clearly shows that chemical modifications between gRNA and donor DNA is tolerated.
Applicant argues that (3; page 19) that Potter demonstrates that sgRNA-ssDNA conjugates do not function. Applicant is the first to demonstrate functional lgRNA-ssDNA conjugates. Applicants point to poster present May 8 2024 and PGPUB 2025/027115.
Regarding Applicant’s arguments, while Potter might not provide the motivation to utilize ssDNA, Lee et al. clearly shows the use of ssDNA and that when conjugated to gRNA it works. The examiner cannot find the USPGUPB Applicants are pointing to in their response as that number does not bring up any references. Perhaps Applicants meant USPGPUB No. 20250027115. This application is the child of the current application and Figure 11 and 12 are the same as in the instant claims. These figures just show the in vitro cleavage data of lgRNA-ssDNA (Fig. 11) and the efficiency of combination of nCas9, DNA polymerase, lgRNA-ssDNA conjugate. But the instant specification does not indicate these results are surprising or unexpected. Furthermore, these results are to a single example. If the art is so unpredictable as Applicant alleges, then support would only be provided for the specific examples taught. Therefore, the examiner cannot agree that there isn’t a reasonable expectation of success in combining Zhong and Lee.
Applicant argues (3; page 20) that claim 38 has been amended to exclude all forms of crRNA-ssDNA conjugates further distinguishing from Lee. Applicant argues that the claims have been amended by excluding crRNA conjugates.
Regarding Applicant’s third argument, the examiner does not understand the argument. While the claim requires the lgRNA to comprise a crRNA, a tracrRNA and at least one non-nucleotide linker, this doesn’t appear to exclude all crRNA-ssDNA conjugates. The one or more molecules can still be covalently linked to the lgRNA by one or more non-nucleotide linkers at the 5’-end of the crRNA as suggested by Lee et al. No where in the instant claims does it exclude the one or molecules from being conjugated at the crRNA. In fact the claims allow for the conjugation at any position on the lgRNA. It appears that Applicants are of the position that unless the crRNA is part of an lgRNA one skilled in the art would now know how to attach the ssDNA donor. The examiner cannot agree. While attachment at the 3’end of the crRNA would not be obvious on an lgRNA as the tracrRNA is attached at the 3’ end of the crRNA via nNt linker. But the 5’ end is still available for conjugation.
Applicant argues (4; page 21); that claim 47 limits the nucleic acid template comprising two sequences which are defined as homolog arms not ssDNA and an aptamer as interpreted by the examiner. Applicants again argue that Lee are gRNAs without internal non-nucleotide linkers.
Regarding Applicants’ fourth argument, nothing in the instant claims limit the two sequences to be homology arms. While the specification teaches “In some embodiments, the said conjugated oligonucleotide is a donor template which comprises two sequences overlapping with the target strand or with the non-target strand of the DNA duplex to be edited, e.g. 5′- and 3′-homology arms, and a gene editing sequence (paragraph 00134). This does not limit the claim to be a specific structure.
Applicant argues that (5; page 21) Lee does not teach the instantly claimed stop codon.
Applicants arguments are considered and persuasive. Therefore, a new grounds of rejection is set forth above addressing this claim.
Applicant argues that (6; page 22) that Donohoue or Lim do not cure the deficiencies of Zhong and Lee et al. Applicant argues that Donohoue does not teach a DNA polymerase using a nucleic acid template. The polymerase activity can be non-templated, e.g. Tdt, which is unsuitable for precise gene editing. Donohoue teaches a first domain from a Cas9 or Cpf1 protein not an entire Cas protein fused for polymerase acidity (or with a DNA polymerase). Applicant points to paragraph 0088 of Donohoue.
