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 .
Please note: The Examiner handling this application has changed and is now Amanda Zahorik in Art Unit 1636.
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 06/30/2025 has been entered.
Application Status
This action is written in response to applicant’s correspondence received 06/30/2025. Claims 1-7, 9-17 and 19-22 are currently pending. No claims are withdrawn from prosecution as being drawn to non-elected subject matter. Accordingly, claims 1-7, 9-17 and 19-22 are examined herein.
Priority
Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 119(e) as follows:
The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
The disclosures of the prior-filed applications, Application Nos. 63/243,985 and 63/339,737, fail to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application.
Claim 1 of the instant application recites a pegRNA comprising phosphonocarboxylate modifications and a fusion protein comprising a nicking Cas9 protein and a reverse transcriptase. Neither of the provisional applications mentioned above provide support or enablement for a method comprising those elements, with the claimed modifications. Each one briefly mentions a pegRNA in the following context:
For example, a gRNA for prime editing (which is commonly referred to as a pegRNA) may comprise a polynucleotide segment that serves as a template sequence for a reverse transcriptase (RT) polypeptide to copy the template-encoded sequence onto the 3’ end of a DNA primer which is a substate for RT activity. The example described here of a gRNA for prime editing may further comprise a polynucleotide segment that is complementary to a nicked strand of a Cas-recognized DNA target sequence (recognized by hybridization of the quide sequence portion of the GRNA} such that the additional polynucleotide segment is substantially RNA and can hybridize to the nicked complementary strand of the DNA target site (nicked by a Cas protein} to produce a substantially RNA:DNA duplex which is recognized and bound by the RT polypeptide to activate its reverse transcriptase activity.
However, this mention of a pegRNA is exemplary, and does not include phosphonocarboxylate modifications. In the description of the invention on pages 4-5, the specification discloses that certain chemical modifications in gRNA can be advantageous for enhancing editing yields, such as one or more MP (2’-O-methyl-3’-phosphonoacetate; see p. 2 for the abbreviations) modifications at the 3’ end. However, the sgRNAs disclosed in Figure 1 appear to be standard ~100 nt gRNAs, not longer pegRNAs. The disclosure supports this conclusion on p. 5 by describing transfection of a mRNA encoding a generic Cas9 and a generic gRNA. Neither of these are disclosed to be anything but a standard Cas9 enzyme and sgRNA. On p. 7, the specification describes targeting HBB with a BE4 (Cas9 nickase and cytidine deaminase) fusion protein and gRNA, but there is no mention of a Cas9 nickase fused to a reverse transcriptase.
Because claim 1 is the independent claim from which all other claims depend, the lack of written support for the above limitations also applies to the dependent claims.
The effective filing date of the instant claims is 09/14/2022.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
Determining the scope and contents of the prior art.
Ascertaining the differences between the prior art and the claims at issue.
Resolving the level of ordinary skill in the pertinent art.
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.
WIPO Publication 2021/138469 A1 to Zhang
Claims 1-7, 9-11, 13, 15-17, 20 and 22 are rejected under 35 U.S.C. 103 as being unpatentable over WIPO Publication 2021/138469 A1 to Zhang (hereinafter ‘Zhang’; published 07/08/2021, priority filing date 12/30/2019), as evidenced by Santa (Santa et al. Frontiers in Immunology, 02/25/2021, Vol 12, pages 1-25; of record).
Regarding claim 1, Zhang teaches a method of prime editing a target region in a nucleic acid in a cell, using a Cas nickase and reverse transcriptase fusion protein and a pegRNA with guide, scaffold, reverse transcriptase template, and a primer-binding region (3’ binding site region) (most relevant portions underlined for emphasis):
[0015] In another aspect, the present invention provides for an engineered or non-naturally occurring composition comprising: a. a Cas polypeptide nickase; b. a reverse transcriptase (RT) polypeptide connected to or otherwise capable of forming a complex with the Cas polypeptide; and c. a guide molecule capable of forming a CRISPR-Cas complex with the Cas polypeptide and comprising: i. a guide sequence capable of directing site-specific binding of the CRISPR- Cas complex to a target sequence of a target polynucleotide; ii. a 3’ binding site region capable of binding to a cleaved upstream strand of the target polynucleotide; iii. a RT template sequence encoding an extended sequence, wherein the extended sequence comprises a variant region and a 3’ homologous sequence capable of hybridization to the downstream cleaved strand of the target polynucleotide; and iv. one or more hairpin structures on the guide molecule.
[0039] …the Cas polypeptide is fused to the reverse transcriptase.
Zhang teaches that guide RNA has two or more phosphorothioate modifications at the 5’ end and two or more thiophosphonocarboxylate (thioPACE) modifications at the 3’ end:
[0138] Examples of guide RNA chemical modifications include, without limitation, incorporation of 2'-0-methyl (M), 2'-0-methyl 3 'phosphorothioate (MS), S-constrained ethyl(cEt), or 2'-0-methyl 3'thioPACE (MSP) at one or more terminal nucleotides…three to five nucleotides at the 5’ and/or the 3’ end of the guide are chemically modified with 2'-0-methyl (M), 2’-0-methyl 3’ phosphorothioate (MS), S-constrained ethyl(cEt), or 2'-O-methyl 3’ thioPACE (MSP)…Such modification can enhance genome editing efficiency (see Hendel et al., Nat. Biotechnol. (2015) 33(9): 985-989).
