DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 08/14/2026 has been entered.
It is noted that the amendment to the claims filed on 08/14/2026 does not comply with the requirements of 37 CFR 1.121(c) because there are markings present in newly added claim 103 (i.e., “comprises”). However, in the interest of compact prosecution, the amendment to the claims has been entered.
Status of the claims: In the amendments filed 08/14/2026, the claims were amended to (a) cancel claim 21, and (b) add 103-104.
Claim(s) 1, 24, 29-30, 41, 53, 60, 65-66, 69-70, 77-78, 81-82, 87, 96-97, and 102-104 are pending.
Applicant’s arguments filed 08/14/2026 have been thoroughly reviewed, but are partially persuasive for the reasons that follow. Any rejections and objections not reiterated in this action have been withdrawn. This action is NON-FINAL.
Election/Restrictions
Applicant’s election without traverse of Group I, drawn to a chemically modified CD33-targeting gRNA, in the reply filed on 12/03/2024 is acknowledged.
Applicant’s election without traverse of species that includes the chemically modified guide RNA of claim 1, wherein (m) the guide sequence comprises the nucleic acid sequence mG*mU*mC*AGUGACGGUACAGGA (SEQ ID NO: 2), wherein m indicates a 2'-O-methyl modification, and wherein * indicates a phosphorothioate modification the modified nucleic acid of SEQ ID NO: 1. The election reads on Group I. Election was made without traverse in the reply filed on 12/03/2024.
Claims 29-30, 41, 53, 60, 65-66, 69-70, 77-78, 81-82, and 87 were withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected subject matter, there being no allowable generic or linking claim.
The species (1) The chemically modified guide RNA of claim 1, selected from the modification or nucleic acid sequences as listed items (a) through (r) from claim 2, and (2) the guide sequence selected from SEQ ID NO: 1 and 16 from claim 21 (now cancelled), were rejoined and are reintroduced into examination in the Office Action mailed 02/27/2025.
Newly added claim 103-104 of the current claim set filed 08/14/2026, reads on Group I.
Claim(s) 1, 24, 96-97, and 102-104 are under consideration.
Priority
Acknowledgement is made that this application is a 371 of PCT/US2020/019370, filed 02/21/2020, which claims priority based on a provisional application filed as 62/809,448 on 02/22/2019.
All claims are given the priority date of 02/22/2019.
Specification
The use of at least the following term(s):
Alt-R (used throughout the specification at numerous locations);
Lipofectamine (p.67, line 9; p.80, line 4);
NEBNext (p. 89, line 20; p.90, line 16);
Puregene (p. 90, line 3);
Graphpad Prism (p. 90, line 27);
which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claim 96 is objected to because of the following informalities: It is understood that the variant S. pyogenes Cas9 is being compared to its own wild-type Cas9, however, for consistency, it would be remedial to add “sp” in front of “Cas9” in “wild-type spCas9 nuclease”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b) - Indefiniteness
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 104 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention. This is a new rejection.
Claim 104 contains the trademark/trade name(s) “SpyFiTM” and “Alt-R”. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe a cas9 variant, accordingly, the identification/description is indefinite.
Response to Arguments – Claim Rejections - 35 USC § 112(b) – Indefiniteness
The previous rejection of claim(s) 1, 24, 96, and 97 under 35 U.S.C 112(b) for the metes and bounds of the claim(s) being indefinite has been withdrawn in view of Applicant’s amendments to the claims filed 08/14/2026.
Claim Rejections - 35 USC § 112(d) – Improper Claim Dependency – New
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 102 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends. This is a new rejection in view of the amendments to the claims filed 08/14/2026.
Claim 102 does not include all the limitations from which it depends upon. Claim 96 requires the sequence of SEQ ID NO: 16. SEQ ID NO: 1 in claim 102 does not contain all of the nucleotides of SEQ ID NO: 16.
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Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Response to Arguments - Claim Rejections - 35 USC § 112(d) – Improper Claim Dependency
The previous rejection of claim 102 under 35 U.S.C 112(d) for improper claim dependency has been withdrawn in view of Applicant’s amendments of the claim filed 08/14/2026.
Claim Rejections - 35 USC § 112(a) – Written Description
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claim 24 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a new rejection.
The fundamental factual inquiry is whether the specification conveys with reasonable clarity to those skilled in the art that, as of the filing date sought, Applicant was in possession of the invention as now claimed. See, e.g., Vas-Cath, Inc., 935 F.2d at 1563-64, 19 USPQ2d at 1117.
Claim 24 is drawn to a genus of “variant Cas9 nuclease comprising reduced off-target activity and maintained or increased on-target activity relative to a wild-type cas9” for complexing with guide RNA. The rejected claims thus comprise a genus of “variant Cas9 nuclease comprising reduced off-target activity and maintained or increased on-target activity relative to a wild-type cas9” and are defined as belonging to the broad class of variant Cas9 nucleases comprising reduced off-target activity and maintained or increased on-target activity relative to a wild-type cas9 and as having the function of complexing with a guide RNA.
To satisfy the written description requirement, MPEP §2163 states, in part “… a patent
specification must describe the claimed invention in sufficient detail that one skilled in the art can reasonably conclude that the inventor had possession of the claimed invention.” Moreover, the written description requirement for a genus may be satisfied through sufficient description of a representative number of species by “… disclosure of relevant, identifying characteristics, i.e.,
structure or other physical and/or chemical properties, by functional characteristics coupled with
a known or disclosed correlation between functional and structure, or by a combination of such
identifying characteristics, sufficient to show the applicant was in possession of the claimed genus.”
The specification envisions the “variant Cas9 nuclease comprising reduced off-target activity and maintained or increased on-target activity relative to a wild-type cas9” as the following:
“RNA-guided nucleases that may be employed by the invention include, without limitation, naturally-occurring Type II CRISPR nucleases such as Cas9, as well as other nucleases derived or obtained therefrom. Exemplary Cas9 nucleases that may be used in the present invention include, but are not limited to, S. pyogenes Cas9 (SpCas9), S. aureus Cas9 (SaCas9), N. meningitidis Cas9 (NmCas9), C. jejuni Cas9 (CjCas9), and Geobacillus Cas9 (GeoCas9). In certain embodiments, the RNA-guided nuclease is a Cas9 nuclease. In certain embodiments, the RNA-guided nuclease is an S. pyogenes Cas9 nuclease or an S. aureus Cas9 nuclease. In certain embodiments, the RNA-guided nuclease is a variant S. pyogenes Cas9 nuclease. In certain embodiments, the Cas9 nuclease is an S. pyogenes Cas9 nuclease or an S.
aureus Cas9 nuclease. In certain embodiments, the Cas9 nuclease is a variant S. pyogenes Cas9
nuclease. In certain embodiments, the variant Cas9 nuclease comprises reduced off-target
activity, and maintained or increased on-target activity relative to a wild-type Cas9. In certain embodiments, the variant Cas9 nuclease is a SpyFiTM Cas9 nuclease.”, (p. 13, lines 21-25; also seen p.44, lines 19-31).
