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 .
Claim Status
Claims 1-18 are examined on the merits.
Priority
This application is a national stage application, of International Patent Application No. PCT/US2022/017578, filed 02/23/2022, which claims priority from U.S. Provisional Application 63152761, filed 02/23/2021 is acknowledged.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-18 are 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.
Claim 1 requires the provision of a genus of “ a) DNA localization components”. The claims encompass any DNA localization component such as Zinc finger proteins, Transcription Activator-like Effectors, DNA-guided Argonaute systems. Thus, the claim encompasses the provision of a genus of DNA localization components that must function to bind DNA but it does not define its structure.
Claim 1 requires the provision of (i) or “an inactivated nuclease domain thereof”. The claim encompasses only an isolated “inactivated nuclease domain” (like HNH or RuvC domains in Cas9), it will not bind a guide RNA on its own.
To provide adequate written description and evidence of possession of a claimed genus, the specification must provide sufficient distinguishing identifying characteristics of the genus. The factors to be considered include disclosure of a complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, and any combination thereof.
The specification envisions a method of producing a plurality of modified human induced pluripotent stem cells (iPSCs) comprising at least one targeted nucleic acid insertion in the genome at a selected site, the method comprising: i) providing to the iPSCs a composition comprising at least one messenger RNA (mRNA) encoding at least one DNA localization component and at least one an effector molecule or a nucleic acid sequence encoding the same, and ii) providing to the iPSCs at least one vector comprising a nucleic acid insertion cassette to allow targeted nucleic acid insertion at the selected site; iii) culturing the iPSCs in conditions sufficient to produce at least one targeted nucleic acid insertion in the genome at the selected site; and wherein greater than 1 % of the plurality of modified iPSCs comprise the targeted nucleic acid insertion in the genome (e.g., paragraph 018). The specification envisions the DNA localization component comprises at least one guide RNA (gRNA), and wherein the effector molecule is a polypeptide comprising a nuclear localization signal and a Clo05 l fused with an inactivated Cas9 (dCas9) or an inactivated nuclease domain thereof (e.g., paragraph 020). The specification envisions, the DNA localization component comprises two guide RNAs (gRNAs), wherein a first gRNA specifically binds to a first strand of a double-stranded DNA target sequence and a second gRNA specifically binds to a second strand of the double stranded DNA target sequence. In some embodiments, the DNA localization component comprises two guide RNAs (gRNAs), wherein a first gRNA specifically binds to at least a first site of the vector and a second gRNA specifically binds to at least a second site of the vector (e.g., paragraph 021). The specification envisions genetically modifying iPSCs using a composition comprising Cas-Clover (NLS-dCas9-Clo05 l-NLS) results in higher insertion rates for large polynucleotide insertions ( e.g. larger than 3 kb in size), in comparison to conventional CRISPR/Cas9 systems. A cut is produced only when the Clo051 portions of the Cas-Clover dimerizes following localization to the genome by sgRNA homology. sgRNA sequences also have homology to the plasmid DNA, which allows tethering of the Cas-Clover with the DNA plasmid, and within the cell to prevent degradation. The distances between sgRNA sequences are optimized to enhance Cas-Clover dimerization in order to allow DNA cleavage (e.g., paragraph 053). The specification envisions that enzymes can be used to create strand breaks in the host genome to facilitate delivery or integration of the transgene. Enzymes can create single-strand breaks or double-strand
breaks. Non-limiting examples of break-inducing enzymes include transposases, integrases, endonucleases, CRISPR/Cas9, transcription activator-like effector nucleases (TALEN), zinc finger nucleases (ZFN), Cas-CLOVER™, and CPF1. Break-inducing enzymes can be
delivered to the cell encoded in DNA, encoded in mRNA, as a protein, or as a nucleoprotein complex with a guide RNA (gRNA) (e.g., paragraph 070).