Zhang teaches that the method comprises in vivo or ex vivo administration:
[0254] Delivery can be to cells (e.g., in vitro or ex vivo administration) or target tissues (e.g., in vivo administration)
Zhang does not explicitly teach that the cell exists ex vivo in the presence of a nuclease-containing fluid. However, Santa evidences that extracellular nucleases DNASE1, DNASE1L1, DNASE1L3, and RNASET3 are expressed in a variety of tissues and cell types (table 1, page 6), including in body fluids which may contain cells of interest for ex vivo applications (page 5, last para).
Zhang does not explicitly exemplify a pegRNA with the claimed end modifications. However, as shown above, Zhang’s disclosures primarily concern CRISPR prime editing systems using reverse transcriptase and pegRNA, as indicated by the abstract:
Systems and methods for targeted gene modification, targeted insertion, perturbation of gene transcripts, and nucleic acid editing. Novel nucleic acid targeting systems comprise components of CRISPR systems, reverse transcriptase, pegRNAs, paired pegRNAs or modified pegRNAs, DNA processing proteins, recombinases, proteins for inhibiting nucleases, and proteins for promoting ssDNA annealing.
Based on that, the skilled artisan would have had a reasonable expectation that Zhang’s recommended modifications could have been applied to the pegRNAs of Zhang’s own disclosure.
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have modified the pegRNA as taught by Zhang by incorporating various combinations of two or more phosphorothioate and/or MSP modifications at the 5’ and 3’ ends of the guide RNA. The skilled artisan would have been motivated to make these modifications based on Zhang’s teachings that such modifications were well-known in the art for enhancing genome editing efficiency. Thus in regard to the limitations of the claims, where the prior art teaches modification of guide RNAs, including pegRNAs, with one to five 2'-0-methyl 3 'phosphorothioate (MS) and/or 2'-0-methyl 3'thioPACE (MSP) at the 5’ and/or 3’ termini of guide RNAs, a person of ordinary skill has good reason to pursue the known options within his or her technical grasp for optimization of the type, number and placement of the terminal MS/MSP modifications. If this leads to the anticipated success, it is likely the product not of innovation but of ordinary skill and common sense to provide routine optimization.
Regarding claim 2, Zhang teaches wherein the phosphorothioate/thiophosphonocarboxylate modifications also comprise 2’-O-methyl (para [0138]).
Regarding claim 3, insofar as Zhang teaches one to five MS at the 5’ and/or 3’ termini of the guide RNA, Zhang teaches embodiments wherein the modified pegRNA comprises at least two consecutive MS within 5 nucleotides of the 5’ end.
Regarding claim 4, Zhang teaches wherein the thiophosphonocarboxylate is thiophosphonoacetate (para [0138]).
Regarding claim 5, Zhang teaches wherein the modified pegRNA comprises at least two consecutive MSP within 5 nucleotides of the 3’ end (see above).
Regarding claim 6, Zhang teaches wherein the modified pegRNA further comprises modified nucleotides located outside of the 5 nucleotides at the termini:
[0139] 12 nucleotides in the tetraloop and 16 nucleotides in the stem-loop region are replaced with 2'-O-methyl analogs.
Please note that the tetraloop and stem-loop regions are located outside of the termini, as shown in Zhang’s FIG. 8A.
Regarding claim 7, Zhang teaches wherein the modified pegRNA is a single guide RNA. See FIG. 8A, which depicts exemplary pegRNA structures with guide regions and the hairpin structures required for Cas9 recruitment.
Regarding claim 9, Zhang teaches wherein the Cas protein and modified pegRNA form a ribonucleoprotein complex
[0453] In particular embodiments, the Cas protein is mixed with guide RNA targeting the gene of interest to form a pre-assembled ribonucleoprotein.
Regarding claim 10, Zhang teaches wherein the mRNA encoding the Cas/RT fusion protein and/or modified pegRNA are provided in a nanoparticle:
Regarding claim 11, Zhang teaches that MS and MSP modifications enhance genome editing efficiency, i.e., wherein the editing occurs with an efficiency higher than that by an unmodified pegRNA which is otherwise identical to the modified pegRNA.
Regarding claim 13, Zhang teaches editing ex vivo in blood (i.e., blood serum):
[0783] Ex Vivo Editing Therapy: The long-standing clinical expertise with the purification, culture and transplantation of hematopoietic cells has made diseases affecting the blood system such as SCID, Fanconi anemia, Wiskott-Aldrich syndrome and sickle cell anemia the focus of ex vivo editing therapy.
Regarding claim 15, Zhang teaches wherein the fluid is cell culture medium:
[0402] Where any treatment is occurring ex vivo, for example in a cell culture
Regarding claim 17, Zhang teaches wherein the cell exists in vivo (para [0254]).