“In certain embodiments, the CRISPR/Cas is derived from a type II CRISPR/Cas system.
In other embodiments, the CRISPR/Cas system is derived from a Cas9 protein. The Cas9 protein
can be from Streptococcus pyogenes, Streptococcus thermophilus, or other species. In certain
embodiments, Cas9 can include: spCas9, Cpfl, CasY, CasX, or saCas9.”, (p.49, lines 6-9).
“In certain embodiments, the RNA-guided nuclease is a Cas9 nuclease. In certain
embodiments, the Cas9 nuclease is an S. pyogenes Cas9 nuclease or an S. aureus Cas9 nuclease.
In certain embodiments, the Cas9 nuclease is a variant S. pyogenes Cas9 nuclease. In certain
embodiments, the variant Cas9 nuclease comprises reduced off-target activity, and maintained or increased on-target activity relative to a wild-type Cas9 nuclease (e.g., a SpyFiTM Cas9 nuclease or a Alt-R HiFi Cas9 nuclease (e.g., a GMP-grade Alt-R HiFi Cas9 nuclease)). In certain embodiments, the RNA-guided nuclease is a variant Cas9 nuclease comprising reduced off-target activity, and maintained or increased on-target activity relative to a wild-type Cas9 nuclease, having an amino acid sequence set forth in SEQ ID NOs: 7-88 disclosed in PCT Publication No. WO2018/068053, the disclosure of which is herein incorporated by reference in its entirety. In
certain embodiments, the RNA-guided nuclease is a variant Cas9 nuclease comprising reduced
off-target activity, and maintained or increased on-target activity relative to a wild-type Cas9
nuclease, having an amino acid sequence set forth in SEQ ID NOs: 141-168 disclosed in PCT
Publication No. WO2019/074542, the disclosure of which is herein incorporated by reference in its entirety. In certain embodiments, the RNA-guided nuclease is a SpyFiTM Cas9 nuclease. In
certain embodiments, the RNA-guided nuclease is an Alt-R HiFi Cas9 nuclease. In certain
embodiments, the RNA-guided nuclease is a GMP-grade Alt-R HiFi Cas9 nuclease.”, (p.51, lines 11-27).
The examples described using spCas9 (p.90, line 13), using TFv2 (an spCas9) or SpyFiTM (p. 95, 29-31). Also see Figure 14-16).
Even if one accepts that the examples described in the specification meet the claim limitations of the rejected claims in regard to structure and function, the examples are only representative of two examples of SpCas9 variants comprising reduced off-target activity and maintained or increased on-target activity relative to a wild-type cas9 (a subgenus of Cas9 variant comprising reduced off-target activity and maintained or increased on-target activity relative to a wild-type cas9) comprising reduced off-target activity and maintained or increased on-target activity relative to a wild-type cas9 used for complexing with guide RNA. These results are not necessarily predictive of all “variant Cas9 nucleases comprising reduced off-target activity and maintained or increased on-target activity relative to a wild-type cas9” capable of complexing with a guide RNA. Thus, it is impossible for one to extrapolate from the two examples of spCas9 variants (subgenus) comprising reduced off-target activity and maintained or increased on-target activity relative to a wild-type cas9 described herein that the genus would necessarily meet the structural/functional characteristics of the rejected claims.
The prior art does not appear to offset the deficiencies of the instant specification in that it does not describe a set of “variant cas9 nuclease comprising reduced off-target activity and maintained or increased on-target activity relative to a wild-type cas9” capable of complexing with guide RNA.
Given its broadest reasonable interpretation, an “ortholog” is also a “variant”.
Yang et al (Superior Fidelity and Distinct Editing Outcomes of SaCas9 Compared with SpCas9 in Genome Editing, Genomics Proteomics Bioinformatics, vol 21, pages 1206-1220, published December 20th, 2022) teaches the comparison between two naturally occurring wild-type Cas9 orthologs, SaCas9 and SpCas9.
Yang et al teaches that “. . . we found that SaCas9 could edit the genome with greater efficiencies than SpCas9. . . Furthermore, SpCas9 exhibited a more substantial bias than SaCas9 for nonhomologous end-joining (NHEJ) +1 insertion at the fourth nucleotide upstream of the protospacer adjacent motif (PAM), indicating a characteristic of a staggered cut. Accordingly, editing with SaCas9 led to higher efficiencies of NHEJ-mediated double-stranded oligodeoxynucleotide (dsODN) insertion or homology-directed repair (HDR)-mediated adeno-associated virus serotype 6 (AAV6) donor knock-in. Finally, GUIDE-seq analysis revealed that SaCas9 exhibited significantly reduced off-target effects compared with SpCas9.”, (abstract).
Accordingly, Yang et al teaches that in Cas9 orthologs, one Cas9 (i.e., saCs9) can have significantly reduced off-target effects compared to SpCas9.
To better understand the genus of “variant Cas9 nuclease comprising reduced off-target activity and maintained or increased on-target activity relative to a wild-type cas9”,
Collias (CRISPR technologies and the search for the PAM-free nuclease, Nat Commun, vol 12, issue 555, pages 1-12, published January 22nd, 2021) teaches, “To date, over 900 distinct Cas9 homologs have been identified in sequenced genomes and metagenomes64, and more homologs likely await discovery with further sequencing efforts. Exploring this expanded set has yielded a wide assortment of Cas9 nucleases with varying PAM profiles, protein sizes, and optimal activity temperature.”, (p.3, col 2, para 2).
Further, Tyumentseva et al (CRISPR/Cas9 Landscape: Current State and Future Perspectives; Int J of Molecular Science, vol 24, issue 16077, pages 1-40; published November 8th, 2023) teaches, “Cas9 proteins are relatively large, with an average length of 1252 ± 165.8 aa (mean ± SD, range 961–1809 aa). More than 10,000 Cas9 orthologs are present among Bacillota, Bacteroidota, Pseudomonadota, and other phylums. Cas9 is most frequently found in microorganisms belonging to the Streptococcus, Listeria, Staphylococcus, Bacteroides, and Neisseria genera (Supplementary Table S3).
All Cas9 proteins can be roughly divided into two types: common-sized Cas9 proteins and miniature Cas9 proteins. Miniature Cas9 proteins are more advantageous in terms of delivery and belong to the Staphylococcaceae, Akkermansiaceae, Lactobacillaceae, Sphingomonadaceae, Corynebacteriaceae, Neisseriaceae, Bacillaceae, Pasteurellaceae, Comamonadaceae, Helicobacteraceae, and Campylobacteraceae families (Supplementary Table S4).
Many Cas9 orthologs (e.g., ScCas9 [177], CjCas9 [333], NmeCas9 [334], FnCas9 [335], SmacCas9 [336], SauriCas9 [337], St1Cas9 [338], St3Cas9 [339], FrCas9 [76], and so on [340,341,342,343,344,345,346,347,348,349,350,351,352,353,354,355,356,357,358]) have been reported to be intrinsically high-fidelity [76,77], and some of them were engineered with a broad PAM range, high fidelity, and/or both [359] (Supplementary Table S4).” ).”, (p.21 para 9 to p.22 para 2).