The working examples disclose that of the protocol to correct sickle cell mutation using a composition of Cas-Clover, two sgRNAs and a DNA plasmid and additionally using PBx with Footprint-Free plasmid. The proportion of iPSC cells with modification (measured by GFP). Cas-Clover shows a 3-fold knock-in efficiency in comparison to CRISPR/Cas9 gene editing. Cas-Clover gene editing resulted in over 5% of modified iPSCs, without any additional purification. In contrast, CRISPR/Cas9 gene editing resulted in only 1.63% of modified iPSCs e.g., paragraph 0571; Fig. 9; Example 5).
The examples described in the specification does disclose that the DNA localization component is a gRNA in the Cas-Clover system. The specification only provides data for gRNA as a DNA localization component, and it is not representative of a broad DNA localization components allowed by the claims. Furthermore, the examples describe in the specification does not meet the limitation of the rejected claim “an inactivated nuclease domain”. There is insufficient guidance provided indicating any elements that are critical to the functioning of the inactivated nuclease domain capable of binding the DNA localization domain.
The state of the art with respect to using inactivated nuclease domain of Cas is underdeveloped and unpredictable. Nishimasu et al. (Cell, 2014) teaches that a crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA, at 2.5 Å resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA (abstract). Nishimatsu teaches that the crystal structure revealed that Cas9 consists of two lobes, a recognition (REC) lobe and a nuclease (NUC) lobe (Figures 1A–D). The REC lobe can be divided into three regions, a long α-helix referred to as the Bridge helix (residues 60–93), the REC1 (residues 94–179 and 308–713) domain, and the REC2 (residues 180–307) domain (Figures 1A–D). The NUC lobe consists of the RuvC (residues 1–59, 718–769 and 909–1098), HNH (residues 775–908), and PAM-interacting (PI) (residues 1099–1368) domains (e.g., paragraph 2nd, page 4; Fig. 1). The sgRNA guide region is primarily recognized by the REC lobe (e.g., paragraph 2nd, page 8; Fig. 5). Nishimatsu teaches that a Cas9 mutant lacking the REC2 domain (Δ175–307) retained ~50% of the wild-type Cas9 activity (Figure 2B), indicating that the REC2 domain is not critical for DNA cleavage. In striking contrast, the deletion of either the repeat-interacting region (Δ97–150) or the anti-repeat-interacting region (Δ312–409) of the REC1 domain abolished the DNA cleavage activity (Figure 2B), indicating that the recognition of the repeat:anti-repeat duplex by the REC1 domain is critical for the Cas9 function (e.g., paragraph 4th, page 4; Fig. 2).
As such, the prior art teaches about the unpredictability of using the NUC lobe (the nuclease domain), the protein lacks the REC lobe required for biding the gRNA, therefore, the nuclease cannot find the DNA target.
The claims encompasses significantly more than what is disclosed in the specification and does not satisfy the written description requirement under 35 U.S.C 112(a).
Therefore, the skilled artisan would have reasonably concluded applicants were not in possession of the claimed invention for claims 1-18.
The claims listed in the statement of rejection but not otherwise discussed are rejected because they are similarly not limited to particular amino acids that are considered to be adequately described by the specification.
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 8, 12 and 15 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
The term “small Cas9” in claim 8 is a relative term which renders the claim indefinite. The term “small” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. One would not know where the cut-off for a small Cas9 lies. Accordingly, the metes and bounds of the claim are unclear. A broad range or limitation together with a narrow range or limitation that falls within the broad range or limitation (in the same claim) may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173.05(c). In the present instance, claim 8 recites the broad recitation “inactivated small Cas9”, and the claim also recites “dSaCas9” which is the narrower statement of the range/limitation. The claim(s) are considered indefinite because there is a question or doubt as to whether the feature introduced by such narrower language is (a) merely exemplary of the remainder of the claim, and therefore not required, or (b) a required feature of the claims. The specification uses the term “dSaCas9” to refer to a Staphylococcus aureus Cas9 (e.g., paragraph [078]). Thus, it is unclear if the claim limitation drawn to inactivated Staphylococcus aureus Cas9 (dSaCas9) is required or merely an example of an inactivated small Cas9.