Regarding Applicant’s sixth argument, firstly, as stated above, if Applicants want me to consider teachings in a reference, any copy of a FOR or NPL document must be provided. As stated in paragraph 0088, the fusion protein refers to a single protein created by joining two or more proteins, protein domains or protein fragments. For example, a first domain form a Cas9 protein and a second domain from a protein other Cas9. Therefore, while the example includes joining two protein domains, Donohoue clearly teaches two or more proteins can be fused. Since the rejection is made under 103, it does not need to exemplify all embodiments, only suggest. “Disclosed examples and preferred embodiments do not constitute a teaching away from the broader disclosure or non-preferred embodiment.” In re Susi, 440 F.2d 442, 169 USPQ 423 (CCPA 1971). MPEP 2123. While the specification might refer to specific embodiments, the claims merely require any Cas protein with nickase activity or Cas protein. The rejection clearly stated that one skilled in the art would have been motivated to select a nickase to reduce NHEJ as taught by Donohoue et al. While Donohoue et al. teaches polymerase in general, there are only two types RNA or DNA. Since the available polymerase choices are so limited one skilled would immediately envision utilizing either. None of Applicants arguments provide for an unexpected or unobvious effect.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 38-56 and 58-70 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-25 of U.S. Patent No. 10059940 in view of Zhong, Lee et al., Feng et al. , Lim et al. and Donohoue et al. Although the conflicting claims are not identical, they are not patentably distinct from each other because both sets of claims overlap in scope.
The instant application claims a conjugate of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas protein-guide RNA(s) complex, comprising (a) an IgRNA-Cas protein (RNP) complex and (b) one or more molecules selected from the group consisting of, antibodies, aptamers, and a nucleic acid template, wherein said one or more molecules are covalently linked to said IgRNA and said IgRNA comprises a crRNA or both a 3’-end truncated crRNA and a 5’-end truncated tracrRNA, wherein said IgRNA comprises at least one non-nucleotide linker located after its first nucleotide and before its last nucleotide.
Patent ‘940 claims an IgRNA for use as one component of a CRISPR-associated protein complex. The IgRNA as claimed includes a spacer of the same length as instantly claimed. As shown in claim 3 the IgRNA has internal nNt-linkers. Claimed is attaching peptides to the IgRNA. The IgRNA has a spacer of an HBV genome. Cationic lipids are claimed. Cell penetrating peptides are claimed. Recombinant engineered CAS9 protein is claimed. nNt-linkers are claimed which read on the instant claims.
Patent ‘940 does not claim one or more molecules of antibodies, aptamers or a nucleic acid template covalently bound to the IgRNA or other limitations of the dependent claims. However, these deficiencies are cured by Zhong, Lee et al., Lim et al. and Donohoue et al.
The teachings of Zhong, Lee et al., Feng et al. Lim et al. and Donohoue et al. are set forth above.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Patent ‘940, Zhong et al., Lee et al., Feng et al., Lim et al. and Donohoue et al. and conjugate (i.e. covalently link) the donor DNA to the crRNA of Patent ‘940 One skilled in the art would have been motivated to form this conjugate as it has been demonstrated that this allows for better transfection than unconjugated as taught by Lee et al.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Patent ‘940, Zhong et al., Lee et al., Feng et al., Lim et al. and Donohoue et al. and utilize a mutated Cas9 where at least two cysteine residues are introduced. One skilled in the art would have been motivated to add cysteine residues in order to provide conjugation sites as taught by Lim et al. Since this type of mutation, i.e. residues which can be mutated which retain activity, is well known as taught by Lim et al. and Donohoue et al. and Zhong teaches the use of cysteine for conjugation there is a reasonable expectation of success. Rendering claims 52 obvious.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Patent ‘940, Zhong et al., Lee et al., Feng et al., Lim et al. and Donohoue et al. and utilize known conjugation groups such as PEG and effector domains (e.g., a transcription activator). One skilled in the art would have been motivated to utilize these types of conjugation groups as Lim et al. teaches that effector domains have yielded transformative technologies and PEG chain may reduce the immunogenicity of the protein. Rendering claim 53 obvious.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Patent ‘940, Zhong et al., Lee et al., Feng et al., Lim et al. and Donohoue et al. and utilize a fusion protein. One skilled in the art would have been motivated to utilize a fusion protein as both Lim et al. and Donohoue et al. teach the use of fusion proteins to provide additional activities. One skilled in the art would have been motivated to utilize a nickase in order to reduce NHEJ (non-homologous end joining) as taught by Donohoue et al. One skilled in the art would have been motivated to utilize a DNA polymerase in order to provide for polymerase activity as suggested by Donohoue et al. Rending claims 58-60, 63, 67-69 obvious.