Therefore, the art does not appear to offset the deficiencies of the specification. Merely describing a “variant Cas9 nuclease comprising reduced off-target activity and maintained or increased on-target activity relative to a wild-type cas9” capable of complexing with a guide RNA without sufficient detail relating to the vast genus of Cas9 nuclease comprising reduced off-target activity and maintained or increased on-target activity relative to a wild-type cas9 in complexing with guide RNA does not allow the skilled artesian to reasonably conclude that the Applicants were in possession of the claimed invention in claim 24.
Amending the claim to recite “variant S. pyogenes Cas9 . . . relative to a wild-type spCas9 nuclease.”
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:
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.
Claim 1 is rejected under 35 U.S.C. 103 as being unpatentable over Gill et al (WO 2017/079400 A1; cited on IDS 11/17/2023) in view of Hendel et al (Chemically modified guide RNAs enhance CRISPR-Cas genome editing in human primary cells, nature biotechnology, vol 33, pages 985-989, published September 1st, 2015). This is a new rejection.
Gill et al teaches using the guide RNAs in CD34+ cells, T-cells, and HSPCs.
Regarding claim 1, Gill et al teaches the base gRNA sequence of SEQ ID NO: 2 (underlined in the figure 4C below) with 100% sequence identity for targeting or knocking out CD33 (pg. 46, lines 21-23; FIG. 4C; see SEQ ID search results; see image below).
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Gill et al teaches, “The sequence of the guide nucleic acid sequence may be within a locus of the gene. In one embodiment, the guide nucleic acid sequence is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or more nucleotides in length.”, (p.24, lines 2-5).
Gill et al teaches that CRISPR/Cas system comprises spacer sequences integrated, transcribed and processed into CRISPR RNAs or crRNAs that anneal to trans-activating crRNAs or tracrRNAs (i.e. interpreted as tracrRNA binding and complementary to the spacer sequence of the crRNA and thereby rendering it an anti-repeat sequence) (pg. 12, lines 21-23).
Gill et al teaches that a seed target recognition sequence (i.e. capable of binding or hybridizing the target gene) is located within the crRNA and a conserved PAM motif (pg. 12, lines 25-27).
Gill et al teaches that CRISPR/Cas system comprises palindromic repeats or short repetition of base sequences that is followed by the spacer region (i.e. indicating the conserved repeat sequence in the crRNA/tracrRNA of gRNA acquired in the bacterial CRISPR system) (pg. 12, lines 14-20).
Gill et al teaches that crRNA-tracrRNA within gRNA contribute to direct Cas9 to cleave sequence of interest (i.e. the endogenous gene encoding CD33) (pg. 12, lines 28-29).
Gill et al does not teach a chemically modified CD-33 targeting guide RNA.
Hendel et al teaches, “[g]uide RNA is composed of two RNAs termed CRISPR RNA (crRNA) and trans-activating crRNA, which can be combined in a chimeric single guide RNA (sgRNA). The sgRNAs are typically about 100 nucleotides (nt) long. Twenty nt at the 5′ end hybridize to a target DNA sequence by Watson-Crick base pairing and guide the Cas endonuclease to cleave the target genomic DNA, and the remaining double-stranded structure at the 3′ side is critical for Cas9 recognition (Fig. 1a). The sgRNA has been delivered into cells as RNA (e.g., prepared by in vitro transcription), or by using a DNA vector expressing the sgRNA.”, (p. 985, col 1, para 1).
Further, Hendel et al goes on to teach, “Although genome editing using the CRISPR-Cas system is highly efficient in human cell lines, CRISPR-Cas genome editing in primary human cells is generally more challenging. The reasons for this remain elusive, but differences in transfection rates, promoter activity, exonuclease activity, interferon production when delivering nucleic acids, and DNA repair fidelity may contribute. Here we demonstrate that chemically synthesized sgRNAs can induce high levels of genome editing and that chemical alterations of the sgRNAs can enhance genome editing in both human primary T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs).”, (p. 985, col 1, para 2 to col 2, para 1).
Regarding claim 1, Hendel et al teaches, “To test the utility of chemically synthesized sgRNAs for genome editing, we synthesized full-length sgRNAs of 100 nt using 2′-O-thionocarbamate-protected nucleoside phosphoramidites We also synthesized sgRNAs with three different chemical modifications at both termini to evaluate their effects on efficacy (Fig. 1a,b and Supplementary Table 1). 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 the 5′ and 3′ ends. We selected these modifications for evaluation owing to their previously reported stability to serum and snake venom phosphodiesterases and for their reported effects on the immunostimulatory properties of nucleic acids.”, (p.985, col 2, para 2).
Hendel et al teaches that MS modified guide RNAs have higher indel rates than unmodified or only 2’-O-methyl modifications in Figure 1 C, E-G and Figure 2 A-D.
Hendel et al teaches, “we show that chemically synthesized sgRNAs can be used effectively for targeted genome editing, and we demonstrate that chemically modified sgRNAs significantly enhance genome editing efficiencies in human primary T cells and CD34+ HSPCs. Chemically synthesized and modified sgRNAs offer advantages over expressed or in vitro transcribed sgRNAs, including (i) increased efficacy, (ii) robust and scalable production of highly pure sgRNAs for biotechnological and therapeutic applications, (iii) greater flexibility in the sgRNA design in contrast to constraints on the first transcribed nucleotides imposed by the U6 or T7 promoters typically used for plasmid expression or in vitro transcription of sgRNAs, respectively, and (iv) enabling of a highly active RNA-only or RNP CRISPR platform with lower cytotoxicity in primary cells than DNA plasmid-based systems.”, (p. 989, col 1, para 2).
Lastly, Hendel et al teaches, “The simplification of the CRISPR-Cas system to a purely RNA or RNP CRISPR system lends itself to formulation in different nano particle vectors for delivery in vivo such that the nuclease will not be expressed continuously, as it would be when delivered as part of a viral vector. Furthermore, we anticipate that chemically modified sgRNAs will enhance multiplexed genome editing as well as high throughput, multiwell experiments. We also anticipate that modified sgRNAs will refine a wide range of CRISPR-associated technologies such as the CRISPRi/CRISPRa systems for inhibition and activation of gene expression, the CRISPR imaging tool for dynamic visualization of genomic loci, and CRISPR-mediated RNA recognition and cleavage. The technology may be further developed to improve intracellular delivery to target cells or tissues, and enable conjugation to various molecules for imaging and biochemical studies.”, (p. 989, col 1, para 3).
Therefore, it would have been obvious to one of skill in the art before the effective filing date of the claimed invention to modify the teachings of Gill et al, i.e., the CD33 guide RNA, with the teachings of Hendel et al, i.e., chemically modifying the three 5’ terminal nucleotides with 2’-O-methyl and phosphorothioate linkages of sgRNA, to yield to predictable results of a CD33 guide RNA with 2’-O-methyl and phosphorothioate modification on the three 5’ terminal nucleotides.
