DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Status of Application/Amendment/Claims
This Office Action is in response to the communications filed on May 26th, 2026.
Currently, claims 1, 3-8, 10-11, 13, 15, 17-18, 20, 22-28, and 30-32 are pending in the instant application. Claims 17, 20, and 31-32 are withdrawn from further consideration as being drawn to a nonelected invention, there being no allowable generic or linking claim. Claim 2, 9, 12, 14, 16, 19, 21, and 29 have been canceled. Accordingly, claims 1, 3-8, 10-11, 13, 15, 17-18, 20, 22-28, and 30 are under examination on the merits in the instant application.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 04/16/2026 and 06/02/2026 were filed after the mailing date of the non-final office action on 02/25/2026. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Response to Amendments
Withdrawn objections and rejections
Any rejections and/or objections not repeated in this Office action are hereby withdrawn.
New Rejections Necessitated by Amendment
CLAIM INTERPRETATION
Amended independent claim 1 recites a guide RNA (gRNA) targeting a fragment of a mutant NF1 gene “between exon 30 and exon 31,” which is further limiting than the original claim 1 wherein the gRNA could target any fragment of a mutant NF1 gene. For the purpose of clarity and establishing a consistent baseline for the record, the examiner notes a duplication and/or overlapping of terminology regarding NF1 gene structure.
In classic nomenclature, the NF1 gene is frequently described as containing 57 constitutional exons, with an alternatively spliced exon located within the GAP-related domain (GRD) designated as “Exon 23a.” Conversely, modern genomic assemblies (such as NCBI Reference Sequence: NG_009018.1) count all exons sequentially, cataloging a total of 58 exons, wherein classic alternative exon 23a corresponds exactly to exon 31.
The specification’s reference to a mutant NF1 comprising “exons 1-57” and targeting “between exon 30 and exon 31” structurally aligns with the intronic regions flanking the alternative exon 31. Accordingly, the examiner interprets the claimed target zone “between exon 30 and exon 31” as structurally equivalent to the intronic sequences immediately adjacent to alternative exon 31, which corresponds to intron 30 and intron 31 of the NCBI RefSeq NG_009018.1. See screenshot of genome browser view displaying BLAT of gRNA SEQ ID NOs: 71-81 and exon 31 from NCBI RefSeq NG_009018.1:
PNG
media_image1.png
208
1172
media_image1.png
Greyscale
In response to the species election requirement mailed 07/10/2025, applicant elected without traverse gRNA SEQ ID NO: 74. Because introns 30 and 31 of the NCBI RefSeq NG_009018.1 represent the same structural region designated by the applicant’s recitation of “between exon 30 and exon 31” in the 57 exon NF1 framework, the applicant’s amendment to claim 1 does not exclude the elected species. The originally elected species is encompassed by the amended claim scope, and the prior art rejections applied below maintain their focus on the merits of claim 1 as reading on elected species SEQ ID NO: 74.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1, 15, 18, 22-25, 28, and 30 are rejected under 35 U.S.C. 103 as being unpatentable over Belmonte et. al., (US-20190225991-A1) in view of Moutal et. al., (PAIN 158(12): 2301-2319; published April 2017), Bai et. al., (Gen. Ther. 26(6): 277-286; published June 2019), and Bednarski et. al., (PLoS One.; 11(8):e0161072; published Aug 15, 2016), and in further view of Li et. al., (Plant Genome Editing with CRISPR Systems. Methods in Molecular Biology, vol 1917, pp 285–296; published Jan 5, 2019) and Ran et. al., (Nature 520: 186-191; published April 9, 2015) as evidenced by NCBI RefSeq (NG_009018.1).
Regarding claim 1, Belmonte teaches a Homology-Independent Targeted Integration (HITI) CRISPR/Cas-based genome editing system comprising a polynucleotide encoding a guide RNA targeting a fragment of a mutant, a polynucleotide encoding a Cas protein, and a polynucleotide encoding donor sequence comprising a fragment of a wild-type gene (claim 58: “a composition comprising a targeting construct comprising a DNA sequence homologous to a wild- type gene or fragment thereof and a targeting sequence, a complementary strand oligonucleotide homologous to the targeting sequence, and a nuclease, wherein the targeting sequence is recognized by the nuclease “for use in treating a genetic disease,” wherein claim 60: “the nuclease is selected from a CRISPR nuclease,” wherein claim 61: “the CRISPR nuclease is…Cas9…,” wherein claim 67: “the genetic disease is…Neurofibromatosis” (NF1)).
Regarding claim 15, Belmonte teaches using the HITI system with a donor sequence comprising one or more exons. Specifically, Belmonte teaches restoring the coding sequence of the Mertk gene by knock-in of wild-type Exon 2 within intron 1 of a mutated Mertk gene while leaving the mutated exon 2 in the endogenous locus.
Regarding claim 23, Belmonte teaches a viral vector comprising the HITI CRISPR/Cas-based genome editing system (claim 71: “…wherein the targeting construct, the complementary strand oligonucleotide, and a polynucleotide encoding the nuclease are contained in a non - viral or viral vector”).