Regarding claim 39, as shown in claim 1 of Zhong, examples of nNt-linkers include:
PNG
media_image2.png
222
574
media_image2.png
Greyscale
which contains an M-1 core, two L wherein one L is L1 with n=6, the other L comprises L4 wherein n is 3 and m is 4, Nuc-2, Nuc-3.
Regarding claim 40, Patent ‘940 teaches CPP. Zhong teaches cell-penetrating peptide (CPP) is conjugated to a purified recombinant Cas9 protein (with appended Cys (i.e. cysteine) residue at the C terminus) (paragraph 0127). This reads on a non-PEG polymer or a peptide being covalently bound to the Cas protein.
Regarding claim 41, as shown in claim 1 of Zhong, the crgRNA comprises nucleotides modified at sugar moieties which includes at least II-6, II-4, II-5, II-7, II-8 and II-3.
Regarding claim 42, as shown in claim 5 of Zhong, the IgRNA comprises a spacer in crgRNA which is 12 to 20 nt in HIV genomes, of which each thymine is replaced with uracil.
Regarding claim 43, Patent ‘940 claims HBV genome, as shown in claim 6 of Zhong, The spacer can be 12 to 20 nt in HBV genomes in which each thymine is replaced with uracil.
Regarding claim 44, as shown in claim 7 of Zhong, the spacer in the crgRNA is 12 to 20 nt in HSV genomes of which each thymine is replaced with uracil.
Regarding claim 45, as shown in claim 8 of Zhong, the spacer in the crgRNA is 12 to 20 nt in EBV genomes, of which each thymine is replaced with uracil.
Regarding claim 46, claim 12 of Zhong indicates that there can be mixtures of IgRNAs with various spacers at different loci of viral genomes. The spacer RNA oligomers have the same sequences as the sense strands of the genomes (see paragraphs 0043-0046).
Regarding claims 47 and 50, Lee et al. teaches that donor DNA can be conjugated wherein this donor DNA contains the desired edit. Lee et al. also teaches that extending the guide RNA by adding aptamer sequences can enable researchers to specifically activate the genes that have been edited. Therefore the use of aptamers and donor DNA which reads on “two sequences” would allow for editing as well as activation of the genes.
Regarding claim 48-49, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Patent ‘940, Zhong et al., Lee et al., Feng et al., Lim et al. and Donohoue et al. and utilize a stop codon and cis-acting transcription control elements in the donor nucleotide sequence. One skilled in the art would have been motivated to utilize cis-acting transcription control elements in order to regulate transcription as taught by Feng et al. One skilled in the art would have been motivated to utilize stop codons in order to prevent the restoration of expression by NHEJ-mediated repair as taught by Feng et al.
Regarding claim 51, Zhong teaches a recombinant Cas9 protein (example 7; claim 10) which is a recombinant engineered class 2 endonuclease (paragraph 0061).
Regarding claims 54 and 70, Zhong teaches cell-penetrating peptide (CPP) is conjugated to a purified recombinant Cas9 protein (with appended Cys (i.e. cysteine) residue at the C terminus) (paragraph 0127). This results in the complex comprising covalently linked molecules for targeted cellular delivery.
Regarding claim 55 and 61-62, Zhong teaches that Cas9 cleaves leaving a blunt end DNA double strand break (paragraph 0061). Taught is the use of the CRISPR-Cas9-IGRNA systems for gene editing (paragraph 0107). As taught by Lee et al., the gRNA was designed to cut the BFP gene and donor DNA was designed to covert the BFP gene into the GFP gene (see Figure 4; page 8). Therefore, the combination of prior art suggest the claimed method of target gene editing.
Regarding claim 56, this claim is directed to the conjugate. The wherein clause is interpreted as what happens to the complex in cells when the complex is encoded by a viral plasmid. Zhong teaches that the Cas9 protein and RNA have been introduced by plasmid transfection (paragraph 0010). Since the same complex is suggested by the cited prior art, the same self-assembly would be present.
Regarding claim 64, Lee et al. teaches the donor DNA includes a short single stranded DNA (Figure 2a; page 4).
Regarding claim 69, Zhong teaches IgRNA incubated with purified Cas9protein and then complexed with cationic lipids (paragraph 0125).