One of skill would be motivated to modify such teachings, i.e., Gill et al with Hendel et al, because (1) Gill et al teaches (a) a sgRNA targeting CD33 comprising a sequence that is 100% identical to instant SEQ ID NO: 2 for use in HSPCs and (b) a guide sequence can be 18 nucleotides in length; and (2) Hendel et al teaches that (c) chemically synthesized and modified sgRNAs offer advantages over expressed or in vitro transcribed sgRNAs (in vitro is what is used in Gill et al), (d) chemically modified sgRNAs significantly enhance genome editing efficiencies in human primary T cells and CD34+ HSPCs (same cell types that are used in Gill et al), (e) chemically modified sgRNAs have increased efficacy, (f) chemically modified sgRNAs have robust and scalable production of highly pure sgRNAs for biotechnological and therapeutic applications, (g) chemically modified sgRNAs have greater flexibility in the sgRNA design in contrast to constraints on the first transcribed nucleotides imposed by the U6 or T7 promoters typically used for plasmid expression or in vitro transcription of sgRNAs, respectively, and (h) chemically modified sgRNAs enable a highly active RNA-only or RNP CRISPR platform with lower cytotoxicity in primary cells than DNA plasmid-based systems.
One of skill would look to the disclosures of both Gill et al and Hendel et al and arrive at the claimed invention with a high likelihood of success.
Accordingly, claim 1 is unpatentable over Gill et al in view of Hendel et al.
Claim 24 is rejected under 35 U.S.C. 103 as being unpatentable over Gill et al (supra) in view of Hendel et al (supra) as applied to claim 1 above, and in further view of Vakulskas et al (A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human haematopoietic stem and progenitor cells, nat med, col 24, issue 8, pages 1216-1224, published August 6th, 2018). This is a new rejection.
Gill et al and Hendel et al teach Cas9, but do not teach a Cas9 variant. Hendel et al teaches Cas9 mRNA or protein, not a plasmid or IVT, complexed to a chemically modified guide RNA, but does not teach that such Cas9 mRNA or protein is a variant Cas9 comprising reduced off-target activity, and maintained or increased on-target activity relative to a wild-type Cas9.
Of note: SpyFiTM and Alt-R Hifi Cas9 are being interpreted by their generic structural modification, which is an R691A mutation compared to wild-type spCas9.
Regarding claim 24, Vakulskas et al teaches, “we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, R691A (HiFi Cas9), retained high on-target activity while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically-relevant loci (HBB, IL2RG, CCR5, HEXB, TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T-cells.”, (abstract).
Accordingly, it would have been obvious to one of skill in the art before the effective filing date of the claimed invention to substitute the Cas9 protein as taught by the combination of Gill et al in view of Hendel et al, with the HiFi Cas9 protein, as taught by Vakulskas et al. Both Cas9 protein and Hifi Cas9 protein were known in the art before the effective filing date of the claimed invention. Vakulskas et al teaches that HiFi Cas9 retained high on-target activity while reducing off-target editing, namely in HSPCs, the same cell type that Gill et al teaches editing in. One of skill in the art could look to the disclosures of Gill et al in view Vakulskas et al and arrived at the claimed invention with a high likelihood of success. One would have been motivated to make such a substitution in order to provide a composition capable of providing editing with reduced off-target activity as taught by Vakulskas et al.
Accordingly, claim 24 is unpatentable over Gill et al in view of Hendel et al in further view of Vakulskas et al .
Claim(s) 96-97 and 104 are rejected under 35 U.S.C. 103 as being unpatentable over Gill et al (WO 2017/079400 A1; cited on IDS 11/17/2023) in view of Vakulskas et al (supra). This is a new rejection.
Of note: SpyFiTM and Alt-R Hifi Cas9 are being interpreted by their generic structural modification, which is an R691A mutation compared to wild-type spCas9.
Gill et al teaches that CRISPR system comprises a Cas9 protein complexed with a guide nucleic acid sequence specific for the endogenous gene wherein the endogenous gene encodes CD33 (pg. 2, lines 26-33).
Gill et al teaches that the guide RNA (gRNA) sequence can be specific for any endogenous gene that is involved in chimeric antigen receptor (CAR) T cell therapy targeting antigens such as CD33 and wherein the gRNA is specific for endogenous gene that encodes CD33 (pg. 24, lines 6-11).
Gill et al teaches that CRISPR/Cas system comprises spacer sequences integrated, transcribed and processed into CRISPR RNAs or crRNAs that anneal to trans-activating crRNAs or tracrRNAs (i.e. interpreted as tracrRNA binding and complementary to the spacer sequence of the crRNA and thereby rendering it an anti-repeat sequence) (pg. 12, lines 21-23).
Gill et al teaches that a seed target recognition sequence (i.e. capable of binding or hybridizing the target gene) is located within the crRNA and a conserved PAM motif (pg. 12, lines 25-27).
Gill et al teaches that CRISPR/Cas system comprises palindromic repeats or short repetition of base sequences that is followed by the spacer region (i.e. indicating the conserved repeat sequence in the crRNA/tracrRNA of gRNA acquired in the bacterial CRISPR system) (pg. 12, lines 14-20).
Gill et al teaches that crRNA-tracrRNA within gRNA contribute to direct Cas9 to cleave sequence of interest (i.e. the endogenous gene encoding CD33) (pg. 12, lines 28-29).
Gill et al teaches that the CRISPR system comprises a Cas9 protein complexed with a guide nucleic (reading on the combination of tracrRNA and crRNA, as taught above) acid sequence specific for the endogenous gene (i.e. CD33) (pg. 2, lines 26-27; and Fig. 9A).
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Gill et al teaches the base gRNA consisting of sequence SEQ ID NO: 16 (highlighted by the line overlaying the sequence with CD33-gRNA) with 100% sequence identity for targeting or knocking out CD33 (pg. 46, lines 21-23; FIG. 4C; see SEQ ID search results; see image below).
Gill et al does not teach a variant S. pyogenes Cas 9 nuclease comprising reduced off-target activity and maintained or increased on-target activity relative to a wild-type Cas9 nuclease.
Regarding claim 96 and 104, Vakulskas et al teaches, “we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, R691A (HiFi Cas9), retained high on-target activity while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically-relevant loci (HBB, IL2RG, CCR5, HEXB, TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T-cells.”, (abstract).
Accordingly, it would have been obvious to one of skill in the art before the effective filing date of the claimed invention to substitute the Cas9 protein as taught by Gill et al, with the HiFi Cas9 protein, as taught by Vakulskas et al. Both Cas9 protein and Hifi Cas9 protein were known in the art before the effective filing date of the claimed invention. Vakulskas et al teaches that HiFi Cas9 retained high on-target activity while reducing off-target editing, namely in HSPCs, the same cell type that Gill et al teaches editing in. One of skill in the art could look to the disclosures of Gill et al in view Vakulskas et al and arrived at the claimed invention with a high likelihood of success. One would have been motivated to make such a substitution in order to provide a composition capable of providing editing with reduced off-target activity as taught by Vakulskas et al.
Accordingly, claim(s) 96-97 and 104 are unpatentable over Gill et al in view of Vakulskas et al.