Regarding claim 24, Belmonte teaches that the viral vector comprising the HITI CRISPR/Cas-based genome editing system is an Adeno-associated virus (AAV) vector (claim 72: “wherein the viral vector is … an adeno-associated virus” (AAV)).
Regarding claim 25, Belmonte teaches that the AAV vector comprising the HITI CRISPR/Cas-based genome editing system is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV-10, AAV-11, AAV-12, AAV-13, or AAVrh.74 vector ([ 0124 ] “all of AAVs were packaged with serotype 8 or serotype 9”).
Regarding claim 28, Belmonte teaches a cell comprising a HITI CRISPR/Cas-based genome editing system (Claim 70: “wherein the composition comprises a non-dividing cell, and claim 75: “wherein the exogenous DNA sequence is integrated into the genome of the non-dividing cell by a virus and a nuclease).
Regarding claim 30, Belmonte teaches a kit comprising a HITI CRISPR/Cas-based genome editing system (claim 82: “A kit comprising a targeting construct comprising an exogenous DNA sequence and at least one targeting sequence, a complementary strand oligonucleotide homologous to the targeting sequence, a nuclease, and instructions for making genetic alterations to non-dividing cells, wherein the targeting sequence is recognized by the nuclease”).
While Belmonte expressly teaches that HITI CRISPR/Cas-based genome editing system is applicable to gene therapy for Neurofibromatosis, Belmonte does not expressly teach that: 1) the mutations in the NF1 gene are responsible for the onset of Neurofibromatosis, 2) the guide RNA of the CRISPR/Cas-based genome editing system targets a fragment of a mutant NF1 gene between exon 30 and exon 31, and 3) that the donor sequence comprises a fragment of a wild-type NF1 gene.
Moutal teaches that “Neurofibromatosis type 1 (NF1) is a rare autosomal dominant disease linked to mutations of the NF1 gene” (see abstract). Moutal teaches that the NF1 gene comprises a variety of mutations throughout the coding region (i.e., in both a 5’ portion and a 3’ portion) (results 3.1: “…more than 1000 pathogenic variants (insertions, microdeletions, copy number alterations) have been reported in the Nf1 gene. Given this wide mutational spectrum and the complexity of genotype-phenotype associations, we selected the C-terminus as it is a region recurrently altered in NF1”). Moutal further teaches single guide RNAs targeting a fragment of the NF1 gene “our strategy was to target the neurofibromin (Nf1) gene using a single guide RNA (sgRNA)” (see methods 2.3).
While Moutal used the CRISPR/Cas system to generate a non-sense mutation (i.e., an early stop codon that results in a truncated protein), Moutal did not use the CRISPR/Cas system to insert a fragment of a wild-type NF1 gene with a gRNA targeting a fragment of a mutant NF1 gene between exon 30 and exon 31.
Among many examples in the art at the time of filing that strategically package domains of wild-type NF1 (i.e., fragments) such as the GRD domain into gene therapy vectors (e.g., AAV vectors), Bai et. el., teaches, “feasibility of using NF1-GRD and AAV for gene replacement therapy in NF1-associated tumors” (see title). Bai states that the results open “up a venue of gene replacement therapy in NF1-related tumors” (see abstract). Bai teaches “expression of various GRD constructs via gene delivery using a panel of adeno-associated virus (AAV) vectors” (see abstract and methods). Bai teaches that limitations of AAV such as the “packaging capacity of up to ~4.7 kb” restricts an artisan to select a fragment of the NF1 gene for packaging into AAV vectors because “NF1’s cDNA is 8.5Kb” which is “too large for AAV vectors” (see introduction). Therefore, Bai describes that “the NF1-GAP-related domain (NF1-GRD),” corresponding to exons 21-27a, is “a ~1kB small subunit of the gene,” and is presumably solely responsible for its tumor suppressive activity; thus, “making neurofibromatosis 1 uniquely suitable for AAV based gene delivery” (see introduction).
While by Bai teaches gene therapy for NF1 using a duel AAV vector system to deliver a wild-type NF1-GRD domain/fragment, Bai does not explicitly disclose the nuclease-mediated targeted integration of a partial wild-type cDNA, “superexon,” donor sequence into an endogenous mutant gene.