Claims 38-56 and 58-70 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 27-42 of copending Application No. 17194312 (USPGPUB No. 20210222165) in view of Zhong, Lee et al., Lim et al., Feng et al. and Donohoue et al. Although the conflicting claims are not identical, they are not patentably distinct from each other because both sets of claims overlap in scope.
This is a provisional nonstatutory double patenting rejection.
Copending ‘312 claims a cell comprising at least one guide RNA wherein said guide RNA comprises at least one internal non-nucleotide linker (aka an IgRNA). The guide RNA comprises a spacer sequence targeting a viral DNA, HBV DNA, HIV DNA, herpesvirus DNA. The cell further comprises a Cas protein complexed with the guide. Claimed is a Cas protein conjugate. Lipids and CPP is claimed.
Copending ‘312 does not claim one or more molecules of antibodies, aptamers or a nucleic acid template covalently bound to the IgRNA or other limitations of the dependent claims. However, these deficiencies are cured by Zhong, Lee et al., Lim et al. and Donohoue et al.
The teachings of Zhong, Lee et al., Lim et al., Feng et al. and Donohoue et al. are set forth above.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Copending ‘312, Zhong et al., Lee et al., Feng et al., Lim et al. and Donohoue et al. and conjugate (i.e. covalently link) the donor DNA to the gRNA of Copending ‘312. One skilled in the art would have been motivated to form this conjugate as it has been demonstrated that this allows for better transfection than unconjugated as taught by Lee et al.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Copending ‘312, Zhong et al., Lee et al., Lim et al., Feng et al. and Donohoue et al. and utilize a mutated Cas9 where at least two cysteine residues are introduced. One skilled in the art would have been motivated to add cysteine residues in order to provide conjugation sites as taught by Lim et al. Since this type of mutation, i.e. residues which can be mutated which retain activity, is well known as taught by Lim et al. and Donohoue et al. and Zhong teaches the use of cysteine for conjugation there is a reasonable expectation of success. Rendering claims 52 obvious.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Copending ‘312, Zhong et al., Lee et al., Lim et al., Feng et al. and Donohoue et al. and utilize known conjugation groups such as PEG and effector domains (e.g., a transcription activator). One skilled in the art would have been motivated to utilize these types of conjugation groups as Lim et al. teaches that effector domains have yielded transformative technologies and PEG chain may reduce the immunogenicity of the protein. Rendering claim 53 obvious.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Copending ‘312, Zhong et al., Lee et al., Feng et al., Lim et al. and Donohoue et al. and utilize a fusion protein. One skilled in the art would have been motivated to utilize a fusion protein as both Lim et al. and Donohoue et al. teach the use of fusion proteins to provide additional activities. One skilled in the art would have been motivated to utilize a nickase in order to reduce NHEJ (non-homologous end joining) as taught by Donohoue et al. One skilled in the art would have been motivated to utilize a DNA polymerase in order to provide for polymerase activity as suggested by Donohoue et al. Rending claims 58-60, 63, 67-69 obvious.
Regarding claim 40, Copending ‘312 teaches CPP. Zhong teaches cell-penetrating peptide (CPP) is conjugated to a purified recombinant Cas9 protein (with appended Cys (i.e. cysteine) residue at the C terminus) (paragraph 0127). This reads on a non-PEG polymer or a peptide being covalently bound to the Cas protein.
Regarding claim 41, as shown in claim 1, the crgRNA comprises nucleotides modified at sugar moieties which includes at least II-6, II-4, II-5, II-7, II-8 and II-3.
Regarding claim 42, as shown in claim 5 of Zhong, the IgRNA comprises a spacer in crgRNA which is 12 to 20 nt in HIV genomes, of which each thymine is replaced with uracil.
Regarding claim 43, Copending ‘312 claims HBV genome, as shown in claim 6 of Zhong, The spacer can be 12 to 20 nt in HBV genomes in which each thymine is replaced with uracil.
Regarding claim 44, as shown in claim 7 of Zhong, the spacer in the crgRNA is 12 to 20 nt in HSV genomes of which each thymine is replaced with uracil.
Regarding claim 45, as shown in claim 8 of Zhong, the spacer in the crgRNA is 12 to 20 nt in EBV genomes, of which each thymine is replaced with uracil.