Regarding claim 20, Zhang teaches wherein the pegRNA and mRNA encoding a fusion protein are provided at a pegRNA:mRNA ratio of 100:1 or less (e.g., 20:1, 5:1, etc.):
[0252] The relative dosages of gene editing components may be important in some applications. In some examples, expression of one or more components of the complex is involved, which may be for example from the same or separate vectors. In the single vector case, it will often be advantageous to vary the effector protein:guide ratio by adjusting the expression levels of the effector protein and guide. In the case of multiple vectors, it will often be advantageous to vary the effector protein:guide ratio by adjusting the doses of the separate vectors and/or the expression levels of the effector protein and guide from the vectors. In certain embodiments, the ratios of vectors for expression of the effector protein and guide are adjusted. For example, the relative doses of an AAV-effector protein expression vector and an AAV-guide expression vector can be adjusted. Usually, the doses are expressed in terms of vector genomes (vg) per ml (vg/ml) or per kg (vg/kg). In certain embodiments, the ratio of vector genomes of the AAV-effector protein and AAV-guide is about 2:1, or about 1:1, or about 1:2, or about 1:4, or about 1:5, or about 1:10, or about 1:20, or from about 2:1 to about 1:1, or from about 2: 1 to about 1 :2, or from about 1 : 1 to about 1 :2 or from about 1 : 1 to about 1 :4, or from about 1 :2 to about 1 :5, or from about 1 :2 to about 1 : 10 or from about 1 :5 to about 1 :20.
Regarding claim 22, Zhang teaches wherein the Cas is a Cas9:
[0020] FIG. 1 - Schematic showing insertion or deletion of DNA using a pair of pegRNAs with a Cas9 nickase. Solid arrows indicate nicking sites. The template is indicated on each pegRNA.
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang evidenced by Santa, as applied to claims 1-7, 9-11, 13, 15-17, 20 and 22 , further in view of Hendel (Hendel et al. "Chemically Modified Guide RNAs Enhanced Crispr-cas Genome Editing in Human Primary Cells, Nature Biotechnology, Vol. 33, No. 9 ,September 2015 ,985-991.; of record, applicant’s submission).
Zhang evidenced by Santa renders obvious the method of claim 1 and 11, from which instantly rejected 12 depends, as described above.
Zhang evidenced by Santa does not teach wherein the editing efficiency is increased by at least 10%.
Hendel teaches sgRNAs modified with MSP, which exhibited 68.0% and 75.7% efficiency compared to 2.4% for unmodified sgRNAs, which is well over a 10% difference (p. 986, Figure 1).
It is also relevant to note that Hendel teaches 3 consecutive modified nucleotides at the 5’ and 3’ termini of the sgRNA (Figure 1a), which is in line with Zhang’s recommendations.
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have, in the course of routine optimization as discussed in the rejection of the claims over Zhang, above, combined the teachings of Zhang with those of Hendel and further optimized the guide RNA by trying various combinations of 3 consecutive MS and/or MSP modifications at the termini at the guide RNA. The skilled artisan would have been motivated to try modifying the same positions modified by Hendel based on the significant increase in editing efficiency compared to unmodified sgRNA. The skilled artisan would have been motivated by both Zhang and Hendel to apply MS and/or MSP modifications to those positions. Given a limited number of positions (6) and modifications (2), the number of possible modification patterns was finite and easy to traverse. Thus in regard to the limitations of the claims, where the prior art teaches modification of three consecutive nucleotides at the termini of guide RNAs, a person of ordinary skill has good reason to pursue the known options within his or her technical grasp for optimization of the type, number and placement of the three terminal MS/MSP modifications. If this leads to the anticipated success, it is likely the product not of innovation but of ordinary skill and common sense to provide routine optimization.
Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang evidenced by Santa, as applied to claims 1-7, 9-11, 13, 15-17, 20 and 22 , further in view of Wickham (Wickham et al. Human cerebrospinal fluid induces neuronal excitability changes in resected human neocortical and hippocampal brain slices. Front. Neurosci., 20 April 2020 Sec. Neural Technology Volume 14.)
Zhang evidenced by Santa renders obvious the method of claim 1, from which instantly rejected 14 depends, as described above.
Zhang evidenced by Santa does not teach wherein the nuclease-containing fluid is CSF.
Wickham teaches an ex vivo model for studying human brain tissue using cerebrospinal fluid (Abstract).
Wickham also provides a suggestion or motivation to design ex vivo experiments mimicking in vivo conditions as closely as possible, to better translate the results back to in vivo (p. 2 right, 1st para).
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have applied the method of gene editing as taught by Zhang to human neurons in Wickham’s ex vivo culture comprising CSF. The skilled artisan would have been motivated by Wickham’s teachings that this ex vivo system would have better mimicked in vivo conditions. This, in turn, would have allowed one of ordinary skill to study the effects of CRISPR prime editing in neurons, in a system which would have allowed for better translation of the results to in vivo conditions.
Claim 19 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang evidenced by Santa, as applied to claims 1-7, 9-11, 13, 15-17, 20 and 22 , further in view of Ryan (Ryan et al. "Improving CRISPR-Cas Specificity With Chemical Modifications In Single-Guide RNAs," Nucleic Acids Research, Vol. 46, No. 2, 2018, 792-803; of record, applicant’s submission and art, published 12/04/2017).
Zhang evidenced by Santa renders obvious the method of claim 1, from which instantly rejected 19 depends, as described above.
Zhang evidenced by Santa does not teach wherein the modified pegRNA comprises a phosphonocarboxylate or thiophosphonocarboxylate modification at nucleotide position 5 or 11, counting from the 5’ end of the pegRNA.
Ryan teaches, regarding chemical modifications to gRNAs, that, “(2’-O-methyl-3’-phosphonoacetate, or ‘MP’) incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes.” (Abstract).
Ryan also specifically recommends incorporating MP at positions 5 or 11, noting, “we observed the greatest specificity enhancement when MP was incorporated at position 5 or 11”.