Bednarski teaches treating large genes with a wide mutational spectrum, which is precisely the problem identified by Moutal for NF1. Bednarski teaches using targeted nucleases to integrate a “super-exon” donor sequence into a mutated gene allowing “for genetic correction of all mutations downstream of the insertion site” (Introduction: “The goal of this study was the development of a targeted genome engineering approach that allows for the genetic correction of the majority of described CF causing mutations…We provide proof that the targeted integration of a super-exon…reinstated expression of functional CFTR that in turn corrected transepithelial characteristics of these cells…our results demonstrate that a super-exon strategy can be applied to correct the majority of CFTR mutations”). Specifically, Bednarski designed the donor construct to replace a “3’ portion” of the CFTR coding sequence (Abstract: “this study proves that the targeted integration of a large super-exon in CFTR…leads to functional correction of CFTR, suggesting that this strategy can be used to functionally correct all CFTR mutations located downstream of the 5’ end of exon 11”). Bednarski further teaches that “a major advantage of locus-specific gene correction over conventional gene therapy is that physiological regulation of gene expression by the endogenous promoter is retained” (Discussion). Bednarski further teaches that the “ZFN based insertion strategy resulted in monoallelic correction of the ΔF508 locus, which corresponds to a heterozygous CFTRwt/CFTRΔF508 locus,” and that the “result is comparable to the outcome of a recent CRISPR/Cas based gene editing approach, in which mainly single allele targeting was observed” (Discussion). Bednarski further discusses the limitation of off target integration using ZNF nucleases (Discussion: “On the other hand, we observed a substantial amount of random integration of donor DNA, even in the presence of ZFNs,” and “we cannot exclude that low specificity of the used ZFNs contributed to the high frequency of improper recombination”). However, Bednarski further teaches that “Alternative designer nucleases with proven high specificity, such as TALENs or second generation CRISPR/Cas systems [53–57], can be used instead” (Discussion).
Regarding claim 18, while Moutal teaches targeting NF1 with gRNA via CRISPR/Cas gene editing and Belmonte teaches the HITI system is suited for Neurofibromatosis gene therapy, neither Belmonte, Moutal, Bai, or Bednarski teach the elected gRNA species, SEQ ID NO: 74 (which targets intron 31 of the NF1 gene sequence as annotated in NCBI RefSeq NG_009018.1), nor do they disclose any of the gRNAs, SEQ ID NOs: 71-81, as claimed in the instant application.
Regarding claim 22, while Belmonte and Moutal teach the use of SpCas9, neither do they nor do Bai and Bednarski teach the use of SaCas9, and more specifically, none of the aforementioned teach SEQ ID NO:20 or SEQ ID NO:21 as claimed in the instant application.
Li teaches “a protocol for obtaining intron-targeted site-specific gene replacements…via the NHEJ pathway using the CRISPR-Cas9 system (Fig. 3), replacing a fragment at the desired locus…” (Introduction). Li’s methods teach an artisan to “identify potential Cas9 target sites in introns adjacent to the location of the target fragment (see Note 4).” Wherein note 4 teaches “when the DSBs created by CRISPR-Cas9 are repaired through the NHEJ pathway, small indels can be created at the break sites. So, target sites should be chosen in introns, and they should be far from the 5′ and 3′ splice sites to avoid interference with the splicing machinery. Target sites can be selected manually by looking for…a PAM…” (Methods and Note 4).
Ran teaches that “the restrictive cargo size (~4.5 kb, excluding the inverted terminal repeats) of AAV presents an “obstacle for packaging the commonly used Streptococcus pyrogenes Cas9 (SpCas9, ~4.2 kb) and its single guide RNA (sgRNA) in a single vector,” while “smaller Cas9 enzymes” especially Staphylococcus aureus (SaCas9), provide “efficient delivery by AAV” because the “small size of SaCas9 enable packaging of” necessary elements for genome editing “into a single AAV vector within the 4.5-kb packaging limit” (see pages 186 and 189).
Ran teaches a CRISPR/Cas-based system wherein the Cas protein is SaCas9 and comprises an amino acid sequence of SEQ ID NO:20 or of SEQ ID NO:21 (Supplementary Information: “Staphylococcus aureus Cas9”). While the S. aureus sequence taught by Ran is a polynucleotide mRNA sequence and not an amino acid sequence as are SEQ ID NOs: 20-21 of the instant application, when the mRNA sequence taught by Ran is translate into its corresponding amino acid sequence, the resulting polypeptide aligns 100% with the claimed and elected species, SEQ ID NO:21, in the instant case, see below for alignment results.
PNG
media_image2.png
112
628
media_image2.png
Greyscale
Ran teaches that “Staphylococcus aureus (SaCas9) produced indels with efficiency comparable to those of SpCas9” and that “SaCas9 achieves the highest editing efficiency in mammalian cells, with guides between 21 and 23 nucleotides long,” wherein “SaCas9 cleaves genomic targets most efficiently with NNGRRT,” which is the SaCas9 PAM sequence (emphasis added, see page 187).
Ran demonstrates in vivo genome editing by incorporating “SaCas9 and its sgRNA into an AAV vector” or by packaging sgRNA into “AAV-SaCas9,” which “is able to mediate efficient and rapid editing” (See pages 189-190).
Ran illustrates a nucleotide sequence alignment between a 21-mer target preceded by NNGRRT PAM sequence and a 21-mer “SaCas9” guide RNA that is designed to be homologous to the target sequence, which is fused to a “tracrRNA” or “SaCas9 sgRNA scaffold,” which is 100% identical to SEO ID N0: 19 as described in the specification of the instant case (see Figure 2a copied below):
PNG
media_image3.png
262
466
media_image3.png
Greyscale
It would have been obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to provide a CRISPR/Cas-based genome editing system comprising a polynucleotide sequence encoding a gRNA that is homologous to a 20-21mer sequence preceded by the NNGRRT PAM sequences within intron 31 of human NF1 (e.g., the elected SEQ ID NO: 74), a polynucleotide sequence encoding a Cas protein, and a donor sequence comprising a fragment of a wild-type NF1 gene.