Regarding claim 46, claim 12 of Zhong indicates that there can be mixtures of IgRNAs with various spacers at different loci of viral genomes. The spacer RNA oligomers have the same sequences as the sense strands of the genomes (see paragraphs 0043-0046).
Regarding claims 47 and 50, Lee et al. teaches that donor DNA can be conjugated wherein this donor DNA contains the desired edit. Lee et al. also teaches that extending the guide RNA by adding aptamer sequences can enable researchers to specifically activate the genes that have been edited. Therefore the use of aptamers and donor DNA which reads on “two sequences” would allow for editing as well as activation of the genes.
Regarding claim 48-49, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Copending ‘312, Zhong et al., Lee et al., Feng et al., Lim et al. and Donohoue et al. and utilize a stop codon and cis-acting transcription control elements in the donor nucleotide sequence. One skilled in the art would have been motivated to utilize cis-acting transcription control elements in order to regulate transcription as taught by Feng et al. One skilled in the art would have been motivated to utilize stop codons in order to prevent the restoration of expression by NHEJ-mediated repair as taught by Feng et al.
Regarding claim 51, Zhong teaches a recombinant Cas9 protein (example 7; claim 10) which is a recombinant engineered class 2 endonuclease (paragraph 0061).
Regarding claims 54 and 70, Zhong teaches cell-penetrating peptide (CPP) is conjugated to a purified recombinant Cas9 protein (with appended Cys (i.e. cysteine) residue at the C terminus) (paragraph 0127). This results in the complex comprising covalently linked molecules for targeted cellular delivery.
Regarding claim 55 and 61-62, Zhong teaches that Cas9 cleaves leaving a blunt end DNA double strand break (paragraph 0061). Taught is the use of the CRISPR-Cas9-IGRNA systems for gene editing (paragraph 0107). As taught by Lee et al., the gRNA was designed to cut the BFP gene and donor DNA was designed to covert the BFP gene into the GFP gene (see Figure 4; page 8). Therefore, the combination of prior art suggest the claimed method of target gene editing.
Regarding claim 56, this claim is directed to the conjugate. The wherein clause is interpreted as what happens to the complex in cells when the complex is encoded by a viral plasmid. Zhong teaches that the Cas9 protein and RNA have been introduced by plasmid transfection (paragraph 0010). Since the same complex is suggested by the cited prior art, the same self-assembly would be present.
Regarding claim 64, Lee et al. teaches the donor DNA includes a short single stranded DNA (Figure 2a; page 4).
Regarding claim 69, Zhong teaches IgRNA incubated with purified Cas9 protein and then complexed with cationic lipids (paragraph 0125).
Claims 38-56 and 58-70 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-31 of copending Application No. 18218659 (USPGPUB No. 20240076661) in view of Zhong, Lee et al., Lim et al. and Donohoue et al. Although the conflicting claims are not identical, they are not patentably distinct from each other because both sets of claims overlap in scope.
This is a provisional nonstatutory double patenting rejection.
Copending ‘659 claims a method of directing a polypeptide to a target nucleic acid using a guide RNA, comprising a) delivering said guide RNA to a targeted cell, and b) binding of said guide RNA to said target nucleic acid in said cell, wherein said polypeptide is: (i) expressed in said cell and the expression is optionally under control, or (ii) separately delivered to said cell either before or after step a), or (iii) co-delivered with said guide RNA in step a), wherein said polypeptide and said guide RNA can be a mixture or their preformed ribonucleoprotein, or (iv) delivered as its encoding nucleic acid either separately or together with said guide RNA, wherein said guide RNA comprises a target specific sequence which recognizes said target nucleic acid by base pairing and a scaffold sequence which binds said polypeptide, and wherein said guide RNA comprises one or more internal non-nucleotide linkers,” “wherein said guide RNA is associated through a covalent bond or base pairings with one or more DNA repair templates selected from the group consisting of ssDNA, RNA, and dsDNA, wherein said ssDNA and RNA can be either linear or circular. CRISPR-associated protein is claimed. DNA polymerase is claimed. A viral vector is claimed. Stop codons and transcription cis-regulator elements are claimed. Covalently linking the guide RNA with molecules such as aptamers, antibodies and oligonucleotides is claimed.