Claim 12 recites that the vector is provided in an amount “at least about” 1 ug. The specification does not provide a definition for determining the scope of “about”; it is not clear if 0.8 ug or 0.9 ug would fall within the scope of the claim, the limitation “at least about” is subjective. The claim does not clarify if this is the total mass in a single reaction, or mass per million cells or per volume of transfection.
The term “highly expressive locus” in claim 15 is a relative term which renders the claim indefinite. The term “highly expressive” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The disclosure does not provide a standard by which to judge a particular locus as being highly expressive or not.
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 15 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. 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. Claim 15 depends from claim 1 and recites, “wherein the selected site is a safe harbor locus, highly expressive locus, temporally expressed locus, or a gene locus for interruption.” Claim 1 recites, “comprising at least one targeted nucleic acid insertion in the genome at a selected site…wherein greater than 2% of the plurality of modified iPSCs comprise the targeted nucleic acid insertion site in the genome.” Thus, the independent claim is drawn to any gene locus for interruption by insertion of a nucleic acid. By reciting “a gene locus for interruption” as one of the alternatives in claim 15, the claim does not further limit the scope of claim 1. This rejection may be overcome by deleting “a gene locus for interruption” from the list of alternatives in claim 15.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 17-18 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Frendewey et al. (“Frendewey”, US 2015/0159174 A1).
Frendewey teaches methods for modifying a genomic locus of interest in a eukaryotic cell, a mammalian cell, a human cell or a non-human mammalian cell using a
large targeting vector (LTVEC) comprising various endogenous or exogenous nucleic acid sequences. Further methods combine the use of the LTVEC with a CRISPR/Cas system (e.g., abstract). Frendewey teaches targeting human iPS cells with an LTVEC. The LTVEC comprised a 16.7 kb nucleic acid comprising mouse Adam6a and Adam6b genes flanked by homology arms containing 34 kb and 105 kb of genomic DNA derived from genomic regions that flank the 4.1 kb sequence of the human ADAM6 locus intended for deletion (It reads on insertion is greater or equal to 3 kb as required by the instant claims) (e.g., paragraph 0984; Fig. 51 [see below]).
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The instant claims are product-by-process claims and therefore read on a cell produced by any means that would provide a cell having the same structural characteristics of a cell produced by the process recited in the claims (see MPEP § 2113). With regard to the structure implied by the process steps recited in the claim, the originally filed specification states: “genetic modulation of pluripotent stem cells using molecular strategies that target specific loci, which result in the stable integration and function of edited genetic material upon stem cell differentiation” (e.g., paragraph 002). In view of this, the skilled artisan would conclude, absent any evidence to the contrary, that the cell of instant claims 17-18 is the same as iPSC homozygous for Glu6Val sickle cell disease and corrected by Cas9 RNP by Martin.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The 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.
Claims 1-5, 8-18 are rejected under 35 U.S.C. 103 as being unpatentable over Yang, et al. (“Yang”, WO 2019/025984 A1) in view of Ostertag et al. (“Ostertag” WO 2018/064681 A1, cited as reference 024 on IDS filed 05/032024).