One of ordinary skill in the art would have been motivated to do so with a reasonable expectation of success in order to knock-in a functional NF1 partial cDNA via a mutation-agnostic “superexon” integration strategy as taught by Bednarski, thereby addressing a majority of the mutations documented in NF1 with a single therapy. Making a CRISPR/Cas-based genome editing system targeting NF1 modeled after the HITI system was an art-recognized goal for treating Neurofibromatosis 1 as taught by Belmonte. Designing the “HITI”-CRISPR/Cas-based genome editing system to knockin “a fragment of wild-type NF1” was an obvious design choice as evidenced by Bai, who taught that the ~8.5Kb NF1 cDNA is too large to fit in a single AAV vector and requires a “split” strategy. Therefore, this “split” strategy necessitates the design of a gRNA that targets a region of NF1 that would allow for a manageable “fragment of a wild-type NF1 gene” to be delivered as a donor sequence via commonly used delivery systems such as AAV. Further, one of ordinary skill in the art would have been motivated to target the introns 30 and 31 adjacent to alternative exon 31 (i.e., between exon 30 and exon 31 in the 57-exon structure/variant of NF1) because Bednarski teaches that a “superexon” (a partial cDNA) can rescue gene function by correcting “all mutations downstream of the insertion site,” and Li teaches that target sites should be chosen in introns” to “avoid interference with the splicing machinery” when “replacing a fragment at the desired locus.” Therefore, intron 31 (i.e., a region between exon 30 and exon 31 in the 57-exon structure/variant of NF1) represents the obvious central genomic split-point necessary to split the ~8.5Kb NF1 cDNA into manageable fragments for delivery, especially if the artisan is contemplating employing AAV vectors, which would accommodate either of the corresponding halves of the NF1 cDNA sequence equaling approximately 4.25Kb each.
It was known in the art that the “small size of SaCas9 enables packaging” of genome editing elements “into a single AAV vector”, thereby overcoming the art-recognized “obstacle for packaging the commonly used Streptococcus pyrogenes Cas9 (SpCas9, ~4.2 kb) and its single guide RNA (sgRNA) in a single vector” as taught by Ran, who also taught that the gRNA sequence for Staphylococcus aureus Cas9 (SaCas9) can be “between 21 and 23 nucleotides long”, wherein “SaCas9 cleaves genomic targets most efficiently with NNGRRT”, which is the PAM sequence for SaCas9. Therefore, SaCas9 represents an obvious choice for the “Cas protein” when employing the HITI system, which utilizes a dual-AAV vector configuration. Specifically, a person of ordinary skill in the art would have been motivated to combine the SaCas9 & gRNA in a single vector configuration to maximize efficiency in a duel-AAV system; by consolidating the Cas protein and the guide into one vector, the entirety of the second AAV vector’s ~4.7Kb capacity is made available to house one of the two 4.25Kb NF1 gene fragments required for full length gene rescue as necessitated by Bai and demonstrated by Bednarski at the CFTR locus.
Regarding claim 18, the entire nucleotide sequence of human NF1 was known in the art at the time of filing as evidenced by public genomic databases (e.g., NCBI RefSeq), and the methodology for identifying a gRNA sequence for SaCas9 was known in the art as taught by Ran, who provided a detailed illustration in Figure 2a copied above. Thus, one of ordinary skill in the relevant art was reasonably equipped with the technical skills and ability to identify gRNA sequences preceding “NNGRRT” PAM sequences within intron 31 of human NF1. As such, one of ordinary skill in the relevant art would have readily identified all “NNGRRT” sequences following a nucleotide sequence of approximately 21 nucleotides long within intron 31 of human NF1, thereby identifying a finite number of gRNA sequences of ~21 nucleotides long preceding the ~54 “NNGRRT” sequences within NF1 intron 31 as demonstrated below, wherein bold nucleotides represent 6-mer PAM sequences and underlined nucleotides represent the 20-mer preceding the “GAGAAT” SaCas9 PAM sequence of the elected species, SEQ ID NO: 74.
PNG
media_image4.png
210
641
media_image4.png
Greyscale
The total number of potential PAM solutions identified using Ran’s teaching are further reduced when applying the teachings of Li when utilizing the HITI technique for knock-in (i.e., that the target sites should be chosen in introns, and they should be far from the 5′ and 3′ splice sites to avoid interference with the splicing machinery). Therefore, "[w]hen there is a design need or market pressure to solve a problem and there are a finite number of identified, predictable solutions, a person of ordinary skill has good reason to pursue the known options within his or her technical grasp." KSR, 550 U.S. at 421, 82 USPQ2d at 1397." MPEP 2143.