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method and pegRNA as taught by Zhang and evidenced by Santa by adding the specific MP modifications at positions 5 and 11, as taught by Ryan. Ryan provides both an explicit suggestion to do so as well as a reasonable expectation of success by showing that placing MP at positions 5 and 11 led to the greatest specificity enhancement.
Claim 21 is rejected under 35 U.S.C. 103 as being unpatentable over Zhang evidenced by Santa, as applied to claims 1-7, 9-11, 13, 15-17, 20 and 22, further in view of U.S. PGPUB 2023/0059368 to Cafferty (published 02/23/2023, priority filing date 06/15/2021)
Zhang evidenced by Santa renders obvious the method of claim 1, from which instantly rejected 21 depends, as described above.
Zhang evidenced by Santa does not teach wherein the pegRNA also comprises 2’-O-methoxyethyl.
Cafferty teaches Cas prime editing systems with prime editing (PE) guide polynucleotides (Abstract), wherein the pegRNA comprises 2’-O-methoxyethyl (2’-MOE) modified nucleotides for improved resistance to RNAse cleavage (para [0288]).
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have modified the pegRNA as taught by Zhang and evidenced by Santa to further comprise 2’-MOE modifications. The skilled artisan would have been motivated by Cafferty’s teachings that this modification would have increased resistance to RNAse cleavage, thus increasing its stability in vivo.
PGPUB 2018/0119140 A1 to Porteus in view of WIPO Publication 2021/138469 A1 to Zhang
Claims 1-7, 9-11, 13, 15-17, 20 and 22 are rejected under 35 U.S.C. 103 as being unpatentable over U.S. PGPUB 2018/0119140 A1 to Porteus (hereinafter ‘Porteus’, of record, shares at least one common inventor and assignee with the instant application; patent issued 04/19/2022 with number 11,306,309) in view of WIPO Publication 2021/138469 A1 to Zhang (hereinafter ‘Zhang’; published 07/08/2021, priority filing date 12/30/2019), as evidenced by Santa (Santa et al. Frontiers in Immunology, 02/25/2021, Vol 12, pages 1-25; of record).
Regarding claim 1, Porteus teaches an in vivo or ex vivo method of CRISPR genome editing using a Cas endonuclease and a guide RNA with a guide sequence and scaffold:
[0007] The present invention provides methods for inducing (e.g., initiating, modulating, enhancing, etc.) gene regulation of a target nucleic acid in a cell. The invention includes using modified single guide RNAs (sgRNAs) that enhance genome editing and/or inhibition or activation of gene expression of a target nucleic acid in a primary cell (e.g., cultured in vitro for use in ex vivo therapy) or in a cell in a subject such as a human (e.g., for use in in vivo therapy).
[0008] In a first aspect, the present invention provides a method for inducing gene regulation of a target nucleic acid in a primary cell, the method comprising:
[0009] introducing into the primary cell:
[0010] (a) a modified single guide RNA (sgRNA) comprising a first nucleotide sequence that is complementary to the target nucleic acid and a second nucleotide sequence that interacts with a CRISPR-associated protein (Cas) polypeptide, wherein one or more of the nucleotides in the first nucleotide sequence and/or the second nucleotide sequence are modified nucleotides; and
[0011] (b) a Cas polypeptide, an mRNA encoding a Cas polypeptide, and/or a recombinant expression vector comprising a nucleotide sequence encoding a Cas polypeptide, wherein the modified sgRNA guides the Cas polypeptide to the target nucleic acid, and
[0012] wherein the modified sgRNA induces gene regulation of the target nucleic acid with an enhanced activity relative to a corresponding unmodified sgRNA.
Porteus further teaches that the guide RNA has two or more phosphorothioate modifications at the 5’ end and two or more thiophosphonocarboxylate (thioPACE) modifications at the 3’ end:
[0199] …chemical modifications comprising 2′-O-methyl (M), 2′-O-methyl 3′phosphorothioate (MS), or 2′-O-methyl 3′thioPACE (MSP) were incorporated at three terminal nucleotides at both 5′ and 3′ ends. These three modifications were selected for evaluation due to their previously reported stability to serum and snake venom phosphordiesterases as well as their wide range of reported effects on the immunostimulatory properties of nucleic acids
Please also note FIG. 1A, which shows the locations of those modifications and that they are consecutive.
Porteus further teaches mRNAs encoding Cas fusion proteins wherein the Cas enzyme is fused to a variety of other enzymes, including transcription factors, DNA modifying enzymes, and the like:
[0189] The method for modulating (e.g., inhibiting or activating) gene expression of a target nucleic acid, e.g., a target DNA, in a cell includes introducing (e.g., electroporating) into the cell the modified sgRNA described herein and either a Cas nuclease (e.g., Cas9 polypeptide) or variant or fragment thereof, an mRNA encoding a Cas nuclease (e.g., Cas9 polypeptide) or variant or fragment thereof, or a recombinant expression vector comprising a nucleotide sequence encoding a Cas nuclease (e.g., Cas9 polypeptide) or variant or fragment thereof. In some embodiments, the Cas nuclease (e.g., Cas9) variant is an endonuclease-deficient Cas (e.g., dCas9) polypeptide. In some instances, the Cas9 variant can have two or more amino acid substitutions compared to the wild-type Cas9 polypeptide. In other instances, the Cas9 variant cannot cleave double-stranded DNA. The Cas nuclease variant can be a Cas (e.g., dCas9) fusion polypeptide. In some embodiments, the fusion polypeptide includes a transcriptional repression domain, a transcriptional activation domain, transcription factor, histone modifying enzyme (e.g., histone deacetylase, histone methyltransferase, histone acetyltransferase), a DNA modifying enzyme (e.g., DNA methyltransferase), and the like.