Claim 103 is rejected under 35 U.S.C. 103 as being unpatentable over Gill et al (supra) in view of Vakulskas et al (supra) as applied to claim 96-97 and 104 above, and further in view of Hendel et al (supra).
Gill et al and Vakulskas et al do not teach a chemically modified CD-33 targeting guide RNA.
Hendel et al teaches, “[g]uide RNA is composed of two RNAs termed CRISPR RNA (crRNA) and trans-activating crRNA, which can be combined in a chimeric single guide RNA (sgRNA). The sgRNAs are typically about 100 nucleotides (nt) long. Twenty nt at the 5′ end hybridize to a target DNA sequence by Watson-Crick base pairing and guide the Cas endonuclease to cleave the target genomic DNA, and the remaining double-stranded structure at the 3′ side is critical for Cas9 recognition (Fig. 1a). The sgRNA has been delivered into cells as RNA (e.g., prepared by in vitro transcription), or by using a DNA vector expressing the sgRNA.”, (p. 985, col 1, para 1).
Further, Hendel et al goes on to teach, “Although genome editing using the CRISPR-Cas system is highly efficient in human cell lines, CRISPR-Cas genome editing in primary human cells is generally more challenging. The reasons for this remain elusive, but differences in transfection rates, promoter activity, exonuclease activity, interferon production when delivering nucleic acids, and DNA repair fidelity may contribute. Here we demonstrate that chemically synthesized sgRNAs can induce high levels of genome editing and that chemical alterations of the sgRNAs can enhance genome editing in both human primary T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs).”, (p. 985, col 1, para 2 to col 2, para 1).
Regarding claim 1, Hendel et al teaches, “To test the utility of chemically synthesized sgRNAs for genome editing, we synthesized full-length sgRNAs of 100 nt using 2′-O-thionocarbamate-protected nucleoside phosphoramidites. We also synthesized sgRNAs with three different chemical modifications at both termini to evaluate their effects on efficacy (Fig. 1a,b and Supplementary Table 1). 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 the 5′ and 3′ ends. We selected these modifications for evaluation owing to their previously reported stability to serum and snake venom phosphodiesterases and for their reported effects on the immunostimulatory properties of nucleic acids.”, (p.985, col 2, para 2).
Hendel et al teaches that MS modified guide RNAs have higher indel rates than unmodified or only 2’-O-methyl modifications in Figure 1 C, E-G and Figure 2 A-D.
Hendel et al teaches, “we show that chemically synthesized sgRNAs can be used effectively for targeted genome editing, and we demonstrate that chemically modified sgRNAs significantly enhance genome editing efficiencies in human primary T cells and CD34+ HSPCs. Chemically synthesized and modified sgRNAs offer advantages over expressed or in vitro transcribed sgRNAs, including (i) increased efficacy, (ii) robust and scalable production of highly pure sgRNAs for biotechnological and therapeutic applications, (iii) greater flexibility in the sgRNA design in contrast to constraints on the first transcribed nucleotides imposed by the U6 or T7 promoters typically used for plasmid expression or in vitro transcription of sgRNAs, respectively, and (iv) enabling of a highly active RNA-only or RNP CRISPR platform with lower cytotoxicity in primary cells than DNA plasmid-based systems.”, (p. 989, col 1, para 2).
Lastly, Hendel et al teaches, “The simplification of the CRISPR-Cas system to a purely RNA or RNP CRISPR system lends itself to formulation in different nano particle vectors for delivery in vivo such that the nuclease will not be expressed continuously, as it would be when delivered as part of a viral vector. Furthermore, we anticipate that chemically modified sgRNAs will enhance multiplexed genome editing as well as high throughput, multiwell experiments. We also anticipate that modified sgRNAs will refine a wide range of CRISPR-associated technologies such as the CRISPRi/CRISPRa systems for inhibition and activation of gene expression19, the CRISPR imaging tool for dynamic visualization of genomic loci20, and CRISPR-mediated RNA recognition and cleavage21. The technology may be further developed to improve intracellular delivery to target cells or tissues, and enable conjugation to various molecules for imaging and biochemical studies.”, (p. 989, col 1, para 3).
Therefore, it would have been obvious to one of skill in the art before the effective filing date of the claimed invention to modify the teachings of Gill et al in view of Vakulskas et al, i.e., the CD33 guide RNA complexed with the Cas9 variant - Hifi-Cas9, with the teachings of Hendel et al, i.e., chemically modifying the three 5’ terminal nucleotides with 2’-O-methyl and phosphorothioate linkages of sgRNA, to yield to predictable results of a CD33 guide RNA with 2’-O-methyl and phosphorothioate modification on the three 5’ terminal nucleotides.
One of skill would be motivated to modify such teachings, i.e., Gill et al in view of Vakulskas et al with Hendel et al, because (1) Gill et al in view of Vakulskas et al teaches (a) a sgRNA targeting CD33 comprising a sequence that is 100% identical to instant SEQ ID NO: 16 for use in HSPCs complexed with a Hifi-Cas9; and (2) Hendel et al teaches that (c) chemically synthesized and modified sgRNAs offer advantages over expressed or in vitro transcribed sgRNAs (in vitro is what is used in Gill et al), (d) chemically modified sgRNAs significantly enhance genome editing efficiencies in human primary T cells and CD34+ HSPCs (same cell types that are used in Gill et al), (e) chemically modified sgRNAs have increased efficacy, (f) chemically modified sgRNAs have robust and scalable production of highly pure sgRNAs for biotechnological and therapeutic applications, (g) chemically modified sgRNAs have greater flexibility in the sgRNA design in contrast to constraints on the first transcribed nucleotides imposed by the U6 or T7 promoters typically used for plasmid expression or in vitro transcription of sgRNAs, respectively, and (h) chemically modified sgRNAs enable a highly active RNA-only or RNP CRISPR platform with lower cytotoxicity in primary cells than DNA plasmid-based systems.
One of skill would look to the disclosures of both Gill et al in view of Vakulskas et al and Hendel et al and arrive at the claimed invention with a high likelihood of success.
Accordingly, claim 103 is unpatentable over Gill et al in view of Vakulskas et al in further view of Hendel et al.
Response to Arguments - Claim Rejections - 35 USC § 103
The previous rejection of claim 21 under 35 U.S.C 103 for being unpatentable over Gill et al in view of Bolen et al (WO2018160768A1; cited on PTO 892 filed 02/27/2025) is moot in in view of Applicant’s cancellation of the claim filed 08/14/2026.
Applicant’s claim amendments and arguments, see page 12 of reply filed 08/14/2026, with respect to the rejection of claim 1, 24, and 96-97 under 35 U.S.C 103 for unpatentable over Gill et al (supra) in view of Bolen et al (supra) have been fully considered and are persuasive. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made in view of newly found prior art references.