Regarding claim 22, It would have been obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to incorporate SaCas9 with SEQ ID NO:21 as taught by Ran into the HITI system taught by Belmonte. The inclusion of SaCas9 with SEQ ID NO:21 in place of SpCas9 in Belmonte’s HITI system represents simple substitution of one known element for another to obtain predictable results as described in KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) and MPEP 2143, as both Cas variants serve the known function of RNA guided nuclease activity. A person of ordinary skill in the art would have been motivated to substitute the SpCas9 with SaCas9 taught by Ran to improve AAV packaging and delivery, thereby achieving increased editing efficiencies in mammalian cells.
Claims 3 and 4 are rejected under 35 U.S.C. 103 as being unpatentable over Belmonte et. al., (US-20190225991-A1) in view of Moutal et. al., (PAIN 158(12): 2301-2319; published April 2017), Bai et. al., (Gen. Ther. 26(6): 277-286; published June 2019), Bednarski et. al., (PLoS One.;11(8):e0161072; published Aug 15, 2016), Li et. al., (Plant Genome Editing with CRISPR Systems. Methods in Molecular Biology, vol 1917, pp 285–296; published Jan 5, 2019) and Ran et. al., (Nature 520: 186-191; published April 9, 2015) as evidenced by NCBI RefSeq (NG_009018.1) as applied to claims 1, 15, 18, 22-25, 28, and 30 above, and further in view of Dombrowski et. al., (US-20170260547-A1).
Teachings of Belmonte et. al., Moutal et. al., Bai et. al., Bednarski et. al., Li et. al., and Ran et. al., as applied to claim 1, have been described above.
Regarding claim 3, Belmonte teaches the HITI system comprises a first vector and a second vector ([0158]: “To attempt improving in vivo HITI efficiency and utility, Cas9, gRNA, and donor DNA were loaded into two AAV vectors. One of the vectors harbored a minimal constitutive hybrid promoter (nEF) driven Cas9 sandwiched by SV40NLS (instead of BPNLS due to the limited cloning capacity of AAV (AAV - Cas9)). The other vector was constructed to accommodate the Tubb3 – gRNA expression cassette, 2 -cut donor and a fluorescent marker (AAV - mTubb3). Both AAVs were packaged with serotype 8, which have previously displayed high infection capability for many organs and therapeutic safety (FIG . 3A)”).
Belmonte, Moutal, Bai, and Bednarski do not explicitly teach the configuration of a first vector comprising the polynucleotide sequences for the gRNA and Cas protein, and a second vector compromising the 2 – cut donor sequence.
Dombrowski teaches a CRISPR/Cas-based system comprising a first vector and a second vector ([0084]: “the vector system may comprise two vectors”), wherein the first vector comprises the polynucleotide sequence encoding the gRNA and the polynucleotide sequence encoding the Cas protein ([0082]: “the nucleotide sequence encoding the guide RNA may be located on the same vector comprising the nucleotide sequence encoding a Cas9 protein), and wherein the second vector comprises the polynucleotide sequence encoding the donor sequence ([0083-0086]: “the nucleotide sequence encoding a Cas9 protein, a nucleotide sequence encoding the guide RNA, and a template may be located on the same or separate vectors…two or more sequences may be located on the same vector… the nucleotide sequence encoding the Cas9 protein and the nucleotide sequence encoding the guide RNA may be located on the same vector…the vector system may further comprise a vector comprising the template described herein”).
Dombrowski teaches a CRISPR/Cas-based system wherein the polynucleotide sequence encoding the gRNA and the polynucleotide sequence encoding the Cas protein are operably linked ([0011]: “a nucleotide sequence encoding the Cas9 protein operably linked to a first promoter,” and “a nucleotide sequence encoding the guide RNA operably linked to a second promoter”).
It would have been obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to configure the two-vector HITI CRISPR/Cas-based genome editing system taught by Belmonte such that polynucleotide sequences encoding the gRNA and Cas protein are located on a first vector and the donor sequence is located on a second vector, as taught by Dombrowski, because this modification represents the simple substitution of one known vector arrangement for another to obtain predictable results, as described in KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) and MPEP 2143. Belmonte teaches the use of a two-vector system to address AAV packaging constraints and optimize delivery in vivo, while Dombrowski teaches the specific arrangement of CRISPR/Cas components between two vectors as an alternative design choice. A person of ordinary skill in the art would have been motivated to select the claimed vector configuration to optimize vector capacity and simplify delivery, thereby improving genome editing efficiency in vivo, with a reasonable expectation of success.
Claims 5-8, 10-11, and 13 are rejected under 35 U.S.C. 103 as being unpatentable over Belmonte et. al., (US-20190225991-A1) in view of Moutal et. al., (PAIN 158(12): 2301-2319; published April 2017), Bai et. al., (Gen. Ther. 26(6): 277-286; published June 2019), Bednarski et. al., (PLoS One.; 11(8):e0161072; published Aug 15, 2016), Li et. al., (Plant Genome Editing with CRISPR Systems. Methods in Molecular Biology, vol 1917, pp 285–296; published Jan 5, 2019) and Ran et. al., (Nature 520: 186-191; published April 9, 2015) as evidenced by NCBI RefSeq (NG_009018.1) as applied to claims 1, 15, 18, 22-25, 28, and 30 above, and further in view of Auricchio et. al., (WO-2020079033-A1; published April 23, 2020).