Regarding claims 1-3, 5, 8, 10, 15, Yang teaches method of making an iPS-derived BCD disease model. Such a method includes obtaining cells from a subject having endogenous mutations in the CYP4V2 gene or cells with no endogenous mutations in the CYP4V2 gene but exogenous CYP4V2 mutation is introduced artificially via gene editing or gene manipulation at this stage or any of the following stages; inducing pluripotency in the cells or reprogramming the cells to produce iPSCs; culturing the iPSCs under conditions that result in differentiation of the iPSCs into desired ocular cells, thereby producing an iPS-derived ocular cell line (e.g., paragraph 3rd, page 13). Yang teaches methods and compositions for generating genetically repaired autologous cells for cell therapy. As used herein, "genetically repaired" or "genetic repair" refers to correction of the CYP4V2 mutations through either gene editing of the patient's genome (e.g., directly on the chromosome using, e.g., CRISPR/Cas9, CRISPR/Cpfl, Zinc Finger, TALEN), or through gene transfer of a healthy copy of the CYP4V2 gene (cDNA, RNA or other form) into the patient cell, which typically does not integrate into the genome (e.g., the CYP4V2 gene therapy as described here) or correcting or compensating for the defective mRNA in the patient's cell (e.g., paragraph 2nd, page 65). Yang teaches CRISPR gene editing therapy involves the use of a CRISPR associated protein (Cas) which is a nuclease and a CRISPR guide RNA. Typically, Cas generates a double-stranded break (DSB) but altered Cas can result in a single-stranded break ( e.g., SpCas9 Nickase (Cas9n D 1 OA)) or no break ( dCas9) (e.g., paragraph 3rd, page 71). Yang teaches that catalytically inactive dCas9 does not cut target DNA but can still attain a sequence replacement without any of the error-prone repair that normally accompanies Cas9 cutting (e.g., paragraph 1st, page 71). Yang teaches that the composition provided that includes: (a) a CRlSPR guide RNA targeting a nucleic acid sequence (It reads on DNA localization component) (the "target sequence") of or within 100 bps to the CYP4V2 gene, and (b) a functional CRlSPR-associated protein (Cas). In some embodiments, such a composition can further include (c) a donor nucleic acid sequence comprising all or a portion of a wild-type sequence or a functional sequence of the CYP4V2 gene for correction, disruption or replacement of CYP4V2 gene or a portion thereof (It reads on instant claim 15) (e.g., paragraph 3rd, page 17). Yang teaches that two sets of gRNAs designed to create DSB at different regions of the CYP4V2 gene can be used to generate a large deletion or a knockout mutation within the CYP4V2 gene or to knockout the entire CYP4V2 gene in cells, thereby generating a BCD cellular model containing a CYP4V2 mutation(s) (e.g., line 18, page 143). Yang teaches that a donor nucleic acid sequence can be provided in the form of a single-stranded DNA (ssDNA, or a single-stranded oligo DNA nucleotide (ssODN) or a vector. In some embodiments, the donor nucleic acid sequence is no more than about 1kb, 800bp, 600bp, 500bp, 400bp, 300bp, 280bp, 260bp, 240bp, 220bp, or 200bp for a donor nucleic acid sequence provided in a ssODN. In some embodiments, the donor nucleic acid sequence is no more than about 25kb, 20kb, 15kb, 10kb, 9kb, 8kb, 7kb, 6kb, 5kb, 4.5kb, 4kb, 3.5kb, or 3kb for a donor nucleic acid sequence provided in a vector. A donor nucleic acid sequence is symmetrical. A donor nucleic acid sequence is asymmetrical. The length of a donor nucleic acid sequence can be adjusted for higher HRD rate (It reads on claims 1-3, 10) (e.g., paragraph 1st, page 73). Yang teaches examples of genetically repaired iPSCs, iPS-RPE cells, iPS-PRCs, iPS-CE cells, iPS-CECs and/or other iPS-ocular cells derived from a BCD patient carrying specific mutation on CYP4V2 (homozygous c.802-8_810dell 7insGC mutation in the CYP4V2 gene) by using CRISPR Cas9 , gRNAs and a donor homology template. After confirming precise correction of the pathogenic mutation with no or minimal off target editing via sequencing, the iPSC corrected by gene editing is used to differentiate into and generate iPS-RPE cells or the other type of iPS-ocular cells (e.g., paragraph 3rd, Examples 19-22; Figs. 15-16).
Regarding claim 8, Yang teaches that the Cas9 comprises a Cas9 ortholog or a mutant Cas9 selected from: Streptococcus pyogenes (SpCas9), SpCas9 nickase (Cas9n D10A), SpCas9 (D1135E), eSpCas9, SpCas9-HFl, SpCas9 VRER, SpCas9 VQR SpCas9EQR Staphylococcus aureus (SaCas9) (e.g., paragraph 2nd, page 18).