Regarding Applicant’s arguments: Applicant points to (a) Gill et al preparing guide RNAs by in vitro transcription (unmodified), (b) Gill et al does not teaching nor suggesting modifying the guide, and (c) Gill et al achieved high editing efficiency using unmodified, in vitro transcribed so a person of skill would have no motivated to introduce recited chemical modifications. Applicant points to (d) Bolen et al only reporting its modification as an incidental, standard synthesis received from a commercial vendor, and (e) Bolen et al does not compare its guides to unmodified guides nor attribute any benefit to the modification, all in all leading to impermissible hindsight.
Examiner agrees with the Applicant for all of the reasons listed above, namely that despite Bolen et al using modified guides to target CD33, Bolen does not teach a motivation to do so. Accordingly, new ground(s) of rejects has been made in view of newly found prior art.
The previous rejection of claim 102 under 35 U.S.C 103 for being unpatentable over Gill et al (supra) in view of Bolen (supra) et al in further view of Fu et al (Improving CRISPR-Cas nuclease specificity using truncated guide RNAs, Nat Biotechnol, Vol 32, Issue 3, page 279-284, Published January 26th, 2014; listed on the PTO 892 mailed 05/14/2026) has been withdrawn in in view of Applicant’s amendments of the claim filed 08/14/2026.
With respect to the rejection of claim 102 under 35 U.S.C. 103, Applicant’s arguments at page 14 in the reply filed 08/14/2026 has been fully considered but is not persuasive.
Applicant argues unexpected results, namely that full-length guide sequences (20 nucleotides) as reflected in claims 96-97 and 103 combined with a variant high-fidelity cas9 nuclease unexpectedly produce the lowest-off target activity among the combinations tested – lower than the corresponding truncated guide (18 nucleotides).
Examiner would like to point out that in the amendments filed 08/14/2026, Applicant amended claim 96 to introduce the limitation of a variant cas9 nuclease. This limitation was not present when the rejection of dependent claim 102 for being unpatentable over Gill et al in view of Bolen et al in further view of Fu et al was made regarding truncated sgRNA’s maintaining optimal guide efficiency and reduce indel frequency. Examiner agrees that this feature of the high-fidelity variant in combination with the 18-nucleotide guide RNA would not be taught by Fu et al because the art explicitly teaches away from high-fidelity spCas9 variants in combination with truncated guides, e.g., that is not unexpected. Kulcsár et al (Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage, Genome Biology, vol 18, issue 190, pages 1-17, published October 6th, 2017) teaches, “In further experiments, we systematically examined and showed, confirming also earlier reports [32, 33], that the routine application of the increased fidelity nucleases is not compatible with modified sgRNAs that are generated by commonly applied approaches such as (i) altering the non-G 5′ end nucleotide to a G, (ii) using the spacers without alteration, i.e., with the 5′ end non-G nucleotide, or (iii) truncating back the guide until a G nucleotide is encountered, resulting in spacers between 19 and 17 nucleotides in length (Additional file 1: Figure S2e, f).”, (p. 3, col 1, para 1).
Thus, in light of the amendment to claim 96 introducing the variant limitation, it would not be obvious to utilize the teachings of Fu et al. Therefore, the rejection of claim 102 has been withdrawn.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claim 1 is rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 10548922 B2 in view of Hendel et al (supra). This is a new rejection.
Claim 1 of 922 recites, “A method for generating a population of modified human hematopoietic stem and progenitor cells (HSPCs) that are resistant to a CD33 CAR-T therapy, comprising: introducing into the HSPCs a CRISPR system comprising a guide nucleic acid, wherein the CRISPR system produces an insertion and/or deletion in an endogenous CD33 coding sequence comprising the nucleic acid sequence set forth in SEQ ID NO: 4, and wherein the insertion and/or deletion is capable of downregulating gene expression of CD33, thereby generating the population of modified human HSPCs.”
Of note: 922 defines guide nucleic acid as “crRNA-tracrRNA fusion transcripts, hereafter referred to as “guide RNAs” or “gRNAs””, (see col 10, lines 7-14).
SEQ ID NO: 4 of 922 is 100% match to instant SEQ ID NO: 2 (unmodified).
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Claim 1 of 922 does not require chemical modifications of the guide RNA.
Hendel et al teaches, “[g]uide RNA is composed of two RNAs termed CRISPR RNA (crRNA) and trans-activating crRNA, which can be combined in a chimeric single guide RNA (sgRNA). The sgRNAs are typically about 100 nucleotides (nt) long. Twenty nt at the 5′ end hybridize to a target DNA sequence by Watson-Crick base pairing and guide the Cas endonuclease to cleave the target genomic DNA, and the remaining double-stranded structure at the 3′ side is critical for Cas9 recognition (Fig. 1a). The sgRNA has been delivered into cells as RNA (e.g., prepared by in vitro transcription), or by using a DNA vector expressing the sgRNA.”, (p. 985, col 1, para 1).
Further, Hendel et al goes on to teach, “Although genome editing using the CRISPR-Cas system is highly efficient in human cell lines, CRISPR-Cas genome editing in primary human cells is generally more challenging. The reasons for this remain elusive, but differences in transfection rates, promoter activity, exonuclease activity, interferon production when delivering nucleic acids, and DNA repair fidelity may contribute. Here we demonstrate that chemically synthesized sgRNAs can induce high levels of genome editing and that chemical alterations of the sgRNAs can enhance genome editing in both human primary T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs).”, (p. 985, col 1, para 2 to col 2, para 1).
Regarding claim 1, Hendel et al teaches, “To test the utility of chemically synthesized sgRNAs for genome editing, we synthesized full-length sgRNAs of 100 nt using 2′-O-thionocarbamate-protected nucleoside phosphoramidites. We also synthesized sgRNAs with three different chemical modifications at both termini to evaluate their effects on efficacy (Fig. 1a,b and Supplementary Table 1). 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 the 5′ and 3′ ends. We selected these modifications for evaluation owing to their previously reported stability to serum and snake venom phosphodiesterases and for their reported effects on the immunostimulatory properties of nucleic acids.”, (p.985, col 2, para 2).
Hendel et al teaches that MS modified guide RNAs have higher indel rates than unmodified or only 2’-O-methyl modifications in Figure 1 C, E-G and Figure 2 A-D.
Hendel et al teaches, “we show that chemically synthesized sgRNAs can be used effectively for targeted genome editing, and we demonstrate that chemically modified sgRNAs significantly enhance genome editing efficiencies in human primary T cells and CD34+ HSPCs. Chemically synthesized and modified sgRNAs offer advantages over expressed or in vitro transcribed sgRNAs, including (i) increased efficacy, (ii) robust and scalable production of highly pure sgRNAs for biotechnological and therapeutic applications, (iii) greater flexibility in the sgRNA design in contrast to constraints on the first transcribed nucleotides imposed by the U6 or T7 promoters typically used for plasmid expression or in vitro transcription of sgRNAs, respectively, and (iv) enabling of a highly active RNA-only or RNP CRISPR platform with lower cytotoxicity in primary cells than DNA plasmid-based systems.”, (p. 989, col 1, para 2).