Teachings of Belmonte et. al., Moutal et. al., Bai et. al., Bednarski et. al., Li et. al., and Ran et. al., as applied to claim 1, have been described above.
Regarding claims 5 and 10, Moutal teaches that the NF1 gene comprises a variety of mutations distributed throughout the coding region, including both 5’ and 3’ portion of the gene, as “more than 1000 pathogenic variants (insertions, microdeletions, copy number alterations) have been reported in the Nf1 gene” (see results 3.1). Belmonte teaches that the HITI system is capable of targeting and repairing mutated genes ([ 0096 ] ”HITI methods disclosed herein, in some embodiments, are capable of…repairing mutations in a host genome or a target genome”), and as described above the HITI system is an ideal gene therapy system to address Neurofibromatosis mutations.
Regarding claims 6, 8, 11, and 13, Belmonte teaches that a unique feature of the HITI system is that the gRNA targets a sequence that is 5’ to the donor sequence and a sequence that is downstream (i.e., 3’) to the donor sequence ([ 0093 ] “In some embodiments of HITI methods disclosed herein, exogenous DNA sequences are fragments of DNA containing the desired sequence to be inserted into the genome of the target cell or host cell; … [0094 ] In some embodiments of HITI methods disclosed herein, the exogenous DNA sequence is flanked by at least one targeting sequence; … [ 0095 ] In some embodiments, in HITI methods disclosed herein, a targeting sequence comprises a nucleotide sequence that is recognized and cleaved by a nuclease”).
Regarding claim 7 and 8, Belmonte further teaches that donor sequences may be operatively linked and followed by expression regulatory elements such as a poly adenylation signal (see FIG. 1).
However, Belmonte, Moutal, Bai, and Bednarski do not teach a NF1 donor sequence comprising one or more stop codon positioned 5’ or 3’ to the donor sequence, wherein the stop codon is placed in-frame with the endogenous gene to terminate translation of a mutant gene product depending on the location of the mutation either in the 5’ or 3’ portion/end of the gene.
Auricchio teaches donor nucleic acid constructs comprising at least one stop codon positioned either upstream or downstream to the exogenous DNA sequence (claim 1 or 4: “the donor sequence comprising: at least one STOP codon…said exogenous DNA sequence”) and a translation regulatory element positioned upstream of the exogenous DNA sequence (claim 4 and FIG. 2A: “a translation initiation sequence (TIS), wherein said TIS is a kozak sequence or an IRES sequence”).
Auricchio also teaches that the gRNA targets a sequence flanking both ends of the construct (claim 1 and FIG. 1: “said donor nucleic acid is flanked at 5' and 3' by inverted targeting sequences”).
Auricchio teaches the functional purpose of a stop codon is to terminate translation as needed (pg. 6 ln 19: “a stop codon in transcribed RNA results in early truncation of a protein translated from the mRNA”). Auricchio teaches in-frame donor designs incorporating a “translation initiation sequence,” such as a Kozak consensus sequence or IRES, to permit expression of the donor sequence following termination of upstream translation (pg. 6 ln 19: “the donor nucleic acid comprises stop codons in three possible frames” and a Kozak consensus sequence “is a motif that functions as translation initiation site in most RNA transcripts, as is recognized by the ribosome as the translational start site, from which a protein is coded by that mRNA molecule” ). For example, Auricchio teaches that when an exogenous wild-type DNA sequence (i.e., a donor sequence) configured to contain at least one stop codon is introduced into a host genome “the host gene is silenced and replaced by a wild-type gene or coding sequence thereof” (see Methods of making changes to genomic DNA, second paragraph).
It would have been obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to configure the donor element of Belmonte’s HITI-based CRISPR/Cas genome editing system, as applied to NF1 detailed in the claim 1 rejection above, to include a donor construct, as taught by Auricchio, comprising one or more stop codons positioned either 5’ or 3’ to the donor sequence, with the stop codon(s) and donor sequence being in the same reading frame of the non-mutated portion of the endogenous NF1 because this configuration represents a combination of prior art elements according to known methods to yield predictable results, as described in KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) and MPEP 2143. Each reference teaches a portion of the claimed system, and their combination applies known genome editing and expression control techniques to achieve their established functions with a reasonable expectation of success. A person of ordinary skill in the art would have been motivated to arrange the stop codon in relation to the donor sequence in a manner appropriate to address various known mutation hotspots within a mutant NF1 gene in order to achieve the intended and predictable control of gene expression (i.e., prevent mutant form expression while simultaneously restoring wild-type expression) following integration.