Copending ‘659 does not claim that the polypeptide is a CAS polypeptide which form a complex with the guide RNA. However, these deficiencies are cured by Zhong, Lee et al., Lim et al. and Donohoue et al.
The teachings of Zhong, Lee et al., Lim et al. and Donohoue et al. are set forth above.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Copending ‘659, Zhong et al., Lee et al., Lim et al. and Donohoue et al. and conjugate (i.e. covalently link) the donor DNA to the gRNA of Copending ‘659. One skilled in the art would have been motivated to form this conjugate as it has been demonstrated that this allows for better transfection than unconjugated as taught by Lee et al.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Copending ‘659, Zhong et al., Lee et al., Lim et al. and Donohoue et al. and utilize a mutated Cas9 where at least two cysteine residues are introduced. One skilled in the art would have been motivated to add cysteine residues in order to provide conjugation sites as taught by Lim et al. Since this type of mutation, i.e. residues which can be mutated which retain activity, is well known as taught by Lim et al. and Donohoue et al. and Zhong teaches the use of cysteine for conjugation there is a reasonable expectation of success. Rendering claims 52 obvious.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Copending ‘659, Zhong et al., Lee et al., Lim et al. and Donohoue et al. and utilize known conjugation groups such as PEG and effector domains (e.g., a transcription activator). One skilled in the art would have been motivated to utilize these types of conjugation groups as Lim et al. teaches that effector domains have yielded transformative technologies and PEG chain may reduce the immunogenicity of the protein. Rendering claim 53 obvious.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Copending ‘659, Zhong et al., Lee et al., Lim et al. and Donohoue et al. and utilize a fusion protein. One skilled in the art would have been motivated to utilize a fusion protein as both Lim et al. and Donohoue et al. teach the use of fusion proteins to provide additional activities. One skilled in the art would have been motivated to utilize a nickase in order to reduce NHEJ (non-homologous end joining) as taught by Donohoue et al. One skilled in the art would have been motivated to utilize a DNA polymerase in order to provide for polymerase activity as suggested by Donohoue et al. Rending claims 58-60, 63, 67-69 obvious.
Regarding claim 40, Zhong teaches cell-penetrating peptide (CPP) is conjugated to a purified recombinant Cas9 protein (with appended Cys (i.e. cysteine) residue at the C terminus) (paragraph 0127). This reads on a non-PEG polymer or a peptide being covalently bound to the Cas protein.
Regarding claim 41, as shown in claim 1 of Zhong, the crgRNA comprises nucleotides modified at sugar moieties which includes at least II-6, II-4, II-5, II-7, II-8 and II-3.
Regarding claim 42, as shown in claim 5 of Zhong, the IgRNA comprises a spacer in crgRNA which is 12 to 20 nt in HIV genomes, of which each thymine is replaced with uracil.
Regarding claim 43, as shown in claim 6 of Zhong, The spacer can be 12 to 20 nt in HBV genomes in which each thymine is replaced with uracil.
Regarding claim 44, as shown in claim 7 of Zhong, the spacer in the crgRNA is 12 to 20 nt in HSV genomes of which each thymine is replaced with uracil.
Regarding claim 45, as shown in claim 8 of Zhong, the spacer in the crgRNA is 12 to 20 nt in EBV genomes, of which each thymine is replaced with uracil.
Regarding claim 46, claim 12 of Zhong indicates that there can be mixtures of IgRNAs with various spacers at different loci of viral genomes. The spacer RNA oligomers have the same sequences as the sense strands of the genomes (see paragraphs 0043-0046).
Regarding claims 47 and 50, Lee et al. teaches that donor DNA can be conjugated wherein this donor DNA contains the desired edit. Lee et al. also teaches that extending the guide RNA by adding aptamer sequences can enable researchers to specifically activate the genes that have been edited. Therefore the use of aptamers and donor DNA which reads on “two sequences” would allow for editing as well as activation of the genes.
Regarding claim 48-49, Copending ‘659 claims one or more stop codons and transcription cis-regulatory elements (claims 12-13).
Regarding claim 51, Zhong teaches a recombinant Cas9 protein (example 7; claim 10) which is a recombinant engineered class 2 endonuclease (paragraph 0061).