Porteus teaches the method using a Cas nickase:
[0108] The CRISPR/Cas system of genome modification includes a Cas nuclease (e.g., Cas9 nuclease) or a variant or fragment thereof, a DNA-targeting RNA (e.g., modified sgRNA) containing a guide sequence that targets the Cas nuclease to the target genomic DNA and a scaffold sequence that interacts with the Cas nuclease (e.g., tracrRNA), and optionally, a donor repair template. In some instances, a variant of a Cas nuclease such as a Cas9 mutant containing one or more of the following mutations: D10A, H840A, D839A, and H863A, or a Cas9 nickase can be used.
Porteus does not teach wherein the guide RNA is a pegRNA with a RT template and a primer binding region, or that the nickase is fused to a reverse transcriptase.
Zhang teaches a method of prime editing a target region in a nucleic acid in a cell, using a Cas nickase and reverse transcriptase fusion protein and a pegRNA with guide, scaffold, reverse transcriptase template, and a primer-binding region (3’ binding site region) (most relevant portions underlined for emphasis):
[0015] In another aspect, the present invention provides for an engineered or non-naturally occurring composition comprising: a. a Cas polypeptide nickase; b. a reverse transcriptase (RT) polypeptide connected to or otherwise capable of forming a complex with the Cas polypeptide; and c. a guide molecule capable of forming a CRISPR-Cas complex with the Cas polypeptide and comprising: i. a guide sequence capable of directing site-specific binding of the CRISPR- Cas complex to a target sequence of a target polynucleotide; ii. a 3’ binding site region capable of binding to a cleaved upstream strand of the target polynucleotide; iii. a RT template sequence encoding an extended sequence, wherein the extended sequence comprises a variant region and a 3’ homologous sequence capable of hybridization to the downstream cleaved strand of the target polynucleotide; and iv. one or more hairpin structures on the guide molecule.
[0039] …the Cas polypeptide is fused to the reverse transcriptase.
Zhang provides a teaching, suggestion or motivation to use prime editing for genome modification because they do not require donor templates and may be used to generate all 12 possible combination swaps:
[0039] Prime editing systems relate to targeted modification of a polynucleotide without generating double stranded breaks or requiring donor templates. Further, prime editing systems may be used to generate all 12 possible combination swaps. Prime editing systems are composed of a Cas polypeptide having nickase activity, a reverse transcriptase, and a guide molecule.
Zhang teaches that their guide RNA may include MSP and MS modifications of three to five nucleotides at the 5’ and/or 3’ ends to enhance genome editing efficiency:
[0138] Examples of guide RNA chemical modifications include, without limitation, incorporation of 2'-0-methyl (M), 2'-0-methyl 3 'phosphorothioate (MS), S-constrained ethyl(cEt), or 2'-0-methyl 3'thioPACE (MSP) at one or more terminal nucleotides…three to five nucleotides at the 5’ and/or the 3’ end of the guide are chemically modified with 2'-0-methyl (M), 2’-0-methyl 3’ phosphorothioate (MS), S-constrained ethyl(cEt), or 2'-O-methyl 3’ thioPACE (MSP)…Such modification can enhance genome editing efficiency (see Hendel et al., Nat. Biotechnol. (2015) 33(9): 985-989).
Zhang does not explicitly exemplify a pegRNA with the claimed end modifications. However, as shown above, Zhang’s disclosures primarily concern CRISPR prime editing systems using reverse transcriptase and pegRNA, as indicated by the abstract:
Systems and methods for targeted gene modification, targeted insertion, perturbation of gene transcripts, and nucleic acid editing. Novel nucleic acid targeting systems comprise components of CRISPR systems, reverse transcriptase, pegRNAs, paired pegRNAs or modified pegRNAs, DNA processing proteins, recombinases, proteins for inhibiting nucleases, and proteins for promoting ssDNA annealing.
Based on that, the skilled artisan would have had a reasonable expectation that Zhang’s recommended modifications could have been applied to the pegRNAs of Zhang’s own disclosure.
Zhang does not explicitly teach that the cell exists ex vivo in the presence of a nuclease-containing fluid. However, Santa evidences that extracellular nucleases DNASE1, DNASE1L1, DNASE1L3, and RNASET3 are expressed in a variety of tissues and cell types (table 1, page 6), including in body fluids which may contain cells of interest for ex vivo applications (page 5, last para).
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have combined the teachings of Porteus and Zhang by applying the modifications taught by both Porteus and Zhang to Zhang’s pegRNA for use in Zhang’s prime editing system. The skilled artisan would have been motivated to apply said modifications based on the combined disclosures of Porteus and Zhang, which both teach that these modifications provide useful characteristics such as enhanced efficiency and stability. The skilled artisan would also have been motivated to apply these modifications to the pegRNAs of Zhang instead of standard gRNAs as taught by Porteus based on Zhang’s teachings that prime editing systems were able to achieve a variety of genome modifications without a donor template. Based on the fact that both Zhang and Porteus teach that these modifications desirable in the context of the two CRISPR systems, the skilled artisan would have had a reasonable expectation that they could successfully be applied to both types of gRNA.