Lastly, Hendel et al teaches, “The simplification of the CRISPR-Cas system to a purely RNA or RNP CRISPR system lends itself to formulation in different nano particle vectors for delivery in vivo such that the nuclease will not be expressed continuously, as it would be when delivered as part of a viral vector. Furthermore, we anticipate that chemically modified sgRNAs will enhance multiplexed genome editing as well as high throughput, multiwell experiments. We also anticipate that modified sgRNAs will refine a wide range of CRISPR-associated technologies such as the CRISPRi/CRISPRa systems for inhibition and activation of gene expression19, the CRISPR imaging tool for dynamic visualization of genomic loci20, and CRISPR-mediated RNA recognition and cleavage21. The technology may be further developed to improve intracellular delivery to target cells or tissues, and enable conjugation to various molecules for imaging and biochemical studies.”, (p. 989, col 1, para 3).
Therefore, it would have been obvious to one of skill in the art before the effective filing date of the claimed invention to modify the teachings of claim 1 of 922 i.e., SEQ ID NO: 4, with the teachings of Hendel et al, i.e., chemically modifying three 5’ terminal nucleotides with 2’-O-methyl and phosphorothioate linkages of sgRNA, to yield to predictable results of a CD33 guide RNA with 2’-O-methyl and phosphorothioate modification on the three 5’ terminal nucleotides.
One of skill would be motivated to modify such teachings, i.e., claim 1 of 922 with Hendel et al, because (1) claim 1 of 922 requires (a) a sgRNA targeting CD33 comprising a sequence that is 100% match to instant SEQ ID NO: 2 for use in HSPCs; and (2) Hendel et al teaches that (c) chemically synthesized and modified sgRNAs offer advantages over expressed or in vitro transcribed sgRNAs, (d) chemically modified sgRNAs significantly enhance genome editing efficiencies in human primary T cells and CD34+ HSPCs (same cell types that are used in claim 1 of 922), (e) chemically modified sgRNAs have increased efficacy, (f) chemically modified sgRNAs have robust and scalable production of highly pure sgRNAs for biotechnological and therapeutic applications, (g) chemically modified sgRNAs have greater flexibility in the sgRNA design in contrast to constraints on the first transcribed nucleotides imposed by the U6 or T7 promoters typically used for plasmid expression or in vitro transcription of sgRNAs, respectively, and (h) chemically modified sgRNAs enable a highly active RNA-only or RNP CRISPR platform with lower cytotoxicity in primary cells than DNA plasmid-based systems.
One of skill would look to the disclosures of both claim 1 of 922 and Hendel et al and arrive at the claimed invention with a high likelihood of success.
Accordingly, Claim 1 is rejected on the ground of nonstatutory double patenting as being unpatentable over U.S. Patent No. 10548922 B2 in view of Hendel et al.
Claim 24 is rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 10548922 B2 in view of Hendel et al (supra) as applied to claim 1 above, in further view of Vakulskas et al (supra). This is a new rejection.
Of note: SpyFiTM and Alt-R Hifi Cas9 are being interpreted by their generic structural modification, which is an R691A mutation compared to wild-type spCas9.
Claim 1 of 922 and Hendel et al teach a CRISPR/Cas system, but do not teach a Cas9 variant. Hendel et al teaches Cas9 mRNA or protein, not a plasmid or IVT, complexed to a chemically modified guide RNA, but does not teach that such Cas9 mRNA or protein is a variant Cas9 comprising reduced off-target activity, and maintained or increased on-target activity relative to a wild-type Cas9.
Regarding claim 24, Vakulskas et al teaches, “we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, R691A (HiFi Cas9), retained high on-target activity while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically-relevant loci (HBB, IL2RG, CCR5, HEXB, TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T-cells.”, (abstract).
Accordingly, it would have been obvious to one of skill in the art before the effective filing date of the claimed invention to substitute the Cas9 protein as taught by the combination of claim 1 of 922 in view of Hendel et al, with the HiFi Cas9 protein, as taught by Vakulskas et al. Both Cas9 protein and Hifi Cas9 protein were known in the art before the effective filing date of the claimed invention. Vakulskas et al teaches that HiFi Cas9 retained high on-target activity while reducing off-target editing, namely in HSPCs, the same cell type that claim 1 of 922 requires editing in. One of skill in the art could look to the disclosures of claim 1 of 922 in view of Hendel et al and Vakulskas et al and arrived at the claimed invention with a high likelihood of success. One would have been motivated to make such a substitution in order to provide a composition capable of providing editing with reduced off-target activity as taught by Vakulskas et al.
Accordingly, claim 24 is rejected on the ground of nonstatutory double patenting as being unpatentable over U.S. Patent No. 10548922 B2 in view of Hendel et al in further view of Vakulskas et al.
Claim(s) 96-97 and 104 is rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 10548922 B2 in view of Vakulskas et al (supra). This is a new rejection.
Claim 1 of 922 recites, “A method for generating a population of modified human hematopoietic stem and progenitor cells (HSPCs) that are resistant to a CD33 CAR-T therapy, comprising: introducing into the HSPCs a CRISPR system comprising a guide nucleic acid, wherein the CRISPR system produces an insertion and/or deletion in an endogenous CD33 coding sequence comprising the nucleic acid sequence set forth in SEQ ID NO: 4, and wherein the insertion and/or deletion is capable of downregulating gene expression of CD33, thereby generating the population of modified human HSPCs.”
Of note: 922 defines guide nucleic acid as “crRNA-tracrRNA fusion transcripts, hereafter referred to as “guide RNAs” or “gRNAs””, (see col 10, lines 7-14).
SEQ ID NO: 4 of 922 is 100% match compared to instant SEQ ID NO: 16.
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Claim 1 of 922 does not require Cas9 in the CRIRSPR system to be a variant spCas 9 nuclease.
Vakulskas et al teaches, “we devised an unbiased bacterial screen to isolate variants that retain activity in the RNP format. Introduction of a single point mutation, R691A (HiFi Cas9), retained high on-target activity while reducing off-target editing. HiFi Cas9 induces robust AAV6-mediated gene targeting at five therapeutically-relevant loci (HBB, IL2RG, CCR5, HEXB, TRAC) in human CD34+ hematopoietic stem and progenitor cells (HSPCs) as well as primary T-cells.”, (abstract).
Accordingly, it would have been obvious to one of skill in the art before the effective filing date of the claimed invention to substitute the Cas9 protein of the CRISPR system as taught by claim 1 of 922, with the HiFi Cas9 protein, as taught by Vakulskas et al. Both Cas9 protein and Hifi Cas9 protein were known in the art before the effective filing date of the claimed invention. Vakulskas et al teaches that HiFi Cas9 retained high on-target activity while reducing off-target editing, namely in HSPCs, the same cell type that claim 1 of 922 teaches editing in. One of skill in the art could look to the disclosures of claim of 922 in view Vakulskas et al and arrived at the claimed invention with a high likelihood of success.
Accordingly, claim 96-97 and 104 are rejected on the ground of nonstatutory double patenting as being unpatentable over U.S. Patent No. 10548922 B2 in view of Vakulskas et al.
Claim 103 is rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1 of U.S. Patent No. 10548922 B2 in view Vakulskas et al (supra) as applied to claims 96-97 and 104 above, in further view of Hendel et al (supra). This is a new rejection.