Regarding claims 13, Yang teaches a donor nucleic acid sequence comprising all or a portion of a wild-type sequence or a functional sequence of the CYP4V2 gene for correction, disruption or replacement of CYP4V2 gene or a portion thereof (e.g., paragraph 3rd, page 17). (e.g., paragraph 2nd, page 28).
Regarding claim 16, Yang teaches the stem cell is an iPC cell, an MSC, an adult stem cell or a tissue specific stem cell. In some embodiments, the iPS cell is reprogrammed using one or more of the OCT4, SOX2, KLF4, and c-MYC transcription factors. In some embodiments, the genetically repaired cells demonstrate one or more of the following: an increase in non-defective CYP4V2 nucleic acid sequence in the cells; an increase in the amount of functional CYP4V2 polypeptides in the cells; and/or improved cell structure, morphology or function, as compared to before genetic repair is performed (e.g., paragraph 3rd, page 22). Yang teaches iPSCs express detectable levels of at least one marker including, without limitation, Oct-4, Sox-2, SSEA4, TRA-1-60, TRA-1-81, AP and/or NANOG (e.g., paragraph 1st, page 59).
Regarding claims 17-18, Yang teaches a method of treating or preventing BCD in a subject or a cell with a mutated CYP4V2 gene is provided. Such a method includes (i) identify the pathologic mutation in the subject or the cell through sequencing; (ii) finding Cas related PAM sites within the region spanning from about 100 bps upstream from the first nucleotide involved in the mutation to about 100 bps downstream from the last nucleotide involved in the mutation; (iii) identity various protospacer element sequences targeting the CYP4V2 sequence relevant to each PAM
site identified in (ii); (iv) assess activity level of each CRISPR guide RNA comprising a protospacer element sequence identified in (iii) and off-target editing profile based on the protospacer element sequence and PAM; (v) select one or more CRISPR guide RNA design based on (iv); (vi) design one or more donor nucleic acid sequence based on homology-based
repair (HDR) for correcting, disrupting or replacing the targeted CYP4V2 mutation; (vii) construct the CRISPR guide RNA, Cas and donor nucleic acid sequence (e.g., paragraph 2nd, page 19).
Yang does not teach the fusion peptide comprising dCas and Clo051 as required by the instant claims. However, this is cured by Ostertag.
Regarding claims 1, 8, Ostertag teaches methods of making and using modified stem-cell memory T cells (e.g., paragraph 03). Ostertag teaches that modified-T cells of the disclosure include, but are not limited to, those cells that express an antigen receptor comprising a protein scaffold of the disclosure. Modified-T cells of the disclosure include, but are not limited to, those cells that express a chimeric antigen receptor (CAR) (i.e. CAR-T cells of the disclosure) (e.g., paragraph 05). Ostertag teaches a method of producing a modified stem memory T cell (TSCM), comprising introducing into a primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a modified T cell, wherein the modified T cell expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a modified stem memory T cell (TSCM). TI1e disclosure provides a method of producing a plurality of modified stem memory T cells (TSCM), comprising introducing into a plurality of primary human T cell (a) a transposon composition comprising a transposon comprising an antigen receptor or a therapeutic protein and (b) a transposase composition comprising a transposase or a sequence encoding the transposase; to produce a plurality of modified T cells, wherein at least 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 65%, 70%, 7Y%, 80%, 85%, 90%, 95%, 99% or any percentage in between of the plurality of modified T cells expresses one or more cell-surface marker(s) of a stem memory T cell (TSCM), thereby producing a plurality of modified stem memory T cells (TSCM) (e.g., paragraph 07). Ostertag teaches methods of homologous recombination of the disclosure; the nuclease domain of a genomic editing composition or construct is capable of introducing a break at a defined location in a genomic sequence: of the primary human T cell. Cas9 is a catalytically inactive or "inactivated" Cas9 (dCas9). The Cas9 is a catalytically inactive or "inactivated" nuclease domain of Cas9. In certain embodiments, the dCas9 is encoded by a shorter sequence that is derived from a full length, catalytically inactivated, Cas9, referred to herein as a "small" dCas9 or dSaCas9 (e.g., paragraph 0232). Ostertag teaches the nuclease domain may comprise, consist essentially of or consist of a dCas9 or a dSaCas9 and a type IIS endonuclease. In certain embodiments of the disclosure, the nuclease domain may comprise, consist essentially of or consist of a dSaCas9 and a type IIS endonuclease, including, but not limited to, AciI, Fokl or Clo051 (e.g., paragraph 0235).