Additionally, in regard to the limitations of the claims concerning various different numbers and configurations of the modifications (i.e., two or more consecutive or non-consecutive), where the prior art teaches modification of guide RNAs, including pegRNAs, with one to five 2'-0-methyl 3 'phosphorothioate (MS) and/or 2'-0-methyl 3'thioPACE (MSP) at the 5’ and/or 3’ termini of guide RNAs, a person of ordinary skill has good reason to pursue the known options within his or her technical grasp for optimization of the type, number and placement of the terminal MS/MSP modifications. If this leads to the anticipated success, it is likely the product not of innovation but of ordinary skill and common sense to provide routine optimization.
Regarding claim 2, Porteus teaches wherein the phosphorothioate/thiophosphonocarboxylate modifications also comprise 2’-O-methyl (para [0199]).
Regarding claim 3, Porteus teaches wherein the modified gRNA comprises at least two consecutive MS within 5 nucleotides of the 5’ end (see FIG. 1A).
Regarding claim 4, Porteus teaches wherein the thiophosphonocarboxylate is thiophosphonoacetate (para [0199]).
Regarding claim 5, Porteus teaches wherein the modified gRNA comprises at least two consecutive MSP within 5 nucleotides of the 3’ end (see above).
Regarding claim 6, Porteus teaches wherein the modified gRNA further comprises modified nucleotides located outside of the 5 nucleotides at the termini:
[0139] In particular embodiments, one or more of the modified nucleotides of the guide sequence and/or one or more of the modified nucleotides of the scaffold sequence of the modified sgRNA include a 2′-O-methyl (M) nucleotide, a 2′-O-methyl 3′-phosphorothioate (MS) nucleotide, a 2′-O-methyl 3′thioPACE (MSP) nucleotide, or a combination thereof.
Please note that the tetraloop and stem-loop regions are located outside of the termini, as shown in FIG. 1A.
Regarding claim 7, Porteus teaches wherein the modified gRNA is a single guide RNA. See FIG. 1A, which depicts an exemplary gRNA structure with a guide region and the hairpin structures required for Cas9 recruitment.
Regarding claim 9, Porteus teaches wherein the Cas protein and modified gRNA form a ribonucleoprotein complex
[0125] In certain instances, the modified sgRNA is complexed with a Cas nuclease (e.g., Cas9 polypeptide) or a variant or fragment thereof to form a ribonucleoprotein (RNP)-based delivery system for introduction into a cell (e.g., an in vitro cell such as a primary cell for ex vivo therapy, or an in vivo cell such as in a patient).
Regarding claim 10, Porteus teaches wherein the mRNA encoding the Cas and/or modified gRNA are provided in a nanoparticle:
[0179] In some embodiments, the components of CRISPR/Cas-mediated gene regulation can be introduced into a cell using a delivery system. In certain instances, the delivery system comprises a nanoparticle
Regarding claim 11, Zhang teaches that MS and MSP modifications enhance genome editing efficiency, i.e., wherein the editing occurs with an efficiency higher than that by an unmodified pegRNA which is otherwise identical to the modified pegRNA (see above).
Regarding claim 13, Porteus teaches editing ex vivo in blood (i.e., blood serum) (para [0175].
Regarding claim 15, Porteus teaches wherein the fluid is cell culture medium (para [0169].
Regarding claim 17, Porteus teaches wherein the cell exists in vivo (para [0007]).
Regarding claim 20, Zhang teaches wherein the pegRNA and mRNA encoding a fusion protein are provided at a pegRNA:mRNA ratio of 100:1 or less (e.g., 20:1, 5:1, etc.):
[0252] The relative dosages of gene editing components may be important in some applications. In some examples, expression of one or more components of the complex is involved, which may be for example from the same or separate vectors. In the single vector case, it will often be advantageous to vary the effector protein:guide ratio by adjusting the expression levels of the effector protein and guide. In the case of multiple vectors, it will often be advantageous to vary the effector protein:guide ratio by adjusting the doses of the separate vectors and/or the expression levels of the effector protein and guide from the vectors. In certain embodiments, the ratios of vectors for expression of the effector protein and guide are adjusted. For example, the relative doses of an AAV-effector protein expression vector and an AAV-guide expression vector can be adjusted. Usually, the doses are expressed in terms of vector genomes (vg) per ml (vg/ml) or per kg (vg/kg). In certain embodiments, the ratio of vector genomes of the AAV-effector protein and AAV-guide is about 2:1, or about 1:1, or about 1:2, or about 1:4, or about 1:5, or about 1:10, or about 1:20, or from about 2:1 to about 1:1, or from about 2: 1 to about 1 :2, or from about 1 : 1 to about 1 :2 or from about 1 : 1 to about 1 :4, or from about 1 :2 to about 1 :5, or from about 1 :2 to about 1 : 10 or from about 1 :5 to about 1 :20.
Regarding claim 22, Zhang teaches wherein the Cas is a Cas9:
[0020] FIG. 1 - Schematic showing insertion or deletion of DNA using a pair of pegRNAs with a Cas9 nickase. Solid arrows indicate nicking sites. The template is indicated on each pegRNA.
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over U.S. PGPUB 2018/0119140 A1 to Porteus (hereinafter ‘Porteus’, of record, shares at least one common inventor and assignee with the instant application) in view of Patent WIPO Publication 2021/138469 A1 to Zhang (hereinafter ‘Zhang’; published 07/08/2021, priority filing date 12/30/2019), as evidenced by Santa (Santa et al. Frontiers in Immunology, 02/25/2021, Vol 12, pages 1-25; of record), further in view of Hendel (Hendel et al. "Chemically Modified Guide RNAs Enhanced Crispr-cas Genome Editing in Human Primary Cells, Nature Biotechnology, Vol. 33, No. 9 ,September 2015 ,985-991.; of record, applicant’s submission).