Regarding claims 54 and 70, Zhong teaches cell-penetrating peptide (CPP) is conjugated to a purified recombinant Cas9 protein (with appended Cys (i.e. cysteine) residue at the C terminus) (paragraph 0127). This results in the complex comprising covalently linked molecules for targeted cellular delivery.
Regarding claim 55 and 61-62, Zhong teaches that Cas9 cleaves leaving a blunt end DNA double strand break (paragraph 0061). Taught is the use of the CRISPR-Cas9-IGRNA systems for gene editing (paragraph 0107). As taught by Lee et al., the gRNA was designed to cut the BFP gene and donor DNA was designed to covert the BFP gene into the GFP gene (see Figure 4; page 8). Therefore, the combination of prior art suggest the claimed method of target gene editing.
Regarding claim 56, this claim is directed to the conjugate. The wherein clause is interpreted as what happens to the complex in cells when the complex is encoded by a viral plasmid. Zhong teaches that the Cas9 protein and RNA have been introduced by plasmid transfection (paragraph 0010). Since the same complex is suggested by the cited prior art, the same self-assembly would be present.
Regarding claim 64, Lee et al. teaches the donor DNA includes a short single stranded DNA (Figure 2a; page 4).
Regarding claim 69, Zhong teaches IgRNA incubated with purified Cas9 protein and then complexed with cationic lipids (paragraph 0125).
Claims 38-56 and 58-70 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-29 of copending Application No. 18628766 (USPGPUB No. 202500271151) in view of Zhong, Lee et al., Lim et al. and Donohoue et al. Although the conflicting claims are not identical, they are not patentably distinct from each other because both sets of claims overlap in scope.
This is a provisional nonstatutory double patenting rejection.
Copending ‘766 claims a conjugate of a CRISPR-Cas protein-guide RNA complex comprising a gRNA-Cas protein (RNP) complex, and a DNA template chemically linked to the gRNA comprising at least one 5-methylcytosine to form a gRNA-DNA conjugate, wherein the conjugate is covalently linked with one or more molecules selected from the group consisting of PEGs, non-PEG polymers, ligands of cellular receptors, lipids, oligonucleotides, polysaccharides, glycans, peptides, aptamers and/or antibodies to form a gRNA conjugate, and the said more molecules can be the same or different, and comprising a gRNA and one or two conjugated ssDNA templates, which comprises two sequences overlapping with the target strand or with the non-target strand of the DNA duplex to be edited and a gene editing sequence, and the said two sequences flanking the said gene editing sequence are optionally chemically modified, wherein said conjugation is by a non-nucleotide linker or an oligonucleotide linker. DNA polymerase are claimed. DNA methyltransferase is claimed.
Copending ‘766 does not claim the specific limitations of the dependent claims. However, these deficiencies are cured by Zhong, Lee et al., Lim et al. and Donohoue et al.
The teachings of Zhong, Lee et al., Lim et al. and Donohoue et al. are set forth above.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Copending ‘766, Zhong et al., Lee et al., Lim et al. and Donohoue et al. and utilize a mutated Cas9 where at least two cysteine residues are introduced. One skilled in the art would have been motivated to add cysteine residues in order to provide conjugation sites as taught by Lim et al. Since this type of mutation, i.e. residues which can be mutated which retain activity, is well known as taught by Lim et al. and Donohoue et al. and Zhong teaches the use of cysteine for conjugation there is a reasonable expectation of success. Rendering claims 52 obvious.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Copending ‘766, Zhong et al., Lee et al., Lim et al. and Donohoue et al. and utilize known conjugation groups such as PEG and effector domains (e.g., a transcription activator). One skilled in the art would have been motivated to utilize these types of conjugation groups as Lim et al. teaches that effector domains have yielded transformative technologies and PEG chain may reduce the immunogenicity of the protein. Rendering claim 53 obvious.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the teachings of Copending ‘766, Zhong et al., Lee et al., Lim et al. and Donohoue et al. and utilize a fusion protein. One skilled in the art would have been motivated to utilize a fusion protein as both Lim et al. and Donohoue et al. teach the use of fusion proteins to provide additional activities. One skilled in the art would have been motivated to utilize a nickase in order to reduce NHEJ (non-homologous end joining) as taught by Donohoue et al. One skilled in the art would have been motivated to utilize a DNA polymerase in order to provide for polymerase activity as suggested by Donohoue et al. Rending claims 58-60, 63, 67-69 obvious.