Regarding claim 9, Ostertag teaches dCas9-Clo051 nuclease domain SEQ ID NO 40 corresponding to SEQ ID NO 10 of the instant claims (e.g., paragraph 0236, see alignment below):
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Regarding claims 10-11, 14, Ostertag teaches plasmid maps for site-specific integration into the AAVS 1 site using either homologous recombination (HR) or MMEJ and corresponding sequences. Donor plasmids for testing stable integration into the genome of human pan T cells via A) site-specific (AAVS1) homologous recombination (HR), B) site-specific (AAVS1) microhomology-mediated end joining (MMEJ) recombination and C) TTAA-specific piggyBacTM transposition. For HR and MMEJ donor plasmids, GFP-2A-DHFR gene expression cassettes were flanked by CRISPR/Cas9 targeting sites (GFP reads on non-naturally occurring protein) (5’ and 3’ CRISPR sites [Fig. 14A below]) and homology arms for AAVS 1 site integration (e.g., paragraph 0115; Fig. 14A [see below])
Fig. 14A:
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Regarding claim 12, Ostertag teaches an example of one protocol for
modifying T cells to express a chimeric antigen receptor (CAR) under conditions that induce or preserve desirable stem-like properties of the T cells (e.g., paragraph 0295, example 1). Ostertag teaches that the amount of both transposon (1 μg) (the transposon comprising an antigen receptor or a therapeutic protein, it reads on targeted insertion in a vector) and transposase (5 μg) stays the same regardless of the number of cells/reaction. Transposition efficiencies remain unchanged between 5xl06 cells/100 μL reaction and 25xl06 cells/100 μL reaction (e.g., paragraph 0305, example 1).
Based on these teachings, it would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to modify the teachings of Yang -a method of making iPSC with a composition comprising a functional CRISPR-Cas9, a gRNA for targeting a DNA sequence and a donor sequence for correction, disruption or replacement of a target gene, wherein the donor sequence is provided by an oligonucleotide or a vector comprising from 0.1 kb to about 30 kb, and culturing the iPSCs under conditions that result in differentiation of the desired iPSCs into desired ocular cells, and substitute the CRISPR Cas9 taught by Yang with the teachings of Ostertag a fused small dSaCas9-Clo051 nuclease domain of SEQ ID NO 40 and a vector that comprises two gRNA sites flanking the 5’ and 3’ homology arms flanking a gene; for someone skilled in the art would have been obvious to use these teachings to achieve the predictable result of developing a method of making iPSC with a composition comprising a functional dCas-Clo051, a gRNA and a donor sequence for gene editing of defective genes in iPSC.
One of ordinary skill in the art before the effective filing date of the invention would have been motivated to try to develop a method for treatment of diseases by genetic modification of iPSC to produce gene-corrected and gene-modified cells for gene therapy.
Claims 6-7 are rejected under 35 U.S.C. 103 as being unpatentable over Yang, et al. (“Yang”, WO 2019/025984 A1) and Ostertag et al. (“Ostertag” WO 2018/064681 A1, cited as reference 024 on IDS filed 05/032024) as applied to claims 1-5, 8-18 above, and further in view of Liszewski K. (“Liszewski”, Genetic Engineering & Biotechnology News, 2018) and Jin et al. (“Jin”, Computational and Structural Biotechnology Journal, 2020).