Porteus, Zhang and Santa render obvious the method of claim 1 and 11, from which instantly rejected 12 depends, as described above.
Porteus, Zhang and Santa render do not teach wherein the editing efficiency is increased by at least 10%.
Hendel teaches sgRNAs modified with MSP, which exhibited 68.0% and 75.7% efficiency compared to 2.4% for unmodified sgRNAs, which is well over a 10% difference (p. 986, Figure 1).
It is also relevant to note that Hendel teaches 3 consecutive modified nucleotides at the 5’ and 3’ termini of the sgRNA (Figure 1a), which is in line with Zhang’s recommendations.
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have, after combining the teachings of Porteus and Zhang and in the course of routine optimization as discussed in the rejection of the claims over Porteus, Zhang and Santa above, combined the teachings of Porteus, Zhang and Santa with those of Hendel and further optimized the guide RNA by trying various combinations of 3 consecutive MS and/or MSP modifications at the termini at the guide RNA. The skilled artisan would have been motivated to try modifying the same positions modified by Hendel based on the significant increase in editing efficiency compared to unmodified sgRNA. The skilled artisan would have been motivated by the combination of Porteus, Zhang and Hendel to apply MS and/or MSP modifications to those positions. Given a limited number of positions (6) and modifications (2), the number of possible modification patterns was finite and easy to traverse. Thus in regard to the limitations of the claims, where the prior art teaches modification of three consecutive nucleotides at the termini of guide RNAs, a person of ordinary skill has good reason to pursue the known options within his or her technical grasp for optimization of the type, number and placement of the three terminal MS/MSP modifications. If this leads to the anticipated success, it is likely the product not of innovation but of ordinary skill and common sense to provide routine optimization.
Claim 14 is rejected under 35 U.S.C. 103 as being unpatentable over PGPUB 2018/0119140 A1 to Porteus (hereinafter ‘Porteus’, of record, shares at least one common inventor and assignee with the instant application) in view of Patent WIPO Publication 2021/138469 A1 to Zhang (hereinafter ‘Zhang’; published 07/08/2021, priority filing date 12/30/2019), as evidenced by Santa (Santa et al. Frontiers in Immunology, 02/25/2021, Vol 12, pages 1-25; of record), further in view of Wickham (Wickham et al. Human cerebrospinal fluid induces neuronal excitability changes in resected human neocortical and hippocampal brain slices. Front. Neurosci., 20 April 2020 Sec. Neural Technology Volume 14.)
Porteus, Zhang and Santa render obvious the method of claim 1, from which instantly rejected 14 depends, as described above.
Porteus, Zhang and Santa do not teach wherein the nuclease-containing fluid is CSF.
Wickham teaches an ex vivo model for studying human brain tissue using cerebrospinal fluid (Abstract).
Wickham also provides a suggestion or motivation to design ex vivo experiments mimicking in vivo conditions as closely as possible, to better translate the results back to in vivo (p. 2 right, 1st para).
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have applied the method of gene editing as taught by Porteus, Zhang and Santa to human neurons in Wickham’s ex vivo culture comprising CSF. The skilled artisan would have been motivated by Wickham’s teachings that this ex vivo system would have better mimicked in vivo conditions. This, in turn, would have allowed one of ordinary skill to study the effects of CRISPR prime editing in neurons, in a system which would have allowed for better translation of the results to in vivo conditions.
Claim 19 is rejected under 35 U.S.C. 103 as being unpatentable over U.S. PGPUB 2018/0119140 A1 to Porteus (hereinafter ‘Porteus’, of record, shares at least one common inventor and assignee with the instant application) in view of Patent WIPO Publication 2021/138469 A1 to Zhang (hereinafter ‘Zhang’; published 07/08/2021, priority filing date 12/30/2019), as evidenced by Santa (Santa et al. Frontiers in Immunology, 02/25/2021, Vol 12, pages 1-25; of record), further in view of Ryan (Ryan et al. "Improving CRISPR-Cas Specificity With Chemical Modifications In Single-Guide RNAs," Nucleic Acids Research, Vol. 46, No. 2, 2018, 792-803; of record, applicant’s submission and art, published 12/04/2017).
Porteus, Zhang and Santa render obvious the method of claim 1, from which instantly rejected 19 depends, as described above.
Porteus, Zhang and Santa do not teach wherein the modified pegRNA comprises a phosphonocarboxylate or thiophosphonocarboxylate modification at nucleotide position 5 or 11, counting from the 5’ end of the pegRNA.
Ryan teaches, regarding chemical modifications to gRNAs, that, “(2’-O-methyl-3’-phosphonoacetate, or ‘MP’) incorporated at select sites in the ribose-phosphate backbone of gRNAs can dramatically reduce off-target cleavage activities while maintaining high on-target performance, as demonstrated in clinically relevant genes.” (Abstract).
Ryan also specifically recommends incorporating MP at positions 5 or 11, noting, “we observed the greatest specificity enhancement when MP was incorporated at position 5 or 11”.