Regarding claim 40, Zhong teaches cell-penetrating peptide (CPP) is conjugated to a purified recombinant Cas9 protein (with appended Cys (i.e. cysteine) residue at the C terminus) (paragraph 0127). This reads on a non-PEG polymer or a peptide being covalently bound to the Cas protein.
Regarding claim 41, as shown in claim 1, the crgRNA comprises nucleotides modified at sugar moieties which includes at least II-6, II-4, II-5, II-7, II-8 and II-3.
Regarding claim 42, copending ‘766 claims a spacer which is 12 to 20 nt in HIV genomes, of which each thymine is replaced with uracil.
Regarding claim 43, Copending ‘766 claims HBV genome, and that spacer can be 12 to 20 nt in HBV genomes in which each thymine is replaced with uracil.
Regarding claim 44, copending ‘766 claims the spacer is 12 to 20 nt in HSV genomes of which each thymine is replaced with uracil.
Regarding claim 45, copending ‘766 claims a spacer that is 12 to 20 nt in EBV genomes, of which each thymine is replaced with uracil.
Regarding claim 46, claim 12 indicates that there can be mixtures of IgRNAs with various spacers at different loci of viral genomes. The spacer RNA oligomers have the same sequences as the sense strands of the genomes (see paragraphs 0043-0046).
Regarding claims 47 and 50, Lee et al. teaches that donor DNA can be conjugated wherein this donor DNA contains the desired edit. Lee et al. also teaches that extending the guide RNA by adding aptamer sequences can enable researchers to specifically activate the genes that have been edited. Therefore the use of aptamers and donor DNA which reads on “two sequences” would allow for editing as well as activation of the genes.
Regarding claim 48-49, copending ‘766 claim one or more transcription cis-regulatory elements and said more elements comprises repetitive sequence separated by absent or more nucleotides in between or different sequences separated by absent or more nucleotides in between
Regarding claim 51, Zhong teaches a recombinant Cas9 protein (example 7; claim 10) which is a recombinant engineered class 2 endonuclease (paragraph 0061).
Regarding claims 54 and 70, Zhong teaches cell-penetrating peptide (CPP) is conjugated to a purified recombinant Cas9 protein (with appended Cys (i.e. cysteine) residue at the C terminus) (paragraph 0127). This results in the complex comprising covalently linked molecules for targeted cellular delivery.
Regarding claim 55 and 61-62, Zhong teaches that Cas9 cleaves leaving a blunt end DNA double strand break (paragraph 0061). Taught is the use of the CRISPR-Cas9-IGRNA systems for gene editing (paragraph 0107). As taught by Lee et al., the gRNA was designed to cut the BFP gene and donor DNA was designed to covert the BFP gene into the GFP gene (see Figure 4; page 8). Therefore, the combination of prior art suggest the claimed method of target gene editing.
Regarding claim 56, this claim is directed to the conjugate. The wherein clause is interpreted as what happens to the complex in cells when the complex is encoded by a viral plasmid. Zhong teaches that the Cas9 protein and RNA have been introduced by plasmid transfection (paragraph 0010). Since the same complex is suggested by the cited prior art, the same self-assembly would be present.
Regarding claim 64, Lee et al. teaches the donor DNA includes a short single stranded DNA (Figure 2a; page 4).
Regarding claim 69, Zhong teaches IgRNA incubated with purified Cas9 protein and then complexed with cationic lipids (paragraph 0125).
Response to Arguments
Applicant's arguments filed November 17 2025 are acknowledged.
Applicant argues that the claims are novel an non-obvious for the reasons set forth in the response to the 103 rejection above. Applicants indicate that terminal disclaimers will be filed upon indication of allowable subject matter.
Regarding Applicant’s arguments, the arguments are not persuasive for the same reasons set forth above. Acknowledgement is made of Applicant indication to file a terminal disclaimer once allowable subject matter has been identified.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ABIGAIL VANHORN whose telephone number is (571)270-3502. The examiner can normally be reached M-Th 6 am-4 pm EST.
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, Neil Hammell can be reached on 571-270-5919. 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.
/ABIGAIL VANHORN/Primary Examiner, Art Unit 1636