It would have also been obvious to position the gRNA target sites upstream and downstream of the stop codon and donor sequence, flaking the entire donor construct at the 5’ and 3’ ends, as required by the HITI system taught by Belmonte and also describe by Auricchio because it would have merely amounted to a simple combination of prior art elements according to known methods to yield predictable results, as described in KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) and MPEP 2143. A person of ordinary skill in the art would have been motivated to flank the donor cassette with gRNA target sequences to comply with the HITI system’s framework, thereby ensuring targeted insertion at the desired target sequence location in the NF1 locus.
It would have further been obvious to include a promoter or other expression control element between the stop codon and the exogenous DNA sequence to enable expression of the exogenous DNA sequence following termination of upstream translation. The inclusion of a promoter in place of a translation initiation sequence to drive exogenous DNA sequence expression represents simple substitution of one known element for another to obtain predictable results, as both elements serve the known function of enabling expression of a downstream coding sequence. As stated in the instant application’s specification, [00025]: “the coding sequence can further include initiation and termination signals operably linked to regulatory elements…the regulatory elements may include, for example, a promoter, an enhancer, an initiation codon, a stop codon, or a polyadenylation signal.” A person of ordinary skill in the art would have been motivated to place a promoter as an alternative expression element upstream of the donor sequence as Auricchio explains that using translation initiation elements (e.g., initiation codon) in that configuration allows for controlled exogenous DNA sequence expression/translation downstream of the included stop codon that simultaneously silences the host gene. Such expression control strategies were well known alternatives in the prior art and acknowledged in the instant application’ specification.
Claim 26 is rejected under 35 U.S.C. 103 as being unpatentable over Belmonte et. al., (US-20190225991-A1) in view of Moutal et. al., (PAIN 158(12): 2301-2319; published April 2017), Bai et. al., (Gen. Ther. 26(6): 277-286; published June 2019), Bednarski et. al., (PLoS One.;11(8):e0161072; published Aug 15, 2016), Li et. al., (Plant Genome Editing with CRISPR Systems. Methods in Molecular Biology, vol 1917, pp 285–296; published Jan 5, 2019) and Ran et. al., (Nature 520: 186-191; published April 9, 2015) as evidenced by NCBI RefSeq (NG_009018.1) as applied to claims 1, 15, 18, 22-25, 28, and 30 above, and further in view of Gersbach et. al., (US- 20180353615-A1).
Teachings of Belmonte et. al., Moutal et. al., Bai et. al., Bednarski et. al., Li et. al., and Ran et. al., as applied to claims 1 and 23-24, have been described above.
While both Belmonte and Bai teach the use of various AAV vectors, and specifically AAV of serotype 8 and 9, neither teach the polynucleotide sequence of the modified AAV vector backbone/plasmid used in the instant application such that it comprises any of the gRNAs, SEQ ID NOs: 71-81, corresponding to SEQ ID NOs: 83-102.
Gersbach teaches a modified AAV expression vector backbone/plasmid sequence comprising standard regulatory elements in the following configuration: a 5’ - ITR, a CMV promoter to drive SaCas9, a SaCas9 ORF, a bGH PolyA tail, a U6 promoter, a guide RNA, a SaCas9 scaffold, and a 3’ ITR which is analogous to the configuration of the AAV vectors claimed in the instant case, for example SEQ ID NO: 89. The only significant difference in the vectors taught by Gersbach is the portion pertaining to the gRNA sequence, which are designed to target the dystrophin gene instead of the NF1 gene.
It would have been obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to reconfigure the modified AAV vector(s) taught by Gersbach to include any of the gRNAs, SEQ ID NOs: 71-81, in order to arrive at corresponding SEQ ID NOs: 83-102 because it is merely a simple substitution of one known element for another to obtain predictable results as described in KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) and MPEP 2143. The AAV vector(s) comprising gRNA taught by Gersbach is/are virtually identical to those claimed in the instant application. For example, SEQ ID NO: 89 of the instant application, has a 99.1% identity score with SEQ ID NO: 39 and SEQ ID NO: 40 of Gersbach (see “search results -01/28/2026;” File Name: 20260127_163902_us-17-921-338-89.rnpbm; RESULT 7 and 8). Analysis of the alignment between the claimed and prior art vectors confirms that the high degree of identity covers the core functional structures of the delivery vehicle. While minor differences exist, specifically mismatches and indels at positions corresponding to 4351, 4354-4356, ~4600-4620, and 4700-4701, these variations are functionally insignificant. The differences at 4351 and 4354-4356 fall within non-coding intergenic linkers, and the differences at 4700-4701 fall at the junction of the gRNA scaffold and the transcriptional termination signal, which a PHOSITA would recognize these as routine length variations in areas that do not alter the function of the vector. The differences at ~4600-4620 correspond to the gRNA insertion site in the vector, and similarly how SEQ ID NO 39 and SEQ ID NO 40 of Gersbach differ from each other only by the inserted gRNA coding sequence, the difference between the claimed SEQ ID NO 89 (among others) and those of Gersbach is the substitution of a dystrophin-targeting gRNA for an NF1-targeting gRNA. As established in the rejection of claim 18 above, the selection of the gRNA sequences, SEQ ID NOs: 71-81, was a matter of routine optimization for the NF1 locus. Therefore, a person of ordinary skill in the art would have been motivated to substitute any of SEQ ID NO: 71-81 into the AAV vector backbone/plasmid taught by Gersbach with a reasonable expectation of success as this modular design allows for easy and time-efficient substitution of desired gRNA sequences to quickly adapt the AAV vector to one’s particular experimental needs while maintaining the fundamental function of the AAV vector as established by Gersbach.