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have modified the method and pegRNA as taught by Porteus, Zhang and Santa by adding the specific MP modifications at positions 5 and 11, as taught by Ryan. Ryan provides both an explicit suggestion to do so as well as a reasonable expectation of success by showing that placing MP at positions 5 and 11 led to the greatest specificity enhancement.
Claim 21 is rejected under 35 U.S.C. 103 as being unpatentable over U.S. PGPUB 2018/0119140 A1 to Porteus (hereinafter ‘Porteus’, of record, shares at least one common inventor and assignee with the instant application) in view of Patent WIPO Publication 2021/138469 A1 to Zhang (hereinafter ‘Zhang’; published 07/08/2021, priority filing date 12/30/2019), as evidenced by Santa (Santa et al. Frontiers in Immunology, 02/25/2021, Vol 12, pages 1-25; of record), further in view of U.S. PGPUB 2023/0059368 to Cafferty (published 02/23/2023, priority filing date 06/15/2021)
Porteus, Zhang and Santa render obvious the method of claim 1, from which instantly rejected 21 depends, as described above.
Porteus, Zhang and Santa do not teach wherein the pegRNA also comprises 2’-O-methoxyethyl.
Cafferty teaches Cas prime editing systems with prime editing (PE) guide polynucleotides (Abstract), wherein the pegRNA comprises 2’-O-methoxyethyl (2’-MOE) modified nucleotides for improved resistance to RNAse cleavage (para [0288]).
It would have been prima facie obvious to a person having ordinary skill in the art before the effective filing date of the claimed invention to have modified the pegRNA as taught by Porteus, Zhang and Santa to further comprise 2’-MOE modifications. The skilled artisan would have been motivated by Cafferty’s teachings that this modification would have increased resistance to RNAse cleavage, thus increasing its stability in vivo.
U.S. PGPUB 2016/0289675 A1 to Ryan/Patent No. 10,900,034 to Ryan in view of WIPO Publication 2021/138469 A1 to Zhang
Claims 1-7, 9-11, 13, 15-17, 20 and 22 are rejected under 35 U.S.C. 103 as being unpatentable over U.S. PGPUB 2016/0289675 A1 to Ryan (hereinafter ‘Ryan’, of record, shares at least one common inventor and assignee with the instant application; patent issued 01/26/2021 with number 10,900,034; of record) in view of WIPO Publication 2021/138469 A1 to Zhang (hereinafter ‘Zhang’; published 07/08/2021, priority filing date 12/30/2019), as evidenced by Santa (Santa et al. Frontiers in Immunology, 02/25/2021, Vol 12, pages 1-25; of record).
Regarding claim 1, Ryan teaches an in vivo or in vitro method of CRISPR genome editing using a Cas endonuclease and a guide RNA with a guide sequence and scaffold:
[0180] As further described below, a guide RNA disclosed herein, including those comprising modified nucleotides and/or modified internucleotide linkages, can be used to perform various CRISPR-mediated functions (including but not limited to editing genes, regulating gene expression, cleaving target sequences, and binding to target sequences) in vitro or in vivo, such as in cell-free assays, in intact cells, or in whole organisms. For in vitro or in vivo applications, the RNA can be delivered into cells or whole organisms in any manner known in the art.
[0042] Shown in FIG. 1 is a diagram of CRISPR-Cas9-mediated sequence-specific cleavage of DNA. The guide RNA is depicted as sgRNA with an exemplary 20-nucleotide (20-nt) guide sequence (other guide sequences may be, for example, from about 15 to about 30 nts in length) within the 5′ domain, an internally positioned base-paired stem, and a 3′ domain. The guide sequence is complementary to an exemplary 20-nt target sequence in a DNA target. The stem corresponds to a repeat sequence in crRNA and is complementary to a sequence in the tracrRNA. The 3′ domain of the guide RNA corresponds to the 3′ domain of the tracrRNA that binds a Cas9 nuclease.
Ryan further teaches that the guide RNA has two or more phosphorothioate modifications at the 5’ end and two or more thiophosphonocarboxylate (thioPACE) modifications at the 3’ end, and that this increases editing efficiency:
[0248] In certain embodiments, the method employs an all-RNA delivery platform. For example, in some such embodiments, the guide RNA and the mRNA encoding the Cas protein are introduced into the cell simultaneously or substantially simultaneously (e.g., by co-transfection or co-nucleofection). In certain embodiments, co-delivery of Cas mRNA and modified gRNA results in higher editing frequencies as compared to co-delivery of Cas mRNA and unmodified gRNA. In particular, gRNA having 2′-O-methyl-3′-phosphorothioate (MS), or 2′-O-methyl-3′-thioPACE (MSP) incorporated at three terminal nucleotides at both the 5′ and 3′ ends, provide higher editing frequencies as compared to unmodified gRNA.
Ryan further teaches mRNAs encoding Cas fusion proteins wherein the Cas enzyme is fused to a variety of other proteins:
[0202] In certain embodiments, the Cas protein is fused to another protein or polypeptide heterologous to the Cas protein to create a fusion protein. In certain embodiments, the heterologous sequence includes one or more effector domains, such as a cleavage domain, a transcriptional activation domain, a transcriptional repressor domain, or an epigenetic modification domain.
Ryan teaches the method using a Cas nickase:
[0198] In some embodiments where one of the nuclease domains is inactive, the mutant is able to introduce a nick into a double-stranded polynucleotide (such protein is termed a “nickase”) but not able to cleave the double-stranded polynucleotide.
Ryans does not teach wherein the guide RNA is