Claim 1 of 922 and Vakulskas et al do not require chemical modifications of the guide RNA.
Hendel et al teaches, “[g]uide RNA is composed of two RNAs termed CRISPR RNA (crRNA) and trans-activating crRNA, which can be combined in a chimeric single guide RNA (sgRNA). The sgRNAs are typically about 100 nucleotides (nt) long. Twenty nt at the 5′ end hybridize to a target DNA sequence by Watson-Crick base pairing and guide the Cas endonuclease to cleave the target genomic DNA, and the remaining double-stranded structure at the 3′ side is critical for Cas9 recognition (Fig. 1a). The sgRNA has been delivered into cells as RNA (e.g., prepared by in vitro transcription), or by using a DNA vector expressing the sgRNA.”, (p. 985, col 1, para 1).
Further, Hendel et al goes on to teach, “Although genome editing using the CRISPR-Cas system is highly efficient in human cell lines, CRISPR-Cas genome editing in primary human cells is generally more challenging. The reasons for this remain elusive, but differences in transfection rates, promoter activity, exonuclease activity, interferon production when delivering nucleic acids, and DNA repair fidelity may contribute. Here we demonstrate that chemically synthesized sgRNAs can induce high levels of genome editing and that chemical alterations of the sgRNAs can enhance genome editing in both human primary T cells and CD34+ hematopoietic stem and progenitor cells (HSPCs).”, (p. 985, col 1, para 2 to col 2, para 1).
Hendel et al teaches, “To test the utility of chemically synthesized sgRNAs for genome editing, we synthesized full-length sgRNAs of 100 nt using 2′-O-thionocarbamate-protected nucleoside phosphoramidites6. We also synthesized sgRNAs with three different chemical modifications at both termini to evaluate their effects on efficacy (Fig. 1a,b and Supplementary Table 1). 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 the 5′ and 3′ ends. We selected these modifications for evaluation owing to their previously reported stability to serum and snake venom phosphodiesterases and for their reported effects on the immunostimulatory properties of nucleic acids7–9.”, (p.985, col 2, para 2).
Hendel et al teaches that MS modified guide RNAs have higher indel rates than unmodified or only 2’-O-methyl modifications in Figure 1 C, E-G and Figure 2 A-D.
Hendel et al teaches, “we show that chemically synthesized sgRNAs can be used effectively for targeted genome editing, and we demonstrate that chemically modified sgRNAs significantly enhance genome editing efficiencies in human primary T cells and CD34+ HSPCs. Chemically synthesized and modified sgRNAs offer advantages over expressed or in vitro transcribed sgRNAs, including (i) increased efficacy, (ii) robust and scalable production of highly pure sgRNAs for biotechnological and therapeutic applications, (iii) greater flexibility in the sgRNA design in contrast to constraints on the first transcribed nucleotides imposed by the U6 or T7 promoters typically used for plasmid expression or in vitro transcription of sgRNAs, respectively, and (iv) enabling of a highly active RNA-only or RNP CRISPR platform with lower cytotoxicity in primary cells than DNA plasmid-based systems.”, (p. 989, col 1, para 2).
Lastly, Hendel et al teaches, “The simplification of the CRISPR-Cas system to a purely RNA or RNP CRISPR system lends itself to formulation in different nano particle vectors for delivery in vivo such that the nuclease will not be expressed continuously, as it would be when delivered as part of a viral vector. Furthermore, we anticipate that chemically modified sgRNAs will enhance multiplexed genome editing as well as high throughput, multiwell experiments. We also anticipate that modified sgRNAs will refine a wide range of CRISPR-associated technologies such as the CRISPRi/CRISPRa systems for inhibition and activation of gene expression19, the CRISPR imaging tool for dynamic visualization of genomic loci20, and CRISPR-mediated RNA recognition and cleavage21. The technology may be further developed to improve intracellular delivery to target cells or tissues, and enable conjugation to various molecules for imaging and biochemical studies.”, (p. 989, col 1, para 3).
Therefore, it would have been obvious to one of skill in the art before the effective filing date of the claimed invention to modify the teachings of claim 1 of 922 in view of Vakulskas et al, i.e., SEQ ID NO: 4 in a CRISPR system complexed with a variant spCas9, with the teachings of Hendel et al, i.e., chemically modifying the three 5’ terminal nucleotides with 2’-O-methyl and phosphorothioate linkages of sgRNA, to yield to predictable results of a CD33 guide RNA with 2’-O-methyl and phosphorothioate modification on the three 5’ terminal nucleotides.
One of skill would be motivated to modify such teachings, i.e., claim 1 of 922 in view of Vakulskas et al, with Hendel et al, because (1) claim 1 of 922 requires (a) a sgRNA targeting CD33 comprising a sequence that is 100% match to instant SEQ ID NO: 2 for use in HSPCs; and (2) Hendel et al teaches that (c) chemically synthesized and modified sgRNAs offer advantages over expressed or in vitro transcribed sgRNAs, (d) chemically modified sgRNAs significantly enhance genome editing efficiencies in human primary T cells and CD34+ HSPCs (same cell types that are used in claim 1 of 922), (e) chemically modified sgRNAs have increased efficacy, (f) chemically modified sgRNAs have robust and scalable production of highly pure sgRNAs for biotechnological and therapeutic applications, (g) chemically modified sgRNAs have greater flexibility in the sgRNA design in contrast to constraints on the first transcribed nucleotides imposed by the U6 or T7 promoters typically used for plasmid expression or in vitro transcription of sgRNAs, respectively, and (h) chemically modified sgRNAs enable a highly active RNA-only or RNP CRISPR platform with lower cytotoxicity in primary cells than DNA plasmid-based systems.
One of skill would look to the disclosures of both claim 1 of 922 in view of Vakulskas et al and Hendel et al and arrive at the claimed invention with a high likelihood of success.
Accordingly, Claim 103 is rejected on the ground of nonstatutory double patenting as being unpatentable over U.S. Patent No. 10548922 B2 in view of Vakulskas et al in further view of Hendel et al.
Response to Arguments – Double Patenting
The previous rejection of claim 21 for Nonstatutory Double Patenting is moot in view of Applicant’s cancellation of the claim filed 08/14/2026.
Applicant’s arguments, see page 15 of the reply filed 08/14/2026, with respect to the rejection(s) of claim(s) 1 and 96 under Nonstatutory Double Patenting have been fully considered and are persuasive. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made in view of newly found prior art.
Reasons for withdrawal can be found under the 35 U.S.C. 103 response to arguments section.
The previous rejection of claim 102 under Nonstatutory Double Patenting for being unpatentable over U.S. Patent No. 10548922 B2 in view of Bolen (supra) et al in further view of Fu et al (supra) has been withdrawn in in view of Applicant’s amendments of the claim filed 08/14/2026.
Reasons for withdrawal can be found under the 35 U.S.C. 103 response to arguments section.
Conclusion
No claims allowed.
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/L.M.T./Examiner, Art Unit 1637
/Jennifer Dunston/Supervisory Patent Examiner, Art Unit 1637
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