Yang and Ostertag do not teach the DNA localization component comprising two gRNAs, as required by the instant claims. However, this is cured by Liszewski and Jin.
Liszewski discloses that Cas-CLOVER™ hybrid gene editing system fuses a functionally inactive Cas9 to the site-specific nuclease, Clo51. Cas-CLOVER is targeted using a set of two distinct gRNAs, operating like CRISPR-Cas9, but the system has the exquisite specificity of type IIS nucleases and causes no or very few off-target mutations (e.g., paragraph 1st, page 5; Fig. [see below]).
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Jin teaches that “deletion of mutant exon 23 (DEx23) (reads on target sequence) with clustered regularly interspaced short palindromic repeats/ CRISPR-associated 9 (CRISPR/Cas9) gene-editing technology can correct dystrophin gene expression in iPSCs. However, successful exon23 deletion and clonal isolation are very inefficient (~3%), and manual selection of each iPSC clone and genotyping to identify DEx23 is labor-intensive. To overcome these obstacles, we added a homology-directed repair (HDR) donor vector, which carries floxed fluorescent protein and antibiotic selection genes, thus allowing us to identify DEx23 iPSC with donor selective gene integration. Our results indicate that the HDR-mediated targeted integration enables DEx23 iPSC identification; the HDR donor vector increased the recognition efficiency of clonal isolation (>90% as confirmed by Sanger sequencing). After removal of the inserted genes by Cre-mediated recombination followed by doxycycline (Dox)-induced MyoD induction, DEx23 iPSC differentiated into MPC with restored dystrophin expression in vitro. Importantly, transplanted DEx23 iPSC-MPC express dystrophin in the muscles of a mouse model of DMD (Mdx mice) (e.g., abstract; Fig. 2). Jin teaches deletion of DMD exon 23 using the combination of CRISPR/Cas9 and HDR. (A) Schematic diagram of CRISPR/Cas9 and HDR mediated exon23 deletions. Step1: The Cas9 nuclease targets intron 22 and intron 23 by two gRNAs (it reads on two gRNAs binding to first and second targeting sites, upstream and downstream of exon 23 respectively). Double-stranded breaks (DSBs) by Cas9 results in the excision of the mutant exon23. Step 2: Through HDR, a portion of the open reading frame (ORF) is replaced with the selection cassette containing GFP and puromycin resistance markers. step 3: The dual-marker cassette, flanked by loxP sites, can be excised from the genome by Cre recombinase. (B) Sorted mouse iPSCsCas9-gRNA-HDR and iPSCsCas9-gRNA-HDR-CRE were imaged by fluorescent microscopy using a GFP filter (scale bar = 200 mm). (C) PCR genotyping analysis of GFP and exon23 (e.g., Fig. 2; [see Fig. 2A below]).
Fig. 2A:
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Based on these teachings, it would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to modify the teachings of Yang and Ostertag -a method of making iPSC with a composition comprising a functional dCas-Clo051, a gRNA and a donor sequence, with the teachings of Liszewski -gene editing system comprise of a functionally inactive Cas9 and Clo051 and for targeting using a set of two distinct gRNAs and the teachings of Acosta a method of modifying iPSC using CRISPR/Cas9 with a dual guide RNAs for gene editing; for someone skilled in the art would have been obvious to use these teachings to achieve the predictable result of developing a method of making iPSC with a composition comprising a functional dCas-Clo051, two gRNAs and a donor sequence for gene editing of defective genes in iPSC.
One of ordinary skill in the art before the effective filing date of the invention would have been motivated to try to develop a method for treatment of diseases by genetic modification of iPSC to produce gene-corrected and gene-modified cells for gene therapy.
Conclusion
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/JULIO WASHINGTON GOMEZ RODRIGUEZ/ Examiner, Art Unit 1637
/Jennifer Dunston/ Supervisory Patent Examiner, Art Unit 1637