Claim 27 is rejected under 35 U.S.C. 103 as being unpatentable over Belmonte et. al., (US-20190225991-A1) in view of Moutal et. al., (PAIN 158(12): 2301-2319; published April 2017), Bai et. al., (Gen. Ther. 26(6): 277-286; published June 2019), Bednarski et. al., (PLoS One.;11(8):e0161072; published Aug 15, 2016), Li et. al., (Plant Genome Editing with CRISPR Systems. Methods in Molecular Biology, vol 1917, pp 285–296; published Jan 5, 2019) and Ran et. al., (Nature 520: 186-191; published April 9, 2015) as evidenced by NCBI RefSeq (NG_009018.1) as applied to claims 1, 15, 18, 22-25, 28, and 30 above, and further in view of Yin et. al., (US-20190144845-A9).
Teachings of Belmonte et. al., Moutal et. al., Bai et. al., Bednarski et. al., Li et. al., and Ran et. al., as applied to claim 1, have been described above.
Neither Belmonte, Moutal, Bai, Bednarski, Li, or Ran teach the molar ratio between the gRNA and the donor sequence is 1:1, or 1:5, or from 5:1 to 1:10, or from 1:1 to 1:5.
Yin teaches a CRISPR/Cas-based system where the ratio of the gRNA and the donor sequence is 1: 1, or 1 :5, or from 5: 1 to 1: 10, or from 1: 1 to 1:5 ([0123]: “the ratio of the repair template to the gRNA and/or to the nucleic acid editing system is optimized for consistent delivery to the target sequence and/or consistent resolution of the disease or disorder…for example… the ratio of Cas9:sgRNA:template is from about 1:1:1 to about 1:1:100. In a further embodiment, the ratio is from about 1:1:2 to about 1:1:90, from about 1:1:5 to about 1:1:75, or from about 1:1:10 to about 1:1:50. In other embodiments, the ratio is about 1:1:1 or below, such as from about 1:1:0.01 to about 1:1:1, from about 1:1:0.02 to about 1:1:0.75, or about 1:1:0.05 to about 1:1:0.5, or about 1:1:0.1 to about 1:1:0.5”).
It would have been obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to modify the HITI system taught by Belmonte to adjust the molar ration between the gRNA and the donor sequence. The claimed ratios represent simple substitution of one known element for another to obtain predictable results as described in KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395-97 (2007) and MPEP 2143, as the claimed ratios merely recite overlapping and routine variations of known parameters. A person of ordinary skill in the art would have been motivated to adjust molar rations of guide RNA and donor template across broad ranges to optimize editing efficiency and/or consistent resolution of the disease or disorder.
Response to Arguments
Applicant's arguments filed 05/26/2026 have been fully considered but they are not persuasive.
Regarding the citation of NCBI RefSeq, while Goldfarb T. et. al., (Nucleic Acids Res. 2025 Jan 6;53(D1):D243-D257) was used to cite the NCBI RefSeq resource as it teaches the publicly available web address (see abstract), history of the NCBI RefSeq resource for the last 25 years (see title and introduction), and the features that the NCBI RefSeq resource comprises (see abstract and rest of article), it does not explicitly teach the identifier for NF1 gene. However, the NF1 gene sequence was well known in the database and is easily accessible by typing in “NF1” into the “gene” search bar, which doing so returns the identifier of, NG_009018.1. This identifier was first available to the public via the NCBI RefSeq resource on February 4th, 2008, which a print screen is now included in the file wrapper for convenience. This entry has undergone numerous updates, but has maintained the same identifier (NG_009018.1) for NF1, and the underlying DNA sequence of intron 31 has not changed since it became publicly available in 2008, see below screenshots of intron 31 (highlighted in blue) from the 2008 original posting vs the 2026 update:
PNG
media_image5.png
1052
789
media_image5.png
Greyscale
In response to applicant’s argument that there is no teaching, suggestion, or motivation to combine the references, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, the prior art explicitly sets forth an art-recognized engineering bottleneck and architectural design principles that directly motivate the combination of the cited references to arrive at the claimed invention.
In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
Regarding applicant’s argument that “there is no indication in any of the references that such a target would be successful,” the prior art does not need to show absolute predictability, but rather a reasonable expectation of success, which the prior art clearly establishes.
Conclusion
No claims are allowed
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to COREY LANE BRETZ whose telephone number is (571)272-7299. The examiner can normally be reached M-F 7:30am - 6:30pm.
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, Ram Shukla can be reached at (571) 272-0735. 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.
/COREY LANE BRETZ/Patent Examiner, 1635
/RAM R SHUKLA/Supervisory Patent Examiner, Art Unit 1635
Prosecution Insights